EFFECTS OF CONTROLLED
BREEDING ON CERVICOVAGINAL
MUCUS AND FERTILITY OF THE
MERINO EWE
Jessie Ward Maddison
A thesis submitted in fulfilment of the requirements for the Degree of Doctor of Philosophy
Faculty of Veterinary Science, The University of Sydney
© 2017 Declaration
Apart from the assistance mentioned in the acknowledgements, the studies contained within this thesis were planned and executed by the author, and have not been previously submitted for any degree to a University or Institution.
Jessie Maddison
B An Vet Bio Sci (Hon I)
Author Attribution Statement
Chapter 2 of this thesis is published as Maddison, JW, Rickard, JP, Mooney, E, Bernecic, NC,
Soleilhavoup, C, Tsikis, G, Druart, X, Leahy, T & de Graaf, SP 2016, 'Oestrus synchronisation and superovulation alter the production and biochemical constituents of ovine cervicovaginal mucus', Animal Reproduction Science, vol. 172, no., pp. 114-122.
I assisted in the design of the study, carried out the experiments, analysed the data and wrote the drafts of the MS.
Chapter 3 of this these is published as Maddison, JW, Rickard, JP, Bernecic, NC, Tsikis, G,
Soleilhavoup, C, Labas, V, Combes-Soia, L, Harichaux, G, Druart, X, Leahy, T & de Graaf,
SP 2017, 'Oestrus synchronisation and superovulation alter the cervicovaginal mucus proteome of the ewe', Journal of Proteomics, vol. 155, no., pp. 1-10.
I assisted in the design of the study, carried out the experiments, analysed the data and wrote the drafts of the MS.
i Acknowledgements
To Simon, Tamara, Xavier and Jess, the supervisor team of the century, thank you all for your support, guidance, patience and encouragement throughout this journey. Simon, thank you, I cannot express how grateful I am for the opportunity you have given me by inviting me into the repro group and setting me on this crazy wonderful experience, I will be forever grateful for the opportunity and I hope that you can be proud of the work we accomplished. Although
I feel I may always be, as you put it…a chicken little…this amazing PhD ride has forever changed me, my confidence in myself as well as directed my journey through life, so I must thank you for what it has brought me, and will continue to do, throughout my life. Tamara, your editing skills, stickers, supportive comments and solutions to sticky experimental issues, of which there have been many, have helped me so much. You brought a new life to the group and I am so glad to have had your support. Xavier, thank you for welcoming me into your lab in France, as well as your home, not once but twice, I had such an amazing time with you all at INRA and appreciate all your patience with me and showing me the French way! Coffees, food, comics, it was an absolute blast, even if all our westerns will never see the light of day!!
Jess, what can I say, you have been such a big part of this journey, from being the honours boss till now, my supervisor. You have helped me with it all and I really treasure the friendship we have developed over these last 5 years. Thanks for your words of wisdom, encouragement, and your unwavering support and belief in me, all the laughs, and a few crazy sleep deprived tears too, Daft Punk will forever remind me of mucus and you # Team Ram!!.
To the other PhD students and now Drs, Danielle and Cassie, thank you all so much for welcoming me into the fold in those early years, you made the transition from honours to PhD
ii life much more enjoyable and manageable with all your wise words and anal lab tendencies!!
Which I will proudly carry on into my new work adventures. To the new kids in town,
Dannielle, Cameron, Taylor and even my own young padawan Naomi, it took a while to adjust to the new office situation but I am so thankful for these last years with you all, our 2:30 pm life chats and animal video distractions will be missed. To my two honours students; Ethan and
Naomi, it was a ride and thank you both for all your hard work, we did good kids. Thank you all for being willing to drop it all and help me collect mucus, wrangle sheep and make lamb babies across the countryside, rain, hail or shine, and at all hours of the day too, I couldn’t have done it without you all.
