The Role of Recombinant Oviduct-Specific Glycoprotein (OVGP1) During Early Events of Fertilization
by
Yuewen Zhao
A thesis submitted to the graduate program in the Department of Biomedical and Molecular Sciences In conformity with the requirements for the degree of Doctor of Philosophy
Queen’s University, Kingston, Ontario, Canada (May 2019)
Copyright ©Yuewen Zhao 2019 Abstract
The mammalian oviductal secretory cells synthesize and secrete a major glycoprotein known as the oviduct-specific glycoprotein (OVGP1) which is in abundance around the time of fertilization. In vitro studies carried out in various mammalian species have shown that native OVGP1 interacts with gametes and early embryos; this interaction can enhance the fertilizing capacity of sperm, increase cleavage rate of early embryos, and prevent polyspermy.
However, functional and mechanistic studies of OVGP1 are hampered due to the difficulty in isolating and purifying OVGP1 from oviductal fluid. In the present study, we successfully produced and purified a secretory form of recombinant hamster OVGP1 (rHamOVGP1) and recombinant human OVGP1 (rHuOVGP1) from stably transfected HEK293 cells. Results from this study showed that both rHamOVGP1 and rHuOVGP1 can bind to their homologous sperm in both the head and tail regions. Both recombinant proteins can enhance sperm capacitation through the increase of tyrosine phosphorylation of sperm proteins. In hamsters, tyrosine phosphorylated (pY) proteins were located in the equatorial segment, mid-piece, and principal piece. One of the major hamster sperm pY proteins is likely to be AKAP83. In human sperm, pY proteins were located predominantly in the fibrous sheath of the principal piece. One of the identified major pY proteins was AKAP3. AKAP proteins play a role in regulating sperm capacitation and motility. Therefore, recombinant OVGP1s are capable of further enhancing sperm capacitation likely through the increase of the signaling events that lead to increased sperm motility. In addition, the presence of recombinant OVGP1s in the capacitating medium was able to further potentiate the sperm to undergo acrosome reaction. Furthermore, the present
i study showed that pre-treatment of the zona pellucida and/or sperm with recombinant OVGP1s can also increase the number of sperm bound to the zona pellucida of oocytes. Cryopreserved human sperm samples were also used in this study and found to benefit from the presence of rHuOVGP1 which resulted in increasing the sperm fertilizing capacity. It is envisaged that supplementing the conditioned medium used in assisted reproductive technology with rHuOVGP1 could be beneficial for enhancing the sperm fertilizing competence.
ii Acknowledgement
First of all, I would like to express my gratitude towards my supervisor, Dr. Frederick
Kan for his tremendous help and continuous mentorship. Studying in the field of reproductive biology, especially assisted reproductive technology has been my long-term interest of research.
I am very thankful for this invaluable opportunity to carry out the current study. In his guidance,
I have not only obtained knowledge in the field, but also learned to be initiative, independent, committed, critical and optimistic to strive for excellence. I would have not been able to complete the thesis without his great support.
I would also like to thank Dr. Xiaojing Yang for her hard work towards the current thesis. Her meticulous work and successful results were the stepping stone for the current study.
I would like to thank the wonderful supervisory committee members and professors from the department, Dr. Chandrakant Tayade, Dr. Richard Oko, Dr. Charles Graham, and Dr.
Stephen Pang, for their advice and inspirations. I would also like to thank Dr. Xiaolong Yang,
Dr. Zongchao Jia, Dr. Shetuan Zhang, Dr. Leda Raptis, and Dr. Richard Oko, as well as their warm-hearted lab members for sharing their lab equipment, providing technique training and consultations and generously lending experimental materials.
I would like to thank all the collaborators outside the department, Dr. Robert Reid and
Dr. Dean Van Vugt from the Kingston General Hospital, Dr. Pierre Leclerc from Laval
University, Dr. Tamer Said from ReproMed, and Dr. Keith Jarvi from the Mont Sinai Hospital,
iii for their generosity to provide us with samples and for their contributions towards the current thesis.
I would like to also thank all the current and past lab members from Dr. Kan’s lab for their inspirations and help in designing the projects and performing the experiments.
Last but not least, I would like to thank my beloved family members. I would like to thank my husband for his great love and support in every aspect of life. I would like to thank my parents for their boundless love and generous financial support. At the end, I would like to thank my lovely son, for being my sunshine, my angel, and my strength to march on.
iv List of Co-Authorship Chapter 3 Published as: Yang X, Zhao Y, Yang X and Kan FW (2015) Recombinant hamster oviductin is biologically active and exerts positive effects on sperm functions and sperm-oocyte binding. PloS One 10 e0123003. YX, ZY, and KFW all designed the experiments and wrote the manuscript. Experiments were mostly done by YX.
Chapter 4 Published as: Zhao Y, Yang X, Jia Z, Reid RL, Leclerc P and Kan FW (2016) Recombinant human oviductin regulates protein tyrosine phosphorylation and acrosome reaction. Reproduction 152 561–573. ZY, YX, and KFW all designed the experiments and wrote the manuscript. YX performed protein purification experiments. YX performed the protein purification experiments and prepared Figure 4- 1 and 2. ZY performed the functional studies and prepared Figure 4- 3, 4 and 5. JZ, RRL and LP assisted with methods and provided human sperm samples.
Chapter 5 Submitted for publication: Zhao Y and Kan FW (2019) Human OVGP1 Enhances Tyrosine Phosphorylation of Proteins in the Fibrous Sheath involving AKAP3 and Increases Sperm-Zona Binding. (Submitted to Journal of Assisted Reproduction and Genetics on March 20, 2019) ZY and KFW designed the experiments and wrote the manuscript. Experiments were performed by ZY.
Chapter 6 In preparation for publication: Zhao Y and Kan FW Effects of Recombinant Human OVGP1 on Protein Tyrosine Phosphorylation and Acrosome Reaction in Cryopreserved Human Sperm. ZY and KFW designed the experiments and wrote the manuscript. Experiments were performed by ZY
v Table of Contents
Abstract ...... i
Acknowledgement ...... iii
List of Co-Authorship ...... v
Table of Contents ...... vi
List of Figures ...... xii
List of Abbreviations ...... xiv
Chapter 1
General Introduction ...... 1
Chapter 2
Literature Review ...... 5
2.1 Oviduct ...... 6
2.1.1 Anatomical Structure of the Human Oviduct ...... 6
2.1.2 Anatomical Structure of the Hamster Oviduct...... 7
2.1.3 Histological Features of the Oviduct ...... 7
2.1.3 Secretions of the Oviduct ...... 9
2.2 Oviductin (OVGP1) ...... 11
2.2.1 General Introduction of OVGP1 ...... 11
2.2.2 Molecular Characterization of OVGP1 ...... 12
2.2.3 Localization and Biological Functions of Mammalian OVGP1 ...... 16
2.2.3.a Localization of OVGP1 in the oviductal epithelium ...... 16
2.2.3.b Localization of OVGP1 in sperm ...... 17
2.2.3.c Localization of OVGP1 in oocytes ...... 18
2.2.3.d Localization of OVGP1 in developing embryos ...... 20
2.2.3.e Extra-oviductal localization of OVGP1 ...... 21
vi 2.2.4 Production of recombinant OVGP1 ...... 23
2.3 Fertilizing Competence of Mammalian Sperm ...... 23
2.3.1 Structure and Composition of Mammalian Sperm ...... 24
2.3.1.a The sperm head ...... 25
2.3.1.b The sperm tail ...... 26
2.3.2 Sperm capacitation ...... 29
2.3.2 Acrosome reaction ...... 30
2.3.3 Zona pellucida of the oocyte and sperm-zona binding ...... 31
2.4 Hypotheses ...... 33
Chapter 3
Recombinant Hamster Oviductin Is Biologically Active and Exerts Positive Effects on
Sperm Functions and Sperm-Oocyte Binding ...... 44
3.1 Abstract ...... 45
3.2 Introduction ...... 47
3.3 Materials and Methods ...... 49
3.3.1 Animals and reagents ...... 49
3.3.2 Plasmid construction ...... 50
3.3.3 Cell culture, transfection, and establishing the rHamOVGP1 stable cell line ...... 51
3.3.4 Purification of secreted rHamOVGP1 from serum-free cell culture medium by
affinity chromatography...... 53
3.3.5 Identification of rHamOVGP1 by immunoblot and mass spectrometric analysis .. 53
3.3.6 Preparation of sperm from the caudal epididymis ...... 54
3.3.7 Tyrosine phosphorylation of sperm proteins during in vitro capacitation ...... 56
3.3.8 SDS-PAGE and immunoblotting of tyrosine phosphorylated sperm proteins ...... 56
3.3.9 Immunofluorescent detection of binding of rHamOVGP1 to sperm ...... 58
3.3.10 Immunofluorescent microscopy of capacitated sperm ...... 58
3.3.11 Assessment of acrosome reaction ...... 59 vii 3.3.12 Superovulation and oocyte retrieval ...... 60
3.3.13 Immunolocalization of rHamOVGP1 in the ZP of ovarian oocytes ...... 60
3.3.14 Sperm-oocyte binding assay ...... 61
3.4 Results ...... 62
3.4.1 Production and purification of rHamOVGP1 ...... 62
3.4.2 Localization of rHamOVGP1 binding sites on hamster sperm ...... 64
3.4.3 rHamOVGP1 enhanced protein tyrosine phosphorylation in hamster sperm ...... 65
3.4.4 Localization of rHamOVGP1-enhanced tyrosine phosphorylated proteins in
hamster sperm ...... 66
3.4.5 Effects of rHamOVGP1 on acrosome reaction ...... 68
3.4.6 Binding of rHamOVGP1 to the ZP of hamster ovarian oocytes ...... 68
3.4.7 Influence of rHamOVGP1 on sperm binding to ZP of oocytes ...... 69
3.5 Discussion...... 70
3.6 Conclusions ...... 77
Chapter 4
Recombinant Human Oviductin Regulates Protein Tyrosine Phosphorylation and
Acrosome Reaction ...... 92
4.1 Abstract ...... 93
4.2 Introduction ...... 94
4.3 Materials and Methods ...... 96
4.3.1 Materials ...... 96
4.3.2 Production of rHuOVGP1 in HEK293 cells ...... 97
4.3.3 Purification of secreted rHuOVGP1 collected from serum-free cell culture medium
...... 99
4.3.4 SDS-PAGE, Western blotting, and immunoprecipitation ...... 100
4.3.5 Trypsin digestion of samples and sequencing of peptides by mass spectrometry 101
viii 4.3.6 Immunofluorescence and confocal microscopy...... 101
4.3.7 Preparation of sperm from fresh semen samples ...... 102
4.3.8 Sperm incubation ...... 103
4.3.9 Binding of rHuOVGP1 to human sperm ...... 103
4.3.10 Binding of rHuOVGP1 to human sperm ...... 104
4.3.11 Evaluation of protein tyrosine phosphorylation ...... 105
4.3.12 Acrosome reaction assays ...... 106
4.3.13 Statistical analysis ...... 107
4.4 Results ...... 107
4.4.1 Establishment of a stable rHuOVGP1-expressing HEK293 cell clone and
purification of rHuOVGP1 ...... 107
4.4.2 Binding of rHuOVGP1 to human sperm ...... 109
4.4.3 Effect of rHuOVGP1 on the expression of phosphotyrosine-containing proteins in
human sperm ...... 111
4.4.4 Effect of rHuOVGP1 on sperm acrosome reaction ...... 113
4.5 Discussion...... 113
Chapter 5
Human OVGP1 Enhances Tyrosine Phosphorylation of Proteins in the Fibrous Sheath involving AKAP3 and Increases Sperm-Zona Binding ...... 129
5.1 Abstract ...... 130
5.2 Introduction ...... 132
5.3 Materials and Methods ...... 134
5.3.1 Materials ...... 134
5.3.2 Production of rHuOVGP1...... 134
5.3.3 Semen preparation and sperm capacitation...... 135
5.3.4 Oocyte collection, storage and recovery ...... 136
5.3.5 Oocyte microbisection ...... 137 ix 5.3.6 Hemizona assay ...... 137
5.3.7 Hemizona index analysis ...... 138
5.3.8 Immunofluorescent imaging of the binding of rHuOVGP1 to oocytes ...... 139
5.3.9 Immunofluorescent labeling of sperm ...... 139
5.3.10 Western Blot Analysis ...... 141
5.3.11 Immunoprecipitation of AKAP3...... 142
5.3.12 Statistical analysis ...... 143
5.4 Results ...... 143
5.4.1 Tyrosine phosphorylated proteins are predominantly localized to the fibrous sheath
(FS) of the sperm tail ...... 143
5.4.2 rHuOVGP1 enhances tyrosine phosphorylation of AKAP3 located in the fibrous
sheath ...... 145
5.4.3 rHuOVGP1 binds to the ZP of oocytes...... 149
5.4.4 rHuOVGP1 increases the number of sperm bound to ZP ...... 149
5.5 Discussion...... 150
5.6 Conclusion ...... 156
Chapter 6
Effects of Recombinant Human OVGP1 on Protein Tyrosine Phosphorylation and
Acrosome Reaction in Cryopreserved Human Sperm ...... 166
6.1 Abstract ...... 167
6.2 Introduction ...... 169
6.3 Materials and Methods ...... 172
6.3.1 Materials ...... 172
6.3.2 Preparation and capacitation of cryopreserved human sperm ...... 172
6.3.3 Immunofluorescent labeling of sperm proteins ...... 173
6.3.4 Western blot ...... 174
x 6.3.5 Acrosome reaction assays ...... 175
6.3.6 Statistical analysis ...... 176
6.4 Results ...... 176
6.4.1 Binding of rHuOVGP1 to the surface of cryopreserved human sperm ...... 176
6.4.2 Effect of rHuOVGP1 on phosphotyrosine-containing protein expression in
cryopreserved sperm ...... 177
6.4.3 Effect of rHuOVGP1 on sperm acrosome reaction ...... 179
6.5 Discussion...... 180
Chapter 7
General Discussion and Future Perspectives ...... 191
7.1 Discussion...... 192
7.2 Future Perspectives: ...... 201
7.3 Overall Summary ...... 204
Reference List ...... 207
Appendices ...... 233
Supplementary Figures and Ethics Approval ...... 233
xi List of Figures Figure 2- 1. Schematic diagram illustrating the human and hamster female reproductive tract.
...... 37 Figure 2- 2. Electron micrograph of hamster oviduct epithelium showing localization of
hamster OVGP1...... 38
Figure 2- 3. Multiple protein sequence alignment of mammalian OVGP1 proteins...... 40
Figure 2- 4. Schematic diagram showing the biological functions of OVGP1 discovered using
native OVGP1 in mammalian species...... 41
Figure 2- 5. Schematic diagram of sperm structures in the hamster and humans...... 42
Figure 2- 6. Schematic diagram of sperm capacitation...... 43
Figure 3- 1:Immunochemical identification of rHamOVGP1...... 80
Figure 3- 2: Purification and analysis of rHamOVGP1...... 81
Figure 3- 3: Confocal microscope imaging of rHamOVGP1 binding sites on hamster sperm
after capacitation...... 83
Figure 3- 4: Dose-dependent effect of rHamOVGP1 on tyrosine phosphorylation of hamster
sperm proteins...... 84
Figure 3- 5: Time course of protein tyrosine phosphorylation in caudal epididymal hamster
sperm...... 85 Figure 3- 6: Western blot analysis of tyrosine phosphorylation of hamster sperm proteins after
demembranation...... 86
Figure 3- 7: Immunolocalization of rHamOVGP1-enhanced tyrosine-phosphorylated proteins
in hamster sperm...... 87
Figure 3- 8: Effect of rHamOVGP1 on acrosome reaction of hamster sperm...... 88
Figure 3- 9: Immunolocalization of rHamOVGP1 in hamster ovarian oocytes...... 90
Figure 3- 10: Effect of rHamOVGP1 (20 μg/mL) on sperm-oocyte binding...... 91
Figure 4- 1: Detection of rHuOVGP1 expression in HuOVGP1 cDNA transfected HEK 293
cells...... 124
Figure 4- 2: Purification of rHuOVGP1 and identity confirmation of the purified protein. . 125
xii Figure 4- 3: Binding of rHuOVGP1 to human sperm during in vitro capacitation...... 126
Figure 4- 4: Effect of rHuOVGP1 on tyrosine phosphorylation of proteins prepared from fresh
human sperm...... 127 Figure 4- 5: Effects of rHuOVGP1 on calcium ionophore-induced acrosome reaction of fresh
sperm...... 128
Figure 5- 1: Tyrosine phosphorylated proteins are predominantly localized to the fibrous sheath
of the sperm tail...... 158
Figure 5- 2: The effect of rHuOVGP1 on tyrosine phosphorylation of AKAP3...... 160 Figure 5- 3: AKAP3 is co-localized with pY proteins in the neck region and in the principal
piece of the sperm tail...... 162
Figure 5- 4: rHuOVGP1 binds to the ZP of human oocytes...... 163
Figure 5- 5: Effects of rHuOVGP1 on sperm-ZP binding...... 165
Figure 6- 1: Binding of rHuOVGP1 to cryopreserved sperm following capacitation...... 187 Figure 6- 2: Effect of rHuOVGP1 on tyrosine phosphorylation of proteins prepared from
cryopreserved sperm...... 188
Figure 6- 3: Tyrosine phosphorylated proteins are localized to the fibrous sheath of the sperm
tail...... 189
Figure 6- 4: Effect of rHuOVGP1 on calcium ionophore-induced acrosome reaction of
cryopreserved sperm...... 190
Figure 7- 1: Schematic diagram of the summary of the role of rHamOVGP1 and rHuOVGP1
in the early events of fertilization...... 206
Appendix Figure- 1. Dose dependent effect of rHuOVGP1 on protein tyrosine phosphorylation.
...... 234 Appendix Figure- 2. Schematic diagram showing the production and purification of
rHamOVGP1...... 235
Appendix Figure- 3. Schematic diagram showing the production and purification of
rHuOVGP1...... 236
xiii List of Abbreviations AKAP A-kinase anchoring protein
AR Acrosome reaction
ART Assisted reproductive technologies
BSA Bovine serum albumin
BWW Biggers-Whitten-Wittingham medium
CEC Cauda epididymal content
DABCO 1,4-Diazabicyclo-(2,2,2)-octane
DMSO Dimethyl sulfoxide
ECL Chemiluminescence
FITC Fluorescein isothiocyanate
FPLC Fast protein liquid chromatography
FS Fibrous sheath
GFP Green fluorescent protein
GH18 Glycoside hydrolase 18
GlcNac β-N-acetylglucosamine
HEK 293 Human embryonic kideney cells 293
HBS Hepes-buffered saline
HPA Helix pomatia agglutinin
HRP Horseradish peroxidase
HZI Hemizona index
IAM Inner acrosomal membrane
ICSI Intracytoplasmic sperm injection iERE Incomplete estrogen-responsive element i.p. Intraperitoneal
xiv IUI Intrauterine insemination
IVF In vitro fertilization
LV Lentivirus-derived vector
MALDI Matrix-Assisted Laser-Desorption
MS Mass spectrometry
MS Mitochondrial sheath
MYH9 Non-muscle myosin IIA
NGS Normal goat serum
ODF Outer dense fibers
OF Oviductal fluid
OVGP1 Oviductin, or oviduct-specific glycoprotein
PFA Paraformaldehyde
PSA Pisum sativum agglutinin
PKA Protein kinase A
PM Plasma membrane
PVDF Polyvinylidene fluoride pY Tyrosine-phophorylated
SDS Sodium dodecyl sulphate
TBS Tris-bufffered saline
TL-HEPES-PVA Tyrode-lactate-HEPES-polyvinyl alcohol
TALP Tyrode’s-Albumin-Lactate-Pyruvate rHamOVGP1 Recombinant hamster OVGP1 rHuOVGP1 Recombinant human OVGP1
VNTR mucin-type variable number of tandem-repeat
ZP Zona pellucida
xv
Chapter 1
General Introduction
1 In the world, about 15% of couples who seek to conceive are suffering from infertility, a condition defined by the failure to achieve a successful pregnancy after 12 months or more of regular unprotected intercourse (Gurunath et al., 2011; Bushnik et al., 2012; Thoma et al.,
2013). Many of the infertile couples used assisted reproductive technologies (ART), which are methods to achieve pregnancy with medical assistance. By 2018, at least 8 million babies were born since 1978 thanks to the invention of ART (European Society of Human Reproduction and Embryology, 2018). In order for the sperm to fertilize the oocytes, different types of ART that can be used include intrauterine insemination (IUI), a treatment that involves placing sperm into a woman’s uterus to facilitate fertilization; conventional in vitro fertilization (IVF), a procedure that sperm and oocytes are incubated in the culture medium; and intracytoplasmic sperm injection (ICSI), a technique that a single sperm is injected directly into the oocyte.
On average, about 27.3% ART cycles result in live-birth (Centers for Disease Control et al., 2016). Total fertilization failure, which is the failure of fertilization in all oocytes, occurs in 5-15% of couples undergoing conventional IVF despite an apparently normal sperm quality
(van der Westerlaken et al., 2005). ICSI is used as a treatment for unexplained total fertilization failure or low fertilization rate after conventional IVF. However, ICSI is an invasive procedure and has been shown to be associated with increased birth defect rates as compared to natural births (Bushnik et al., 2012; Simpson, 2014). As the particular sperm is selected objectively by an individual lab personnel, not selected by the oocyte per se, human error can affect the outcome of the procedure. Given the potential adverse effects of ICSI on the offspring, investigations to improve the conventional IVF success rate are warranted in order to
2 encourage patients with normal and mild male factor infertility to pursue the less invasive conventional IVF.
The culture medium plays an important role for the advancement of ART in the past few decades. Freshly ejaculated sperm are not capable of fertilizing oocytes in ex vivo conditions. Sperm are required to undergo a physiological process named “capacitation” prior to fertilization (Chang, 1951). Capacitation is broadly defined as the functional and physiological modifications rendering sperm to become competent to fertilize the egg, including the change of sperm motility from progressive to hyperactivated, gaining the ability to bind to the zona pellucida and undergo acrosome reaction, and the capacity to fuse with the oocyte (Yanagimachi, 1994; Bailey, 2010; Signorelli et al., 2012). In the native condition, sperm that have reached and resided in the oviducts acquire the maximum level of fertilizing capacity. Therefore, the physiological environment and molecular components of oviductal fluid have been shown to play an essential role in the early events of fertilization. The incubating medium that mimics the physiological condition of oviductal fluid has made the
IVF possible.
Among numerous components of the oviductal fluid, a major glycoprotein, named oviductin, or oviduct-specific glycoprotein (OVGP1) has been identified and characterized in many mammalian species. Previous studies demonstrated the ability of OVGP1 to bind to sperm and zona pellucida of their own species and increase sperm motility and viability (Abe et al., 1995), enhance sperm capacitation (King et al., 1994; Saccary et al., 2013), enhance
3 sperm-egg binding and penetration rates (Boatman and Magnoni, 1995; O’Day-Bowman et al.,
1996), decrease polyspermy (Coy et al., 2008), and increase cleavage rates of embryos and the number of embryos reaching the blastocyst stage (Hill et al., 1997; Kouba et al., 2000).
Especially, the purified native hamster OVGP1 has been well studied in our laboratory. The glycoprotein has been shown to be regulated by estrogen (McBride et al., 2004a), localized to zona pellucida as well as perivitelline space of hamster oocytes (McBride et al., 2004b), bind to hamster sperm and increase sperm capacitation (Saccary et al., 2013). Accumulating evidences suggest that supplementing medium with OVGP1 could be beneficial for the increase of successful rate of IVF. However, to obtain large quantities of OVGP1 from oviductal fluids to study the functional mechanism of OVGP1 involved in these events is technically difficult, particularly in humans, to obtain human oviducts for collecting sufficient amount of OVGP1 is ethically impossible. In order to circumvent this difficulty and in the hope of improving the fertilization rate of IVF, the present study, was designed to produce recombinant hamster and human OVGP1 using recombinant DNA technologies. It was hypothesized that these recombinant OVGP1s can bind to homologous sperm and enhance sperm fertilizing capacity. Furthermore, recombinant OVGP1 were used to explore its mechanism in enhancing sperm capacitation. In addition to using fresh human sperm sample, in the present study, cryopreserved human sperm samples were also employed for the purpose of investigating the beneficial role of OVGP1 in IVF using cryopreserved sperm.
4
Chapter 2
Literature Review
5 2.1 Oviduct
2.1.1 Anatomical Structure of the Human Oviduct
The female reproductive tract is composed of the external genitalia, vagina, uterus/uteri, the oviducts, and the ovaries (Fig 2-1). In humans, the oviduct is also named the Fallopian tube.
The present thesis focuses on studies involving the function of the oviduct. The paired oviducts are supported by ligaments and mesenteries and with openings into the peritoneal cavity near the ovary. Each oviduct is 10-12 cm in length in humans with four regions described as the following (Eddy and Pauerstein, 1980; Mescher, 2016): 1) the infundibulum is the funnel- shaped distal portion of the oviduct, approximately 1 cm in length, with fimbriated projections at the end and extensively folded (Patek et al., 1972a, b); 2) the ampulla is the longest portion of the human oviduct with 5-8 cm in length with the luminal diameter varies from more than 1 cm near the distal end to 1-2 mm at its junction with the isthmus (Eddy and Pauerstein, 1980);
3) the isthmus is the most proximal portion of the extrauterine oviduct with an average length of 2-3 cm and a diameter ranging from 0.1-1.0 c; and 4) the intramural segment of the oviduct passes through the wall of the uterus and opens into the uterine cavity. The ovarian artery and uterine artery anastomose to form the blood supply of the oviduct and are parallel to the venous drainage. The anatomical structure of the blood supply provides a counter-current mechanism that involves the local transfer of ovarian steroids (Leese, 1988). The oviduct is innervated by both sympathetic and parasympathetic nervous system that controls the contractility of the smooth muscles of the oviduct and may function to mix and transport the contents of the oviductal fluid, ova, spermatozoa and early embryos (Leese, 1988).
6 2.1.2 Anatomical Structure of the Hamster Oviduct
The 3 to 4 mm long, oval-shaped hamster ovaries are located dorsolateral to the kidneys and are completely enclosed within membranous bursae ovaricae. The hamster oviduct presents a tightly convoluted shape and averaging about 15 mm in length (Hoffman et al., 1968;
Murray, 2012). Like in the rat, the oviduct winds around the ovary and forms several loops
(Hebel and Stromberg, 1986). The proximal cone-shaped infundibulum contains the ostium and projects into the bursae ovaricae. After leaving the bursa, the oviduct, suspended by a long mesosalpinx, runs along the ovary and attaches to the wall of the bursae ovaricae. Following its final loop, the oviduct is directed caudally before it penetrates the tip of the uterus.
2.1.3 Histological Features of the Oviduct
. The wall of the oviduct consists of a thin serosa covered by visceral peritoneum with mesothelium, a muscularis with inner circular and outer longitudinal smooth muscle layers, and a mucosa with a pattern of complex longitudinal folds (Mescher, 2016). The oviductal mucosa is lined by simple columnar epithelium that consists of interspersed ciliated cells and non-ciliated secretory cells. The infundibulum and ampulla region of the oviduct are more densely ciliated than the isthmus(Eddy and Pauerstein, 1980).
The ciliated cells are characterized by long cilia extending into the lumen of the oviduct.
The secretory cells are elongated and have short microvilli on the apical cell membrane. A well-developed Golgi apparatus is located close to the nucleus of the secretory cells. Secretory granules largely occupy the cytoplasm of these cells and contain molecules that ultimately secreted into the oviductal lumen(Kan et al., 1989).
7 Variations in the type and number of the secretory granules are found in different regions of the oviductal epithelium, which could be important for the reproductive events that occur in the different segments of the oviduct (Abe and Oikawa, 1991). Marked differences in the morphological characteristics of the secretory granules were also reported in different species.
In cattle, during the follicular phase of the estrous cycle, non-ciliated cells of the ampulla and fimbriae contain large amount of secretory granules, while in the isthmus the number of cytoplasm granules is smaller and show different structural characteristics (Killian, 2011).
Secretions from the ampullary segment where fertilization occurs may contribute to sperm-egg interaction, whereas those from the isthmic region might contribute to capacitation of spermatozoa and/or maintenance of embryos (Abe and Oikawa, 1991). Estradiol induces hypertrophy of both the ciliated and secretory cells and formation of secretory granules within the secretory cells (Malette et al., 1995a; Verhage et al., 1997a; Mescher, 2016). Conversely, progesterone induces the regression of the oviductal epithelial cells (Verhage et al., 1997b;
Mescher, 2016).
Once entering the isthmic region of the oviduct, sperm tend to directly contact and adhere to the epithelium, which becomes a reservoir of sperm (Smith, 1998; Suarez, 1998). The interaction between sperm and the epithelium may maintain sperm viability and modulate the timing of capacitation. Once capacitated, sperm detach from the epithelium and leave the isthmus (Smith and Yanagimachi, 1991). Individual sperm seem to be capacitated at different rate and detach from the isthmic epithelium, a few at a time, over an extended period
(Yanagimachi, 1994). The sperm reservoir appears to coordinate with ovulation to ensure the
8 presence of fertile sperm in the ampullary portion of the oviduct at the time of fertilization
(Suarez, 1998; Wagner et al., 2002).
2.1.3 Secretions of the Oviduct
The mammalian oviduct and oviductal fluid (OF) provide a dynamic micro-environment for gamete transport, oocyte final maturation, sperm selection and capacitation, prevention of polyspermy, fertilization, early embryonic cleavage, embryonic genome activation, maternal immunologic response to gametes and developing embryos, as well as transport of embryo to the uterus (Pérez-Cerezales et al., 2018). The component of OF is contributed by secretions from oviductal non-ciliated epithelial cells and selective transudation from blood plasma
(Leese, 1988; Buhi et al., 2000; Aviles et al., 2010). The OF contains metabolic components that include glucose, lactate, pyruvate and amino acids with different respective concentrations comparing to those of the uterine fluid and plasma (Aviles et al., 2010). A large group of proteins have been identified in OF, including growth factors, cytokines and receptors, hormones and receptors, proteases and inhibitors, antioxidant protective agents, defense agents, glycosidases and glycosyl transferases, chaperones and heat shock proteins, mucins, glycosaminoglycans and proteoglycans (Buhi et al., 2000; Killian, 2004; Aviles et al., 2010;
Soleilhavoup et al., 2016), which have been shown to contribute to the development of the optimal environment for the different processes occurring in the oviduct. Analogs of the proteins present in the OF have been found across different mammalian species, suggesting that the functions of the oviduct are conserved (Seytanoglu et al., 2008; Cerny et al., 2015;
Soleilhavoup et al., 2016; Acuña et al., 2017). More than 1090 orthologous genes have been
9 found common to the oviduct transcriptome of humans, pigs, and cows (Tone et al., 2008;
Mondéjar et al., 2012; Cerny et al., 2015; Pérez-Cerezales et al., 2018).
Proteins of the OF and the oviductal epithelium are found differentially expressed according to the specific stages of the estrous cycle (Pérez-Cerezales et al., 2018). Likely under the influence of estrogen production during the menstrual cycle in the human, not only the volume of the OF from both oviducts increased drastically from approximately 0.1 mL around menstruation to 9.5 mL during the periovulatory phase, but also the amount of total protein was changed from less than 15 mg to around 180 mg in average with the highest during the periovulatory phase (Lippes et al., 1981). The production of a large amount of oviductal proteins during this phase is thought to benefit sperm capacitation, oocyte maturation, sperm- oocyte binding, and reduce maternal immune reaction towards the sperm and developing embryo (Pérez-Cerezales et al., 2018). Differential regulation of oviductal proteins during the estrous cycle was found in cows (Bauersachs et al., 2004; Cerny et al., 2015), pigs (Acuña et al., 2017), sheep (Soleilhavoup et al., 2016) and humans (Tone et al., 2008; George et al.,
2011). An analysis of the gene expression in bovine oviductal epithelial cells at estrus and diestrus showed that 972 and 597 transcripts were up- and down-regulated, respectively, in the epithelial cells of the ampulla region, while 946 and 817 transcripts were up- and down- regulated, respectively, in the epithelial cells of the isthmus region (Bauersachs et al., 2004).
The up-regulated transcripts were found largely involved in cholesterol biosynthesis and cell cycle regulations, while the down-regulated transcripts were found to be involved in inflammatory response pathways (Bauersachs et al., 2004). Another study carried out in ewes
10 have detected a set of 291 proteins out of 940 proteins from the reproductive tract that was differentially regulated throughout the estrus cycle (Soleilhavoup et al., 2016).
2.2 Oviductin (OVGP1)
2.2.1 General Introduction of OVGP1
Among the differentially regulated proteins in the oviductal fluid, one major glycoprotein was identified and named as oviductin, or oviductal glycoprotein 1 (OVGP1), which produced specifically by the oviductal secretory cells (Fig 2-2). Since the first discovery in the hamster that the oviductal originated antigen was able to bind to the zona pellucida of ovulated hamster oocytes (Fox and Shivers, 1975), OVGP1 has been identified and characterized in many mammalian species including mice (Kapur and Johnson, 1985; 1986), hamsters (Leveille et al.,
1987), rabbits (Oliphant and Ross, 1982; Oliphant et al., 1984), dogs (Saint-Dizier et al., 2014), cats (Hachen et al., 2012), sheep (Sutton et al., 1984; 1986; Gandolfi et al., 1989), pigs (Buhi et al., 1990), cows (Malayer et al., 1988; Boice et al., 1990a), rhesus monkeys (Verhage et al.,
1997a), baboons (Fazleabas and Verhage, 1986; Verhage et al., 1989, 1990), and humans
(Verhage et al., 1988), showing the ability of OVGP1 to bind to the sperm and zona pellucida of their own species. Recent proteomic study suggests that OVGP1 is quantitatively the most abundant protein in the oviductal fluid in the sheep with a 1.7-5.5 fold increase in estrus stage compared to diestrus phase (Soleilhavoup et al., 2016). OVGP1 has been found beneficial to multiple processes during fertilization.
11 2.2.2 Molecular Characterization of OVGP1
OVGP1 has been characterized in humans (Arias et al., 1994), baboons (Donnelly et al.,
1991), rhesus monkeys (Verhage et al., 1997a), hamsters (Suzuki et al., 1995), mice (Sendai et al., 1995), rabbits (Yong et al., 2002), cats (Hachen et al., 2012), cows (Sendai et al., 1994), pigs (Buhi et al., 1996), sheep (DeSouza and Murray, 1995) and goats (Pradeep et al., 2011).
These OVGP1s share high sequence identity (70-78%) and similarity (76-87%) in the N- terminal region of the protein and low sequence identity (37–63%) and similarity (50–75%) in the C-terminal region (Buhi, 2002). The glycosylated OVGP1 varies in molecular weights (90-
95 kDa in domestic animals; 120 kDa in primates; 160-350 kDa in rodents); however, the core protein size of several species studied to date is approximately the same (~70kDa). The variability in molecular weights is attributed to differences in glycosylation patterns (Roux et al., 1997; Verhage et al., 1997a). Hamster OVGP1 has a molecular weight of 71 kDa for the core protein. The OVGP1 purified from hamster oviductal fluid appears to be a group of high molecular weight and heterogeneous glycoproteins with a molecular weight of 160-250 kDa
(Robitaille et al., 1988; St-Jacques and Bleau, 1988; Paquette et al., 1995). In humans, the core protein is 75 kDa whereas the secreted form is about 110-130 kDa (O’Day-Bowman et al.,
1995).
The protein sequence alignment of the human, hamster, mouse, monkey, baboon, cow, goat, and sheep OVGP1s are shown in Figure 2-3. In silico motif scan of OVGP1 protein revealed a domain that resembled glycoside hydrolase 18 (GH18) family of chitinases in the
N-terminal region (Malette et al., 1995b). Chitins, the polymers of β-N-acetylglucosamine
12 (GlcNAc), are the major components of the scaffold of cell walls of fungi and the exoskeletons of arthropods, as well as the constituent of nematode cuticle and egg shells (Huang et al., 2012).
However, the catalytic domain of OVGP1 lacks an essential glutamic acid residue that donates a proton for the hydrolysis reaction, making OVGP1 an inactive chitinase (Huang et al., 2012).
Hydrophobic cluster analysis indicates that amino acids 386–525 in pig OVGP1 correspond to a C-terminal chitin binding domain (Buhi et al., 1996). Exon arrangement analysis also revealed the chitin binding domain in the human and mouse OVGP1 (Huang et al., 2012).
Owing to the divergence in the C-terminal domain, this region is thought to constitute both a binding domain and a species-specific recognition domain (Buhi, 2002).
The C-terminal region of OVGP1 contains many Ser-Thr residues for O-glycosylation
(Buhi et al., 1996). The C-terminal region of hamster OVGP1 contains 6 repeats of mucin-type variable number of tandem-repeat (VNTR) sequences with 15 amino acids in each repeat
(Paquette et al., 1995) while a similar region in the mouse OVGP1 contains 21 repeat sequences, each composed of 7 amino acids (Sendai et al., 1995). The C-terminal region of OVGP1 contains only 4 tandem-repeat sequences in the human, baboon and rhesus monkey, whereas that contains incomplete or no tandem-repeat sequences in cattle, sheep and pigs (Buhi, 2002).
Taking into consideration that these VNTR regions are usually heavily O-glycosylated, hamster and mouse OVGP1 that have more VNTR repeats may be more closely related to mucins than their counterparts in other species. In addition to O-linked glycosylation, human
OVGP also has four potential N-linked glycosylation sites near the C-terminus (Malette et al.,
1995b).
13 Glycosylation plays an important role in the biological activity of OVGP1. The glycans of
OVGP1 appear to be essential for maintaining the hydrophilicity of the protein in the extracellular environment, as complete deglycosylation of OVGP1 resulted in an insoluble protein (Satoh et al., 1995). Removal of sialic acid or N-linked glycans from bovine OVGP1 significantly reduces the ability of bovine OVGP1 to maintain sperm viability (Satoh et al.,
1995). Furthermore, addition of human OVGP1 inhibited hamster sperm binding to hamster oocytes, while hamster homologous OVGP1 enhanced sperm binding (Schmidt et al., 1997).
Similarly, baboon OVGP1 inhibited binding of human sperm to human ZP whereas human
OVGP1 enhanced it, even though baboon OVGP1 shares 95% sequence similarity with human
(O’Day-Bowman et al., 1996). Differences in tandem repeats, the presence and distribution of
N- and O-linked carbohydrate chains, the diversity of side chains, and the length of the C- terminal region may confer species specificity, mediate specific recognition events and regulate the biological activity of OVGP1.
The expression of OVGP1 appears to be regulated by steroid hormones. An incomplete estrogen-responsive element (iERE) (5′-GGTCANNNTGACT-3′) was found at the position
150-162 bp before the start codon of the promoter region of human OVGP1 (HuOVGP1) gene
(Agarwal et al., 2002). DNA sequence analysis of the Human OVGP1 putative promoter revealed little homology with its hamster and mouse OVGP1 gene counterparts; however, the same iERE site was found in the promoter region of OVGP1 of hamster and mouse. The iERE sequence of HuOVGP1 was capable of binding to estrogen receptor ER but not ER.
Evidence of estrogen regulated OVGP1 expression was discovered in several species,
14 including the human (Verhage et al., 1988; Arias et al., 1994; O’Day-Bowman et al., 1995;
Lok et al., 2002), baboon (Verhage et al., 1990; Arias et al., 1994; Jaffe et al., 1996), sheep
(Sutton et al., 1984; Buhi et al., 1991), cow (Malayer et al., 1988; Boice et al., 1990a), pig
(Buhi et al., 1990, 1996), and hamster (McBride et al., 2004a). In species with long ovulatory cycles, the maximum level of OVGP1 mRNA and protein expressions is found during the late follicular phase of the menstrual cycle in humans (Arias et al., 1994; O’Day-Bowman et al.,
1995; Lok et al., 2002) or during the first two days of the estrous cycle in pigs (Buhi et al.,
1996). OVGP1 mRNA and protein expression levels are very low or absent during the luteal phase or diestrus (Buhi et al., 1991, 1996; Arias et al., 1994; O’Day-Bowman et al., 1995;
Verhage et al., 1998; Lok et al., 2002). Hormone replacement with estrogen after ovariectomy restored oviductin mRNA and protein expression; conversely, treatment with progesterone suppressed both mRNA and protein expressions of OVGP1 (Buhi, 2002). In hamsters, the levels of OVGP1 mRNA were found to be relatively the same throughout the estrous cycle
(Komiya et al., 1996); however, a cyclic variation in glycosylated OVGP1 concentrations in the hamster oviduct epithelium was found with the highest levels during estrus and the lowest during diestrus (McBride et al., 2004a). A corresponding increase in glycosyltransferase activity in the hamster oviduct at the time of ovulation suggests that glycosylation of OVGP1 may be necessary for its full functions during the process of fertilization (McBride et al., 2004a).
Therefore, the protein expression of OVGP1 is under the influence of estrogen, although the mode of regulation can be different among species.
15 2.2.3 Localization and Biological Functions of Mammalian OVGP1
The biological functions of OVGP1 during early events of fertilization that are known to date are summarized in Figure 2-4 and explained in detail in the following sections. Briefly, In vitro studies from various mammalian species have shown that native OVGP1 can bind to sperm and oocyte and functions to enhance sperm capacitation, increase sperm motility and viability, enhance sperm-egg binding, increase penetration rate and fertilization rate, decrease polyspermy, increase embryo quality, and increase embryo development into blastocyst.
2.2.3.a Localization of OVGP1 in the oviductal epithelium
OVGP1 protein is expressed and secreted by secretory cells in the oviductal epithelium
(St-Jacques and Bleau, 1988; Maines-Bandiera et al., 2010; Saint-Dizier et al., 2014). Northern blot analysis has shown that mRNA transcripts for oviductin were found exclusively in the oviduct and not in any other tissues in hamsters (Suzuki et al., 1995). The production of
OVGP1 protein expression gradually increases as the animal becomes sexually mature
(Malette et al., 1995a). Electron microscopic immunocytochemistry revealed the presence of
OVGP1 within the secretory granules and Golgi saccules in secretory cells but not ciliated cells of the oviductal epithelium in hamsters (Kan et al., 1989; McBride et al., 2004a) (Fig 2-2).
Quantitative immunocytochemical analysis of OVGP1 in various species showed maximal production of OVGP1 around the time of ovulation (Abe and Oikawa, 1993; O’Day-Bowman et al., 1995). An increased level of protein expression during the peri-ovulatory phase suggests an important role of OVGP1 in the fertilization process.
16 2.2.3.b Localization of OVGP1 in sperm
OVGP1 has been shown to bind to sperm and has positive effects on sperm capacitation, viability, motility and acrosome reaction (King et al., 1994; Abe et al., 1995; Killian, 2004).
Binding of OVGP1 to sperm in vitro has been demonstrated by several research groups.
Binding of purified hamster OVGP1 to the head region of homologous sperm has been reported
(Boatman and Magnoni, 1995). The pattern of OVGP1 binding to sperm showed regional and temporal modifications during sperm capacitation. For the un-capacitated sperm, OVGP1 binds to the acrosomal region of sperm head, whereas after capacitation, OVGP1 binds to the equatorial segment and the post-acrosomal region (Boatman and Magnoni, 1995). The binding of OVGP1 to the equatorial segment also increases in intensities following capacitation as shown by immunolabeling (Kan and Esperanzate, 2006). As OVGP1 is the major glycoprotein secreted by the oviductal epithelium, binding of OVGP1 to the head region of sperm is thought to be important in the sperm-oviduct interaction.
The localization of OVGP1 to sperm varies among species. In the bovine, OVGP1 binds to the posterior region of the sperm head and the mid-piece of sperm tail (Abe et al., 1995). In humans, however, the binding of OVGP1 to sperm was less clear. An earlier study detected the binding of the oviduct-specific glycoprotein over the surface of the head region (Lippes and Wagh, 1989), however, in vitro capacitation of human sperm with partially purified
OVGP1 was unable to detect the binding of the glycoprotein to sperm surfaces (O’Day-
Bowman et al., 1996).
17 The binding of OVGP1 to sperm appears to exert positive effects on sperm functions.
Incubating bovine spermatozoa with purified OVGP1 in conditioned medium can enhance sperm capacitation, increase the potential for sperm to proceed to acrosome reaction, enhance the ability of sperm to fertilize bovine oocytes (King et al., 1994; Satoh et al., 1995), and increase sperm motility and viability in a dose-dependent manner (Abe et al., 1995). Purified bovine OVGP1 was found to increase the potential of bull sperm to proceed to acrosome reaction. Partially purified porcine OVGP1 added in the incubating medium at low concentrations was able to increase viability of porcine sperm (McCauley et al., 2003).
Purified Hamster OVGP1 from the estrus stage was shown to enhance sperm capacitation through the increase of levels of tyrosine phosphorylation of sperm proteins in a time dependent manner (Saccary et al., 2013). Therefore, the above findings of OVGP1 binding to sperm and enhance sperm fertilizing competence suggest that supplementing the incubating medium with
OVGP1 can be beneficial for sperm biological functions.
2.2.3.c Localization of OVGP1 in oocytes
Localization of OVGP1 to the zona pellucida of oviductal oocytes was one of the first features discovered for this glycoprotein (Araki et al., 1987). OVGP1 has been demonstrated to bind to the zona pellucida of ovarian oocytes in the hamster (St-Jacques and Bleau, 1988;
Abe and Oikawa, 1990; McBride et al., 2004b), specifically associated with the dense filamentous structures comprising the zona matrix (McBride et al., 2004b). OVGP1 is absent in the ovarian follicles in the ovary (McBride et al., 2004b). The presence of the glycoprotein was also detected on the microvilli of the post-ovulatory oocyte, in the perivitelline space, and 18 in the multi-vesicular bodies of early embryos (Kan et al., 1993; McBride et al., 2004b).
Similarly, in baboons, incubating oocytes with extracted oviductal fluid or purified homologous OVGP1 also showed binding of OVGP1 to the zona pellucida (O’Day-Bowman et al., 2002).
Binding of OVGP1 to the zona pellucida appears to modify the functions of zona pellucida.
Co-incubating human sperm and hemizonae in the presence of human OVGP1 and co- incubation of hamster sperm and ovarian oocytes in the presence of hamster OVGP1 enhanced sperm-zona binding and sperm-egg penetration (Boatman and Magnoni, 1995; O’Day-
Bowman et al., 1996). Despite the high homology between human and baboon OVGP1, addition of baboon OVGP1 in the human hemizona binding assay showed a decrease in binding of sperm to hemizonae, indicating the importance of species specificity in sperm-egg binding
(O’Day-Bowman et al., 1996).
