INVESTIGATION OF METHODS AND FACTORS
INFLUENCING MOUSE OOCYTE QUALITY, IN VITRO
FERTILIZATION AND EMBRYO DEVELOPMENT
______
A Dissertation presented to the Faculty of the Graduate School University of Missouri-Columbia ______
In partial fulfillment
of the requirements for the Degree
Doctor of Philosophy
______
By LIGA WURI
Dr. Yuksel Agca, Dissertation Supervisor
DECEMBER 2018
The undersigned, appointed by the dean of the Graduate School, have examined the
dissertation entitled
INVESTIGATION OF METHODS AND FACTORS INFLUENCING MOUSE OOCYTE QUALITY, IN VITRO FERTILIZATION AND EMBRYO DEVELOPMENT
Presented by LIGA WURI,
A candidate for the degree of doctor of philosophy
And hereby certify that, in their opinion, it is worthy of acceptance.
Professor Yuksel Agca
Professor Tumen Wuliji
Professor William R. Lamberson
Professor Heide Schatten
ACKNOWLEDGEMENTS
I am so grateful for everyone who helped me to complete my PhD program in
Animal Science here at Mizzou. Specifically, I would like to thank my committee members, Dr. Agca, Dr. Wuliji, Dr. Lamberson, and Dr. Schatten, who helped me tremendously to reach this point. Their knowledge, guidance, patience, and encouragement have been integral in helping me complete my degree. I am so thankful for all of their support and help. I could not have made it this far without them.
I would also like to thank Lincoln University of Missouri provided me the graduate student assistantship through its USDA capacity grant project (Accession Number
1008620 Feasibility study: An out-of-season breeding system for organic fall lamb production) and SARE grant project (LNC14-363: Detection and Prevention of Footrot
Outbreak in Sheep and Goats) for most my study, and the Division of Animal Sciences at
Mizzou provided scholarships for the last couple of months so that I’ve been able to live and complete my research.
In addition, I would like to thank the staff at Cytology Core at the University of
Missouri-Columbia for their assistance helping me to learn confocal imaging, so that I was able to collect my images and data.
Finally, I am very grateful to the entire faculty and staff at the Division of Animal
Sciences and Comparative Medicine Program at Mizzou, along with Lincoln University of Missouri for their encouragement, guidance, assistance, and help. Their names are numerous and their helps’ invaluable. Thank you all!!!
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TABLE OF CONTENTS
ACKNOWLEDGEMENTS ...... ii
LIST OF TABLES ...... vi
LIST OF FIGURES ...... vii
ABBREVIATIONS ...... xii
ABSTRACT ...... xv
Chapter One
Literature Review...... 1
Introduction ...... 1
Subcellular and molecular changes associated with oocyte development ...... 4
Cortical Granules ...... 8
Mitochondria ...... 13
Meiotic Spindles ...... 15
F-actin ...... 16
Euthanasia methods ...... 18
Superovulation methods ...... 19
Oocyte cryopreservation ...... 20
Chapter two
Euthanasia via CO2 inhalation Causes Premature Cortical Granule Exocytosis and
Influences In-vitro Fertilization and Embryo Development in MII Mouse Oocytes ...... 25
iii
Abstract ...... 25
Introduction ...... 28
Materials and Methods...... 31
Results ...... 36
Discussion ...... 39
Chapter Three
Morphometric, Subcellular, In-Vitro Fertilization and Embryonic Developmental
Assessment of MII Mouse Oocytes Following Superovulation with Anti-Inhibin Serum or Pregnant Mare Serum Gonadotropin ...... 56
Abstract ...... 56
Introduction ...... 58
Materials and Methods...... 62
Results ...... 66
Discussion ...... 69
Chapter Four
Cryo-survival and Subcellular Structural Characterization of MII Mouse Cumulus Oocyte
Complexes Following Vitrification via Cryoloop Method ...... 91
Abstract ...... 91
Introduction ...... 92
Materials and Methods...... 97
iv
Results ...... 101
Discussion ...... 103
LITERATURE CITED ...... 122
VITA ...... 150
v
LIST OF TABLES
Chapter Two
Table 2. 1. In-vitro fertilization and pre-implantation embryonic development of MII mouse oocytes derived from CD and L CO2 euthanasia methods...... 55
Chapter Four
Table 4. 1. Post-thaw morphologic integrity of mouse oocytes following vitrification with
Cryoloop or 0.25 mL French straws (n=8)...... 109
Table 4. 2. In-vitro fertilization and pre-implantation embryonic development competence of fresh and vitrified mouse COCs to blastocyst stage...... 109
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LIST OF FIGURES
Chapter One
Figure 1. 1. Generalized Model of The Mechanism of Intracellular Calcium Release
Leading To Cortical Granule Exocytosis...... 11
Chapter Two
Figure 2. 1. Two-cell (A), morula (B) and blastocyst (C) development of oocytes collected from CD, L CO2 and H CO2 euthanized donors following in-vitro fertilization with frozen-thawed sperm and subsequent in-vitro embryo culture...... 45
Figure 2. 2. Two-cell (A), morula (B) and blastocyst (C) development of MII mouse oocytes collected from CD, L CO2 and H CO2 euthanized donors following in-vitro fertilization with fresh sperm and subsequent in-vitro embryo culture...... 46
Figure 2. 3. Representative bright field images of two-cell embryos from CD (A), L CO2
(B) and H CO2 (C)...... 47
Figure 2. 4. Plasma anion gap measurements of MII mouse oocyte donors euthanized by using CD, L CO2 or H CO2...... 48
Figure 2. 5. Representative confocal images of the localization of the cortical granules
(green) by Lectin conjugated with FITC, F-actin (red) was detected by phalloidin-TRITC and DNA (blue) was stained by DAPI in the oocytes. Confocal images of DNA (A, E, I),
Cortical granules (B, F, J), F-actin (C, G, K) and merged (D, H, L) representative oocytes.
(A-D) oocytes from cervical dislocation (n=16), (E-H) oocytes from H CO2 (n=10), (I-L) oocytes from L CO2 (n=10)...... 49
Figure 2. 6. Cortical granules fluorescence intensity measurement of MII mouse oocytes derived from CD, L CO2 and H CO2 euthanized donors...... 50
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Figure 2. 7. F-actin fluorescence intensity of MII mouse oocytes derived from CD, L CO2 and H CO2 euthanized donors...... 51
Figure 2. 8. Representative confocal images of the localization of the cortical granules
(green) by using lectin conjugated with FITC, F-actin (red) were detected by phalloidin-
TRITC. Confocal images of cortical granules (A, D), F-actin (B, E) and merged (C, F) representative oocytes. (A-C) oocytes from cervical dislocation (n=16), (D-F) oocytes from 5 minutes after cervical dislocation (n=36)...... 52
Figure 2. 9. F-actin fluorescence intensity of MII mouse oocytes collected from donors that were euthanized by using CD and recovered 5 minutes after CD...... 53
Figure 2. 10. Cortical granules fluorescence intensity of MII mouse oocytes collected from donors that were euthanized by using CD and recovered 5 minutes after CD...... 54
Chapter Three
Figure 3. 1. Average number of MII mouse oocytes obtained from each donor female after either AIS or PMSG superovulation methods...... 75
Figure 3. 2. The average number of morphologically normal MII mouse oocytes from each donor female after either AIS or PMSG superovulation methods (A). The average percent of morphologically normal MII mouse oocytes from each donor female after either AIS or PMSG superovulation methods (B)...... 76
Figure 3. 3. A representative image of morphologically abnormal oocytes (A). The average number of morphologically abnormal MII mouse oocytes from each donor female after AIS or PMSG superovulation methods (B). The average percent of morphologically abnormal oocytes from each female donor after AIS or PMSG superovulation (C)...... 77
viii
Figure 3. 4. The average thickness of the ZP of MII mouse oocytes derived from either
AIS or PMSG superovulation methods...... 78
Figure 3. 5. The average PVS of the MII mouse oocytes derived from AIS and PMSG superovulation methods...... 79
Figure 3. 6. The average diameter of MII mouse oocytes derived from AIS or PMSG superovulation methods...... 80
Figure 3. 7. The representative confocal images of oocyte microtubules from AIS and
PMSG groups. The microtubules from all of the MII oocytes were maintained in normal shape. PMSG microtubules (A) microtubules (green) with DNA (blue), AIS (B) microtubules (green) with DNA (blue)...... 81
Figure 3. 8. The microtubules fluorescence intensity measurement of MII oocyte derived from AIS or PMSG superovulation methods...... 82
Figure 3. 9. Representative confocal images of F-actins of the MII mouse oocytes derived from AIS (A) or PMSG (B) superovulation methods...... 83
Figure 3. 10. The F-actin fluorescence intensity measurement of MII mouse oocytes derived from AIS or PMSG superovulation methods...... 84
Figure 3. 11. Representative confocal image of cortical granules of MII mouse oocytes derived from either PMSG (A) or AIS (B) superovulation methods...... 85
Figure 3. 12. Cortical granules fluorescence intensity measurement of MII mouse oocytes derived from AIS or PMSG superovulation methods...... 86
Figure 3. 13. Representative images of mitochondria distribution PMSG (A) and AIS (B).
Both groups also had a normal mitochondria distribution with more homogenous
ix distribution in the PMSG and were more peripherally distributed in the AIS superovulation method...... 87
Figure 3. 14. Mitochondria fluorescence intensity measurement of MII mouse oocytes derived from AIS or PMSG superovulation methods...... 88
Figure 3. 15. Two-cell (A), morula (B) and blastocyst (C) development of AIS or PMSG derived oocytes following in-vitro fertilization with fresh sperm and subsequent in-vitro embryo culture...... 89
Figure 3. 16. Two-cell (A), morula (B) and blastocyst (C) development of AIS or PMSG derived oocytes following in-vitro fertilization with frozen-thawed sperm and subsequent in-vitro embryo culture...... 90
Chapter Four
Figure 4. 1. Representative confocal images of Metaphase-II spindle (green fluorescence) of fresh (A) and vitrified-warmed (B) mouse MII oocytes...... 110
Figure 4. 2. Percentages of normal microtubule spindle fiber configuration of fresh and vitrified-warmed MII mouse oocytes...... 111
Figure 4. 3. Microtubules fluorescence intensity measurement of fresh and vitrified- warmed MII mouse oocytes...... 112
Figure 4. 4. Representative normal confocal images of fresh (A) and vitrified-warmed (B)
MII oocytes as well as a representative abnormal (C) F-actin distribution...... 113
Figure 4. 5. Percentages of normal F-actin distribution for fresh and vitrified-warmed MII mouse oocytes...... 114
Figure 4. 6. F-actins fluorescence intensity of fresh and vitrified-warmed MII oocytes.
...... 115
x
Figure 4. 7. Representative normal confocal images of cortical granule vesicle from fresh
(A) and vitrified-warmed (B) oocytes and a representative image of an abnormal (C) distribution of cortical granule vesicles...... 116
Figure 4. 8. Percentage of normal cortical granule vesicle distribution of fresh and vitrified-warmed MII mouse oocytes...... 117
Figure 4. 9. Cortical granule vesicle fluorescence intensity of fresh and vitrified-warmed
MII mouse oocytes...... 118
Figure 4. 10. Representative confocal images of mitochondria of fresh (A) and vitrified- warmed (B) MII mouse oocytes...... 119
Figure 4. 11. Percentage of normal mitochondria distribution of fresh and vitrified- warmed MII mouse oocytes...... 120
Figure 4. 12. Mitochondrial fluorescence intensity measurement of fresh and vitrified- warmed MII mouse oocytes...... 121
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ABBREVIATIONS
ARTs Assisted Reproductive Technologies
ICSI Intracytoplasmic Sperm Injection
SCNT Somatic Cell Nuclear Transfer
FSH Follicle Stimulating Hormone
PMSG Pregnant Mare Serum Gonadotropin
AIS Anti-inhibin Serum
PCGE Premature Cortical Granule Exocytosis
PGCs Primordial Germ Cells
GV Germinal Vesicle
GVBD Germinal Vesicle Breakdown
CGs Cortical Granules
CO2 Carbon Dioxide hCG Human Chorionic Gonadotropin
IVF In Vitro Fertilization
ZP Zona Pellucida
PVS Perivitelline Space
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CD Cervical Dislocation
LH Luteinizing Hormone
L CO2 Low Flow Carbon Dioxide
H CO2 High Flow Carbon Dioxide cAMP Cyclic Adenosine Mono-Phosphate
AC Adenylate Cyclase
CGFD Cortical Granule Free Domain
COCs Cumulus Oocyte Complexes
LN2 Liquid Nitrogen
PGCs Primordial Germ Cells
PB Polar Body
MPF Maturation Promoting Factor
CSF Cytostatic Factor
GTPases Guanosine Triphosphatase
Cdc42 Cell Division Cycle 42 siRNA small interference RNA
MI Metaphase I
xiii
MII Metaphase II
DAG Diacyl Glycerol
ER Endoplasmic Reticulum
PIP2 Phosphatidylinositol 4, 5-Bisphosphate
DMSO Dimethyl Sulfoxide
PROH Propane Diol
EG Ethylene Glycol
PLC Phospholipase C
IP3 Inositol 1,4,5-Triphosphate
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INVESTIGATION OF METHODS AND FACTORS INFLUENCING MOUSE OOCYTE QUALITY, IN VITRO FERTILIZATION AND EMBRYO DEVELOPMENT
Liga Wuri
Dr. Yuksel Agca, dissertation advisor
ABSTRACT
Mice are one of the most commonly used rodent species as a model for biomedical research to better understand molecular, genetic, and cellular causes of human disease and disorders. The production of good quality oocytes is one of the important determinant factors for successful assisted reproductive technologies (ARTs) clinical outcome as well as reproductive biology research. Mouse oocyte quality, morphology and functions are influenced by a variety of the factors such as euthanasia methods of female donors, superovulation regimes and cryopreservation. The objectives of these studies were mainly to investigate the methods and factors that are influencing oocyte quality, in-vitro fertilization (IVF) and embryonic development.
First, how different euthanasia methods including cervical dislocation (CD), high flow rate CO2 (H CO2) and low flow CO2 (L CO2) would affect the quality and integrity of the metaphase II (MII) oocytes have been investigated. Cumulus oocyte complexes (COCs) were collected from female donors that were euthanized by three different methods and then the oocytes’ subcellular structures including microtubules, F-actin, cortical granules
(CGs) and mitochondria integrities were detected by specific fluorescence dyes. The
xv results showed that L CO2 caused significant increase in the incidence of premature cortical granule exocytosis (PCGE) which might be responsible for significantly reducing the in-vitro fertilization (IVF) and embryonic development rate compared to CD and H
CO2. Secondly, how the superovulation methods would affect the resulting oocyte morphology, quality, IVF competence and embryonic development was investigated. The anti-inhibin serum (AIS) superovulation method produced a significantly higher number of oocytes compared to the pregnant mare serum gonadotrophin (PMSG). Overall, both methods yielded oocytes with similar sizes and comparable subcellular structures including microtubules, F-actin, cortical granules and mitochondria. However, superovulation with AIS produced significantly thinner zona pellucide than PMSG and the perivitelline space of the oocytes generated from AIS were significantly larger than
PMSG. There were no differences in terms of two-cell embryo development, or morula and blastocyst formation rates between AIS and PMSG when the oocytes from two methods were in-vitro fertilized with fresh sperm. Morula and blastocyst development rates were significantly higher for AIS compared to PMSG when oocytes were fertilized with frozen-thawed sperm. Thirdly, clutches of mouse cumulus oocytes complexes
(COCs) were cryopreserved by the cryoloop vitrification method after PMSG superovulation. The cryo-survival rate and integrity and distribution of subcellular structures including the meiotic spindles, F-actin, cortical granules and mitochondria were examined and compared with fresh MII oocytes. The vitrified-warmed oocytes maintained their subcellular structures to a high degree and resulted in acceptable IVF and embryonic development.
xvi
In conclusion, for optimal research and clinical outcome, considerations should be given regard to euthanasia methods of oocyte donor mice and type of superovulation regimes.
Despite of its high oocyte yield, superovulation of mice with AIS provides comparable quality oocytes to the PMSG method. Cryopreservation of the clutches of mouse COCs via cryo-loop vitrification should be considered for genome banking of genetically modified mice and biomedical research.
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Chapter One
Literature Review
Introduction
Mice are the most often used laboratory rodents for biomedical research and germline genetic manipulation in order to model human diseases and disorders (1-3). Generation of good quality and sufficient numbers of mouse oocytes would minimize the number of donor animals needed for genetic manipulation and genome banking as well as rare species propagation and preservation. It is also important for biomedical research purposes in terms of a wide array of assisted reproductive technologies (ARTs) (4) including in-vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), transgenic animal production, germplasm cryo-banking and somatic cell nuclear transfer (SCNT).
However, mouse oocyte quality, morphology and subcellular structures might be affected by the euthanasia methods, superovulation methods and cryopreservation procedure.
There has been some evidence suggesting that euthanasia via CO2 inhalation causes reduced fertilization rates, change in blood pH, respiration rate and heart rate, and blood pressure (5) and hypothermia (6). It has also been reported that CO2 acidification under in-vitro conditions may cause permeability of the zona pellucida, interfering with proper fertilization due to premature cortical granule exocytosis (PCGE) and subsequent in-vitro embryo development competence as well as fetal development following embryo transfer
1
(7). Therefore, in this research, we performed a series of experiments to determine how euthanasia methods influence the oocyte subcellular structures subsequently causing decreased IVF and embryonic development.
Follicle-stimulating hormone (FSH) is a glycoprotein polypeptide hormone that is synthesized and produced from the anterior pituitary gland by the gonadotropic cells.
Pregnant mare’s serum gonadotropin (PMSG) is widely used for superovulation of laboratory rodents including mice and rats since PMSG mimics the function of FSH (8) which selects and stimulates multiple immature ovarian follicles to grow and mature in mice.
It was demonstrated that the administration of AIS to immune-neutralize inhibin in sexually mature cycling rats has successfully increased the production of FSH (9). Thus, immunization of inhibin has been shown to be a very effective way for superovulation
(10). Usually, after 48 hours of PMSG or AIS administration, followed by an injection of human chronic gonadotrophin (hCG) which mimics the function of luteinizing hormone
(LH) (8) in most species to stimulate final maturation of ovarian follicles and result is ovulation. It has been reported that passive immunization of inhibin through AIS produced 1.5 to 3.2 times more oocytes than PMSG administration in mice (11). Given this large quantity of oocytes obtained from AIS, we hypothesized that there may be potential morphometric, subcellular and fertilization and embryo development characteristics differences between these two sources of oocytes which may ultimately affect their quality and developmental potential.
2
If successful, oocyte cryopreservation is preferable to embryo cryopreservation since it avoids the controversy concerning ethical, legal and religious concerns in human reproductive medicine (12). Research for successful oocyte cryopreservation was demanded due to legislation that restricted cryopreserving excessive embryos in some countries such as in Italy (13). Unfortunately, unlike embryo and sperm cryopreservation, oocyte cryopreservation has not been as successful (12, 14-17).
Oocytes are relatively more difficult to cryopreserve compared to sperm and embryos due to their low surface area to volume ratio and high vulnerability to lethal intracellular ice formation (18). Therefore, there is an urgent need for oocyte cryopreservation for a wide range of infertility treatments and preserving fertility for female oncology patients who are undergoing cancer treatments (e.g. chemotherapy or radiotherapy) since both chemo- and radiotherapy cause severe damage to ovarian follicular reserves (19).
Despite significant progress made over the last decade, oocyte cryopreservation is still a challenging area of reproductive cryobiology and it is less successful than sperm and embryo cryopreservation (16, 17). Additionally, post-thaw survival, fertilization rate and embryonic development of the cryopreserved oocytes are relatively poor (15, 20).
Unfortunately, no standard cryopreservation protocol was been established for oocytes from many mammalian species (21) and the currently available cryopreservation protocols are still not satisfactory (22). The nylon cryoloop has been used as vitrification carrier by suspending a droplet of cryoprotectant containing the oocytes or embryos and directly plunging into liquid nitrogen (LN2) (23). The vitrification by cryoloop method
3 has been relatively successful for the cryopreservation of porcine spermatozoa (24) and mouse embryos (25). One of the advantages of the cryoloop method is the high cooling rate (~20,000 °C/min) which is one of the required conditions to achieve a glassy state by avoiding intracellular ice formation (25, 26).
Subcellular and molecular changes associated with oocyte development
In mammals, after implantation of the blastocyst in the uterus, a small portion of the cell in the epiblast of the inner cell mass eventually differentiates into precursors of the primordial germ cells (PGCs) which ultimately form gonads and produce male or female gametes. After formation of ovaries during fetal development, mammalian growing immature oocytes are first arrested at the diplotene stage of the prophase I stage of meiosis I within fetal ovaries and remaining tetraploid until puberty by an inhibitory signal from the surrounding somatic cells (mural granulosa) in ovarian follicle (27, 28).
This developmentally arrested oocyte is called the germinal vesicle (GV) stage oocyte due to the characteristic of a large vesicular nucleus in which the chromosomes are decondensed state in the oocyte (29). Molecular modification in the cytoplasm, such as elevated cyclic adenosine mono-phosphate (cAMP) prevent the germinal vesicle breakdown (GVBD) (27), cAMP is a second messenger that is produced from ATP through the enzyme called adenylate cyclase (AC) which is located inside the oolemma.
The GV stage oocyte progresses to the next developmental stage when the female reaches puberty wherein individuals who are sexually mature and secrete gonadotropins from the pituitary gland to stimulate the ovarian follicles to cause GVBD (28, 30).
4
The decreased cAMP causes resumption of meiosis that triggers GVBD (31). The GVBD also can be considered an indication of the resumption of meiosis (32) and, during the course of GVBD, chromosomes change from decondensed to condensed state and meiotic spindles are formed (29). The first meiotic spindle forms in the vicinity of the condensing chromosomes at the center of the oocyte about 2 hours after GVBD (33).
During prometaphase, the spindles are usually slightly off center and, in most cases, migrate toward the nearest cortex region of the oocyte just before 1st polar body (PB) extrusion (34). Cytoplasmic maturation of the oocyte after GVBD can be evaluated by the following processes: 1) re-distribution of F-actin and microtubules such as their migration to the cortical region of the oocytes at metaphase stage of the oocyte; and 2) cortical granule (CGs) re-location to the cortex region of the Metaphase II stage of the oocyte with the support of the astral microtubules and F-actins. The process of GV oocyte maturation is also controlled by the maturation promoting factor (MPF) which is present as a complex form of Cdk 1(p34cdc2) and cyclin B in immature oocytes and
MPF reaches its peak in MI oocytes then decreases at anaphase I and telophase1. High content of MPF is restored in the cytostatic factor (CSF) arrested oocyte which is also called MII-arrested oocyte; then, MPF completely disappears after fertilization (35, 36).