To the rest of the repro team, Ros and Angela, thank you for all the wise words from down the hall, they will be missed!! To the Camden boys, past and present, Byron, Keith, Sam and
Cameron, thank you for being so accommodating with my requests, you made my life so much easier and I appreciate you all going above and beyond for me time and time again. To the lab veterans, Andrew and Kim, thank you both for all your help and tolerance, of yet another PhD student to come in and take over the lab, your knowledge and skill were crucial over the years for me in many situations. Kim your countless hours running bloods for me, answering all manner of questions and hallway chats have helped me get through these years, so a special thanks to you! To the French boys, Guilluame and Clement, you were both so welcoming and helpful during both of my INRA visits, I have many fond memories of my time there, I dedicate all my westerns to you both! A special thankyou has to go out to my Team Ram buddies….You know who you are. We started as an awesome foursome and have grown to the dominating force it is today, you are a special bunch of people and I am very proud to have been a part of it all with you.
iii Last but by no means least, I would like to thank my friends and family for all their support throughout my PhD, especially to Brad and my father Anthony. Bradley James, thank you for supporting and encouraging me throughout this crazy ride, even if it meant me occasionally checking out of our life, not making numerous occasions of climbing and weekend frivolities, your love has helped me so much the last few years, muchos gracias mi amore.
Pa, my rock, thank you for the numerous chats, cries, laughs, pep talks and also for just caring about me and all of this so so much, to have you backing me through this whole uni journey of mine has been immeasurably important to me and I am so very grateful that you are my dad. I couldn’t have done it, any of it without you..smashalacken is complete!! You have both lived this mucus-laden journey from the get go and we can celebrate its completion together!!
iv Summary
This thesis examines the impact of oestrus synchronisation and superovulation on the cervicovaginal (CV) mucus of ewes during their oestrous cycle. The quantity, biochemistry and protein composition of ovine CV mucus produced under these controlled breeding regimes are investigated. Artificial methods of mucus modification (in vitro) and their impact on sperm interaction with this important biological fluid are also examined.
The abundance, composition, physiochemical characteristics and proteome of cervicovaginal mucus collected during oestrus and the luteal phase were compared between naturally cycling, progesterone synchronised, superovulated and prostaglandin-F2α synchronised Merino ewes.
Oestrus CV mucus was more abundant, clearer in colour and less proteinaceous than luteal phase mucus. Superovulation resulted in a marked increase in CV mucus production and total protein concentration while synchronisation using Prostandin-F2a significantly reduced mucus volume. Contrary to popular theory, mucus pH (oestrus 6.2-6.5), biochemical profile and penetration by spermatozoa were largely unchanged by superovulation and/or oestrus synchronisation. Quantitative and qualitative proteomic analysis of mucus identified 60 proteins to be more abundant during oestrus, whilst 127 were more abundant during the luteal phase. Furthermore, specific proteins were found to be only present during oestrus (27 proteins; one example being the mucolytic enzyme neuraminidase (NEU1)) or the luteal phase (40 proteins). Superovulation and/or oestrus synchronisation also greatly altered the proteome of
CV mucus increasing and decreasing the abundance of a variety of proteins during oestrus when compared to CV mucus obtained during the oestrus of naturally cycling ewes. This study represents the most comprehensive proteomic description of the changes to cervicovaginal
v mucus of the ewe both over the oestrous cycle and the first to detail the effects of controlled breeding practices on the CV mucus proteome.
The effects of proteomic changes of key structural mucus proteins was investigated in an in vitro setting to ascertain if such changes might influence sperm motility and viability when incubated in a cervicovaginal mucus simulant (CMS). Specifically, variation in the percent composition of mucin, the gel forming protein which provides mucus with its structural characteristics, and variation in the amount of the enzyme neuraminidase (NEU1) which acts to denature mucin proteins and alter the gel structure of mucus, was investigated. Increasing mucin levels of a CMS caused increased mucus viscosity, but interestingly did not affect sperm motility, and resulted in more frozen-thawed spermatozoa migrating through CMS. The inclusion of mucin into CMS resulted in altered sperm velocity, but not always in a dose dependent manner. Spermatozoa had reduced membrane damage and reacted acrosomes when incubated in CMS with 2% or 4% mucins, or CMS pre-treated with NEU1, compared to CMS containing no NEU1 or mucins. These results show that variable mucin and neuraminidase concentrations alters the viscosity of a cervicovaginal mucus simulant, the motility and viability of spermatozoa, and the mucus migrating ability of frozen-thawed spermatozoa.