In livestock, polyspermy is a common phenomenon that affects the in vitro fertilization rate (Suzuki et al., 2003). OVGP1 has been shown to decrease the polyspermy rate in these species. Pretreatment of bovine and porcine ovarian oocytes prior to incubation with the respective sperm has shown positive effects on fertilization. Bovine oocytes preincubated with homologous OVGP1 resulted in a significant increase in fertilization rates, especially a smaller number of sperm was used to fertilize the oocytes (Martus et al., 1998). In pigs, polyspermy was significantly reduced when oocytes were treated with low concentrations of OVGP1 while high penetration and fertilization rates were maintained (McCauley et al., 2003). Similarly, in another study, porcine OVGP1 added in the IVF medium at low concentration (<20μg/mL)
19 maintained high penetration rates with a significant decrease in polyspermy (Kouba et al.,
2000); however, treating porcine sperm with high concentrations of OVGP1 reduced sperm penetration and binding of sperm to oocytes. Goat OVGP1 was found to be capable of increasing the resistance of zona pellucida to pronase digestion in a dose dependent manner; however, low concentrations of OVGP1 in the incubating medium resulted in higher cleavage rate of the fertilized oocytes, and more embryos developed to morula and blastocyst stages
(Pradeep et al., 2011). Taken together, these studies demonstrated that OVGP1 have positive effects on both sperm and oocytes albeit with varying degree among the species.
2.2.3.d Localization of OVGP1 in developing embryos
Endocytosis of OVGP1 by the oocytes suggests that OVGP1 may also play a role in the embryo development. A study carried out by Kan and Roux (1995) detected oviductin in the perivitelline space and multi-vesicular bodies of developing young blastocysts suggesting that the blastomeres internalize the oviductin shedding from the ZP. Incubation of ovine spermatozoa and oocytes with OVGP1 during fertilization showed a significant increase in cleavage rate and in the number of embryos developed to blastocysts (Hill et al., 1996a); however, addition of OVGP1 during culture after in vitro fertilization showed no effect on cleavage rates (Hill et al., 1996b). During in vitro fertilization in the pig in the presence of porcine OVGP1, although no effects on cleavage rates were noted, there was an increase in the number of embryos that developed to blastocyst stage (Kouba et al., 2000). Similarly, in the sheep, while addition of OVGP1 in the culture medium during in vitro fertilization had no effects on cleavage rates, a high blastocyst yield and a shorter time for fertilized oocytes to 20 develop to blastocyst stage were observed (Hill et al., 1997). Therefore, results obtained from the above studies indicate that the uptake of OVGP1 by oocytes plays an embryo-trophic role during the early events of fertilization.
2.2.3.e Extra-oviductal localization of OVGP1
Aside from the expression of OVGP1 by the oviductal epithelium, to a lesser extent,
OVGP1 was also found on the apical surface of the endometrial epithelial lining in hamsters.
Upon release from the oviductal secretory cells, hamster OVGP1 binds to the microvilli of endometrial epithelial cells and becomes internalized (Martoglio and Kan, 1996; Roux et al.,
1997; McBride et al., 2004b). During early gestation in hamsters, immunolabeling of OVGP1 over the cell surface of the uterine epithelium was found to decrease in intensity from day 1 to day 6 (Roux et al., 1997). At day 4 of gestation, the decrease in immunoreactivity was evident.
By day 6, around the time of embryo implantation, the signal was almost undetectable (Roux et al., 1997). In mice, OVGP1 expression at uterine epithelium was gradually increased following conception (Laheri et al., 2017). The level of expression was the most evident in early day 5 and subsequently decreased afterwards. The alteration of OVGP1 expression levels at the uterine epithelium around the time of implantation suggests a possible role of OVGP1 in regulating uterine receptivity. A study using an OVGP1 expressing human endometrial epithelial adenocarcinoma cell line demonstrated that the knockdown of OVGP1 mRNA down- regulated the expression of genes related to endometrial-receptivity as well as factors related to decidualization and extra-cellular matrix remodeling (Laheri et al., 2017). The culture medium derived from OVGP1 knockdown epithelial cells reduced the in vitro adhesiveness of 21 trophoblast cells (Laheri et al., 2017). Therefore, OVGP1 not only plays a role in fertilization but also is involved in regulating embryo implantation.
In rabbits and monkeys, OVGP1 was also detected in the cervical epithelium (Hendrix et al., 2001; Slayden et al., 2018). The level of OVGP1 expression in rabbit endocervix was up- regulated by increased estradiol levels (Hendrix et al., 2001). OVGP1 expressed by epithelial cells of the macaque cervix was up-regulated in the proliferative phase and down-regulated in the secretory phase in a menstrual cycle (Slayden et al., 2018). The evidence that OVGP1 in the endocervix is expressed in concert with the level of estradiol during the female reproductive cycle indicates that OVGP1 may regulate sperm fertilizing competence during sperm transit through the cervix.
Although OVGP1 expression is absent in normal human ovarian tissues (Maines-Bandiera et al., 2010), an increased level of OVGP1 was found in benign ovarian tumor fluids (Poersch et al., 2016), borderline tumors, stage I and II serous carcinomas, and mucinous carcinomas
(Woo et al., 2004) as compared to late stage ovarian carcinomas, suggesting the use of OVGP1 as a marker for detection of early events in ovarian tumorigenesis.
In order to study the physiological significance of this glycoprotein, Araki and colleagues produced Ovgp1 gene null mice (Araki et al., 2003). Evidence showing sub-fertility (i.e. smaller litter sizes from the Ovgp1-null females and a decreased ability of sperm to fertilize
Ovgp1-null oocytes) was reported; however, Ovgp1 knockout mice remained fertile as compared to the wild-type mice. Therefore, in mice, although OVGP1 may not be essential for in vivo reproduction, its absence may result in reproductive deficits.
22 2.2.4 Production of recombinant OVGP1
It is technically difficult to isolate and purify a large amount of native OVGP1 from oviductal fluid for extensive studies of biological functions and mechanisms of actions of the glycoprotein. Therefore, recombinant protein technology has been attempted to produce
OVGP1 from cell cultures for further studies. A recent study using recombinant porcine
OVGP1 demonstrated that although both full length and truncated OVGP1 were able to bind to porcine zona pellucida, full length OVGP1 was able to penetrate deeper into the zona pellucida as compared to the truncated form of recombinant porcine OVGP1 (Algarra et al.,
2016). The C-terminal region of porcine OVGP1 was shown to be essential for penetration of the glycoprotein into the zona pellucida and its endocytosis by in vitro matured oocytes. The study also showed that only full-length recombinant porcine OVGP1 is able to increase the efficient rate of in vitro fertilization (Algarra et al., 2018). Supplementing recombinant OVGP1 in the conditioned medium used in the in vitro fertilization procedure can be beneficial to increase the fertilization rate.
2.3 Fertilizing Competence of Mammalian Sperm
Fertilization is the process by which two haploid gametes, namely the spermatozoon and oocyte, unite to produce a genetically distinct individual. In mammals, fertilization involves a number of sequential steps, including sperm migration through the female reproductive tract, sperm penetration through the cumulus mass surrounding the oocyte, binding of sperm to the zona pellucida, acrosome reaction, penetration of sperm into the zona pellucida and fusion of the sperm and oocyte plasma membranes (PM) (Yanagimachi, 1994). Freshly ejaculated
23 mammalian spermatozoa lack the capability to fertilize the oocytes. It was discovered by Chang and Austin in 1951 that, in vivo, freshly ejaculated mammalian sperm were unable to fertilize the oocytes unless sperm had resided in the female reproductive tract for a period of time and acquired the ability through a process known as “capacitation” (Chang, 1951). Capacitation is broadly defined as the functional and physiological modifications of sperm that normally occur in the female reproductive tract rendering sperm competent to fertilize the egg, including the change of motility from progressive to hyperactivated, gaining the ability to bind to the zona pellucida and undergo acrosome reaction, and the capacity to fuse with the oocyte
(Yanagimachi, 1994; Bailey, 2010; Signorelli et al., 2012).
2.3.1 Structure and Composition of Mammalian Sperm
Mammalian spermatozoa are produced in the seminiferous tubules in the testis through a process termed spermatogenesis. The newly formed cells mature in the cauda epididymis prior to deposition into the female reproductive tract (Lui et al., 2003). Although millions of mature sperm cells are deposited into the female reproductive tract at the time of coitus, after travelling through physical and chemical barriers, only a small number of sperm cells are able to reach the ampulla of oviducts to fertilize the oocytes (Eddy et al., 1994).
The main components of a sperm are the head and flagellum (tail), joined by the connecting piece (neck) (Toshimori and Eddy, 2015). As explained in the book “Knobil and Neill's
Physiology of Reproduction (4th edition)” and summarized in Figure 2-5, the head region of a spermatozoon contains the nucleus that has highly condensed chromatin and is capped anteriorly by the acrosome, a membrane-enclosed cytoplasmic vesicle containing hydrolytic
24 enzymes and molecules required for fertilization. The connecting piece contains centriolar derivatives and the segmented columns. The flagellum is divided successively into the mid- piece, principal piece, and end-piece regions. The flagellum contains a central complex of microtubules forming the axoneme, surrounded by outer dense fibers extending from the neck into the principal piece. The mid-piece contains the mitochondrial sheath, a tightly wrapped helix of mitochondrial sheath surrounding the outer dense fibers and axoneme. The majority of the length of the tail is the principal piece, defined by the presence of a fibrous sheath (FS) surrounding the outer dense fibers and axoneme. The end-piece is the final segment and contains only axonemal microtubules. The sperm cell is tightly enclosed by a continuous PM covering from head to tail and contains a sparse amount of cytoplasm.
2.3.1.a The sperm head
Substantial structural differences reside in spermatozoa from different species in terms of the size and length, and the shape of the head. The sperm head from rodent species is usually hook-shaped (falciform), while ungulate, carnivore, and primate species usually have sperm head with a spatula-shaped (spatulate) form (Sutovsky and Manandhar, 2006). Inside the sperm head resides the compact haploid nucleus that contains the highly condensed chromosomes surrounded by a nuclear envelope (Fig. 2-5). The perinuclear theca is a detergent resistant capsule that surrounds the mature sperm nucleus. Apically, perinuclear theca resides between the inner acrosomal membrane (IAM) and the nuclear envelope making up the subacrosomal layer, while caudally, it resides between PM and the nuclear envelope making up the postacrosomal sheath (Oko and Maravei, 1994; Oko, 1995). The posterior region contains 25 factors essential for activating oocytes at the moment of sperm-oocyte fusion (Oko and
Sutovsky, 2009; Aarabi et al., 2014). The acrosome, a derivative from the Golgi apparatus during spermiogenesis, forms a cap in the anterior region of the head and contains proteases and lysozymes, as well as proteins required for sperm interaction with the zona pellucida of the oocyte (Tulsiani et al., 1998). Upon binding to the zona pellucida, the PM and outer acrosomal membrane (OAM) fuse and become vesiculated resulting in releasing the acrosome contents that are high in lysozymes and proteases to digest the outer coverings of the egg
(Toshimori and Eddy, 2015).
2.3.1.b The sperm tail
The tail or flagellum of the mammalian sperm consists of four distinct regions: the connecting piece (neck region), the mid-piece, the principal piece, and the end-piece (Fig. 2-5)
(Toshimori and Eddy, 2015). The flagellum attaches to the base of the nucleus envelope at the structure called the basal plate. The connecting piece comprises the capitulum (the dense fibrous plate-like structure) that attaches to the basal plate proximally and the striated columns distally. Within the connecting piece resides the centrosomes. Special to sperm centrosome is that the only one of the centrioles is remained in the mature spermatozoa (proximal centriole) and locates next to the basal plate (Schatten and Sun, 2009). The distal centriole is partially degenerated during spermiogenesis (Manandhar et al., 2000). The connecting piece contains components that are essential for the anchorage and assembly of axoneme, condensation of the chromosomes in the nuclear, and regulators of cell cycle checkpoints (Toshimori and Eddy,
2015). 26 The main structural components within the flagellum is the microtubule filament complex named axoneme. Cytoskeletal tubulin and motor protein dynein are the major components. The axoneme is composed of 9 outer doublets and 2 central singlet microtubules. In most mammals, the central pair of microtubules appears to extend from the capitulum in the connecting piece to the end-piece of the tail (Toshimori and Eddy, 2015). Peripheral to axoneme is the cytoskeletal structures named the outer dense fibers (ODF). The fibers run longitudinally throughout the length of the mid-piece and most of the principal piece of the mammalian sperm tail. In the mid-piece segment, 9 fibers correspond and bind to the adjacent microtubule doublets of the axoneme. The fibers are not uniform in size. They gradually reduce in size and terminate along the length of the tail. Smaller sized fibers terminate in the principal piece as the longitudinal columns of the FS and larger sized ones are the last to terminate (Toshimori and Eddy, 2015). The fibers are composed of two main ODF proteins, ODF1 and ODF2
(Petersen et al., 1999). The ODFs function to improve the bending torque of the tail and likely protecting the sperm tail against shear forces during movement and transport (Lindemann,
1996).
Characteristic to the mid-piece of the sperm tail is that a mitochondrial sheath surrounds the ODFs (Fawcett, 1970; Toshimori and Eddy, 2015). Sperm mitochondria are arranged circumferentially in an end-to end fashion to form a helically coiled sheath. Where the mid- piece terminates, a ring-like structure, named the annulus, covers the ODFs and is followed immediately by the FS in the principal piece.
27 The FS is a cytoskeletal sheath-like structure unique to sperm that surrounds the outer dense fibers and axoneme in the principal piece of the sperm tail. Structurally, FS is a tapering cylinder formed by two longitudinal columns connected by circumferential ribs. FS is stabilized by disulfide bonds and is resistant to detergent solubilizing (Calvin et al., 1973;
Olson et al., 1976). The FS provides a scaffold for proteins in signaling and metabolic pathways that are required for sperm capacitation and motility (Eddy et al., 2003; Krisfalusi et al., 2006;
Eddy, 2007). Two major components of the FS are A-kinase anchoring proteins (AKAP) 3 and
4 (Eddy et al., 2003; Eddy, 2007). AKAP4 is the major component of the FS in mice (Carrera et al., 1994), however, in human sperm, AKAP3 seems to be the predominant type of AKAPs
(Vijayaraghavan et al., 1999). AKAP proteins bind to the regulatory subunits of protein kinase
A (PKA) via an amphipathic helix in the PKA-binding domain and confine PKA to distinct subcellular locations within the cell via a localization sequence to regulate the cAMP-PKA pathway (Tasken and Aandahl, 2004; Luconi et al., 2005). Tyrosine phosphorylation of
AKAP3 results in an increased binding of PKA and AKAP3 compartmentalization in the fibrous sheath, leading to increased sperm motility (Luconi et al., 2005). AKAP proteins are found to be essential for the integrity of the FS and the movement of sperm tail. AKAP4 knockout male mice are infertile and have reduced sperm motility (Miki et al., 2002). In these mice, the principal piece of the tail was found to be malformed with thin circumferential ribs of the FS. A case study of an asthenozoospermic patient with FS dysplasia in the sperm tail demonstrated intragenic partial deletions of AKAP3 and 4 (Baccetti et al., 2005). Absence of
28 AKAP4 protein was also found in a case of necrospermia (Moretti et al., 2006). Therefore, a properly formed FS is critical for sperm function.
2.3.2 Sperm capacitation
As aforementioned, capacitation refers to the physiological process rendering sperm capable of fertilizing the oocyte. The mechanism of sperm capacitation is summarized in Figure
2-6. One of the first changes described in capacitating mammalian spermatozoa was the loss of cholesterol from the sperm PM (Davis et al., 1979). Cholesterol is a decapacitation factor that serves to stabilize the PM of the spermatozoa during epididymal transit and prevent premature capacitation (Davis et al., 1980). Cholesterol is removed from the sperm surface by proteinaceous acceptor molecules such as albumin, high-density lipoproteins, and apolipoproteins to the extracellular space. Cholesterol efflux alters the protein-lipid organization of the sperm PM and increases its fluidity. Membrane fluidization is synchronized
- 2+ with an influx of HCO3 and Ca , which onsets a cascade of signal transduction events that lead to sperm capacitation.
The key to induce sperm capacitation is the activation of cyclic adenosine monophosphate (cAMP) dependent PKA signaling pathway. The influx of bicarbonate activates the bicarbonate-dependent soluble adenylyl cyclase that subsequently converts ATP into cAMP. The cAMP binds to the regulatory subunit of PKA, which results in the release and activation of the catalytic subunits of PKA (Walsh et al., 1968; Luconi et al., 2011). PKA is a tetrameric enzyme consisting of two catalytic subunits and two regulatory subunits. Upon binding of cAMP to PKA, the catalytic subunits are released as active serine-threonine kinases
29 and can actively phosphorylate their specific substrates, initiating a cascade of signaling events inside the cell (Luconi et al., 2011; Taylor et al., 2012).
Phosphorylation of PKA substrates finally leads to protein phosphorylation in tyrosine residues. This massive increase in tyrosine phosphorylation has been shown to be a characteristic feature of capacitation in mammalian species including the human (Naz et al.,
1991; Leclerc et al., 1996), bovine (Galantino-Homer et al., 1997), porcine (Flesch et al., 1999), equine (Pommer et al., 2003), hamster (Visconti et al., 1999), mouse (Visconti et al., 1995a), and rat (Lewis and Aitken, 2001). However, how PKA activation results in the increase in Tyr phosphorylation is still not well understood. Advances made in proteomics have revealed that the proteins undergoing tyrosine phosphorylation during capacitation include ion channels, metabolic enzymes, and structural proteins (Ficarro et al., 2003; Baker et al., 2010a; Nixon et al., 2010). Two prominent tyrosine phosphorylated proteins are AKAP 3 and 4 (Baker et al.,
2010a). Phosphorylated AKAPs sequester PKA in the tail region by binding to the regulatory subunit of PKA (Miki and Eddy, 1998; Michel and Scott, 2002; Tasken and Aandahl, 2004).
Consequently, PKA can be localized in proximity with cAMP or target proteins to enhance its activity during capacitation (Burton and McKnight, 2007).
2.3.2 Acrosome reaction
Capacitated sperm can then undergo acrosome reaction, which is the prerequisite step before sperm fertilize the oocyte. When acrosome reaction is triggered, the acrosome become vesiculated by the fusion of sperm PM with the OAM, resulting in the release of acrosome
30 content and expose IAM which are rich in enzymes responsible for breaking through the oocyte coating and allowing fertilization to occur.
The presence of the acrosome and its ability to function properly is essential for the fertilizing potential of the spermatozoon, and spermatozoa without an acrosome are unable to penetrate the egg vestments (Schill, 1974). The inducibility of the acrosome reaction can correlate with sperm penetration of the zona pellucida (Liu & Baker, 1996b). Therefore, the inducibility of the acrosome reaction can predict the fertilizing potential of a sperm sample
(Tasdemir et al., 1993).
2.3.3 Zona pellucida of the oocyte and sperm-zona binding
Mammalian oocytes are surrounded by a ~7-20 µm thick porous extracellular matrix named zona pellucida (ZP) composed of glycoproteins (Chiu et al., 2014). The ZP provides both a barrier to prevent polyspermy, a condition in which more than one sperm fertilize the oocyte, as well as a binding site for sperm to fertilize the oocyte. Like the egg coat in avian and reptile species, the ZP also provides protections for the fragile oocytes and preimplantation embryos against physical damages (Noakes, 1995).
The acellular ZP is comprised of either three or four ZP family members designated as
ZP1, ZP2, ZP3 and ZP4. The mouse ZP is composed of three ZP glycoproteins, ZP1, ZP2 and
ZP3 (Bleil and Wassarman, 1980), while ZP4 is a pseudogene and does not express the corresponding protein (Lefievre et al., 2004; Goudet et al., 2008). The ZP of pigs (Hedrick and
Wardrip, 1987), cows (Noguchi et al., 1994) and dogs (Goudet et al., 2008) are made of ZP2,
ZP3 and ZP4. Humans, other primates, rats and hamsters express all four ZP glycoproteins
31 (Wassarman et al., 2001; Chiu et al., 2008; Izquierdo-Rico et al., 2009; Gupta and Bhandari,
2011; Claw and Swanson, 2012). Studies on the mouse ZP structure suggest that ZP is a non- covalently assembled structure composed of ZP2-ZP3 dimers that polymerize into filaments crosslinked by ZP1 (Greve and Wassarman, 1985; Wassarman et al., 1999). The study on the ability of wheat germ agglutinin, which binds to N-acetyl-D-glucosamine and sialic acid, in blocking fertilization leads to the thought that glycosylation is essential for the biological functions of ZP (Oikawa et al., 1973). Each of the ZP glycoproteins is heterogeneously glycosylated with N-linked and O-linked glycans with varying degrees of sialylation and sulfation. In mice, the relevance of O-linked glycans for functional activity of ZP glycoproteins is unclear. Elective removal of the O-linked glycans of the mouse ZP3 by alkaline hydrolysis abrogates its sperm binding activity (Florman and Wassarman, 1985). However, transgenic mice with specific deletion of 1-3 galactosyltransferase, an enzyme required for biosynthesis of core 1 O-linked glycans, which is the only O-linked glycan on ZP3, retains normal fertility
(Williams et al., 2007). Studies on the contribution of N-linked glycans on ZP sperm binding activity, on the other hand, was first found out to be not involved in the action of ZP3 inducted acrosome reaction in mice (Florman and Wassarman, 1985). However, inactivation of N- acetylglucosaminyltransferase I that is responsible for the synthesis of hybrid and complex N- linked glycans using transgenic mouse models resulted in severely decreased fertility (Shi et al., 2004). Evidence points to the theory that the initial sperm-egg binding depends on the interaction of a sperm surface protein with a supramolecular complex of the three mouse ZP glycoproteins (Clark, 2011).
32 Binding of sperm to the ZP is an essential step during fertilization. Defective sperm-ZP binding and ZP penetration are major causes of low fertilization rate in clinical in vitro fertilization (IVF).
2.4 Hypotheses
Accumulating events suggest that supplementing OVGP1 can be beneficial for the increase of successful rate of IVF. However, to obtain a large quantity of pure OVGP1 to study the functional mechanism of OVGP1 involved in these events from oviductal fluids is technically difficult, particularly in humans. Therefore, to obtain human oviducts for collecting sufficient amount of OVGP1 is ethically impossible. Based on these premises, my objective was to produce recombinant hamster and human OVGP1 and examine the effects of these recombinant proteins in enhancing sperm fertilizing competence during in vitro capacitation.
The present investigation was carried out to test the following hypotheses:
Hypothesis 1: Recombinant hamster OVGP1 (rHamOVGP1) can be produced from stably transfected HEK293 cells and is capable of enhancing hamster sperm fertilizing competence.
Published in Yang X et. al. (2015) PLOS ONE 10 e0123003.
Hypothesis 2: Recombinant human OVGP1 (rHuOVGP1) can be produced from stably transfected HEK293 cells and is capable of enhancing human sperm fertilizing competence.
Published in Zhao Y, et. al. (2016) Reproduction 152: 561-573.
33
Hypothesis 3: Recombinant human OVGP1 (rHuOVGP1) enhances tyrosine phosphorylation of proteins in the fibrous sheath involving AKAP3 and increases sperm-zona binding. Zhao Y and Kan FWK (2019) Journal of Assisted Reproduction and Genetics (submitted and in revision).
Hypothesis 4: Recombinant human OVGP1 (rHuOVGP1) is capable of enhancing the fertilizing capacity of cryopreserved human sperm. (Manuscript in preparation)
34
Figures
35 A
B
36
Figure 2- 1. Schematic diagram illustrating the human and hamster female reproductive tract. (A) The human female reproductive tract is composed of the ovaries, oviducts (uterine tubes), uterus, vagina and external genitalia (Modified from Mescher, 2016). (B) The ovary (O) is enclosed within the ovarian bursa that is surrounded by a fat pad (FP). The infundibulum (In) and fimbriae (not shown) are contained within the bursa. The ampulla (A) and isthmus (I) of the oviduct are highly coiled in the hamster. A small part of one horn of the uterus (U) is also illustrated (Modified from Boatman et al., 1994).
37
Figure 2- 2. Electron micrograph of hamster oviduct epithelium showing localization of hamster OVGP1. OVGP1, indicated by gold particles, is specifically located in the secretory granules (SG) of non-ciliated secretory cells (SC). The membrane of the secretory granules (short arrows), the apical microvillar membrane (arrowheads), and the lateral cell membrane (long arrows) are devoid of OVGP1. The ciliated cells (CC) does not contain OVGP1 but it is sparsely bound to the cilia (C) extending into the lumen of the oviduct. (Modified from McBride et al., 2004b)
38 Glycosyl hydrolases family 18 domain T/S and K rich glycosylation region
AAI36407Homo sapiens 1 MWKLLLWVGLVLVLKHHDGAAHKLVCYFTNWAHSRPGPASILPHDLDPFLCTHLIFAFAS MNNNQIVAKDLQDEKILYPE 80 MesocricetusNP_001268266auratus 1 MGRLLLWVGLVLLMKPNDGTAYKLVCYFTNWAHSRPVPASILPRDLDPFLCTHLIFAFAS MSNNQIVANNLQDEKILYPE 80 AAI37996Mus musculus 1 MGRLLLLAGLVLLMKHSDGTAYKLVCYFTNWAHSRPGPASIMPHDLDPFLCTHLIFAFAS MSNNQIVAKNLQDENVLYPE 80 MacacaNP_001036252mulatta 1 MWKLLLWVGLVLVLKHHDGAAHKLVCYFTNWAHSRPGPASILPHDLDPFLCTHLIFAFAS MNNNQIVAKDLQDEKILYPE 80 NP_001106087Papio anubis 1 MWKLLLWVGLVLVLKHHDGAAHKLVCYFTNWAHSRPGPASILPHDLDPFLCTHLIFAFAS MNNNQIVAKDLQDEKILYPE 80 NP_001073685Bos taurus 1 ------MSNNQIVPKDPQDEKILYPE 20 ABF20534Capra hircus 1 MGKLLLWIGLLLMLKHHDGAAHKLVCYFTNWAFSRPSPASILPRDLDPFLCTHLVFAFAS MNNNQIVPKDPLDEKILYPE 80 NP_999235Sus scrofa 1 MGKLLLWVGLVLVLKHHNGAAHKLVCYFANWAFSRPGPASILPRDLDPFLCTHLVFAFAS MNDSQIVAKDARDESIFYPE 80 NP_001009779Ovis aries 1 MGKLLLWVGLLLMLKHHDGAAHKLVCYFTNWAFSRPGSASILPRDLDPFLCTHLVFAFAS MNNNQIVPKDPLDEKILYPE 80
AAI36407Homo sapiens 81 FNKLKERNRELKTLLSIGGWNFGTSRFTTMLSTFANREKFIASVISLLRTHDFDGLDLFFLYPGLRGSPMHDRWTFLFLI 160 MesocricetusNP_001268266auratus 81 FNKLKERNRALKTLLSVGGWNFGTSRFTTMLSTLASREKFIGSVVSFLRTHGFDGLDLFFLYPGLRGSPINDRWNFLFLI 160 AAI37996Mus musculus 81 FNKLKERNRELKTLLSIGGWNFGTSRFTAMLSTLANREKFIDSVISFLRIHGFDGLDLFFLYPGLRGSPPHDRWNFLFLI 160 MacacaNP_001036252mulatta 81 FNKLKERNRELKTLLSIGGWNFGTSRFTTMLSTFANREKFIASVISLLRIHDFDGLDLFFLYPGLRGSPMHDRWTFLFLI 160 NP_001106087Papio anubis 81 FNKLKERNRELKTLLSIGGWNFGTSRFTTMLSTFANREKFIASVISLLRTHDFDGLDLFFLYPGLRGSPMHDRWTFLFLI 160 NP_001073685Bos taurus 21 FNKLKERNRGLKTLLSIGGWNFGTVRFTTMLSTFSNRERFVSSVIALLRTHGFDGLDLFFLYPGLRGSPARDRWTFVFLL 100 ABF20534Capra hircus 81 FNKLKERNRGLKTLLSVGGWNFGTSRFTKMLSTFSNRERFVNSVIALLRTHGFDGLDLFFLYPGLRGSPARDRWTFVFLL 160 NP_999235Sus scrofa 81 FNQLKERNEKLKTLLSIGGWNFGTSRFTTMLSTFTNREKFIRSAIGLLRTHGFDGLDLFFLYPGLRGSPRRDRWNFLFLL 160 NP_001009779Ovis aries 81 FNKLKERNRGLKTLLSVGGWNFGTSRFTKMLSTFSNRERFVKSVIALLRTHGFDGLDLFFLYPGLRGSPARDRWTFVFLL 160 * * AAI36407Homo sapiens 161 EELLFAFRKEALLTMRPRLLLSAAVSGVPHIVQTSYDVRFLGRLLDFINVLSYDLHGSWERFTGHNSPLFSLPEDPKSSA 240 MesocricetusNP_001268266auratus 161 EELQFAFEKEALLTQRPRLLLSAAVSGIPYIIQTSYDVHLLGRRLDFINVLSYDLHGSWEKSTGHNSPLFSLPEDPKSSA 240 AAI37996Mus musculus 161 EELQFAFEREALLTQHPRLLLSAAVSGIPSIIHTSYDALLLGRRLDFINVLSYDLHGSWEKFTGHNSPLFSLPEDSKSSA 240 MacacaNP_001036252mulatta 161 EELLFAFRKEALLTMRPRLLLSAAVSGVPHIVQTSYDVRFLGRLLDFINVLSYDLHGSWEKFTGHNSPLFSLPEDPKSSA 240 NP_001106087Papio anubis 161 EELLFAFRKEALLTMRPRLLLSAAVSGVPHIVQTSYDVRFLGRLLDFINVLSYDLHGSWEKFTGHNSPLFSLPEDPKSSA 240 NP_001073685Bos taurus 101 EELLQAFKNEAQLTMRPRLLLSAAVSGDPHVVQKAYEARLLGRLLDFISVLSYDLHGSWEKVTGHNSPLFSLPGDPKSSA 180 ABF20534Capra hircus 161 EELLQAFKNEAQLTMRPRLLLSAAVSGDPHVIQKAYDARLLRRLLDFISVLSYDLHGSWEKVTGHNSPLFSLPGDPKSSA 240 NP_999235Sus scrofa 161 EELLLAFRREAQLTMRPRLLLSAAVSADPHVIQKAYDVRLLGRLLDFINVLSYDLHGSWEKVTGHNSPLFSLSDDPKSSA 240 NP_001009779Ovis aries 161 EELLQAFKNEAQLTMRPRLLLSAAVSGDPHVIQKAYDARLLGRLLDFISVLSYDLHGSWEKVTGHNSPLFSLPGDPKSSA 240
AAI36407Homo sapiens 241 YAMNYWRKLGAPSEKLIMGIPTYGRTFRLLKASKNGLQARAIGPASPGKYTKQEGFLAYFEICSFVWGAKKHWIDYQYVP 320 MesocricetusNP_001268266auratus 241 FAMNYWRNLGAPADKLLMGFPAYGRTFHLLRESKNGLQAASMGPASPGKYTKQAGFLAYYEVCSFIQRAEKHWIDHQYVP 320 AAI37996Mus musculus 241 YAMNYWRKLGTPADKLIMGFPTYGRNFYLLKESKNGLQTASMGPASPGKYTKQSGFLAYYEVCSFVQRAKKHWIDYQYVP 320 MacacaNP_001036252mulatta 241 YAMNYWRKLGAPSEKLIMGIPTYGRTFRLLKASKNGLQATAVGPASPGKYTKQAGFLAYFEICSFVWGAKKHWIDYQYVP 320 NP_001106087Papio anubis 241 YAMNYWRKLGAPSEKLIMGIPTYGRTFRLLKASKNGLQATAIGPASPGKYTKQAGFLAYFEICSFVWGAKKHWIDYQYVP 320 NP_001073685Bos taurus 181 YAMNYWRQLGVPPEKLLMGLPTYGRTFHLLKASQNELRAQAVGPASPGKYTKQAGFLAYYEICCFVRRAKKRWINDQYVP 260 ABF20534Capra hircus 241 YAMSYWRQLGVPPEKLLMGLPTYGRTFHLLRASQNELGAGAVGPASPGKYTKQAGFLAYYEVCSFVQRAKKRWINDQYVP 320 NP_999235Sus scrofa 241 YTMNYWRKLGAPPEKLLMGFPTYGRTFRLLKASKNELGAEAVGPASPGKYTKQAGFLAYYEVCSFVQRAKKRWIDHQYVP 320 NP_001009779Ovis aries 241 YAMSYWRQLGVPPEKLLMGLPTYGRTFHLLRASQNELGAGAAGPASPGKYTKQAGFLAYYEVCSFVQRAKKRWINDQYVP 320
AAI36407Homo sapiens 321 YANKGKEWVGYDNAISFSYKAWFIRREHFGGAMVWTLDMDDVRGTFCGTGPFPLVYVLNDILVRAEFSSTSLPQFWLSSA 400 MesocricetusNP_001268266auratus 321 YAYKGKEWVGYDDAVSFSYKAMFVKKEHFGGAMVWTLDMDDVRGTFCGNGPFPLVHILNELLVRAEFNSTPLPQFWFTLP 400 AAI37996Mus musculus 321 YAFKGKEWLGYDDTISFSYKAMYVKREHFGGAMVWTLDMDDVRGTFCGNGPFPLVHILNELLVQTESNSTPLPQFWFTSS 400 MacacaNP_001036252mulatta 321 YANKGKEWVGYDDAISFSYKAWFIRREHFGGAMVWTLDMDDVKGTFCGTGPFPLVYVLNDILVRAEFSSTSLPQFWLSSA 400 NP_001106087Papio anubis 321 YANKGKEWVGYDDAISFSYKAWFIRREHFGGAMVWTLDMDDVRGTFCGTGPFPLVYVMNDILVRAEFSSTSLPQFWLSSA 400 NP_001073685Bos taurus 261 YAFKGKEWVGYDDAISFGYKAFFIKREHFGGAMVWTLDLDDFRGYFCGTGPFPLVHTLNNLLVNDEFSSTPSPKFWFSTA 340 ABF20534Capra hircus 321 YAFKGKEWVGYDDAISFGYKAFFIKREHFGGAMVWTLDLDDFRGNFCGTGPFPLVHTLNNLLVNDEFSSTPSPKFWFSTA 400 NP_999235Sus scrofa 321 YAYRGKEWVGYDDDISFSYKAFFIKKEHFGGAMVWTLDLDDVRGTFCGTGPFPLVYMLNDLLLKAEVSSTLSPGFGLSTT 400 NP_001009779Ovis aries 321 YAFKGKEWVGYDDAISFGYKAFFIKREHFGGAMVWTLDLDDFRGNFCGTGPFPLAHTLNNLLVNDEFSSTPSPKFWFSTA 400
AAI36407Homo sapiens 401 VNSSSTDPERLAVTTAWTTD-SKILPPGGEAGVTEIHGKCENMTITPRGTTVTP------TKETVSLGKHTVALGEKT-- 471 MesocricetusNP_001268266auratus 401 VNSSGPGSESLPATEELTTDtVKILPPGGEAMATEVHRKYEKVTTIPNGGFVTPagttspTTHAVALERNAMAPGAKTtt 480 AAI37996Mus musculus 401 VNASGPGSENTALTEVLTTDtIKILPPGGEAMTTEVHRRYENMTTVPSDGSVTPggtaspRKHAVTPENNTMAAEAKTms 480 MacacaNP_001036252mulatta 401 VNSSSTEPERLAVTKAWTTD-IKILPPGGEAGVTEINGKCENMTITPRVTIVTP------TKETVSLGKHTVALGEKT-- 471 NP_001106087Papio anubis 401 VNSSSTDPERLAVTKAWTTD-IKILPPGGEAGVTEIHGKCENMTITPRVTIVTP------TKETVSLGKHTVALGEKT-- 471 NP_001073685Bos taurus 341 VNSSRIGPEMPTMTRDLTTG-LGILPPGGEAVATETHRKSETMTITPKGEIATP------TRTPLSFGRRTAAPEGKT-- 411 ABF20534Capra hircus 401 VNSSRIGPEMPTMTRDLTTG-LGILPLGGEAMATETHRKSATMTTTPRGETATP------TRTPLSFGRRTAAPEGKT-- 471 NP_999235Sus scrofa 401 VNSSRTCPESLAVTKDLTTD-LGILPLGGEAVATETHGRSDNMTVTPGGGLVAP------TRPTLSFGKLTVAPEGKT-- 471 NP_001009779Ovis aries 401 VNSSRIGPEMPTMTRDLTTG-LGILPLGGEAVATETHRKSATMTTTPRGETATP------TRTPLSSGRRTAAPEGKT-- 471
AAI36407Homo sapiens 472 ------EITGAMTMTSVGHQSMTPGEKALTPVGHQ------500 MesocricetusNP_001268266auratus 481 sldllsetmtgm--tvtvqtQTAGRETMTTVGNQSVTPGGETMTTVGNQSvtpg------GETMTT 538 AAI37996Mus musculus 481 tldffsktttgvsktttgisKTTTGVSKTTTG-ISKTTTGVSKTTTGVSKatagisktipeiskatagvsktitGVSKTT 559 MacacaNP_001036252mulatta 472 ------EITGATTMTSVGHPSMTPGEKALTPVGHQSelpg------KKTLTP 511 NP_001106087Papio anubis 472 ------EITGATTMTSVGHQSMTPGEKALTPVGHQSelpg------KKTLTP 511 NP_001073685Bos taurus 412 ------ESPGEKPLTTVGHLAVSPGGIAVGPVRLQT------GQKVTP 447 ABF20534Capra hircus 472 ------ESPGEKPLTTVGHLVVSPGGIAVGPVHLQT------GQKVTP 507 NP_999235Sus scrofa 472 ------ESPGEKAMTPVGHPSVTPGDMSVPPVPIQT------GDRITP 507 NP_001009779Ovis aries 472 ------ESPGEKPLTSVGRLAVSPGGIAVGPVHLQI------GQKVTP 507
AAI36407Homo sapiens 501 ----SVTTGQKTLTSVGYQSVT-PGEKTL------TPVGHQSVTPV------SHQS 539 MesocricetusNP_001268266auratus 539 VGNQSVTPGGETVTIVGNKSVTpVGETV------TIVGNKSVTPG------gqttatvGSQS 588 AAI37996Mus musculus 560 TGVSKITTGVSKTTTGISKTTTgISQTTTgisktttdiskTTTGISKTTPGiskttpgmtvivqtqaneaettatmDHQS 639 MacacaNP_001036252mulatta 512 VGHQSVTTGQKTLISVGYHSVTpPGEKTL------TPVDHPSVTPV------SHQS 555 NP_001106087Papio anubis 512 VGHQSVTTGQKTLISVGYHSVT-PGEKTL------TPVGHPSVTPV------SHQS 554 NP_001073685Bos taurus 448 PGRKAGVPEKVTTPS------GKMTV------TPDGRAETLER------RL-- 480 ABF20534Capra hircus 508 PGRKAGVPEKVTTPS------GKMTV------TPDGRAETLER------L-- 539 NP_999235Sus scrofa 508 PRRQAVAPEKMTLPS------GK------RSD------527 NP_001009779Ovis aries 508 PGRKAGVPEKVTIPS------GKMTV------TPDGRAETLER------L-- 539
AAI36407Homo sapiens 540 VSPGGTTMTPVHFQTETLRQNTVApRRKAVAREKVTVPSRNISVTPEGQTMPLRGENLTSEVGTHPRMGnlglqmeaenr 619 MesocricetusNP_001268266auratus 589 VTPPGMDTTLVYLQTMTLSEKGTS-SKKAVVLEKVTVPPREISVMPNEQNTALNRENLIAEVESYSQDG------656 AAI37996Mus musculus 640 VTPTGMDTTLFYLKTMTPSEKETS-RKKTMVLEKATVSPREMSATPNGQSKTLKWASLITEVETYSQDG------707 MacacaNP_001036252mulatta 556 VSPGGMTMTPVHFQTETLRQNTMApRRKAVAREKVTVPSRKISVTPEGQTVPLRGEYLTSETGTHPQGG------624 NP_001106087Papio anubis 555 VSPGGMTMTPVHFQTETLRQNTMApRRKAVAHEKVTVPSRKISVTPEGQTVPLRGEYLTSETGTHPQDG------623 NP_001073685Bos taurus ------ABF20534Capra hircus ------NP_999235Sus scrofa ------NP_001009779Ovis aries ------
AAI36407Homo sapiens 620 mmlssspviqlpeqtplafdnrfvpiygnhssvnsvtpqtsplslkkeipensavdeea 678 MesocricetusNP_001268266auratus ------AAI37996Mus musculus ------MacacaNP_001036252mulatta ------NP_001106087Papio anubis ------NP_001073685Bos taurus ------ABF20534Capra hircus ------NP_999235Sus scrofa ------NP_001009779Ovis aries ------
AAI36407.1 Oviductal glycoprotein 1, 120kDa [Homo sapiens] Related Information NP_001268266.1 oviduct-speci39fic gl ycoprotein precursor [Mesocricetus auratus] Related Information AAI37996.1 Ovgp1 protein [Mus musculus] Related Information NP_001036252.1 oviduct-specific glycoprotein precursor [Macaca mulatta] Related Information NP_001106087.1 oviduct-specific glycoprotein precursor [Papio anubis] Related Information NP_001073685.1 oviduct-specific glycoprotein [Bos taurus] Related Information ABF20534.1 oviductin [Capra hircus] Related Information NP_999235.1 oviduct-specific glycoprotein precursor [Sus scrofa] Related Information NP_001009779.1 oviduct-specific glycoprotein precursor [Ovis aries] Related Information
Figure 2- 3. Multiple protein sequence alignment of mammalian OVGP1 proteins. The top schematic diagram shows two main regions of the OVGP1 protein with the N-terminal conserved glycosyl hydrolases family 18 (GH18 or chitinase) domain and C-terminal variable glycosylation region. Below shows the alignment of OVGP1 amino acid sequences of the human (homo sapiens, Accession number: AAI36407.1), hamster (mesocricetus auratus, NP_001268266.1), mouse (mus musculus, AAI37996.1), monkey (macaca mulatta, NP_001036252), baboon (papio anubis, NP_001106087), cow (bos taurus, NP_001073685), goat (capra hircus, ABF20534), pig (sus scrofa, NP_999235), and sheep (ovis aries, NP_001009779) using Constraint-based Multiple Alignment Tool. The red colored sequences are identical sequences and the blue colored sequences are less conserved sequences. Gray is for columns containing gaps. Where less than 50% of the sequences contain gaps, they are shown in gray uppercase, greater than 50% are in gray lowercase. The black box indicates where the predicted enzymatic sites of chitinases locate on OVGP1s; however, Glu and Asp residues essential for the glycohydrolytic activity of the enzyme of chitinases are marked by an asterisk (*).
40
Figure 2- 4. Schematic diagram showing the biological functions of OVGP1 discovered using native OVGP1 in mammalian species. Briefly, the synthesis and secretion of OVGP1 are influenced by the level of estrogen (E2). In vitro studies from various mammalian species have shown that native OVGP1 can bind to sperm and oocyte and functions to enhance sperm capacitation, increase sperm motility and viability, enhance sperm-egg binding, increase penetration rate and fertilization rate, decrease polyspermy, increase embryo quality, and increase embryo development into blastocyst.
41
A A
B B Plasma membrane Acrosome OAM
Head IAM Nucleus Equatorial segment Perinuclear theca
Neck Centriole
Mitochondrial sheath
Axoneme Mid-piece Outer dense fiber
Fibrous sheath
Principal piece
FigureFigu 2r-e 52.- Schematic5. Schema tdiagramic diagra ofm spermof spe rstructuresm structu rines thein thamsterhe hams tander a nhuman.d hum an. (A)( ADiagram) Diagr aofm hamsterof hams tspermer spe rwithm w ilabeledth labe lstructures,ed structur adaptedes, adap tfromed fr oVadnaism Vadn etai sal. et, a2014l., 20. 1(B)4. (DiagramB) Diagr am of o humanf hum a spermn spe r withm w i labeledth labe lestructures.d structur e IAM:s. IA M inner: in n acrosomaler acrosom membrane.al membra n OAM:e. OA M outer: ou t acrosomer acrosalom al membrane.membra ne.
42 42
Albumin
Sperm PM
- 2+ HCO3 Ca
sAC
ATP cAMP
C PKA RII PKA RII C PKA RII PKA RII AKAP AKAP P P C
C
Hyperactivation ↑Protein Tyrosine Tyrosine Kinases Capacitation Phosphorylation Acrosome Reaction
Figure 2- 6. Schematic diagram of sperm capacitation. Briefly, Sperm capacitation is a series of physiological changes and molecular modifications that render sperm capable of fertilizing the oocyte. In the beginning of capacitation, membrane cholesterols are removed by albumin present in oviductal fluid or capacitating medium, which results in an increase in - 2+ membrane destabilization. This increased membrane fluidity then leads to an influx of HCO3 and Ca ions. They can subsequently bind to and activate a soluble adenylyl cyclase (sAC) that produces cAMP from ATP. Binding of cAMP to the regulatory subunit of PKA causes a release and activation of the catalytic subunit (C). Phosphorylated AKAPs sequester PKA in the tail region by binding to the regulatory subunit of PKA. Consequently, PKA can be localized in proximity to cAMP or target proteins to enhance its activity during capacitation Activated PKA can subsequently activate tyrosine kinases, which lead to a timely increase of the levels of tyrosine phosphorylation of sperm proteins. Capacitated sperm adopt a hyperactivated motility where the beating of the tail changes from a symmetrical, small amplitude manner to an asymmetrical, large amplitude manner. Capacitated sperm can then undergo acrosome reaction, which is the prerequisite step before sperm fertilize the oocyte. Acrosome is a cap-like structure over the anterior surface of sperm head. When acrosome reaction is triggered, plasma membrane (PM) and the outer acrosomal membrane (OAM) of the sperm fuse and form vesicles, resulting in the release of acrosome content and expose the inner acrosomal membrane (IAM) which are rich in enzymes responsible for breaking through the oocyte coating in order to fertilize the oocyte.
43
Chapter 3
Recombinant Hamster Oviductin Is Biologically Active and Exerts Positive Effects on Sperm Functions and Sperm-Oocyte Binding
Published as:
Zhao Y, Yang X, Jia Z, Reid RL, Leclerc P and Kan FW (2016) Recombinant human oviductin regulates protein tyrosine phosphorylation and acrosome reaction. Reproduction 152 561–573.
44 3.1 Abstract
Studies carried out in several mammalian species suggest that oviductin, also known as oviduct-specific glycoprotein or OVGP1, plays a key role in sperm capacitation, fertilization, and development of early embryos. In the present study, we used recombinant DNA technology to produce, for the first time, recombinant hamster OVGP1 (rHamOVGP1) in human embryonic kidney 293 (HEK293) cells. rHamOVGP1 secreted in the culture medium was purified by affinity chromatography. The resulting protein migrated as a poly-dispersed band of 160-350 kDa on SDS-PAGE corresponding to the molecular mass of the native HamOVGP1.