Small guanosine triphosphatase (GTPases) and formins also regulate the cytoskeleton during meiotic maturation (28). F-actins, myosin motors, and formin-2 are necessary for
5 meiotic spindle migration (37). It was determined that the spindle migration in MI stage oocytes was faulty in oocytes collected from formin-2 knockout mice since they failed to correctly organize the metaphase spindles and thus, are unable to extrude the 1st PB (38).
Cell division cycle 42 (Cdc42), which is a member of the Rho family of small GTPase
(39), also regulates meiotic spindles and central spindles (28). In the mouse oocytes,
Cdc42 is essential for oocyte polarization that causes an asymmetrical division and meiotic spindle formation in Meiosis I (37). There are high levels of expression in GV oocytes and injecting small interference RNA (siRNA) of Cdc42 into GV stage oocytes decreased both mRNA expression level and production of Cdc42 which caused reduction in the 1st PB extrusion rates and thus, showed the importance of Cdc42 on oocyte polarization (39). With the onset of puberty, luteinizing hormone (LH) binds to its receptors on the surrounding somatic cells (mural granulosa cells), causing the resumption of meiosis and oocytes proceed to the second metaphase state which is then become competent to be fertilized by spermatozoa (27, 40). LH is the most important hormone for oocyte maturation and induction of ovulation because only LH anti-serum can block ovulation (32). LH is the physiological stimulation for the prophase-to- metaphase transition (27, 41). LH decreases the mural granulosa cells cGMP so there is less cGMP available to transfer from the somatic cells into the ooplasm (27). Either inhibiting a guanylyl cyclase or stimulating cGMP phosphodiesterase also causes reduction of cGMP (27). Furthermore, LH causes a significantly reduced guanylate cyclase activity in the cytosol and particulate fractions which might be due to the
6 decreased level of cGMP (42). A decreased level of cGMP causes low cAMP in oocytes that resumes the meiotic arrest through GVBD (31).
The migration of the first meiotic spindle relies highly on F-actin, but not microtubules, which was demonstrated by treating oocytes with either cytochalasin D or nocodazole
(33, 34, 43-45). During oocyte development, there are changes in the structure, organization and composition of the oolemma and cortical cytoplasm at the cortex region close to MI or MII chromosomes associated with their respective meiotic spindle (45).
However, there are no CGs at the cortex region overlying the metaphase spindles in mouse eggs (46), thickening the F-actins at the cortex region overlying the metaphase spindles (46). For the oolemma, there are no microvilli, reducing the cell surface glycoproteins overlying the metaphase spindles (46). All of these changes indicate an apparent polarity of the preovulatory oocyte through structural differentiation of the ooplasm and oolemma that are over the metaphase spindles (43, 47).
Mitochondria number increases dramatically during the oocyte maturation process. In primordial germ cells (PGCs), there are about a few hundred mitochondria and they increase to 200,000 in fully grown oocytes (48). Even though the mitochondria replication start from PGCs, the major increase is observed during the late stages of folliculogenesis (48). Since the early stages of ovarian follicle growth, the Golgi apparatus start to produce immature small vesicles which migrate to the cortex region of
7 the oocyte. These vesicles are bound to one another to form mature cortical granules (49-
51).
After GVBD, oocytes develop to the metaphase I (MI) stage at which the tetrads migrate to a plane which is called the metaphase plate. In the mouse oocyte, the MI stage usually lasts about 6-11 hours and spindle migration of the chromosomes to the oocyte cortex region happens during this time (52, 53). Then, oocytes enter anaphase I at which microtubules and chromosomes are already moving to the cortical region of the oocyte which is followed by telophase I at which the 1st PB is extruded. At the time of ovulation, meiosis I is completed and initially extrudes 1st PB (28) and then arrests again at Metaphase II until post-ovulation induction, which is the fertilization event (30).
Cortical Granules
Cortical granules (CGs) are regulatory secretory membrane bound organelles located in the cortex region of unfertilized metaphase II stage oocytes. Fertilization takes place following mutual recognition between male and female gametes. In mice, fertilizable oocytes are ovulated as cumulus oocyte complexes (COCs) in the ampulla of the oviduct and interact with sperm to get fertilized. These oocytes are at the Metaphase II stage with the presence of first polar body in the perivitelline space between the oolemma and the zona pellucid (a proteinaceous matrix surrounding the oocyte) that is surrounded by many layers of cumulus cells (54). The CGs are initially located at the center and but then translocate to the cortex region of the oocytes (55). In immature (germinal vesicle stage)
8 oocytes, CGs are found in the entire cytoplasm, both in the cortex cytoplasm and the inner cytoplasm. In fully matured oocytes, more specifically in mice and hamsters, there is an asymmetric distribution of CGs in the cortex region of the oocyte (56).
Proper migration of CGs is one of the criteria for cytoplasmic maturation of the oocytes
(57) and plays a major role for blocking polyspermic fertilization (58). There are two ways to block polyspermy: firstly, when the first sperm fuses with the oolemma, it causes depolarization of the oolemma, and the shedding of the oocyte’s sperm receptor folate receptor 4 known as Juno (54). Secondly, through the CGs vesicles releasing their contents into the perivitelline space; CGs contain enzymes that harden the zona pellucida which prevent polyspermic fertilization (54, 55). Cortical granules prevent polyspermy through these two processes: first, it removes the sperm receptors from the oolemma, and second, it hardens the zona pellucida and forms a fertilization envelope to prevent polyspermy. Polyspermy can cause aneuploidy which will result in miscarriages in humans (59). Therefore, one oocyte has to be fertilized with only one sperm in order for the conceptus to develop normally.
The zona pellucida (ZP) is the extracellular matrix (ECM) and is comprised of three glycoproteins, ZP1, ZP2 and ZP3 (56, 60, 61) that surround the plasma membrane of the unfertilized mammalian oocyte. ZP plays a critical role in blocking polyspermy and CGs are playing an important role in modifying the structure of ZPs. The structural changes of the ZP that prevent polyspermy are achieved by exocytosis of CGs from the cortex region
9 of the oocyte. CGs are regulatory secretory organelles that are located along the cortex of oocytes and which release their contents such as ovoperoxidase in extracellular matrix of oocytes to cross link the tryrosin residues in the ZP to harden it to prevent polyspermy.
The CGs contain proteinases, ovoperoxidase, calreticulin, N-acetyglucosaminidase, p32 and peptidylarginine deiminase and high percentages of glycosylated components such as mannosylated proteins, some of which form the CG envelope structure.
Since the early stages ovarian follicle growth, the Golgi apparatus starts to produce small immature vesicles which migrate to the cortex region of the oocyte; these vesicles are bound to one another to form mature CGs (49-51). For hamsters, the small vesicles from the Golgi fuse with secreted vesicles from the rough endoplasmic reticulum (ER) to form mature CGs (56). In mammalian oocytes, CGs are continuously produced and translocated by the cytoskeleton (such as F-actins) to the cortex region (62) of the oocyte until ovulation.
When a spermatozoa penetrates to the oolemma, sperm releases its content into the cytoplasm of the oocytes, one of which is the phospholipase C (PLC) which catalyzes phosphatidylinositol 4, 5-bisphosphate (PIP2) to diacyl glycerol (DAG) and inositol
1,4,5-triphosphate (IP3). IP3 from IP2 will bind to its receptor on membrane of the ER in the oocytes. This binding will lead to calcium release from ER; then, these increased intracellular calcium releases will cause CGs exocytosis. In this process, the single layer of CGs will fuse with double membrane oolemma and subsequently release its contents
10 into extracellular matrix, some of the protein such as ovoperoxidase will cross link the tryrosin residues in ZP that harden it to prevent polyspermy. The following figure shows the schematic view of CGs exocytosis:
Figure 1. 1. Generalized Model of The Mechanism of Intracellular Calcium Release
Leading To Cortical Granule Exocytosis.
The Biology of Cortical Granules (63)
The morphology of CGs varies in different species in terms of size, shape, number and density. For example, CGs of the purple sea urchin oocyte contain a striking spiral structure while CGs in Lytechinus species contain a mosaic substructure in Arbacia species is stellate (64).
11
In mouse oocytes, the CGs increase in number and migrate to the cortex during oocyte maturation. Cortical granules in the cortex region usually accumulate together to a final count of around 6000-8000, at a density of 14 granules per 100 µm² and their size is around 0.1-0.5 um. For sea urchins, the cortex of the egg contains around 15,000 docked
CGs (65).There are both light and dark CGs in mature oocytes, indicating the maturation condition of CGs content (55).
There is evidence of cross species reactivity in mammalian sperm-oocyte binding. For example, sperm from rabbits, mice and hamsters readily bind to human oocytes. Human sperm bind to the other higher primates (66). However, there is no report of zygote produced from these cross species oocyte-sperm binding, probably because of ethical reasons. Interestingly, for mouse and hamster oocytes, there are CG free domains
(CGFDs) over the metaphase I and metaphase II spindles associated with nuclear maturation (67). This CGFDs is specific for rodents because other species including feline (67), equine (57) , bovine (68), porcine (69), and human (70) oocytes do not have this domain.
The CGFD forms over the metaphase II meiotic spindles which is unique to rodents and is associated with partial CGs exocytosis during the oocyte maturation process (65, 71).
Due to this partial CGs exocytosis, there are around 4,000 CGs in one metaphase-II arrested oocyte compared to the original 6,000-8,000 in the fully grown oocytes (65). The
12 formation of the CGFD also creates of a microvillar-free domain and cortical actin cap, which is F-actin thickening over the metaphase spindle (44, 65).
In humans, around 10% of spontaneous abortions are caused by triploidy due to polyspermy penetration of the oocyte (72). Consistent with these data, in-vitro fertilization data also show that around 10% of fertilized oocytes have three or more pronuclei indicating a failure to block polyspermy (73). High concentrations of cryoprotectants such as dimethylsulfoxide (DMSO) and propanediol (PROH) trigger 70-
80% of the CGs exocytosis in all human and mouse oocytes (74, 75).
Mitochondria
Mitochondria in mammalian oocytes play very important roles for energy production, oocyte maturation, fertilization and embryonic development as well as contributes DNA to the next generations. Furthermore, mitochondria play critical roles in oocytes’ functioning and they are one of the important indicators of oocyte quality that is vital to successful fertilization and embryo development (76). One of the important functions of mitochondria is energy production and thus they are called the energy factories of the cells. They are double membrane cellular organelles that produce ATP which is also called energy currency for the cellular function. Mitochondria also play important roles in cell ageing, apoptosis, metabolism and many diseases (76, 77).
13
Mitochondria are the subcellular structure that contain DNA called mitochondrial DNA
(mtDNA) it was originally derived from bacterial genes, and it is the only animal cellular organelle that contains genetic materials (78). mtDNA is vulnerable to mutation compared to DNA in nuclear because there is no histone protection as in nucleus DNA
(78). mtDNA encodes several key genes of electron transfer chain which are very important for mitochondrial energy production (48).
Previous studies have not been consistent about the number of the mitochondria in the fully grown oocytes. According to Cummins et al (77), a fully grown mammalian oocyte contains around 100,000 copies of mitochondria that are inherited uniparentally from the females, as sperm mitochondrial proteins are ubiquitinated and degraded through proteasomes and autophagy after the sperm penetrates the oocyte (77). However, according to St John et al (48), in primordial germ cells (PGCs), there are a few hundred mitochondria and they increase to 200,000 in fully grown oocytes. Even though mitochondria replication starts from PGCs, the major increase is observed during the late stages of folliculogenesis (48).
In addition to their well-known energy production function, mitochondria have other critical functions including calcium homeostasis (79, 80), cell signaling, apoptosis (76)
(81). Since mitochondria are inherited from maternal parents (76, 77), mitochondrial
14 dysfunction causing diabetes and deafness (82), Leigh’s Disease (83), and NARP syndrome (84) are inherited through the oocyte.
Meiotic Spindles
A fertilizable haploid oocyte is produced from one diploid genome content of a germline precursor converted to a haploid state gamete by dumping ¾ of the duplicated chromosomes through two meiotic cell divisions known as meiosis I and II (85, 86).
Incorrect meiotic divisions cause aneuploid eggs which develop into nonviable or impaired embryos that carry an in-correct number of chromosomes which result in spontaneous abortion, infertility or birth defects in humans (86-88). Chromosome segregation during meiosis I and II in mammalian oocytes is determined by a microtubule spindle with no centrosomes (89). Chromosome alignment and segregation depend on this non centrosomes spindle-shaped structure that is made from microtubules, 25 nm diameter tubes containing of the a-tubulin and b-tubulin dimmers (86).
Meiotic spindles are essential for meiotic division to produce haploid gamete cells (90).
Derive in vitro maturation, the oocyte resumes meiosis after GVBD, the meiotic spindle starts to form in the center of the oocyte and translocates to the cortex region in metaphase I (MI). After first polar body extrusion, the oocyte is arrested in metaphase II
(MII) until fertilization (91).
15
Assessing the integrity of the microtubules is the one of the most important criteria to evaluating the quality of an oocyte. The meiotic spindles are composed of microtubules that are assembled by polymerization of the a-tubulin and b-tubulin (92). Microtubules are organized by the microtubule-organizing centers at the two ends and anchor chromosomes with the kinetochores at the equatorial plane of the meiotic spindles, forming a barrel shape (93-95). Microtubules play important roles for alignment of the chromosomes in Metaphase II stage oocytes and correct segregation of the sister chromatids during anaphase II (96, 97), errors in chromosome segregation can cause aneuploidy which is a major cause of spontaneous abortions and pregnancy loss in humans (98). It had also been reported that temperature at 0 °C can cause depolymerization of microtubular tubulin and the intense alteration of spindle in human oocytes (99), vitrification can cause disturbance of the microtubules of bovine oocytes
(100).
F-actin
Capturing each and every chromosomes by spindle apparatus during the cell division is very important to avoid chromosome loss and subsequent aneuploidy (101). However, this mechanism is not sufficient for larger oocytes from marine species (101). It has been reported that in starfish oocytes, chromosome capture and transport were dependent both microtubule assembly and F-actin-driven transport (101). This coordination between spindle assembly and F-actin is essential because early capture of the chromosome by
16 meiotic spindles which are not synchronous to capture by microtubules would lead to incorrect chromosome separation and aneuploidy oocytes formation (101). This F-actin driven mechanism is a prerequisite to preventing aneuploidy in the oocyte and normal embryonic development (102).
In addition, F-actin is one of the most important cytoskeletal components. They contribute to maintaining the oocytes’ shape, migration of meiotic spindles and chromosomes, cytoplasm, polar body extrusion, spindle positioning and cell division, and mammalian embryogenesis. During the process of generating the MII mouse oocytes, one primary oocyte produces a functional haploid gamete through meiosis I and meiosis II and two relatively small polar bodies with minimal cytoplasmic content through asymmetric division, which yields a relatively larger volume of oocyte containing a haploid genome and most cytoplasm content, which is important for providing the nutrition and energy for successful fertilization and further early embryonic development
(91). F-actin play a very important role for this asymmetric division by pulling out the half of the genetic material but relatively small cytoplasmic content, and maintaining the shape of oocyte. When inhibits the F-actin, it leads to the failure of the extrusion of the polar body (103). In mammalian oocyte meiotic division, spindle position and movement, cortical translocation to the cortex region, and cytokinesis depend on F-actins (91, 104-
106). F-actins at the cortical region reorganize and form an F-actin enriched domain known as the actin cap above the spindle. At anaphase, F-actins form a nascent contractile ring to extrude the polar body (103). In addition, F-actin are important for the
17 distribution, movement and exocytosis of cortical granules during oocyte maturation and fertilization, respectively (107). The actin cap forms at the cortex region over the meiotic spindles where the polar body will be extruded (95). The altered distribution of F-actins may cause decreased IVF, lower cleavage and reduced embryo development rate (108).
Euthanasia methods
Euthanasia via carbon dioxide (CO2) inhalation is the most commonly used method for laboratory animals including mice and rats. However, there are increasing numbers of reports suggesting that euthanasia via CO2 inhalation causes reduced fertilization rates, change in blood pH, blood pressure, respiration and heart rate (5) and hypothermia (6).
In addition, CO2 inhalation has an anesthetic effect on mice, which may cause depression of respiratory and cardiovascular systems (6). However, there are still debates about whether euthanasia via CO2 inhalation is more humane and would actually yield an optimal research outcome (5, 109). According to the 2013 edition of the American
Veterinary Medical Association (AVMA) guidelines (110), CO2 inhalation with 10% to
30% volume displacement per minute is required for rodent euthanasia (111) unless cervical dislocation (CD) is scientifically justified (6). It was suggested that while fast displacement of 100% CO2 decreases the duration of the stress mice undergo, slower displacement of CO2, prolongs the time period of experiencing distress, ataxia, stress on breathing (111) and decreases the duration of dyspnea (112).
18
Superovulation methods
Pregnant mare’s serum gonadotropin (PMSG) is a unique gonadotropin among mammals and is widely used for superovulation of laboratory rodents. PMSG acts similar to follicle stimulating hormone (FSH) (8) which select and stimulate multiple ovarian follicles to grow and mature by forming functional complexes with both FSH and LH and receptors.
Human chorionic gonadotropin (hCG), on the other hand, appear to form more prompt and stable hormone-receptor complexes than PMSG. Inhibins are glycoprotein hormones composed of two molecular forms, inhibin A and inhibin B (113). They are mainly produced from ovarian follicles and inhibit the production of FSH from the anterior pituitary gland (10). Using anti inhibin serum (AIS) to immune-neutralize inhibin in cycling rats was shown to increase the production of the FSH (9). Injection of female mice with AIS causes an increase in endogenous production of FSH that stimulates multiple follicular growth in the ovaries (4). Thus, immunization of the inhibin has been determined to be a very effective way of superovulation (10). To date, superovulation via immune-neutralization of inhibin was been successfully used for several species including mice (114), rats (115), hamsters (116), guinea pigs (117), cows (118), mares
(119), ewes (120) and goats (121). The AIS has been reported to be a very effective way to produce a large number of oocytes from limited number of genetically modified and wild mouse species along with various inbred and outbred mice strains (11).
Approximately after 48 hours of PMSG or AIS injection, followed by an injection of human chronic gonadotrophin (hCG) which mimics the function of luteinizing hormone
(LH) (8) most animals undergo final maturation of ovarian follicles and ovulate.
19
Oocyte cryopreservation
Human oocyte cryopreservation can avoid a number of ethical, social and religious problems associated with embryo cryopreservation (12). Increasing numbers of reproductive aged women choose to cryopreserve their oocytes due to medical, social or personal reasons. With delayed child bearing age and improved cancer survival rates, more women desire to preserve their oocytes for future fertility opportunities. For reproduction, there is an obvious differences between men and women because men are usually able to reproduce much older than women. In most developed and developing countries, except for medical reasons women also desire to delay their motherhood either due to their career or not having a suitable partner. Thus, oocyte cryopreservation also provides fertility options for these women who wish to preserve their fertility first and then decide when and with whom they wish to have a child (12).
The very first birth from human frozen oocytes achieved through slow cooling method was reported in Australia in 1986 (122). A twin pregnancy was achieved after in-vitro fertilization of cryopreserved oocytes and transfer of the embryos into the uterus (122).
However, MII stage oocytes might be vulnerable to cryopreservation since, at this stage, there is no nuclear membrane and the chromosomes are loosely attached to the meiotic spindles (123). The integrity of the meiotic spindle in oocytes is very important for normal meiotic division and equal haploid chromosome complements (123). The meiotic spindles capture, organize and stabilize chromosomes through kinetochores (124). Any
20 spindle damage can cause abnormal chromosome division and aneuploidy which will cause failure of subsequent embryo and fetal development (123). On the other hand, there are recent reports which indicate that the success rate of cryopreserved human oocytes is comparable to fresh control. In-vitro fertilization outcomes from randomized control trials also showed that cryosurvival of human oocytes is greatly improving (12).
After age of 35, the fertility of women starts to decline dramatically mostly due to a decreased follicular numbers as well as poorer oocyte quality. Even though older women have the ability to conceive, they take significant risk of miscarriages or having a child with a genetic anomaly such as Down syndrome (12). Family size is also affected by women’s age as there is a 90% of chance to have only one child if they start conceive at age of 35 versus a chance of having 3 children family if they start have children at 28
(12).
Oocyte cryopreservation plays an important role oocytes donation programs for oocyte banks (125). There are also oocyte cryopreservation implications for some other reasons.
For example, women with endometriosis may experience a reduced ovarian follicle reserve post-surgery. It is recommended as a preoperative fertility preservation option for young women who are suffering from endometriosis (126).
21
There is a case report which demonstrates successful cryopreservation of oocytes of a 25- year old nulliparous woman who was suffering from severe endometriosis and low antral follicle count (126). Women with autoimmune diseases are also advised to cryopreserve their oocytes for fertility preservation before they are going through gonadotoxic treatment (127). Furthermore, women with premature ovarian failure due to a variety of reasons such as genetic aberrations and viral infections may also consider cryopreserving their oocytes for fertility preservation (128). Other applications may be due to male fertility problems, for example, the male partner fails to produce sperm on the day of oocyte retrieval (129). There has been a report of on emergency oocyte vitrification for women when sperm extraction from the male partner was not successful on the day of oocyte retrieval, 117 oocytes from 15 women were vitrified and warmed, post-warmed survival rate was 84.6 with 83.8% fertilization rate by ICSI. The pregnancy rate was
53.3% and 9 healthy babies were delivered from 8 pregnancies (130). Cryopreservation via vitrification is also used to cryopreserve accumulated oocytes from multiple ovarian stimulations for low responders to achieve successful IVF and embryo transfer (131).
Unlike embryo and sperm cryopreservation, for most species oocyte cryopreservation has not been as successful (12, 14-17). Therefore, oocyte cryopreservation is a potential assisted reproductive technology (ART) for long-term germplasm preservation and a wide range of infertility treatments and preserving fertility for women who are undergoing cancer treatments (e.g. chemotherapy or radiotherapy). Both chemo- and radiotherapy are known to cause severe damage to ovarian follicular reserves (19). Even though there are several reports about human pregnancy and childbirth (122, 132, 133).