This thesis culminated in an investigation of hormonal oestrus synchronisation (Progesterone or Prostaglandin-F2a) on the ability of fresh, chilled and cryopreserved ram spermatozoa to traverse the cervix. Ewes synchronised using prostaglandin-F2α had significantly lower pregnancy rates compared to both naturally cycling ewes and progesterone synchronised ewes
(6%, 17% and 25%, respectively). Unexpectedly, progesterone synchronised ewes had comparable pregnancy rates to naturally cycling ewes. While ewes inseminated with cryopreserved semen had lower pregnancy rates than those inseminated with chilled or fresh
vi semen, differences were not significant (10%, 20% and 17%, respectively), probably due to the overall low pregnancy rate. No significant interactions between semen type (fresh, chilled or cryopreserved semen) and synchronisation method (NAT, P4 or PGF2α) were identified.
Whilst not all results followed expected trends, they undoubtedly indicate that oestrus synchronisation using prostaglandin-F2α markedly limits the ability of ram spermatozoa of any type to traverse the cervix, as indicated by fertility data.
Combined, the findings of this thesis confirm that use of exogenous hormones for both oestrus synchronisation and superovulation procedures result in altered cervicovaginal mucus production and characteristics. Specifically, exogenous hormones result in a significantly altered cervicovaginal environment, which may contribute to the decreased ability of spermatozoa to traverse the cervix in vivo. Whilst results reported herein clearly show that exogenous hormone usage for common controlled breeding practices alter the cervical environment and in some cases fertility of ewe, further research is necessary to identify other causes of the limited fertility usually achieved following cervical artificial insemination with cryopreserved ram spermatozoa. Success of this endeavour would hopefully allow for the
Australian sheep industry to fully capitalise on the rapid genetic improvement made possible through the widespread use of artificial insemination.
vii Table of Contents
DECLARATION ...... I
AUTHOR ATTRIBUTION STATEMENT ...... I
ACKNOWLEDGEMENTS ...... II
SUMMARY ...... V
LIST OF ABBREVIATIONS ...... XII
LIST OF TABLES ...... XVI
LIST OF FIGURES ...... XVII
LIST OF PUBLICATIONS ...... XXIII
CHAPTER 1 THE OVINE CERVIX AND ITS SECRETIONS: A REVIEW...... 25 1.1. ABSTRACT ...... 25 1.2. INTRODUCTION ...... 26
1.3. THE OVINE CERVIX ...... 27 1.3.1. Anatomy ...... 27 1.3.2. Influence of cervical anatomy on artificial insemination ...... 29 1.4. CERVICAL AND CERVICOVAGINAL MUCUS ...... 34 1.4.1. Composition ...... 34 1.4.2. Mucus pH ...... 35 1.4.3. Mucus proteome ...... 36 1.4.4. Mucins; their production and release ...... 37 1.4.5. The function of cervical and cervicovaginal mucus ...... 40 1.5. VARIATION IN CERVICAL AND CERVICOVAGINAL MUCUS ...... 44 1.5.1. Across the oestrous cycle ...... 44 1.5.2. Effect of controlled breeding practices ...... 47 1.6. INTERACTION OF SPERMATOZOA WITH THE CERVIX ...... 49
1.7. CONCLUDING REMARKS AND OBJECTIVES OF THE CURRENT STUDY ...... 51
CHAPTER 2. OESTRUS SYNCHRONISATION AND SUPEROVULATION ALTER THE PRODUCTION AND BIOCHEMICAL CONSTITUENTS OF OVINE CERVICOVAGINAL MUCUS . 54 2.1. ABSTRACT ...... 54