Subsequent mass spectrometric analysis of the purified rHamOVGP1 confirmed its identity as
HamOVGP1. Immunocytochemistry demonstrated binding of rHamOVGP1 to the mid-piece and head of hamster sperm and to the zona pellucida (ZP) of ovarian oocytes. In vitro functional experiments showed that addition of rHamOVGP1 in the capacitation medium further enhanced tyrosine phosphorylation of two sperm proteins of approximately 75 kDa and
83 kDa in a time-dependent manner. After 3 hours of incubation in the presence of rHamOVGP1, a significant increase in acrosome reaction was measured. Pretreatment of either sperm or oocyte with 20 μg/mL of rHamOVGP1 prior to sperm-egg binding assay significantly increased the number of sperm bound to the ZP. Addition of rHamOVGP1 in the medium during sperm-egg binding with either oocyte or sperm pretreated with rHamOVGP1 also saw an increase in the number of sperm bound to ZP. In all experimental conditions, the effect of rHamOVGP1 on sperm-oocyte binding was negated by the addition of monoclonal anti-
HamOVGP1 antibody. The successful production and purification of a biologically active
45 rHamOVGP1 will allow further exploration of the function of this glycoprotein in reproductive function.
46 3.2 Introduction
The mammalian oviduct is a strategic site in the female reproductive tract where it provides a luminal microenvironment for gamete transport and maturation, sperm capacitation, and fertilization as well as early embryo development by secreting an oviductal fluid consisted of a combination of plasma exudates and secretory components from oviductal epithelial cells
(Leese, 1988; Buhi et al., 2000; Coy et al., 2012). A major component of the secretory products of the oviduct is a high-molecular-weight, oviduct-specific and estrogen-dependent glycoprotein known as oviductin or oviduct-specific glycoprotein (OVGP1). OVGP1 has been identified in a variety of species, including the mouse (Kapur and Johnson, 1988; Sendai et al.,
1995), hamster (Araki et al., 1987; Oikawa et al., 1988; Robitaille et al., 1988; Suzuki et al.,
1995), rabbit (Oliphant and Ross, 1982), cow (Boice et al., 1990a; Sendai et al., 1994), pig
(Buhi et al., 1990, 1996), baboon (Verhage et al., 1989; Jaffe et al., 1996), rhesus monkey
(Verhage et al., 1997a), goat (Pradeep et al., 2011), and human (Verhage et al., 1988; Arias et al., 1994). OVGP1 belongs to the glycosyl hydrolase family 18 that lacks chitinase enzyme activity (DeSouza and Murray, 1995). OVGP1 cDNAs have been cloned from several mammalian species and notable conservation has been found within the N-terminal amino acid sequences of different mammalian OVGP1 cDNAs whereas sequence divergence and low identity between species exist within the C-terminal regions (Verhage et al., 1988; Killian,
2004; 2011). Previous studies have shown that, upon its secretion into the lumen of the oviduct, mammalian OVGP1 becomes intimately associated with the zona pellucida (ZP) of postovulatory oocytes in oviductal transit (Buhi, 2002), but is absent in ovarian oocytes (Araki
47 et al., 1987; Kan et al., 1989; Abe and Oikawa, 1990). Results from in vitro functional studies indicated that OVGP1 has positive effects on sperm capacitation, sperm-ovum binding, sperm penetration of the ovum, polyspermy prevention, and early embryo development (Killian, 2004;
2011). For examples, bovine OVGP1 has been shown to enhance sperm capacitation and increase in vitro fertilization rates (King et al., 1994). In vitro, Hamster OVGP1 (HamOVGP1) has been shown to increase the sperm penetration rate three-fold as compared to the penetration rate in the absence of HamOVGP1 (Boatman and Magnoni, 1995). The presence of partially purified human OVGP1 during sperm-hemizona binding was found to enhance the binding of sperm to the outer ZP by a factor of 3.7, and the effect can be blocked by preincubation of human OVGP1 with antibody against human OVGP1 (O’Day-Bowman et al., 1996). The presence of goat OVGP1 in the culture medium was found to enhance embryo cleavage rate and blastocyst formation and prevent polyspermy (Pradeep et al., 2011). The secretion of equine oviductal epithelial cells was shown to increase the rate of in vitro fertilization (Mugnier et al., 2009). Incubation of porcine in vitro-matured oocytes in oviductal fluid rendered the ZP resistant to proteolytic digestion and increased the incidence of monospermy (Coy et al., 2008).
Recent studies in our laboratory have demonstrated that estrus stage-specific HamOVGP1 enhances tyrosine phosphorylation of a sub-set of sperm proteins during in vitro capacitation
(Saccary et al., 2013). These accumulative findings all point to several important roles played by OVGP1 during the early events of mammalian reproduction. Despite the identification of
OVGP1 in various mammalian species, further exploration of its roles in mammalian reproduction and elucidation of the mechanism underlying its functions are hampered by the
48 limited availability of sufficient amounts of purified OVGP1. To circumvent the problem of obtaining adequate amounts of native OVGP1 for future studies, the production of recombinant
OVGP1 can be envisaged as an alternative.
In the present study, we successfully employed recombinant DNA technology to produce recombinant HamOVGP1 (rHamOVGP1) in human embryonic kidney (HEK293) cells. We further purified rHamOVGP1 from culture medium by lectin-affinity purification. To find out if rHamOVGP1 is biologically active, we examined the effect of rHamOVGP1 on capacitation by determining whether rHamOVGP1 can enhance tyrosine phosphorylation of sperm proteins which is a hallmark of capacitation. The effect of rHamOVGP1 on acrosome reaction of hamster sperm was also investigated. An additional aim of our study was to determine whether rHamOVGP1 could bind to the ZP of hamster ovarian oocytes and influence sperm-egg binding. Information gained from the present study shows that rHamOVGP1 is biologically active and that the large scale production of rHamOVGP1 may prove useful for further understanding of its role in fertilization and early embryo development as well as for elucidating the mechanism that regulates its functions.
3.3 Materials and Methods
3.3.1 Animals and reagents
Golden hamsters (Mesocricetus auratus) were purchased from Charles River (St. Constant,
Quebec, Canada). Male and female hamsters of 7 to 9 weeks of age were housed in a temperature-controlled room with exposure to light 12 hours/day (6:00 a.m.-6:00 p.m.). All
49 experiments carried out with the hamsters were approved by The University Animal Care
Committee of Queen’s University in accordance with the guidelines stipulated by the Canadian
Council on Animal Care. All reagents and chemicals were of molecular biology grade and were purchased from Fisher Scientific Co., Sigma-Aldrich, Invitrogen, or New England BioLabs
Inc., unless otherwise specified. The monoclonal antibody (IgG1.k) against HamOVGP1 used in the present study was a gift from Dr. Gilles Bleau of the University of Montreal. The monoclonal antibody recognizes an antigen, an oviduct-specific glycoprotein the molecular weight of which obtained by SDS-PAGE under reducing conditions is between 160-350 kDa
(St-Jacques and Bleau, 1988). This glycoprotein contains a high proportion of sugar residues
(85%) which account for the antigenic determinants recognized by the monoclonal antibody
(Robitaille et al., 1988). Tissue-specificity of the monoclonal antibody has been previously documented (St-Jacques and Bleau, 1988).
3.3.2 Plasmid construction
HamOVGP1-pGEM-T was a gift from Dr. Gilles Bleau of the University of Montreal.
HamOVGP1 cDNA possessing a partial open reading frame of HamOVGP1 with nucleotides
15-1766 of the sequence (Paquette et al., 1995) was first amplified by PCR using HamOVGP1- pGEM-T plasmid as a template, digested by Pme I, and subsequently cloned into the Pme I site of WPI lentiviral vector, which bicistonically expresses green fluorescent protein (GFP). The
HamOVG1-WPI plasmid was confirmed by DNA sequencing (ACGT CORP., Toronto, ON,
Canada). The following primers were used for PCR: forward primer (5-GA TAC CAT
50 GTTTAAAC ATG CAT CAT CAC CAT CAC CAC GCT GAG ATG GGG AGG CTG CTG
CTG-3) and reverse primer (5’-GA AGT CAT GTTTAAAC CAC TGT GGC TGT GAT CTG
TC-3’).
3.3.3 Cell culture, transfection, and establishing the rHamOVGP1 stable cell line
The human embryonic kidney 293 cells (HEK293T; ATCC) were cultured at 37oC and 5%
CO2 in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% Penicillin and Streptomycin. Lentiviruses were produced by Lipofectamine 2000- mediated transfection of HEK 293 as previously described by Chow et al. (Chow et al., 2010).
One day prior to transfection, 1.5 x 107 HEK 293 cells were seeded in a 15 cm Petri dish in 25 mL of DMEM with 10% FBS. The next day, the cells were co-transfected with the lentiviral transfer plasmid pWPI plus packaging plasmid psPAX and envelop plasmid pMD2G at a ratio of 4:3:1 (w/w/w) according to manufacturer’s protocol. At 24 hours (h) post-transfection, the medium was replaced with 15 mL of warm OPTI-MEM I medium (serum free) containing 10 mM sodium butyrate. The medium was harvested at 48 h post-transfection and centrifuged at
500 xg for 10 minutes (min). The supernatant was carefully removed, filtered (0.45 µm membrane) to remove cellular debris, centrifuged at 1000 xg for 30 min and stored at –80oC as lentivirus stock solution for subsequent infection.
For the lentivirus infection of HEK 293 cells, a total of 1 x 105 cells per well were seeded in a 6-well plate the night prior to infection and the cells were 40-50% confluent on the day of infection. The medium was removed and replaced with OPTI-MEM with polybrene (8 µg/mL)
51 o containing lentivirus stock solution. The cells were incubated in 5% CO2 at 37 C. At 48 h post- infection, 3 mL of growth medium (DMEM with 10%FBS, 1% Penicillin and Streptomycin) per well was added. The infected cells were sub-cultured into 10 cm dishes after they were confluent and the green fluorescent protein (GFP)-positive cells were monitored by fluorescence microscopy. About 2 weeks after the passage, GFP-positive cell clones were apparent. Using cloning cylinders individual GFP-positive clones were transferred to 96-well plates and grown in growth medium until confluence. The cells were then passaged into 24- well plates and continually grown in growth medium for 1-2 additional passages with daily monitoring of GFP-positive cell clones. The clones were screened for expression levels of rHamOVGP1 by immunoblot analysis of culture supernatants using monoclonal antibody against hamster OVGP1 (St-Jacques and Bleau, 1988). HEK293 cells derived from clones stably expressing high levels of rHamOVGP1 were selected and maintained in the medium and
GFP expression was monitored on a daily basis.
For large scale production of rHamOVGP1, the stable rHamOVGP1-expressing HEK293 cells were cultured in 15 cm dishes in CD293 serum-free medium supplemented with 4 mM
o L-glutamine, penicillin (50 U/mL), and streptomycin (50 µg/mL) at 37 C and 5% CO2. After
7-10 days, serum-free culture medium containing the recombinant glycoprotein was collected and centrifuged at 1000 xg for 5 min at 4oC. The culture supernatant was collected from each dish and stored at –70oC for further purification. Secretion of rHamOVGP1 was monitored weekly by Western blot analysis of culture supernatants.
52 3.3.4 Purification of secreted rHamOVGP1 from serum-free cell culture medium by affinity chromatography
Purification of rHamOVGP1 was performed using lectin-affinity chromatography as previously described (Malette and Bleau, 1993). Briefly, the culture supernatant was dialyzed overnight in the cold room against a buffer containing 150 mM NaCl, 50 mM Tris-HCL (pH
8.0) and 0.02% NaN3. The buffer-exchanged solution was then concentrated by ultra-filtration using Amicon Ultra-15 centrifugal filter with 50 kDa cut-off membrane (EMD Millipore). The concentrated samples were loaded on Helix pomatia agglutinin (HPA)-agarose (5 mL; 1.5 mg/mL of lectin) column pre-equilibrated with the same buffer. Samples were incubated with
HPA-agarose for 1 h at 4oC and the column was then washed extensively with 10 bed volume of buffer consisted of 300 mM NaCl, 50 mM Tris-HCl (pH 8.0), and 0.02% NaN3. rHamOVGP1 was eluted from HPA-agarose column with 3 bed volume of buffer consisted of
150 mM NaCl, 200 mM N-Acetyl-D-galactosamine (-D-GalNAc, MP Biomedicals), 200 mM glycine-HCl (pH 2.5), and 0.02% NaN3. The flow rate of the column was controlled at 10 mL/h.
The eluates were neutralized, desalted and concentrated by ultra-filtration using Amicon Ultra-
15 centifugal filter with 50 kDa cut-off membrane.
3.3.5 Identification of rHamOVGP1 by immunoblot and mass spectrometric analysis
The protein samples were size-fractionated by SDS-PAGE on a 6.0% gel. The proteins were visualized with silver staining. For Western blot analysis, the protein samples were mixed with reducing SDS sample buffer (2% SDS, 10% glycerol, 63 mM Tris HCl (pH 6.8), 0.1% -
53 mercaptoethanol, 0.0025% bromophenol blue), boiled for 5 min, separated on the gel, and electrophoretically transferred to polyvinylidene fluoride (PVDF) membrane. After blocking for 1 h with blocking buffer (5% nonfat milk and 0.1% Tween 20 in Tris-buffered saline (TBS) containing 50 mM Tris and 150 mM NaCl, pH 7.5), blots were probed sequentially with mouse monoclonal antibody against HamOVGP1 at a final concentration of 1 µg/mL, and then with peroxidase-labeled goat anti-mouse IgG at a final concentration of 0.02 µg/mL. Labeling was visualized by enhance chemiluminescence (ECL, PerkinElmer).
Purified rHamOVGP1 was further identified by mass spectrometry (MS). Briefly, the samples were run on SDS-PAGE gels. After brief staining of the gels, protein bands were cut from the gel and digested using the Micromass MassPREP Robotic Protein Handling System
(PerkinElmer). The trypsin-digested sample was subjected to analysis using the SCIEX
Voyager DE Pro Matrix-Assisted Laser-Desorption (MALDI) mass spectrometer at the Protein
Function Discovery Facility of Queen’s University, Ontario, Canada. Data from peptide mass fingerprinting were acquired over the mass range m/z 700 to 4000. MS data were processed using Applied Biosystems Data Explorer version 5.1 and submitted to the Genebio Aldente search engine for comparison against the Swiss-Prot database.
3.3.6 Preparation of sperm from the caudal epididymis
Motile hamster sperm were isolated and prepared following the procedure described by
Bavister (Bavister, 1989) with some modifications. Briefly, male hamsters at 7 to 9 weeks of age were anesthetized with an intraperitoneal injection of 150 μl sodium pentobarbital (McGill
54 University, Montreal, Quebec, Canada) and then sacrificed by cervical dislocation. The epididymides were excised and placed in a 6 mm plastic petri-dish containing mineral oil extracted with Tyrode-lactate-HEPES-polyvinyl alcohol (TL-HEPES-PVA) buffer. The viscous cauda epididymal contents (CECs) were released into the oil by repeatedly puncturing the cauda epididymis with a 23-gauge syringe needle. CECs were then transferred into a polypropylene tube containing 5 mL of TL-HEPES-PVA medium and resuspended gently, and incubated for 5 min at 37oC. During this period, sperm were allowed to swim-up. The top layer
(3 mL) of the suspension was withdrawn with a pipette and centrifuged at 500 xg for 5 min.
After centrifugation, the supernatant was immediately removed and discarded. The sperm pellet was resuspended in Tyrode’s-Albumin-Lactate-Pyruvate (TALP) medium. TALP is a modified Tyrode’s medium containing NaCl (114 mM), KCl (3.16 mM), CaCl2 (2 mM), MgCl2
(0.5 mM), NaHCO2 (25 mM), NaH2PO4 (0.35 mM), sodium lactate (10 mM), glucose (5 mM), and phenol red (0.001g/100 mL, pH 7.5). TALP medium was always supplemented with PVA
(0.1g/100 mL) and freshly made just before use or stored at 4oC and used within a month.
Immediately prior to use, sodium pyruvate (0.1 mM) and bovine serum albumin (BSA) (3
o mg/mL) were added and the medium was allowed to equilibrate at 37 C in 5% CO2 for 1 h.
The motility stimulators PHE (100 x stock solution) namely D-penicillamine (2 mM), hypotaurine (10 mM) and epinephrine (100 M) were added at 1/100 dilution in the medium equilibration. The vast majority of sperm (averaged 70-80%) were found to be motile when aliquots of the samples were viewed on the light microscope. The sperm count was determined using motile sperm obtained with the swim-up method.
55 3.3.7 Tyrosine phosphorylation of sperm proteins during in vitro capacitation
A dose-dependent experiment was first carried out to determine the optimal concentration of rHamOVGP1 to be used throughout the investigation. Aliquots of the sperm suspension (2 x 106 cells) were capacitated in vitro by incubating sperm in TALP-PVA medium containing
PHE in the presence of different concentrations of rHamOVGP1 (0, 10, 20, 40, 60 μg/mL) for
o 4 h at 37 C in 5% CO2 with 100% humidity. The effect of different concentrations of rHamOVGP1 on enhancement of tyrosine phosphorylation of sperm proteins was assessed by
Western blot analysis following the procedures described below. Results showed that rHamOVGP1 at a concentration of 20 μg/mL yielded the optimal results (see Results section).
Accordingly, rHamOVGP1 at a concentration of 20 μg/mL was used throughout the rest of the investigation. To determine the time-dependent effects of rHamOVGP1 on tyrosine phosphorylation of sperm proteins, sperm were incubated in the capacitating medium in the presence of 20 µg/mL of rHamOVGP1 for 0-6 h. Aliquots of the sperm suspension were taken at different time points and processed for SDS-PAGE or indirect immunofluorescence described below. All experiments were repeated three to four times using different male hamsters.
3.3.8 SDS-PAGE and immunoblotting of tyrosine phosphorylated sperm proteins
At the end of capacitation, sperm suspensions were collected and washed by centrifugation twice at 800 ×g for 5 min in D-PBS (Dulbecco’s phosphate-buffered saline, pH 7.4, containing
137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.47 mM KH2PO4, 0.9 mM CaCl2, 0.5 mM
56 MgCl2) with 1 mM sodium orthovanadate as a tyrosine phosphatase inhibitor. Sperm pellets were mixed with reducing SDS sample buffer (80 mM Tris HCl, pH6.8, 10% glycerol, 2%
SDS, 5% -mercaptoethanol, 0.02% bromophenol blue) and boiled for 5 min. In addition, demembranation of sperm was performed according to the procedure previously described
(Oko, 1988). The membrane was extracted twice for 15 min with shaking at 4oC in DT (2%
Triton X-100, 5 mM DTT, and 50 mM Tris-HCl, pH9.0). Each extraction was followed by a centrifugation at 1,000 xg for 10 min. After the last extraction, the demembranated sperm pellet was washed twice in 50 mM Tris-HCl (pH9.0) and used for immunoblot analysis. Samples were analyzed on 7.5% SDS-polyacrylamide gels. Following SDS-PAGE the resolved proteins were transferred to PVDF. After blocking with the blocking solution (5% nonfat milk and 0.1%
Tween 20 in TBS) for 1 h, blots were probed overnight at 4oC with gentle agitation with anti- phosphotyrosine antibody (Clone 4G10, Millipore) diluted in blocking solution at a final concentration of 0.1 µg/mL,). Afterwards, the membrane was washed thoroughly with TBS-T and then incubated for 1 h at room temperature with agitation in horseradish-peroxidase conjugated goat anti-mouse IgG diluted in blocking solution at a final concentration of 0.02
µg/mL. The membrane was then washed four times for 10 min each with TBS-T. Finally, the blot was developed using ECL detection kit according to the manufacturer’s instruction. The blot was stripped and re-probed using mouse anti--tubulin antibody at a final concentration of 0.02 µg/mL as internal control. Band intensity levels were measured by ImageJ software
(NIH, USA). Statistical analysis was performed using the Student’s two-tailed t-test. In experiments where mouse anti-AKAP82-antibdy (BD Transduction LaboratoriesTM, San Jose,
57 California) was employed, the blot was probed with the antibody at a final concentration of
0.01 µg/mL for 3 h at 4oC followed by incubation with horseradish-peroxidase conjugated goat anti-mouse IgG (0.02 µg/mL). The blot was developed using the ECL-kit.
3.3.9 Immunofluorescent detection of binding of rHamOVGP1 to sperm
Aliquots of the sperm capacitated in the presence of 20 µg/mL of rHamOVGP1 for 0, 1, and 3 h, respectively, were used to examine the ability of rHamOVGP1 to bind to sperm cell surfaces. Following incubation as described above, aliquots of 20 μl of sperm were smeared onto superfrost plus microscope slides, air-dried, and fixed for 15 min in 4% paraformaldehyde.
The fixed sperm samples were then labeled with a monoclonal antibody against HamOVGP1
(1 µg/mL) or with the same buffer but in the absence of the primary antibody followed by incubation in a solution of goat anti-mouse IgG-FITC at a final concentration of 2 µg/mL. After washing, the samples were mounted with 1% DABCO (1,4-diazobicyclo-[2,2,2]-octane) in 90% glycerol/PBS and then viewed on a Leica TCS-SP2 Multiphoton Confocal Laser Scanning
Microscope (TCS-MP, Heidelberg, Germany).
3.3.10 Immunofluorescent microscopy of capacitated sperm
Capacitated sperm (1 x 105 cells) prepared as described above were smeared onto the surface of superfrost plus microscope slides, air dried, and fixed for 15 min in 4% paraformaldehyde, rinsed in PBS and then permeabilized with 0.1% Triton in PBS at 4oC overnight. In addition, samples of demembranated sperm prepared as described above were smeared onto slides, air-dried, fixed with cold (-20oC) 100% methanol for 1 min, and air-dried.
58 Slides were then washed with DPBS containing 1% normal goat serum (NGS) and blocked with 5% NGS in DPBS for 1 h. Mouse monoclonal anti-phosphotyrosine antibody Clone 4G10
(diluted in blocking solution at a final concentration of 1 µg/mL) was applied to the slides for immunolabeling for 2 h at room temperature. Slides were then washed and incubated with goat anti-mouse IgG-FITC at a final concentration of 1 µg/mL for 1 h in the dark at room temperature. Subsequently, the slides were washed three times with DPBS containing 1% NGS and air-dried, followed by mounting with 1% DABCO in 90% glycerol/PBS. Fluorescent microscopy was performed using a Leica TCS-SP2 Multiphoton Confocal Laser Scanning
Microscope (TCS-MP, Heidelberg, Germany).
3.3.11 Assessment of acrosome reaction
Sperm (2 x 105 cells) were incubated in TALP-PVA medium in the presence (20 µg/mL) or absence of rHamOVGP1 for different time intervals (0-6 h). Sperm were washed twice with
1mL of HBSS (5.33 mM KCl, 0.44 mM KH2PO4, 137.93 mM NaCl, 0.34 mM Na2HPO4, 5.56 mM D-glucose), centrifuged at 500 xg for 5 min and fixed in 100% methanol on ice for 30 min.
Fixed sperm were smeared onto superfrost plus microscope slides and dried on the slide warmer. To assess the acrosomal status, samples of acrosome-reacted sperm were stained with
FITC-conjugated Pisum sativum agglutinin (PSA) at a final concentration of 75 µg/mL.
Coverslips were mounted on slides with 90% glycerol/PBS containing 1% (w/v) DABCO as an anti-bleaching agent and observed under an immunofluorescent microscope. The status of acrosome reaction was then evaluated according to the fluorescent pattern of the acrosome.
59 Experiments were carried out in triplicates and at least 200 sperm per slide were evaluated for acrosomal status.
3.3.12 Superovulation and oocyte retrieval
Four mature female hamsters weighing approximately 110 to 130g were each superovulated by intraperitoneal (i.p.) injection of 40 IU of pregnant mare’s serum gonadotropin in 100 μl saline solution (0.9% NaCl) between 9:30 and 10:00 a.m. on the day of postovualotry discharge (metestrus). Sixty hours later, the females each received an i.p. injection of 40 IU of hCG in 100 μl saline solution. Superovulated female hamsters were sacrificed 13.5 hr post-hCG injection by cervical dislocation under anesthesia after receiving an i.p. injection of sodium pentobarbital (60 mg/kg body weight). Ovaries were removed under aseptic procedures and placed in a culture dish containing TALP-HEPES-PVA. Ovarian oocytes with an expanded cumulus were collected by repeated follicle puncture using a 28- gauge needle. The oocytes with surrounding cumulus cells were transferred to TALP-HEPES-
PVA medium containing hyaluronidase (1 mg/mL) and soybean trypsin inhibitor (0.01 mg/mL). Cumulus cells were removed by using a narrow-bore glass pipette, and oocytes were
o then washed three times in TALP-PVA medium previously equilibrated at 37 C in 5% CO2 in air.
3.3.13 Immunolocalization of rHamOVGP1 in the ZP of ovarian oocytes
A total of 48 oocytes from mature ovarian follicles were isolated and incubated in TALP-
PVA medium in the absence (24 oocytes) or presence (24 oocytes) of immunopurified
60 o rHamOVGP1 at a concentration of 20 μg/mL for 3 h at 37 C in 5% CO2. After incubation, oocytes were washed three times with TL-HEPES buffer, fixed for 30 min in a fixative containing 2% glutaraldehyde and 2% formaldehyde in PBS, blocked with 5% normal goat serum (NGS) in PBS for 1 h prior to incubation in primary antibody (mouse monoclonal anti- hamster OVGP1 antibody diluted in 1% NGS in PBS at a final concentration of 1 µg/mL) overnight at 4oC. Following incubation, oocytes were washed three times for 5 min each in
PBS containing 1% NGS. Oocytes were then incubated in fluorescent (FITC)-conjugated goat anti-mouse IgG at a final concentration of 1µg/mL for 1 h in the dark at room temperature.
Subsequently, oocytes were washed as above and air-dried, followed by mounting using 1%
DABCO in 90% glycerol/PBS. Fluorescent microscopy was performed using a Leica TCS-SP2
Multiphoton Confocal Laser Scanning Microscope (TCS-MP, Heidelberg, Germany).
3.3.14 Sperm-oocyte binding assay
Ovarian oocytes were incubated in 100 l TALP-PVA with 5,000 capacitated sperm under mineral oil for 30 min. The oocytes were then washed three times with a wide-bore glass pipette to remove loosely attached sperm, and immediately fixed in a fixative containing 2% glutaraldehyde and 2% formaldehyde in PBS. Sperm bound to the oocytes were counted under the light microscope (Zeiss A x 10) at x20, and photographs were taken for documentation. In order to investigate the effect of rHamOVGP1 on sperm binding to ovarian oocytes, ovarian oocytes or sperm were preincubated with rHamOVGP1 (20 µg/mL) for 1 h (prior to sperm binding) and washed thrice with TALP-PVA medium between the preincubation and sperm-
61 oocyte binding steps. Eleven different groups were included in this experiment as follows -
Group A: capacitated sperm were incubated with oocytes for 30 min in TALP-PVA alone;
Group B: pretreatment of oocytes alone with rHamOVGP1 for 1 h prior to sperm-oocyte binding; Group C: pretreatment of sperm alone with rHamOVGP1 prior to sperm-oocyte binding; Group D: rHamOVGP1 was present only during sperm-oocyte binding without prior pretreatment of oocytes or sperm; Group E: rHamOVGP1 was present during sperm-oocyte binding with pre-treated oocytes and untreated sperm; Group F: rHamOVGP1 was present during sperm-oocyte binding with pretreated sperm and untreated oocytes; Groups G to K
(controls) were carried out in the same manner as Groups B to F except that monoclonal antibody against hamster OVGP1 (0.1 µg/mL) was added to the culture medium during sperm- egg binding . A total of 165 ovarian oocytes were retrieved from six animals. The 165 oocytes were randomly assigned to each of the experimental or control groups with 15 ovarian oocytes in each group.
3.4 Results
3.4.1 Production and purification of rHamOVGP1
As aforementioned, oviductin (OVGP1) has been identified and characterized in several mammalian species. However, further exploration of the biological roles of OVGP1 has been hampered by the unavailability of sufficient amounts of native OVGP1 for functional and mechanistic studies. To overcome this drawback, an alternative would be to produce recombinant OVGP1 that is biologically active. It is now generally agreed that the stability and
62 level of transgene expression from integrated lentivirus-derived vectors (LVs) are better than the traditional oncoretroviral vectors. In the present study, LV-mediated gene transfer was applied to the establishment of stable cell lines co-expressing rHamOVGP1and GFP. HEK 293 cells were infected with the transfer vector WPI-HamOVGP1. At 2-week post-infection, the individual GFP-positive clones were isolated and grown in the growth medium. The clones were screened for expression levels of rHamOVGP1 by immunoblot analysis of culture supernatants using monoclonal antibody against HamOVGP1. HEK293 cells derived from clones stably expressing high levels of rHamOVGP1 were selected and maintained in the medium and GFP expression was monitored on a daily basis. Over 90% of the cells remained
GFP-positive for 12 weeks. Since rHamOVGP1 was expected to be secreted into the culture medium by HEK293 cells, we probed the presence of rHamOVGP1 in the culture medium by immunoblot analysis. The rHamOVGP1 migrated as a polydispersed band of 160-350 kDa during SDS-PAGE under reducing condition (Fig. 3-1) consistent with results previously obtained with purified HamOVGP1 (Malette and Bleau, 1993).
At the end of the production, the culture medium was harvested and stored as described in Materials and Methods. Previous study showed that HamOVGP1 is a highly glycosylated protein that contains terminal -D-GalNAc residues recognized by Helix pomatia agglutinin
(HPA) (Malette and Bleau, 1993). This carbohydrate moiety of HamOVGP1 allowed the purification of rHamOVGP1 to homogeneity using HPA-agarose for the affinity purification of rHamOVGP1. Bound rHamOVGP1 (approx. 160-350 kDa) was eluted (Fig. 3-2A, lane 2).
Indeed, using this approach we were able to produce considerably amount of rHamOVGP1
63 from HEK293 cells. In our hands, a typical yield from 1L of the conditional medium varied between 0.25 to 0.5 mg of rHamOVGP1. The purity of rHamOVGP1 was assessed by SDS-
PAGE (Fig. 3-2A, lane 2) and Western blot analyses (Fig. 3-2B, lane 1). As shown in Figure
3-2A, purified rHamOVGP1 revealed predominantly a polydispersed band of the expected molecular mass of 160-350 kDa under reducing conditions (lane 2). Immunoblot analysis of
HPA-purified rHamOVGP1 (Fig. 3-2B, lane 1) was consistent with results obtained with HPA- purified HamOVGP1 from hamster oviducts prepared from the estrus stage (Fig. 3-2B, lane 2).
The identity of the purified recombinant glycoprotein was verified to be HamOVGP1 by mass spectrometric analysis (Fig. 3-2C).
3.4.2 Localization of rHamOVGP1 binding sites on hamster sperm
Previous immunolocalization studies carried out with OVGP1 showed the binding of
HamOVGP1 to the acrosomal region of hamster sperm (Boatman and Magnoni, 1995; Saccary et al., 2013) and the binding of bovine OVGP1 to the head and tail regions of bovine sperm
(King et al., 1994). Having successfully produced rHamOVGP1, our next step was to determine if rHamOVGP1 has the ability to bind to homologous sperm like the native
HamOVGP1. Indeed, indirect immunoflurescence showed the localization of rHamOVGP1 mainly to the head and mid-piece of hamster epididymal sperm after 1 and 3 h of capacitation in a time-dependent manner (Fig. 3-3). Immunostaining was practically absent at both time intervals when sperm were capacitated in the absence of rHamOVGP1. At 1 h, a strong immunoreaction was detected in the mid-piece of the sperm tails and in the equatorial segment
64 region of the sperm heads (Fig. 3-3). At 3 h, a strong immunoreaction remained associated with the mid-piece of the sperm tails with a weaker staining intensity over the principal piece.
Interesting, immunostaining of the equatorial segment region previously seen at 1 h disappeared at the 3 h-time interval (Fig. 3-3). In controls where the primary antibody was omitted, sperm samples incubated in capacitating medium even in the presence of rHamOVGP1 showed a negative immunoreaction demonstrating the specificity of the antibody
(results not shown).
3.4.3 rHamOVGP1 enhanced protein tyrosine phosphorylation in hamster sperm
A dose-dependent experiment was carried out where hamster sperm were incubated in capacitation medium (TALP-PVA) alone or supplemented with different concentrations of rHamOVGP1 (0, 10, 20, 40 and 60 g/mL) for 4 h. Immunoblot analysis using anti- phosphotyrosine antibody indicated that the overall pattern of protein tyrosine phosphorylation in rHamOVGP1-treated and control hamster sperm was very similar (Fig. 3-4A). But six tyrosine-phosphorylated proteins (35, 63, 75, 83, 160 and 180 kDa) exhibited an increase in labeling intensity in the treated-sperm (Fig. 3-4A). Of the six enhanced tyrosine phosphorylated proteins the 75- and 83-kDa proteins were the most intensely enhanced phosphorylated proteins
(Fig. 3-4A). Quantification of the intensity of the major proteins showed that rHamOVGP1 at a concentration of 20 g/mL enhanced the major tyrosine phosphorylated bands of 63, 75, 83 and 180 kDa whereas rHamOVGP1 at a concentration of 40 and 60 g/mL only enhanced the
63kDa and 83 kDa protein bands (Fig. 3-4B). Based on the results obtained from the dose-
65 dependent experiment, rHamOVGP1 at a concentration of 20 µg/mL was considered the optimal concentration and was chosen for use in the time-course experiment and for the rest of the study. Our results showed that hamster sperm following incubation in TALP-PVA alone or supplemented with 20 µg/mL of rHamOVGP1 for 0-6 h exhibited a time-dependent increase in protein tyrosine phosphorylation (Fig. 3-5). Of all the proteins, the 83 kDa protein was the most intensely phosphorylated in capacitated sperm following SDS-PAGE and immunoblot analysis (Fig. 3-5). In this study, attempts were made to further characterize the 83 kDa protein.
For this purpose, the extracts containing proteins from intact or demembranated sperm were resolved by gel electrophoresis and detected by immunoblot analysis using anti- phosphotyrosine antibody followed by stripping and re-probing using a mouse anti-AKAP82 antibody since the 83 kDa is known to be a hamster homologue of mouse AKAP82 (Jha and
Shivaji, 2002). Enrichment of sperm capacitation-associated tyrosine phosphorylated proteins by DT extraction revealed that most of them were non-membranous in nature (Fig. 3-6A, lanes
3 and 4). A predominant 83 kDa protein was detected by anti-AKAP82 antibody (Fig. 3-6B) corresponding to the 83 kDa protein band detected by anti-phosphotyrosine antibody (Fig. 3-
6A).
3.4.4 Localization of rHamOVGP1-enhanced tyrosine phosphorylated proteins in hamster sperm
To further validate the Western blot data, sperm were subjected to immunocytochemistry using anti-phosphotyrosine antibody. Figure 3-7 showed a time-dependent increase of
66 immunofluorescence signal for tyrosine phosphorylated proteins in sperm treated with 20
µg/mL of rHamOVGP1 during capacitation. Localization of tyrosine phosphorylated proteins was detected mainly in the equatorial segment region of the sperm head and along the mid- piece as well as principal piece (Figs. 3-7 B2, D2 and F2). Immunoreaction started to appear at 0 h but was localized only to the equatorial segment region of the sperm heads and not in other places (Fig. 3-7B2). A strong intensity of immunostaining began to appear over the mid- piece with a weaker immunoreaction over the principal piece of the sperm tails at the 2 h-time interval (Fig. 3-7D2) and this immunostaining pattern of the sperm tail persisted at 4 h of capacitation (Fig. 3-7F2). Interestingly, immunoreaction over the equatorial segment seen at both the 0 h- and 2 h-time intervals disappeared at the 4 h-time interval (Fig. 3-7F2). The untreated groups showed a much weaker immunofluorescence signal (Figs. 3-7A2, C2 and E2).
To substantiate the localization data, rHamOVGP1 treated-sperm were demembranated using dithiothrietol-triton which consistently removes all the membranous components leaving the cytoskeletal components of the sperm flagellum including the outer dense fibers, fibrous sheath, and axoneme intact. Demembranated sperm exhibited an intense signal in the principal piece of the sperm tail but only a faint immunofluresence signal was observed in the mid-piece (Fig.
3-7G). These findings indicated that capacitation-associated tyrosine phosphorylated proteins are associated with membranous structures in the head region and mid-piece region, but with non-membranous components, in particular, the fibrous sheath, in the principal piece.
67 3.4.5 Effects of rHamOVGP1 on acrosome reaction
To find out if rHamOVGP1 has any positive effects on acrosome reaction, hamster sperm were incubated in TALP-PVA alone or in the same medium supplemented with 20 μg/mL of rHamOVGP1 for different time intervals (0-6 h). The percentage of sperm undergoing spontaneous acrosome reaction was then measured. Hamster sperm incubated in TALP-PVA alone showed a time-dependent increase in the percentage of acrosome-reacted sperm (Fig. 3-
8). A further increase in the number of acrosome-reacted sperm was noted after 3-5 h of capacitation in the presence of rHamOVGP1 compared to the controls in the absence of rHamOVGP1 (Fig. 3-8). There was no significant difference in acrosome reaction at 6 h of capacitation since acrosome reaction of sperm incubated in TALP-PVA alone reached 80%.
Therefore, rHamOVGP1 can exert a positive effect in facilitating and accelerating the process of acrosome reaction.
3.4.6 Binding of rHamOVGP1 to the ZP of hamster ovarian oocytes
As expected, immunofluorescence signal was absent in the ZP of ovarian oocytes incubated in TALP-PVA medium alone (Fig. 3-9). Bright immunofluorescence was detected throughout the entire thickness of the ZP when ovarian oocytes were incubated in culture medium containing rHamOVGP1 (Fig. 3-9). The immunofluorescent labeling observed in the oocyte proper is considered unspecific since this was observed in both experimental and control groups.
68 3.4.7 Influence of rHamOVGP1 on sperm binding to ZP of oocytes
In order to determine if rHamOVGP1 affects sperm-oocyte binding, pre-treatment of sperm and/or oocytes with rHamOVGP1 were carried prior to incubation in TALP-PVA medium in the presence or absence of rHamOVGP1. The mean number of hamster sperm bound to hamster ovarian oocytes in TALP-PVA medium alone was 10 1 sperm/oocyte (Fig.
3-10: Group A). A significant increase in the number of sperm bound to the oocytes was found when either oocytes (Fig. 3-10: Group B) or sperm (Fig. 3-10: Group C) were pre-treated with rHamOVGP1 prior to incubation in TALP-PVA alone (22 ± 2 and 22 ± 3 sperm/oocytes, respectively, with p<0.05). Interestingly, when both previously untreated oocytes and sperm were incubated in TALP-PVA in the presence of rHamOVGP1 (Fig. 3-10: Group D), the number of sperm bound per oocytes (12 ± 2) was comparable to that of the control (10 ± 1) with no significant increase. A significant increase was noted when either pre-treated oocytes
(Fig. 3-10: Group E) or sperm (Fig. 3-10: Group F) were incubated in the medium in the presence of rHamOVGP1 albeit with a larger increase in the pre-treated oocyte group (31 ± 3, p<0.01 vs. 18 ± 4, p<0.05). Addition of monoclonal antibody against hamster OVGP1 to the medium during sperm-oocyte binding incubation in experimental groups B to F abolished the enhancement of sperm binding to oocyte by rHamOVGP1 (Fig. 3-10: Groups G to K). In addition, we also performed an experiment in our study where both sperm and oocytes were pre-treated with rHamOVGP1 prior to sperm-oocyte binding assay. However, the increase was found to be not statistically significant as compared to the control (result not shown).
69 3.5 Discussion
Mammalian oviductal fluid consists of a complex mixture originating from plasma transudation and glycoproteins secreted by oviduct epithelial cells (Aviles et al., 2010).
Accumulated evidence has shown that OVGP1, also known as oviduct-specific glycoprotein or oviductin, a major glycoprotein secreted by the oviductal epithelium, plays important roles during the early events of fertilization and early embryo development (Buhi, 2002). Although
OVGP1 has been identified and characterized in many mammalian species including the human, further elucidation of its biological functions and the study of the mechanism regulating its functions have been hampered by the limited quantities of OVGP1 that can be purified from oviductal fluid and/or oviductal cells.
In the present study, we used recombinant technology to successfully produce rHamOVGP1. It has been previously shown that HamOVGP1 contained terminal -D-GalNAc residues (Araki et al., 1987) and can be purified using HPA-agarose affinity columns (Malette and Bleau, 1993). Using the similar approach, we purified from HEK293 culture medium the rHamOVGP1 that displayed a polydispersed band of 160-350 kDa on SDS-PAGE gel, which is consistent with the molecular mass of purified native protein previously described (Malette and Bleau, 1993). The identity of the recombinant glycoprotein was further confirmed by immunoblot analysis using antibody generated against HamOVGP1 and subsequently by mass spectrometric analysis.
The main goal of producing rHamOVGP1 is to obtain sufficient quantities of the recombinant glycoprotein for functional and mechanistic studies, which, otherwise, cannot be
70 attained with limited amount of native HamOVGP1. Therefore, having successfully produced rHamOVGP1 it would be imperative to determine if the newly generated rHamOVGP1 is biologically active. First, we set out with an experiment to determine if rHamOVGP1 has the ability to bind to the sperm surfaces since previous studies showed the binding of native hamster (Kan and Esperanzate, 2006) and bovine (King and Killian, 1994) OVGP1, respectively, to the head regions of corresponding homologous sperm. Indeed, our results with indirect immunofluorescence showed localization of rHamOVGP1 mainly to the sperm head and to the middle piece of the sperm tail with a lesser extent to the principal piece. The present results suggest that rHamOVGP1 may exert its function through its binding to both the head and tail regions of hamster sperm. This notion is particularly appealing since a recent study carried out in our laboratory showed that native HamOVGP1 purified from hamster oviducts can enhance sperm capacitation by potentiating tyrosine phosphorylation of several sperm proteins known to be involved in sperm-egg binding and sperm motility that are associated with the sperm head and tail, respectively (Saccary et al., 2013). In the present rHamOVGP1 binding experiment, it was also observed that the immunostaining of the head region previously seen at 1 h greatly diminished or disappeared at the 3 h-time interval. This could be attributed to the high percentage of sperm undergoing spontaneous acrosome reaction in the hamster species as previously reported (Siva et al., 2006). As shown in Figure 3-8, 45 to 55 percent of hamster sperm were found to be acrosome-reacted between the 3 h- and 4-h time intervals.
Therefore, the reduction or disappearance of the immunostaining at the 3 h-time interval was likely due to the acrosome-reacted status of the sperm heads.
71 In order to become fertilizing competent, mammalian sperm freshly coming from the epididymis must undergo a process called “capacitation” which occurs either normally in the female reproductive tract or in vitro under an appropriate environment (Austin, 1951; Chang,
1951). Capacitation is characterized by sperm exhibiting hyperactivated motility, and is
2+ - associated with several molecular changes including increases in intracellular Ca , HCO3 , and cAMP levels, an efflux of cholesterol, and changes in protein kinase and phosphatase activities
(Bailey, 2010; Aitken and Nixon, 2013). Among these physiological changes characteristic of sperm capacitation, an increase in global tyrosine phosphorylation of sperm proteins has been shown to be a hallmark of capacitation (Visconti et al., 1995b; Naz and Rajesh, 2004).
Mammalian fertilization resulting from fusion of an oocyte with a capacitated sperm represents the standard endpoint of capacitation. Accumulated evidence points to the fact that acquisition of hyperactivation by sperm during capacitation, an event necessary for fertilization, is under the influence of OVGP1 (Aviles et al., 2010). We have recently demonstrated, for the first time, that native estrus-associated HamOVGP1 can enhance sperm capacitation by increasing tyrosine phosphorylation of a subset of sperm proteins in a time-dependent manner (Saccary et al., 2013). Therefore, in the present study an experiment was carried out to determine if rHamOVGP1 has the ability to enhance sperm capacitation by increasing tyrosine phosphorylation of sperm proteins similar to what we previously observed with the native
HamOVGP1 (Saccary et al., 2013). As expected, our results demonstrated that the rHamOVGP1 that we produced is biologically potent and capable of enhancing tyrosine phosphorylation of sperm proteins like the native HamOVGP1. Particularly rHamOVGP1 at a
72 concentration of 20 μg/mL appears to be the optimal concentration which significantly enhanced several proteins of 63, 75, 83 and 180 kDa in a time-dependent manner with a peak at 4 h of capacitation (Figs. 3-5 and 6). The present results demonstrate clearly that the effect of rHamOVGP1 on protein tyrosine phosphorylation during capacitation is dose and time- dependent.
Having obtained the results above, it would be of interest to identify the enhanced tyrosine phosphorylated proteins that are under the influence of rHamOVGP1. Of all the tyrosine phosphorylated proteins enhanced by rHamOVGP1, the 83 kDa protein was found to be the most intensely phosphorylated reaching a peak at 4 h of capacitation. This protein was subsequently revealed by immunoblot analysis to be a kinase anchoring protein (AKAP83, also called AKAP4), a major component of the fibrous sheath previously shown to be a major tyrosine phosphorylated protein in capacitated hamster sperm (Jha and Shivaji, 2002).
Importantly, we have now reported, for the first time, that this protein (AKAP83) is further enhanced by rHamOVGP1 during capacitation substantiating the involvement of AKAP83 in capacitation and its possible role in sperm motility due to its localization in the principle piece of the sperm tail. In our study, immunofluorescent staining of demembranated sperm localized the rHamOVGP1-enhanced, tyrosine phosphorylated proteins exclusively to the principal piece of hamster sperm flagella. Therefore, the present immuno-localization results confirm the identity of the rHamOVGP1-enhanced 83 kDa protein as a fibrous sheath protein. Prior experimental evidence suggests that the fibrous sheath not only acts as a passive elastic structural element of the sperm tail but may also be an active regulator of flagellar movement
73 essential for sperm motility (Si and Okuno, 1999). The authors of the study postulated that tyrosine phosphorylation of fibrous sheath components of the sperm tail could lead to a decrease in the stiffness of fibrous sheath and thus enabling the flagellum to generate greater bends leading to sperm hyperactivation. To date, despite extensive study of mammalian sperm motility, the mechanism underlying sperm hyperactivation is still not fully understood. Our present experimental results revealing the 83 kDa protein as the AKAP83 fibrous sheath protein provide further, albeit indirect, support for a role of tyrosine phosphorylation of sperm proteins in sperm hyperactivation. More importantly, the present results showing the rHamOVGP1-enhanced tyrosine phosphorylation level of several major proteins, in particular the 83 kDa protein, reinforces the notion that the oviductal lumen, where OVGP1 is present in vivo, provides a suitable milieu for sperm to prepare their way for fertilization.