22
Despite significant progress made over the last decade, oocyte cryopreservation is still a challenging area in the reproductive cryobiology field and less successful compared to sperm and embryo cryopreservation (16, 17). The post-thaw survival, fertilization rate and embryonic development of the cryopreserved oocytes are relatively poor (15, 20).
Unfortunately, there is no standard cryopreservation protocol for oocytes from many mammalian species (21) and the current available cryopreservation protocols are still not satisfactory (22).
Vitrification has been widely used for cryopreservation of oocytes and embryos since it is more effective, easier to perform, and a less time-consuming than the traditional slow cooling method (134). In order to avoid formation of lethal intracellular ice, several methods have been used to perform cryopreservation by vitrification. These protocols used very small amounts (1-3 µl) of vitrification solution to achieve ultra-rapid cooling when the samples are rapidly plunged into LN2. So-called “minimum drop vitrification” systems have resulted in higher survival rates of bovine and porcine oocytes cryopreservation (135). Other vitrification techniques utilized a few micro liters of vitrification solution in open pulled plastic French straws (136), cryoloop (137), and cryotop (138). The nylon cryoloop has been used as a vitrification carrier where oocytes or embryos were suspended in a droplet of cryoprotectant solution followed by directly plunging into LN2 for long term storage (23). The cryoloop method has been relatively successful for the cryopreservation of porcine spermatozoa (24) and mouse embryos (25).
23
One of the advantages of the cryoloop method is the generation of a high cooling rate
(20,000 °C/min) to achieve optimal vitrification (25, 26).
24
Chapter Two
Euthanasia via CO2 inhalation Causes Premature Cortical
Granule Exocytosis and Influences In-vitro Fertilization and
Embryo Development in MII Mouse Oocytes
Abstract
The production of good quality of oocytes is one of the essential steps for successful outcome of assisted reproductive technologies (ART) and biomedical reproductive research. Cervical dislocation (CD) or CO2 inhalation are two commonly used methods for mouse euthanasia. The objectives of our research were to assess the impact of CD and
CO2 euthanasia on metaphase II (MII) oocyte, in-vitro fertilization (IVF), embryo development, and subcellular structure, as well as female donor blood clinical chemistry.
In a preliminary study, the superovulated C57BL/6 female mice were euthanized by either CD or low flow CO2 (15% displacement; L/ per min, L CO2). The collected oocytes were subjected to IVF and an embryo culture procedure. The oocytes collected from CD donor females resulted in significantly higher two-cell development rates than those of L CO2 euthanized donors (48.7% vs. 30.30%, respectively; p = 0.028).
Similarly, the oocytes collected from CD donor females resulted in higher embryo development rates than those of L CO2 euthanized donors (42.0% vs. 29.0%, respectively; p = 0.027).
25
In a further experiment, the superovulated C57BL/6 female mice were euthanized by either CD, L CO2 (15% displacement) or high flow (100% displacement; H CO2). The cumulus-oocyte complexes (COCs) derived from each euthanasia method were then in- vitro fertilized by using either fresh or frozen-thawed C57BL/6 sperm. After IVF with frozen-thawed sperm, the oocytes collected from CD euthanized female donors resulted in a higher two-cell and morula development rates (68.7%) than the L CO2 (43.7%) euthanized donors (P=0.028). The oocytes collected from CD donor females resulted in a higher blastocyst (51.6%) development rates than those of H CO2 (29.3%) and L CO2
(14.0%) euthanized donors (P<0.001). Although two-cell development between H CO2
(53.7%) and L CO2 (43.7%) was not statistically different (P=0.443), blastocyst formation rate (P=0.039) was higher for H CO2 (29.3%) than L CO2 (14.0%). After IVF with fresh sperm, the oocytes collected from CD euthanized female donors resulted in significantly higher two-cell (83.1%, 64.6% and 55.3%), morula (74.3% 56.1%, 41.2%) and blastocyst formation (64.7%, 41.4%, and 31.6%) rates than both H CO2 ( P<0.005,
P<0.003, P<0.001) and L CO2 (P<0.001) euthanized donors, respectively. Although oocytes derived from L CO2 euthanized donors resulted in lower 2-cell (P=0.189 and blastocyst (P=0.156) formation than H CO2 euthanized donors, the difference was not statistically significant.
In order to understand the mechanisms underlying decreased IVF and embryo development rate in the CO2 euthanasia groups, ICR female mice were randomly assigned to three experimental groups, high flow CO2 (H CO2), low flow CO2 (L CO2) and CD. Response variables were: anion gap, cortical granules (CGs), and F-actin. The
26 results showed that the plasma anion gap was increased (P<0.001) in the L CO2 group compared to H CO2. The CGs integrity were measured by using fluorescence intensity analysis revealed significantly higher rate of premature CG exocytosis (PCGE) for oocytes derived from the mice euthanized by L CO2 than either CD or H CO2 (P< 0.001).
There was also a higher incidence of PCGE in oocytes derived from L CO2 than H CO2 euthanized mice (P< 0.001). The incidence of PCGE in oocytes derived from H CO2 euthanized mice was higher than those from CD, but lower than those from L CO2 euthanized mice (P< 0.001). In oocytes derived from CD mice, the integrity of F-actins were preserved at a higher level than oocytes derived from both L CO2 and H CO2 euthanized mice (P<0.001). However, there was no difference in F-actins integrity between L CO2 and H CO2 (P=0.988).
These data collectively suggest that, as compared to CD, euthanasia by either L CO2 or H
CO2 inhalation causes PCGE and consequently produces lower fertilization and embryo development rates. Therefore, we conclude that CD is the optimal method of euthanasia for the purpose of obtaining good quality MII oocytes for mouse IVF and other reproductive studies.
Key Words: Mouse, Euthanasia, CO2, Cervical Dislocation, Cortical Granules, F-actin,
Anion Gap, IVF
27
Introduction
Mice are the most commonly used laboratory animals for biomedical research, for production of gametes and embryos, and as genetically engineered animal models for human disease and disorders (1-3). Carbon dioxide (CO2) inhalation is a widely used euthanasia method for laboratory animals including mice and rats. However, there is an increasing body of evidence suggesting that euthanasia via CO2 inhalation causes reduced fertilization rates, and before death changes blood pH, respiration rate, heart rate, blood pressure (5) and hypothermia (6).
In addition, CO2 inhalation has an anesthetic effect on mice, which may cause depression of respiratory and cardiovascular systems (6). However, whether euthanasia via CO2 inhalation is more humane and would actually yield an optimal research outcome has been controversial (5, 109). According to the 2013 edition of the American Veterinary
Medical Association (AVMA) guidelines (110), the CO2 inhalation method with 10% to
30% volume displacement per minute is required for rodent euthanasia (111) unless CD is scientifically justified (6). It was suggested that while fast displacement of 100% CO2 decreases the duration of the stress mice undergo, slow displacement of CO2 prolongs the time period of experiencing distress, ataxia, stress on breathing (111) and decreases the duration of dyspnea (112).
There were an estimated tens of millions of laboratory rodents euthanized by CO2 inhalation (5), thus potential stress and pain caused by CO2 should be taken into consideration (139). To this end, a recent study found that a darkened chamber with
28 slow displacement of CO2 may alleviate stress during CO2 euthanasia (140). A few studies have reported that euthanasia via CD significantly increased the IVF and embryo development rate as compared to CO2 euthanasia (6). Roustan et al., (2012) recommended CD as the optimal method for collecting good quality MII mouse oocytes for biomedical research (3). The authors suggested that L CO2 can cause decreased pH and body temperature which subsequently decreases IVF and later embryo development rate (3, 141).
It has been also suggested that CO2 acidification may cause permeability of the zona pellucida, interfering with proper fertilization due to premature cortical granule exocytosis (PCGE) and subsequently embryo in-vitro developmental competence, as well as fetal development following embryo transfer (7). Several early studies concluded that pH outside the optimal range can also adversely affect the IVF success rate and recommended the optimal pH for the mouse oocytes to be fertilized in-vitro to be between 7.3 to 7.7 (142, 143).
The CG are membrane bound regulatory secretory organelles located in the cortex of unfertilized MII oocytes of most mammalian species (56, 144). Under normal conditions, upon fertilization, a single membrane of CGs fuses with the oolemma and releases its content into the perivitelline space of the oocyte and hardens the zona pellucida to block additional sperm penetration or prevent polyspermy (145). However, CGs exocytosis might also occur prematurely in conditions such as the membrane permeability of due to hypothermia, and toxicity and osmatic shocks caused by cryoprotectant. Cooling-induced
29 increased intracellular Ca2+ might also cause PCGE (145, 146). Cryoprotective agents
(e.g. PROH and DMSO) and cryopreservation procedures can also result in extensive
PCGE (74, 145). MII oocytes might be activated due to osmotic shock which possibly cause, PCGE and second polar body extrusion during the cryopreservation process due to relatively higher concentrations of cryoprotectant solutions (147). Due to the important function of the CGs for blocking polyspermy during the fertilization, it becomes imperative to examine their integrity after various euthanasia methods.
F-actins are one of the cytoskeletal components important for maintaining oocytes shape, migration of meiotic spindles and chromosomes, polar body extrusion, spindle positioning and cell division, during mammalian embryogenesis. F-actin are also involved in the distribution, movement and exocytosis of CGs during oocyte maturation and fertilization (107). The altered distribution of F-actin may cause decreased IVF, lower cleavage and reduced embryo development rates (108). Since there has been no report at this point of the effect of CO2 euthanasia on oocyte subcellular structure changes
(e.g. cortical granules and F-actin) that might cause a decreased IVF and embryo development rate, our objective was to investigate the oocyte subcellular changes associated with decreased IVF rates and embryo development in H CO2 and L CO2 euthanasia methods and directly comparing to CD. Our overall hypotheses was that different euthanasia methods will influence MII mouse oocytes’ fertilization and subcellular integrity including F-actin CGs.
30
Materials and Methods
Animals: In this study, 8-10 week old inbred C57BL/6 (n=36) and eight-week old outbred ICR mice (n=40) were used (Charles River Wilmington, MA). All of the animal studies performed were approved by the University of Missouri’s Animal Care and Use
Committee and were in accordance with the guidelines of the Institute for Laboratory
Animal Research Guide for the Care and Use of Laboratory Animals. On arrival at the
University of Missouri, mice were housed in microisolator caging at temperature range of
20-25oC and lighting (10-h dark/14 h light) controlled environment and provided free access to water and standard pelleted rodent chow. Animals were checked daily by the animal care staff.
Chemicals: All chemicals used in these studies were obtained from Sigma Chemical
Company (St. Louis, MO) or ThermoFisher Scientific unless stated otherwise.
Superovulation and oocyte collection: For superovulation, each female mouse was injected with 7.5 IU of pregnant mare serum gonadotropin intraperitoneally (IP) followed by injection of 7.5 IU human chorionic gonadotropin (hCG) 48 h later. The clutches of cumulus-oocyte complexes (COCs) were collected from the oviducts 14-15 hours after hCG injection.
31
Sperm collection and freezing: The cauda epididymides of proven males were dissected out post-mortem and placed in 1.2 mL 18% raffinose pentahydrate (w/v) and 3% skimmed milk (w/v) freezing solution as previously described (148). Each epididymis, a small cut was made by using sterile micro-scissors was secured with a fine tweezers.
After 10 min, freezing solution containing the sperm was loaded into 0.25 mL French straws, and straws were placed and cooled in vapor phase of LN2 on a wire mesh 8 inches above the LN2 in a Styrofoam box for 5 min, and then the straws were plunged into LN2 for long term storage.
For fresh sperm collection, a sexually mature proven male was euthanized by cervical dislocation and the cauda epididymides were gently dissected out and cleaned of any blood and fat on a clean tissue paper and placed in mineral oil next to the FERTIUP sperm pre-incubation drop. With the aid of a fine tweezers a small cut was made in each epididymis. A sterile 27g needle was used to squeeze out dense sperm and immediately pulled into a 90 L drop of FERTIUP for sperm capacitation for 30 min before IVF. The
o procedure took place in an atmosphere of 5% CO2 in air at 37 C.
In Vitro Fertilization: The straws containing frozen sperm were removed from LN2 and thawed in a water bath at 37oC. The content of the straw was gently expelled on top of 1 mL FHM (holding media) containing BSA (4 mg/mL) in an Eppendorf tube and then centrifuged for 5 min at 300 G. The supernatant was removed and the sperm pellet was gently pipetted out and placed into pre-equilibrated 95 mL FERTIUP drop for sperm capacitation. An IVF procedure was performed as previously described (149, 150). Both
FERTIUP and CARD (for in-vitro fertilization) media were pre-equilibrated in a
32
o humidified incubator containing 5% CO2 in air under mineral oil at 37 C in 35 mm Petri dishes (Falcon 351008). For IVF, previously dissected oviducts from each donor females were separately placed into mineral oil next to a CARD drop and under a stereomicroscope. The clutches of COCs from each donor were first released into mineral oil with the aid of fine tweezers and a 28 g needle and then gently pulled into 90 L
CARD drops. For IVF, frozen-thawed or fresh sperm capacitated in FERTIUP were then gently added (~350x105 motile sperm/mL) into each CARD drop containing the clutches of COCs. After about 6 hours, sperm and egg co-incubation the presumptive zygotes were washed 3 times in FHM media containing BSA (4 mg/mL) and transferred into
KSOM (embryo culture media) amino acid culture drops (151, 152) to determine embryo stage.
Euthanasia Methods: Male mice for the collection of fresh sperm or freezing were euthanized by using cervical dislocation. Female mice used as oocyte or blood donors were euthanized by using either CD or by using CO2 inhalation in a clear acrylic plastic chamber measuring (38.5 cm L, 19.5 cm W, 19.5 cm H) with medical grade (100%) CO2.
Each mouse was euthanized individually and the CO2 chamber was ventilated between the mice by using a hair dryer in order to clear residual CO2. Low flow rate CO2 (L CO2) euthanasia was performed at a displacement rate of 15% CO2 per minute in the chamber via Western Medica CO2 flow meter (Westlake, OH), according to AVMA 2016 guidelines (153). High flow rate CO2 (H CO2) euthanasia was done by using a standard
CO2 regulator providing 100% CO2 into the gas chamber according to AVMA 2013
33 guidelines (110). Once the breathing ceased, each mouse was subjected to CD to ensure death.
Cervical dislocation: For cervical dislocation, a trained person simply grasped the skin on the back of the neck by the thumb and forefinger and immediately pulled on the base of the tail in an opposite upward direction from the head in accordance to 2013 AVMA guidelines (110). The immediate dislocation of the spinal column from the brain ensured death within a few seconds.
Clinical chemistry: Blood collection was performed by using a 1 mL syringe with a 25- gauge needle via intracardiac puncture immediately after euthanasia was confirmed. The withdrawn blood was transferred into a tube containing sodium heparin and centrifuged
10 minutes at 1,000 x G. The resulting plasma was immediately transferred to a clean polypropylene tube in 0.5 mL aliquots and placed in the freezer at -20°C until further analysis. The plasma bicarbonate, sodium, chloride and potassium were determined to calculate the anion gap (Na - (Cl + HCO3) for each mouse.
Immunofluorescence staining and confocal microscopy: For fluorescence staining, fresh COCs were dispersed by using 1 mg/ml hyaluronidase to remove the cumulus cells and 5 mg/ml protease to remove the zona pellucida. Oocytes were fixed with 3.7% formaldehyde in PBS for 30 min at 37°C. After two washes in PBS containing 0.3% polyvinylpyrrolidone (PVP), the oocytes were transferred into blocking buffer which is
34 composed of 1% BSA and 0.01% Triton X-100 in PBS for one hour. For CGs staining after three washes in 0.3% PVP/PBS containing 0.1% Tween 20 and 0.01% Triton X-100 for 5 min each, oocytes in each group were stained with LCA-FITC (Lectin from Arachis hypogaea conjugated with FITC, Sigma, Catalog number: L7381) (10l stock solution+
990 l PBS) for 1 hour at room temperature.
For F-actin staining, oocytes in each group were incubated in 1g/ml of phalloidin-
TRITC (Rhodamine Phalloidin, Invitrogen, catalog number # R415) at room temperature for 30 min. After three washes in PBS containing 0.3% PVP, the oocytes were transferred into a small drop of prolong Antifade mounting medium containing 4, 6 -diamidino-2- phenylindole (DAPI) on a microscope slide and covered with a coverslip. Confocal laser- scanning images were obtained by using Leica Confocal microscope (Leica, Germany).
Statistical analyses
One-way ANOVA with Tukey post-test comparison were used to compare the effects of euthanasia methods on fertilization and embryo development and anion gap by using a
Generalized linear model procedure (GLM) in SAS 9.4 for Windows (SAS Institute Inc.,
Cary, NC). Fluorescence intensity of all images were measured by using Fiji ImageJ software and then all of the data were analyzed by using either one way ANOVA or t-test by SigmaPlot 14.0 (Systat Software Inc.) and graphs were created based on the analyzed results.
35
Results
In order to determine whether the method of euthanasia used for female donor mice influence oocyte quality and subsequent IVF and embryo development competence, we first compared CD with CO2 inhalation (15% displacement; L CO2) on the C57BL/6 oocytes by using frozen-thawed sperm. The results of IVF and pre-implantation embryonic development competence of oocytes obtained from either L CO2 inhalation or
CD euthanasia are shown in Table 2.1. There was a decrease (18.5%) (P<0.05) in 2-cell and pre-implantation embryonic development rate (13%) (P<0.05) for the L CO2 treatment compared to CD. In a further experiment, the superovulated C57BL/6 female mice were euthanized by either CD, L CO2 (15% displacement) or high flow (100% displacement; H CO2) and fertilized by using either fresh or frozen-thawed sperm.
Figure 2.1 shows two-cell, morula and blastocyst development of oocytes collected from
CD, L CO2 and H CO2 euthanized donors following in-vitro fertilization with frozen- thawed sperm and subsequent in-vitro embryo culture. The oocytes collected from CD donor females resulted in higher two-cell (83.1%, 64.6% and 55.3%), morula (74.3%
56.1%, 41.2%) and blastocyst formation (64.7%, 41.4%, and 31.6%) rates than both H
CO2 ( P<0.005, P<0.003, P<0.001) and L CO2 (P<0.001) euthanized donors, respectively.
Although oocytes derived from L CO2 euthanized donors resulted in lower 2-cell
(P=0.189 and blastocyst (P=0.156) formation than H CO2 euthanized donors, the difference were not statistically significant.
36
Figure 2.2 shows two-cell, morula and blastocyst development of oocytes collected from
CD, L CO2 and H CO2 euthanized donors following in-vitro fertilization with fresh sperm and subsequent in-vitro embryo culture. The oocytes collected from CD donor females resulted in higher two-cell (68.7% vs. 43.7%, P=0.028) and morula (61.9% vs. 36.9%,
P=0.027) and blastocyst (51.6% vs. 14.0%, P< 0.01) development rates than the L CO2 euthanized donors. The morula development rates decreased for the L CO2 (36.9%) compared to CD (61.9%, P=0.027) but were not significantly different for CD vs. H CO2
(P=0.148) and H CO2 vs. L CO2 (P=0.529). The oocytes collected from CD donor females resulted in higher blastocyst (51.6%) development rates than those H CO2
(29.3%) and L CO2 (14.0%) euthanized donors (P<0.001). Although two-cell development rates did not reach statistical difference between H CO2 (53.7%) and L CO2
(43.7%, P=0.443), blastocyst formation rate was higher for H CO2 (29.3%) than L CO2
(14.0%, P<0.001). Two-cell embryo development rates were not different between HCO2
(53.7%) and CD (68.7%, P=0.196).
In order to gain fundamental understanding about underlying causes for decreased IVF and embryo development rates for the oocytes derived from the L CO2 treatment, we conducted a series of experiments to determine the relationship between euthanasia methods (H CO2, L CO2, CD) and oocyte subcellular structural characteristics including
CGs and F-actin. While euthanasia by CD was within few seconds, it took an average of
41.1± 0.88 seconds or 4.71± 0.16 min for H CO2, and L CO2 respectively. We determined and compared the plasma anion gap, CG integrity, F-actin distribution, and two-cell, morula and blastocyst stage embryo development rates among the three
37 euthanasia methods. The anion gap for the L CO2 (13.30 ±1.02) was higher compared to
H CO2 (7.63 ± 1.02) (Figure 2.4, P=0.001) while no statistical difference was detected between either CD (10.54 ± 0.88) and H CO2, P=0.117) or CD and L CO2 (P=0.093).
Severe PCGE was observed in oocytes obtained from L CO2 euthanized donors as manifest by reduced fluorescence intensity compared to H CO2 and CD (Figure 2.5J and
Figure 2.6). There were also some disrupted CGs in the H CO2 (Figure 2.5F) oocyte group compared with CD while all oocytes obtained from CD had intact CGs (Figure
2.5B). There was a significant decrease in CG fluorescence intensity for both L CO2 and
H CO2 compared to CD and the CG fluorescence intensity for H CO2 was higher than the
L CO2 treatment group (P<0.001). Although most oocytes from L CO2, H CO2 and CD seemingly maintained the intact F-actins (Figure 2.5C, G, K) there were significant decreases in F-actin fluorescence intensity for L CO2 and H CO2 compared to CD
(P<0.001, Figure 2.7). There was no difference in F-actins intensity detected between L
CO2 and H CO2 (P=0.988).
In order to determine if longer duration of death observed in L CO2 (4.7 min) alone has any adverse effects on the oocytes, the oviducts containing the COCs were left in the body for about 5 min after CD, then dissected out and then subjected to CGs and F-actin integrity assessment. Although apparently intact CGs (Figure 2.8A, D) and F-actin distribution (Figure 2.8B, E) in both groups, there was a slight but statistically significant decrease in F-actin fluorescence intensity 5 minute after CD compared to right after CD
38
(P=0.01) Figure 2.9). However, there was also difference in terms of CGs fluorescence intensity between the groups (P=0.031, Figure 2.10).
Discussion
Rodent models are one of the most commonly used animal experimentation models for life sciences and biomedical researchers (3, 154, 155). One of the most important considerations for animal experimentation is to obtain optimal quality cells or tissues post-mortem for reliable outcomes. Thus, it becomes extremely important to determine the optimal method of euthanasia prior to blood, cell and tissue collection because the method of euthanasia may impose profound adverse effects on the viability and functionality of the cells or tissues collected. The main principles of animal use in biomedical research are defined as 3Rs namely, replacement, reduction and refinement.