viii 2.1.1. KEY WORDS ...... 55
2.2. INTRODUCTION ...... 55
2.3. MATERIALS AND METHODS ...... 57
2.3.1. EXPERIMENTAL DESIGN ...... 57
2.3.2. HORMONE ADMINISTRATION ...... 58
2.3.3. OESTRUS DETECTION ...... 59
2.3.4. CERVICAL MUCUS COLLECTION AND HANDLING ...... 60
2.3.5. ASSESSMENT OF CERVICAL MUCUS CHARACTERISTICS ...... 61
2.3.6. CHEMICAL ASSESSMENTS ...... 61
2.3.7. PROTEIN CONCENTRATION ...... 62
2.3.8. PH ...... 62
2.3.9. SPERM MIGRATION ...... 62
2.3.10. STATISTICAL ANALYSIS ...... 63
2.4. RESULTS ...... 64
2.4.1. VOLUME ...... 64
2.4.2. COLOUR ...... 65
2.4.3. SPINNBARKEIT ...... 66
2.4.4. CHEMICAL ASSESSMENT ...... 66
2.4.5. PROTEIN CONCENTRATION ...... 67
2.4.6. PH ...... 67
2.4.7. SPERM MIGRATION IN CV MUCUS ...... 68
2.5. DISCUSSION ...... 68
2.5.1. MUCUS VOLUME INCREASES DURING THE FOLLICULAR PHASE AND IS ALTERED BY
SUPEROVULATION AND PROSTAGLANDIN-F2Α SYNCHRONISATION ...... 69
2.5.2. PROTEIN CONCENTRATION PEAKS DURING THE LUTEAL PHASE AND AFTER
SUPEROVULATION ...... 71
2.5.3. MUCUS COLOUR, SPINNBARKEIT AND PENETRABILITY; OVER THE OESTROUS CYCLES
AND AFTER SYNCHRONISATION AND SUPEROVULATION ...... 73
2.5.4. MUCUS PH IS UNALTERED BY EXOGENOUS HORMONES ...... 75
2.6. ACKNOWLEDGEMENTS ...... 77
CHAPTER 3. OESTRUS SYNCHRONISATION AND SUPEROVULATION ALTER THE CERVICOVAGINAL MUCUS PROTEOME OF THE EWE ...... 78 3.1. ABSTRACT ...... 78
ix 3.2. INTRODUCTION ...... 79
3.3. MATERIALS AND METHODS ...... 81
3.3.1. HORMONE ADMINISTRATION ...... 81
3.3.2. OESTRUS DETECTION ...... 82
3.3.3. CERVICOVAGINAL MUCUS COLLECTION ...... 83
3.3.4. SAMPLE PREPARATION ...... 83
3.3.5. 1D-SDS PAGE OF QUALITATIVE SAMPLES ...... 84
3.3.6. PROTEIN DIGESTION...... 84
3.3.6.1 IN-GEL DIGESTION OF QUALITATIVE SAMPLES ...... 84
3.3.6.1 IN-SOLUTION DIGESTION OF QUANTITATIVE SAMPLES ...... 85
3.3.7. NANOLC- MS/MS ...... 85
3.3.8. PROTEIN IDENTIFICATION AND VALIDATION ...... 86
3.3.9. LABEL-FREE PROTEIN QUANTIFICATION USING SPECTRAL COUNTING ...... 86
3.3.10. MOLECULAR FUNCTION ANALYSIS ...... 87
3.4. RESULTS ...... 87
3.4.1. QUALITATIVE WHOLE OESTROUS CYCLE ANALYSIS OF MUCUS ...... 87
3.4.2. QUANTITATIVE COMPARISON OF OESTRUS AND MID-LUTEAL PHASE MUCUS ...... 89
3.4.3. QUANTITATIVE COMPARISON OF NAT, P4 AND SOV MUCUS PRODUCED DURING
OESTRUS ...... 94
3.5. DISCUSSION ...... 96
3.6. ACKNOWLEDGEMENTS ...... 104
CHAPTER 4. MUCIN CONTENT AND NEURAMINIDASE IMPACTS CERVICOVAGINAL MUCUS VISCOSITY AND RAM SPERM MOTILITY, MIGRATING ABILITY AND VIABILITY...... 106 4.1. ABSTRACT ...... 106
4.1.1. KEYWORDS ...... 107
4.2. INTRODUCTION ...... 107
4.3. MATERIALS AND METHODS ...... 110
4.3.1. EXPERIMENTAL DESIGN ...... 110
4.3.2. ARTIFICIAL MUCUS ...... 110
4.3.3. SEMEN COLLECTION AND FREEZING ...... 111
4.3.4. VISCOSITY OF CMS ...... 112
4.3.5. SPERM MIGRATION IN COMPOSITIONALLY ALTERED MUCUS ...... 112
4.3.6. MOTILITY AND KINEMATIC PARAMETERS ...... 113
x 4.3.7. MEMBRANE VIABILITY AND ACROSOME INTEGRITY ...... 114
4.3.8. STATISTICAL ANALYSIS ...... 114
4.4. RESULTS ...... 115
4.4.1. VISCOSITY ...... 115
4.4.2. SPERM MIGRATION IN MUCUS ...... 117
4.4.3. MOTILITY PARAMETERS ...... 118
4.4.4. MEMBRANE VIABILITY AND ACROSOME INTEGRITY ...... 121
4.5. DISCUSSION ...... 123
4.6. ACKNOWLEDGEMENTS ...... 127