Since capacitation prepares sperm to undergo acrosome reaction, a study was undertaken to determine if prior capacitation of hamster sperm in the presence rHamOVGP1 can increase the number of acrosome-reacted sperm as compared to their counterpart capacitated in the absence of rHamOVGP1. The spontaneous acrosome reaction in active motile sperm can be used as an indication of the completion of capacitation in hamster sperm. Indeed, using this parameter the present results showed a significant and time-dependent increase in the number of acrosome-reacted sperm when sperm were capacitated in TALP-PVA medium in the presence 20 µg/mL of rHamOVGP1 at 3 h, 4h and 5 h time intervals.
Previous studies using oviductal fluid or partially purified OVGP1 from various species, including the hamster, showed the binding of OVGP1 to the ZP of corresponding post-
74 ovulatory oocytes (Kan et al., 1989; Boice et al., 1990a; Malette and Bleau, 1993; Buhi, 2002).
Endogenous form of HamOVGP1 has been found to associate with the ZP of ovulated oocytes once it is released from the oviductal epithelial cells (Kan et al., 1989). Oocytes isolated from mature ovarian follicles incubated with immunopurified endogenous HamOVGP1 displayed a strong immunofluorescent signal localized in the ZP of hamster oocytes (Malette and Bleau,
1993; Boatman and Magnoni, 1995). Therefore, if the rHamOVGP1 that we produced is biologically active, it should also possess the ZP-binding property. Indeed, results of our immunostaining experiments showed that rHamOVGP1 behaved like its endogenous counterpart and bound uniformly throughout the entire thickness of the ZP of hamster ovarian oocytes that had not been previously exposed to the oviductal environment (Fig. 3-9). The immunofluorescent signal observed in the oocyte proper was likely due to nonspecific binding of the secondary antibody to the oocyte proper since this signal was observed regardless of the presence or absence of rHamOVGP1 and the monoclonal anti-HamOVGP1 antibody.
Having demonstrated that rHamOVGP1 is able to bind the ZP of ovarian oocytes, we hypothesized that rHamOVGP1 can also increase sperm-oocyte binding. In the present study, pre-treatment of either oocytes or sperm with rHamOVGP1 prior to incubation in TALP-PVA alone significantly increased the number of sperm bound per oocyte. This increase was also observed when rHamOVGP1 was present during both oocyte or sperm pre-incubation and sperm-oocyte binding. The addition of rHamOVGP1 in TALP-PVA medium without pre- treatment of oocytes and sperm, however, did not seem to affect sperm-oocyte binding.
Therefore, the positive effect of rHamOVGP1 on sperm-oocyte binding appears to be more
75 associated with the oocyte since the largest increase in the number of sperm bound to oocytes was noted when the oocytes were pre-exposed to rHamOVGP1. The addition of monoclonal
HamOVGP1 antibody in the medium negated the positive effect of rHamOVGP1 on sperm- oocyte binding thus confirming the specificity of rHamOVGP1. The present results are in agreement with the overall concept that the effects of mammalian OVGP1 on reproductive events are mediated primarily through interactions with the oocyte (Buhi, 2002). It is noteworthy to mention OVGP1-null mice have been reported to be fertile in vivo (Araki et al.,
2003). However, protein expression of mouse OVGP1 was not carried out in that study (Araki et al., 2003). Furthermore, information on mouse OVGP1 is relatively limited compared to that found in other mammals including the human, particularly at the protein level. Contrary to other mammals where OVGP1 is detected in the ZP of postovulatory oocytes, an oviduct- derived glycoprotein reactive with wheat germ agglutinin was found in the perivitelline space but not in the ZP of mouse oocytes and early embryos (Kapur and Johnson, 1986). Although a latter study using antibody against human OVGP1 localized the corresponding antigen to both the ZP and perivitelline space of mouse oocytes (Lyng and Shur, 2009), the identity and biological role of OVGP1 in the mouse species remain unclear.
Importantly, in the present study our results directly link the increase in tyrosine phosphorylation of sperm proteins to the beneficial effect of OVGP1 on sperm capacitation.
Although capacitation of mammalian sperm, including human sperm, can be achieved in vitro under appropriate conditions, an exhaustive search on major suppliers of commercially available capacitating media reveals that commercially available capacitating media do not
76 contain OVGP1. Capacitation is a fundamental process that normally occurs in the lumen of the female reproductive tract where OVGP1 is present. Our data showing that pre-treatment of sperm and/or oocytes with OVGP1 can enhance sperm capacitation through stimulation of tyrosine phosphorylation of sperm proteins and increase sperm-oocyte binding are novel and important findings that reinforce the biological significance of OVGP1 in vitro. It is envisaged that future development and refinement of recombinant OVGP1 in other mammalian species, including the human, will allow us to improve and optimize culture conditions for early embryo development and in vitro fertilization procedures.
3.6 Conclusions
In summary, results obtained with our newly produced rHamOVGP1 showed that addition of rHamOVGP1 in the capacitation medium further enhanced tyrosine phosphorylation of a sub-set of sperm proteins and significantly increased acrosome reaction. Interestingly, one of the most enhanced tyrosine phosphorylated protein during sperm capacitation under the influence of rHamOVGP1 is AKAP83, a major fibrous sheath protein. Furthermore, results obtained in the present study validated our hypothesis that rHamOVGP1 is biologically active and that pre-treatment of oocytes and sperm with rHamOVGP1 can increase sperm-oocyte binding. The successful large-scale production of rHamOVGP1 and the positive effects of rHamOVGP1 on sperm functions and sperm-oocyte binding will pave way for further identification and characterization of capacitation-associated and rHamOVGP1-enhanced tyrosine phosphorylated proteins and for further elucidation of its role in the early events of fertilization. Future studies are also warranted to investigate the mechanisms that regulate the
77 physiological function of rHamOVGP1, for instance, the role of glycan derivatives of
HamOVGP1 on early events of fertilization.
78
Figures
79
Figure 3- 1:Immunochemical identification of rHamOVGP1. Culture supernatant from control (lane 1) and from rHamOVGP1-transduced HEK293 cells (lane 2) were subjected to SDS-PAGE (6.0%) followed by transfer to PVDF membranes. Western blot analysis was performed as described in Materials and Methods.
80
A B kDa 1 2 kDa 1 2
250 ¬ HamOVGP1 ® 250 - 150 150 - 100
100 - 75 - 50 75
37 50 -
25
C
MGRLLLWVGLVLLMKPNDGTAYKLVCYFTNWAHSRPVPASILPRDLDPFLCTHLIFAFASMSN NQIVANNLQDEKILYPEFNKLKERNRALKTLLSVGGWNFGTSRFTTMLSTLASREKFIGSVVS FLRTHGFDGLDLFFLYPGLRGSPIDNRWNFLFLIEELQFAFEKEALLTQRPRLLLSAAVSGIPYI IQTSYDVHLLGRRLDFINVLSYDLHGSWEKSTGHNSPLFSLPEDPKSSAFAMNYWRNLGAPAD KLLMGFPAYGRTFHLLRESKNGLQAASMGPASPGKYTKQAGFLAYYEVCSFIQRAEKHWID HQYVPYAYKGKEWVGYDDAVSFSYKAMFVKKEHFGGAMVWTLDMDDVRGTFCGNGPFPL VHILNELLVRAEFNSTPLPQFWFTLPVNSSGPGSESLPATEELTTDTVKILPPGGEAMATEVHR KYEKVTTIPNGGFVTPAGTTSPTTHAVALERNAMAPGAKTTTSLDLLSETMTGMTVTVQTQT AGRETMTTVGNQSVTPGGETMTTVGNQSVTPGGETMTTVGNQSVTPGGETVTIVGNKSVTP VGETVTIVGNKSVTPGGQTTATVGSQSVTPPGMDTTLVYLQTMTLSEKGTSSKKAVVLEKVT VPPREISVMPNEQNTALNRENLIAEVESYSQDG
Figure 3- 2: Purification and analysis of rHamOVGP1. (A) The samples were analyzed using 6% SDS-PAGE under reducing conditions followed by silver staining. Lane 1: protein ladder; lane 2: purified rHamOVGP1 (1 µg/lane) using HPA-agarose; (B) Samples were subjected to 6% SDS-PAGE followed by immunoblot analysis as described in Materials and Methods. Lane 1: purified rHamOVGP1 (100 ng/lane) using HPA-agarose; lane 2: purified HamOVGP1 (100 ng/lane) from homogenates of estrus-stage hamster oviducts using HPA-agarose; (C) Complete sequence of HamOVGP1 with the coverage of peptides (bold red) identified from the trypsin digest by MS.
81
PP
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82
Figure 3- 3: Confocal microscope imaging of rHamOVGP1 binding sites on hamster sperm after capacitation. Epididymal sperm were incubated in capacitating medium in the absence (-) or presence (+) of 20 μg/mL rHamOVGP1 for 0-, 1- and 3-h, respectively. Immunofluorescent signal was practically absent at all three time intervals in the absence of rHamOVGP1. In the presence of rHamOVGP1, immunostaining started to appear at 1 h mainly over the equatorial segment (ES) region of the sperm head (see insert for a higher magnification of the same sperm head indicated by double-head arrow in red) and the mid-piece (MP) of the sperm tail with a relatively weaker immunostaining over the principle piece (not shown). The pattern of immunostaining of the mid-piece (MP) and principle piece (PP) persisted at the 3 h time interval except that the previously seen immunofluorescent signal over the equatorial segment region now diminished or disappeared. Scale bars = 50 μm, Scale bar of insert = 140 μm
83 kDa 1 2 3 4 5 A 180 - 135 - 100 -
75-
63-
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Figure 3- 4: Dose-dependent effect of rHamOVGP1 on tyrosine phosphorylation of hamster sperm proteins. Hamster caudal epididymal sperm, cultured for 4 h in modified TALP medium alone or supplemented with different concentration of rHamOVGP1 (10, 20, 40, and 60 g/mL), were processed for SDS- PAGE, transferred to PVDF membrane and probed with anti-phosphotyrosine antibody. A: representative blot; B: summary of data. Asterisks (*) indicate values that are significantly different (p<0.05) from that obtained with untreated sperm.
84
Figure 3- 5: Time course of protein tyrosine phosphorylation in caudal epididymal hamster sperm. Sperm were incubated in modified TALP medium in the absence or presence of 20 g/mL rHamOVGP1 for different time intervals (0-6 h). Equal numbers of sperm were solubilized for SDS-PAGE and immunoblotting with anti-phosphotyrosine antibody.
85
B A
kDa 1 2 3 4 1 2 3 4 180 - 135 -
100 - ¬AKAP83 75-
63-
48-
35- rHamOVGP1 - + - + - + - + (20 μg/ml)
Figure 3- 6: Western blot analysis of tyrosine phosphorylation of hamster sperm proteins after demembranation. (A) Hamster caudal epididymal sperm, cultured for 4 h in modified TALP medium alone (lane 1) or supplemented with 20 g/mL of rHamOVGP1 (lane 2), were solubilized in SDS-loading buffer and subjected to immunoblot analysis with anti-phosphotyrosine antibody. Lanes 3 and 4 corresponded to lanes 1 and 2, respectively, except that sperm were demembranated with DTT-Triton X as described in Materials and Methods prior to immunoblotting. (B) The immunoblot was stripped and re-probed with anti-AKAP82 antibody as described in Materials and Methods.
86 A1 A2 B1 B2 MP
MP MP ES ES PP
ES ES C1 C2 D1 D2
PP PP
MP MP ES ES PP MP MP PP ES ES
E1 E2 F1 F2
MP MP ES MP ES MP ES
PP PP PP PP
G1 G G2 H H PP MP MP MP PP PP PP PP PP MP H MP H MP
Figure 3- 7: Immunolocalization of rHamOVGP1-enhanced tyrosine-phosphorylated proteins in hamster sperm. Sperm, after being cultured in modified TALP medium alone (A, C, E) or supplemented with 20 g/mL rHamOVGP1 (B, D, F) for 0 h (A, B), 2 h (C, D) and 4 h (E, F), were subjected to indirect immunofluorescence as described in Materials and Methods. Immunofluorescent staining of tyrosine phosphorylated proteins was observed in the mid-piece (MP) and principal piece (PP). (A1-F1) are corresponding bright field photomicrographs of (A2-F2) respectively. (G) shows intense immunostaining over the principle piece (PP) with a weaker staining intensity over the mid-piece (MP) after sperm were demembranated with 2% Triton X-100 for 10 min following incubation for 4 h in modified TALP medium supplemented with 20 g/mL rHamOVGP1. Scale bars = 50 μm
87
*** *
**
0 1 2 3 4 5 6
Figure 3- 8: Effect of rHamOVGP1 on acrosome reaction of hamster sperm. Hamster caudal epididymal sperm were incubated in modified TALP medium alone or supplemented with 20 g/mL rHamOVGP1 for 0 to 6 h. Results are expressed as mean SEM as compared to the control group (n=4 different experiments for each group carried out under the same conditions). * p<0.05; ** p<0.01; *** p<0.001.
88 +
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O I F O
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O
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Z
P
89 Figure 3- 9: Immunolocalization of rHamOVGP1 in hamster ovarian oocytes. Confocal microscope imaging of rHamOVGP1 binding sites in hamster ovarian oocytes following 3 h of incubation in the absence or presence of 20 g/mL rHamOVGP1. Column 1: immunofluorescent confocal microscope images; column 2: phase contrast confocal microscope images; column 3: merge of phase contrast and immunofluoresent confocal microscope images. ZP: zona pellucida; O: oocyte proper. Scale bars = 30 μm
90
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Figure 3- 10: Effect of rHamOVGP1 (20 μg/mL) on sperm-oocyte binding. Group A: control (medium without additive). Group B: hamster ovarian oocytes were pretreated with rHamOVGP1 for 1 h prior to sperm-oocyte binding assay in the absence of rHamOVGP1. Group C: hamster caudal epididymal sperm were pretreated with rHamOVGP1 for 1 h prior to sperm-oocyte binding assay in the absence of rHamOVGP1. Group D: Sperm-oocyte binding assay was performed in the presence of rHamOVGP1 without prior pretreatment of either oocytes or sperm. Group E: Sperm- oocyte binding assay was performed in the presence of rHamOVGP1 with pretreated oocytes and untreated sperm. F: Sperm-oocyte binding assay was performed in the presence of rHamOVGP1 with pretreated sperm and untreated oocytes. Groups G to K correspond to Group B to F, respectively, except that monoclonal antibody against hamster OVGP1 was added to the culture medium during sperm- oocyte binding. Data are presented as Mean SEM of the number of sperm bound per oocyte. A total of 165 oocytes were obtained from six animals. Fifteen ovarian oocytes were present in each group. Group B to F are compared to Group A; Group G to K are compared to Group B to F, respectively. * p<0.05; ** p<0.001.
91
Chapter 4
Recombinant Human Oviductin Regulates Protein Tyrosine Phosphorylation and Acrosome Reaction
Published as:
Zhao Y, Yang X, Jia Z, Reid RL, Leclerc P and Kan FW (2016) Recombinant human oviductin regulates protein tyrosine phosphorylation and acrosome reaction. Reproduction 152 561–573.
92 4.1 Abstract
The mammalian oviduct synthesizes and secretes a major glycoprotein known as oviductin
(OVGP1) which has been shown to interact with gametes and early embryos. We report here the use of recombinant DNA technology to produce, for the first time, the secretory form of human OVGP1 in HEK293 cells. HEK293 colonies stably expressing recombinant human
OVGP1 (rHuOVGP1) were established by transfecting cells with an expression vector pCMV6-Entry constructed with OVGP1 cDNA. Large quantities of rHuOVGP1 were obtained from the stably transfected cells using the CELLSPIN cell cultivation system. A two-step purification system was carried out to yield rHuOVGP1 with purity of >95%. Upon gel electrophoresis, purified rHuOVGP1 showed a single band corresponding to the 120-150 kDa size range of human OVGP1. Mass spectrometric analysis of the purified rHuOVG1 revealed its identity as human oviductin. Immunofluorescence showed the binding of rHuOVGP1 to different regions of human sperm cell surfaces in various degrees of intensity. Prior treatment of sperm with 1% Triton X-100 altered the immunostaining pattern of rHuOVGP1 with an intense immunostaining over the equatorial segment and post-acrosomal region as well as along the length of the tail. Addition of rHuOVGP1 in the capacitating medium was found to further enhance tyrosine phosphorylation of sperm proteins in a time-dependent manner. After
4 hours of incubation in the presence of rHuOVGP1, the number of acrosome-reacted sperm induced by calcium ionophore significantly increased. The successful production of rHuOVGP1 can now facilitate the study of the role of human OVGP1 in fertilization and early embryo development.
93
4.2 Introduction
The mammalian oviduct undergoes endocrine-induced morphological, biochemical and physiological changes during the estrous or menstrual cycle. These changes establish an essential microenvironment within the lumen of the oviduct for final maturation of gametes, capacitation of sperm, transport of gametes and embryos, fertilization and early cleavage-stage embryonic development (Hunter, 2003). Early investigations in many mammalian species, including the mouse (Kapur and Johnson, 1985, 1986), hamster (Leveille et al., 1987), rabbit
(Oliphant and Ross, 1982; Oliphant et al., 1984), sheep (Sutton et al., 1984, 1986; Gandolfi et al., 1989), pig (Buhi et al., 1990), cow (Malayer et al., 1988; Boice et al., 1990a), baboon
(Fazleabas and Verhage, 1986; Verhage and Fazleabas, 1988; Verhage et al., 1989), and the human (Verhage et al., 1988) identified and characterized a high molecular weight glycoprotein secreted by secretory cells of the oviduct collectively known as oviductin or oviduct-specific glycoprotein (OVGP1). Subsequent in vitro studies carried out in different animal models and in the human showed specific association of OVGP1 with the zona pellucida (ZP) of ovulated ova and early embryos (Kapur and Johnson, 1988; Kan et al., 1989,
1993; Boice et al., 1990b; Gandolfi et al., 1991; Wegner and Killian, 1991; Buhi et al., 1993;
Abe et al., 1995) and correlated the secretion of OVGP1 with the estrogen-dominated stage of estrous (King and Killian, 1994; Boatman and Magnoni, 1995) and menstrual cycles (Verhage and Fazleabas, 1988; Verhage et al., 1989). The nucleotide and amino acid sequence of OVGP1 is highly conserved among mammalian species. Expression of OVGP1 appears to be estrogen-
94 dependent or estrous cycle-associated in most species but differences have been noted between species (Buhi et al., 1991; Murray, 1993; Arias et al., 1994; Malette et al., 1995a; Briton-Jones et al., 2001, 2004, McBride et al., 2004a, b). Previous results obtained from in vitro studies suggested several beneficial effects of OVGP1 for gametes and embryos. Bovine OVGP1 was shown to increase the motility and viability of homologous sperm (Abe et al., 1995) and enhance sperm capacitation and fertilizing capabilities in vitro (King et al., 1994). The presence of OVGP1 in the incubation medium has been shown to increase sperm-egg binding and penetration rates (Boatman and Magnoni, 1995; O’Day-Bowman et al., 1996) and decrease polyspermy (Coy et al., 2008). OVGP1 from different mammalian species has been shown to increase cleavage rates of embryos and the number of embryos reaching the blastocyst stage
(Hill et al., 1997; Kouba et al., 2000). In our laboratory, we have recently shown that hamster
OVGP1 prepared from the estrus stage of the estrous cycle can regulate the expression of tyrosine-phosphorylated proteins in hamster sperm during capacitation in vitro (Saccary et al.,
2013). We also successfully produced a biologically active recombinant hamster oviductin that can exert positive effects on sperm functions and sperm-oocyte binding (Yang et al., 2015).
Taken together, these results indicate the beneficial effects of OVGP1 and the importance of its presence in the luminal milieu of the oviduct during the process of fertilization and early embryo development.
Despite the findings from animal models supporting a role for OVGP1 in fertilization and early embryo development, the molecular mechanisms underlying the mode of action of
OVGP1 are not fully understood. Information concerning the biological role of human OVGP1
95 is lacking behind its counterpart in other mammalian species. This is mainly due to the ethical issue and technical difficulty in obtaining sufficient amount of human oviductal fluid to prepare purified human OVGP1 for functional studies. In the present study, our objective was to produce a secretory form of recombinant human OVGP1 (rHuOVGP1) in human embryonic kidney 293 (HEK293) cells. We also aimed at evaluating the effects of adding rHuOVGP1 in the capacitating medium on tyrosine phosphorylation of sperm proteins and acrosome reaction.
We report here, for the first time, the successful production of rHuOVGP1 of high purity.
Experiments were carried out to show binding of rHuOVGP1 to sperm cell surfaces and its association with structural components that are Triton resistant. In the present study, in vitro functional studies demonstrated that the presence of rHuOVGP1 in the capacitating medium can further enhance tyrosine phosphorylation of human sperm proteins, a hallmark of sperm capacitation, in a time-dependent manner, and significantly increase the number of acrosome- reacted sperm induced by ionophore A23187. Taken together, the present findings confirm the biologically active nature of our newly produced rHuOVGP1 creating opportunities for further exploration of the role of human OVGP1 in reproductive functions.
4.3 Materials and Methods
4.3.1 Materials
The following materials were purchased from the sources indicated: Dulbecco’s Modified
Eagle Medium (DMEM), CD293, fetal bovine serum (FBS), penicillin/streptomycin,
LipofectaminTM 2000, Geneticin, L-glutamine (Invitrogen, Carlsbad, CA, USA); open reading frame (ORF) clone of human oviductal glycoprotein 1 (OVGP1, oviductin) (ORIGENE,
96 Rockville, MD, USA); goat anti-oviductin antibody (P-20), oviductin immunizing peptide (P-
20)P, horseradish peroxidase (HRP)-linked donkey anti-goat IgG, goat anti-rabbit IgG-HRP, goat anti-mouse IgG-HRP, fluorescein isothiocyanate (FITC)-linked goat anti-mouse IgG, donkey anti-goat IgG-FITC (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); Rabbit anti-human oviductal glycoprotein polyclonal antibody was a gift from Dr. Patricia
Mavrogianis, University of Illinois, Chicago, USA (O'Day-Bowman et al., 1996); mouse anti- phosphotyrosine antibody (clone 4G10®) (EMD Millipore, Bedford, MA, USA); mouse anti-
α-tubulin antibody (clone B-5-1-2) (Sigma-Aldrich, St. Louis, MO, USA); Western Lighting-
Enhanced Chemiluminescence Substrate, Micromass MassPREP Robotic Protein Handling
System (PerkinElmer, Waltham, MA, USA ); Amicon Ultra-4 and -15 centrifugal filter units with a 30 kDa molecular-weight cut-off (EMD Millipore, Bedford, MA, USA); Superdex 200
(XK 16/60), GammaBind plus Sepharose (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).
All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).
4.3.2 Production of rHuOVGP1 in HEK293 cells
HEK293 cells were a gift from Dr. Leda Raptis, Queen’s University, Ontario, Canada. The cells were cultured in DMEM supplemented with 10% FBS, penicillin (50 U/mL) and streptomycin (50 µg/mL) (growth medium). To establish stable cell line producing rHuOVGP1,
HEK293 cells were transfected with 4 µg of human OVGP1 cDNA ORF clone using
LipofectaminTM 2000 as described by the manufacturer. Briefly, 0.25 106 cells were passaged into a 35 mm dish and grown until 70-80% confluency. The cells were incubated in DMEM
o supplemented with 10% FBS at 37 C in 5% CO2 for 6 hours (hrs). Human OVGP1 cDNA and
97 LipofectamineTM 2000 reagent were diluted, respectively, with DMEM serum-free medium without antibiotics. Diluted cDNA was added to diluted LipofectamineTM 2000 reagent at 1:2.5 ratio (µg/µl). The transfection mixture was incubated at room temperature for 20 minutes (min)
o and then added dropwise to the cells. The cells were incubated at 37 C in 5% CO2. After 48 hrs of transfection, the medium was replaced with fresh growth medium containing 1 mg/mL
Geneticin (G418) (selective medium). The selective medium was changed every 4-5 days.
After 2 weeks of incubation in the selective medium, the individual clones were transferred using cloning cylinders to 96-well plates and grown in selection medium until confluency. The cells were then passaged into 24-well plates and continually grown in the selective medium for
1-2 additional passages. Clones were screened for expression level by Western blot analysis of culture supernatants using the anti-oviductin antibody (P-20). HEK293 cells stably expressing rHuOVGP1 were maintained in the above medium supplemented with 200 g/mL Geneticin.
In order to produce large amount of rHuOVGP1, the stable rHuOVGP1-expressing HEK293 cell clone was grown to confluence in 100 mm dishes in the above medium in the presence of
200 µg/mL Geneticin and passaged into 500mL spinner flasks on a stirring platform at 75 rpm
(Cellspin Cell Cultivation System, Integra Biosciences AG, Zizers, Switzerland) at a density of 2.5 105 cells/mL. During production of rHuOVGP1, cells were grown in suspension in
CD293 serum-free medium supplemented with 4 mM L-glutamine, penicillin (50 U/mL),
o streptomycin (50 µg/mL), and 200 µg/mL Geneticin at 37 C and 8% CO2. The cell count and viability were determined daily by Trypan Blue staining. When the viable cell count reached
1.5 106 cells/mL, the cells were diluted to 2.5-3.0 105 cells/mL with warm (37oC) CD293
98 medium. After 7-10 days, serum-free culture medium containing the recombinant glycoprotein was collected and centrifuged at 1000 ×g for 5 min at 4oC. The supernatant was collected and stored at –70oC for further purification. Production of rHuOVGP1 was monitored weekly by
Western blot analysis of culture supernatants using the anti-human OVGP1 antibody (P-20).
4.3.3 Purification of secreted rHuOVGP1 collected from serum-free cell culture medium
The culture supernatant was concentrated by dialysis against poly(ethylene glycol) 20K at
o 4 C. The first purification step was carried out by ammonium sulfate ((NH4)2SO4) precipitation
(Duong-Ly and Gabelli, 2014). Briefly, (NH4)2SO4 was added slowly to the concentrated
o culture supernatant at 4 C with constant stirring until 30% (NH4)2SO4 saturation. After 1 hr of incubation at 4oC, the sample was centrifuged for 30 min at 10,000 ×g at 4oC. The supernatant fraction was collected and subjected to a second saturation step until the sample reached 70%
(NH4)2SO4 saturation. After 3 hrs of incubation in the cold room, the precipitate was spun down
o by centrifugation for 30 min at 10,000 ×g at 4 C. The pellet obtained from 70% (NH4)2SO4 saturation was dissolved in PBS and subsequently dialyzed overnight against a dialyzing buffer containing 0.05 M sodium phosphate and 0.15 M NaCl, pH 7.2, in the cold room. The buffer- exchanged solution was then concentrated by filtration using Amicon Ultra-15 centrifugal filter with a 30 kDa cut-off membrane. The concentrated samples were further purified using gel filtration chromatography. Gel filtration was performed on a fast protein liquid chromatography (FPLC) system (Pharmacia) using a Superdex 200 (XK16/60) column (120-
124 mL bed volume) equilibrated with the dialyzing buffer. All solutions were de-gassed and filtered through 0.22 µm filter. The sample was centrifuged at 10,000 ×g for 10 min at 4oC
99 prior to applying to the column. Absorbance at 280 nm was monitored and recorded. The sample was eluted isocratically from the column using the above buffer at the flow rate of 1.0 mL/min. The eluate was collected in 1-mL fractions and the peak fractions containing rHuOVGP1 were collected and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified rHuOVGP1 was desalted in PBS and sterile- filtered. The protein concentration of the samples at each purification step was determined by the Bradford protein assay using bovine serum albumin (BSA) as the standard. To assess the purity level of rHuOVGP1, 3 µg of the recombinant glycoprotein were used for SDS-PAGE followed by Coomassie staining.
4.3.4 SDS-PAGE, Western blotting, and immunoprecipitation
The culture supernatants were collected as above. The protein samples (20 μl each) were size-fractionated by SDS-PAGE on a 7.5% gel. The proteins were visualized with Coomassie
Brilliant Blue R-250. For Western blot analysis, the protein samples were mixed with reducing
SDS sample buffer (63 mM Tris HCl, pH 6.8, 10% glycerol, 2% SDS, 0.1% -mercaptoethanol,
0.0025% bromophenol blue), boiled for 5 min, separated on the gel, and electrophoretically transferred to polyvinylidene fluoride (PVDF). After blocking with blocking buffer [5% nonfat milk Tris-buffered saline (150 mM NaCl, 50 mM Tris, pH 7.5) with 0.05% Tween 20 (TBST)] for 1 hr, blots were probed sequentially with goat anti-oviductin antibody (P-20) at a dilution of 1:1000, and then with donkey anti-goat IgG-HRP at a 1:2000 dilution. Alternatively, the blots were probed sequentially with a polyclonal antibody against human oviductin (a gift from
100 Dr. P. Mavrogianis) at a dilution of 1:50 and goat anti-rabbit IgG-HRP at a 1:10000 dilution.
Labeling was visualized by enhanced chemiluminescence.
Immunoprecipitation was performed on culture supernatants from the stable rHuOVGP1- expressing HEK293 cells. Samples (100 µl) that contained high concentrations of rHuOVGP1 were pre-cleared with 20 µl GammaBind Plus Sepharose for 1 hr at 4oC. Samples were then immunoprecipitated overnight at 4oC with 30 µl GammaBind Plus Sepharose containing 0.2
µg/µl polyclonal anti-human oviductin antibody [Oviductin (P-20)] and subsequently analyzed by SDS-PAGE and Western blotting as described above.
4.3.5 Trypsin digestion of samples and sequencing of peptides by mass spectrometry
Samples of purified rHuOVGP1 were electrophoretically separated by SDS-PAGE. After
Coomassie staining of the gels, protein bands were cut from the gels and digested using the
Micromass MassPREP Robotic Protein Handling System. The tryptically-digested sample was subjected to the SCIEX Voyager DE Pro Matrix-Assisted Laser-Desorption (MALDI) mass spectrometer at the Protein Function and Discovery Facility of Queen’s University, Ontario,
Canada. Data from peptide mass fingerprinting were acquired over the mass range between m/z 700 to 4000. Mass spectrometry (MS) data were processed using Applied Biosystems Data
Explorer version 5.1 and submitted to the Genebio Aldente search engine for comparison against the Swiss-Prot database.
4.3.6 Immunofluorescence and confocal microscopy
HEK293 cells transfected with HuOVGP1 cDNA and mock-transfected cells were grown, respectively, on glass coverslips in culture dishes in DMEM growth medium. Cells grown to
101 80% confluency on the coverslips were washed 3 times with PBS and then fixed in 2% paraformaldehyde in PBS for 10 min at room temperature. After fixation, cells were washed with PBS once for 5 min and permeabilized with 0.5% Triton X-100 in PBS for 20 min at room temperature. Nonspecific binding was blocked with 1% BSA in PBS for 45 min at room temperature. The cells were probed with goat polyclonal anti-human oviductin antibody (P-
20), and subsequently stained with donkey anti-goat IgG-FITC and DAPI (nuclei staining).
Cells were washed and mounted in Mowiol 4-88/glycerol solution before imaging using a
Leica TCS-SP2 Multiphoton Confocal Laser Scanning Microscope (TCS-MP, Heidelberg,
Germany).
4.3.7 Preparation of sperm from fresh semen samples
Studies with human semen were approved by the ethics committee for research on human subjects from the Laval University Medical Center and by Queen’s University Health Sciences and Affiliated Teaching Hospitals Research Ethics Board. Human semen samples were obtained from healthy donors by masturbation with consent after 3 days of sexual abstinence.
Upon receiving the samples, they were liquefied in a 37oC water bath. Semen viscosity and volume, sperm motility and sperm concentration were assessed according to the WHO laboratory manual for the examination and processing of human semen (WHO, 2010). Only normozoospermic samples were selected for the present study. Liquefied semen was layered on the top of a gradient composed of 2-mL fractions each of 20%, 40% and 65% and 0.1 mL of 95% Percoll and then centrifuged at 1000 ×g for 30 min to isolate sperm from the seminal plasma. Percoll was made isotonic in HEPES-buffered saline (HBS; 25mM HEPES, 130mM
102 NaCl, 4mM KCL, 0.4 mM MgCl2, 14 mM fructose, pH 7.6). Following centrifugation, sperm from the 65-95% interface and within the 95% Percoll fraction representing the highly motile population were pooled and their concentration was determined by a hematocytometer count.
4.3.8 Sperm incubation
Sperm were incubated at a concentration of 2 107 cells/mL in the presence or absence of
o 10, 25, 50, or 75 µg/mL of rHuOVGP1 for 0, 1, 2, 3, or 4 hrs, respectively, at 37 C in 5% CO2 with 100% humidity in modified Biggers-Whitten-Wittingham medium (BWW; 10 mM
HEPES, 94.6 mM NaCl, 4.8 mM KCl. 1.7 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25.1 mM NaHCO3, 5.6 mM D-glucose, 21.6 mM Na lactate, 0.25 mM Na pyruvate, 0.1mg/mL phenol red, pH 7.4) supplemented with 0.3% fatty acid-free BSA.
4.3.9 Binding of rHuOVGP1 to human sperm
A dose-dependent experiment was carried out first to determine the effect of various concentrations of rHuOVGP1 on enhancement of protein tyrosine phosphorylation and acrosome reaction of sperm after 4 hrs of capacitation (see methods described for evaluation of protein tyrosine phosphorylation and acrosome reaction below). At a concentration of 50
µg/mL was found to be optimal for significantly enhancing the level of protein tyrosine phosphorylation and chemical-induced acrosome reaction. Based on these initial results, rHuOVGP1 at a concentration of 50 µg/mL was used for the binding study. Sperm incubated in the presence of 50 µg/mL of rHuOVGP1 for 1 hr and 4 hrs, respectively, were washed with
HBS twice and resuspended in phosphate-buffered 1% Triton X-100 solution with protease inhibitors for 1 hr at 4 °C. After Triton treatment, the samples were centrifuged at 10,000 ×g
103 for 10 min. The cell pellets were washed in PBS twice and fixed in phosphate-buffered 1% paraformaldehyde solution for 1 hr at room temperature. Samples were then transferred onto poly-L-lysine pre-coated slides allowing sperm to attach. Excess of fixative solution on the slides was washed off with PBS. The samples were blocked with 5% donkey serum in PBS for
1 hr at room temperature and incubated overnight at 4°C with goat anti-oviductin (P-20) primary antibody at a dilution of 1:100 in 1% donkey serum in PBS. Following incubation, the slides were washed three times each for 10 min with PBS. The samples were then stained for
1 hr at room temperature with donkey anti-goat IgG-FITC at a dilution of 1:200 in 1% donkey serum in PBS. After washing with PBS, coverslips were mounted on slides with 90% glycerol containing 1% (w/v) 1,4-Diazabicyclo-(2,2,2)-octane (DABCO) as an anti-bleaching agent.
Fluorescent signals were detected by confocal microscopy.
4.3.10 Binding of rHuOVGP1 to human sperm
At the end of each time interval, sperm were washed with 1mL Hepes-buffered saline
(HBS) and centrifuged at 1000 ×g for 5 min. Sperm proteins were solubilized in electrophoresis sample buffer (2% SDS, 10% glycerol, 50 mM dithiothreitol, 62.5 mM Tris-
HCl, pH6.8) and denatured at 100oC for 5 min. Sperm proteins were resolved by SDS-PAGE and transferred onto PVDF. Non-specific binding on the PVDF membrane was blocked with
5% (w/v) skim milk in TBST. The PVDF membrane was then incubated with a mouse anti- phosphotyrosine antibody diluted 1:10000 in TBST for 1 hr at room temperature. Following several washes with TBST, the PVDF membrane was incubated for 1 hr with goat anti-mouse
IgG-HRP diluted 1:10000 with 5% milk in TBST. After five washes in TBST, positive
104 immunoreactive bands were detected using the ECL kit according to the manufacturer’s instructions.
To ensure that an equal amount of protein was loaded in each well of the gel apparatus, the same membrane was reprobed with a mouse anti-α-tubulin antibody diluted 1:50000 with
5% milk in TBST. After incubation, the membrane was labeled with goat anti-mouse-HRP diluted 1:10000 with 5% milk in TBST. Immunoreactive bands were detected as outlined above.
The band intensities were quantified by densitometry using ImageJ software (National
Institutes of Health, Bethesda, Maryland, USA). The band intensities of tyrosine- phosphorylated proteins were normalized to the ones of -tubulin. The level of tyrosine phosphorylation at each time point was further compared to the 0-hr time point and expressed as fold increase.
4.3.11 Evaluation of protein tyrosine phosphorylation
At the end of each time interval, sperm were washed with 1mL Hepes-buffered saline
(HBS) and centrifuged at 1000 ×g for 5 min. Sperm proteins were solubilized in electrophoresis sample buffer (2% SDS, 10% glycerol, 50 mM dithiothreitol, 62.5 mM Tris-
HCl, pH6.8) and denatured at 100oC for 5 min. Sperm proteins were resolved by SDS-PAGE and transferred onto PVDF. Non-specific binding on the PVDF membrane was blocked with
5% (w/v) skim milk in TBST. The PVDF membrane was then incubated with a mouse anti- phosphotyrosine antibody diluted 1:10000 in TBST for 1 hr at room temperature. Following several washes with TBST, the PVDF membrane was incubated for 1 hr with goat anti-mouse
IgG-HRP diluted 1:10000 with 5% milk in TBST. After five washes in TBST, positive
105 immunoreactive bands were detected using the ECL kit according to the manufacturer’s instructions.
To ensure that an equal amount of protein was loaded in each well of the gel apparatus, the same membrane was reprobed with a mouse anti-α-tubulin antibody diluted 1:50000 with
5% milk in TBST. After incubation, the membrane was labeled with goat anti-mouse-HRP diluted 1:10000 with 5% milk in TBST. Immunoreactive bands were detected as outlined above.
The band intensities were quantified by densitometry using ImageJ software. The band intensities of tyrosine-phosphorylated proteins were normalized to the ones of -tubulin. The level of tyrosine phosphorylation at each time point was further compared to the 0-hr time point and expressed as fold increase.
4.3.12 Acrosome reaction assays
Sperm after 4 hrs of incubation in the capacitating medium in the presence or absence of rHuOVGP1 were divided into two aliquots; both were washed with 1 mL HBS and then centrifuged at 1000 ×g for 5 min before being resuspended in BWW. Calcium ionophore
A23187 was added (10 µM final concentration) to one of the two aliquots to induce the acrosome reaction while dimethyl sulfoxide (DMSO) was added to the other aliquot which served as a control for spontaneous acrosome reaction. Subsequently, both samples were
o incubated for 1 hr at 37 C in 5% CO2 with 100% humidity after which sperm were washed with 1 mL HBS and centrifuged at 1000 ×g for 5 min. Sperm pellet was resuspended in 100% methanol for 30 min on ice, spread on slides, and dried at 37oC on a heated stage. The acrosome of sperm was stained for 30 min with Pisum sativum agglutinin conjugated with fluorescein
106 isothiocyanate (psa-FITC) (75 μg/mL in PBS) in a humid chamber. The slides were washed with water and air-dried. Coverslips were mounted on slides with 25 μl DABCO. The acrosomal status was determined using fluorescence microscopy and evaluated based on the immunostaining pattern of psa-FITC as previously described (Cross et al., 1986). For each sample more than 200 sperm were counted to obtain the percentage of acrosome-reacted sperm.
4.3.13 Statistical analysis
The band intensities of tyrosine phosphorylated proteins and the number of acrosome- reacted sperm between groups was compared using Student’s paired or unpaired t-test. The statistical analysis was performed using Prism 6 (GraphPad Software, La Jolla California,
USA). Data are expressed as mean ± SE. P-values equal to or less than 0.05 with 95% confidence intervals were considered statistically significant.
4.4 Results
4.4.1 Establishment of a stable rHuOVGP1-expressing HEK293 cell clone and purification of rHuOVGP1
To produce the rHuOVGP1, we used the HEK293 cells transfected with the ORF clone of human OVGP1. Selection of cell clone stably expressing human OVGP1 was performed using
Geneticin. The clone that stably produced the recombinant glycoprotein was isolated and cultured. Immunofluorescent staining was used in conjunction with confocal microscopy to confirm protein expression and subcellular localization of rHuOVGP1 in the HEK293 cells.
Confocal microscopy revealed a strong immunoreaction in the perinuclear region and the cytoplasmic region (Fig. 4-1A).
107 Since OVGP1 is a secretory glycoprotein, HEK293 cells are expected to secrete the mature form of rHuOVGP1 into the culture medium. We probed the presence of rHuOVGP1 in the culture medium by Western blot analysis using a polyclonal anti-human oviductin antibody
(oviductin [P-20]). As expected the antibody detected a polydispersed band (120-150kDa) in the supernatant fraction (Fig. 4-1B: lanes 2 and 3) corresponding to the molecular weight of human OVGP1. These immunoreactive protein bands (Fig. 4-1B: lanes 2 and 3) were abrogated in the presence of the blocking peptide (oviductin[P-20]P) (Fig. 4-1B: lanes 5 and 6) confirming the specificity of the antibody. Supernatant fractions from the mock transfection were not reactive to the antibody (Fig. 4-1B: lanes 1 and 4). To further confirm the identity of the glycoprotein detected by the aforementioned antibody, another primary antibody known to recognize human OVGP1 (a gift from Dr. P. Mavrogianis) was used to probe the presence of rHuOVGP1 in the culture medium prepared from HEK293 cells. Again, a polydispersed protein band of approximately 110-150 kDa was detected (Fig. 4-1C). Supernatant fractions of the culture medium collected from duplicated experiments of HuOVGP1 cDNA transfection
(Fig. 4-1B: lane 2 and 3, 5 and 6; Fig. 4-1C: lane 2 and 3) were assessed by Western blot analysis for the reproducibility of the transfection procedure. Subsequently, individual rHuOVGP1 transfected cell clones were selected for establishing stable transfected cell clone.
Expression of rHuOVGP1 in stable cell clone was also confirmed by Western blotting using the oviductin (P-20) antibody (data not shown).
In order to obtain purified rHuOVGP1, CELLSPIN, a large-scale cell cultivation system, was used to collect secreted rHuOVGP1 stably produced by HEK293 cells in the culture
108 medium. After ammonium sulphate precipitation followed by size exclusion chromatography, rHuOVGP1 was obtained with a purity of over 95% based on a conservative estimation using densitometric analysis of the SDS-PAGE resolved purified glycoprotein. Using this approach a considerable amount of rHuOVGP1 protein was produced from HEK293 cells stably expressing rHuOVGP1. In our laboratory, a typical yield from 1L of the conditioned medium varied between 1.8 to 2.7 mg of rHuOVGP1. The purity of OVGP1 was assessed by SDS-
PAGE in association with Coomassie Blue staining (Fig. 4-2A) and Western blot analyses (Fig.
4-2B). As shown in Figs. 4-2A and B, purified OVGP1 revealed predominantly a single band of the expected molecular mass of 120-150 kDa under reducing conditions. The identity of the purified recombinant protein was verified by mass spectrometric analysis (Fig. 4-2C). The peptide mass fragment spectra of the 120-150 kDa protein demonstrated that 19 of 30 peptides at 50 ppm mass accuracy were matched to human OVGP1 protein with 23% sequence coverage, thus confirming the identity of rHuOVGP1 as human OVGP1.
4.4.2 Binding of rHuOVGP1 to human sperm
Having obtained the purified rHuOVGP1, we then investigated the binding of this glycoprotein to sperm. First, Western blot analysis was performed to confirm the presence of rHuOVGP1 in sperm protein extractions after incubation with rHuOVGP1 in capacitating medium. After 4 hrs of incubation, a fair amount of rHuOVGP1 was present in the Triton X-
100 soluble fraction, indicating binding of the glycoprotein to sperm (Fig. 4-3A). Noticeably, an evident amount of rHuOVGP1 was still present in the Triton insoluble fraction even after extensive washing (Fig. 4-3A). As a positive control, the supernatant recovered from the
109 medium showed the presence of rHuOVGP1 in the capacitating medium after 4 hrs of incubation. The HBS wash following incubation was found to be negative in immunoreaction for rHuOVGP1, and the PBS wash after Triton extraction was also found to be negative in immunoreaction for rHuOVGP1. Samples from sperm incubated in the absence of rHuOVGP1 were used as negative controls. These results provide evidence for the binding of rHuOVGP1 to both Triton soluble and insoluble fractions of sperm protein extractions.
To determine the specific sites of binding of rHuOVGP1 to human sperm, indirect immunofluorescence was performed. At 1 hr of capacitation in the presence of rHuOVGP1, immunoreaction of rHuOVGP1was detected over the head, mid-piece and, and to a lesser extent, over the principal piece of the tail (Fig. 4-3Bi). In the sperm head, immunoreaction exhibited as a punctated staining pattern and was mainly found over the acrosomal region and the equatorial segment but the immunostaining extended to the post-acrosomal region in some cells. The connecting piece in the sperm neck and the mid-piece were found to be intensely stained whereas the principal piece was weakly stained in all cells examined. Sperm incubated for 4 hrs in the presence of rHuOVGP1 displayed a similar immunostaining pattern with a persistent staining intensity (Fig. 4-3Bii). The aforementioned immunostaining was not seen at both time intervals when sperm were incubated in the absence of rHuOVGP1 (Fig. 3Biii).
Sperm cells also showed a negative immunoreaction when the primary antibody was omitted in the staining process of the sperm incubated in the presence of rHuOVGP1 (result not shown).
Interestingly, when sperm were treated with 1% Triton X-100 prior to immunostaining for rHuOVGP1, the signal was more intense over the equatorial segment and postacrosomal
110 region (Fig. 4-3Biv), which was not seen in sperm without Triton treatment. Also after treatment with 1% Triton, while the mid-piece remained strongly stained, the fluorescent signal was found relatively uniform throughout the sperm tail including the mid-piece and the principal piece (Fig. 4-3Biv). This altered pattern of immunostaining after Triton treatment persisted after 4 hrs of incubation of sperm in the presence of rHuOVGP1 (Fig. 4-3Bv). The immunostaining was also not seen at both time intervals when sperm were incubated in the absence of rHuOVGP1 (Fig. 4-3Bvi).
4.4.3 Effect of rHuOVGP1 on the expression of phosphotyrosine-containing proteins in human sperm
As one of the characteristic features of sperm capacitation is the increase of protein tyrosine phosphorylation, the effect of rHuOVGP1 on enhancing sperm protein tyrosine phosphorylation following capacitation was evaluated. A dose-dependent experiment was first carried out where sperm samples were incubated for 4 hrs in BWW medium supplemented with different concentrations of rHuOVGP1 (0, 10, 25, 50, and 75 g/mL). Western blot analysis was performed to detect the level of protein tyrosine phosphorylation expression by probing with an anti-phosphotyrosine antibody. Our result indicated that two proteins migrated at 105 kDa (p105) and 81 kDa (p81) exhibited an increase in tyrosine phosphorylation in a dose-dependent manner in the presence of rHuOVGP1 at 4 hrs of incubation (Fig. 4-4A). The immunolabeling intensity of protein tyrosine phosphorylation was almost undetectable at 0 hr for uncapacitated sperm. The strongest labeling intensity of the two proteins was detected when rHuOVGP1 was used at a concentration of 50 g/mL. Statistical analysis was also performed
111 confirming that the levels of tyrosine phosphorylation of p105 and p81 were significantly increased in the presence of rHuOVGP1 at a concentration of 50 g/mL as compared to incubation in the absence of the protein (Appendix Figure-1). The levels of tyrosine phosphorylation of p105 and p81 were also found to be the highest at a concentration of 50
g/mL as compared to other concentrations (10, 25, and 75 g/mL). Based on these results, rHuOVGP1 at 50 g/mL was considered the optimal concentration to be used for enhancing protein tyrosine phosphorylation during sperm capacitation.