These considerations are collectively implemented to improve animal welfare and scientific quality where the use of animals cannot be avoided (156). To this end, the method of euthanasia at the completion of the animal experiments or for future in-vitro or in-vivo studies warrant optimal quality of cells or tissue retrieved. In other word, the collected cells or tissues should not be affected by the euthanasia method to which donor animal are subjected to ensure reliability and reproducibility of the data generated. This will not only assure minimal numbers of animals used but also reliability of future experiments as well as reproducibility of the data.
39
Mammalian oocytes are extremely sensitive cell types which may lose functional integrity when they are subjected to suboptimal conditions under in-vitro conditions and prevent them from undergoing proper fertilization and further embryonic development
(145, 157). For assisted reproductive techniques, such as IVF, sperm micro injection, and cryopreservation, it is important to obtain good quality oocytes from each female donor in order to minimize animal use for genetically engineered mice in particular (6). To date, there has been a very limited number of studies which investigated the influence of euthanasia procedure on post-mortem oocyte and embryo quality (3, 6, 7, 158).
In this study, we first conducted a preliminary experiment on C57BL/6 inbred donor female mice which are most commonly used strain to create genetic modifications for human disease models (154, 155). The C57BL/6 mice were euthanized the animals either by L CO2 or CD euthanasia, and determined the IVF and embryo development rates were determined. The results of this experiment showed that L CO2 euthanasia method caused significant reduction in two-cell development (30.30% vs. 48.7%) and further embryo development (29.0% vs. 42.0%). This reduction may be attributed to hypothermia and all acidic environment during the euthanasia process since CO2 has all anesthesia effect on mice which may have decreased their body temperature due to the long duration (4.71 min) of L CO2 euthanasia and may have lowered the pH during CO2 inhalation (6).
Since the H CO2 method was a previously allowable method of euthanasia based on
AVMA guidelines before 2013, we designed an experiment to compare IVF and embryo
40 development rates of C57BL/6 oocytes collected from the mice that were euthanized by either H CO2, L CO2 or CD. The result of this experiment were consistent with our preliminary study in that L CO2 euthanasia method resulted in significantly lower
(43.7%) 2-cell development compared to CD (68.7%) and H CO2 (53.7%). Further, in- vitro embryonic development competence also showed a similar pattern in which L CO2 resulted in a significantly lower (14.0%) morula formation rate than from CD (51.6%) or
H CO2 (29.3%).
Because euthanasia by both H CO2 and L CO2 inhalation resulted in a lower fertilization and embryonic development rates than CD, we conducted all in-depth investigation to determine the underlying mechanism involved in this phenomenon by examining the changes in oocytes’ crucial subcellular structures that are responsible for proper fertilization (cortical granules) and subsequent mitotic cell division (F-actin). The integrity of CGs is measured by the level of fluorescence intensity within the oocytes.
While oocytes emitting high fluorescence intensity are considered to possess intact CGs, oocytes emitting low intensity are considered as pre-mature cortical granule exocytosis
(PCGE). The fluorescence intensity of CGs of the oocytes collected from L CO2 euthanasia method significantly decreased compared to both H CO2 and CD. The oocytes revealed severe PCGE for L CO2 and moderate PCGE for H CO2 euthanasia method while the vast majority of oocytes derived from CD maintained their integrity.
41
These reductions in IVF and 2-cell development rates for the L CO2 group can be explained by a higher incidence of PCGE which is responsible for blocking more than one sperm from penetrating the oolemma during a normal fertilization event (56). We also analyzed the blood plasma clinical chemistry (total CO2, Na, K, Cl) of the oocyte donors to determine anion gap among H CO2, L CO2 and CD euthanasia methods. The anion gap is increased in L CO2 compared to H CO2 and CD, which indicates the possibility of acidification in the blood circulation that subsequently caused PCGE. A previous study reported that euthanasia via gas (isoflurane) inhalation causes increased abnormality of the oocytes (3) which might be indicating increased accumulation of gas in the oviduct activating the oocytes and PCGE. However, the L CO2 group in our study preserved intact F-actin which indicates that F-actin distribution was not affected by the L
CO2 euthanasia method.
Both two-cell embryo development and morula formation rate were also significantly decreased (25%) in the L CO2 euthanized mice compared to the CD, it is most likely because the increased anion gap caused acidic environment which subsequently caused the severe PCGE of oocytes. These results are consist with the previous reported studies about the adverse effect of CO2 euthanasia methods on IVF and embryo development (6,
7). It was also reported that MII mouse oocytes acidified by 20% CO2 (pH=6.9) for one hour could have altered oocyte structures that increase the permeability of the zona pellucida which subsequently allow multiple sperm penetration and degeneration of the resulting embryos (7). However, in their study, the oocytes were not affected by the alkaline conditions, but were still able to develop into two-cell embryos and blastocyst stage embryos (7).
42
It was reported that the optimal pH for the mouse oocyte IVF was in a range between 7.3 to 7.7 (143). The blood pH of the female C57BL/6 mice euthanized by using CO2 at a displacement rate of 20%/ L/min significantly lowered the pH to 6.5 compared to the CD
(7.31) method (6), which might explain the increased anion gap indicating acidosis in this study for L CO2 mice. In our preliminary study, the H CO2 euthanasia method caused a significant increase in PCGE as compared to CD, but did not show a statistically significant reduction in terms of two-cell embryo development or morula formation.
These results suggest that euthanasia by H CO2 caused a less acidic environment with a significantly smaller anion gap compared to the L CO2 group, and thus, preserved the majority of the CGs in the cortex region of the oocytes.
A previous related study investigated the quality of zygote stage embryos derived from various strains of mice C57BL/6, FVB/N, and B6SJLF1 mice that were euthanized by either H CO2 (100% displacement) or CD (158). There was no difference between H CO2 and CD when they compared the mean viable egg yield among the strains examined.
Furthermore, their study found no significant difference for in-vitro development potential of the zygotes to blastocyst stage between the H CO2 and CD euthanasia methods. It has been well documented that fertilization is the most critical period of embryonic development in mice. Thus, the exposure of oocytes or sperm to suboptimal conditions (i.e. hypothermia, various physical stress) before or during the course of fertilization could inflict PCGE or pre-mature acrosome reaction in respective cell types
(159-161). These potential insults could ultimately cause a significant reduction in
43 fertility and produce lower embryonic development (161). On the other hand, once fertilization completed, development to advanced stages (two-cell to blastocyst) is relatively less intricate and may be tolerated despite minor insults to already fertilized eggs (162).
George and Doe, (163) examined the influence of delayed post CD dissection of oviducts on mouse oocyte viability and embryo developmental competence in-vitro. They determined that leaving the MII oocytes or pre-implantation embryos in the oviduct up to
30 minutes resulted in lower oocyte viability upon recovery and adversely affected their developmental potential. Although length of euthanasia with the use of L CO2 in our study was much shorter (4.7 min) than their study (30 min) such a delay may also contribute to lower fertilization and embryo development.
Overall, this study clearly demonstrated that CD euthanasia method was able to preserve the intact distribution CGs and F-actin of oocytes with significantly higher two-cell embryo development, and morula and blastocyst formation rate compared to the either H
CO2 or L CO2. We conclude that the main reason for the higher oocyte quality was because CD allows the fastest tissue recovery, and maintains blood pH and the integrity of oocytes CGs and F-actins, which collectively safeguards optimal fertilization and embryonic development in-vitro. Furthermore, these results suggest that the CD euthanasia method is the best euthanasia method for obtaining good quality oocytes for
IVF or other reproductive studies in biomedical research.
44
Figure legends
Figure 2. 1. Two-cell (A), morula (B) and blastocyst (C) development of oocytes collected from CD, L CO2 and H CO2 euthanized donors following in-vitro fertilization with frozen-thawed sperm and subsequent in-vitro embryo culture.
45
Figure 2. 2. Two-cell (A), morula (B) and blastocyst (C) development of MII mouse oocytes collected from CD, L CO2 and H CO2 euthanized donors following in-vitro fertilization with fresh sperm and subsequent in-vitro embryo culture.
46
Figure 2. 3. Representative bright field images of two-cell embryos from CD (A), L CO2
(B) and H CO2 (C).
47
Figure 2. 4. Plasma anion gap measurements of MII mouse oocyte donors euthanized by using CD, L CO2 or H CO2.
48
Figure 2. 5. Representative confocal images of the localization of the cortical granules
(green) by Lectin conjugated with FITC, F-actin (red) was detected by phalloidin-TRITC and DNA (blue) was stained by DAPI in the oocytes. Confocal images of DNA (A, E, I),
Cortical granules (B, F, J), F-actin (C, G, K) and merged (D, H, L) representative oocytes.
(A-D) oocytes from cervical dislocation (n=16), (E-H) oocytes from H CO2 (n=10), (I-L) oocytes from L CO2 (n=10).
49
Figure 2. 6. Cortical granules fluorescence intensity measurement of MII mouse oocytes derived from CD, L CO2 and H CO2 euthanized donors.
50
Figure 2. 7. F-actin fluorescence intensity of MII mouse oocytes derived from CD, L CO2 and H CO2 euthanized donors.
51
Figure 2. 8. Representative confocal images of the localization of the cortical granules
(green) by using lectin conjugated with FITC, F-actin (red) were detected by phalloidin-
TRITC. Confocal images of cortical granules (A, D), F-actin (B, E) and merged (C, F) representative oocytes. (A-C) oocytes from cervical dislocation (n=16), (D-F) oocytes from 5 minutes after cervical dislocation (n=36).
52
Figure 2. 9. F-actin fluorescence intensity of MII mouse oocytes collected from donors that were euthanized by using CD and recovered 5 minutes after CD.
53
Figure 2. 10. Cortical granules fluorescence intensity of MII mouse oocytes collected from donors that were euthanized by using CD and recovered 5 minutes after CD.
54
Table 2. 1. In-vitro fertilization and pre-implantation embryonic development of MII mouse oocytes derived from CD and L CO2 euthanasia methods.
Euthanasia method No. oocytes % 2-cell embryos % Blastocyst Cervical dislocation 119 48.70±7.9a 42.0±4.6a b b Low CO2 inhalation 89 30.30±7.6 29.0±5.1 a,b Values with different letters within a column are different (P<0.05).
55
Chapter Three
Morphometric, Subcellular, In-Vitro Fertilization and
Embryonic Developmental Assessment of MII Mouse Oocytes
Following Superovulation with Anti-Inhibin Serum or
Pregnant Mare Serum Gonadotropin
Abstract
The production of sufficient numbers of oocytes or embryos from donor females is one of the limitations for assisted reproductive technologies (ARTs) such as in-vitro fertilization
(IVF), intracytoplasmic sperm injection (ICSI), transgenic animal production, germplasm cryo-banking and somatic cell nuclear transfer (SCNT). This study compared morphometric, subcellular characteristics, and IVF and in-vitro developmental potential of Metaphase II (MII) mouse oocytes derived from either Anti-Inhibin Serum (AIS) or
Pregnant Mare Serum Gonadotropin (PMSG) superovulated donor females. Comparisons were made between the two treatments in terms of quantity, quality, thickness of zona pellucida (ZP), perivitelline space (PVS), diameter (OD), microtubules, F-actin, cortical granules (CGs), and mitochondria. Furthermore, fertilization and developmental potential to blastocyst stage between AIS and PMSG superovulation methods were compared. The AIS superovulation method produced higher numbers of oocytes compared to the PMSG treatment (P=0.002). The number of morphologically normal oocytes produced from the AIS was significantly higher than the PMSG treatment
56
(P=0.01). There was no significant difference with regards to morphologically abnormal oocytes collected from either AIS or PMSG treated females (P=0.425). Additionally, the morphometric measurements of the oocytes showed no significant difference in diameter between AIS and PMSG treated females (P=0.289). However, the thickness of the ZP of oocytes collected from AIS treated females was significantly decreased compared to
PMSG treated females (P<0.0001). The PVS of oocytes derived from the AIS treated females was larger than for PMSG treated females (P<.0001).
To determine the integrity of subcellular structures, in addition to subjective examination, fluorescence intensity analysis was performed. The microtubules in both AIS and PMSG groups were normal even though there was an increased fluorescence intensity in the AIS group (P<0.001). The F-actin in both the AIS and PMSG groups were normal and the fluorescence intensity was not significantly different (P=0.330). The CGs in both the
PMSG and AIS groups also showed normal distribution with no significant difference in fluorescence intensity. While the oocytes derived from PMSG treated females had more homogenously distributed mitochondria, AIS showed a more peripheral distribution with no significant fluorescence intensity differences between the two treatment groups.
Embryonic development rate after IVF with fresh sperm showed no significant difference between AIS and PMSG for two-cell (P=0.103), morula (P=0.203) and blastocyst
(P=0.235). Embryonic development to 2-cell after IVF with frozen-thawed sperm also did not show significant differences between AIS and PMSG. However, there were decrease in morula (P=0.01) and blastocyst formation (P=0.032) in the PMSG group compared to AIS method.
57
Overall, these data suggest that AIS produced around 1.81 times more morphologically normal oocytes with higher two-cell embryo development, morula and blastocyst formation rates than PMSG. The AIS method not only produce high numbers of morphologically normal oocytes, but they possessed normal subcellular structures with good morphological characteristics of oocytes with high in-vitro embryonic developmental potential.
Keywords: Superovulation, Anti Inhibin Serum (AIS), Pregnant Mare's Serum
Gonadotropin (PMSG), Microtubules, F-actin, Cortical Granules, Mitochondria, Zona
Pellucida, morphometric
Introduction
The production of a sufficient quantity of good quality oocytes is very critical for biomedical research and assisted reproductive technologies (ARTs) (4) such as in-vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), transgenic animal production, germplasm cryo-banking (14) and somatic cell nuclear transfer (SCNT).
Therefore, the availability of effective superovulation procedure is very important for producing an adequate number of oocytes and reducing the number of the animals to be euthanized particularly for genetically modified rare species and low- responder female strains (4, 11).
58
Pregnant mare’s serum gonadotropin (PMSG) is a unique gonadotropin among mammals and most widely used for superovulation of laboratory rodents. PMSG acts similar to follicle stimulating hormone (FSH) (8) which selects and stimulates multiple ovarian follicles to grow and mature by forming functional complexes with both FSH and LH receptors. Human chorionic gonadotropin (hCG), on the other hand, appears to form more prompt and stable hormone-receptor complexes than PMSG. Inhibins are glycoprotein hormones composed of two molecular forms denoted inhibin A and inhibin
B (113). They are mainly produced from ovarian follicles that are potentially inhibiting the production of the FSH from the anterior pituitary gland (10). Using AIS to immune- neutralize inhibin in cycling rats was known to increase the production of FSH (9).
Immunization of inhibin is a very effective way for superovulation (10). It was demonstrated that the immune-neutralization of inhibin superovulation method works in several species including mice (114), rats (115), hamsters (116), guinea pigs (117), cows
(118), mares (119), ewes (120) and goats (121). Injection of female mice with AIS causes an increase in endogenous production of FSH that stimulates follicular growth in the ovaries (4). It is a very effective way to produce adequate numbers of oocytes from genetically modified rare species, and various inbred and outbred mice strains (11).
Generally 48 hours after PMSG or AIS injection, followed by an injection of human chronic gonadotrophin (hCG) which mimics the function of luteinizing hormone (LH)
(8), most species stimulate final maturation of ovarian follicles and trigger ovulation.
59
Oocytes quality is one of the most important determining factors for successful fertilization and embryo development, both of which are critical for successful pregnancy for infertility treatments. Thus, examining and selecting the morphological normal, good quality oocytes is very important for successful execution of ARTs. The integrity of the microtubules is one of the most important determining factors for the quality of the oocyte. Microtubules play important roles for the course of the alignment of chromosomes of Metaphase II stage oocytes and correct segregation of the sister chromatids during anaphase II (96, 97). Errors in chromosome segregation can cause aneuploidy which is a major cause of spontaneous abortions and pregnancy loss in humans (98). Therefore, the integrity of the microtubules is very important for successful
IVF or micro-insemination, cleavage and embryonic development.
Similar to microtubules, F-actin distribution is also very critical during fertilization, cleavage and proper pre-implantation embryo development. F-actin are one of the most important cytoskeletal components that contribute to maintaining oocytes’ shape, migration of meiotic spindles and chromosomes, cytoplasm, polar body extrusion, spindle positioning and cell division during preimplantation embryo development and thereafter. In addition, F-actins are important for the distribution, movement and exocytosis of cortical granules (CGs) during oocyte maturation and fertilization (107).
The altered distribution of F-actin may cause decreased IVF, lower cleavage and reduced pre-implantation embryo development rate (108).
60
The CGs are the Golgi derivative membrane bound regulatory secretory organelles that range from 0.2 m to 0.6 m in diameter and are found in the cortex of unfertilized MII oocytes of most species (56, 144). During fertilization, an increased influx of Ca2+ cause the single membrane of the cortical granules to merge with the oocyte membrane and release their substances into the perivitelline space. Thus change the structure of ZP which block the polyspemic fertilization (145).
Mitochondria are considered as one of the important indicators of oocyte quality and are closely related to successful fertilization and embryonic development (76). Mitochondria contain genetic materials that contributed from the female oocytes to the offspring.
Mitochondrial dysfunction inherited through the maternal oocyte can cause diabetes and deafness (82), Leigh’s Disease (83), NARP syndrome (84) etc. in the offspring.
The ZP is an oocyte extracellular matrix that is composed of at least three or four glycoproteins named ZP1, ZP2, ZP3 and ZP4 (164, 165). They collectively play an important role during the fertilization and embryo development and implantation. It has been reported that thickness of the ZP in human oocytes is related to the quality of the oocytes and fertilization rate (166). Relatively thinner (16.6 m) ZP had higher IVF rate and the ZP equal to or thicker than 22 m might need ICSI. For rabbit oocytes, the thicker ZP (19.2 ± 0.3 m) also causes failed fertilization compared to thinner ZP (18.5 ±
0.3 m) of successfully fertilized oocytes (167).
61
The perivitelline space (PVS) is the cavity between the oocyte plasma membrane and the
ZP, and is rich in extra-cellular matrix components which are essential for fertilization, implantation and embryo development. For humans, a systematic review after screening large numbers (1,622) of MII human oocytes from 160 ovarian stimulation cycles and
126 oocyte donors (168) revealed that oocyte PVS larger than normal have a 1.8 times higher chance to develop into good quality of embryos.
Materials and Methods
Animals: In this study, 8-12 week old (n= 58) outbred ICR mice were used (Charles
River Wilmington, MA). All of the animal studies were approved by the University of
Missouri’s Animal Care and Use Committee and were in accordance with the guidelines of the Institute for Laboratory Animal Research Guide for the Care and Use of
Laboratory Animals. On arrival at the University of Missouri. Mice were housed in microisolator caging at a temperature range of 20-25oC and lighting (10-h dark/14 h light) controlled environment and provided free access to water and standard pelleted rodent chow. Animals were checked daily by the animal care staff.
Chemicals: All chemicals used in this experiment were obtained from Sigma Chemical
Company (St. Louis, MO) or ThermoFisher Scientific unless stated otherwise.
62
Superovulation and oocyte collection: Each mouse was injected intraperitoneally (I.P) with either 7.5 or 10 IU of PMSG or 100 l of AIS (CARD institute, Japan) followed by human chorionic gonadotropins (hCG, 10 IU) injection 48 h later. The AIS had a titer of
1:1000000 as defined by the final dilution of the antiserum required to bind 50% of 125I- labeled bovine 32-kDa inhibin. The clutches of cumulus-oocyte complexes (COCs) were collected from the oviducts 14-15 hours after hCG injection.
Sperm collection and freezing: The cauda epididymides were dissected out and placed in 1.2 mL 18% raffinose pentahydrate (w/v) and 3% skimmed milk (w/v) freezing solution as previously described (148). A small cut was made by using sterile micro- scissors by securing each epididymis with a fine tweezer. After 10 min freezing solution containing the sperm were loaded into 0.25 mL French straws and placed in a Styrofoam box containing LN2. Straws were placed and cooled in vapor phase of LN2 on a mash wire 8 inches above LN2 for 5 min, and then the straws were plunged into LN2 for long term storage.
In Vitro Fertilization (IVF) and embryo culture: For IVF with frozen-thawed sperm, the straws containing frozen sperm were removed from LN2 and thawed in a water bath at
37oC. The content of the straw was gently expelled on top of 1 mL FHM media containing BSA (4 mg/mL) in an Eppendorf tube and then centrifuged for 5 min at 300
G. The supernatant was removed and the sperm pellet was gently pipetted out and placed into a pre-equilibrated 95 L FERTIUP drop for sperm capacitation. For IVF with fresh
63 sperm, an adult proven male mouse was euthanized by cervical dislocation and the cauda epididymides were gently dissected out and cleaned of any blood and fat on clean tissue paper and placed in mineral oil next to the FERTIUP sperm pre-incubation drop. With the aid of a fine tweezers a small cut was made in each epididymis. A sterile 27g needle was used to squeeze out dense sperm and immediately pulled into a 90 L drop of
FERTIUP for sperm capacitation for 30 min before IVF procedure. The procedure was
o conducted in an atmosphere of 5% CO2, in air at 37 C.
IVF procedure was performed as previously described (149, 150). Both FERTIUP and
CARD (for in-vitro fertilization) media were pre-equilibrated in a humidified incubator
o containing 5% CO2 in air under mineral oil at 37 C in 35 mm Petri dishes (Falcon
351008). For IVF, previously dissected oviducts from each donor female were separately placed into mineral oil next to a CARD drop and under a stereomicroscope. The clutches of COCs from each donor were first released into the mineral oil with the aid of fine tweezers and 28 g needle and then gently pulled into 90 L CARD drops. For IVF, frozen-thawed sperm were capacitated in FERTIUP and then gently added (~350x105 motile sperm/mL) into each CARD drop containing the clutches of COCs. After about 6 hours of sperm and egg co-incubation, the presumptive zygotes were washed 3 times in
FHM media containing BSA (4 mg/mL) and transferred into KSOM amino acid culture drops (151, 152) for determination of two-cell, morula and blastocyst development.