CHAPTER 5. OESTRUS SYNCHRONISATION USING PROSTAGLANDIN-F2Α DECREASES FERTILITY IN THE MERINO EWE ...... 128 5.1. ABSTRACT ...... 128
5.2. INTRODUCTION ...... 129
5.3. MATERIALS AND METHODS ...... 130
5.3.1. ANIMALS ...... 131
5.3.2. HORMONE ADMINISTRATION ...... 131
5.3.3. OESTRUS DETECTION ...... 133
5.3.4. SEMEN COLLECTION AND PROCESSING ...... 133
5.3.5. INSEMINATION ...... 134
5.3.6. STATISTICAL ANALYSIS ...... 135
5.4. RESULTS ...... 135
5.5. DISCUSSION ...... 139
5.6. ACKNOWLEDGEMENTS ...... 143
CHAPTER 6. GENERAL DISCUSSION AND CONCLUSIONS ...... 144
REFERENCES ...... 152
APPENDIX 1: CONFERENCE PROCEEDINGS ...... 176
APPENDIX 2: SUPPLEMENTARY FILES ...... 179
xi
List of Abbreviations
°C degrees centigrade
1D-PAGE one-dimensional polyacrylamide gel electrophoresis
2D-PAGE two-dimensional polyacrylamide gel electrophoresis
AI artificial insemination
AM artificial mucus
ALH amplitude of lateral head displacement
ANOVA analysis of variance
AV artificial vagina
BCF beat cross frequency
BCS body condition score
BSA bovine serum albumin
CASA computer aided sperm analysis drug release device
CIDR controlled internal drug releasing device
CHILLED ram spermatozoa chilled to 5°C
CMS cervicovaginal mucus simulant
CMMT cervical mucus migration test cp centipoise
CV cervicovaginal cm centimetres d days
xii Da Daltons
FITC/PNA fluorescein isothiocyanate conjugated peanut agglutinin
FRESH fresh ram spermatozoa
FROZEN cryopreserved ram spermatozoa
FSH follicle stimulating hormone g grams
GLMM generalised liner mixed model
HIV human immunodeficiency virus h hours
ID internal diameter
IU international units
IVF in vitro fertilisation
KDa kilodalton
LAP AI laparoscopic intrauterine artificial insemination
LIN linearity mg milligrams min minimum mL mililitres mm millimetres mM millimolar
MW Molecular weight m/z mass to charge ratio
NAT naturally cycling
NEU1 neuraminidase-1 ng/µl nanograms per microlitre
xiii ng/ml nanograms per millilitre nm nanometres
NWS normalised weighted spectra
PANTHER Protein ANalysis THrough Evolutionary Relationships
Pa. s pascal second
PBS phosphate buffered saline
PEN penicillamine
PGF2α prostaglandin F2-alpha pH hydrogen ion concentration, -log 10 of
PI propidium iodide
PMSG pregnant mare serum gonadotrophin ppm parts per million
P4 progesterone
REML residual maximum likelihood rpm revolutions per minute
RT room/ambient temperature
S.E.M standard error of the mean
SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
SOV superovulated
STR straightness
TALP Tyrode’s medium with albumin, lactate and pyruvate
TCAI transcervical artificial insemination
UVJ uterovaginal junction
μ (prefix) micro (× 10-6)
µl micro litres
xiv µm micro meters
µg/ml micro gram per litre v/v volume/volume
VAP average path velocity
VCL curvilinear velocity vs versus
VSL straight-line velocity w/v weight/volume
xv List of Tables
2.1 Levels of sodium (Na), magnesium (Mg), calcium (Ca), potassium (K) and chloride
(Cl) in the cervicovaginal mucus of naturally cycling (NAT), progesterone
synchronised (P4), superovulated (SOV) and prostaglandin-F2α synchronised (PGF2α)
ewes during oestrus (n=5 ewes/treatment, N=8
aspirations/ewe).……………………………………………………………...... 67
3.1 Proteins (NWS >5, fold change between oestrus and luteal NWS ≥1.5) identified within
ovine cervicovaginal mucus found to be significantly (p <0.05) more abundant during