A time-dependent experiment was subsequently carried out where the sperm samples were incubated for 0-4 hrs in BWW medium alone or supplemented with 50 g/mL of rHuOVGP1.
Results obtained with Western blot analysis demonstrated a time-dependent increase in tyrosine phosphorylation of p105 and p81 during 4 hrs of capacitation that was further potentiated in the presence of rHuOVGP1 (Fig. 4-4B). Variability in the amplitude of increased levels of tyrosine phosphorylation during capacitation is commonly found among sperm samples from different donors, as observed in Fig. 4-4A (lane 2 from left) as compared to Fig.
4-4B (lane 8 from left). Nonetheless, densitometric image analysis of labeling intensities of p105 and p81 indicated that the level of tyrosine phosphorylation of p105 and p81 was significantly increased at 3 hrs and 4 hrs, respectively, when incubated in the presence of rHuOVGP1 as compared to their corresponding counterparts obtained from sperm without rHuOVGP1 treatment (Fig. 4-4C).
112 4.4.4 Effect of rHuOVGP1 on sperm acrosome reaction
The ability of sperm to undergo acrosome reaction is essential for the subsequent sperm- oocyte binding and successful fertilization. In order to examine if rHuOVGP1 exerts any positive effects on acrosome reaction, fresh sperm were incubated in BWW medium alone or in the presence of different concentrations of rHuOVGP1 (10, 25, 50, and 75 g/mL). The percentage of acrosome-reacted sperm was measured and compared between spontaneous
(DMSO) and chemically-induced (A23187) acrosome reaction (Fig. 4-5). Spontaneous acrosome reaction of sperm remained low and was not significantly different among samples incubated with various concentrations of rHuOVGP1 or with the medium alone. Sperm treated with A23187 showed an increase in acrosome reaction in the presence of rHuOVGP1 in a dose- dependent manner. The percentage of acrosome-reacted sperm was significantly increased after incubation with rHuOVGP1 at the concentration of 50 g/mL compared to sperm incubated without rHuOVGP1. The increase of percentage of acrosome-reacted sperm after incubation with 50 g/mL of rHuOVGP1 was at the highest level compared to those obtained with other concentrations (10, 25, and 75 g/mL).
4.5 Discussion
Accumulating evidence indicates that mammalian OVGP1 exerts positive effects during early events of fertilization and early embryo development, but the molecular mechanisms of the physiological function of OVGP1 involved in these events are not clearly understood, especially in the human. In order to carry out functional and mechanistic studies, two approaches can be envisaged for obtaining purified OVGP1. One approach is to purify
113 sufficient amount of the native glycoprotein from oviductal fluid and an alternative is to utilize recombinant DNA technology to produce a recombinant oviductin. In our laboratory, we have previously isolated and purified hamster oviductin (HamOVGP1) from oviducts at different stages of the estrous cycle and showed binding of the estrus stage-associated HamOVGP1 to the cell surfaces of homologous sperm (Kan and Esperanzate, 2006). The estrus stage-specific
HamOVGP1 has also been found to enhance, during in vitro capacitation, tyrosine phosphorylation of a subset of sperm proteins known to play important functions in fertilization
(Saccary et al., 2013). We have recently produced a biologically active recombinant hamster
OVGP1 (rHamOVGP1) and demonstrated that this recombinant glycoprotein can enhance tyrosine phosphorylation of hamster sperm proteins, acrosome reaction, and sperm-egg binding
(Yang et al., 2015). However, isolating and purifying large amounts of native OVGP1 from human Fallopian tubes is not feasible mainly due to ethical reasons and limited accessibility to human tissue samples. In the present study, we have successfully produced a biologically active rHuOVGP1 protein using the recombinant DNA approach and a two-step protein purification procedure. We have found that rHuOVGP1 can enhance tyrosine phosphorylation of two human sperm proteins with a molecular weight of 105 kDa and 81 kDa, respectively, during capacitation. As well, rHuOVGP1 has been found to increase the number of acrosome-reacted sperm after in vitro capacitation.
In the beginning of our study, we attempted to produce rHuOVGP1 in the immortalized human oviductal cell line OE6/E7 (Lee et al., 2001). We have previously demonstrated that this cell line expresses endogenous human OVGP1 mRNA and protein (Ling et al., 2005) .
114 While we were able to overexpress human OVGP1 mRNA in the transfected OE6/E7 cells, only small amounts of secreted rHuOVGP1 were obtained from the culture medium. In order to increase the secretion levels of rHuOVGP1, HEK293 cells were used instead. In the past,
HEK293 cells have been successfully used for the production of several recombinant human glycoproteins such as cathepsin E (Cappiello et al., 2004) and IFNα2b (Loignon et al., 2008).
The use of HEK293 cells is also compatible with the large scale cell cultivating system
CELLSPIN as these cells can be grown in suspension. Our present results showed the establishment of a HEK293 cell clone stably expressing and secreting glycosylated rHuOVGP1 and that the cells can be cultured to produce large amounts of rHuOVGP1 using the CELLSPIN system. rHuOVGP1 was further purified from serum-free culture medium by a two-step procedure starting with ammonium sulphate precipitation followed by gel filtration chromatography on Superdex 200. The identity of the glycoprotein was confirmed by Western blot analysis using both a commercially available antibody generated against a peptide mapping near the C-terminus of human OVGP1 and an antibody against the full sequence of partially purified native HuOVGP1. Mass spectrometric analysis further confirmed the purified glycoprotein as human OVGP1.
OVGP1 has been shown previously to bind to the acrosomal region of hamster sperm
(Boatman and Magnoni, 1995), and to the head and mid-tail regions of bovine sperm (King et al., 1994). Using the purified rHuOVGP1, results of the present study demonstrated, for the first time, its binding to human sperm. Our Western blot results showed that not only rHuOVGP1 was present in the Triton soluble fraction, but it was also detected in the insoluble
115 fraction. These findings suggest the association of rHuOVGP1 not only with the plasma membrane of sperm but also with certain structural elements underlying the cell surfaces.
Results obtained with immunofluorescence further revealed the sites of localization of rHuOVGP1 in human sperm during capacitation. On the membrane-intact sperm cells, rHuOVGP1 was found to bind primarily to the connecting piece and mid-piece and, to a lesser extent, to the principal piece. Immunostaining of rHuOVGP1 with a punctate pattern was observed over the acrosomal and equatorial regions of the sperm head. However, when treated with 1% Triton X-100, a relatively intense immunostaining of rHuOVGP1 was found associated with the equatorial and post-acrosomal regions in the sperm head and throughout the mid-piece and principal piece of the tail. As Triton X-100 is a nonionic surfactant which can be used to solubilize cell membranes and peripheral membrane bound proteins, the present results indicate that rHuOVGP1 binds not only to the cell surfaces of human sperm, but also to certain structural elements that are Triton resistant. Our immunofluorescent results are supported by findings of previous studies showing binding of OVGP1 to capacitated sperm and that the binding sites can be visualized on membrane-permeabilized sperm from bovine and monkey (King and Killian, 1994; Natraj et al., 2002). In the monkey, the N-terminal conserved region of OVGP1 has been found to be associated with a cytoskeletal protein, non- muscle myosin IIA (MYH9) (Kadam et al., 2006). MYH9 is also expressed on the cell surface
(Olden et al., 1976; Arii et al., 2010) and is enriched in detergent-resistant membrane skeleton and lipid rafts (Li et al., 1994; Nebl et al., 2002). Furthermore, MYH9 is thought to be a functional viral glycoprotein receptor for entry of viruses into cells (Arii et al., 2010; Xiong et
116 al., 2015). Based on these premises, we speculate that once rHuOVGP1 binds to the sperm surfaces, it becomes incorporated into detergent-resistant lipid rafts that could possibly bind to
MYH9. Future experiments are necessary to verify the presumptive association of OVGP1 with
MYH9. Conversely, Reuter et al. were not able to detect binding of native human OVGP1 to human sperm using partially purified OVGP1 from hydrosalpinx fluid (Reuter et al., 1994).
The discrepancy in OVGP1 binding to human sperm may be due to the use of partially purified protein by Reuter’s group versus pure recombinant protein used in the present study. The use of rHuOVGP1 with high purity in the present study for examining its binding ability to the sperm surface is also considered more specific as compared to the use of partially purified human OVGP1 previously reported.
A previous in vitro study showed that incubation of bovine sperm in culture medium in the presence of bovine oviductal fluid enhanced sperm capacitation (King et al., 1994). A time- dependent increase in protein tyrosine phosphorylation of sperm proteins is associated with in vitro sperm capacitation and this is a common occurrence found in various mammalian species including the human (Visconti et al., 1995b; Leclerc et al., 1996). Since OVGP1 is present in the luminal milieu of mammalian oviducts with the highest expression level during the peri- ovulatory phase, therefore, it was of our interest to find out if addition of rHuOVGP1 in capacitating medium can further enhance tyrosine phosphorylation of sperm proteins as this glycoprotein is lacking in commercially available capacitating medium or in capacitating medium prepared in the laboratory setting. In the present study, Western blot analysis revealed two proteins of 105 kDa and 81 kDa both of which were found to be significantly increased in
117 tyrosine phosphorylation level after 3 and 4 hrs, respectively, of incubation of sperm in capacitating medium in the presence of 50 μg/mL rHuOVGP1. The molecular weight of these two tyrosine-phosphorylated proteins correspond, respectively, to that of the A Kinase Anchor
Protein (AKAP) 3 and 4 that have been shown to be tyrosine-phosphorylated during sperm capacitation (Luconi et al., 2011). AKAP 3 and 4 are mainly located in the principal piece of the sperm tail (Brown et al., 2003a). Tyrosine phosphorylation of sperm proteins following capacitation occurs primarily in the principal piece, and to a lesser extent in the neck, equatorial and acrosome regions (Sati et al., 2014). Since rHuOVGP1 binds to sperm cell surfaces, it is plausible that binding of rHuOVGP1 to the tail could impact the local signal transduction cascade leading to an increase in the level of tyrosine phosphorylation of a subset of sperm proteins, including the 105 kDa- and 81 kDa proteins that were found to be under the influence of rHuOVGP1. The time discrepancy between the increase of tyrosine phosphorylation levels of p105 (at 3 hr) and p81 (at 4 hr) could be due to mechanisms that regulate phosphorylation and de-phosphorylation of tyrosine residues at a precise time during sperm capacitation. For example, AKAP3 protein has been shown to undergo de-phosphorylation during capacitation that leads to degradation of the protein (Vizel et al., 2015). Since capacitation can occur in vitro, we believe that capacitation and the events associated with this process are controlled intrinsically by sperm itself as long as specific requirements are met in vitro. This intrinsic control likely involves a series of phosphorylation and de-phosphorylation that occur at precise timing to prepare the sperm for successful sperm-egg binding, acrosome reaction and the
118 subsequent fertilization. Future studies are warranted to examine if human OVGP1 is involved in regulating the signaling mechanism of tyrosine phosphorylation of sperm proteins.
Acrosome reaction is a critical event that follows capacitation and precedes fertilization.
The percentage of acrosome-reacted sperm induced by A23187 correlates with the fertilizing capacity of the sperm sample (Yovich et al., 1994; Liu and Baker, 1998). Concordantly, the present results showed that exposure of human sperm to rHuOVGP1 for four hours prior to induction of acrosome reaction with calcium ionophore significantly increased the number of acrosome-reacted sperm. Therefore, rHuOVGP1 can likely increase the fertilizing capacity of the sperm following capacitation. Our results showed that the presence of rHuOVGP1 in the incubating medium did not alter the percentage of spontaneous acrosome reaction, which is consistent with previous studies indicating that human sperm normally have very low percentage of spontaneous acrosome reaction following in vitro capacitation (Byrd and Wolf,
1986; Fénichel et al., 1991). Our dose-dependent experiments showed that rHuOVGP1 at a concentration of 50 μg/mL significantly increased the percentage of calcium ionophore- induced acrosome reaction. At a higher concentration of 75 μg/mL, the increase in acrosome reaction became insignificant compared to incubation without rHuOVGP1 suggesting that a moderate concentration of rHuOVGP1 is able to potentiate acrosome reaction. We also observed the similar effect on hamster sperm in a previous study carried out in our laboratory when hamster recombinant OVGP1 was used at a high concentration (Yang et al., 2015). Taken together, higher concentrations of OVGP1 seem to alleviate the effect of OVGP1 on sperm capacitation as shown in the human sperm in the present study and in the hamster sperm as we
119 previously reported. Although our findings indicate that rHuOVGP1 significantly increased tyrosine phosphorylation of two sperm proteins during capacitation and significantly increased chemical-induced acrosome reaction, at this time, it is not clear whether the increase in acrosome reaction is directly linked to the increase in protein tyrosine phosphorylation during sperm capacitation under the influence of rHuOVGP1. It will be of interest to examine, in the future, the in vitro effects of rHuOVGP1 on sperm hyperactivated motility since hyperactivation is an important feature of sperm capacitation (Kulanand and Shivaji, 2001).
In summary, we have successfully produced and purified, for the first time, a secreted form of recombinant human oviductin from HEK293 cells. Sufficient amount of this recombinant glycoprotein has been purified for in vitro functional studies as demonstrated in the present study with respect to the effect of rHuOVGP1 on enhancement of tyrosine phosphorylation of sperm proteins and acrosome reaction. As OVGP1 is a secretory glycoprotein, the biological functions of OVGP1 may be attributable to its carbohydrate components. A direct comparison of N- and O-linked glycans between native human OVGP1 (HuOVGP1) and rHuOVGP1 by mass spectrometry is not feasible due to the unavailability of sufficient amount of purified native HuOVGP1. Based on the premise that the immortalized human oviductal cells display phenotype characteristics of primary human oviduct cells and that the former cells express both mRNA transcripts and protein of HuOVGP1 (Ling et al., 2005), we employed an alternative approach and compared the biosynthetic pathways of N- and O-glycosylation in HEK293 cells, where rHuOVGP1 is produced, with those of immortalized human oviductal cells. As expected, we found the two cell lines are very similar in the pathways of N- and O-linked glycosylation
120 (Yang et al., 2012). In view of these findings, it is reasonable to assume that the rHuOVGP1 described in the present study is very similar to the native HuOVGP1.
In the past, despite the fact that OVGP1 has been identified and characterized in many mammalian species including the human, further understanding of the roles played by OVGP1 in human reproduction has been hampered by the unavailability of purified native HuOVGP1.
The successful production and purification of rHuOVGP1 will now make it possible to determine the mechanisms that regulate its biological activity. For example, since human
OVGP1 is a secretory glycoprotein, it will be of interest to examine if N- and/or O-glycans and specific carbohydrates of rHuOVGP1 are responsible for mediating the functions of rHuOVGP1. The fact that rHuOVGP1 binds to different regions of sperm during capacitation raises the question of the presence of potential rHuOVGP1 receptors on sperm cell surfaces.
The rHuOVGP1 that we produced could also have clinical significance. Failed sperm binding and lack of fertilization is a major limitation of human in vitro-fertilization due mainly to male factor indications. Since rHuOVGP1 is able to enhance human sperm capacitation presumably through the increase of protein tyrosine-phosphorylation, and potentiate the subsequent acrosome reaction, exploration of the use of rHuOVGP1 in enhancing sperm fertilizing competence in order to improve the rate of sperm-egg binding during in vitro fertilization is also warranted.
121
Figures
122 ! Figure'1' '
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123 Figure 4- 1: Detection of rHuOVGP1 expression in HuOVGP1 cDNA transfected HEK 293 cells. (A) Immunofluorescent staining results showing transfection of HEK 293 cells with HuOVGP1 cDNA ORF clone (lower panel). Mock transfection (upper panel) was used as control. Cells were probed with a goat anti-HuOVGP1 polyclonal antibody [Oviductin (P-20)] followed by a donkey anti-goat IgG- FITC secondary antibody (green) and DAPI (blue nuclei staining). Scale bar = 30 μm. (B) Western blot results showing the expression of rHuOVGP1 in HEK293 cell culture supernatant. Lanes 1 and 4 represent results obtained from mock transfection and lanes 2, 3, 5 and 6 represent results obtained from OVGP1 human cDNA ORF clone transfection. The blot was probed with a goat anti-HuOVGP1 polyclonal antibody (Oviductin [P-20]) (lanes 1-3) or with the same antibody in the presence of the antibody blocking peptide (lanes 4-6). (C) A similar blot was probed with a rabbit anti-HuOVGP1 polyclonal antibody and similar results were obtained. Mock transfection (lane 1), OVGP1 human cDNA ORF clone transfection (lanes 2 and 3).
124
Figure 2 A B 1 2 3 4 5 kDa kDa 1 2 3 4 250- 250- 150- ¬rHuOVGP1 150- 100- 100- 75- 75- 50-
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Figure 4- 2: Purification of rHuOVGP1 and identity confirmation of the purified protein. (A) Coomassie-stained SDS-PAGE analysis: lane 1 is a molecular mass ladder; lane 2 represents culture supernatant; lane 3 is the concentrated culture supernatant; lanes 4-5 are purified rHuOVGP1 from different fractions of the gel filtration chromatography. (B) Western blot analysis showing rHuOVGP1 expression levels in main fractions from the separation process: culture supernatant (lane 1); proteins captured by 70% ammonium sulfate saturation (lane 2); purified rHuOVGP1 from different fractions of the gel filtration chromatography (lanes 3 and 4). The extra band with lower molecular weight in lane 1 is considered as non-specific found in the culture medium since this band is absent in lane 2 and in the purified fractions (lanes 3 and 4). (C) Mass spectrometry analysis showing the entire sequence of HuOVGP1 with the coverage of peptides (bold red) identified from the trypsin digest of rHuOVGP.
125 0 4 hrs A - - 10 25 50 75 μg/mL rHuOVGP1
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Figure 4- 3: Binding of rHuOVGP1 to human sperm during in vitro capacitation. (A) Western blot showing the presence of rHuOVGP1 in 1% Triton X-100 soluble fractions and insoluble fractions of sperm after 4 hrs of capacitation. Lanes from left to right: supernatant from medium, HBS wash following incubating with capacitating medium, Triton soluble fractions, PBS wash after Triton solubilization, and Triton insoluble cell pellets. Minus “-” and plus “+” indicate incubation in the absence or presence of rHuOVGP1 respectively. (B) Confocal microscopy imaging of human sperm incubated in the presence of rHuOVGP1 shows binding of the recombinant glycoprotein to membrane intact sperm (i and ii) and 1% Triton X-100 treated sperm (iv and v) following 1 hrs (i and iv) and 4 hrs (ii and v) of capacitation. Controls shows negative immunostaining when sperm were incubated in the absence of rHuOVGP1 for the membrane intact sperm (iii) and Triton-treated sperm (vi). Scale bar = 10 μm. Inserts: high magnifications of sperm cells revealing various immunolabeled structures. Ac: acrosomal region; ES: equatorial segment; PA: post-acrosomal region; MP: mid-piece; PP: principal piece. Scale bar = 5 μm.
126
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Figure 4- 4: Effect of rHuOVGP1 on tyrosine phosphorylation of proteins prepared from fresh human sperm. (A) Western blot showing dose-dependent effect of rHuOVGP1 on tyrosine phosphorylation of proteins from sperm incubated for 4 hrs in BWW medium alone or in BWW medium supplemented with different concentrations of rHuOVGP1 (10, 25, 50, and 75 g/mL) (upper panels: proteins migrated at 105 and 81 kDa that detected by Western blot using anti-phosphotyrosine antibody; lower panel: Western blot using anti-α-tubulin antibody), (B) Western blot showing protein tyrosine phosphorylation in the absence (-) or presence (+) of 50 g/mL rHuOVGP1 following 0-, 1-, 2, 3- and 4-hrs of capacitation, (C) statistical analysis of the levels of tyrosine phosphorylation of 105 kDa and 81 kDa proteins obtained with Western blot (n = 6 samples each from individual donors for each time point, *P < 0.05).
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Figure 4- 5: Effects of rHuOVGP1 on calcium ionophore-induced acrosome reaction of fresh sperm. Percentage of acrosome-reacted sperm (mean±S.E.M., unpaired t-test) obtained under different experimental conditions was indicated by the blank or shaded symbols as shown in the histogram. Sperm were assessed for acrosome reaction after incubation in capacitating medium for 0 hr or 4 hrs in the absence or presence of different concentrations of rHuOVGP1 (n = 5 samples each from individual donors, *p<0.05).
128
Chapter 5
Human OVGP1 Enhances Tyrosine Phosphorylation of Proteins in the Fibrous Sheath involving AKAP3 and Increases Sperm-Zona Binding
129 5.1 Abstract
Purpose: To investigate if the recombinant human OVGP1 (rHuOVGP1)-enhanced tyrosine- phosphorylated (pY) proteins are components of specific structure(s) of the sperm tail and if rHuOVGP1 binds to the oocyte and enhances sperm-egg binding.
Methods: Immunofluorescent staining and confocal microscopy were performed to examine the localization of pY proteins, outer dense fiber (ODF), and A-Kinase Associated Protein 3
(AKAP3) in human sperm during capacitation. Western blot and immunoprecipitation were employed to analyze protein levels of pY proteins and AKAP3. Immunofluorescent staining was performed to examine the binding of rHuOVGP1 to human oocytes. The effect of rHuOVGP1 on enhancing sperm-zona binding was examined using hemi-zona assay.
Results: pY proteins were detected mainly in the fibrous sheath (FS) surrounding the ODF with a relatively weak immunoreaction in the neck and mid-piece. Western-blot analysis revealed co-migration of the pY 105 kDa protein with AKAP3, which was further confirmed by immunoprecipitation correlating immunofluorescent results of co-localization of pY proteins with AKAP3 in the sperm tail. rHuOVGP1 binds specifically to the zona pellucida
(ZP) of human oocytes. Prior incubation of sperm and/or ZP with rHuOVGP1 increased sperm- egg binding.
Conclusions: The present study revealed that one of the major rHuOVGP1-enhanced pY proteins could be AKAP3 of the FS and that rHuOVGP1 is capable of binding to human ZP and its presence in the medium results in an increase in sperm-zona binding. Supplement of
130 rHuOVGP1 in in vitro fertilization media could be beneficial for enhancement of the fertilizing ability of human sperm.
131 5.2 Introduction
The mammalian oviduct provides an essential microenvironment in its lumen for maturation and transport of gametes, capacitation of sperm, fertilization and early embryo development (Hunter, 2003). A high-molecular-weight glycoprotein secreted by secretory cells of the oviduct collectively known as oviductin or oviduct-specific glycoprotein (OVGP1) has been identified and characterized in many mammalian species, including the mouse (Kapur and Johnson, 1985, 1986), hamster (Leveille et al., 1987), rabbit (Oliphant and Ross, 1982;
Oliphant et al., 1984), dog (Saint-Dizier et al., 2014), cat (Hachen et al., 2012), sheep (Sutton et al., 1984, 1986; Gandolfi et al., 1989), pig (Buhi et al., 1990), cow (Malayer et al., 1988;
Boice et al., 1990a), rhesus monkey (Verhage et al., 1997a), baboon (Fazleabas and Verhage,
1986; Verhage et al., 1989), and the human (Verhage et al., 1988). The amino acid sequence of the N-terminal region of OVGP1 is highly conserved among mammalian species; however, the C-terminal region shows low degree of homology with variable glycosylation sites (Aviles et al., 2010). Expression of OVGP1 appears to be estrogen-dependent or estrous cycle- associated in most species (Buhi et al., 1991; Murray, 1993; Arias et al., 1994; Malette et al.,
1995a; Briton-Jones et al., 2001, 2004, McBride et al., 2004a, 2005; Chen et al., 2013). Results from functional studies carried out mainly in vitro indicate several beneficial effects of OVGP1 and the importance of its presence in the luminal milieu of the oviduct during the process of fertilization and early embryo development. For examples, bovine OVGP1 was shown to increase the motility and viability of homologous sperm (Abe et al., 1995) and enhance sperm capacitation and fertilizing capabilities in vitro (King et al., 1994). The presence of native
132 OVGP1 in the incubation medium was shown to increase sperm-egg binding and penetration rates in the hamster (Boatman and Magnoni, 1995), baboon, and human (O’Day-Bowman et al., 1996) and decrease polyspermy in the pig (Kouba et al., 2000). Ovine and porcine
OVGP1were found to increase cleavage rates of embryos and the number of embryos reaching the blastocyst stage (Hill et al., 1997; Kouba et al., 2000). Previous studies from our laboratory demonstrated that native hamster OVGP1 prepared from the estrus stage can regulate the expression of tyrosine-phosphorylated proteins in hamster sperm during in vitro capacitation
(Saccary et al., 2013). Recombinant hamster OVGP1 has also been found to enhance tyrosine phosphorylation of a subset of sperm proteins during sperm capacitation and increase the number of sperm bound to oocytes (Yang et al., 2015). Recombinant human OVGP1
(rHuOVGP1), recently produced in our laboratory, is capable of binding to human sperm, enhancing sperm capacitation through the increase in the level of tyrosine phosphorylation of sperm proteins, and potentiating acrosome reaction (Zhao et al., 2016). Two of the major tyrosine-phosphorylated proteins that are enhanced by the presence of rHuOVGP1 migrate at
105 kDa (p105) and 81 kDa (p81), respectively (Zhao et al., 2016). However, the localization and the identity of these tyrosine-phosphorylated proteins during capacitation remained to be elucidated.
The aim of the present study was two-fold: firstly, to investigate if the rHuOVGP1-enhanced tyrosine phosphorylated proteins detected in our previous study are components of specific structure(s) of the sperm tail, and secondly, to investigate if rHuOVGP1 can bind to the zona pellucida (ZP) and enhance sperm-zona binding. The present findings suggest that the major
133 rHuOVGP1-enhanced tyrosine phosphorylated proteins are associated with A-Kinase
Associated Proteins (AKAPs) of the fibrous sheath and that rHuOVGP1 is capable of binding to human ZP in addition to sperm as previously reported. The presence of rHuOVGP1 in the culture medium was also found to increase the number of sperm bound to the oocytes as compared to similar conditions but in its absence.
5.3 Materials and Methods
5.3.1 Materials
The following materials were purchased from the sources indicated: goat anti-human oviductin antibody (P-20), goat anti-AKAP3 antibody (C-20), horseradish peroxidase (HRP)- conjugated donkey anti-goat IgG, goat anti-mouse IgG-HRP, fluorescein isothiocyanate
(FITC)-linked goat anti-mouse IgG, donkey anti-goat IgG-CFL555 (Santa Cruz Biotechnology
Inc., Santa Cruz, CA, USA); mouse anti-phosphotyrosine antibody (clone 4G10®) (EMD
Millipore, Bedford, MA, USA); mouse anti-α-tubulin antibody (clone B-5-1-2), rabbit anti-
ODF antibody (Sigma-Aldrich, St. Louis, MO, USA); goat anti-rabbit IgG-Alexa568 antibody
(Invitrogen, Burlington, ON, Canada); Western Lighting-Enhanced Chemiluminescence
Substrate, Micromass MassPREP Robotic Protein Handling System (PerkinElmer, Waltham,
MA, USA ). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).
5.3.2 Production of rHuOVGP1
The production of rHuOVGP1 from stably transfected HEK293 cells in our laboratory using recombinant DNA technology has been described elsewhere (Zhao et al., 2016). We used
134 a two-step purification system to yield rHuOVGP1 with a purity of ˃95%. The resulting protein showed a single band of 120-150 kDa size range on SDS-PAGE corresponding to the molecular mass of the native human OVGP1, and subsequent mass spectrometric analysis of the purified rHuOVGP1 confirmed its identity as human OVGP1 (Zhao et al., 2016).
To determine the optimal concentration of purified rHuOVGP1 for use in in vitro functional studies, a dose-dependent experiment was carried out where human sperm samples were incubated for 4 hrs in BWW medium supplemented with different concentrations of rHuOVGP1 (i.e. 0, 10, 25, 50 and 75 µg/mL). Western blot analysis was carried out to detect the level of protein tyrosine phosphorylation expression using an anti-phosphotyrosine antibody (Zhao et al., 2016). Results showed that the strongest labeling intensity of two proteins migrated at 105 kDa and 81 kDa was detected when rHuOVGP1 was used at a concentration of 50 µg/mL (Zhao et al., 2016). Based on the results obtained from the dose- dependent experiment, rHuOVGP1 at a final concentration of 50 µg/mL was used in all the in vitro functional assays in the present study.
5.3.3 Semen preparation and sperm capacitation
Studies with human semen were approved by the Ethics Committee for Research on
Human Subjects from the Queen’s University Health Sciences and Affiliated Teaching
Hospitals Research Ethics Board. Human semen samples were obtained from healthy donors by masturbation after 3 days of sexual abstinence. The sperm samples were prepared and capacitated as previously described (Zhao et al., 2016). Briefly, after the samples were liquefied in a 37oC water bath, semen viscosity and volume, sperm motility and sperm
135 concentration were assessed according to the WHO laboratory manual for the examination and processing of human semen (WHO, 2010). Normozoospermic samples were selected for experiments in the current study. Liquefied semen samples were separated by centrifugation using 20-40-65-95% Percoll gradients in HEPES buffered saline (HBS; 25mM HEPES,
130mM NaCl, 4mM KCL, 0.4 mM MgCl2, 14 mM fructose, pH 7.6). Sperm from the 65-95% interface and in the 95% Percoll fraction representing the highly motile population were pooled and washed with HEPES. Sperm concentration was determined by use of a hemocytometer.
Subsequently, sperm were incubated at a concentration of 2 107 cells/mL in the presence or absence of 50 µg/mL of recombinant human OVGP1 (rHuOVGP1) for up to 4 hrs at 37oC in
5% CO2 with 100% humidity in modified Biggers-Whitten-Wittingham medium (BWW; 10 mM HEPES, 94.6 mM NaCl, 4.8 mM KCl. 1.7 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4,
25.1 mM NaHCO3, 5.6 mM D-glucose, 21.6 mM Na lactate, 0.25 mM Na pyruvate, 0.1mg/mL phenol red, pH 7.4) supplemented with 0.3% fatty acid-free BSA.
5.3.4 Oocyte collection, storage and recovery
Human oocytes that were immature (GV and MI stage) or not qualified for intracytoplasmic sperm injection procedure were generously provided by Dr. Tamer Said and collected with ethical approval from patients at the ReproMed fertility clinic (Toronto, ON,
Canada). Oocytes were stored and transported in high salt solution (1.5 M MgCl2, 0.1% PVP
(MW 40,000), 40 mM sodium HEPES, pH 7.4) at 4°C. One day before carrying out the experiments, the oocytes were recovered by washing successively in 3 dishes of fresh and pre-
136 equilibrated BWW medium and stored overnight at 37°C with 5% CO2 in droplets of BWW medium covered with oil.
5.3.5 Oocyte microbisection
Individual oocyte was transferred into mineral oil covered-BWW medium in the absence of BSA and washed in successive droplets of the same medium using oocyte-denuding pipettes until the oocyte settled on the bottom of the culture dish. On the platform of an inverted microscope (Leica WILD M3Z), a glass blade with a sharp angled-tip, held by hand, was lowered vertically into the medium droplet. By pressing down the glass blade steadily and firmly in the median line of the oocyte and moving along the cutting axis, the oocyte was bisected into two equal halves. Subsequently, the hemizona pairs were washed using denuding pipettes to remove the residual cytoplasm. Mineral oil was removed and the hemizona pairs were transferred into BWW medium droplets supplemented with BSA. The hemizona pairs were incubated overnight at 37°C with 5% CO2 in BWW medium droplets covered with mineral oil.
5.3.6 Hemizona assay
Hemizona assay was performed following the procedure previously described by
Oehninger et al. (2013) with modifications. Three different experimental conditions were carried out with 8-9 oocytes used in each condition to examine the effect of rHuOVGP1 on enhancement of sperm-zona binding. 1) Pre-treatment of sperm and hemizonae, respectively, with rHuOVGP1: hemizonae were incubated in BWW medium droplets in the presence of 50
μg/mL rHuOVGP1 for 1 hour at 37°C with 5% CO2. Sperm were suspended at a concentration
137 of 2 x 106/mL in BWW medium in the presence of 50 μg/mL rHuOVGP1. 2) Pre-treatment of sperm with rHuOVGP1: sperm were incubated in BWW medium in the presence of 50 μg/mL rHuOVGP1 and incubated at 37°C with 5% CO2 for 1 hr. The sperm sample was washed with
HBS solution followed by centrifugation. The sperm sample was then diluted in fresh BWW medium at a concentration of 2 x 106/mL. Hemizonae were used without prior treatment with rHuOVGP1. 3) Pre-treatment of hemizonae with rHuOVGP1: hemizonae were incubated in
BWW medium droplets in the presence of 50 μg/mL rHuOVGP1 for 1 hr at 37°C with 5%
6 CO2. Sperm suspension was prepared at a concentration of 2 x 10 /mL in fresh BWW medium without prior treatment with rHuOVGP1. For all experiments, droplets of 25 μL of sperm suspension were prepared on a culture dish and covered with mineral oil. Hemizonae were added into the sperm droplets and incubated for 4 hrs at 37°C with 5% CO2. Control hemizonae were incubated in BWW medium in the absence of rHuOVGP1, transferred into sperm droplets in BWW medium in the absence of rHuOVGP1, and incubated in the same manner as described above.
5.3.7 Hemizona index analysis
Following sperm-hemizona incubation, the hemizonae were washed 5 times in PBS and then transferred into 1% paraformaldehyde (PFA)-PBS droplets covered with oil.
Photomicrographs were captured with different focal planes that covered all zona-bound sperm.
The number of bound sperm from all focal planes was counted and hemizona index (HZI) was calculated using the equation below (Oehninger et al., 2013). If the total number of sperm was
138 < 20 for the hemizona or the hemizona was saturated with sperm, the assay results for that particular experimental hemizona and the control half were not included when reporting results. Number of sperm bound in the test sample HZI = × 100 Number of bound sperm in the control sample
5.3.8 Immunofluorescent imaging of the binding of rHuOVGP1 to oocytes
Oocytes were recovered and incubated in pre-equilibrated BWW medium overnight at
37°C with 5% CO2. The oocytes were then transferred into BWW medium droplets covered with mineral oil in the presence or absence of 50 μg/mL rHuOVGP1 and incubated for 4 hrs at
37°C with 5% CO2. After incubation, oocytes were washed in PBS and then fixed in 1% PFA-
PBS for 30 min at room temperature. The oocytes were washed with PBS and transferred into oil-covered droplets of 5% donkey serum-PBS and incubated for 30 min. Subsequently, the oocytes were incubated at 4°C overnight with goat polyclonal anti-OVGP1 antibody at a concentration of 2 μg/mL in 30 μL of 1% donkey serum-PBS covered with oil. Thereafter, the oocytes were washed with PBS and incubated with donkey anti-goat IgG-FITC antibody at a concentration of 1 μg/mL in 1% donkey serum-PBS for 1hr at room temperature in the dark.
The oocytes were then washed with PBS, transferred onto glass slides and mounted with 1%
1,4-Diazabicyclo-(2,2,2)-octane (DABCO) in PBS with 90% glycerol. Images of oocytes were captured using a Zeiss Axiovert 200M Fluorescence Imaging Microscope.
5.3.9 Immunofluorescent labeling of sperm
Sperm samples were capacitated in BWW medium in the presence or absence of 50 μg/mL rHuOVGP1, washed with HBS, and then spread on positively charged glass slides. For detecting tyrosine-phosphorylated (pY) proteins, sperm samples on slides were fixed with 1%
139 PFA-PBS for 30 min and washed with PBS, treated with 90% ethanol for 30 min in a humid chamber, and followed by PBS washes. The sperm slides were first incubated with 10% normal goat serum (NGS)-PBS containing 0.05% Tween-20 (NGS-PBST) for 1 hr at room temperature followed by incubation with mouse anti-phosphotyrosine antibody (0.5 μg/mL) in 5 % NGS-
PBST at 4°C overnight. Subsequently, the slides were washed with PBST, and then incubated with goat anti-mouse IgG-FITC antibody (1 μg/mL) in 5 % NGS-PBST at room temperature for 1 hr. After being washed with PBST and mounted with 1% DABCO in PBS and 90% glycerol, the slides were examined on a Zeiss Axiovert 200M Fluorescence Imaging
Microscope. For confocal microscopic examination of the localization of pY proteins and outer dense fiber (ODF), sperm samples were extracted with Triton-DTT buffer (2% TritonX-100,
5mM DTT, 50mM Tris-HCl, pH 9.0, and protease inhibitors) for 15 min at room temperature with agitation. Extracted samples were centrifuged and washed with Tris-HCl (pH 9.0). The pellets were resuspended with PBS and spread on glass slides. The cell smear was fixed immediately and washed. The sperm slides were then blocked with 10% NGS-PBST followed by incubation with mouse anti-phosphotyrosine antibody (0.5 μg/mL) and rabbit anti-ODF antibody (2 μg/mL) in 5% NGS-PBST, and subsequently with goat anti-mouse IgG-FITC antibody (1 μg/mL) and goat anti-rabbit IgG-Alexa568 antibody (8 μg/mL) in 5 % NGS-PBST for 1 hr following the procedure as mentioned earlier. After the successive incubations, the sperm slides were washed with PBS, mounted with DABCO, and examined on a Leica TCS
SP2 confocal microscope.
140 For co-localization of AKAP3 and pY proteins, the sperm sample was first divided into a membrane-intact fraction and fixed with 1% PFA-PBS after capacitation, and a membrane- extracted fraction that was first treated with Triton-DTT buffer and then fixed with 1% PFA-
PBS. In each case, after preparing sperm smears on glass slides as described above, sperm slides were treated with 90% ethanol, washed with PBS and blocked with 3% BSA-PBST for
1 hr at room temperature followed by incubation overnight at 4°C with goat anti-AKAP3 antibody (4 μg/mL) and mouse anti-phosphotyrosine antibody (0.5 μg/mL) in 1% BSA-PBST.
After being washed with PBST, the sperm slides were incubated with donkey anti-goat IgG-
CFL555 (1 μg/mL) in 1% BSA-PBST for 1 hr at room temperature followed by several washes with PBST. The sperm slides were then incubated with goat anti-mouse IgG-FITC (1 μg/mL) in 1% BSA-PBST for 1 hr at room temperature, washed with PBST, and mounted with
DABCO. Fluorescent images were captured by confocal microscopy.
5.3.10 Western Blot Analysis
Detection of the level of protein tyrosine phosphorylation and AKAP3 protein expression was carried out as previously described (Zhao et al., 2016). Briefly, capacitated and non-capacitated sperm samples were solubilized in electrophoresis sample buffer (2% SDS,
10% glycerol, 50 mM dithiothreitol, 62.5 mM Tris-HCl, pH6.8) and boiled at 100oC for 5 min.
Sperm proteins were resolved by SDS-PAGE and transferred onto a PVDF membrane. To compare the migration distance of the pY proteins and AKAP3, the membrane was cut along the mid-line of the lane with sperm sample that was capacitated for 4 hrs in the presence of rHuOVGP1. The membrane halves were blocked with 5% (w/v) skim milk in Tris-buffered
141 saline (150 mM NaCl, 50 mM Tris, pH 7.5) containing 0.05% Tween 20 (TBST). Half of the membrane was incubated with mouse monoclonal anti-phosphotyrosine antibody (0.1 μg/mL in TBST) for 1 hr at room temperature followed by several washes with TBST. The membrane was then incubated for 1 hr with goat anti-mouse IgG-HRP (0.02 μg/mL) in TBST containing
5% skim milk and washed with TBST. The other half of the membrane was incubated overnight at 4°C with goat anti-AKAP3 antibody (1 μg/mL in TBST containing 5% skim milk). After incubation, the membrane was washed several times with TBST and blotted with donkey anti- goat IgG-HRP (0.1 μg/mL in TBST with 5% skim milk) for 1 hr followed by several washes with TBST. The two halves of the membrane were matched and aligned along the cutting edge, and the immunoreactivity was revealed using the Electrogenerated chemiluminescence (ECL) kit according to the manufacturer’s instruction. The same membranes were probed with mouse monoclonal anti-α-tubulin antibody (0.1 μg/mL in TBST) for 30 min at room temperature followed by goat anti-mouse IgG-HRP (0.02 μg/mL). The intensities of immunoreaction were quantified using ImageJ software. Intensities of pY proteins and AKAP3 were normalized to the ones of -tubulin.
5.3.11 Immunoprecipitation of AKAP3
Immunoprecipitation of AKAP3 was performed as previously described by Luconi, et al, with modifications(Luconi et al., 2005). Briefly, each sample containing 50 million of sperm cells were subjected to capacitation. sperm samples were lysed in SDS-lysis buffer (20 mM
Tris-HCl, pH 7.4, 1% SDS, 1 mM PMSF, 1mM Na3VO4) by vortexing for 1 min and then rotating for 30 min at 4 C. After 5 min of boiling at 95 C, samples were kept for 10 min on
142 ice and centrifuged at 16,000 g for 5 min at room temperature. Extracted supernatants were diluted 10 times with 20 mM Tris-HCl, pH 7.4 buffer and subjected to AKAP3 immunoprecipitation by first incubating overnight using 1.5 µg of goat anti-AKAP3 antibody followed by 2 hrs of incubation at 4 C with 15 µL of protein G-Sepharose beads. The protein beads were washed three times in 20 mM Tris-HCl, pH 7.4 by centrifugation at 1,000 g at
4C for 5 min and then resuspended in sample buffer and subjected to SDS-PAGE followed by Western blot analysis.
5.3.12 Statistical analysis
Statistical analysis was performed using Prism 6 (GraphPad Software, La Jolla
California, USA). Data are expressed as mean ± SE. P-values equal to or less than 0.05 with
95% confidence intervals were considered statistically significant.
5.4 Results
5.4.1 Tyrosine phosphorylated proteins are predominantly localized to the fibrous sheath
(FS) of the sperm tail
One characteristic feature during sperm capacitation is the time-dependent increased level of tyrosine phosphorylation of sperm proteins (Visconti et al., 1995a; Bailey, 2010; Signorelli et al., 2012). We have previously shown that supplement of rHuOVGP1 at an optimal concentration of 50 g/mL in the capacitating medium can significantly increase the level of tyrosine phosphorylation of a subset of proteins in human sperm (Zhao et al., 2016). In order to further investigate the role of rHuOVGP1 in regulating human sperm function, immunofluorescent experiments were carried out in the present study to examine the specific
143 site of localization of pY proteins following capacitation. At least 200 sperm cells were examined on each slide. Photomicrographs representative of the immuno-detection of pY proteins in human sperm are best illustrated in Fig. 5-1A where immunofluorescent and overlay images showed that the tyrosine-phosphorylated (pY) proteins were predominantly localized in the principal piece of the sperm tail. A relatively weak immunoreactivity for pY proteins was also detected in the neck region immediately below the sperm head with no labeling in the mid-piece region.
As the immunoreactivity for the pY proteins was found to be mainly associated with the principal piece of the sperm tail, the possible association of pY proteins with the underlying cytoskeletal elements in the tail region were further explored by removing the sperm plasma membrane (PM) and mitochondrial sheath (MS) using Triton-DTT extraction buffer followed by co-localization of the pY proteins and the outer dense fiber (ODF) (Fig. 5-1B). The pattern of immunolabeling of pY proteins in sperm after extraction of PM and MS (Fig. 5-1B: pY) was similar to that of the intact sperm in the tail region (Fig. 5-1A: pY). After extraction with Triton-
DTT, the immunoreactivity remained with the entire principal piece of the tail (Fig. 5-1B: pY).
The labeling appeared to be stronger along the outer edges and weaker along the midline of the principal piece. The neck region immediately below the sperm head was strongly labeled and the immunoreactivity gradually decreased towards the mid-piece region (Fig. 5-1B: pY).
Immunolabeling of ODF, however, displayed a different pattern than that of pY proteins. In general, immunoreactivity of ODF was detected in all sperm examined with an intact tail. ODF was found throughout the neck region, mid-piece, and principal piece, albeit with a higher
144 intensity in the neck region and the proximal segment of the mid-piece (Fig. 5-1B: ODF). The intensity of immunoreaction decreased towards the end of principal piece. The overlay image of the immunolabeling of pY proteins and ODF revealed that the mid-piece houses the proximal segment of the ODF but was devoid of pY proteins. The pY proteins and ODF were co-localized along the midline of the principal piece where ODF is located. However, the majority of the pY proteins was localized to the periphery of the principal piece where the FS is present. The pY proteins appeared to form a sheath in the principal piece enclosing the ODF
(Fig. 5-1B: Overlay). The immunostaining pattern described above was similar in all sperm cells examined. One characteristic feature of the principal piece is the FS that surrounds the bundle of ODF. Therefore, the majority of the up-regulated pY proteins following capacitation is likely associated with the FS of the sperm tail. The immunofluorescent signals were essentially absent in the negative controls for both pY proteins and ODF (Fig. 5-1B: Lower panel).
5.4.2 rHuOVGP1 enhances tyrosine phosphorylation of AKAP3 located in the fibrous sheath
We previously reported that one of the most abundantly tyrosine-phosphorylated human sperm proteins migrates at 105 kDa (p105) the phosphorylation level of which was further enhanced by the presence of optimally 50 µg/mL rHuOVGP1 in the capacitating medium
(Zhao et al., 2016). Based on our previous results and the initial findings of association of pY proteins with the FS as well as current information available in the literature concerning proteins in the FS (Carrera et al., 1994; Fulcher et al., 1995; Mandal et al., 1999;
145 Vijayaraghavan et al., 1999), we postulated that the p105 could be A-Kinase Anchoring Protein
3 (AKAP3). Western blot analysis was carried out to examine whether, in human sperm,
AKAP3 co-migrates with the tyrosine-phosphorylated p105. As shown in Fig. 5-2A, two protein bands were detected in the sperm protein lysate using goat anti-AKAP3 antibody. The protein band corresponding to AKAP3 co-migrated with p105 at the same distance, indicating that the tyrosine-phosphorylated p105 during capacitation that we previously reported is likely the AKAP3. A second protein band with a weaker immunoreactivity co-migrated with tyrosine-phosphorylated p81 (Fig. 5-2A). Based on the sequence similarities at the C-terminal regions of AKAP3 and AKAP4 and the fact that AKAP4 is known to be tyrosine- phosphorylated during capacitation (Baker et al., 2010b), the tyrosine-phosphorylated p81 that we previously reported is likely AKAP4. Immunoprecipitation experiments were carried out to determine the identity of p105 using goat anti-AKAP3 antibody (Fig 5-2B). The anti-
AKAP3 antibody was able to sufficiently pull down AKAP3 from the sperm lysate. Stripping and reprobing the same membrane with anti-phosphotyrosine antibody showed that the immunoprecipitated AKAP3 was also tyrosine phosphorylated that migrated at the same level as compared to p105 in the whole cell lysate. The blocking peptide for the anti-AKAP3 antibody successfully blocked the immunoprecipitation of AKAP3 as well as the tyrosine phosphorylation signals from AKAP3 (Fig 5-2B). Having shown that the 105 kDa protein
(p105) corresponds to the tyrosine phosphorylated AKAP3, further experiment was carried out to determine the effect of rHuOVGP1 on the tyrosine phosphorylation level of AKAP3 following capacitation. Similar to results that we recently reported (Zhao et al., 2016), in the
146 present study, the tyrosine phosphorylation level of p105 in the cell lysate was found to be increased in sperm capacitated in the presence of rHuOVGP1 as compared to sperm capacitated in the absence of rHuOVGP1 (p = 0.04, Fig. 5-2C and D). For the uncapacitated sperm, the tyrosine phosphorylation level of AKAP3 (ph-AKAP3) was found to be very low (Figs. 5-2C and D). Following 4 hrs of capacitation, the level of ph-AKAP3 was largely increased.