Immunofluorescence staining and Confocal Microscopy
64
For fluorescence staining, oocytes from both AIS and PMSG cumulus oocyte complexes were dispersed by using 1 mg/ml hyaluronidase to remove the cumulus cells and 5 mg/ml protease to remove the ZP. Denuded oocytes were then fixed with 3.7% formaldehyde for
30 min and then permeabilized with 0.3% PVP+0.1% Tween-20 + 0.01% Triton X-100 for 20 min. This was followed by blocking in 1% BSA in PBS containing 0.1% Triton for
1 hour. Then, the oocytes in each group were incubated with anti-α-tubulin−FITC antibody, mouse monoclonal (clone DM1A, purified from hybridoma cell culture from
Sigma, Catalog number: F2168) diluted 1:200 with blocking buffer for 60 min at room temperature. For F-actin staining, the oocytes in each group were incubated in 1g/ml of phalloidin-TRITC (Rhodamine Phalloidin, Invitrogen, catalog number # R415) at room temperature for 30 min. For cortical granules staining, the oocytes in each group were stained with LCA-FITC (Lectin from Arachis hypogaea conjugated with FITC, Sigma,
Catalog number: L7381) (10 stock solution+ 990 l PBS) for 1 hour at room temperature.
After three washes in PBS containing 0.3% polyvinylpyrrolidone (PVP), the oocytes were transferred into a small drop of prolong Antifade mounting medium containing 4, 6
–diamidino-2-phenylindole (DAPI) on a microscope slide and covered with a coverslip.
Confocal laser-scanning images were obtained by Leica Confocal (Leica, Germany). For mitochondria staining, the oocytes in each group were incubated with rabbit polyclonal customized phosphate carrier primary antibody overnight, and then stained with goat anti-rabbit IgG secondary antibody, Alexa Flour plus 647 (Invitrogen) at a dilution of
1:1000 for 1hr at RT. After three washes in PBS containing 0.3% polyvinylpyrrolidone
(PVP), the oocytes were transferred into a small drop of prolong Antifade mounting medium containing 4, 6 –diamidino-2-phenylindole (DAPI) on a microscope slide and
65 covered with a coverslip. Confocal laser-scanning images were obtained by using Leica
Confocal (Leica, Germany).
Statistical analyses. All embryonic development, ZP thickness, perivitelline space, oocyte diameter, F-actin fluorescence intensity, microtubules fluorescence intensity, CGs fluorescence intensity, and mitochondria fluorescence intensity data from AIS and PMSG superovulation groups were analyzed by t-test comparison by using SigmaPlot 14.0
(Systat Software Inc.) and graphs were created based on the analyzed results.
Results
Figure 3.1 shows the comparison of the average number of the oocytes obtained per donor female after either AIS or PMSG administration. A total of 744 oocytes were collected from 14 donors after AIS and the total number of 500 oocytes from 17 donors after PMSG. The mean number of the oocytes obtained from AIS (n=53.14) was higher than the PMSG (n=29.4, P=0.002). While 90.2% (671/744) of the oocytes were morphologically normal in the AIS method, 87.4% (437/500) with the use of PMSG.
Figure 3.2 shows the average number of morphologically normal oocytes from each donor produced from either AIS or PMSG. The average number of morphologically normal oocytes per donor with the AIS (n=44.7) was higher than PMSG (n=29.1,
P=0.01).
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Figure 3.3 shows the average number of morphologically abnormal (lysed, fragmented) of oocytes from each donor. There was no significant difference in the average number of the morphologically abnormal oocytes between AIS (n=4.9) and PMSG (n=4.2,
P=0.425).
In addition to gross morphological evaluation, we performed morphometric analysis by measuring the diameter, zona pellucida (ZP) thickness and perivitelline space (PVS) of the oocyte derived from AIS or PMSG stimulation. Figure 3.4 shows the average thickness of the ZP of oocytes derived from either AIS or PMSG. The ZP thickness of oocytes collected from AIS (7.92 m) was thinner (8.74 m) than those ZP of PMSG derived oocytes (P<0.0001). Figure 3.5 shows the average PVS between the AIS and
PMSG methods. The PVS of oocytes derived from AIS (10.98 m) was larger than those obtained from the PMSG (7.16 m) method (P<0.001). Figure 3.6 shows the average diameter of oocytes derived from either the AIS or PMSG methods. There was no statistical difference in oocyte diameter between AIS (71.17 ± 0.28 m) PMSG (73.15 ±
0.30 m, P=0.289).
Further investigation was performed to determine if AIS and PMSG stimulation produces oocytes with similar subcellular organelle properties including microtubules, F-actin, cortical granules and mitochondria. Figure 3.7 depicts the representative confocal images of microtubules of the oocyte derived from AIS or PMSG stimulation. Although the oocytes from both AIS (n=47) and PMSG (n=100) maintained normal microtubule, structural integrity, AIS derived oocytes had significantly higher fluorescence intensity
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(Figure 3.8) than PMSG (P<0.001). Figure 3.9 depicts the representative confocal images of F-actins of the oocytes derived from AIS or PMSG stimulation. The F-actin in both groups had normal distributions and there was no difference in fluorescence intensity
(Figure 3.10) between AIS or PMSG stimulation (P=0.33). Figure 3.11 depicts the representative confocal images of cortical granules of oocytes derived from either the
AIS or PMSG superovulation procedure. Both AIS and PMSG derived oocytes had normal CGs distribution with no significant difference in fluorescence intensity (P=0.13,
Figure 3.12).
Figure 3.13 depicts representative images of the mitochondrial distribution of the oocytes derived from AIS or PMSG. While the oocytes obtained from PMSG had more homogenously distributed mitochondria, AIS derived oocytes showed a more peripheral distribution within the cytoplasm. However, there was no statistical difference with regard to the fluorescence intensity (Figure 3.14) between AIS and PMSG derived oocytes (P=0.137).
In order to determine if there is a relation among morphometric, subcellular characteristics, fertility and embryonic development of oocytes obtained after AIS or
PMSG stimulation, IVF and in-vitro embryo cultures were performed. Figure 3.15 shows two-cell, morula and blastocyst development of AIS or PMSG derived oocytes following in-vitro fertilization with fresh sperm and subsequent in-vitro embryo culture. There were no difference in 2-cell embryo development between AIS (86.2%) and PMSG (75.9%) stimulated oocytes (P=0.103). There was also no significant difference in morula
68 formation rates (94.3% vs 90%, P=0.235) and blastocyst formation rates (96.8% vs
94.7%, P=0.203) between AIS and PMSG, respectively.
Figure 3.16 shows two-cell, morula and blastocyst development of AIS or PMSG derived oocytes following in-vitro fertilization with frozen-thawed sperm and subsequent in-vitro embryo culture. Although there was a 10.6% higher 2-cell embryo development rate for the AIS (71.5%), derived oocytes, it was not statistically significant compared to PMSG
(60.9%, P=0.189). However, there was a significantly higher rate of morula (91.0% vs
86.9%, P=0.01) and blastocysts formation (95.4% vs 89.9%, P=0.031) for AIS compared to the PMSG, respectively.
Discussion
It has been reported that passive immunization of inhibin through AIS will produce 1.5 to
3.2 times more mouse oocytes than PMSG administration (11). Given this large numbers of oocytes obtained from AIS, we hypothesized that there may be potential morphometric, subcellular and embryo development characteristics between these two sources of oocytes which may affect their quality their developmental potential. To our best knowledge, this is the first comprehensive study which investigated and compared the oocyte diameter, zona pellucida thickness and perivitelline space, oocytes subcellular structures including microtubules, F-actin, cortical granules, mitochondria, between AIS and PMSG superovulation protocols for mice.
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The previous studies also investigated the superovulation efficiencies of AIS-hCG and
PMSG-hCG protocols on mice demonstrating lower superovaulatory response such as
Japanese wild-derived Mus musculus molossinus (169) as well as commonly used inbred
(A/J, BALB/cByJ, C3HeJ, DBA/2J, FVB/NJ, BALB/cA, C57BL/6), outbred (ICR) and hybrid (B6D2F1) strains (4, 11). It has been reported that superovulation by AIS produces a similar or an increased number of good quality oocytes as compared to PMSG as determined by developmental potential of 2-cell embryos after IVF and live offspring
(4).
In this study, we first measured the oocyte diameter, ZP thickness, PVS, microtubule integrity, F-actin, cortical granules and mitochondria distribution from oocytes derived from either AIS or PMSG. Interestingly, we found that the ZP of oocytes produced from
AIS (7.92 m) was significantly thinner than the ZP (8.74 m) of oocytes obtained from
PMSG (P<0.0001). This is might be indicating a higher potential for in-vitro fertilization rate according to previous studies on human and rabbits. For MII human oocytes, it was reported that the thickness of the ZP was related to the quality of the oocytes and in-vitro fertilization rate (166). A relatively thinner (16.6 m) ZP had the higher IVF rate and the
ZP thicker or equal to or thicker than 22 m might need ICSI. Similarly, in rabbits, oocytes with thicker ZP (19.2 ± 0.3 m) caused failed fertilization while the oocytes having thinner ZP (18.5 ± 0.3 m) resulted in successful fertilization (167).
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On the other hand, the PVS (10.98 m) of the oocytes produced from the AIS group were larger (P<0.001) than the PVS (7.16 m) of oocytes induced from the PMSG method.
This might be representing a greater chance to develop into good quality of embryos based on a systematic review of human studies which reported that oocyte PVS larger than normal have a 1.8 times higher chance to develop into good quality of embryos after screening 1,622 MII human oocytes from 160 ovarian stimulation cycles from 126 oocyte donors (168). For oocyte diameter, in our study, these two groups produce a similar size of the oocytes. There were no statistically significant differences in terms of the diameter of oocytes from AIS vs. PMSG methods (P=0.289).
In this study, although the microtubules of oocytes derived from both superovulation methods had good integrity, AIS derived oocytes showed significantly higher fluorescence intensity. This suggests the good quality and in-vitro developmental potential of the oocytes with AIS in this study since integrity of the microtubules plays an important role for alignment of chromosomes in the MII stage oocytes and correct segregation of the sister chromatids during anaphase II (96, 97).
In both AIS and PMSG derived oocytes, the distribution of F-actin were also normal with no significant difference in fluorescence intensity detected. This additionally demonstrates the decent quality of the oocytes of both groups since F-actin are one of the most important cytoskeleton components that contribute to maintaining oocytes’ shape,
71 migration of meiotic spindles and chromosomes, cytoplasm, polar body extrusion, spindle positioning and cell division, mammalian embryogenesis (170) (106).
Additionally, the cortical granules integrity of both AIS and PMSG derived oocytes were comparable and no differences in fluorescence intensity was detected. This further validates the potential good quality of oocytes in both AIS and PMSG groups since CGs play an important role in blocking polyspermy and embryonic development for ensuring normal fertilization and development of the oocytes (65) (145).
Mitochondria are one of the important indicators of the oocyte quality closely related to successful fertilization and embryonic developments (76). In this study, the mitochondria were more homogenously distributed in the oocytes derived from PMSG whereas more peripherally distributed in the oocytes derived from AIS. However, the fluorescence intensity of mitochondria in two methods were not statistically significant which provides further evidence for production of good quality of mouse oocytes in both superovulation methods.
In order to have a better understanding about the quality and developmental potential of the oocytes derived from AIS and PMSG, we performed quantitative (number of oocyte per donor) and qualitative (fragmentation and lysis) evaluations as well as two-cell embryo development following IVF with either fresh or frozen-thawed sperm, and monitored 2-cell, morula and blastocyst formation rates. The mean number of the oocytes
72 ovulated from each animal in the AIS superovulation method (n=49.6) was significantly
(P=0.002) higher than the mean number of the oocytes ovulated from each clutch in the
PMSG superovulation group (n=29.4) which is consistent with the results of the previous studies (4) which found significantly increased oocyte yield from AIS in inbred
(BALB/cA, C57BL/6), outbred (ICR) and hybrid (B6D2F1) strains, and wild-derived strain (Mus musculus molossinus) (169).
With fresh sperm fertilization, we found that there was no significant difference in terms of the 2-cell embryo formation between AIS (86.2%) and PMSG (75.9%) groups which was consistent with the results from a previous study (4) that of a similar percentage oocytes from the AIS and PMSG were able to develop into two-cell embryos. There was also no significant (P=0.203) difference in terms of the morula formation rate between the AIS (94.3%) and PMSG (90%) methods. In terms of the blastocyst formation rate, even though it was slightly higher in the AIS group, it is not statistically different between AIS (96.8%) and PMSG (94.7%).
Since the oocytes are more often fertilized with frozen-thawed sperm clinically, we evaluated how the frozen-thawed sperm would affect the 2-cell, morula and blastocyst formation rate. With frozen-thawed sperm fertilization, our result showed that even though there was a 10.6% increased 2-cell embryo formation rate in the AIS group, the difference was not significant. However, there was a significantly higher morula formation rate found in the AIS (91%) method than the PMSG (86.9%) procedure. In
73 terms of the blastocyst formation rate, there was also a significant increase in AIS
(95.4%) group compared to the PMSG (89.9%) method. All of these indicated that the oocytes produced from AIS method had a higher potential for embryonic development and offspring (4, 171).
In conclusion, our results showed that normal microtubules, F-actin, cortical granules, and mitochondria, and especially significantly thinner ZP and larger PVS, contributed to the slightly higher two cell embryo development, morula and blastocyst formation in the
AIS method than PMSG procedure. But when the oocytes were fertilized with frozen- thawed sperm, there were significantly higher morula and blastocyst formation in the AIS group than the PMSG group. Our results further showed that the AIS method is a very efficient superovulation method for producing high developmental potential oocytes with the detailed morphology characteristics and subcellular structures in ICR mice. Thus, the
AIS method can be a very good reference for usage on efficient superovulation for low- responding and genetically rare species animals for biomedical research.
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Figure Legends
Figure 3. 1. Average number of MII mouse oocytes obtained from each donor female after either AIS or PMSG superovulation methods.
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Figure 3. 2. The average number of morphologically normal MII mouse oocytes from each donor female after either AIS or PMSG superovulation methods (A). The average percent of morphologically normal MII mouse oocytes from each donor female after either AIS or PMSG superovulation methods (B).
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Figure 3. 3. A representative image of morphologically abnormal oocytes (A). The average number of morphologically abnormal MII mouse oocytes from each donor female after AIS or PMSG superovulation methods (B). The average percent of morphologically abnormal oocytes from each female donor after AIS or PMSG superovulation (C).
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Figure 3. 4. The average thickness of the ZP of MII mouse oocytes derived from either
AIS or PMSG superovulation methods.
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Figure 3. 5. The average PVS of the MII mouse oocytes derived from AIS and PMSG superovulation methods.
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Figure 3. 6. The average diameter of MII mouse oocytes derived from AIS or PMSG superovulation methods.
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Figure 3. 7. The representative confocal images of oocyte microtubules from AIS and
PMSG groups. The microtubules from all of the MII oocytes were maintained in normal shape. PMSG microtubules (A) microtubules (green) with DNA (blue), AIS (B) microtubules (green) with DNA (blue).
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Figure 3. 8. The microtubules fluorescence intensity measurement of MII oocyte derived from AIS or PMSG superovulation methods.
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Figure 3. 9. Representative confocal images of F-actins of the MII mouse oocytes derived from AIS (A) or PMSG (B) superovulation methods.
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Figure 3. 10. The F-actin fluorescence intensity measurement of MII mouse oocytes derived from AIS or PMSG superovulation methods.
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Figure 3. 11. Representative confocal image of cortical granules of MII mouse oocytes derived from either PMSG (A) or AIS (B) superovulation methods.
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Figure 3. 12. Cortical granules fluorescence intensity measurement of MII mouse oocytes derived from AIS or PMSG superovulation methods.
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Figure 3. 13. Representative images of mitochondria distribution PMSG (A) and AIS (B).
Both groups also had a normal mitochondria distribution with more homogenous distribution in the PMSG and were more peripherally distributed in the AIS superovulation method.
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Figure 3. 14. Mitochondria fluorescence intensity measurement of MII mouse oocytes derived from AIS or PMSG superovulation methods.
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Figure 3. 15. Two-cell (A), morula (B) and blastocyst (C) development of AIS or PMSG derived oocytes following in-vitro fertilization with fresh sperm and subsequent in-vitro embryo culture.
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Figure 3. 16. Two-cell (A), morula (B) and blastocyst (C) development of AIS or PMSG derived oocytes following in-vitro fertilization with frozen-thawed sperm and subsequent in-vitro embryo culture.
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Chapter Four
Cryo-survival and Subcellular Structural Characterization of
MII Mouse Cumulus Oocyte Complexes Following
Vitrification via Cryoloop Method
Abstract
Oocyte cryopreservation is one of the fertility preservation options for animals and humans. Vitrification is the most commonly used method to cryopreserve oocytes and embryos, which uses ultra-rapid cooling and eliminates intracellular ice formation which is often detrimental to oocyte post-thaw survival. However, due to the exposure to relatively high concentration of cryoprotective agents, vitrification might cause potential damage to meiotic spindle, F-actin alteration, premature cortical granules exocytosis and altered distribution of mitochondria. The objective of this study was to investigate post- warming survival rate of ICR metaphase II (MII) mouse cumulus oocyte complexes
(COCs), subcellular structures of cryo-survived oocytes, and pre-implantation embryonic development potentials, following in-vitro fertilization (IVF). First, whole clutches of
COCs from superovulated mice were vitrified, in either cryoloops or 0.25 mL French straws in vitrifcation solution containing 30% ethylene glycol and 0.5 M sucrose. The survival rate for oocytes in French straws was significantly lower (19%) than the cryoloop (75%) vitrification method. (P<0.05). Since the oocyte survival rate with the use of French straws method was dismal, we compared fertilization and blastocyst formation rates of fresh and cryoloop-vitrified COCs. Both IVF (78% vs. 98.4%) and blastocyst
91 formation (56.1% vs. 90.6%) rates for cryoloop method were significantly lower than fresh control, respectively COCs (P<0.05). We then investigated the integrity of the abovementioned subcellular structures in order to have a better understanding of the decreased fertilization and blastocysts formation rate of the cryoloop-vitrified oocytes.
For microtubule integrity, 100% of the vitrified-warmed oocytes (n=100) had normal MII spindle. Ninety-six percent of the cortical granules of vitrified-warmed oocytes (n=100) maintained the normal distribution and all of the vitrified-warmed oocytes (n=100) maintained the normal mitochondrial distribution. However, only 62% of vitrified oocytes maintained normal distribution of F-actin (n=100). These results showed cryoloop vitrification of mice COCs had high post-thaw survival with high integrity of subcellular structures, in spite of decreased IVF and embryo development rates compared to fresh controls. Thus, the cryoloop vitrification method might be an effective way to cryopreserve COCs to preserve mouse strains for genome banking and can be a good model for cryopreserving human oocytes for fertility preservation.
Key words: Cryoloop vitrification, mouse oocytes, microtubules, F-actin, cortical granules, mitochondria
Introduction
Unlike embryo and sperm cryopreservation, oocyte cryopreservation has not been as successful (12, 14-17). Oocyte cryopreservation is a very in demand assisted reproductive technology (ART) for long-term germplasm preservation and a wide range of infertility treatments, as well as preserving fertility for women with cancer who are undergoing
92 cancer treatments (e.g. chemotherapy or radiotherapy). Both chemo- and radiotherapy are known to cause severe damage to ovarian follicular reserves (19).
Despite significant progress made over the last decade, oocyte cryopreservation is still a challenging area in the reproductive cryobiology field (16, 17). The post-thaw survival, fertilization rate and embryonic development of the cryopreserved oocytes are relatively poor (15, 20). Unfortunately, there is no standard cryopreservation protocol for oocytes for many mammalian species (21) and the currently available cryopreservation protocols are still not satisfactory (22).
Cryopreservation by vitrification has been widely used for cryopreservation of oocyes and embryos since it is more effective, easier to perform, and less time-consuming than the slow cooling method (134). In order to avoid formation of lethal intracellular ice, several methods have been used to perform cryopreservation by vitrification. These protocols use very small amounts (1-3 µl) of vitrification solution to achieve ultra-rapid cooling when the samples are rapidly plunged into LN2. So-called “minimum drop vitrification” systems have resulted in higher survival rates for bovine and porcine oocytes (135). Other vitrification techniques utilized a few micro liters of vitrification solution in open pulled plastic French straws (136), cryoloop (137), and cryotop (138).
The cryoloop has been used as a vitrification carrier. The loop is made of nylon which suspends a droplet of cryoprotectant containing the oocytes or embryos for directly
93 plunging into LN2 (23). The cryoloop method has been relatively successful for the cryopreservation of porcine spermatozoa (24) and mouse embryos (25). One of the advantages of the cryoloop method is the high cooling rate (20,000 °C/min) which is a very fast vitrification speed that minimizes the ice crystal formation (25, 26).
The subcellular organelles of the MII oocytes are known to be susceptible to cryopreservation procedures. Microtubules damage and disappearance occur in the MII mouse oocyte during the process of cryopreservation (19). The premature cortical granule exocytosis (PCGE) (172-174), change in mitochondrial membrane potential (22, 175) and mitochondrial distribution (176) have also been reported for various species including
MII human, bovine and mouse oocytes. To this end, assessing the integrity of the microtubules is one of the most important criteria to evaluate the development potential of oocyte following cryopreservation. The meiotic spindles are composed of microtubules that are assembled by polymerization of the α-tubulin and β-tubulin dimmers (92). Microtubules are organized by the microtubule-organizing centers at the two ends and anchor chromosomes with the kinetochores at the equatorial plane of the meiotic spindles, forming a barrel shape (93, 94).
Microtubules play important roles for alignment of chromosomes of Metaphase II stage oocytes and correct segregation of the sister chromatids during anaphase II (96, 97), errors in chromosome segregation can cause aneuploidy which is a major cause of spontaneous abortions and pregnancy loss in humans (98). It has been previously
94 reported that hypothermic conditions (0 - 4°C) can cause depolymerization of microtubular tubulin and alteration of spindle in human oocytes (99), and vitrification can cause disruption of the microtubules of bovine MII oocytes (100).Therefore, examination of the microtubule integrity in vitrified-warmed MII oocytes is very important for predicting euploid embryo yield (177).
F-actin are one of the most important cytoskeletal components as they contribute to maintaining oocyte shape, migration of meiotic spindles and chromosomes, cytoplasm, polar body extrusion, spindle positioning and cell division, and mammalian embryogenesis (178, 179). In addition, F-actins are important for the distribution, movement and cortical granules exocytosis during oocyte maturation and fertilization
(107, 180). It was reported that the vitrification procedure alters F-actin structures of MII human oocytes, which can be repaired 3 hours after warming (108). Since the altered distribution of F-actins may cause reduction in fertilization, cleavage and result in lower pre-implantation embryo development, it is imperative to evaluate F-actin distribution in vitrified-warmed oocytes.