oestrus when compared to the mid-luteal phase……………………………...... 89
3.2 Proteins identified within ovine cervicovaginal mucus found only during oestrus…..93
5.1 Pregnancy rates of naturally cycling (NAT), progesterone synchronised (P4) and
prostaglandin-F2α (PGF2α) synchronised ewes after intracervical artificial insemination
with fresh ram semen (FRESH), ram semen chilled for 12 h at 5°C (CHILLED) and
frozen-thawed ram semen (FROZEN)……………………………………………...137
xvi List of Figures
1.1 Figure 1 Classification of common external os types of the ovine cervix; (a) duckbill,
(b) slit, (c) rose, (d) papilla and (e) flap (Kershaw et al. 2005)………………………28
1.2 Silicone cast of the cervical lumen of the lamb cervix. Illustrates the caudally directed
rings/folds that comprise the cervix (Naqvi et al. 2005)……………………………..29
1.3 Classification of cervical grade in the ewe (a) grade 1, (b) grade 2 & (c) grade 3.
Illustrates the variation in misalignment and interdigitating of the cervical rings. Arrow
indicates direction and maximum insertion depth of an inseminating pipette (Kershaw
et al. 2005)………………………………………………………………...... 29
− 1.4 Proposed roles of bicarbonate ( HCO3 ) in mucin release from goblet cell mucin
granules, taken from Muchekehu and Quinton (2010)………………………………39
2.1 Average volume per ewe (a), colour (b), spinnbarkeit (c) and protein concentration (d)
of cervicovaginal mucus from naturally cycling (NAT; white bars), progesterone
synchronised (P4; light grey bars), superovulated (SOV; dark grey bars) and
prostaglandin-F2α synchronised (PGF2α; black bars) ewes (n=5 ewes/treatment) during
the follicular (N=24 aspirations/ewe) and luteal phases (N=8 aspirations/ewe) of the
oestrous cycle. Values without common superscripts differ significantly (p
<0.05)…………………………………………………………………………………65
2.2 pH of cervicovaginal mucus from naturally cycling (NAT; white bars), progesterone
synchronised (P4; light grey bars) and superovulated (SOV; dark grey bars) ewes (n=11
xvii ewes/treatment) during oestrus (N=2 observations/ewe) and the mid-luteal phase (N=2
observations) of the oestrous cycle. Values without common superscripts differ
significantly (p <0.05)……...…………………………………………………………68
3.1 Molecular functions and GO annotations of proteins identified in ovine cervicovaginal
mucus pooled from the entire oestrous cycle (follicular and luteal phases). Molecular
function and GO annotations determined by PANTHER analysis………………….88
3.2 Proteins (gene names listed) in the cervicovaginal mucus of ewes which differed markedly
in abundance between oestrus and the mid-luteal phase of the oestrous cycle (based on
fold change (FC) comparison of average normalised weighted spectra (NWS), FC 0 =
green, 5 = black, 35 ≥ red, 70 most disparate proteins displayed)……………………91
3.3 Proteins identified (gene names listed) in ovine cervicovaginal mucus of progesterone
synchronised (P4), and progesterone synchronised then superovulated (SOV) ewes that
were most disparate in abundance (based on fold change (FC) comparison of average
normalised weighted spectra (NWS), FC value; 0 = blue, red ≥ 15, grey = present only
in corresponding sample, 20 most disparate proteins displayed) when compared to
levels in mucus of naturally cycling ewes (NAT)……………………...... 95