However, in the presence of rHuOVGP1, the level of ph-AKAP3 was found to be about the same as compared to sperm capacitated in the absence of rHuOVGP1 (p = 0.17, Figs. 5-2C and
D).
Although a trend of increase of AKAP3 protein levels with a peak at 4 hrs in the absence of rHuOVGP1 and a peak at 3 hrs in the presence of rHuOVGP1 was noted, the protein levels of AKAP3 are not statistically different between the hours of capacitation (Figs. 5-2E and F).
A marginal decrease of AKAP3 level at 4 hrs of capacitation in the presence of rHuOVGP1 was observed as compared to capacitation in the absence of rHuOVGP1 (p = 0.059, Fig. 5-2F).
Surely, a larger sample size has to be used in the future to determine unequivocally whether these observed trends are meaningful.
To further examine whether AKAP proteins co-localized with pY proteins in human sperm, double immunostaining experiments were performed using anti-AKAP3 and anti- phosphotyrosine antibodies. Both membrane-intact and Triton-DTT extracted sperm were examined for AKAP3 and pY co-localization. At least 200 sperm cells were examined in each condition. Whereas all sperm examined having an intact tail structure displayed immunoreactivity of AKAP3, the intensity of immunostaining for tyrosine phosphorylation
147 varied among sperm cells. In membrane-intact sperm, immunoreaction for pY was found to be specifically associated with the neck region and the entire principal piece of the tail (Fig. 5-3a).
After extraction with Triton-DTT, immunoreaction over the neck region became more intense and extended into the mid-piece while the staining pattern remained the same in the principal piece before and after extraction (Fig. 5-3b). In membrane-intact sperm, immunoreactivity of
AKAP3 was found in the acrosomal region, the neck region, the mid-piece and the principal piece (Fig. 5-3c). After extraction with Triton-DTT, immunoreactivity was absent in the sperm head and became weaker in the neck region (Fig. 5-3d). Immunostaining for AKAP3 persisted in the tail with an intense and uniformly distributed immunoreaction over the mid-piece after extraction with Triton-DTT (Fig. 5-3d). The overlay image demonstrated a high degree of co- localization of AKAP3 and pY proteins in the neck region and in the principal piece of sperm with or without Triton-DTT extraction. However, the co-localization appeared to be more pronounced in the neck region after treatment with Triton-DTT (Fig. 5-3: compare f with e).
The previously observed immunoreactivity in the membrane-intact sperm and in the Triton-
DTT extracted sperm was abolished when a blocking peptide specific for anti-AKAP3 antibody was added to the antibody prior to immunostaining (Figs. 5-3g and h). Based on these findings, the detergent-soluble AKAP3 in the mid-piece and in the acrosome region does not appear to be co-localized with tyrosine-phosphorylated proteins, whereas the opposite is true of detergent-insoluble AKAP3 associated with the neck region and the principal piece.
148 5.4.3 rHuOVGP1 binds to the ZP of oocytes
Following our previous study reporting that rHuOVGP1 binds to the cell surface and underlying cytoskeletal elements of human sperm (Zhao et al., 2016), in the present study, we went further to investigate the effect of rHuOVGP1 on early events of fertilization. We first examined the ability of rHuOVGP1 to bind to the zona pellucida (ZP) of human oocytes.
Human oocytes recovered from high-salt storage buffer and incubated in BWW in the presence of 50 μg/mL of rHuOVGP1 followed by labeling with anti-human OVGP1 antibody showed immunoreaction uniformly distributed throughout the ZP surrounding the oocyte (Fig. 5-4).
The oocyte cytoplasm was also immunoreactive to the antibody. However, the cytoplasmic immunoreaction was later found to be nonspecific since oocytes incubated in BWW in the absence of rHuOVGP1 also showed similar non-specific immunoreactivity in the oocyte cytoplasm whereas the ZP displayed no immunoreactivity (Fig. 5-4). The ZP was also devoid of immunostaining in negative controls where human oocytes were incubated with rHuOVGP1 in the presence of a specific blocking peptide or where oocytes were incubated with rHuOVGP1 but with the omission of the primary antibody. Therefore, our results demonstrated the capability of rHuOVGP1 in binding to the ZP of human oocytes in addition to human sperm as we previously reported (Zhao et al., 2016). Immunostaining of the oocyte cytoplasm is considered non-specific due to a false-positive immunoreaction from the secondary antibody.
5.4.4 rHuOVGP1 increases the number of sperm bound to ZP
Carried forward from our previous study showing that rHuOVGP1 can potentiate acrosome reaction (Zhao et al., 2016), the role of rHuOVGP1 in sperm-egg binding was
149 investigated in the present study by performing three different experimental conditions of hemizona binding assay (HZA). Treatment of both the sperm and hemizonae with 50 μg/mL of rHuOVGP1 prior to HZA yielded an increase of the hemizona index (HZI) by 2.37 fold
(p<0.005) as compared to the untreated conditions (Fig. 5-5). The HZI was increased by 1.64 fold (p<0.001) when sperm were treated with 50 μg/mL of rHuOVGP1 prior to incubation with the untreated hemizonae as compared to that of the untreated sperm incubated with untreated hemizonae (Fig. 5-5). Similarly, prior treatment of the hemizonae with rHuOVGP1 before incubation with untreated sperm yielded an increase in the HZI by 1.75 fold (p<0.001) as compared to the untreated hemizonae (Fig. 5-5). As a result, pre-treatment of sperm and/or ZP can enhance the number of sperm bound to ZP albeit at different levels.
5.5 Discussion
During capacitation in human sperm, protein tyrosine-phosphorylation in the principal piece but not the acrosome, equatorial segment, or the neck region is correlated with fertilization rates (Sakkas et al., 2003). However, the subcellular localization and identity of these tyrosine-phosphorylated (pY) proteins in human sperm remained to be elucidated.
Recently, we have shown that during in vitro capacitation, the presence of rHuOVGP1 in the capacitating medium can further enhance the level of tyrosine phosphorylation of two sperm proteins migrated at 105 kDa and 81 kDa, respectively, on SDS-PAGE (Zhao et al., 2016). In the present study, we further demonstrated that the tyrosine-phosphorylated proteins are predominantly localized in the fibrous sheath (FS) of the principal piece of the sperm tail in both membrane-intact and Triton-DTT-treated sperm. A relatively weak immunofluorescent
150 signal was also found in the neck region of the membrane-intact sperm. Interestingly, after extraction with Triton-DTT, the intensity of immunostaining of pY proteins was found to increase in the neck region, and the immunoreactivity extended towards the mid-piece. In human sperm, a tightly packed and well-organized mitochondrial sheath envelops the cytoskeletal tubules of the mid-piece. Triton-DTT treatment has been used to remove the plasma membrane and the underlying mitochondria in the mid-piece of mouse sperm (Oko,
1988). Removal of the plasma membrane and mitochondrial sheath allows the anti- phosphotyrosine antibody to gain access to the underlying pY proteins in the mid-piece as shown in the present study. Our results showed that immunostaining for the outer dense fiber
(ODF) is present throughout the neck region, mid-piece, and principal piece of the sperm tail.
The finding of immunoreactivity of ODF decreasing towards the end piece is in agreement with earlier findings that the majority of the ODF extends from the mid-piece downward to reach up to 60% of the entire length of the principal piece (Petersen et al., 1999). Overlay immunofluorescent images of pY proteins and ODF showed co-localization of pY proteins with ODF in the mid-line of principal piece but not in the neck region or in the mid-piece. In the principal piece, the immunostaining of pY proteins appeared to be weaker in the mid-line but intensely stained along the outer edges, forming a sheath surrounding the ODF. This suggests that the pY proteins are predominantly localized to the FS of the sperm tail. In the rat and hamster, the outer dense fiber protein has been reported to be tyrosine-phosphorylated during sperm capacitation (Baker et al., 2010a; Mariappa et al., 2010). The present results
151 showing co-localization of pY proteins and ODF in the principal piece of human sperm during capacitation corroborates with observations made in the rat and hamster (51, 52).
Previous proteomic studies have revealed that the proteins undergoing tyrosine phosphorylation during sperm capacitation include ion channels, metabolic enzymes, and structural proteins (Ficarro et al., 2003; Baker et al., 2010a; Nixon et al., 2010). Two prominent tyrosine phosphorylated proteins are A-kinase anchoring protein (AKAP) 3 and 4 (Baker et al.,
2010a). Phosphorylated AKAPs sequester a cyclic adenosine monophosphate (cAMP) dependent protein kinase A (PKA), a key enzyme which induces sperm capacitation, to subcellular compartments by binding to the regulatory subunit RII of PKA (Miki and Eddy,
1998; Michel and Scott, 2002; Tasken and Aandahl, 2004). AKAP3 and 4 are known to be the main components in the FS of the sperm tail (Carrera et al., 1994; Fulcher et al., 1995; Mandal et al., 1999; Vijayaraghavan et al., 1999). In the present study, AKAP3 was found to co-migrate with the tyrosine-phosphorylated p105 (Fig. 5-2A). The AKAP3 antibody, produced against the C-terminus of human AKAP3 protein, also detected a lower band that co-migrated with p81. The C-terminus of human AKAP3 and AKAP4 shares a homology domain with high protein sequence identity (NCBI Reference Sequence: NP_001265238.2 and NP_003877.2). It is likely that the same antibody can also detect AKAP4 protein. In the present study, immunoprecipitation experiment using anti-AKAP3 antibody showed that the tyrosine- phosphorylated sperm protein p105 corresponds to AKAP3, which is in agreement with the findings reported by Luconi et al. (2005) that AKAP3 is the major tyrosine-phosphorylated sperm protein following capacitation. However, contrary to a significant increase in the level
152 of tyrosine phosphorylated p105 at 4 hrs of capacitation in the presence of rHuOVGP1, immunoprecipitation experiments revealed similar levels of tyrosine phosphorylated AKAP3 regardless of the presence or absence of rHuOVGP1. It is noteworthy to mention that previous studies in bovine sperm have shown that tyrosine phosphorylated AKAP3 was degraded through proteasomal degradation following 4 hrs of incubation in the capacitation medium
(Hillman et al., 2013; Vizel et al., 2015). However, in our present study using human sperm, we did not observe significant alterations of the protein level of AKAP3 up to 4 hrs of capacitation in the presence or absence of rHuOVGP1. Whether rHuOVGP1 exerts a positive influence on enhancement of the level of tyrosine phosphorylation of AKAP3 during capacitation needs to be confirmed in the future using a larger sample size, especially in view of the well-documented fact that large intra- and inter-individual variability exists in various parameters of human sperm (Overstreet, 1994). Since enhancement of tyrosine phosphorylation is one of the most important physiological changes during sperm capacitation, it will be of interest in the future to utilize high-throughput phosphoproteome analysis to investigate the effect of rHuOVGP1 on the overall phosphorylation events during sperm capacitation in humans. Phosphoproteome analysis has been used to identify and characterize tyrosine phosphoproteins during capacitation in human sperm, including AKAP3 and AKAP4, and mapped eight phosphorylation sites of these proteins (Ficarro et al., 2003). This approach has been used to study phosphoprotein alterations in sperm between healthy and asthenospermic individuals and between capacitated and non-capacitated sperm (Cao et al.,
2018). Therefore, phosphoproteomic analysis with high-throughput ability will be a useful tool
153 for determining the effect of rHuOVGP1 on tyrosine phosphorylation so sperm proteins during capacitation.
The FS is a cytoskeletal structure unique to sperm that surrounds the ODF and axoneme in the principal piece of the sperm tail (Eddy et al., 2003). Functionally, FS provides a scaffold for glycolytic and signaling enzymes during capacitation that is important for hyperactivated motility (Miki et al., 2002; Eddy et al., 2003; Krisfalusi et al., 2006). AKAP3 has been reported to localize only to the principal piece region of mouse sperm (Brown et al., 2003b; Xu et al.,
2016). In human sperm, antibodies against variants of AKAP3 have detected disparate localization patterns in head and tail structures (Lefevre et al., 1999; Mandal et al., 1999). The present study, using an antibody against the C-terminal region of AKAP3, is the first to show the localization of AKAP3 to both acrosomal and tail regions of human sperm, which corroborated results previously found in bovine sperm (Vijayaraghavan et al., 1999). Our double immunolabeling experiment of pY proteins and AKAP3 showed that only AKAP3 in the principal piece and the neck region was co-localized with pY proteins while AKAP3 in the acrosomal region and mid-piece was not. One possible explanation for this difference in co- localization of pY proteins and AKAP3 between the principle piece/neck region and the acrosome reaction could be that human AKAP3 retains the dual-binding specificity properties found in the mouse sperm where AKAP3 binds to both PKA regulatory subunits, the RI, which is present in the acrosomal region, and the RII, which is exclusively found in the tail region
(Xu and Qi, 2014).
154 The present study also showed the binding of rHuOVGP1 to the ZP of human oocytes, which is consistent with results previously reported by the use of partially purified native human OVGP1 (O’Day-Bowman et al., 1996). Importantly, the number of sperm bound to the
ZP was significantly increased in the presence of rHuOVGP1. Pre-treatment of either sperm or hemi-zonae alone with rHuOVGP1 increased the number of sperm bound to hemi-zonae.
However, pre-treatment of both sperm and hemi-zonae prior to co-incubation yielded the highest increase of the HZI by 2.37 fold. The present results indicate that OVGP1 possesses dual-binding specificity towards sperm and zona pellucida resulting in the enhancement of sperm capacitation as well as sperm-oocyte binding. The presence of rHuOVGP1 in the incubating medium during the co-incubation of sperm and oocytes represents the optimal condition and mimics the in vivo situation where sperm fertilize the oocyte in the oviduct. Our results are consistent with studies conducted in the hamster (Yang et al., 2015) and porcine
(Yang et al., 2015; Algarra et al., 2016) where recombinant OVGP1 of the respective species was found to bind to the ZP and enhance sperm-egg binding. In the porcine, the N-terminal region of recombinant OVGP1 is responsible for the binding of OVGP1 to the ZP while the C- terminal region influences the capacity of the full-length OVGP1in penetrating the ZP, its sensitivity to pronase digestion as well as the subsequent fertilization efficiency (Algarra et al.,
2016). Different functional properties of the N-terminus and C-terminus of rHuOVGP1 remain to be elucidated.
155 5.6 Conclusion
Results from the present study showed that, during human sperm capacitation, tyrosine- phosphorylated proteins are predominantly located in the FS. AKAP3 has been detected in the acrosome, neck region, mid-piece and principal piece of the human sperm tail. AKAP3 co- migrates with the tyrosine-phosphorylated p105. Double labeling experiments indicate that
AKAP3 in the neck region and principal piece are co-localized with tyrosine-phosphorylated proteins whereas AKAP3 in the acrosomal region and mid-piece is not. Taken together, results from our previous study (Zhao et al., 2016) and the present findings suggest that rHuOVP1 enhances sperm capacitation, in part, through the increase in the level of tyrosine phosphorylation of sperm proteins that are associated with AKAP3 in the sperm tail. In vitro experiments in the present study showed that rHuOVGP1 binds to the ZP of human oocytes and increases sperm-zona binding. Based on these results, supplement of rHuOVGP1 in capacitating medium in standard in vitro fertilization procedures could be beneficial for increasing the fertilization success rate. What remains to be studied in the future is the mechanism of rHuOVGP1 that regulates the increase in tyrosine phosphorylation of sperm proteins during capacitation and the enhancement of human sperm-egg binding.
156
Figures
157 Figure 5- 1: Tyrosine phosphorylated proteins are predominantly localized to the fibrous sheath of the sperm tail. (A) Microscopic images showing the immunofluorescent labeling of sperm proteins that are tyrosine phosphorylated (pY) following capacitation. Scale bar = 50 μm. (B) Confocal fluorescent images showing the immunofluorescent labeling of tyrosine phosphorylated sperm proteins (upper left), the ODF protein (upper middle), the overlay image (upper right), and corresponding negative controls (lower panel). Inserts: high magnifications of sperm cells within the framed boxes revealing various immunolabeled structures. Scale bar = 10 μm. N, neck; MP, mid-piece; PP, principal piece.
158 IB: AKAP3 IB: pY A 0 2 2 4 4 4 4 4 0 hrs - - + - + + + - - rHuOVGP1 AKAP3 à ßp105 ßp81 IB: α-tubulin Blocking peptide to - - + - B AKAP3 antibody AKAP3 pY
Cell Lysate IP: AKAP3
C D CL: p105 IP: ph-AKAP3/ AKAP3 10 1.5
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159 Figure 5- 2: The effect of rHuOVGP1 on tyrosine phosphorylation of AKAP3. (A) Western blot showing co-migration of AKAP3 (left) and tyrosine phosphorylated proteins (PY: right) from human sperm after incubation in capacitating medium in the presence or absence of 50 μg/mL rHuOVGP1 from 0-4 hrs. Lower panel shows the immunoblotting of α-tubulin. (B) Experiment of AKAP3 immunoprecipitation where sperm extracts were immunoprecipitated using anti-AKAP3 antibody alone or in the presence of a blocking peptide specific for the anti-AKAP3 antibody. Western blot analysis using anti-AKAP3 antibody (AKAP3) or stripping and re-probing with anti- phosphotyrosine antibody (p105) is shown. (C) Comparison of the levels of AKAP3 and tyrosine- phosphorylated p105 in the presence or absence of 50 µg/mL rHuOVGP1 following 4 hrs of capacitation in the sperm cell lysate as well as the levels of immunoprecipitated AKAP3 and the tyrosine-phosphorylated AKAP3 (ph-AKAP3) from the respective sperm cell lysates. (D) Statistical analysis of the intensities of protein bands from Western blots showing the levels of AKAP3 in cell lysates, p105 in cell lysates, and ph-AKAP3/AKAP3 in the eluates of immunoprecipitation (data represent means SEM obtained from four separate experiments). (E) Western blot showing the levels of AKAP3 following capacitation in the presence or absence of 50 µg/mL rHuOVGP1. (F) Statistical analysis of the intensities of protein bands from Western blots showing the protein level of AKAP3 during 4 hours of capacitation in the presence (blue) or absence (red) of rHuOVGP1 (data represent means SEM obtained from seven separate experiments).
160
161
Figure 5- 3: AKAP3 is co-localized with pY proteins in the neck region and in the principal piece of the sperm tail. Confocal microscopic images of immunofluorescent labeling of pY proteins (a and b) and AKAP3 (c and d) on capacitated sperm with intact membrane (a, c, and e) and Triton-DTT extracted (b, d, and f) sperm. Bottom panel (g and h) shows the negative control of AKAP3 labeling. Inserts: high magnifications of sperm cells within the framed boxes revealing various immunolabeled structures. Scale bar = 10 m. N, neck region; MP, mid-piece, PP, principal piece; Ac, acrosome.
162 Figure 5- 4: rHuOVGP1 binds to the ZP of human oocytes. Microscopic images showing the immunofluorescent labeling of rHuOVGP1 in human oocytes following incubation of oocytes in the presence (+) or absence (-) of rHuOVGP1, with the blocking peptide specific for the OVGP1 antibody, or in the absence (-) of the primary antibody during immunolabeling, respectively. Scale bar = 50 μm.
163
164 Figure 5- 5: Effects of rHuOVGP1 on sperm-ZP binding. (A) Representative microscopic images showing the hemizona binding assays from the three experimental conditions: (b) pretreatment of sperm with rHuOVGP1, (d) pretreatment of hemizonae with rHuOVGP1, and (f) pretreatment of both sperm and hemizonae with rHuOVGP1, as compared to their corresponding control assays (a, c, and e) in the absence of rHuOVGP1. (B) Histogram showing statistical analysis of hemizona index of the aforementioned assays. Data are shown as the mean ± SEM; (n) indicates the number of hemizona pairs for each experimental condition; ** P < 0.005; *** P < 0.001
165
Chapter 6
Effects of Recombinant Human OVGP1 on Protein Tyrosine Phosphorylation and Acrosome Reaction in Cryopreserved Human Sperm
166 6.1 Abstract
Cryopreservation of sperm is an effective way to preserve reproductive function in men undergoing chemo- or radiotherapy treatments. However, the freezing and thawing processes are known to cause structural and functional changes in the post-thawed human sperm.
Oviduct-specific glycoprotein 1 (OVGP1), a major glycoprotein produced by the mammalian oviduct, has been shown to play an important role in several events during mammalian reproduction, including sperm capacitation, sperm-egg binding, and early embryo development.
The present study was carried out to determine if addition of recombinant human OVGP1
(rHuOVGP1) in the capacitating medium has any effect on tyrosine phosphorylation of proteins and acrosome reaction of previously cryopreserved human sperm. Immunofluorescent experiments showed that rHuOVGP1 binds to the sperm head and tail regions of post-thaw sperm during capacitation. Western blot analysis revealed an increased level of tyrosine- phosphorylated (pY) p105 and p81 in a time-dependent manner with a significant increase at
4 hrs after capacitation when post-thaw sperm were incubated in the presence of rHuOVGP1 as compared to sperm incubated in the absence of rHuOVGP1 where the increase plateaued after 1 hr of capacitation. pY proteins were found predominantly in the fibrous sheath of the sperm tail. Although the level of spontaneous acrosome reaction in the post-thaw sperm was higher than that found previously in the fresh sperm following capacitation, 20-30% of the post-thaw sperm can be further induced to undergo acrosome reaction comparable to results previously obtained with fresh sperm. Furthermore, addition of rHuOVGP1 was also found to significantly potentiate acrosome reaction in the post-thaw sperm. Taken together, results of
167 the present study showed that addition of rHuOVGP1 in the capacitation medium can improve the quality of post-thaw human sperm.
168 6.2 Introduction
Mammalian sperm are only marginally stable after collection. Cryopreservation of sperm has been used to facilitate transportation and for long-term storage. This technique is now widely used in assisted reproductive technologies to preserve male fertility and to avoid damaging effects on the germ cells that are irreversible after radio- or chemotherapy treatments
(Sanger et al., 1992; Di Santo et al., 2012). However, freeze-thawing of sperm is known to damage the integrity of the sperm membrane and chromatin structure and cause DNA fragmentation (Medeiros et al., 2002; Gandini et al., 2006; Gosálvez et al., 2011; Boitrelle et al., 2012; Sharma et al., 2015). Studies also showed that post-thawing of cryopreserved sperm results in alteration of cell morphology and changes in mitochondrial functionality and oxidative stress that lead to lipid peroxidation (Verheyen et al., 1993; Wang et al., 1997;
Schuffner et al., 2001; O’Connell et al., 2002). The viability and mobility of previously cryopreserved sperm are also severely affected and compromised by freeze-thawing procedures (Hammadeh et al., 1999; Donnelly et al., 2001; O’Connell et al., 2002). In order to preserve sperm quality, investigators have examined the use of a variety of cryoprotectants and freezing extenders during cryopreservation. Examples include the use of glycerol, ethylene glycerol, dimethyl sulphoxide (DMSO), and 1,2-propanediol as cryoprotectants (Di Santo et al., 2012), and the use of animal or plant-based lipids, growth factors, antioxidants, and other micronutrients as freezing extenders (Lenzi et al., 2004; Kumaresan et al., 2005; Di Santo et al., 2012; Banihani et al., 2014; Amidi et al., 2016; Liu et al., 2016; Najafi et al., 2016;
Saeednia et al., 2016; Hatef et al., 2017; Aliabadi et al., 2018). While the use of cryoprotective
169 agents can reduce, to a certain degree, damage to sperm caused by the cryopreservation, information on factors that can benefit the thawing process and improve the quality of sperm after post-thawing is lacking in the field.
Capacitation is a prerequisite physiological process that renders sperm competent to fertilize the egg (Austin, 1952; Yanagimachi, 1994). During capacitation, sperm adopt hyperactivated motility, acquire the potential to undergo acrosome reaction and subsequently the capability to bind to the zona pellucida (Yanagimachi, 1994; Bailey, 2010). One characteristic and essential molecular event associated with capacitation is the time-dependent increased level of tyrosine phosphorylation of sperm proteins (Urner and Sakkas, 2003; Naz and Rajesh, 2004; Barbonetti et al., 2008; Bailey, 2010; Baker et al., 2010a; Aitken and Nixon,
2013). However, unlike the capacitation process of fresh sperm samples, immediate tyrosine phosphorylation of sperm proteins is found in the frozen-thawed sperm and is thought to be non-capacitation related (Naresh and Atreja, 2015). Whether frozen-thawed human sperm are able to carry out capacitation-related protein tyrosine phosphorylation remains unclear.
Furthermore, effective acrosome reaction is essential for the sperm to bind and penetrate the zona pellucida and subsequently fuse with the oocyte (Yanagimachi, 1994; Fraser, 1998; Liu and Baker, 1998). Frozen-thawed sperm are prone to have damaged acrosome integrity that could compromise the fertilizing capacity of sperm (Cross and Hanks, 1991). Information concerning the improvement of the potential of the post-thaw sperm to undergo acrosome reaction is also lacking in the field.
170 Oviduct-specific glycoprotein 1 (OVGP1) is a high-molecular-weight glycoprotein synthesized and secreted by non-ciliated secretory cells of the oviduct and has been identified in many mammalian species including the mouse (Kapur and Johnson, 1985, 1986), hamster
(Leveille et al., 1987), rabbit (Oliphant and Ross, 1982; Oliphant et al., 1984), dog (Saint-
Dizier et al., 2014), cat (Hachen et al., 2012), sheep (Sutton et al., 1984, 1986; Gandolfi et al.,
1989), pig (Buhi et al., 1990), cow (Malayer et al., 1988; Boice et al., 1990a), rhesus monkey
(Verhage et al., 1997a), baboon (Fazleabas and Verhage, 1986; Verhage et al., 1989, 1990), and the human (Verhage et al., 1988). OVGP1 has been shown to play a beneficial role during the process of fertilization and early embryo development (King and Killian, 1994; Abe et al.,
1995; Boatman and Magnoni, 1995; O’Day-Bowman et al., 1996; Hill et al., 1997; Kouba et al., 2000; Aviles et al., 2010; Saccary et al., 2013; Yang et al., 2015; Algarra et al., 2016). Our laboratory has recently produced a biologically active recombinant human OVGP1
(rHuOVGP1) which can enhance sperm capacitation through an increase in the level of tyrosine phosphorylation of sperm proteins, and potentiate the increase in acrosome reaction in fresh human sperm (Zhao et al., 2016). The present study was carried out to examine the effect of rHuOVGP1 on tyrosine phosphorylation of sperm proteins and acrosome reaction during the post-thaw step in previously cryopreserved human sperm as compared to that of the untreated control. Western blot analysis was used to measure the level of tyrosine phosphorylation of sperm proteins. The Pisum sativum agglutinin (PSA) lectin was used to evaluate the status of acrosome reaction. Immunofluorescent experiments were also performed
171 to examine the binding of rHuOVGP1 to human sperm and the localization of sperm proteins in rHuOVGP1-treated and untreated samples of cryopreserved sperm.
6.3 Materials and Methods
6.3.1 Materials
The following materials were purchased from the sources indicated: human oviductin antibody raised in goat [oviductin (P-20)], horseradish peroxidase (HRP)-conjugated donkey anti-goat IgG (DAG-HRP), fluorescein isothiocyanate (FITC)-linked goat anti-mouse IgG
(GAM-FITC) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); mouse anti- phosphotyrosine antibody (clone 4G10®) (EMD Millipore, Bedford, MA, USA); mouse anti-
α-tubulin antibody (clone B-5-1-2), rabbit anti-outer dense fiber (ODF) antibody (Sigma-
Aldrich, St. Louis, MO, USA); goat anti-rabbit IgG-Alexa568 antibody (Invitrogen, Burlington,
ON, Canada); Western Lighting-Enhanced Chemiluminescence Substrate (PerkinElmer,
Waltham, MA, USA ); All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO,
USA).
6.3.2 Preparation and capacitation of cryopreserved human sperm
Studies with human sperm were approved by the Ethics Committee for Research on
Human Subjects from the Queen’s University Health Sciences and Affiliated Teaching
Hospitals Research Ethics Board. Cryopreserved sperm samples from proven fertile donors were provided by ReproMed Ltd., Toronto, Ontario, Canada. Samples were thawed at 37°C in
172 a water bath for 4 mins and then layered on a gradient composed of 1.5 mL-fractions each of
45% and 90% SpermGradTM in HEPES-buffered saline (HBS; 25mM HEPES, 130mM NaCl,
4mM KCL, 0.4 mM MgCl2, 14 mM fructose, pH 7.6) and centrifuged at 1000xg for 30 mins.
Sperm from the 90% SpermGrad fraction were pooled and washed with HBS. The sperm pellet was resuspended and subjected to cell counting using a hemacytometer. The sperm samples were capacitated as described previously and the optimal concentration of rHuOVGP1 was determined to be 50 µg/mL as previously reported (Zhao et al., 2016). Briefly, sperm cells were incubated in the presence or absence of 50 µg/mL of rHuOVGP1 for up to 4 hrs at 37°C in 5% CO2 with 100% humidity in modified Biggers-Whitten-Wittingham medium (BWW; 10 mM HEPES, 94.6 mM NaCl, 4.8 mM KCl. 1.7 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4,
25.1 mM NaHCO3, 5.6 mM D-glucose, 21.6 mM Na lactate, 0.25 mM Na pyruvate, 0.1mg/mL phenol red, pH 7.4) supplemented with 0.3% fatty acid-free BSA.
6.3.3 Immunofluorescent labeling of sperm proteins
Following capacitation, sperm samples were washed with HBS, and then spread on positively charged glass slides. Sperm samples on slides were fixed with 1% paraformaldehyde
(PFA)-PBS for 30 mins and washed with PBS. For the detection of binding of rHuOVGP1 to sperm surfaces, sperm samples on slides were blocked with 3% BSA-PBS overnight at 4°C followed by incubation overnight at 4°C with goat anti-human oviductin antibody at a dilution of 4 μg/mL in 1% BSA-PBS. Following incubation, the slides were washed with PBS containing 0.05% Tween-20 (PBST) and stained for 2 hrs at room temperature with donkey
173 anti-goat IgG-FITC at a dilution of 1 μg/mL in 1% BSA-PBS. After washing with PBST buffer, coverslips were mounted on slides with 90% glycerol containing 1% (w/v) 1,4-diazobicyclo-
(2,2,2)-octane (DABCO) as an anti-bleaching agent.
For examination of the localization of tyrosine-phosphorylated (pY) proteins and outer dense fiber (ODF), sperm samples were extracted with Triton-DTT buffer (2% TritonX-100,
5mM DTT, 50mM Tris-HCl, pH 9.0, and protease inhibitors) for 15 mins at room temperature with agitation. Extracted samples were centrifuged and washed with Tris-HCl (pH 9.0). The pellets were resuspended with PBS and spread on glass slides. The cell smear was fixed immediately and washed as described above. The sperm slides were then blocked with 10%
NGS-PBST followed by incubation with mouse anti-phosphotyrosine antibody (0.5 μg/mL) and rabbit anti-ODF antibody (2 μg/mL) in 5% NGS-PBST, and subsequently with goat anti- mouse IgG-FITC antibody (1 μg/mL) and goat anti-rabbit IgG-Alexa568 antibody (8 μg/mL) in 5 % NGS-PBST for 1 hr following the procedure as mentioned earlier. After the successive incubations, the sperm slides were washed with PBS, mounted with DABCO, and examined on a Leica TCS SP2 confocal microscope.
6.3.4 Western blot
At the end of each time interval, sperm cells were washed with HBS and centrifuged at
1000xg for 5 min. Sperm proteins were solubilized in electrophoresis sample buffer (2% sodium dodecyl sulphate [SDS], 10% glycerol, 50 mM dithiothreitol, 62.5 mM Tris-HCl, pH6.8) and denatured at 100oC for 5 mins. Sperm proteins were resolved by SDS-PAGE and
174 transferred onto PVDF. The PVDF membrane was blocked with 5% (w/v) skim milk in Tris- buffered saline containing Tween-20 (TBST: 150 mM NaCl, 20 mM Tris-HCl, 0.1% Tween-
20, pH 7.4) and subsequently incubated for 1 hr at room temperature with an anti- phosphotyrosine antibody in TBST at a final concentration of 0.1 μg/mL. Following several washes with TBS-T, the PVDF membrane was incubated for 1 hr with goat anti-mouse IgG conjugated to horseradish peroxidase (GAM-HRP) in TBST at a final concentration of 0.02
μg/mL. After washes in TBST, positive immunoreactive bands were detected using the ECL
Western blotting kit according to the manufacturer’s instructions. The same membrane was reprobed with a monoclonal antibody against alpha-tubulin in TBST at a final concentration of
0.01 μg/mL. After incubation, the membrane was labeled with GAM-HRP diluted at 0.02
μg/mL with TBST. Immunoreactive bands were detected as outlined above.
6.3.5 Acrosome reaction assays
Following 4 hrs of capacitation in the presence or absence of rHuOVGP1, sperm samples were divided into two aliquots; both were washed with HBS and then centrifuged at
1000xg for 5 min before being resuspended in BWW. Calcium ionophore A23187 (10 µM final concentration) was added to one of the two aliquots to induce the acrosome reaction while
DMSO was added to the other aliquot serving as a control for spontaneous acrosome reaction.
Subsequently, both samples were incubated for 1 hr at 37°C in 5% CO2 after which sperm samples were washed with HBS and centrifuged at 1000xg for 5 mins. Sperm pellet was resuspended in 100% methanol for 30 mins on ice, spread on slides, and dried at 37°C on a
175 heated stage. The acrosome of the sperm cells was stained for 30 mins with Pisum sativum agglutinin (PSA) conjugated with FITC (75 μg/mL in PBS) in a humid chamber. The slides were washed with water and air-dried. Coverslips were mounted on slides with 25 μl DABCO and observed under UV illumination. For each sample more than 200 sperm cells were counted to obtain the percentage of acrosome-reacted sperm.
6.3.6 Statistical analysis
Statistical analysis was performed using Prism 6 (GraphPad Software, La Jolla
California, USA). The band intensities of tyrosine phosphorylated proteins and the number of acrosome-reacted sperm between groups was compared using Student’s t-test. Data are expressed as mean ± SE. P values equal to or less than 0.05 with 95% confidence intervals were considered statistically significant.
6.4 Results
6.4.1 Binding of rHuOVGP1 to the surface of cryopreserved human sperm
To determine if rHuOVGP1 is capable of binding to cell surfaces of frozen-thawed human sperm, thawed sperm samples were capacitated for 1 hr and 4 hrs in the presence or absence of rHuOVGP1 and subjected to indirect immunofluorescent staining. At least 200 sperm cells were examined for each condition. At 1 hr of capacitation in the presence of rHuOVGP1, only a few sperm cells displayed an immunoreaction for rHuOVGP1 binding.
Among the immunoreactive cells, indirect immunofluorescence showed the localization of
176 rHuOVGP1 mainly to the head, neck region and mid-piece of the spermatozoa (Fig. 6-1: A1 and A2). After 4 hrs of capacitation, more sperm cells displayed immunostaining for rHuOVGP1. At this time, a strong immunoreaction remained associated with the mid-piece and the neck region with a weaker immunostaining intensity over the principal piece (Fig. 6-1:
B1 and B2). However, it was noted that the staining intensity of binding of rHuOVGP1 to different regions varied among individual sperm within the same sample (Fig. 6-1: B1 and B2).
The immunostaining of the sperm head previously seen at 1 hr also diminished (Fig. 6-1: B1 and B2) with a small number of sperm cells (~5% of all sperm examined) displayed binding of rHuOVGP1 to the acrosomal and post-acrosomal regions. Immunostaining was practically absent at both time intervals when sperm were incubated in the absence of rHuOVGP1 (Fig.
6-1C). Sperm cells also showed a negative immunoreaction when the primary antibody was omitted in the staining process of the sperm incubated in the presence of rHuOVGP1 (results not shown).
6.4.2 Effect of rHuOVGP1 on phosphotyrosine-containing protein expression in cryopreserved sperm
As one of the characteristic features of sperm capacitation is the time-dependent increase of pY sperm proteins, the effect of rHuOVGP1 on enhancing tyrosine phosphorylation of sperm proteins was evaluated in the cryopreserved samples using Western blot analysis.
Two major pY proteins migrated at 105 kDa (p105) and 81kDa (p81), respectively, were detected (Fig. 6-2A). An additional protein band at a molecular weight higher than p105 was
177 also observed in the frozen-thawed sperm samples. Following 4 hrs of capacitation, the level of tyrosine phosphorylation of both p105 and p81 from sperm samples capacitated in the presence of rHuOVGP1 was significantly increased in comparison with that obtained from sperm samples capacitated in the absence of rHuOVGP1 (Figs. 6-2B1 and B2). No significant difference was found in the other time intervals (1 hr, 2 hrs, and 3 hrs) between the treated and non-treated samples. Confocal microscopy of immunostaining of tyrosine phosphorylated (pY) proteins revealed that pY proteins was associated with the principal piece of the sperm tail but predominantly localized to the periphery of the principal piece (Fig. 6-3A) where the fibrous sheath is normally found. A relatively weak immunoreaction was also found in the neck region whereas the mid-piece was practically devoid of immunofluorescent signal (Fig. 6-3A). To examine if pY proteins are co-localized with any cytoskeletal structures in the sperm tail of human sperm, a double-immunolabeling experiment was carried out on sperm samples after extraction with Triton-DTT. We first examined immunolocalization of the outer dense fiber
(ODF) since they are major cytoskeletal structures that surround the axoneme in the middle piece and principal piece of the sperm tail. Immunostaining of outer dense fiber was found in the neck region, mid-piece, and throughout the length of the principal piece of the tail with a relatively high intensity of immunostaining in the neck and in the proximal region of the mid- piece (Fig. 6-3B). Results obtained with double-immunolabeling showed that pY proteins co- localized with ODF in the neck region and, to a lesser extent, in the center of the principal piece in a punctate pattern (Figs. 6-3C and 3D). The immunostaining of pY proteins in the periphery of the principal piece is likely associated with the fibrous sheath (FS) surrounding the ODF in
178 the principle piece of the tail. The ODF in the mid-piece was also tyrosine-phosphorylated, albeit to a lesser extent, in the frozen-thawed sperm samples. Therefore, the majority of the increased level of pY proteins in the post-thaw human sperm at 4 hrs of capacitation shown in
Figure 6-2 is likely associated with the FS of the sperm tail.
6.4.3 Effect of rHuOVGP1 on sperm acrosome reaction
While effective acrosome reaction is essential for successful fertilization, the freeze- thaw procedure tends to damage the acrosome integrity (Cross and Hanks, 1991). Based on our previous results obtained with fresh human sperm showing the tyrosine phosphorylation level of sperm proteins at the highest at 4 hrs of capacitation under the influence of rHuOVGP1, the effect of rHuOVGP1 on potentiating the acrosome reaction of previously cryopreserved sperm was examined by evaluating the status of the chemically-induced acrosome reaction following
4 hrs of capacitation in the presence or absence of rHuOVGP1. A dose response study of rHuOVGP1 (0, 25, 50, and 75 μg/mL) in capacitating medium was performed and the percentage of acrosome-reacted sperm determined at 4 hrs of capacitation was compared between the sperm sample treated with the ionophore A23187 and the control treated with
DMSO. In the A23187-treated samples, among the various concentrations of rHuOVGP1 used, the percentage of acrosome-reacted sperm was significantly increased in the presence of 50
μg/mL rHuOVGP1 as compared to that of sperm incubated in the absence of rHuOVGP1 (Fig.
6-4). No significant differences were observed in the percentage of acrosome-reacted sperm between control samples treated with DMSO and incubated either in the presence or absence
179 of rHuOVGP1 and the non-capacitated group (Fig. 6-4). Therefore, supplementing the capacitating medium with 50 μg/mL of rHuOVGP1 further potentiates the capacity of cryopreserved sperm to undergo acrosome reaction.
6.5 Discussion
Cryopreserved sperm samples are frequently used in IVF procedures. Although several studies suggested that the cryopreserved sperm sufficiently retain the fertilizing capability
(Yogev et al., 1999, 2010; Marcus-Braun et al., 2004), the post-thaw sperm suffer from significantly reduced motility and viability, and damaged chromatin integrity (Di Santo et al.,
2012). Our laboratory has recently produced a biologically active recombinant human oviductin (rHuOVGP1) [53]. We have shown that addition of rHuOVGP1 in the capacitation medium can further enhance the level of tyrosine phosphorylation of sperm proteins and potentiate the increase in acrosome reaction of human sperm freshly collected from donors.
Since tyrosine phosphorylation is a biochemical hall mark of capacitation, and capacitation and acrosome reaction are two membrane events that are essential for successful fertilization, we decided to examine if supplementation of the capacitation medium with rHuOVGP1 has any effect on tyrosine phosphorylation of sperm proteins and acrosome reaction in previously cryopreserved human sperm.
In the present study, we sought first to find out if rHuOVGP1 binds to frozen-thawed human sperm. Our results showed that the immunostaining pattern of binding of rHuOVGP1 to previously cryopreserved human sperm is similar to that of freshly collected sperm cells we
180 have recently reported (Zhao et al., 2016) albeit with some variations. In the post-thaw human sperm following capacitation in the presence of rHuOVGP1, the glycoprotein was found to be associated with the neck region and mid-piece, and to a lesser extent, to the principal piece.
The intensity of immunostaining detected in the different sperm membrane domains varied among individual sperm cells within the same sample with rHuOVGP1 detected over the acrosomal and postacrosomal regions in about five percent of all sperm cells examined.
Previously shown in the fresh sperm cells, a high intensity of immunostaining for rHuOVGP1 was detected over the mid-piece and, to a lesser extent, the principal piece of the sperm tail
(Zhao et al., 2016). A punctate immunostaining pattern of rHuOVGP1 was also found to be associated with the acrosomal region and equatorial segment of the sperm head (Zhao et al.,
2016). Based on the results obtained in the present study, it appears that the pattern of binding of rHuOVGP1 to the cell surfaces of frozen-thawed human sperm is very similar to that observed in fresh human sperm although there was no apparent variation regarding the intensity or the pattern of the immunostaining in capacitated fresh sperm samples. The variation of the intensity and pattern of immunostaining of binding of rHuOVGP1 in the post-thaw sperm is likely due to changes that might have occurred at the level of the sperm plasma membrane during cryopreservation, as the loss of plasma membrane integrity and alteration in membrane components have been found to be associated with cryopreservation of human sperm
(Schuffner et al., 2001; Desrosiers et al., 2006). It has also been reported that cryopreservation of human sperm results in a marked increase in intra- and inter-individual differences in semen parameters (Keel, 2006).
181 Knowing that rHuOVGP1 has the ability to bind to previously cryopreserved human sperm, we then sought to examine if supplementing the capacitation medium with rHuOVGP1 has any positive effect on post-thaw sperm cells. We decided to examine two sperm parameters, capacitation and acrosome reaction, two events that are critical for successful fertilization.
During in vitro capacitation, a time-dependent increase in protein tyrosine phosphorylation of sperm proteins is considered as a biochemical hallmark of sperm capacitation and this membrane phenomenon is commonly found in various mammalian species including the human (Visconti et al., 1995a; Leclerc et al., 1996). We have previously shown that, in freshly collected human normozoospermia samples, the level of tyrosine phosphorylation of two major proteins migrated at 105 kDa (p105) and 81 kDa (p81) increased, respectively, in a time- dependent manner during capacitation (Zhao et al., 2016). The presence of rHuOVGP1 in the capacitation medium is able to significantly enhance tyrosine phosphorylation of p105 and p81 following 3 hrs and 4 hrs of capacitation (Zhao et al., 2016). In the present study, the level of tyrosine phosphorylation of p105 and p81 increased in a time-dependent manner in the presence of 50 μg/mL rHuOVGP1 (Fig. 6-2B). In the absence of rHuOVGP1, the tyrosine phosphorylation level of p105 and p81 of post-thaw human sperm plateaued immediately following 1 hr of capacitation. The tyrosine phosphorylation level of both proteins was significantly increased at 4 hrs of capacitation in the presence of rHuOVGP1 as compared to capacitation in the absence of rHuOVGP1 (Fig. 6-2B). It has been reported that the thawing process can cause an increase in tyrosine phosphorylation of cryopreserved sperm not related to capacitation and that the capacity of cryopreserved sperm to undergo further increase in
182 tyrosine phosphorylation normally associated with capacitation process is limited or restricted
(Naresh and Atreja, 2015). Our present results showed that, in the absence of rHuOVGP1, the increase in the level of tyrosine phosphorylation of p105 and p81 is not significant following
1 hr of capacitation in the post-thaw sperm cells. However, the presence of rHuOVGP1 appears to benefit the capacitation process through a significant enhancement of tyrosine phosphorylation of sperm proteins at 4 hrs of incubation as shown in the present study. An additional pY protein band with a molecular weight higher than that of p105 was detected in post-thaw sperm cells at both the 3-hr and 4-hr time points during incubation (Fig. 6-2A), This additional protein band was not found in our previous results obtained with fresh sperm cells
(Zhao et al., 2016). Further studies are warranted for identifying the p105, p81 proteins and the additional pY protein as well as their function in relation to the fertilizing capacity of cryopreserved human sperm.
Images obtained with confocal microscopy revealed that pY proteins in the frozen- thawed sperm cells following capacitation were found mainly in the principal piece of the sperm tail with a relatively weak signal of tyrosine phosphorylation in the neck region while immunoreaction is absent in the mid-piece (Fig. 6-3). Co-localization of phosphorylated tyrosine residues and outer dense fiber (ODF) was detected in a punctate pattern along the mid- line of the principal piece. Unique to the principal piece is a fibrous sheath that surrounds the
ODF and axoneme of the sperm tail (Eddy et al., 2003). Fibrous sheath is a cytoskeletal structure that provides a scaffold for glycolytic and signaling enzymes during capacitation and is important for hyperactivated motility (Miki et al., 2002; Eddy et al., 2003; Krisfalusi et al.,
183 2006). It is attempting to speculate that rHuOVGP1 could further enhance capacitation of previously cryopreserved human sperm through the increase of tyrosine phosphorylation of fibrous sheath proteins. The mechanism of how rHuOVGP1 regulates tyrosine phosphorylation of fibrous sheath proteins and other proteins during capacitation remains to be elucidated.