The cortical granules (CGs) are originally Golgi derivative membrane bound regulatory secretory organelles that range from 0.2 µm to 0.6 µm in diameter and are located in the cortex of MII oocytes of most species (56, 144). During the course of the fertilization event, an increase influx of Ca2+ causes the single membrane of CGs to merge with oolemma and release their content into the perivitelline space which causes structural
95 changes in the zona pellucida and ultimately blocks the penetration of additional spermatozoa (145).
It has been well documented that mitochondria are one of the important indicators of the oocyte quality and closely related to successful fertilization and embryonic developments
(76). Since mitochondria contain genetic materials contributed from oocytes to the next generations, maternal mitochondrial dysfunction can cause various diseases in the resulting offspring including diabetes and deafness (82), Leigh’s disease (83), and
NARP syndrome (84).
Although it has been widely used, the vitrification procedure can have many adverse effects on oocytes including altered F-actin distribution (108), premature cortical granules exocytosis (PCGE) (172), change in the mitochondria membrane potential (22,
175, 181), disruption of the mitochondria distribution (176) and subsequent cell death
(182). The origins of these adverse effects have been mainly attributed to osmotic shock due to sudden exposure of oocytes to high concentrations of vitrification solution (183,
184) and sudden cold shock (185), chemical toxicity (183) and the relative large size of oocyte (70-120 μm) and thus, high water content in the oocyte (186-188).
Mouse models have been commonly used for oocyte cryobiology studies due to their relative availability and because they provide a good model for development (189, 190).
These previous reports overall suggest that oocyte cryopreservation by vitrification can
96 be improved if necessary modification are made to avoid potential harmful effects. The significance of our study was the use of a relatively lower concentration of permeating cryoprotectant EG which may cause less toxicity and osmotic stress to the whole clutches of COCs and also achieve ultra-fast cooling and warming rates, which collectively prevent the oocytes from direct osmotic injury and chemical toxicity. This is one of the most comprehensive studies which examines various vital subcellular organelles of MII mouse oocytes after vitrification and warming. Therefore, the information gained from the current study will provide broad understanding about potential damages on the subcellular structures of vitrified-warmed MII mouse oocytes and their impact on the subsequent in-vitro fertilization and embryonic development competence.
Materials and Methods
Animals: In this study 7-8 weeks old (n=80) outbred ICR mice were used (Charles River
Wilmington, MA). All of the animal studies were approved by the University of
Missouri’s Animal Care and Use Committee and were in accordance with the guidelines of the Institute for Laboratory Animal Research Guide for the Care and Use of
Laboratory Animals. On arrival at the University of Missouri, mice were housed in microisolator caging with a temperature range of 20-25oC and lighting controlled environment (10-h dark/14 h light) and provided free access to water and standard pelleted rodent chow. Animals were checked daily by the animal care staff.
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Chemicals: All chemicals used in this experiment were obtained from the Sigma
Chemical Company (St. Louis, MO) or ThermoFisher Scientific unless stated otherwise.
Superovulation and oocyte collection: Each mouse was intraperitoneally injected with
10 IU PMSG followed by 10 IU human chorionic gonadotropins (hCG) 48 h later. The clutches of cumulus-oocyte complexes (COCs) were collected from the oviducts 14-15 hours after hCG injection.
Vitrification and warming procedure:
The COCs were pulled out from the ampulla of each oviduct and pulled into vitrification solution 1 (VS1: PBS containing 15% EG + 20% fetal calf serum (FCS)) to equilibrate for 5 min at 25°C. They were then transferred to vitrification solution 2 (VS2: PBS containing 30% EG + 0.5 M sucrose 20% FCS) for 1 min at 25°C and immediately loaded in the cryoloop or French straws and directly plunged into the liquid nitrogen
(LN2). For warming, cryoloops or French straws containing the vitrified COCs were taken out from LN2 and the contents were transferred into PBS containing 0.5 M sucrose
+ 20% FCS for 5 min at 36°C, then transferred into a washing solution of FHM with 4 mg/mL BSA and then used for post-thaw survival, IVF or confocal microscopy analysis.
Sperm collection and capacitation. The cauda epididymides were dissected out from a proven male after euthanization by cervical dislocation and cleaned of any blood, fat on
98 clean tissue paper and placed in mineral oil next to the FERTIUP sperm pre-incubation drop. With the aid of fine tweezers a small cut was made in each epididymis, a sterile 27g needle was used to squeeze out dense sperm and immediately pulled into 90 L drop of
FERTIUP for sperm capacitation for 30 min before the IVF procedure. The procedure
o was conducted in an atmosphere of 5% CO2, in air at 37 C.
In Vitro Fertilization (IVF) and embryo culture: The IVF procedure was performed as previously described (149, 150). Both FERTIUP and CARD (for in-vitro fertilization) media were pre-equilibrated in a humidified incubator containing 5% CO2 in air under mineral oil at 37oC in 35 mm Petri dishes (Falcon 351008). To perform IVF with fresh
COCs, dissected oviducts from each donor female were separately placed into mineral oil next to a CARD drop and under a stereomicroscope. The clutches of COCs from each donor were first released into the mineral oil with the aid of fine tweezers and 28 g needle and then gently pulled into 90 L CARD drops. The clutches of vitrified-warmed COCs were washed three times with HEPES-buffered FHM (4 mg/mL BSA) solution and then transferred into CARD drops. Both freshly collected and vitrified-warmed COCs were then fertilized by fresh capacitated sperm by gently adding into each CARD drops (~350 x105 motile sperm/mL) having the fresh or vitrified-warmed COCs. After about 6 hours of sperm and egg co-incubation the presumptive zygotes were washed 3 times in FHM media containing BSA (4 mg/mL) and transferred into KSOM amino acid culture drops
(151, 152) for determination of two-cell and blastocyst development.
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Immunofluorescence staining and Confocal Microscopy
For fluorescence staining, both fresh and vitrified COCs were dispersed by using 1 mg/ml hyaluronidase to remove the cumulus cells and 5 mg/ml protease to remove the zona pellucida. Denuded oocytes were then fixed with 3.7% formaldehyde for 30mins and then permeabilized with 0.3% PVP+0.1% Tween-20 + 0.01% Triton X-100 for 20min. This was followed by blocking in 1% BSA in PBS containing 0.1% Triton for 1hour. Then,
100 oocytes in each group were incubated with Anti-α-Tubulin−FITC antibody, Mouse monoclonal (clone DM1A, purified from hybridoma cell culture from Sigma, Catalog number: F2168) diluted 1:200 with blocking buffer for 60 min at room temperature. For
F-actin staining, 100 oocytes in each group were incubated in 1μg/ml of phalloidin-
TRITC (Rhodamine Phalloidin, Invitrogen, catalog number # R415) at room temperature for 30 min. For cortical granules staining, 100 oocytes in each group were stained with
LCA-FITC (Lectin from Arachis hypogaea conjugated with FITC, Sigma, Catalog number: L7381) (10 stock solution+ 990 μl PBS) for 1 hour at room temperature. After three washes in PBS containing 0.3% polyvinylpyrrolidone (PVP), the oocytes were transferred into a small drop of prolong Antifade mounting medium containing 4, 6 – diamidino-2-phenylindole (DAPI) on a microscope slide and covered with a coverslip.
Confocal laser-scanning images were obtained by using Leica Confocal (Leica,
Germany). For mitochondria staining, 100 oocytes in each group were incubated with
Rabbit polyclonal customized phosphate carrier antibody overnight, and then stained with goat anti-rabbit IgG secondary antibody, Alexa Flour Plus 647 (Invitrogen) at a dilution of 1:1000 for 1hr at RT. After three washes in PBS containing 0.3% polyvinylpyrrolidone
(PVP), the oocytes were transferred into a small drop of prolong Antifade mounting
100 medium containing 4, 6 –diamidino-2-phenylindole (DAPI) on a microscope slide and covered with a coverslip. Confocal laser-scanning images were obtained by using Leica
Confocal (Leica, Germany).
Statistical analysis
Fertilization and embryonic development data were analyzed by one way ANOVA (SAS
9.4) for Windows (SAS Institute Inc., Cary, NC). Fluorescence intensity data of microtubules, F-actin, CGs and mitochondria were collected by Fiji ImageJ and then analyzed by using SigmaPlot 14.0 (Systat Software Inc.) and graphs were created based on the analyzed results.
Results
Table 4.1 shows post-warming cryosurvival rates of MII stage mouse COCs after vitrification with either a cryoloop or 0.25 mL French straws. Cryo-survival rates of
COCs vitrified in the French straws were lower (19%) than those vitrified in French straws (75%, P<0.05). Since the cryo-survival rates with the use of French straws were very low, we compared in-vitro fertilization (IVF) and embryo development rates of fresh
COCs and cryoloop-vitrified COCs. Table 4.2 shows in-vitro fertilization and pre- implantation embryonic development competence of fresh and cryoloop-vitrified mouse
COCs to blastocyst stage. Two cell development rates of cryoloop-vitrified COCs (78%) were significantly lower than those that were fresh control COCs (98.4%) (P<0.05).
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Blastocyst formation rates of COCs from the cryoloop method (56.1%) was also lower than fresh control (90.6%) (P<0.05).
In order to have a fundamental understanding of decreased IVF and embryonic development rate of the oocytes from cryoloop-vitrifed COCs, further investigation was performed to determine the integrity of oocyte subcellular organelles including microtubules, cortical granules, mitochondria, and F-actins.
Figure 4.1 depicts representative confocal images of Metaphase-II spindle (green fluorescence) from fresh vitrified oocytes. The integrity of microtubules of cryo-survived vitrified oocytes were equal to that of controls (100%) (n=100) (Figure 4.2). Figure 4.3 shows microtubules fluorescence intensity measurement of fresh and vitrified-warmed
MII oocytes. The microtubule fluorescence intensity for vitrified-warmed oocytes were higher in the vitrified oocytes (P<0.001).
Figure 4.4 depicts representative normal confocal images of fresh and vitrified oocytes as well as a representative image of an abnormal F-actin distribution. Although 62%
(62/100) vitrified-warmed oocytes had normal F-actins distribution (Figure 4.5), the F- actins fluorescence intensity (Figure 4.6) for vitrified-warmed oocytes was higher than fresh oocytes (P<0.001).
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Figure 4.7 depicts representative confocal images of cortical granules from fresh and vitrified-warmed oocytes as well as a representative image of an abnormal cortical granule distribution. The percentage of oocytes with normal cortical granules distribution between fresh (99/100, 99%) and vitrified (96/100, 96%) oocytes were comparable
(Figure 4.8), but there was a significant decrease in cortical granules fluorescence intensity in the vitrified-warmed oocytes compared to fresh control (Figure 4.9, P<0.001).
Figure 4.10 depicts representative confocal images of mitochondria from fresh and vitrified-warmed oocytes. The mitochondria from all fresh (100/100, 100%) and vitrified- warmed (100/100, 100%) oocytes showed a normal distribution (Figure 4.11) and the difference for mitochondria fluorescence intensity between fresh and vitrified-warmed oocytes was not significant (P=0.076, Figure 4.12).
Discussion
There have been several studies which investigated the cryopreserved MII oocyte subcellular structures including cortical granules in humans (191-195), meiotic spindles in mice (194, 196, 197), mitochondria distribution in humans and domestic cats (195,
198, 199) and F-actin in humans (108). Our study simultaneously characterized the vitrified-warmed MII mouse oocyte subcellular structures including meiotic spindles, F- actin, CGs and mitochondria. In addition to subcellular examination, we performed IVF and monitored the embryonic developmental potential of cryoloop-vitrified MII oocytes up to the blastocyst stage.
103
Our initial experiment comparing cryo-survival of oocytes in a cryoloop versus French straw showed that cryoloop vitrification was superior to French straw vitrification. It has been suggested that ice formation is potentially more lethal to oocytes due to their high surface to volume ratio (200). Vitrification is the transition of an aqueous solution from liquid to glass state (solid), bypassing the crystalline solid state (201). To achieve vitrification in the context of cell cryopreservation, the solutions and container in which the cells are suspended must provide sufficient cooling rates to achieve intracellular and extra cellular vitrification and avoid lethal intracellular ice formation. This lower cryosurvival for French straws might be due to the much slower cooling rates with
French straws (5,000 °C/min) than cryoloop (~20,000 °C/min) (25, 26).
In addition to cooling rates, there are several bottlenecks for the vitrification approach including injury as a result of osmotic stress to the oolemma and subcellular structures, and chemical toxicity due to high CPA concentration. Furthermore, there is a biological limit to the concentration of CPAs that the subcellular organelles tolerate. Cytoskeletal elements are made up of protein filaments such as microtubules, F-actins and intermediate filaments.
Microtubules are one of the most important subcellular structures of the MII oocyte as they play important roles for completion of the meiosis during fertilization and post- fertilization embryonic development (179, 202). The main function of the meiotic spindle in the MII oocyte is to segregate sister chromatids equally to the pronucleus and the
104 second polar body during the fertilization process (178, 179). Therefore, the microtubule is critical to producing a normal embryo that contains the correct number of genetic chromosomes (203, 204). Previous studies have shown that both human and mouse MII oocyte spindles are extremely sensitive to slow cooling temperatures (205-210). When exposed to hypothermic temperatures (0-4 oC), MII meiotic spindles depolymerize very rapidly (206, 208). It has also been reported for MII mouse oocytes that even slightly below room temperatures might cause abnormal spindle configuration (207, 211).
It also has been suggested that CPA can actually stabilize the microtubule structures and protect them from depolymerization in the lower temperatures (209, 212-214). The current study resulted in a high level of microtubule integrity for vitrified-warmed MII oocytes derived from outbred ICR mice using a cryoloop as a carrier. This is consistent with a previous study in that all vitrified-warmed B6D2F1 hybrid MII mouse oocytes maintained normal microtubule configuration after cryopreservation in the presence of a ethylene glycol (EG) and dimethylsulphoxide (DMSO) combination using a cryotop as a carrier (197).
F-actin is very important for cell migration and division, spindle positioning and asymmetric division in mammalian oocytes (104, 178). The structures of F-actin in an
MII oocyte is that there will be a formation of the actin cap which is an increased intensity network of F-actins on top of the spindles and DNA to position the microtubules which will ensure asymmetric division that extrude the second polar body with a
105 relatively small amount of the cytoplasmic during the fertilization process. This can be an indication of vitrified-warmed oocytes have the full potential to extrude the second polar body. Previous studies reported that vitrification altered the F-actin structure of human oocytes but normal structure was recovered after three hours of warming (108).
F-actin of oocytes were considered normal when a uniformly stained actin with a strong
F-actin networks at the CGs free region were found. In this study, we found reduction
(62%) in normal F-actin distribution in vitrified-warmed oocytes as compared to fresh ones (100%) although relatively normal F-actin networks around microtubules of vitrified-warmed oocytes were observed. This lower F-actin may explain the lower IVF and subsequent embryonic development rates in vitrified-warmed oocytes.
Cortical granule vesicles play a critical role in blocking polyspermy (178, 202). In that study, a majority (96%) of the vitrified-warmed MII oocytes had intact CGs vesicles with normal structure and located in the peripheral region with the exception of the cortical granule free domain (178). This suggests that CGs of MII mouse oocytes maintain their integrity at a high level throughout the vitrification procedure including during CPA exposure and ultra-rapid cooling. These findings are consistent with a previous study which also found no apparent CGs exocytosis in MII human oocytes after slow cooling
(175). However, it has also been reported that a slow freezing process caused a reduction in CGs density and possibly is the cause of zona hardening in human MII oocytes (191-
193). Contrary to our study, previous studies used slow cooling and also transmission electron microscopy (TEM) method to examine CGs and microvilli although the same
CPA, ethylene glycol, was used in this study as well. A latter study by (192) showed that
106 choice of carrier used to perform vitrification also influenced the degree of CGs exocytosis in MII human oocytes. They determined that using a cryoleaf as a carrier better maintained CGs vesicle integrity than the cryoloop. Imaging was by using TEM in that study while confocal microscopy was used in the current study.
Mitochondria are the primary source of ATP production in MII oocytes and pre- implantation embryos (215). In the current study, we used a polycolonal mitochondrial phosphate carrier which correlated with ATP level to locate the mitochondria distribution
(216). We determined that all cryo-survived vitrified-warmed MII mouse oocytes examined had a normal distribution of mitochondria with a slightly increased intensity near peripheral region, especially where meiotic spindles and chromosomes are located.
No difference in mitochondria distribution between fresh and cryopreserved germinal vesicle (GV) and MII human oocytes by using Mitotracker Red CMXRos stainings (198).
It has been reported for both GV and MII stage domestic cat oocytes that vitrification does not disturb the mitochondrial distribution, but causes mitochondrial aggregation and a decreased fluorescence intensity when evaluated using the MitoTracker Red CMXRos staining (199).
In conclusion, the current studies collectively provide a more comprehensive perspective for understanding in-vitro developmental competence and subcellular structural analysis of the CG, meiotic spindles, actin-filaments and mitochondria of MII mouse COCs following cryoloop vitrification. Overall, our results suggest that cryoloop-vitrification of clutches of mouse COCs provides acceptable cryo-survival, subcellular structural
107 integrity, and in-vitro embryonic development. Therefore, the cryoloop vitrification method might be an effective means to cryopreserve COCs to preserve genetically modified mouse strains for genome banking. These results also are very encouraging for further studies in order to optimize the cryopreservation conditions and methods for human MII oocytes.
108
Table 4. 1. Post-thaw morphologic integrity of mouse oocytes following vitrification with
Cryoloop or 0.25 mL French straws (n=8).
No. Oocytes % Oocyte survived
French Straw 129 25 (19.37 ± 2.0)a
Cryoloop 131 99 (75.57 ± 2.0)b
a,b Values with different letters within a column are different (P<0.05).
Table 4. 2. In-vitro fertilization and pre-implantation embryonic development competence of fresh and vitrified mouse COCs to blastocyst stage.
No. oocytes % Fertilized % 2-cell % Blastocysts
Fresh 82 78.0±2.5a 98.4±2.1a 90.6±2.8a
Vitrified 115 71.3±2.2a 78.0±1.8b 56.1±2.4b
a,b Values with different letters within a column are different (P<0.05).
109
Figure Legends
Figure 4. 1. Representative confocal images of Metaphase-II spindle (green fluorescence) of fresh (A) and vitrified-warmed (B) mouse MII oocytes.
110
Figure 4. 2. Percentages of normal microtubule spindle fiber configuration of fresh and vitrified-warmed MII mouse oocytes.
111
Figure 4. 3. Microtubules fluorescence intensity measurement of fresh and vitrified- warmed MII mouse oocytes.
112
Figure 4. 4. Representative normal confocal images of fresh (A) and vitrified-warmed (B)
MII oocytes as well as a representative abnormal (C) F-actin distribution.
113
Figure 4. 5. Percentages of normal F-actin distribution for fresh and vitrified-warmed MII mouse oocytes.
114
Figure 4. 6. F-actins fluorescence intensity of fresh and vitrified-warmed MII oocytes.
115
Figure 4. 7. Representative normal confocal images of cortical granule vesicle from fresh
(A) and vitrified-warmed (B) oocytes and a representative image of an abnormal (C) distribution of cortical granule vesicles.
116
Figure 4. 8. Percentage of normal cortical granule vesicle distribution of fresh and vitrified-warmed MII mouse oocytes.
117
Figure 4. 9. Cortical granule vesicle fluorescence intensity of fresh and vitrified-warmed
MII mouse oocytes.
118
Figure 4. 10. Representative confocal images of mitochondria of fresh (A) and vitrified- warmed (B) MII mouse oocytes.
119
Figure 4. 11. Percentage of normal mitochondria distribution of fresh and vitrified- warmed MII mouse oocytes.
120
Figure 4. 12. Mitochondrial fluorescence intensity measurement of fresh and vitrified- warmed MII mouse oocytes.
121
LITERATURE CITED
1. Berthelot-Ricou A, Perrin J, Di Giorgio C, De Meo M, Botta A, Courbiere B.
Comet assay on mouse oocytes: an improved technique to evaluate genotoxic risk on female germ cells. Fertility and sterility. 2011;95(4):1452-7.
2. Kulandavelu S, Qu D, Sunn N, Mu J, Rennie MY, Whiteley KJ, et al. Embryonic and neonatal phenotyping of genetically engineered mice. Ilar j. 2006;47(2):103-17.
3. Roustan A, Perrin J, Berthelot-Ricou A, Lopez E, Botta A, Courbiere B.
Evaluating methods of mouse euthanasia on the oocyte quality: cervical dislocation versus isoflurane inhalation. Laboratory animals. 2012;46(2):167-9.
4. Hasegawa A, Mochida K, Inoue H, Noda Y, Endo T, Watanabe G, et al. High-
Yield Superovulation in Adult Mice by Anti-Inhibin Serum Treatment Combined with
Estrous Cycle Synchronization. Biology of reproduction. 2016;94(1):21.
5. Conlee KM, Stephens ML, Rowan AN, King LA. Carbon dioxide for euthanasia: concerns regarding pain and distress, with special reference to mice and rats. Laboratory animals. 2005;39(2):137-61.
6. Hazzard KC, Watkins-Chow DE, Garrett LJ. Method of euthanasia influences the oocyte fertilization rate with fresh mouse sperm. Journal of the American Association for
Laboratory Animal Science : JAALAS. 2014;53(6):641-6.
7. Puissant F, Degueldre M, Buisson L, Leroy F. Effects of carbon dioxide acidification of mouse oocytes before in vitro fertilization, culture, and transfer. Gamete
Research. 1986;13(3):223-30.
122
8. Behringer R, Gertsenstein M, Nagy KV, Nagy A. Administration of
Gonadotropins for Superovulation in Mice. Cold Spring Harbor protocols.
2018;2018(1):pdb.prot092403.
9. Gordon A, Aguilar R, Garrido-Gracia JC, Guil-Luna S, Sanchez Cespedes R,
Millan Y, et al. Immunoneutralization of inhibin in cycling rats increases follicle- stimulating hormone secretion, stimulates the ovary and attenuates progesterone receptor- dependent preovulatory luteinizing hormone secretion. Neuroendocrinology.