3.4 Differences in molecular function (MF) of the proteins identified in ovine cervicovaginal
mucus significantly affected (p <0.05, Fold change ≤1.5) by progesterone
synchronisation (A; P4) and superovulation (B; SOV) in comparison to a natural cycle
(NAT). Proteins that were more abundant (red bar for each MF category) and less
xviii abundant (blue bar for each MF category) in comparison to levels in NAT mucus are
shown. Molecular function determined by PANTHER analysis...... 95
4.1 Viscosity of cervicovaginal mucus simulant (CMS) containing (A) varying percent of
mucins (black bar; CMS, dark grey bar; CMS with 1% mucins, light grey bar; CMS
with 2% mucins, lighter grey bar; CMS with 4% mucins), and (B) CMS pre-treated
with varying levels of Neuraminidase (NEU1) (black bar; CMS with no mucins or
NEU1, dark grey bar; CMS with 2% mucins and no NEU1, light grey bar; CMS with
2% mucins and 1 unit NEU1/ml CMS, and lighter grey bar; CMS with 2% mucins and
2 units NEU1/ml CMS). Significant differences between viscosity of CMS is denoted
by differing superscripts…………………………………………………………….115
4.2 Viscosity of cervicovaginal mucus simulant (CMS) following incubation with spermatozoa
(fresh and frozen-thawed results pooled) when CMS contains (A) varying percent of
mucins (black bar; CMS, dark grey bar; CMS with 1% mucins, light grey bar; CMS
with 2% mucins, lighter grey bar; CMS with 4% mucins), and (B) CMS pre-treated
with varying levels of Neuraminidase (NEU1) (black bar; CMS with no mucins or
NEU1, dark grey bar; CMS with 2% mucins and no NEU1, light grey bar; CMS with
2% mucins and 1 unit NEU1/ml CMS, and lighter grey bar; CMS with 2% mucins and
2 units NEU1/ml CMS). Significant differences between the viscosity of CMS
following incubation with spermatozoa is denoted by differing
superscripts……………………………………………………………………….....116
4.3 Concentration of fresh and frozen-thawed sperm that migrated into cervicovaginal mucus
simulant (CMS) containing (A) varying percent of mucins (black bar; 0% mucins, dark
xix grey bar; 1% mucins, light grey bar; 2% mucins, lighter grey bar; 4% mucins), and (B)
CMS pre-treated with varying levels of Neuraminidase (NEU1) ( black bar; no
MUCINS: artificial mucus media with no mucins or NEU1, dark grey bar; no
NEU1:CMS with 2% mucins and no NEU1, light grey bar; NEU1-1: CMS with 2%
mucins and 1 unit NEU1/ml CMS, and lighter grey bar; NEU1-2: CMS with 2% mucins
and 2 units NEU1/ml CMS). Significant difference in sperm concentration between
treatments denoted by differing superscripts for each graph………………………...117
4.4 Motility of sperm (fresh and frozen-thawed pooled) following incubation (1:1; v/v) with
cervicovaginal mucus simulant (CMS) containing (A) varying percent of mucins (black
bar; CMS, dark grey bar; CMS with 1% mucins, light grey bar; CMS with 2% mucins,
lighter grey bar; CMS with 4% mucins), and (B) CMS pre-treated with varying levels
of Neuraminidase (NEU1) (black bar; CMS with no mucins or NEU1, dark grey bar;
CMS with 2% mucins and no NEU1, light grey bar; CMS with 2% mucins and 1 unit
NEU1/ml CMS, and lighter grey bar; CMS with 2% mucins and 2 units NEU1/ml
CMS). Significant difference between motility of sperm in CMS denoted by differing
superscripts.…………………………………………………………………………119