Acrosome reaction is a prerequisite for fertilization and the freeze-thaw process is known to damage the acrosome integrity (Cross and Hanks, 1991; Nijs and Ombelet, 2001). In the present study, the percentage of spontaneous acrosome reaction of the frozen-thawed sperm immediately following capacitation was found to be around 30% (Fig. 6-4), which is much higher than the 5% of spontaneous acrosome reaction that we previously observed in the fresh sperm (Zhao et al., 2016). Chemical induction of acrosome reaction with A23187 was found to further increase the number of acrosome-reacted sperm by 20-30%, which is similar to previous findings obtained with fresh sperm cells (Zhao et al., 2016). Therefore, although cryopreservation of human sperm resulted in an increase in percentage of sperm cells undergoing spontaneous acrosome reaction and while increased spontaneous acrosome reaction is usually associated with compromised fertilizing capacity in humans (Fénichel et al.,
1991), our findings that cryopreserved human sperm exhibit the potential of undergoing chemically-induced acrosome reaction similar to those found in fresh sperm cells corroborate with observations that cryopreserved human spermatozoa retain their in vitro fertilizing capacity (Yogev et al., 1999, 2010; Marcus-Braun et al., 2004). Our dose-dependent experiments showed that rHuOVGP1 at a concentration of 50 μg/mL significantly increased
184 the percentage of A23187-induced acrosome reaction for cryopreserved sperm, which is consistent with previous findings obtained with fresh sperm cells (Zhao et al., 2016).
Recently, the production of recombinant OVGP1 in different species including the human(Zhao et al., 2016), hamster(Yang et al., 2015), and pig(Algarra et al., 2016) has provided opportunities to further explore its role in improving sperm quality in vitro. The present study is the first to show that supplementation of capacitation medium with rHuOVGP1 can further enhance the level of tyrosine phosphorylation of proteins, a biochemical hallmark of capacitation, and potentiate the increase of acrosome reaction in frozen-thawed human sperm. Future studies are warranted to examine if addition of rHuOVGP1 to the capacitation medium can reduce lipid peroxidation, mitochondrial damage, and DNA fragmentation and the underlying mechanisms in post-thaw human sperm.
185
Figures
186 FITC Overlay
1 hr +rHuOVGP1
4 hr +rHuOVGP1
4 hr -rHuOVGP1
Figure 6- 1: Binding of rHuOVGP1 to cryopreserved sperm following capacitation. Confocal microscopic images showing binding of rHuOVGP1 to cryopreserved sperm during 1-hr (A1 and A2) and 4-hr (B1 and B2) of capacitation. Immunostaining for rHuOVGP1 was absent in control samples where rHuOVGP1 was omitted during capacitation (C1 and C2). Inserts: high magnifications of sperm cells indicated by double-head arrows revealing various immunolabeled structures. Ac: acrosome; PA: post-acrosomal region; MP: mid-piece; N: neck; PP: principal piece; Scale bars = 10 µm.
187
Figure 6- 2: Effect of rHuOVGP1 on tyrosine phosphorylation of proteins prepared from cryopreserved sperm. (A) Immunoblot showing tyrosine phosphorylation of sperm proteins at 0-, 1-, 2-, 3-, and 4-hr of capacitation in the absence (-) or presence (+) of 50 μg/mL of rHuOVGP1. Histograms showing comparison of the relative intensities of immunoreaction of the p105 (B1) and p81 (B2) proteins obtained with sperm samples incubated in the absence or presence of rHuOVGP1 during the 4 hrs of capacitation. Data represent means SEM obtained from 10 separate experiments. n.s. = nonsignificant.
188
Figure 6- 3: Tyrosine phosphorylated proteins are localized to the fibrous sheath of the sperm tail. (A) Confocal microscopic image showing that tyrosine phosphorylated proteins (pY, green fluorescence) are predominantly associated with the fibrous sheath (FS) of the sperm tail. A weak immunoreaction is also seen in the neck region. (B) Immunostaining of the outer dense fiber (ODF, red fluorescence) is seen in the neck region, the mid-piece and throughout the principal piece. (C) Double- immunostaining of pY proteins and ODF reveals that pY proteins (green fluorescence) are mainly associated with the fibrous sheath and co-localized with the ODF in a punctate pattern (orange fluorescence) in the center of the principal piece. Fig.3D is a merged image of bright field and double- immunostaining of pY proteins and ODF. N: Neck; MP: Mid-piece; PP: Principal piece. Scale bar for all panels = 10 μm.
189
p=0.023 AR%
m 60
r
e
p
S
d e
t 40
c
a
e R
Non capacitated e -rHuOVGP1 DMSO m 20
o -rHuOVGP1 A23187 s
o +rHuOVGP1 DMSO r
c +rHuOVGP1 A23187 A
% 0 0 7 7 7 7 7 O 8 O 8 O 8 O 8 O 8 0T S 01 S 110 S 215 S 510 S 715 μg/mL rHuOVGP1 3 M 3 M 3 M 3 M 3 M 2 2 2 2 2 D D D D D 01 A 4 0 A 4 5 A 4 0 A 4 5 A 4 hrs P 1 1 0 2 5 5 0 7 5 G P 4 1 4 2 4 5 4 7 V G T 4 T 4 T 4 T 4 O V T T T T - O 4 - T 4 T
Figure 6- 4: Effect of rHuOVGP1 on calcium ionophore-induced acrosome reaction of cryopreserved sperm. Histogram showing the effect of rHuOVGP1 on calcium ionophore-induced acrosome reaction of cryopreserved sperm. Percentage of acrosome-reacted sperm was assessed for acrosome reaction after incubation in capacitating medium for 0 hr or 4 hrs in the absence or presence of different concentrations of rHuOVGP1 under different experimental conditions indicated by the blank or shaded symbols as shown in the histogram. n = 12 samples from individual donors, *p<0.05.
190
Chapter 7
General Discussion and Future Perspectives
191 7.1 Discussion
Mammalian oviductal fluid consists of a complex mixture originating from plasma transudation and glycoproteins secreted by oviduct epithelial cells (Aviles et al., 2010). Freshly ejaculated sperm need to undergo capacitation in order to fertilize the oocytes. In the native condition, sperm that have reached and resided in the oviducts acquire the maximum level of fertilizing capacity. Therefore, the physiological environment and molecular components of oviductal fluid have been shown to play an essential role in the early events of fertilization.
The incubating medium that mimics the physiological condition of oviductal fluid has made the in vitro fertilization (IVF) possible.
About 15% of infertility rate is happening in the world among the couples who seek to conceive. Infertile couples often seek assisted reproduction as a treatment. However, total fertilization failure occurs in 5 -15% of couples undergoing conventional IVF despite an apparently normal sperm quality (van der Westerlaken et al., 2005). Intracytoplasmic sperm injection (ICSI) procedure is used as a treatment for unexplained total fertilization failure or low fertilization rate after conventional IVF. However, ICSI is an invasive procedure and has been shown to be associated with increased birth defect rates as compared to natural births
(Bushnik et al., 2012; Simpson, 2014). As the particular sperm is selected objectively by an individual lab personnel, not selected by the oocyte per se, human error can affect the outcome of the procedure. Given the potential adverse effects of ICSI on the offspring, investigations to
192 improve the conventional IVF success rate are warranted in order to encourage patients with normal and mild male factor infertility to pursue the less invasive conventional IVF.
Accumulating evidence indicates that mammalian oviduct-specific glycoprotein
(OVGP1) exerts positive effects during early events of fertilization and early embryo development, however, the molecular mechanisms of the physiological function of OVGP1 involved in these events are not clearly understood, mainly due to the fact that it is technically difficult to obtain a large quantity of pure OVGP1 from oviductal fluids, and especially in humans, it is ethically impossible to obtain human oviducts for collecting sufficient amount of
OVGP1 for functional and mechanistic studies. Therefore, in our laboratory, we attempted to produce recombinant OVGP1 using recombinant DNA technology in order to study the role of
OVGP1 in early events of fertilization including sperm capacitation, acrosome reaction, and sperm binding to zona pellucida (ZP) during in vitro incubation. As previously demonstrated in our laboratory that purified native hamster OVGP1 was capable of enhancing tyrosine phosphorylation of hamster sperm proteins (Saccary et al., 2013), we first attempted to produce recombinant hamster OVGP1 (rHamOVGP1) to determine the role of rHamOVGP1 in regulating hamster sperm functions. We went further to translate the results obtained with rHamOVGP1 to human situation in order to examine the role of human OVGP1 in enhancing human sperm functions, thereby a recombinant human OVGP1 (rHuOVGP1) was produced and purified. In addition to using fresh human sperm sample in the present study, cryopreserved human sperm were also employed to examine the role of rHuOVGP1 in enhancing sperm functions as cryopreserved sperm are frequently used in the clinical practice. The freeze-thaw
193 process is known to reduce sperm membrane integrity and sperm capacitation (Medeiros et al.,
2002; O’Connell et al., 2002). In the present investigation, we sought to examine whether rHuOVGP1 can enhance the fertilizing capacity of cryopreserved sperm.
As shown in the present study, our laboratory has successfully produced, for the first time, biologically active rHamOVGP1 and rHuOVGP1. Both proteins were produced from
HEK293 cells with stable expression of high levels of OVGP1 protein. The production and purification procedures are described in Figures 2 and 3 in the Appendix and in Chapters 3 and
4. The production of rHamOVGP1 was achieved by transfecting HEK293 cells with lentivirus that carried full length HamOVGP1 cDNA, whereas production of rHuOVGP1 was attained by transfecting HEK293 cells with a bacterial plasmid that carried full length HuOVGP1 cDNA. Glycosylation plays an important role in the biological functions of OVGP1 (Satoh et al., 1995). Therefore, we intended to collect the glycosylated forms of full length OVGP1 that was secreted into the culture medium by HEK293 cells. As HamOVGP1 is a highly glycosylated protein that contains terminal -D-GalNAc residues recognized by Helix pomatia agglutinin (HPA) (Malette and Bleau, 1993), affinity purification columns with HPA-agarose were used to purify rHamOVGP1. The purification of rHuOVGP1 was performed by size exclusion chromatography. The resulting purified rHamOVGP1 and rHuOVGP1 had similar molecular weight as compared to their native counterparts as shown, respectively, in Figures
3-2 and 4-2. The identities of the purified recombinant proteins were further confirmed by mass spectrometry. The techniques that we employed in the present study have been shown to be
194 effective in producing sufficient amount of purified recombinant hamster and human OVGP1 to be used for biological functional studies.
Glycosylation of OVGP1 is important for its biological functions. In another study performed in our laboratory, the glycan structures and the glycosylation pathway of HEK293 cells were examined as compared to that of immortalized human oviductal cells which express human OVGP1 (Yang et al., 2012). Mass spectrometry revealed a broad range of many simple and complex O- and N-linked glycans of rHuOVGP1. Glycosyltransferase activities involved in the assembly of O- and N-glycans in HEK293 cells exhibited similar spectrum as compared to those from the immortalized human oviductal cells that can synthesize elongated and sialylated O-glycans with core 1 and 2 structures, as well as complex multiantennary N-glycans.
The molecular weight of rHuOVGP1 correspond to the molecular weight of human OVGP1.
Therefore, it is reasonable to assume that rHuOVGP1 produced from HEK293 cells is very similar to the native human OVGP1.
In order to examine the role of recombinant OVGP1 in regulating sperm functions, we first examined the binding of rHamOVGP1 and rHuOVGP1 to hamster and human sperm, respectively, during in vitro capacitation. The native hamster OVGP1 has been shown in an earlier study that the glycoprotein is capable of binding to the head region of the homologous sperm (Kan and Esperanzate, 2006). Similarly, binding of rHamOVGP1 to the head region was also observed in the present study initially at 1 hr of capacitation; however, the localization in the head region disappeared following 3 hrs of capacitation, possibly due to high percentage of
195 spontaneous acrosome reaction in the sperm. Moreover, localization of rHamOVGP1 was also found in the middle piece of the sperm tail with a lesser extent in the principal piece. Binding of rHamOVGP1 to both head and tail region suggests that this glycoprotein may exert its functions on different biological events in fertilization. Similar observations were found with rHuOVGP1. In the membrane intact sperm, binding of rHuOVGP1 was found over the head region, neck region, mid-piece, and to a lesser extent, the principal piece. Unlike the rHamOVGP1, rHuOVGP1 binding to the head region persisted following 4 hrs of capacitation, possibly due to the intact acrosome in human sperm during capacitation. Interestingly, binding of rHuOVGP1 to underlying sperm structures was detected in the post-acrosomal region and throughout the tail when the plasma membrane was removed. In the monkey, the N-terminal conserved region of OVGP1 has been found to be associated with a cytoskeletal protein, non- muscle myosin IIA (Kadam et al., 2006). It is possible that rHuOVGP1 binds to cytoskeletal structures in human sperm and regulates sperm capacitation. As for the cryopreserved human sperm, binding of rHuOVGP1 to sperm during capacitation was mainly found in the mid-piece, and to a lesser extent, the principal piece. The localization of rHuOVGP1 to the head region is highly variable in intensity and pattern within the same sample, which is likely due to changes that might have occurred at the level of the sperm plasma membrane during cryopreservation.
Loss of plasma membrane integrity and alteration in membrane components have been found to be associated with cryopreservation of human sperm (Schuffner et al., 2001; Desrosiers et al., 2006).
196 After knowing that the recombinant OVGP1s produced in our laboratory are capable of binding to sperm, we sought to determine whether the glycoproteins were capable of enhancing sperm capacitation. During sperm capacitation, a characteristic post-translational modification event is the time-dependent increase in global tyrosine phosphorylation of sperm proteins (Visconti et al., 1995b; Naz and Rajesh, 2004). An earlier study carried out in our lab showed that native HamOVGP1 can enhance sperm capacitation by increasing the level of tyrosine phosphorylation of several sperm proteins known to play important roles in sperm function (Saccary et al., 2013). In the present study, rHamOVGP1 with an optimal concentration at 20 μg/mL was capable of further enhancing tyrosine phosphorylation of hamster sperm proteins at 63, 75, 83, and 180 kDa in a time-dependent manner with a peak at
4 hr of capacitation. The 83 kDa protein was found to be the most intensely phosphorylated among these proteins. Immunoblot analysis revealed the identity of this protein is likely the A kinase anchoring protein (AKAP) 83, also known as AKAP4. AKAP4 is a major component of the fibrous sheath (FS) in the mouse and hamster sperm tail that is tyrosine-phosphorylated during capacitation and plays an important role in sperm motility (Jha and Shivaji, 2002; Miki et al., 2002; Brown et al., 2003a; Moretti et al., 2007; Luconi et al., 2011). Tyrosine- phosphorylated proteins were localized predominantly in the principal piece of the sperm tail revealed by immunofluorescent labeling of phosphotyrosine residues on the de-membraned sperm. FS, a unique structure exclusively found in the principal piece that provides a scaffold for proteins in signaling and metabolic pathways that are required for sperm capacitation, is also essential for sperm motility (Si and Okuno, 1999; Eddy et al., 2003; Krisfalusi et al., 2006;
197 Eddy, 2007). AKAP proteins bind to the regulatory subunits of protein kinase A (PKA) and confine PKA to distinct subcellular locations within the cell to regulate the cAMP-PKA pathway (Tasken and Aandahl, 2004). The cAMP-PKA signaling pathway is the key for sperm capacitation. Therefore, the fact that enhanced tyrosine phosphorylation of the 83 kDa protein in the presence of rHamOVGP1 during sperm capacitation suggests that rHamOVGP1 could enhance sperm capacitation partially through the increase in signaling of proteins involved in sperm motility.
In fresh human sperm samples, the levels of two major tyrosine-phosphorylated proteins migrated at 105 (p105) and 81 (p81) kDa were further enhanced by rHuOVGP1 (used at an optimal concentration of 50 μg/mL) with a peak, respectively, at 3 and 4 hrs of capacitation. In cryopreserved sperm samples, the levels of p105 and p81 were both further enhanced by rHuOVGP1 at 4 hrs of capacitation. The tyrosine-phosphorylated proteins were again found predominantly located in the fibrous sheath in the principal piece of the human sperm tail, in particular, mainly in the peripheral region of the sperm tail encompassing the outer dense fibers. In humans, the major form of AKAP proteins found in the fibrous sheath is
AKAP3, which is also tyrosine-phosphorylated and involved in the increase of human sperm motility during capacitation (Luconi et al., 2005, 2011). In the present study, immunoblotting and immunoprecipitation of AKAP3 demonstrated that AKAP3 is tyrosine-phosphorylated following capacitation and migrates at the same molecular weight as the tyrosine- phosphorylated p105 protein. However, the levels of tyrosine-phosphorylated AKAP3 was not significantly different in the presence or absence of rHuOVGP1, unlike the significantly
198 increase in the levels of p105 following sperm capacitation in the presence of rHuOVGP1 as compared to capacitation in the absence of rHuOVGP1. A larger sample size will be needed in the future to examine the regulation of the level of tyrosine-phosphorylated AKAP3 by rHuOVGP1 during capacitation, especially considering the fact that large intra- and inter- individual variability exists in various parameters of human sperm (Overstreet, 1994). Despite the limitation of the relatively small sample size used in the present study, the present results showed that rHuOVGP1 is capable of further enhancing sperm capacitation through the increase in the level of tyrosine-phosphorylation of sperm proteins predominantly located in the FS of sperm tail possibly involving AKAP3 protein.
Capacitated sperm gains the capacity to undergo acrosome reaction which is the prerequisite for fertilization to occur. In rodents such as mice and hamsters, sperm undergo spontaneous acrosome reaction in the in vitro capacitating medium (Byrd and Wolf, 1986;
Fénichel et al., 1991). There is a significant increase in the number of acrosome-reacted sperm when sperm are first capacitated in the presence of rHamOVGP1 as compared to their counterpart capacitated in the absence of rHamOVGP1 at 3, 4 and 5 hr time intervals. The occurrence of spontaneous acrosome reaction is rare in human sperm that are capacitated in vitro (Byrd and Wolf, 1986; Fénichel et al., 1991). Calcium ionophore-induced acrosome reaction has been employed to assess the sperm quality and fertilizing capacity (Yovich et al.,
1994; Liu and Baker, 1998; Makkar et al., 2003). Low spontaneous acrosome reaction rate and high calcium ionophore-induced acrosome reaction are an indicator of better sperm quality with higher fertilization successful rate (Fénichel et al., 1991). In the present study, sperm
199 incubated in the presence of rHuOVGP1 at a concentration of 50 µg/mL significantly increased the percentage of ionophore-induced acrosome reaction as compared to sperm capacitated in the absence of rHuOVGP1. A higher percentage of spontaneous reaction was observed in the cryopreserved sperm as compared to fresh sperm. This could be due to the damage of acrosome integrity during the freeze-thaw process (Cross and Hanks, 1991; Nijs and Ombelet, 2001).
However, the presence of rHuOVGP1 can further increase the percentage of ionophore- induced acrosome reaction. Therefore, taken together our findings indicate that supplementing the culture medium with recombinant OVGP1 can further potentiate sperm to undergo acrosome reaction which is linked to the increase in fertilization rate.
Sperm binding to ZP is a critical step before sperm can fertilize the oocyte. Defective sperm-ZP binding and penetration are the most common causes of fertilization failure in the conventional IVF (Liu and Baker, 2000, 2003, 2004; Aitken, 2006). Based on the above findings that recombinant OVGP1s produced in our laboratory are capable of enhancing sperm capacitation and potentiating acrosome reaction, we next investigated whether recombinant
OVGP1s can also increase sperm-ZP binding, which is another indicator of sperm fertilizing capacity. Firstly, rHamOVGP1 and rHuOVGP1 both are able to bind to ZP of hamster and human oocytes, respectively. In hamsters, pre-treatment of hamster oocytes with rHamOVGP1 followed by supplementing the incubating medium with rHamOVGP1 during sperm-egg binding yields the largest number of sperm bound to the ZP. In humans, pre-treatment of both hemi-zonae and sperm prior to the hemi-zona assay gives the highest number of sperm bound to hemi-zonae. Our results showed the binding of rHuOVGP1 to the ZP of human oocytes,
200 which is consistent with results previously reported by other researchers using partially purified native human OVGP1 (O’Day-Bowman et al., 1996). The present results indicate that OVGP1 possesses dual-binding specificity towards sperm and ZP resulting in the enhancement of sperm capacitation as well as sperm-oocyte binding. Our results are consistent with studies conducted in the hamster (Yang et al., 2015) and porcine (Yang et al., 2015; Algarra et al.,
2016) where recombinant OVGP1 of the respective species was found to bind to the ZP and enhance sperm-egg binding.
7.2 Future Perspectives:
In the future, in order to understand the mechanism of OVGP1 in enhancing sperm fertilizing capacity, it will be of interest to identify the receptor of OVGP1 on sperm cell surfaces. One study carried out by immunoprecipitation suggested that the N-terminal region of monkey OVGP1 binds to a cytoskeletal protein, non-muscle myosin IIA (Kadam et al.,
2006). We have not been able to successfully immunoprecipitated the receptor for rHuOVGP1 using full-length protein and non-cross-linking immunoprecipitation. It will be of interest to carry out cross-linking immunoprecipitation experiment in the future to identify the binding partner of OVGP1. It will also be desirable to design and produce different lengths of OVGP1 to determine the specific region that is responsible for the binding of OVGP1 to sperm by immunoprecipitation. In the porcine, the N-terminal region of recombinant porcine OVGP1 is responsible for the binding of OVGP1 to the ZP while the C-terminal region influences the capacity of the full-length OVGP1in penetrating the ZP, its sensitivity to pronase digestion as
201 well as the subsequent fertilization efficiency (Algarra et al., 2016). Different functional properties of the N-terminus and C-terminus of rHuOVGP1 remain to be elucidated.
Both human and hamster OVGP1 are glycoproteins with O- and N-linked glycans.
Previous studies have shown that glycans in glycoproteins play important roles in the regulation of various cellular processes during fertilization (Florman and Wassarman, 1985;
Capone et al., 1999; Rosati et al., 2000; Caputo et al., 2005; Seppälä et al., 2007). Since human and hamster OVGP1 are both heavily glycosylated proteins, it will be of interest in the future to examine the effect of enhancement of tyrosine phosphorylation of sperm proteins by rHuOVGP1 or rHamOVGP1 is mediated by specific glycans. To date, the role of specific glycans of mammalian OVGP1 in sperm capacitation is not known. Future studies will help to identify the specific glycans/glycopeptides that are responsible for exerting the specific effect of OVGP1.
In the future, it will be of interest to examine the effect of rHuOVGP1 on tyrosine phosphorylation of sperm proteins by more advanced and sensitive technique such as image- based flow cytometry to examine tyrosine phosphorylation of sperm proteins during capacitation in different regions of sperm. This technique is a semi-automatized segmentation method allowing for unbiased quantification and simultaneous localization of post- translational changes in an extended sperm population (Matamoros-Volante et al., 2018). This will be an alternative strategy to study signaling markers associated with capacitation, such as protein tyrosine phosphorylation, in a fast and quantitative manner. Phosphoproteomics is
202 another powerful technique that can be used to identify the rHuOVGP1-enhanced tyrosine phosphorylated sperm proteins. Phosphopeptide enrichment coupled with label-free quantitative mass spectrometry are suited for measuring changes in protein phosphorylation resulting from the sperm capacitation process. Phosphoproteome analysis has been used successfully to determine capacitation-associated changes in the phosphorylation status of sperm proteins in humans (Ficarro et al., 2003), mice (Platt et al., 2009; Baker et al., 2010a) and hamsters (Kota et al., 2009). Identification and localization of novel phosphoproteins enhanced by rHuOVGP1 will create new avenues for future functional studies of the regulatory roles of these phosphorylated protein substrates through the use of protein overexpression, gene knockout, or addition of enzyme inhibitors.
It is well known that hyperactivation of sperm motility is closely associated with capacitation. The processes regulated by protein phosphorylation include capacitation and hyperactivated motility of sperm (Urner and Sakkas, 2003). In the present study, we have shown the beneficial effect of rHuOVGP1 and rHamOVGP1 as an effector of sperm capacitation through its stimulatory effect on tyrosine phosphorylation of sperm proteins. It will be of interest in the future to determine if both rHuOVGP1 and rHamOVGP1 can further induce sperm hyperactivation. The motility of human or hamster sperm treated with rHuOVGP1 and rHamOVGP1, respectively, can be evaluated by a computer-aided sperm analyzer system for the sperm swimming pattern and the percentage of sperm that exhibit hypermotility.
203 The fresh human sperm samples that we used for the present study are from normozoospermia samples for the purpose of producing consistent results. After obtaining results showing that rHuOVGP1 can effectively enhance the fertilizing capacity of sperm, in the future, it will be of great interest to study the effect of OVGP1 on subfertile sperm samples, such as asthenozoospermia with reduced sperm motility, or oligozoospermia with a low sperm count. It will also be of interest to determine if rHuOVGP1 can increase the number of subfertile sperm bound to ZP, in the hope of improving the fertilization rate of the conventional
IVF procedure.
7.3 Overall Summary
In the present investigation, as summarized in Figure 7-1, we have successfully produced, for the first time, biologically active rHamOVGP1 and rHuOVGP1 from HEK 293 cells. The availability of recombinant OVGP1 circumvents the limited availability of native
OVGP1 for studying the beneficial role of these glycoproteins during early fertilization. We have demonstrated that both recombinant proteins are capable of 1) enhancing homologous sperm capacitation through the increase in the level of protein tyrosine phosphorylation; 2) further potentiating the sperm to undergo acrosome reaction; and 3) increasing the number of sperm binding to zonae pellucidae of oocytes. The tyrosine-phosphorylated proteins are predominantly located in the FS of the sperm tail, possibly involving AKAP proteins that play an important role in sperm motility related to capacitation. Supplement of rHuOVGP1 in capacitating medium in standard IVF procedures could be beneficial for increasing the fertilization success rate. Cryopreserved sperm can benefit from medium containing
204 rHuOVGP1 with increased fertilizing capacity. Inclusion of rHuOVGP1 in the capacitating medium may be particularly useful for treatment of male infertility with mild cases of male factor. Further studies on the mechanism of the recombinant OVGP1 in regulating sperm capacitation is warranted in the future.
205
Recombinant human and hamster OVGP1
Bind to sperm and zona pellucida
↑Tyrosine phosphorylation of sperm proteins
↑Sperm capacitation
↑Acrosome reaction ↑Sperm-egg binding
Figure 7- 1: Schematic diagram of the summary of the role of rHamOVGP1 and rHuOVGP1 in the early events of fertilization.
206
Reference List
207 Aarabi M, Balakier H, Bashar S, Moskovtsev SI, Sutovsky P, Librach CL and Oko R (2014) Sperm-derived WW domain-binding protein, PAWP, elicits calcium oscillations and oocyte activation in humans and mice. The FASEB Journal 28 4434–4440.
Abe H and Oikawa T (1990) Ultrastructural evidence for an association between an oviductal glycoprotein and the zona pellucida of the golden hamster egg. The Journal of Experimental Zoology 256 210–221.
Abe H and Oikawa T (1991) Regional differences in the ultrastructural features of secretory cells in the golden hamster (Mesocricetus auratus) oviductal epithelium. Journal of Anatomy 175 147–158.
Abe H and Oikawa T (1993) Effects of estradiol and progesterone on the cytodifferentiation of epithelial cells in the oviduct of the newborn golden hamster. The Anatomical Record 235 390–398.
Abe H, Sendai Y, Satoh T and Hoshi H (1995) Bovine oviduct-specific glycoprotein: a potent factor for maintenance of viability and motility of bovine spermatozoa in vitro. Molecular Reproduction and Development 42 226–232.
Acuña OS, Avilés M, López-Úbeda R, Guillén-Martínez A, Soriano-Úbeda C, Torrecillas A, Coy P and Izquierdo-Rico MJ (2017) Differential gene expression in porcine oviduct during the oestrous cycle. Reproduction, Fertility and Development 29 2387–2399.
Agarwal A, Yeung WSB and Lee K-F (2002) Cloning and characterization of the human oviduct-specific glycoprotein (HuOGP) gene promoter. Molecular Human Reproduction 8 167–175.
Aitken RJ (2006) Sperm function tests and fertility. International Journal of Andrology 29 69–75.
Aitken RJ and Nixon B (2013) Sperm capacitation: a distant landscape glimpsed but unexplored. Molecular Human Reproduction 19 785–793.
Algarra B, Han L, Soriano-Úbeda C, Avilés M, Coy P, Jovine L and Jiménez-Movilla M (2016) The C-terminal region of OVGP1 remodels the zona pellucida and modifies fertility parameters. Scientific Reports 6 srep32556.
Algarra B, Maillo V, Avilés M, Gutiérrez-Adán A, Rizos D and Jiménez-Movilla M (2018) Effects of recombinant OVGP1 protein on in vitro bovine embryo development. Journal of Reproduction and Development 64 433–443.
Aliabadi E, Jahanshahi S, Talaei-Khozani T and Banaei M (2018) Comparison and evaluation of capacitation and acrosomal reaction in freeze-thawed human ejaculated
208 spermatozoa treated with L-carnitine and pentoxifylline. Andrologia 50 e12845.
Amidi F, Pazhohan A, Shabani Nashtaei M, Khodarahmian M and Nekoonam S (2016) The role of antioxidants in sperm freezing: a review. Cell and Tissue Banking 17 745– 756.
Araki Y, Kurata S, Oikawa T, Yamashita T, Hiroi M, Naiki M and Sendo F (1987) A monoclonal antibody reacting with the zona pellucida of the oviductal egg but not with that of the ovarian egg of the golden hamster. Journal of Reproductive Immunology 11 193–208.
Araki Y, Nohara M, Yoshida-Komiya H, Kuramochi T, Ito M, Hoshi H, Shinkai Y and Sendai Y (2003) Effect of a null mutation of the oviduct-specific glycoprotein gene on mouse fertilization. The Biochemical Journal 374 551–557.
Arias EB, Verhage HG and Jaffe RC (1994) Complementary deoxyribonucleic acid cloning and molecular characterization of an estrogen-dependent human oviductal glycoprotein. Biology of Reproduction 51 685–694.
Arii J, Goto H, Suenaga T, Oyama M, Kozuka-Hata H, Imai T, Minowa A, Akashi H, Arase H, Kawaoka Y et al. (2010) Non-muscle myosin IIA is a functional entry receptor for herpes simplex virus-1. Nature 467 859–862.
Austin CR (1951) Observations on the penetration of the sperm in the mammalian egg. Australian Journal of Scientific Research.Ser.B: Biological Sciences 4 581–596.
Austin CR (1952) The capacitation of the mammalian sperm. Nature 170 326.
Aviles M, Gutierrez-Adan A and Coy P (2010) Oviductal secretions: will they be key factors for the future ARTs? Molecular Human Reproduction 16 896–906.
Baccetti B, Collodel G, Estenoz M, Manca D, Moretti E and Piomboni P (2005) Gene deletions in an infertile man with sperm fibrous sheath dysplasia. Human Reproduction 20 2790–2794.
Bailey JL (2010) Factors regulating sperm capacitation. Systems Biology in Reproductive Medicine 56 334–348.
Baker MA, Smith ND, Hetherington L, Taubman K, Graham ME, Robinson PJ and Aitken RJ (2010a) Label-free quantitation of phosphopeptide changes during rat sperm capacitation. Journal of Proteome Research 9 718–729.
Baker MA, Reeves G, Hetherington L and Aitken RJ (2010b) Analysis of proteomic changes associated with sperm capacitation through the combined use of IPG-strip pre- fractionation followed by RP chromatography LC-MS/MS analysis. Proteomics 10 482– 495. 209 Banihani S, Agarwal A, Sharma R and Bayachou M (2014) Cryoprotective effect of l- carnitine on motility, vitality and DNA oxidation of human spermatozoa. Andrologia 46 637–641.
Barbonetti A, Vassallo MR, Cinque B, Antonangelo C, Sciarretta F, Santucci R, D’Angeli A, Francavilla S and Francavilla F (2008) Dynamics of the global tyrosine phosphorylation during capacitation and acquisition of the ability to fuse with oocytes in human spermatozoa. Biology of Reproduction 79 649–656.
Bauersachs S, Rehfeld S, Ulbrich SE, Mallok S, Prelle K, Wenigerkind H, Einspanier R, Blum H and Wolf E (2004) Monitoring gene expression changes in bovine oviduct epithelial cells during the oestrous cycle. Journal of Molecular Endocrinology 32 449– 466.
Bavister BD (1989) A consistently successful procedure for in vitro fertilization of golden hamster eggs. Gamete Research 23 139–158.
Bleil JD and Wassarman PM (1980) Structure and function of the zona pellucida: identification and characterization of the proteins of the mouse oocyte’s zona pellucida. Developmental Biology 76 185–202.
Boatman DE and Magnoni GE (1995) Identification of a sperm penetration factor in the oviduct of the golden hamster. Biology of Reproduction 52 199–207.
Boatman DE, Felson SE and Kimura J (1994) Changes in morphology, sperm penetration and fertilization of ovulated hamster eggs induced by oviductal exposure. Human Reproduction 9 519–526.
Boice ML, Geisert RD, Blair RM and Verhage HG (1990a) Identification and characterization of bovine oviductal glycoproteins synthesized at estrus. Biology of Reproduction 43 457–465.
Boice ML, McCarthy TJ, Mavrogianis PA, Fazlebas AT and Verhage HG (1990b) Localization of oviductal glycoproteins within the zona pellucida and perivitelline space of ovulated ova and early embryos in baboons (Papio anubis). Biology of Reproduction 43 340–346.
Boitrelle F, Albert M, Theillac C, Ferfouri F, Bergere M, Vialard F, Wainer R, Bailly M and Selva J (2012) Cryopreservation of Human Spermatozoa Decreases the Number of Motile Normal Spermatozoa, Induces Nuclear Vacuolization and Chromatin Decondensation. Journal of Andrology 33 1371–1378.
Briton-Jones C, Lok IH, Yuen PM, Chiu TT, Cheung LP and Haines C (2001) Regulation of human oviductin mRNA expression in vivo. Fertility and Sterility 75 942–946.
210 Briton-Jones C, Lok IH, Cheung CK, Chiu TT, Cheung LP and Haines C (2004) Estradiol regulation of oviductin/oviduct-specific glycoprotein messenger ribonucleic acid expression in human oviduct mucosal cells in vitro. Fertility and Sterility 81 Suppl 1 749–756.
Brown PR, Miki K, Harper DB and Eddy EM (2003a) A-kinase anchoring protein 4 binding proteins in the fibrous sheath of the sperm flagellum. Biology of Reproduction 68 2241–2248.
Brown RL, August SL, Williams CJ and Moss SB (2003b) AKAP7gamma is a nuclear RI- binding AKAP. Biochem Biophys Res Commun 306 394–401.
Buhi WC (2002) Characterization and biological roles of oviduct-specific, oestrogen- dependent glycoprotein. Reproduction 123 355–362.
Buhi WC, Alvarez IM, Sudhipong V and Dones-Smith MM (1990) Identification and characterization of de novo-synthesized porcine oviductal secretory proteins. Biology of Reproduction 43 929–938.
Buhi WC, Bazer FW, Alvarez IM and Mirando MA (1991) In vitro synthesis of oviductal proteins associated with estrus and 17 beta-estradiol-treated ovariectomized ewes. Endocrinology 128 3086–3095.
Buhi WC, O’Brien B, Alvarez IM, Erdos G and Dubois D (1993) Immunogold localization of porcine oviductal secretory proteins within the zona pellucida, perivitelline space, and plasma membrane of oviductal and uterine oocytes and early embryos. Biology of Reproduction 48 1274–1283.
Buhi WC, Alvarez IM, Choi I, Cleaver BD and Simmen FA (1996) Molecular cloning and characterization of an estrogen-dependent porcine oviductal secretory glycoprotein. Biology of Reproduction 55 1305–1314.
Buhi WC, Alvarez IM and Kouba AJ (2000) Secreted proteins of the oviduct. Cells, Tissues, Organs 166 165–179.
Burton KA and McKnight GS (2007) PKA, Germ Cells, and Fertility. Physiology 22 40– 46.
Bushnik T, Cook JL, Yuzpe AA, Tough S and Collins J (2012) Estimating the prevalence of infertility in Canada. Human Reproduction 27 738–746.
Byrd W and Wolf DP (1986) Acrosomal status in fresh and capacitated human ejaculated sperm. Biology of Reproduction 34 859–869.
Calvin HI, Yu CC and Bedford JM (1973) Effects of epididymal maturation, zinc (II) and copper (II) on the reactive sulfhydryl content of structural elements in rat spermatozoa. 211 Experimental Cell Research 81 333–341.
Cao X, Cui Y, Zhang X, Lou J, Zhou J, Bei H and Wei R (2018) Proteomic profile of human spermatozoa in healthy and asthenozoospermia individuals. Reproductive Biology and Endocrinology 16 16.
Capone A, Rosati F and Focarelli R (1999) A 140-kDa glycopeptide from the sperm ligand of the vitelline coat of the freshwater bivalve Unio elongatulus, only contains O-linked oligosaccharide chains and mediates sperm-egg interaction. Molecular Reproduction and Development 54 203–207.
Cappiello MG, Wu Z, Scott BB, McGeehan GM and Harrison RK (2004) Purification and characterization of recombinant human cathepsin E expressed in human kidney cell line 293. Protein Expression and Purification 37 53–60.
Caputo M, Riccio S, Paglierucci P, Tretola L, Diglio C, Carotenuto R, De Marco N and Campanella C (2005) Sulphated glycoconjugates are powerful inhibitors of spermatozoa binding to the vitelline envelope in amphibian eggs. Biology of the Cell 97 435–444.
Carrera A, Gerton GL and Moss SB (1994) The Major Fibrous Sheath Polypeptide of Mouse Sperm: Structural and Functional Similarities to the A-Kinase Anchoring Proteins. Developmental Biology 165 272–284.
Centers for Disease Control, and Prevention AS for R, Medicine S for AR and Technology (2016) 2016 Assisted Reproductive Technology National Summary Report. Atlanta, GA: US Dept of Health and Human Services.
Cerny KL, Garrett E, Walton AJ, Anderson LH and Bridges PJ (2015) A transcriptomal analysis of bovine oviductal epithelial cells collected during the follicular phase versus the luteal phase of the estrous cycle. Reproductive Biology and Endocrinology 13 84.
Chang MC (1951) Fertilizing capacity of spermatozoa deposited into the fallopian tubes. Nature 168 697–698.
Chen S, Einspanier R and Schoen J (2013) In vitro mimicking of estrous cycle stages in porcine oviduct epithelium cells: estradiol and progesterone regulate differentiation, gene expression, and cellular function. Biology of Reproduction 89 54.
Chiu PCN, Wong BST, Lee CL, Pang RTK, Lee K-F, Sumitro SB, Gupta SK and Yeung WSB (2008) Native human zona pellucida glycoproteins: purification and binding properties. Human Reproduction 23 1385–1393.
Chiu PCN, Lam KKW, Wong RCW and Yeung WSB (2014) The identity of zona pellucida receptor on spermatozoa: an unresolved issue in developmental biology.
212 Seminars in Cell & Developmental Biology 30 86–95.
Chow A, Hao Y and Yang X (2010) Molecular characterization of human homologs of yeast MOB1. International Journal of Cancer 126 2079–2089.
Clark GF (2011) Molecular models for mouse sperm-oocyte binding. Glycobiology 21 3–5.
Claw KG and Swanson WJ (2012) Evolution of the Egg: New Findings and Challenges. Annual Review of Genomics and Human Genetics 13 109–125.
Coy P, Canovas S, Mondejar I, Saavedra MD, Romar R, Grullon L, Matas C and Aviles M (2008) Oviduct-specific glycoprotein and heparin modulate sperm-zona pellucida interaction during fertilization and contribute to the control of polyspermy. Proceedings of the National Academy of Sciences of the United States of America 105 15809–15814.
Coy P, Garcia-Vazquez FA, Visconti PE and Aviles M (2012) Roles of the oviduct in mammalian fertilization. Reproduction 144 649–660.
Cross NL and Hanks SE (1991) Effects of cryopreservation on human sperm acrosomes. Human Reproduction 6 1279–1283.
Cross NL, Lambert H and Samuels S (1986) Sperm binding activity of the zona pellucida of immature mouse oocytes. Cell Biology International Reports 10 545–554.
Davis BK, Byrne R and Hungund B (1979) Studies on the mechanism of capacitation. II. Evidence for lipid transfer between plasma membrane of rat sperm and serum albumin during capacitation in vitro. Biochimica et Biophysica Acta 558 257–266.
Davis BK, Byrne R and Bedigian K (1980) Studies on the mechanism of capacitation: albumin-mediated changes in plasma membrane lipids during in vitro incubation of rat sperm cells. Proceedings of the National Academy of Sciences of the United States of America 77 1546–1550.
DeSouza MM and Murray MK (1995) An estrogen-dependent secretory protein, which shares identity with chitinases, is expressed in a temporally and regionally specific manner in the sheep oviduct at the time of fertilization and embryo development. Endocrinology 136 2485–2496.
Desrosiers P, Legare C, Leclerc P and Sullivan R (2006) Membranous and structural damage that occur during cryopreservation of human sperm may be time-related events. Fertility and Sterility 85 1744–1752.
Donnelly KM, Fazleabas AT, Verhage HG, Mavrogianis PA and Jaffe RC (1991) Cloning of a Recombinant Complementary DNA to a Baboon (Papio anubis) Estradiol- Dependent Oviduct-Specific Glycoprotein. Molecular Endocrinology 5 356–364.
213 Donnelly ET, McClure N and Lewis SE (2001) Cryopreservation of human semen and prepared sperm: effects on motility parameters and DNA integrity. Fertility and Sterility 76 892–900.
Eddy EM (2007) The scaffold role of the fibrous sheath. Society of Reproduction and Fertility Supplement 65 45–62.
Eddy CA and Pauerstein CJ (1980) Anatomy and physiology of the fallopian tube. Clin Obstet Gynecol 23 1177–1193.
Eddy EM, Toshimori K and O’Brien DA (2003) Fibrous sheath of mammalian spermatozoa. Microscopy Research and Technique 61 103–115.
European Society of Human Reproduction and Embryology (2018) More than 8 million babies born from IVF since the world’s first in 1978: European IVF pregnancy rates now steady at around 36 percent, according to ESHRE monitoring. In Science Daily.
Fawcett DW (1970) A comparative view of sperm ultrastructure. Biology of Reproduction. Supplement 2 90–127.
Fazleabas AT and Verhage HG (1986) The detection of oviduct-specific proteins in the baboon (Papio anubis). Biology of Reproduction 35 455–462.
Fénichel P, Donzeau M, Farahifar D, Basteris B, Ayraud N and Hsi BL (1991) Dynamics of human sperm acrosome reaction: relation with in vitro fertilization. Fertility and Sterility 55 994–999.
Ficarro S, Chertihin O, Westbrook VA, White F, Jayes F, Kalab P, Marto JA, Shabanowitz J, Herr JC, Hunt DF et al. (2003) Phosphoproteome analysis of capacitated human sperm. Evidence of tyrosine phosphorylation of a kinase-anchoring protein 3 and valosin-containing protein/p97 during capacitation. The Journal of Biological Chemistry 278 11579–11589.
Flesch FM, Colenbrander B, van Golde LM and Gadella BM (1999) Capacitation induces tyrosine phosphorylation of proteins in the boar sperm plasma membrane. Biochemical and Biophysical Research Communications 262 787–792.
Florman HM and Wassarman PM (1985) O-linked oligosaccharides of mouse egg ZP3 account for its sperm receptor activity. Cell 41 313–324.
Fox LL and Shivers CA (1975) Detection and localization of specific antigens in the reproductive tracts of cycling, pregnant, and ovariectomized hamsters. Fertility and Sterility 26 579–598.
Fraser LR (1998) Sperm capacitation and the acrosome reaction. Human Reproduction 13 Suppl 1 9–19. 214 Fulcher KD, Mori C, Welch JE, O’Brien DA, Klapper DG and Eddy EM (1995) Characterization of Fsc1 cDNA for a mouse sperm fibrous sheath component. Biol Reprod 52 41–49.
Galantino-Homer HL, Visconti PE and Kopf GS (1997) Regulation of protein tyrosine phosphorylation during bovine sperm capacitation by a cyclic adenosine 3’5’- monophosphate-dependent pathway. Biology of Reproduction 56 707–719.
Gandini L, Lombardo F, Lenzi A, Spanò M and Dondero F (2006) Cryopreservation and sperm DNA integrity. Cell and Tissue Banking 7 91–98.
Gandolfi F, Brevini TA, Richardson L, Brown CR and Moor RM (1989) Characterization of proteins secreted by sheep oviduct epithelial cells and their function in embryonic development. Development 106 303–312.
Gandolfi F, Modina S, Brevini TA, Galli C, Moor RM and Lauria A (1991) Oviduct ampullary epithelium contributes a glycoprotein to the zona pellucida, perivitelline space and blastomeres membrane of sheep embryos. European Journal of Basic and Applied Histochemistry 35 383–392.
George SHL, Greenaway J, Milea A, Clary V, Shaw S, Sharma M, Virtanen C and Shaw PA (2011) Identification of abrogated pathways in fallopian tube epithelium from BRCA1 mutation carriers. Journal of Pathology 225 106–117.
Gosálvez J, Núñez R, Fernández JL, López-Fernández C and Caballero P (2011) Dynamics of sperm DNA damage in fresh versus frozen-thawed and gradient processed ejaculates in human donors. Andrologia 43 373–377.
Goudet G, Mugnier S, Callebaut I and Monget P (2008) Phylogenetic analysis and identification of pseudogenes reveal a progressive loss of zona pellucida genes during evolution of vertebrates. Biology of Reproduction 78 796–806.
Greve JM and Wassarman PM (1985) Mouse egg extracellular coat is a matrix of interconnected filaments possessing a structural repeat. Journal of Molecular Biology 181 253–264.
Gupta SK and Bhandari B (2011) Acrosome reaction: relevance of zona pellucida glycoproteins. Asian Journal of Andrology 13 97–105.
Gurunath S, Pandian Z, Anderson RA and Bhattacharya S (2011) Defining infertility—a systematic review of prevalence studies. Human Reproduction Update 17 575–588.
Hachen A, Jewgenow K and Braun BC (2012) Sequence analysis of feline oviductin and its expression during the estrous cycle in the domestic cat (Felis catus). Theriogenology 77 539–549.