2010;91(4):291-301.
10. Medan MS, Arai KY, Watanabe G, Taya K. Inhibin: Regulation of reproductive function and practical use in females. Animal Science Journal. 2007;78(1):16-27.
11. Takeo T, Nakagata N. Immunotherapy using inhibin antiserum enhanced the efficacy of equine chorionic gonadotropin on superovulation in major inbred and outbred mice strains. Theriogenology. 2016;86(5):1341-6.
12. Argyle CE, Harper JC, Davies MC. Oocyte cryopreservation: where are we now?
Human reproduction update. 2016;22(4):440-9.
13. Benagiano G, Gianaroli L. The new Italian IVF legislation. Reproductive biomedicine online. 2004;9(2):117-25.
14. Agca Y. Genome resource banking of biomedically important laboratory animals.
Theriogenology. 2012;78(8):1653-65.
15. Mandawala AA, Harvey SC, Roy TK, Fowler KE. Cryopreservation of animal oocytes and embryos: Current progress and future prospects. Theriogenology.
2016;86(7):1637-44.
123
16. Morrell JM, Mayer I. Reproduction biotechnologies in germplasm banking of livestock species: a review. Zygote (Cambridge, England). 2017;25(5):545-57.
17. Dolmans M-M, Manavella DD. Recent advances in fertility preservation. Journal of Obstetrics and Gynaecology Research.0(0).
18. Paynter SJ, Cooper A, Gregory L, Fuller BJ, Shaw RW. Permeability characteristics of human oocytes in the presence of the cryoprotectant dimethylsulphoxide. Human reproduction (Oxford, England). 1999;14(9):2338-42.
19. Tamura AN, Huang TT, Marikawa Y. Impact of vitrification on the meiotic spindle and components of the microtubule-organizing center in mouse mature oocytes.
Biology of reproduction. 2013;89(5):112.
20. Li XH. [Study and applications of human oocyte cryopreservation]. Beijing Da
Xue Xue Bao Yi Xue Ban. 2004;36(6):664-7.
21. Prentice JR, Anzar M. Cryopreservation of Mammalian oocyte for conservation of animal genetics. Vet Med Int. 2010;2011.
22. Lei T, Guo N, Liu JQ, Tan MH, Li YF. Vitrification of in vitro matured oocytes: effects on meiotic spindle configuration and mitochondrial function. Int J Clin Exp
Pathol. 2014;7(3):1159-65.
23. Ghasem S, Negar K. Effect of vitrification on number of inner cell mass in mouse blastocysts in conventional straw, closed pulled straw, open pulled straw and cryoloop carriers. JPMA The Journal of the Pakistan Medical Association. 2013;63(4):486-9.
24. Arraztoa CC, Miragaya MH, Chaves MG, Trasorras VL, Gambarotta MC,
Pendola CH, et al. Porcine sperm vitrification I: cryoloops method. Andrologia.
2017;49(7).
124
25. AbdelHafez F, Xu J, Goldberg J, Desai N. Vitrification in open and closed carriers at different cell stages: assessment of embryo survival, development, DNA integrity and stability during vapor phase storage for transport. BMC biotechnology.
2011;11:29.
26. Shaw JM, Jones GM. Terminology associated with vitrification and other cryopreservation procedures for oocytes and embryos. Human reproduction update.
2003;9(6):583-605.
27. Norris RP, Ratzan WJ, Freudzon M, Mehlmann LM, Krall J, Movsesian MA, et al. Cyclic GMP from the surrounding somatic cells regulates cyclic AMP and meiosis in the mouse oocyte. Development. 2009;136(11):1869-78.
28. Kwon S, Lim HJ. Small GTPases and formins in mammalian oocyte maturation: cytoskeletal organizers. Clinical and experimental reproductive medicine. 2011;38(1):1-
5.
29. Tosti E, Boni R, Gallo A, Silvestre F. Ion currents modulating oocyte maturation in animals. Systems biology in reproductive medicine. 2013;59(2):61-8.
30. Chen ZQ, Ming TX, Nielsen HI. Maturation arrest of human oocytes at germinal vesicle stage. J Hum Reprod Sci. 2010;3(3):153-7.
31. Schultz RM, Montgomery RR, Belanoff JR. Regulation of mouse oocyte meiotic maturation: implication of a decrease in oocyte cAMP and protein dephosphorylation in commitment to resume meiosis. Developmental biology. 1983;97(2):264-73.
32. Törnell J, Billig H, Hillensjö T. REVIEWRegulation of oocyte maturation by changes in ovarian levels of cyclic nucleotides. Human Reproduction. 1991;6(3):411-22.
125
33. Verlhac MH, Lefebvre C, Guillaud P, Rassinier P, Maro B. Asymmetric division in mouse oocytes: with or without Mos. Curr Biol. 2000;10(20):1303-6.
34. Verlhac M-H, Lefebvre C, Guillaud P, Rassinier P, Maro B. Asymmetric division in mouse oocytes: with or without Mos. Current Biology. 2000;10(20):1303-6.
35. Nebreda AR, Ferby I. Regulation of the meiotic cell cycle in oocytes. Current
Opinion in Cell Biology. 2000;12(6):666-75.
36. Tunquist BJ, Maller JL. Under arrest: cytostatic factor (CSF)-mediated metaphase arrest in vertebrate eggs. Genes Dev. 2003;17(6):683-710.
37. Na J, Zernicka-Goetz M. Asymmetric positioning and organization of the meiotic spindle of mouse oocytes requires CDC42 function. Curr Biol. 2006;16(12):1249-54.
38. Leader B, Lim H, Carabatsos MJ, Harrington A, Ecsedy J, Pellman D, et al.
Formin-2, polyploidy, hypofertility and positioning of the meiotic spindle in mouse oocytes. Nat Cell Biol. 2002;4(12):921-8.
39. Cui XS, Li XY, Kim NH. Cdc42 is implicated in polarity during meiotic resumption and blastocyst formation in the mouse. Molecular reproduction and development. 2007;74(6):785-94.
40. Mehlmann LM, Jones TLZ, Jaffe LA. Meiotic Arrest in the Mouse Follicle
Maintained by a Gs Protein in the Oocyte. Science. 2002;297(5585):1343-5.
41. Mehlmann LM, Kalinowski RR, Ross LF, Parlow AF, Hewlett EL, Jaffe LA.
Meiotic resumption in response to luteinizing hormone is independent of a Gi family G protein or calcium in the mouse oocyte. Developmental biology. 2006;299(2):345-55.
126
42. Patwardhan VV, Lanthier A. Cyclic GMP phosphodiesterase and guanylate cyclase activities in rabbit ovaries and the effect of in-vivo stimulation with LH. J
Endocrinol. 1984;101(3):305-10.
43. Longo FJ, Chen DY. Development of cortical polarity in mouse eggs: involvement of the meiotic apparatus. Developmental biology. 1985;107(2):382-94.
44. Longo FJ. Fine structure of the mammalian egg cortex. American journal of anatomy. 1985;174(3):303-15.
45. Van Blerkom J, Bell H. Regulation of development in the fully grown mouse oocyte: chromosome-mediated temporal and spatial differentiation of the cytoplasm and plasma membrane. Journal of embryology and experimental morphology. 1986;93:213-
38.
46. Nicosia SV, Wolf DP, Inoue M. Cortical granule distribution and cell surface characteristics in mouse eggs. Developmental biology. 1977;57(1):56-74.
47. Johnson MH, Eager D, Muggleton-Harris A, Grave HM. Mosaicism in organisation concanavalin A receptors on surface membrane of mouse egg. Nature.
1975;257(5524):321-2.
48. St John J. The control of mtDNA replication during differentiation and development. Biochim Biophys Acta. 2014;1840(4):1345-54.
49. Gulyas BJ. Cortical Granules of Mammalian Eggs. In: Bourne GH, Danielli JF,
Jeon KW, editors. International review of cytology. 63: Academic Press; 1980. p. 357-92.
50. Szollosi D. Development of cortical granules and the cortical reaction in rat and hamster eggs. The Anatomical record. 1967;159(4):431-46.
127
51. Abbott AL, Ducibella T. Calcium and the control of mammalian cortical granule exocytosis. Frontiers in bioscience : a journal and virtual library. 2001;6:D792-806.
52. Polanski Z. Genetic background of the differences in timing of meiotic maturation in mouse oocytes: a study using recombinant inbred strains. J Reprod Fertil.
1997;109(1):109-14.
53. Polanski Z. In-vivo and in-vitro maturation rate of oocytes from two strains of mice. J Reprod Fertil. 1986;78(1):103-9.
54. Cheeseman LP, Boulanger J, Bond LM, Schuh M. Two pathways regulate cortical granule translocation to prevent polyspermy in mouse oocytes. Nat Commun.
2016;7:13726.
55. Gulyas BJ. Cortical granules of mammalian eggs. International review of cytology. 1980;63:357-92.
56. Liu M. The biology and dynamics of mammalian cortical granules. Reproductive biology and endocrinology : RB&E. 2011;9:149.
57. Carneiro GF, Liu IK, Hyde D, Anderson GB, Lorenzo PL, Ball BA.
Quantification and distribution of equine oocyte cortical granules during meiotic maturation and after activation. Molecular reproduction and development.
2002;63(4):451-8.
58. Cheeseman LP, Boulanger J, Bond LM, Schuh M. Two pathways regulate cortical granule translocation to prevent polyspermy in mouse oocytes. Nature Communications.
2016;7:13726.
128
59. Hassold T, Chen N, Funkhouser J, Jooss T, Manuel B, Matsuura J, et al. A cytogenetic study of 1000 spontaneous abortions. Annals of human genetics.
1980;44(2):151-78.
60. Bleil JD, Wassarman PM. Structure and function of the zona pellucida: identification and characterization of the proteins of the mouse oocyte's zona pellucida.
Developmental biology. 1980;76(1):185-202.
61. Boja ES, Hoodbhoy T, Fales HM, Dean J. Structural characterization of native mouse zona pellucida proteins using mass spectrometry. The Journal of biological chemistry. 2003;278(36):34189-202.
62. Wessel GM, Conner SD, Berg L. Cortical granule translocation is microfilament mediated and linked to meiotic maturation in the sea urchin oocyte. Development.
2002;129(18):4315-25.
63. Wessel GM, Brooks JM, Green E, Haley S, Voronina E, Wong J, et al. The biology of cortical granules. International review of cytology. 2001;209:117-206.
64. Wessel GM. Cortical granule-specific components are present within oocytes and accessory cells during sea urchin oogenesis. J Histochem Cytochem. 1989;37(9):1409-20.
65. Wessel GM, Brooks JM, Green E, Haley S, Voronina E, Wong J, et al. The biology of cortical granules. International review of cytology. 209: Academic Press;
2001. p. 117-206.
66. Bedford JM. Sperm/egg interaction: the specificity of human spermatozoa. The
Anatomical record. 1977;188(4):477-87.
129
67. Byers AP, Barone MA, Donoghue AM, Wildt DE. Mature domestic cat oocyte does not express a cortical granule-free domain. Biology of reproduction.
1992;47(5):709-15.
68. Long CR, Damiani P, Pinto-Correia C, MacLean RA, Duby RT, Robl JM.
Morphology and subsequent development in culture of bovine oocytes matured in vitro under various conditions of fertilization. J Reprod Fertil. 1994;102(2):361-9.
69. Wang W-H, Sun Q-Y, Hosoe M, Shioya Y, Day BN. Quantified Analysis of
Cortical Granule Distribution and Exocytosis of Porcine Oocytes during Meiotic
Maturation and Activation1. Biology of reproduction. 1997;56(6):1376-82.
70. Santella L, Alikani M, Talansky BE, Cohen J, Dale B. Is the human oocyte plasma membrane polarized? Human reproduction (Oxford, England). 1992;7(7):999-
1003.
71. Ducibella T, Rangarajan S, Anderson E. The development of mouse oocyte cortical reaction competence is accompanied by major changes in cortical vesicles and not cortical granule depth. Developmental biology. 1988;130(2):789-92.
72. Hassold T, Chen N, Funkhouser J, Jooss T, Manuel B, Matsuura J, et al. A cytogenetic study of 1000 spontaneous abortions. Annals of human genetics. 1980;44(Pt
2):151-78.
73. van der Ven HH, Al-Hasani S, Diedrich K, Hamerich U, Lehmann F, Krebs D.
Polyspermy in in vitro fertilization of human oocytes: frequency and possible causes.
Ann N Y Acad Sci. 1985;442:88-95.
130
74. Schalkoff ME, Oskowitz SP, Powers RD. Ultrastructural observations of human and mouse oocytes treated with cryopreservatives. Biology of reproduction.
1989;40(2):379-93.
75. Vincent C, Pickering SJ, Johnson MH. The hardening effect of dimethylsulphoxide on the mouse zona pellucida requires the presence of an oocyte and is associated with a reduction in the number of cortical granules present. J Reprod Fertil.
1990;89(1):253-9.
76. Schatten H, Sun Q-Y, Prather R. The impact of mitochondrial function/dysfunction on IVF and new treatment possibilities for infertility. Reproductive
Biology and Endocrinology. 2014;12(1):111.
77. Cummins JM. Mitochondria: potential roles in embryogenesis and nucleocytoplasmic transfer. Human reproduction update. 2001;7(2):217-28.
78. Babayev E, Seli E. Oocyte mitochondrial function and reproduction. Current opinion in obstetrics & gynecology. 2015;27(3):175-81.
79. Raffaello A, Mammucari C, Gherardi G, Rizzuto R. Calcium at the Center of Cell
Signaling: Interplay between Endoplasmic Reticulum, Mitochondria, and Lysosomes.
Trends in Biochemical Sciences. 2016;41(12):1035-49.
80. Santulli G, Xie W, Reiken SR, Marks AR. Mitochondrial calcium overload is a key determinant in heart failure. Proceedings of the National Academy of Sciences of the
United States of America. 2015;112(36):11389-94.
81. Danial NN, Korsmeyer SJ. Cell Death: Critical Control Points. Cell.
2004;116(2):205-19.
131
82. Murphy R, Turnbull DM, Walker M, Hattersley AT. Clinical features, diagnosis and management of maternally inherited diabetes and deafness (MIDD) associated with the 3243A>G mitochondrial point mutation. Diabet Med. 2008;25(4):383-99.
83. Murphy JV, Craig L. Leigh's disease: significance of the biochemical changes in brain. Journal of Neurology, Neurosurgery, and Psychiatry. 1975;38(11):1100-3.
84. Thorburn DR, Rahman J, Rahman S. Mitochondrial DNA-Associated Leigh
Syndrome and NARP. In: Adam MP, Ardinger HH, Pagon RA, Wallace SE, Bean LJH,
Stephens K, et al., editors. GeneReviews((R)). Seattle (WA): University of Washington,
Seattle
University of Washington, Seattle. GeneReviews is a registered trademark of the
University of Washington, Seattle. All rights reserved.; 1993.
85. Severson AF, von Dassow G, Bowerman B. Oocyte Meiotic Spindle Assembly and Function. Current topics in developmental biology. 2016;116:65-98.
86. Dumont J, Desai A. Acentrosomal spindle assembly and chromosome segregation during oocyte meiosis. Trends in cell biology. 2012;22(5):241-9.
87. Hassold T, Hunt P. Maternal age and chromosomally abnormal pregnancies: what we know and what we wish we knew. Current opinion in pediatrics. 2009;21(6):703-8.
88. Webster A, Schuh M. Mechanisms of Aneuploidy in Human Eggs. Trends in cell biology. 2017;27(1):55-68.
89. Schuh M, Ellenberg J. Self-organization of MTOCs replaces centrosome function during acentrosomal spindle assembly in live mouse oocytes. Cell. 2007;130(3):484-98.
90. Bennabi I, Terret ME, Verlhac MH. Meiotic spindle assembly and chromosome segregation in oocytes. The Journal of cell biology. 2016;215(5):611-9.
132
91. Lu Y, Zhang Y, Pan MH, Kim NH, Sun SC, Cui XS. Daam1 regulates fascin for actin assembly in mouse oocyte meiosis. Cell cycle (Georgetown, Tex).
2017;16(14):1350-6.
92. Zhou J, Shu HB, Joshi HC. Regulation of tubulin synthesis and cell cycle progression in mammalian cells by gamma-tubulin-mediated microtubule nucleation.
Journal of cellular biochemistry. 2002;84(3):472-83.
93. Maro B, Johnson MH, Webb M, Flach G. Mechanism of polar body formation in the mouse oocyte: an interaction between the chromosomes, the cytoskeleton and the plasma membrane. Journal of embryology and experimental morphology. 1986;92:11-32.
94. Chen SU, Lien YR, Chao KH, Ho HN, Yang YS, Lee TY. Effects of cryopreservation on meiotic spindles of oocytes and its dynamics after thawing: clinical implications in oocyte freezing--a review article. Mol Cell Endocrinol. 2003;202(1-
2):101-7.
95. Verlhac M-H, Breuer M. Cytoskeletal Correlates of Oocyte Meiotic Divisions. In:
Coticchio G, Albertini DF, De Santis L, editors. Oogenesis. London: Springer London;
2013. p. 195-207.
96. Tomari H, Honjo K, Kunitake K, Aramaki N, Kuhara S, Hidaka N, et al. Meiotic spindle size is a strong indicator of human oocyte quality. Reproductive Medicine and
Biology. 2018;17(3):268-74.
97. Vogt E, Kirsch-Volders M, Parry J, Eichenlaub-Ritter U. Spindle formation, chromosome segregation and the spindle checkpoint in mammalian oocytes and susceptibility to meiotic error. Mutation research. 2008;651(1-2):14-29.
133
98. Bond D, Chandley A. Aneuploidy, Oxford Monographs on Medical Genetics.
1983.
99. Zenzes MT, Bielecki R, Casper RF, Leibo SP. Effects of chilling to 0 degrees C on the morphology of meiotic spindles in human metaphase II oocytes. Fertility and sterility. 2001;75(4):769-77.
100. Rho GJ, Kim S, Yoo JG, Balasubramanian S, Lee HJ, Choe SY. Microtubulin configuration and mitochondrial distribution after ultra-rapid cooling of bovine oocytes.
Molecular reproduction and development. 2002;63(4):464-70.
101. Burdyniuk M, Callegari A, Mori M, Nédélec F, Lénárt P. F-Actin nucleated on chromosomes coordinates their capture by microtubules in oocyte meiosis. The Journal of cell biology. 2018;217(8):2661-74.
102. Bun P, Dmitrieff S, Belmonte JM, Nedelec FJ, Lenart P. A disassembly-driven mechanism explains F-actin-mediated chromosome transport in starfish oocytes. eLife.
2018;7.
103. Maddox AS, Azoury J, Dumont J. Polar body cytokinesis. Cytoskeleton
(Hoboken, NJ). 2012;69(11):855-68.
104. Almonacid M, Terret ME, Verlhac MH. Actin-based spindle positioning: new insights from female gametes. Journal of cell science. 2014;127(Pt 3):477-83.
105. Woolner S, O'Brien LL, Wiese C, Bement WM. Myosin-10 and actin filaments are essential for mitotic spindle function. The Journal of cell biology. 2008;182(1):77-88.
106. Azoury J, Verlhac MH, Dumont J. Actin filaments: key players in the control of asymmetric divisions in mouse oocytes. Biology of the cell. 2009;101(2):69-76.
134
107. Kim NH, Day BN, Lee HT, Chung KS. Microfilament assembly and cortical granule distribution during maturation, parthenogenetic activation and fertilisation in the porcine oocyte. Zygote (Cambridge, England). 1996;4(2):145-9.
108. Ci Q, Li M, Zhang Y, Ma S, Gao Q, Shi Y. Confocal microscopic analysis of the microfilament configurations from human vitrification-thawed oocytes matured in vitro.
Cryo letters. 2014;35(6):544-8.
109. Leach MC, Bowell VA, Allan TF, Morton DB. Aversion to gaseous euthanasia agents in rats and mice. Comparative medicine. 2002;52(3):249-57.
110. Leary SL, Underwood W, Anthony R, Gwaltney-Brant S, Poison A, Meyer R, editors. AVMA guidelines for the euthanasia of animals: 2013 edition2013: American
Veterinary Medical Association Schaumburg, IL.
111. Creamer-Hente MA, Lao FK, Dragos ZP, Waterman LL. Sex- and Strain-related
Differences in the Stress Response of Mice to CO(2) Euthanasia. Journal of the American
Association for Laboratory Animal Science : JAALAS. 2018;57(5):513-9.
112. Moody CM, Chua B, Weary DM. The effect of carbon dioxide flow rate on the euthanasia of laboratory mice. Laboratory animals. 2014;48(4):298-304.
113. Muttukrishna S. Role of inhibin in normal and high-risk pregnancy. Seminars in reproductive medicine. 2004;22(3):227-34.
114. Wang H, Herath CB, Xia G, Watanabe G, Taya K. Superovulation, fertilization and in vitro embryo development in mice after administration of an inhibin-neutralizing antiserum. Reproduction (Cambridge, England). 2001;122(5):809-16.
135
115. Ishigame H, Medan MS, Watanabe G, Shi Z, Kishi H, Arai KY, et al. A New
Alternative Method for Superovulation Using Passive Immunization Against Inhibin in
Adult Rats1. Biology of reproduction. 2004;71(1):236-43.
116. Kishi H, Okada T, Otsuka M, Watanabe G, Taya K, Sasamoto S. Induction of superovulation by immunoneutralization of endogenous inhibin through the increase in the secretion of follicle-stimulating hormone in the cyclic golden hamster. J Endocrinol.
1996;151(1):65-75.
117. Shi F, Mochida K, Suzuki O, Matsuda J, Ogura A, Ozawa M, et al. Ovarian
Localization of Immunoglobulin G and Inhibin α-Subunit in Guinea Pigs after
Passive Immunization against the Inhibin α-Subunit. Journal of Reproduction and
Development. 2000;46(5):293-9.
118. Takedomi T, Kaneko H, Aoyagi Y, Konishi M, Kishi H, Watanabe G, et al.
Effects of passive immunization against inhibin on ovulation rate and embryo recovery in holstein heifers. Theriogenology. 1997;47(8):1507-18.
119. Nambo Y, Kaneko H, Nagata S, Oikawa M, Yoshihara T, Nagamine N, et al.
Effect of passive immunization against inhibin on FSH secretion, folliculogenesis and ovulation rate during the follicular phase of the estrous cycle in mares. Theriogenology.
1998;50(4):545-57.
120. Bartlewski P, Beard A, Cook S, Chandolia R, Honaramooz A, Rawlings N.
Ovarian antral follicular dynamics and their relationships with endocrine variables throughout the oestrous cycle in breeds of sheep differing in prolificacy. Reproduction
(Cambridge, England). 1999;115(1):111-24.
136
121. Medan MS, Watanabe G, Sasaki K, Nagura Y, Sakaime H, Fujita M, et al. Effects of passive immunization of goats against inhibin on follicular development, hormone profile and ovulation rate. Reproduction (Cambridge, England). 2003;125(5):751-7.
122. Chen C. Pregnancy after human oocyte cryopreservation. Lancet.
1986;1(8486):884-6.
123. Bernard A, Fuller BJ. Cryopreservation of human oocytes: a review of current problems and perspectives. Human reproduction update. 1996;2(3):193-207.
124. Stachecki JJ, Munne S, Cohen J. Spindle organization after cryopreservation of mouse, human, and bovine oocytes. Reproductive biomedicine online. 2004;8(6):664-72.
125. Domingues TS, Aquino AP, Barros B, Mazetto R, Nicolielo M, Kimati CM, et al.
Egg donation of vitrified oocytes bank produces similar pregnancy rates by blastocyst transfer when compared to fresh cycle. J Assist Reprod Genet. 2017;34(11):1553-7.
126. Elizur SE, Chian R-C, Holzer HEG, Gidoni Y, Tulandi T, Tan SL.
Cryopreservation of oocytes in a young woman with severe and symptomatic endometriosis: a new indication for fertility preservation. Fertility and sterility.
2009;91(1):293.e1-.e3.
127. Elizur SE, Chian RC, Pineau CA, Son WY, Holzer HE, Huang JY, et al. Fertility preservation treatment for young women with autoimmune diseases facing treatment with gonadotoxic agents. Rheumatology (Oxford). 2008;47(10):1506-9.
128. Goswami D, Conway GS. Premature ovarian failure. Human reproduction update.
2005;11(4):391-410.
137
129. Okohue JE, Ikimalo JI, Onuh SO. Ejaculation failure on the day of oocyte retrieval for IVF: A report of five cases. Middle East Fertility Society Journal.
2011;16(2):159-62.
130. Song WY, Sun YP, Jin HX, Xin ZM, Su YC, Chian RC. Clinical outcome of emergency egg vitrification for women when sperm extraction from the testicular tissues of the male partner is not successful. Systems biology in reproductive medicine.
2011;57(4):210-3.
131. Cobo A, Garrido N, Crespo J, Jose R, Pellicer A. Accumulation of oocytes: a new strategy for managing low-responder patients. Reproductive biomedicine online.
2012;24(4):424-32.
132. van Uem JF, Siebzehnrubl ER, Schuh B, Koch R, Trotnow S, Lang N. Birth after cryopreservation of unfertilized oocytes. Lancet. 1987;1(8535):752-3.
133. Kuleshova L, Gianaroli L, Magli C, Ferraretti A, Trounson A. Birth following vitrification of a small number of human oocytes: case report. Human reproduction
(Oxford, England). 1999;14(12):3077-9.
134. Zhou CJ, Wang DH, Niu XX, Kong XW, Li YJ, Ren J, et al. High survival of mouse oocytes using an optimized vitrification protocol. Sci Rep. 2016;6:19465.
135. Arav A, Shehu D, Mattioli M. Osmotic and cytotoxic study of vitrification of immature bovine oocytes. Journal of reproduction and fertility. 1993;99(2):353-8.
136. Vajta G, Lewis I, Kuwayama M, Greeve T, Callesen H. Sterile application of the open pulled straw (OPS) vitrification method. Cryo-letters. 1998;19(6):389.
138
137. Lane M, Schoolcraft WB, Gardner DK. Vitrification of mouse and human blastocysts using a novel cryoloop container-less technique. Fertility and sterility.
1999;72(6):1073-8.
138. Kuwayama M, Vajta G, Kato O, Leibo SP. Highly efficient vitrification method for cryopreservation of human oocytes. Reproductive BioMedicine Online (Reproductive
Healthcare Limited). 2005;11(3):300-8.
139. Thomas AA, Flecknell PA, Golledge HD. Combining nitrous oxide with carbon dioxide decreases the time to loss of consciousness during euthanasia in mice--refinement of animal welfare? PloS one. 2012;7(3):e32290.
140. Powell K, Ethun K, Taylor DK. The effect of light level, CO2 flow rate, and anesthesia on the stress response of mice during CO2 euthanasia. Lab animal.
2016;45(10):386-95.
141. Hazzard KC, Watkins-Chow DE, Garrett LJ. Method of Euthanasia Influences the
Oocyte Fertilization Rate with Fresh Mouse Sperm. Journal of the American Association for Laboratory Animal Science : JAALAS. 2014;53(6):641-6.
142. Miyamoto H, Toyoda Y, Chang MC. Effect of hydrogen-ion concentration on in vitro fertilization of mouse, golden hamster, and rat eggs. Biology of reproduction.
1974;10(4):487-93.
143. Miyamoto H, Toyoda Y, Chang MC. Effect of Hydrogen-Ion Concentration on in
Vitro Fertilization of Mouse, Golden Hamster, and Rat Eggs. Biology of reproduction.
1974;10(4):487-93.
144. Cran DG, Esper CR. Cortical granules and the cortical reaction in mammals.
Journal of reproduction and fertility Supplement. 1990;42:177-88.
139
145. Ghetler Y, Skutelsky E, Ben Nun I, Ben Dor L, Amihai D, Shalgi R. Human oocyte cryopreservation and the fate of cortical granules. Fertility and sterility.
2006;86(1):210-6.
146. Ben-Yosef D, Oron Y, Shalgi R. Low temperature and fertilization-induced Ca2+ changes in rat eggs. Molecular reproduction and development. 1995;42(1):122-9.
147. Inagaki N, Suzuki S, Kuji N, Kitai H, Nakatogawa N, Nozawa S. Egg activation induced by osmotic pressure change and the effects of amiloride on the cryopreservation of mouse oocytes. Molecular human reproduction. 1996;2(11):835-43.
148. Takeo T, Nakagata N. Reduced glutathione enhances fertility of frozen/thawed
C57BL/6 mouse sperm after exposure to methyl-beta-cyclodextrin. Biology of reproduction. 2011;85(5):1066-72.
149. Nakagata N. Cryopreservation of mouse spermatozoa and in vitro fertilization.
Methods in molecular biology (Clifton, NJ). 2011;693:57-73.
150. Takeo T, Hoshii T, Kondo Y, Toyodome H, Arima H, Yamamura K, et al.
Methyl-beta-cyclodextrin improves fertilizing ability of C57BL/6 mouse sperm after freezing and thawing by facilitating cholesterol efflux from the cells. Biology of reproduction. 2008;78(3):546-51.
151. Ho Y, Wigglesworth K, Eppig JJ, Schultz RM. Preimplantation development of mouse embryos in KSOM: augmentation by amino acids and analysis of gene expression.
Molecular reproduction and development. 1995;41(2):232-8.
152. Summers MC. A brief history of the development of the KSOM family of media.
Journal of assisted reproduction and genetics. 2013;30(8):995-9.
140
153. Leary S, Underwood W, Anthony R, Corey D, Grandin T, Gwaltney-Brant S.
AVMA Guidelines for the humane sluaghter of animals: 2016 Edition. Association
AVM, editor: AVMA. 2016:64.
154. Mohammed-Ali Z, Cruz GL, Lu C, Carlisle RE, Werner KE, Ask K, et al.
Development of a Model of Chronic Kidney Disease in the C57BL/6 Mouse with
Properties of Progressive Human CKD. Biomed Res Int. 2015;2015:172302.
155. Huang L, Scarpellini A, Funck M, Verderio EA, Johnson TS. Development of a chronic kidney disease model in C57BL/6 mice with relevance to human pathology.
Nephron Extra. 2013;3(1):12-29.
156. Fenwick N, Danielson P, Griffin G. Survey of Canadian animal-based researchers' views on the Three Rs: replacement, reduction and refinement. PloS one.
2011;6(8):e22478-e.
157. Hyttel P, Vajta G, Callesen H. Vitrification of bovine oocytes with the open pulled straw method: ultrastructural consequences. Molecular reproduction and development. 2000;56(1):80-8.
158. Howell RL, Donegan CL, Pinkert CA. Mouse embryo yield and viability after euthanasia by CO2 inhalation or cervical dislocation. Comparative medicine.
2003;53(5):510-3.
159. Varisli O, Uguz C, Agca C, Agca Y. Various physical stress factors on rat sperm motility, integrity of acrosome, and plasma membrane. Journal of andrology.
2009;30(1):75-86.
141
160. Li SJ, Su WD, Qiu LJ, Wang X, Liu J. [Resveratrol protects human sperm against cryopreservation-induced injury]. Zhonghua nan ke xue = National journal of andrology.
2018;24(6):499-503.
161. Fuku EJ, Liu J, Downey BR. In vitro viability and ultrastructural changes in bovine oocytes treated with a vitrification solution. Molecular reproduction and development. 1995;40(2):177-85.
162. Jackson KV, Kiessling AA. Fertilization and cleavage of mouse oocytes exposed to the conditions of human oocyte retrieval for in vitro fertilization. Fertility and sterility.
1989;51(4):675-81.
163. George MA, Doe BG. The influence of handling procedures during mouse oocyte and embryo recovery on viability and subsequent development in vitro. Journal of in vitro fertilization and embryo transfer : IVF. 1989;6(2):69-72.
164. Stetson I, Aviles M, Moros C, Garcia-Vazquez FA, Gimeno L, Torrecillas A, et al. Four glycoproteins are expressed in the cat zona pellucida. Theriogenology.
2015;83(7):1162-73.
165. Moros-Nicolas C, Chevret P, Izquierdo-Rico MJ, Holt WV, Esteban-Diaz D,
Lopez-Bejar M, et al. Composition of marsupial zona pellucida: a molecular and phylogenetic approach. Reproduction, fertility, and development. 2017.
166. Bertrand E, Van Den Bergh M, Englert Y. Fertilization and early embryology:
Does zona pellucida thickness influence the fertilization rate? Human Reproduction.
1995;10(5):1189-93.
142
167. Marco-Jimenez F, Naturil-Alfonso C, Jimenez-Trigos E, Lavara R, Vicente JS.
Influence of zona pellucida thickness on fertilization, embryo implantation and birth.
Animal reproduction science. 2012;132(1-2):96-100.
168. Ten J, Mendiola J, Vioque J, de Juan J, Bernabeu R. Donor oocyte dysmorphisms and their influence on fertilization and embryo quality. Reproductive biomedicine online.
2007;14(1):40-8.
169. Hasegawa A, Mochida K, Matoba S, Yonezawa K, Ohta A, Watanabe G, et al.
Efficient production of offspring from Japanese wild-derived strains of mice (Mus musculus molossinus) by improved assisted reproductive technologies. Biology of reproduction. 2012;86(5):167, 1-7.
170. Lenart P, Bacher CP, Daigle N, Hand AR, Eils R, Terasaki M, et al. A contractile nuclear actin network drives chromosome congression in oocytes. Nature.
2005;436(7052):812-8.
171. Takeo T, Nakagata N. Superovulation using the combined administration of inhibin antiserum and equine chorionic gonadotropin increases the number of ovulated oocytes in C57BL/6 female mice. PloS one. 2015;10(5):e0128330.
172. Fuku E, Xia L, Downey BR. Ultrastructural changes in bovine oocytes cryopreserved by vitrification. Cryobiology. 1995;32(2):139-56.
173. Bianchi V, Macchiarelli G, Borini A, Lappi M, Cecconi S, Miglietta S, et al. Fine morphological assessment of quality of human mature oocytes after slow freezing or vitrification with a closed device: a comparative analysis. Reproductive biology and endocrinology : RB&E. 2014;12:110.
143
174. Marco-Jimenez F, Casares-Crespo L, Vicente JS. Porcine oocyte vitrification in optimized low toxicity solution with open pulled straws. Zygote (Cambridge, England).
2014;22(2):204-12.
175. Jones A, Van Blerkom J, Davis P, Toledo AA. Cryopreservation of metaphase II human oocytes effects mitochondrial membrane potential: implications for developmental competence. Human reproduction (Oxford, England). 2004;19(8):1861-6.
176. Chen ZJ, Li M, Ma JL, Li Y, Ma SY, Gao X. [The cryopreservation of human oocytes at different maturity stages]. Beijing Da Xue Xue Bao Yi Xue Ban.
2004;36(6):571-4.
177. Kola I, Kirby C, Shaw J, Davey A, Trounson A. Vitrification of mouse oocytes results in aneuploid zygotes and malformed fetuses. Teratology. 1988;38(5):467-74.
178. Wassarman PM, Litscher ES. Mammalian fertilization: the egg's multifunctional zona pellucida. The International journal of developmental biology. 2008;52(5-6):665-76.
179. Wassarman PM, Albertini. DF. "The Mammalian Ovum." The Physiology of
Reproduction (2nd edition). 1994:79-122.
180. Eroglu A, Toth TL, Toner M. Alterations of the cytoskeleton and polyploidy induced by cryopreservation of metaphase II mouse oocytes. Fertility and sterility.
1998;69(5):944-57.
181. Lei T, Guo N, Tan MH, Li YF. Effect of mouse oocyte vitrification on mitochondrial membrane potential and distribution. J Huazhong Univ Sci Technolog
Med Sci. 2014;34(1):99-102.
144
182. Dai J, Wu C, Muneri CW, Niu Y, Zhang S, Rui R, et al. Changes in mitochondrial function in porcine vitrified MII-stage oocytes and their impacts on apoptosis and developmental ability. Cryobiology. 2015;71(2):291-8.
183. Zhou D, Shen X, Gu Y, Zhang N, Li T, Wu X, et al. Effects of dimethyl sulfoxide on asymmetric division and cytokinesis in mouse oocytes. BMC Developmental Biology.
2014;14(1):28.
184. Marques CC, Santos-Silva C, Rodrigues C, Matos JE, Moura T, Baptista MC, et al. Bovine oocyte membrane permeability and cryosurvival: Effects of different cryoprotectants and calcium in the vitrification media. Cryobiology. 2018;81:4-11.
185. Nottola SA, Albani E, Coticchio G, Palmerini MG, Lorenzo C, Scaravelli G, et al.
Freeze/thaw stress induces organelle remodeling and membrane recycling in cryopreserved human mature oocytes. Journal of Assisted Reproduction and Genetics.
2016;33(12):1559-70.
186. Agca Y, Liu J, Critser ES, McGrath JJ, Critser JK. Temperature-dependent osmotic behavior of germinal vesicle and metaphase II stage bovine oocytes in the presence of Me2SO in relationship to cryobiology. Molecular reproduction and development. 1999;53(1):59-67.
187. Agca Y, Liu J, McGrath JJ, Peter AT, Critser ES, Critser JK. Membrane permeability characteristics of metaphase II mouse oocytes at various temperatures in the presence of Me2SO. Cryobiology. 1998;36(4):287-300.
188. Mullen SF, Agca Y, Critser JK. Modeling the Probability of MII Spindle
Disruption in Bovine Oocytes as a Function of Total Osmolality Using Logistic
145
Regression and Its Application toward Improved CPA Addition and Removal
Procedures. Cell Preservation Technology. 2004;2(2):145-55.
189. Matson PL, Graefling J, Junk SM, Yovich JL, Edirisinghe WR. Cryopreservation of oocytes and embryos: use of a mouse model to investigate effects upon zona hardness and formulate treatment strategies in an in-vitro fertilization programme. Human
Reproduction. 1997;12(7):1550-3.
190. Cheng KR, Fu XW, Zhang RN, Jia GX, Hou YP, Zhu SE. Effect of oocyte vitrification on deoxyribonucleic acid methylation of H19, Peg3, and Snrpn differentially methylated regions in mouse blastocysts. Fertility & Sterility. 2014;102(4):1183-90.e3.
191. Nottola SA, Coticchio G, De Santis L, Macchiarelli G, Maione M, Bianchi S, et al. Ultrastructure of human mature oocytes after slow cooling cryopreservation with ethylene glycol. Reproductive biomedicine online. 2008;17(3):368-77.
192. Nottola SA, Coticchio G, Sciajno R, Gambardella A, Maione M, Scaravelli G, et al. Ultrastructural markers of quality in human mature oocytes vitrified using cryoleaf and cryoloop. Reproductive biomedicine online. 2009;19 Suppl 3:17-27.
193. Nottola SA, Macchiarelli G, Coticchio G, Bianchi S, Cecconi S, De Santis L, et al. Ultrastructure of human mature oocytes after slow cooling cryopreservation using different sucrose concentrations. Human reproduction (Oxford, England).
2007;22(4):1123-33.
194. Zhou CJ, Wang DH, Niu XX, Kong XW, Li YJ, Ren J, et al. High survival of mouse oocytes using an optimized vitrification protocol. Scientific Reports. 2016;6.
146
195. Jones A, Van Blerkom J, Davis P, Toledo AA. Cryopreservation of metaphase II human oocytes effects mitochondrial membrane potential: implications for developmental competence. Human Reproduction. 2004;19(8):1861-6.
196. Chang CC, Lin CJ, Sung LY, Kort HI, Tian XC, Nagy ZP. Impact of phase transition on the mouse oocyte spindle during vitrification. Reproductive biomedicine online. 2011;22(2):184-91.
197. Chang C-C, Nel-Themaat L, Nagy ZP. Cryopreservation of oocytes in experimental models. Reproductive biomedicine online. 2011;23(3):307-13.
198. Stimpfel M, Vrtacnik-Bokal E, Virant-Klun I. No difference in mitochondrial distribution is observed in human oocytes after cryopreservation. Arch Gynecol Obstet.
2017;296(2):373-81.
199. Sowinska N, Muller K, Nizanski W, Jewgenow K. Mitochondrial characteristics in oocytes of the domestic cat (Felis catus) after in vitro maturation and vitrification.
Reproduction in domestic animals = Zuchthygiene. 2017;52(5):806-13.
200. Mazur P, Pinn IL, Kleinhans FW. Intracellular ice formation in mouse oocytes subjected to interrupted rapid cooling. Cryobiology. 2007;55(2):158-66.
201. Fahy GM, MacFarlane D, Angell C, Meryman H. Vitrification as an approach to cryopreservation. Cryobiology. 1984;21(4):407-26.
202. Wassarman PM. Mammalian fertilization: molecular aspects of gamete adhesion, exocytosis, and fusion. Cell. 1999;96(2):175-83.
203. Gadde S, Heald R. Mechanisms and molecules of the mitotic spindle. Curr Biol.
2004;14(18):R797-805.
147
204. Dumont J, Desai A. Acentrosomal Spindle Assembly & Chromosome
Segregation During Oocyte Meiosis. Trends in cell biology. 2012;22(5):241-9.
205. Keefe D, Liu L, Wang W, Silva C. Imaging meiotic spindles by polarization light microscopy: Principles and applications to IVF. Reproductive biomedicine online.
2003;7(1):24-9.
206. Magistrini M, Szollosi D. Effects of cold and of isopropyl-N-phenylcarbamate on the second meiotic spindle of mouse oocytes. European Journal of Cell Biology.
1980;22(2):699-707.
207. Pickering SJ, Johnson MH. The influence of cooling on the oranization of meiotic spindle of the mouse oocyte. Human Reproduction. 1987;2(3):207-16.
208. Sathananthan AH, Kirby C, Trounson A, Philipatos D, Shaw J. The effects of cooling mouse oocytes. Journal of Assisted Reproduction and Genetics. 1992;9(2):139-
48.
209. Van Der Elst J, Van Den Abbeel E, Jacobs R, Wisse E, Van Steirteghem A. Effect of 1,2-propanediol and dimethylsulphoxide on the meiotic spindle of the mouse oocyte.
Human Reproduction. 1988;3(8):960-7.
210. Wang WH, Meng L, Hackett RJ, Odenbourg R, Keefe DL. Limited recovery of meiotic spindles in living human oocytes after cooling-rewarming observed using polarized light microscopy. Human Reproduction. 2001;16(11):2374-8.
211. Van der Elst J, Van den Abbeel E, Jacobs R, Wisse E, Van Steirteghem A. Effect of 1,2-propanediol and dimethylsulphoxide on the meiotic spindle of the mouse oocyte.
Human reproduction (Oxford, England). 1988;3(8):960-7.
148
212. George MA, Johnson MH. Cytoskeletal organization and zona sensitivity to digestion by chymotrypsin of frozen-thawed mouse oocytes. Human Reproduction.
1993;8(4):612-20.
213. Joly C, Bchini O, Boulekbache H, Testart J, Maro B. Effects of 1,2-propanediol on the cytoskeletal organization of the mouse oocyte. Human reproduction (Oxford,
England). 1992;7(3):374-8.
214. Chang CC, Sung LY, Lin CJ, Kort HI, Yang X, Tian XC, et al. The oocyte spindle is preserved by 1,2-propanediol during slow freezing. Fertility and sterility.
2010;93(5):1430-9.
215. Dumollard R, Duchen M, Carroll J. The role of mitochondrial function in the oocyte and embryo. Current topics in developmental biology. 2007;77:21-49.
216. Gutierrez-Aguilar M, Douglas DL, Gibson AK, Domeier TL, Molkentin JD,
Baines CP. Genetic manipulation of the cardiac mitochondrial phosphate carrier does not affect permeability transition. J Mol Cell Cardiol. 2014;72:316-25.
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VITA
Liga Wuri is from Inner Mongolia, China. She was born in a very conventional
Mongolian family. She speaks Mongolian, Chinese (Mandarin) and English. She went to traditional Mongolian school from primary school through her undergraduate. She did her undergraduate in Mongolian majored in Animal Science at Inner Mongolia Agricultural
University from 2006-2010. Before she finished her undergraduate, due to her outstanding academic performance, she was only one in her class who was recommended by her department to pursue her master’s degree in the area of animal genetics, reproduction and breeding at Inner Mongolia Agricultural University with the exempt of national graduate student entry examination.
In 2013, she received her M.S. from Inner Mongolia Agricultural University. She volunteered in the rural communities and farms for two years in Inner Mongolia before she move to Columbia, Missouri in May 2015 to pursue her Ph.D in animal sciences at
Mizzou.
In 2018, she will complete her doctorate degree in Animal Sciences from University of
Missouri-Columbia.
150