215 Hammadeh ME, Askari AS, Georg T, Rosenbaum P and Schmidt W (1999) Effect of freeze-thawing procedure on chromatin stability, morphological alteration and membrane integrity of human spermatozoa in fertile and subfertile men. International Journal of Andrology 22 155–162.
Hatef B, Taromchi A, Nejatbakhsh R, Farrokhi A and Shokri S (2017) Supplementation of freezing media with stromal cell-derived factor-1α preserves human sperm from cryodamage. Cryobiology 79 37–42.
Hebel R and Stromberg MW (1986) Anatomy and Embryology of the Laboratory Rat. Wörthsee: BioMed.
Hedrick JL and Wardrip NJ (1987) On the macromolecular composition of the zona pellucida from porcine oocytes. Developmental Biology 121 478–488.
Hendrix E, Hewetson A, Mansharamani M and Chilton BS (2001) Oviductin (Muc9) is expressed in rabbit endocervix. Endocrinology 142 2151.
Hill JL, Walker SK, Brown GH and Nancarrow CD (1996a) The effects of an estrus- associated oviductal glycoprotein on the in vitro fertilization and development of ovine oocytes matured in vitro. Theriogenology 46 1379–1388.
Hill JL, Walker SK, Brown GH and Nancarrow CD (1996b) The effects of an ovine oviductal estrus-associated glycoprotein on early embryo development. Theriogenology 46 1367–1377.
Hill JL, Wade MG, Nancarrow CD, Kelleher DL and Boland MP (1997) Influence of ovine oviducal amino acid concentrations and an ovine oestrus-associated glycoprotein on development and viability of bovine embryos. Molecular Reproduction and Development 47 164–169.
Hillman P, Ickowicz D, Vizel R and Breitbart H (2013) Dissociation between AKAP3 and PKARII promotes AKAP3 degradation in sperm capacitation. PloS One 8 e68873.
Hoffman RA, Robinson PF and Magalhaes H (1968) The Golden Hamster: Its Biology and Use in Medical Research. Iowa State University Press.
Huang Q-S, Xie X-L, Liang G, Gong F, Wang Y, Wei X-Q, Wang Q, Ji Z-L and Chen Q-X (2012) The GH18 family of chitinases: Their domain architectures, functions and evolutions. Glycobiology 22 23–34.
Hunter RH (2003) Reflections upon sperm-endosalpingeal and sperm-zona pellucida interactions in vivo and in vitro. Reproduction in Domestic Animals 38 147–154.
Izquierdo-Rico MJ, Jimenez-Movilla M, Llop E, Perez-Oliva AB, Ballesta J, Gutierrez- Gallego R, Jimenez-Cervantes C and Aviles M (2009) Hamster zona pellucida is 216 formed by four glycoproteins: ZP1, ZP2, ZP3, and ZP4. Journal of Proteome Research 8 926–941.
Jaffe RC, Arias EB, O’Day-Bowman MB, Donnelly KM, Mavrogianis PA, Verhage HG, O’Day-Bowman MB, Donnelly KM, Mavrogianis PA, Verhage HG et al. (1996) Regional distribution and hormonal control of estrogen-dependent oviduct-specific glycoprotein messenger ribonucleic acid in the baboon (Papio anubis). Biology of Reproduction 55 421–426.
Jha KN and Shivaji S (2002) Identification of the major tyrosine phosphorylated protein of capacitated hamster spermatozoa as a homologue of mammalian sperm a kinase anchoring protein. Molecular Reproduction and Development 61 258–270.
Kadam KM, D’Souza SJ, Bandivdekar AH and Natraj U (2006) Identification and characterization of oviductal glycoprotein-binding protein partner on gametes: epitopic similarity to non-muscle myosin IIA, MYH 9. Molecular Human Reproduction 12 275– 282.
Kan FW and Esperanzate PW (2006) Surface mapping of binding of oviductin to the plasma membrane of golden hamster spermatozoa during in vitro capacitation and acrosome reaction. Molecular Reproduction and Development 73 756–766.
Kan FW, St-Jacques S and Bleau G (1989) Immunocytochemical evidence for the transfer of an oviductal antigen to the zona pellucida of hamster ova after ovulation. Biology of Reproduction 40 585–598.
Kan FW, Roux E and Bleau G (1993) Immunolocalization of oviductin in endocytic compartments in the blastomeres of developing embryos in the golden hamster. Biology of Reproduction 48 77–88.
Kapur RP and Johnson L V (1985) An oviductal fluid glycoprotein associated with ovulated mouse ova and early embryos. Developmental Biology 112 89–93.
Kapur RP and Johnson L V (1986) Selective sequestration of an oviductal fluid glycoprotein in the perivitelline space of mouse oocytes and embryos. The Journal of Experimental Zoology 238 249–260.
Kapur RP and Johnson L V (1988) Ultrastructural evidence that specialized regions of the murine oviduct contribute a glycoprotein to the extracellular matrix of mouse oocytes. The Anatomical Record 221 720–729.
Keel BA (2006) Within- and between-subject variation in semen parameters in infertile men and normal semen donors. Fertility and Sterility 85 128–134.
Killian G. (2004) Evidence for the role of oviduct secretions in sperm function, fertilization
217 and embryo development. Animal Reproduction Science 82–83 141–153.
Killian G (2011) Physiology and endocrinology symposium: evidence that oviduct secretions influence sperm function: a retrospective view for livestock. Journal of Animal Science 89 1315–1322.
King RS and Killian GJ (1994) Purification of bovine estrus-associated protein and localization of binding on sperm. Biology of Reproduction 51 34–42.
King RS, Anderson SH and Killian GJ (1994) Effect of bovine oviductal estrus-associated protein on the ability of sperm to capacitate and fertilize oocytes. Journal of Andrology 15 468–478.
Komiya H, Onuma T, Hiroi M and Araki Y (1996) In situ localization of messenger ribonucleic acid for an oviduct-specific glycoprotein during various hormonal conditions in the golden hamster. Biology of Reproduction 55 1107–1118.
Kota V, Dhople VM and Shivaji S (2009) Tyrosine phosphoproteome of hamster spermatozoa: role of glycerol-3-phosphate dehydrogenase 2 in sperm capacitation. Proteomics 9 1809–1826.
Kouba AJ, Abeydeera LR, Alvarez IM, Day BN and Buhi WC (2000) Effects of the porcine oviduct-specific glycoprotein on fertilization, polyspermy, and embryonic development in vitro. Biology of Reproduction 63 242–250.
Krisfalusi M, Miki K, Magyar PL and O’Brien DA (2006) Multiple glycolytic enzymes are tightly bound to the fibrous sheath of mouse spermatozoa. Biology of Reproduction 75 270–278.
Kulanand J and Shivaji S (2001) Capacitation-associated changes in protein tyrosine phosphorylation, hyperactivation and acrosome reaction in hamster spermatozoa. Andrologia 33 95–104.
Kumaresan A, Ansari MR and Garg A (2005) Modulation of post-thaw sperm functions with oviductal proteins in buffaloes. Animal Reproduction Science 90 73–84.
Laheri S, Modi D and Bhatt P (2017) Extra-oviductal expression of oviductal glycoprotein 1 in mouse: Detection in testis, epididymis and ovary. Journal of Biosciences 42 69–80.
Leclerc P, de Lamirande E and Gagnon C (1996) Cyclic adenosine 3’,5’monophosphate- dependent regulation of protein tyrosine phosphorylation in relation to human sperm capacitation and motility. Biology of Reproduction 55 684–692.
Lee YL, Lee KF, Xu JS, Wang YL, Tsao SW and Yeung WS (2001) Establishment and characterization of an immortalized human oviductal cell line. Molecular Reproduction and Development 59 400–409. 218 Leese HJ (1988) The formation and function of oviduct fluid. Journal of Reproduction and Fertility 82 843–856.
Lefevre A, Duquenne C, Rousseau-Merck MF, Rogier E and Finaz C (1999) Cloning and characterization of SOB1, a new testis-specific cDNA encoding a human sperm protein probably involved in oocyte recognition. Biochemical and Biophysical Research Communications 259 60–66.
Lefievre L, Conner SJ, Salpekar A, Olufowobi O, Ashton P, Pavlovic B, Lenton W, Afnan M, Brewis IA, Monk M et al. (2004) Four zona pellucida glycoproteins are expressed in the human. Human Reproduction 19 1580–1586.
Lenzi A, Sgrò P, Salacone P, Paoli D, Gilio B, Lombardo F, Santulli M, Agarwal A and Gandini L (2004) A placebo-controlled double-blind randomized trial of the use of combined L-carnitine and L-acetyl-carnitine treatment in men with asthenozoospermia. Fertility and Sterility 81 1578–1584.
Leveille MC, Roberts KD, Chevalier S, Chapdelaine A and Bleau G (1987) Uptake of an oviductal antigen by the hamster zona pellucida. Biology of Reproduction 36 227–238.
Lewis B and Aitken RJ (2001) A redox-regulated tyrosine phosphorylation cascade in rat spermatozoa. Journal of Andrology 22 611–622.
Li D, Miller M and Chantler PD (1994) Association of a cellular myosin II with anionic phospholipids and the neuronal plasma membrane. Proceedings of the National Academy of Sciences of the United States of America 91 853–857.
Lindemann CB (1996) Functional significance of the outer dense fibers of mammalian sperm examined by computer simulations with the geometric clutch model. Cell Motility and the Cytoskeleton 34 258–270.
Ling L, Lee YL, Lee KF, Tsao SW, Yeung WS and Kan FW (2005) Expression of human oviductin in an immortalized human oviductal cell line. Fertility and Sterility 84 Suppl 2 1095–1103.
Lippes J and Wagh P V. (1989) Human oviductal fluid (hOF) proteins. IV. Evidence for hOF proteins binding to human sperm. Fertility and Sterility 51 89–94.
Lippes J, Krasner J, Alfonso LA, Dacalos ED and Lucero R (1981) Human oviductal fluid proteins. Fertility and Sterility 36 623–629.
Liu DY and Baker HW (1998) Calcium ionophore-induced acrosome reaction correlates with fertilization rates in vitro in patients with teratozoospermic semen. Human Reproduction 13 905–910.
Liu DY and Baker HW (2000) Defective sperm-zona pellucida interaction: a major cause of 219 failure of fertilization in clinical in-vitro fertilization. Human Reproduction 15 702–708.
Liu DY and Baker HWG (2003) Frequency of defective sperm-zona pellucida interaction in severely teratozoospermic infertile men. Human Reproduction 18 802–807.
Liu DY and Baker HWG (2004) High frequency of defective sperm-zona pellucida interaction in oligozoospermic infertile men. Human Reproduction 19 228–233.
Liu T, Gao J, Zhou N, Mo M, Wang X, Zhang X, Yang H, Chen Q, Ao L, Liu J et al. (2016) The effect of two cryopreservation methods on human sperm DNA damage. Cryobiology 72 210–215.
Loignon M, Perret S, Kelly J, Boulais D, Cass B, Bisson L, Afkhamizarreh F and Durocher Y (2008) Stable high volumetric production of glycosylated human recombinant IFNalpha2b in HEK293 cells. BioMed Central Biotechnology 8 65.
Lok IH, Briton-Jones CM, Yuen PM and Haines CJ (2002) Variable expression of oviductin mRNA at different stages of human reproductive cycle. Journal of Assisted Reproduction and Genetics 19 569–576.
Luconi M, Porazzi I, Ferruzzi P, Marchiani S, Forti G and Baldi E (2005) Tyrosine phosphorylation of the a kinase anchoring protein 3 (AKAP3) and soluble adenylate cyclase are involved in the increase of human sperm motility by bicarbonate. Biology of Reproduction 72 22–32.
Luconi M, Cantini G, Baldi E and Forti G (2011) Role of a-kinase anchoring proteins (AKAPs) in reproduction. Frontiers in Bioscience 16 1315–1330.
Lyng R and Shur BD (2009) Mouse oviduct-specific glycoprotein is an egg-associated ZP3- independent sperm-adhesion ligand. Journal of Cell Science 122 3894–3906.
Maines-Bandiera S, Woo MM, Borugian M, Molday LL, Hii T, Gilks B, Leung PC, Molday RS and Auersperg N (2010) Oviductal glycoprotein (OVGP1, MUC9): a differentiation-based mucin present in serum of women with ovarian cancer. International Journal of Gynecological Cancer 20 16–22.
Makkar G, Ng EHY, Yeung WSB and Ho PC (2003) The significance of the ionophore- challenged acrosome reaction in the prediction of successful outcome of controlled ovarian stimulation and intrauterine insemination. Human Reproduction 18 534–539.
Malayer JR, Hansen PJ and Buhi WC (1988) Secretion of proteins by cultured bovine oviducts collected from estrus through early diestrus. Journal of Experimental Zoology 248 345–353.
Malette B and Bleau G (1993) Biochemical characterization of hamster oviductin as a sulphated zona pellucida-binding glycoprotein. Biochemical Journal 295 437–445. 220 Malette B, Filion B, St-Jacques S, Kan FW and Bleau G (1995a) Hormonal control of the biosynthesis of hamster oviductin. Microscopy Research and Technique 31 470–477.
Malette B, Paquette Y, Merlen Y and Bleau G (1995b) Oviductins possess chitinase- and mucin-like domains: a lead in the search for the biological function of these oviduct- specific ZP-associating glycoproteins. Molecular Reproduction and Development 41 384–397.
Manandhar G, Simerly C and Schatten G (2000) Highly degenerated distal centrioles in rhesus and human spermatozoa. Human Reproduction 15 256–263.
Mandal A, Naaby-Hansen S, Wolkowicz MJ, Klotz K, Shetty J, Retief JD, Coonrod SA, Kinter M, Sherman N, Cesar F et al. (1999) FSP95, a testis-specific 95-kilodalton fibrous sheath antigen that undergoes tyrosine phosphorylation in capacitated human spermatozoa. Biology of Reproduction 61 1184–1197.
Marcus-Braun N, Braun G, Potashnik G and Har-vardi I (2004) Effect of cryopreservation on quality and fertilization capacity of human sperm. European Journal of Obstetrics Gynecology and Reproductive Biology 116 63–66.
Mariappa D, Aladakatti RH, Dasari SK, Sreekumar A, Wolkowicz M, Van Der Hoorn F and Seshagiri PB (2010) Inhibition of tyrosine phosphorylation of sperm flagellar proteins, outer dense fiber protein-2 and tektin-2, is associated with impaired motility during capacitation of hamster spermatozoa. Molecular Reproduction and Development 77 182–193.
Martoglio AM and Kan FW (1996) Immunohistochemical localization of oviductin in the endometrial lining of the golden hamster (Mesocricetus auratus) during the estrous cycle and early gestation. The Histochemical Journal 28 449–459.
Martus NS, Verhage HG, Mavrogianis PA and Thibodeaux JK (1998) Enhancement of bovine oocyte fertilization in vitro with a bovine oviductal specific glycoprotein. Journal of Reproduction and Fertility 113 323–329.
Matamoros-Volante A, Moreno-Irusta A, Torres-Rodriguez P, Giojalas L, Gervasi MG, Visconti PE and Treviño CL (2018) Semi-automatized segmentation method using image-based flow cytometry to study sperm physiology: the case of capacitation- induced tyrosine phosphorylation. Molecular Human Reproduction 24 64–73.
McBride DS, Boisvert C, Bleau G and Kan FW (2004a) Evidence for the regulation of glycosylation of golden hamster (Mesocricetus auratus) oviductin during the estrous cycle. Biology of Reproduction 70 198–203.
McBride DS, Boisvert C, Bleau G and Kan FW (2004b) Detection of nascent and/or mature forms of oviductin in the female reproductive tract and post-ovulatory oocytes
221 by use of a polyclonal antibody against recombinant hamster oviductin. The Journal of Histochemistry and Cytochemistry 52 1001–1009.
McBride DS, Brockhausen I and Kan FW (2005) Detection of glycosyltransferases in the golden hamster (Mesocricetus auratus) oviduct and evidence for the regulation of O- glycan biosynthesis during the estrous cycle. Biochimica et Biophysica Acta 1721 107– 115.
McCauley TC, Buhi WC, Wu GM, Mao J, Caamano JN, Didion BA and Day BN (2003) Oviduct-specific glycoprotein modulates sperm-zona binding and improves efficiency of porcine fertilization in vitro. Biology of Reproduction 69 828–834.
Medeiros CMO, Forell F, Oliveira ATD and Rodrigues JL (2002) Current status of sperm cryopreservation: Why isn’t it better? Theriogenology 57 327–344.
Mescher AL (2016) The Female Reproductive System. In Junqueira’s Basic Histology, 14e. New York, NY: McGraw-Hill Education.
Michel JJ and Scott JD (2002) AKAP mediated signal transduction. Annual Review of Pharmacology and Toxicology 42 235–257.
Miki K and Eddy EM (1998) Identification of tethering domains for protein kinase A type Ialpha regulatory subunits on sperm fibrous sheath protein FSC1. The Journal of Biological Chemistry 273 34384–34390.
Miki K, Willis WD, Brown PR, Goulding EH, Fulcher KD and Eddy EM (2002) Targeted disruption of the Akap4 gene causes defects in sperm flagellum and motility. Developmental Biology 248 331–342.
Mondéjar I, Acuña O, Izquierdo-Rico M, Coy P and Avilés M (2012) The Oviduct: Functional Genomic and Proteomic Approach. Reproduction in Domestic Animals 47 22–29.
Moretti E, Baccetti B, Scapigliati G and Collodel G (2006) Transmission electron microscopy, immunocytochemical and fluorescence in situ hybridisation studies in a case of 100% necrozoospermia: case report. Andrologia 38 233–238.
Moretti E, Scapigliati G, Pascarelli NA, Baccetti B and Collodel G (2007) Localization of AKAP4 and tubulin proteins in sperm with reduced motility. Asian Journal of Andrology 9 641–649.
Mugnier S, Kervella M, Douet C, Canepa S, Pascal G, Deleuze S, Duchamp G, Monget P and Goudet G (2009) The secretions of oviduct epithelial cells increase the equine in vitro fertilization rate: are osteopontin, atrial natriuretic peptide A and oviductin involved? Reproductive Biology and Endocrinology 7 129.
222 Murray MK (1993) An estrogen-dependent glycoprotein is synthesized and released from the oviduct in a temporal- and region-specific manner during early pregnancy in the ewe. Biology of Reproduction 48 446–453.
Murray KA (2012) Chapter 27 - Anatomy, Physiology, and Behavior. In The Laboratory Rabbit, Guinea Pig, Hamster, and Other Rodents, 1st ed, pp 753–763. Eds M Suckow, K Stevens and R Wilson. Boston: Academic Press.
Najafi A, Asadi E, Moawad AR, Mikaeili S, Amidi F, Adutwum E, Safa M and Sobhani AG (2016) Supplementation of freezing and thawing media with brain-derived neurotrophic factor protects human sperm from freeze-thaw-induced damage. Fertility and Sterility 106 1658–1665.e4.
Naresh S and Atreja SK (2015) The protein tyrosine phosphorylation during in vitro capacitation and cryopreservation of mammalian spermatozoa. Cryobiology 70 211– 216.
Natraj U, Bhatt P, Vanage G and Moodbidri SB (2002) Overexpression of monkey oviductal protein: purification and characterization of recombinant protein and its antibodies. Biology of Reproduction 67 1897–1906.
Naz RK and Rajesh PB (2004) Role of tyrosine phosphorylation in sperm capacitation / acrosome reaction. Reproductive Biology and Endocrinology 2 75.
Naz RK, Ahmad K and Kumar R (1991) Role of membrane phosphotyrosine proteins in human spermatozoal function. Journal of Cell Science 99 157–165.
Nebl T, Pestonjamasp KN, Leszyk JD, Crowley JL, Oh SW and Luna EJ (2002) Proteomic analysis of a detergent-resistant membrane skeleton from neutrophil plasma membranes. The Journal of Biological Chemistry 277 43399–43409.
Nijs M and Ombelet W (2001) Cryopreservation of human sperm. Human Fertility 4 158– 163.
Nixon B, Bielanowicz A, Anderson AL, Walsh A, Hall T, McCloghry A and Aitken RJ (2010) Elucidation of the signaling pathways that underpin capacitation-associated surface phosphotyrosine expression in mouse spermatozoa. Journal of Cellular Physiology 224 71–83.
Noakes DE (1995) The Physiology of Reproduction, 2nd Edn, E. Knobil, J.D. Neill (Eds.), Raven Press, New York (1994). British Veterinary Journal 151 114.
Noguchi S, Yonezawa N, Katsumata T, Hashizume K, Kuwayama M, Hamano S, Watanabe S and Nakano M (1994) Characterization of the zona pellucida glycoproteins from bovine ovarian and fertilized eggs. Biochimica et Biophysica Acta
223 1201 7–14.
O’Connell M, McClure N and Lewis SEM (2002) The effects of cryopreservation on sperm morphology, motility and mitochondrial function. Human Reproduction 17 704– 709.
O’Day-Bowman MB, Mavrogianis PA, Fazleabas AT and Verhage HG (1995) A human oviduct-specific glycoprotein: synthesis, secretion, and localization during the menstrual cycle. Microscopy Research and Technique 32 57–69.
O’Day-Bowman MB, Mavrogianis P a, Reuter LM, Johnson DE, Fazleabas AT and Verhage HG (1996) Association of oviduct-specific glycoproteins with human and baboon (Papio anubis) ovarian oocytes and enhancement of human sperm binding to human hemizonae following in vitro incubation. Biology of Reproduction 54 60–69.
O’Day-Bowman MB, Mavrogianis PA, Minshall RD and Verhage HG (2002) In vivo versus in vitro oviductal glycoprotein (OGP) association with the zona pellucida (ZP) in the hamster and baboon. Molecular Reproduction and Development 62 248–256.
Oehninger S, Morshedi M and Franken D (2013) The Hemizona Assay for Assessment of Sperm Function. Methods in Molecular Biology 927 91–102.
Oikawa T, Yanagimachi R and Nicolson GL (1973) Wheat germ agglutinin blocks mammalian fertilization. Nature 241 256–259.
Oikawa T, Sendai Y, Kurata S and Yanagimachi R (1988) A glycoprotein of oviductal origin alters biochemical properties of the zona pellucida of hamster egg. Gamete Research 19 113–122.
Oko R (1988) Comparative analysis of proteins from the fibrous sheath and outer dense fibers of rat spermatozoa. Biology of Reproduction 39 169–182.
Oko RJ (1995) Developmental expression and possible role of perinuclear theca proteins in mammalian spermatozoa. Reproduction, Fertility, and Development 7 777–797.
Oko R and Maravei D (1994) Protein composition of the perinuclear theca of bull spermatozoa. Biology of Reproduction 50 1000–1014.
Oko R and Sutovsky P (2009) Biogenesis of sperm perinuclear theca and its role in sperm functional competence and fertilization. Journal of Reproductive Immunology 83 2–7.
Olden K, Willingham M and Pastan I (1976) Cell surface myosin in cultured fibroblasts. Cell 8 383–390.
Oliphant G and Ross PR (1982) Demonstration of production and isolation of three sulfated glycoproteins from the rabbit oviduct. Biology of Reproduction 26 537–544.
224 Oliphant G, Reynolds AB, Smith PF, Ross PR and Marta JS (1984) Immunocytochemical localization and determination of hormone-induced synthesis of the sulfated oviductal glycoproteins. Biology of Reproduction 31 165–174.
Olson GE, Hamilton DW and Fawcett DW (1976) Isolation and characterization of the fibrous sheath of rat epididymal spermatozoa. Biology of Reproduction 14 517–530.
Paquette Y, Merlen Y, Malette B and Bleau G (1995) Allelic polymorphism in the hamster oviductin gene is due to a variable number of mucin-like tandem repeats. Molecular Reproduction and Development 42 388–396.
Patek E, Nilsson L and Johannisson E (1972a) Scanning electron microscopic study of the human fallopian tube. Report I. The proliferative and secretory stages. Fertility and Sterility 23 549–565.
Patek E, Nilsson L and Johannisson E (1972b) Scanning electron microscopic study of the human fallopian tube. Report II. Fetal life, reproductive life, and postmenopause. Fertility and Sterility 23 719–733.
Pérez-Cerezales S, Ramos-Ibeas P, Acuña OS, Avilés M, Coy P, Rizos D and Gutiérrez- Adán A (2018) The oviduct: from sperm selection to the epigenetic landscape of the embryo†. Biology of Reproduction 98 262–276.
Petersen C, Füzesi L and Hoyer-Fender S (1999) Outer dense fibre proteins from human sperm tail: molecular cloning and expression analyses of two cDNA transcripts encoding proteins of approximately 70 kDa. Molecular Human Reproduction 5 627– 635.
Platt MD, Salicioni AM, Hunt DF and Visconti PE (2009) Use of differential isotopic labeling and mass spectrometry to analyze capacitation-associated changes in the phosphorylation status of mouse sperm proteins. Journal of Proteome Research 8 1431– 1440.
Poersch A, Grassi ML, Carvalho VP de, Lanfredi GP, Palma C de S, Greene LJ, de Sousa CB, Carrara HHA, Candido Dos Reis FJ and Faça VM (2016) A proteomic signature of ovarian cancer tumor fluid identified by highthroughput and verified by targeted proteomics. Journal of Proteomics 145 226–236.
Pommer AC, Rutllant J and Meyers SA (2003) Phosphorylation of protein tyrosine residues in fresh and cryopreserved stallion spermatozoa under capacitating conditions. Biology of Reproduction 68 1208–1214.
Pradeep MA, Jagadeesh J, De AK, Kaushik JK, Malakar D, Kumar S, Dang AK, Das SK and Mohanty AK (2011) Purification, sequence characterization and effect of goat oviduct-specific glycoprotein on in vitro embryo development. Theriogenology 75
225 1005–1015.
Robitaille G, St-Jacques S, Potier M and Bleau G (1988) Characterization of an oviductal glycoprotein associated with the ovulated hamster oocyte. Biology of Reproduction 38 687–694.
Rosati F, Capone A, Giovampaola CD, Brettoni C and Focarelli R (2000) Sperm-egg interaction at fertilization: glycans as recognition signals. International Journal of Developmental Biology 44 609–618.
Roux E, Bleau G and Kan FW (1997) Fate of hamster oviductin in the oviduct and uterus during early gestation. Molecular Reproduction and Development 46 306–317.
Saccary L, She YM, Oko R and Kan FW (2013) Hamster oviductin regulates tyrosine phosphorylation of sperm proteins during in vitro capacitation. Biology of Reproduction 89 38.
Saeednia S, Shabani Nashtaei M, Bahadoran H, Aleyasin A and Amidi F (2016) Effect of nerve growth factor on sperm quality in asthenozoosprmic men during cryopreservation. Reproductive Biology and Endocrinology 14 29.
Saint-Dizier M, Marnier C, Tahir MZ, Grimard B, Thoumire S, Chastant-Maillard S and Reynaud K (2014) OVGP1 is expressed in the canine oviduct at the time and place of oocyte maturation and fertilization. Molecular Reproduction and Development 81 972–982.
Sakkas D, Leppens-Luisier G, Lucas H, Chardonnens D, Campana A, Franken DR and Urner F (2003) Localization of Tyrosine Phosphorylated Proteins in Human Sperm and Relation to Capacitation and Zona Pellucida Binding1. Biology of Reproduction 68 1463–1469.
Sanger WG, Olson JH and Sherman JK (1992) Semen cryobanking for men with cancer-- criteria change. Fertility and Sterility 58 1024–1027.
Di Santo M, Tarozzi N, Nadalini M and Borini A (2012) Human Sperm Cryopreservation: Update on Techniques, Effect on DNA Integrity, and Implications for ART. Advances in Urology 2012 e854837.
Sati L, Cayli S, Delpiano E, Sakkas D and Huszar G (2014) The pattern of tyrosine phosphorylation in human sperm in response to binding to zona pellucida or hyaluronic acid. Reprodoctive Sciences 21 573–581.
Satoh T, Abe H, Sendai Y, Iwata H and Hoshi H (1995) Biochemical characterization of a bovine oviduct-specific sialo-glycoprotein that sustains sperm viability in vitro. Biochimica et Biophysica Acta 1266 117–123.
226 Schatten H and Sun Q-Y (2009) The role of centrosomes in mammalian fertilization and its significance for ICSI. Molecular Human Reproduction 15 531–538.
Schmidt A, Mavrogianis PA, O’Day-Bowman MB and Verhage HG (1997) Species- specific effect of oviductal glycoproteins on hamster sperm binding to hamster oocytes. Molecular Reproduction and Development 46 201–207.
Schuffner A, Morshedi M and Oehninger S (2001) Cryopreservation of fractionated, highly motile human spermatozoa: effect on membrane phosphatidylserine externalization and lipid peroxidation. Human Reproduction 16 2148–2153.
Sendai Y, Abe H, Kikuchi M, Satoh T and Hoshi H (1994) Purification and molecular cloning of bovine oviduct-specific glycoprotein. Biology of Reproduction 50 927–934.
Sendai Y, Komiya H, Suzuki K, Onuma T, Kikuchi M, Hoshi H and Araki Y (1995) Molecular cloning and characterization of a mouse oviduct-specific glycoprotein. Biology of Reproduction 53 285–294.
Seppälä M, Koistinen H, Koistinen R, Chiu PCN and Yeung WSB (2007) Glycosylation related actions of glycodelin: gamete, cumulus cell, immune cell and clinical associations. Human Reproduction Update 13 275–287.
Seytanoglu A, Stephen Georgiou A, Sostaric E, Watson PF, Holt W V. and Fazeli A (2008) Oviductal cell proteome alterations during the reproductive cycle in pigs. Journal of Proteome Research 7 2825–2833.
Sharma R, Kattoor AJ, Ghulmiyyah J and Agarwal A (2015) Effect of sperm storage and selection techniques on sperm parameters. Systems Biology in Reproductive Medicine 61 1–12.
Shi S, Williams SA, Seppo A, Kurniawan H, Chen W, Ye Z, Marth JD and Stanley P (2004) Inactivation of the Mgat1 gene in oocytes impairs oogenesis, but embryos lacking complex and hybrid N-glycans develop and implant. Molecular and Cellular Biology 24 9920–9929.
Si Y and Okuno M (1999) Role of tyrosine phosphorylation of flagellar proteins in hamster sperm hyperactivation. Biology of Reproduction 61 240–246.
Signorelli J, Diaz ES and Morales P (2012) Kinases, phosphatases and proteases during sperm capacitation. Cell and Tissue Research 349 765–782.
Simpson JL (2014) Birth defects and assisted reproductive technologies. Seminars in Fetal and Neonatal Medicine 19 177–182.
Siva AB, Yeung C-H, Cooper TG and Shivaji S (2006) Antimicrobial drug ornidazole inhibits hamster sperm capacitation, in vitro. Reproductive Toxicology 22 702–709. 227 Slayden OD, Friason FKE, Bond KR and Mishler EC (2018) Hormonal regulation of oviductal glycoprotein 1 (OVGP1; MUC9) in the rhesus macaque cervix. Journal of Medical Primatology 47 362–370.
Smith TT (1998) The modulation of sperm function by the oviductal epithelium. Biology of Reproduction 58 1102–1104.
Smith TT and Yanagimachi R (1991) Attachment and release of spermatozoa from the caudal isthmus of the hamster oviduct. Journal of Reproduction and Fertility 91 567– 573.
Soleilhavoup C, Riou C, Tsikis G, Labas V, Harichaux G, Kohnke P, Reynaud K, de Graaf SP, Gerard N and Druart X (2016) Proteomes of the Female Genital Tract During the Oestrous Cycle. Molecular & Cellular Proteomics 15 93–108.
St-Jacques S and Bleau G (1988) Monoclonal antibodies specific for an oviductal component associated with the hamster zona pellucida. Journal of Reproductive Immunology 12 247–261.
Suarez SS (1998) The oviductal sperm reservoir in mammals: mechanisms of formation. Biology of Reproduction 58 1105–1107.
Sutovsky P and Manandhar G (2006) Mammalian spermatogenesis and sperm structure: anatomical and compartmental analysis. In The Sperm Cell, pp 1–30. Eds CJ De Jonge and C Barratt. Cambridge: Cambridge University Press.
Sutton R, Nancarrow CD, Wallace AL and Rigby NW (1984) Identification of an oestrus- associated glycoprotein in oviducal fluid of the sheep. Journal of Reproduction and Fertility 72 415–422.
Sutton R, Nancarrow CD and Wallace AL (1986) Oestrogen and seasonal effects on the production of an oestrus-associated glycoprotein in oviducal fluid of sheep. Journal of Reproduction and Fertility 77 645–653.
Suzuki K, Sendai Y, Onuma T, Hoshi H, Hiroi M and Araki Y (1995) Molecular characterization of a hamster oviduct-specific glycoprotein. Biology of Reproduction 53 345–354.
Suzuki H, Saito Y, Kagawa N and Yang X (2003) In vitro fertilization and polyspermy in the pig: factors affecting fertilization rates and cytoskeletal reorganization of the oocyte. Microscopy Research and Technique 61 327–334.
Tasken K and Aandahl EM (2004) Localized effects of cAMP mediated by distinct routes of protein kinase A. Physiological Reviews 84 137–167.
Taylor SS, Ilouz R, Zhang P and Kornev AP (2012) Assembly of allosteric 228 macromolecular switches: lessons from PKA. Nature Reviews. Molecular Cell Biology 13 646–658.
Thoma ME, McLain AC, Louis JF, King RB, Trumble AC, Sundaram R and Buck Louis GM (2013) Prevalence of infertility in the United States as estimated by the current duration approach and a traditional constructed approach. Fertility and Sterility 99 1324–1331.e1.
Tone AA, Begley H, Sharma M, Murphy J, Rosen B, Brown TJ and Shaw PA (2008) Gene expression profiles of luteal phase fallopian tube epithelium from BRCA mutation carriers resemble high-grade serous carcinoma. Clinical Cancer Research 14 4067– 4078.
Toshimori K and Eddy EM (2015) The Spermatozoon. In Knobil and Neill’s Physiology of Reproduction (Fourth Edition), pp 99–148. Eds TM Plant and AJ Zeleznik. San Diego: Academic Press.
Tulsiani DR, Abou-Haila A, Loeser CR and Pereira BM (1998) The biological and functional significance of the sperm acrosome and acrosomal enzymes in mammalian fertilization. Experimental Cell Research 240 151–164.
Urner F and Sakkas D (2003) Protein phosphorylation in mammalian spermatozoa. Reproduction 125 17–26.
Vadnais ML, Lin AM and Gerton GL (2014) Mitochondrial fusion protein MFN2 interacts with the mitostatin-related protein MNS1 required for mouse sperm flagellar structure and function. Cilia 3 5.
Verhage HG and Fazleabas AT (1988) The in vitro synthesis of estrogen-dependent proteins by the baboon (Papio anubis) oviduct. Endocrinology 123 552–558.
Verhage HG, Fazleabas AT and Donnelly K (1988) The in vitro synthesis and release of proteins by the human oviduct. Endocrinology 122 1639–1645.
Verhage HG, Boice ML, Mavrogianis P, Donnelly K and Fazleabas AT (1989) Immunological characterization and immunocytochemical localization of oviduct- specific glycoproteins in the baboon (Papio anubis). Endocrinology 124 2464–2472.
Verhage HG, Mavrogianis PA, Boice ML, Li W and Fazleabas AT (1990) Oviductal epithelium of the baboon: hormonal control and the immuno-gold localization of oviduct-specific glycoproteins. American Journal of Anatomy 187 81–90.
Verhage HG, Mavrogianis PA, Boomsma RA, Schmidt A, Brenner RM, Slayden O V and Jaffe RC (1997a) Immunologic and molecular characterization of an estrogen- dependent glycoprotein in the rhesus (Macaca mulatta) oviduct. Biology of Reproduction
229 57 525–531.
Verhage HG, Fazleabas AT, Mavrogianis PA, O’Day-Bowman MB, Schmidt A, Arias EB and Jaffe RC (1997b) Characteristics of an oviductal glycoprotein and its potential role in fertility control. Journal of Reproduction and Fertility.Supplement 51 217–226.
Verhage HG, Mavrogianis PA, O’Day-Bowman MB, Schmidt A, Arias EB, Donnelly KM, Boomsma RA, Thibodeaux JK, Fazleabas AT and Jaffe RC (1998) Characteristics of an oviductal glycoprotein and its potential role in the fertilization process. Biology of Reproduction 58 1098–1101.
Verheyen G, Pletincx I and Van Steirteghem A (1993) Andrology: Effect of freezing method, thawing temperature and post-thaw dilution/washing on motility (CASA) and morphology characteristics of high-quality human sperm. Human Reproduction 8 1678– 1684.
Vijayaraghavan S, Liberty GA, Mohan J, Winfrey VP, Olson GE and Carr DW (1999) Isolation and molecular characterization of AKAP110, a novel, sperm-specific protein kinase A-anchoring protein. Molecular Endocrinology 13 705–717.
Visconti PE, Moore GD, Bailey JL, Leclerc P, Connors SA, Pan D, Olds-Clarke P and Kopf GS (1995a) Capacitation of mouse spermatozoa. II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway. Development 121 1139–1150.
Visconti PE, Bailey JL, Moore GD, Pan D, Olds-Clarke P and Kopf GS (1995b) Capacitation of mouse spermatozoa. I. Correlation between the capacitation state and protein tyrosine phosphorylation. Development 121 1129–1137.
Visconti PE, Stewart-Savage J, Blasco A, Battaglia L, Miranda P, Kopf GS, Tezón JG and Tezon JG (1999) Roles of bicarbonate, cAMP, and protein tyrosine phosphorylation on capacitation and the spontaneous acrosome reaction of hamster sperm. Biology of Reproduction 61 76–84.
Vizel R, Hillman P, Ickowicz D, Breitbart H and Biochim Biophys A (2015) AKAP3 degradation in sperm capacitation is regulated by its tyrosine phosphorylation. Biochimica et Biophysica Acta 1850 1912–1920.
Wagner A, Ekhlasi-Hundrieser M, Hettel C, Petrunkina A, Waberski D, Nimtz M and Töpfer-Petersen E (2002) Carbohydrate-based interactions of oviductal sperm reservoir formation-studies in the pig. Molecular Reproduction and Development 61 249–257.
Walsh DA, Perkins JP and Krebs EG (1968) An adenosine 3’,5’-monophosphate- dependant protein kinase from rabbit skeletal muscle. The Journal of Biological Chemistry 243 3763–3765.
230 Wang Y, Sharma RK and Agarwal A (1997) Effect of cryopreservation and sperm concentration on lipid peroxidation in human semen. Urology 50 409–413.
Wassarman P, Chen J, Cohen N, Litscher E, Liu C, Qi H and Williams Z (1999) Structure and function of the mammalian egg zona pellucida. Journal of Experimental Zoology 285 251–258.
Wassarman PM, Jovine L and Litscher ES (2001) A profile of fertilization in mammals. Nature Cell Biology 3 E59–E64.
Wegner CC and Killian GJ (1991) In vitro and in vivo association of an oviduct estrus- associated protein with bovine zona pellucida. Molecular Reproduction and Development 29 77–84. van der Westerlaken L, Helmerhorst F, Dieben S and Naaktgeboren N (2005) Intracytoplasmic sperm injection as a treatment for unexplained total fertilization failure or low fertilization after conventional in vitro fertilization. Fertility and Sterility 83 612– 617.
WHO (2010) Examination and processing of human semen. World Health Edition, V 286.
Williams SA, Xia L, Cummings RD, McEver RP and Stanley P (2007) Fertilization in mouse does not require terminal galactose or N-acetylglucosamine on the zona pellucida glycans. Journal of Cell Science 120 1341–1349.
Woo MMM, Gilks CB, Verhage HG, Longacre TA, Leung PCK and Auersperg N (2004) Oviductal glycoprotein, a new differentiation-based indicator present in early ovarian epithelial neoplasia and cortical inclusion cysts. Gynecologic Oncology 93 315– 319.
Xiong D, Du Y, Wang H-B, Zhao B, Zhang H, Li Y, Hu L-J, Cao J-Y, Zhong Q, Liu W- L et al. (2015) Nonmuscle myosin heavy chain IIA mediates Epstein–Barr virus infection of nasopharyngeal epithelial cells. Proceedings of the National Academy of Sciences 112 11036–11041.
Xu K and Qi H (2014) Sperm-specific AKAP3 is a dual-specificity anchoring protein that interacts with both protein kinase a regulatory subunits via conserved N-terminal amphipathic peptides. Molecular Reproduction and Development 81 595–607.
Xu Y, Xie J, Chen R, Cao Y, Ping Y, Xu Q, Hu W, Wu D, Gu L, Zhou H et al. (2016) Fluorescence- and magnetic-activated cell sorting strategies to separate spermatozoa involving plural contributors from biological mixtures for human identification. Scientific Reports 6 36515.
Yanagimachi R (1994) Mammalian fertilization. In The Physiology of Reproduction, pp
231 189–317. Eds E Knobil and JD Neil. New York: Raven Press.
Yang X, Tao S, Orlando R, Brockhausen I and Kan FWK (2012) Structures and biosynthesis of the N- and O-glycans of recombinant human oviduct-specific glycoprotein expressed in human embryonic kidney cells. Carbohydrate Research 358 47–55.
Yang X, Zhao Y, Yang X and Kan FW (2015) Recombinant hamster oviductin is biologically active and exerts positive effects on sperm functions and sperm-oocyte binding. PloS One 10 e0123003.
Yogev L, Gamzu R, Paz G, Kleiman S, Botchan A, Hauser R and Yavetz H (1999) Pre- freezing sperm preparation does not impair thawed spermatozoa binding to the zona pellucida. Human Reproduction 14 114–117.
Yogev L, Kleiman SE, Shabtai E, Botchan A, Paz G, Hauser R, Lehavi O, Yavetz H and Gamzu R (2010) Long-term cryostorage of sperm in a human sperm bank does not damage progressive motility concentration. Human Reproduction 25 1097–1103.
Yong P, Gu Z, Luo JP, Wang JR and Tso JK (2002) Antibodies against the C-terminal peptide of rabbit oviductin inhibit mouse early embryo development to pass 2-cell stage. Cell Research 12 69–78.
Yovich JM, Edirisinghe WR and Yovich JL (1994) Use of the acrosome reaction to ionophore challenge test in managing patients in an assisted reproduction program: a prospective, double-blind, randomized controlled study. Fertility and Sterility 61 902– 910.
Zhao Y, Yang X, Jia Z, Reid RL, Leclerc P and Kan FW (2016) Recombinant human oviductin regulates protein tyrosine phosphorylation and acrosome reaction. Reproduction 152 561–573.
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Appendix
Supplementary Figures and Ethics Approval
233
Appendix Figure- 1. Dose-dependent effect of rHuOVGP1 on protein tyrosine phosphorylation. Dose-dependent effect of rHuOVGP1 on protein tyrosine phosphorylation after 4 hours of incubation in capacitating medium and statistical analysis of the levels of tyrosine phosphorylation of 105 kDa and 81 kDa proteins obtained with Western blotting. Samples incubated in the presence of various concentrations of rHuOVGP1 were compared with samples incubated in the absence of rHuOVGP1 (n = 6 samples each from individual donors for each time point, *p < 0.05, **p < 0.01, *** p < 0.001).
234
Speci fic Aim 1: Recombinant Hamster OVGP1
(rHam OVGP1) Purified from HEK293 Cells
HamOVGP1 cDNA lentivirus Colonies stably express rHamOVGP1 gene
G418R GFP
HEK293 cells Large Scale Cell Culture
Western Blot
Mass Spec
Confirmation of Protein Identity Centrifugation with MW cutoff
1 HPA affinity purification
Appendix Figure- 2. Schematic diagram showing the production and purification of rHamOVGP1. Briefly, the plasmid containing the cDNA of hamster OVGP1 was constructed into lentivirus. The lentivirus was then used to transduce HEK293 cells. The clones that stably over-expressed rHamOVGP1 plasmid were selected and cultured in a large scale. The cell culture medium that contained secreted rHamOVGP1 was concentrated and buffer-exchanged by centrifugation. The rHamOVGP1 was then purified by lectin affinity purification. Western blotting and mass-spectrometry were performed to confirm the identity of purified rHamOVGP1.
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Specific Aim 1: Recombinant Human OVGP1 (rHuOVGP1)
Purified from HEK293 Cells
HuOVGP1 cDNA Colonies stably express rHuOVGP1 gene
G418R GFP
HEK293 cells Large Scale Cell Culture
Western Blot Ammonium Sulfate Mass Spec Precipitation
Confirmation of Protein Identity
Size Exclusion Chromatography
2
Appendix Figure- 3. Schematic diagram showing the production and purification of rHuOVGP1. Briefly, the cDNA of human OVGP1 was cloned and inserted into a plasmid and subsequently transfected into HEK293 cells. The clones that stably over-expressed rHuOVGP1 plasmid were selected and cultured in a large scale. The cell culture medium that contained secreted rHuOVGP1 protein was collected and subjected to a two-step purification involving ammonium sulphate precipitation and size exclusion chromatography. Western blotting and mass-spectrometry confirmed the identity of purified rHuOVGP1.
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QUEEN'S UNIVERSITY HEALTH SCIENCES & AFFILIATED TEACHING HOSPITALS RESEARCH ETHICS BOARD (HSREB)
HSREB Renewal of Ethics Clearance
March 05, 2019
Dr. Frederick W. K. Kan Department of Biomedical and Molecular Sciences Queen's University
TRAQ #: 6012029 Department Code: DBMS-030-14 Study Title: "The Role of Recombinant Human Oviductal Glycoprotein (rHuOVGP1) in Human Sperm Function" Review Type: Delegated Date Ethics Clearance Effective: March 05, 2019 Ethics Clearance Expiry Date: March 05, 2020
Dear Dr. Kan:
The Queen's University Health Sciences & Affiliated Teaching Hospitals Research Ethics Board (HSREB) has reviewed the application. This study, including all currently approved documentation has been granted ethical clearance until the expiry date noted above.
Prior to the expiration of your ethics clearance, you will be reminded to submit your renewal report through TRAQ. Any lapses in ethical clearance will be documented below.
Lapses in Ethics Clearance: N/A
Yours sincerely,
Albert F. Clark, PhD Chair, Queen's University Health Sciences and Affiliated Teaching Hospitals Ethics Board
The HSREB operates in compliance with, and is constituted in accordance with, he requirements of the Tri-Council Policy Statement: Ethical Conduct for Research Involving Humans (TCPS 2); the international Conference on Harmonisation Good Clinical Practice Consolidated Guideline (ICH GCP); Part C, Division 5 of the Food and Drug Regulations; Part 4 of the Natural Health Product Regulations; Part 3 of the Medical Devices Regulations, and the provisions of the Ontario Personal Health Information Protection Act (PHIPA 2004) and its applicable regulations. The HSREB is qualified through the CTO REB Qualification Program and is registered with the U.S. Department of Health and Human Services (DHHS) Office for Human Research Protection (OHRP). Federalwide Assurance Number: FWA#: 00004184, IRB#: 00001173
HSREB members involved in the research project do not participate in the review, discussion or decision.
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