4.5 Velocity parameters (average path velocity; solid line, straight line velocity; dotted line
and curvilinear velocity; dashed line) of sperm (fresh and frozen-thawed pooled)
incubated 1:1 (v/v) with cervicovaginal mucus simulant (CMS) containing (A) varying
percent content of mucins; CMS with no mucins (CMS), CMS with 1% mucins (CMS
+1% MUCINS), CMS with 2% mucins (CMS + 2% MUCINS) or CMS with 4% mucins
(CMS + 4% MUCINS), and (B) CMS pre-treated with or without varying levels of
Neuraminidase; CMS with no mucins or NEU1 (CMS), CMS with 2% mucins but no
xx NEU1 (CMS + 2% MUCINS), CMS with 2% mucins and 1 unit NEU1/ml CMS (CMS
+ 2% MUCINS + NEU1-1), or CMS with 2% mucins and 2 units NEU1/ml CMS (CMS
+ 2% MUCINS + NEU1-2). Significant differences between types of CMS denoted by
asterix superscript………………………...…………………………………………120
4.6 Percent of membrane viable/acrosome intact (solid line; PI-, FITC/PNA-), non-viable
membranes/acrosome intact (dashed line; PI+, FITC/PNA-) and non-viable
membranes/acrosome reacted (dotted line; PI+, FITC/PNA+) spermatozoa incubated 1:1
(v/v) in cervicovaginal mucus simulant (CMS) containing (A) varying percent content
of mucins; CMS with no mucins (CMS), CMS with 1% mucins (CMS +1% MUCINS),
CMS with 2% mucins (CMS + 2% MUCINS) or CMS with 4% mucins (CMS + 4%
MUCINS), and (B) CMS pre-treated with or without varying levels of Neuraminidase;
CMS with no mucins or NEU1 (CMS), CMS with 2% mucins but no NEU1 (CMS +
2% MUCINS), CMS with 2% mucins and 1 unit NEU1/ml CMS (CMS + 2% MUCINS
+ NEU1-1), or CMS with 2% mucins and 2 units NEU1/ml CMS (CMS + 2% MUCINS
+ NEU1-2). Significant difference in viability between types of CMS denoted by asterix
superscript for each experiment…………………………………………………..…123
5.1 Subjective Motility of fresh ram semen (FRESH; n = 32), ram semen chilled for 12 h at
5°C (CHILLED; n = 40) and frozen-thawed ram semen (FROZEN; n = 37) used for
intracervical and laparoscopic intrauterine artificial insemination. Values without
common superscripts differ significantly (p <0.05)………………………………...136
5.2 Pregnancy rates of naturally cycling (NAT; n = 106), progesterone synchronised (P4; n
=132) and prostaglandin-F2α synchronised (PGF2α; n = 126) merino ewes after
xxi intracervical artificial insemination. Semen type data pooled. Values without common
superscripts differ significantly (p <0.05)…………………………………………..137
5.3 Pregnancy rates of ewes inseminated by intracervical artificial insemination with fresh
ram semen (FRESH; n = 112), ram semen chilled for 12 h at 5°C (CHILLED; n = 126)
and frozen-thawed ram semen (FROZEN; n = 126). Data pooled for all oestrus
synchronisation methods. Values without common superscripts differ significantly (p
<0.05)………………………………………………………………………………..138
5.4 Pregnancy rates of ewes inseminated by intracervical artificial insemination (Intra-
cervical AI; n = 46) or laparoscopic intrauterine artificial insemination (Laparoscopic
Intrauterine AI; n = 31) methods with frozen-thawed (FROZEN) ram semen at
progesterone synchronised oestrus. Values without common superscripts differ
significantly (p <0.05)………………………………………………………………139
xxii
List of Publications
The work described in this thesis has resulted in, or contributed to, the following publications and presentations.
Journal Publications
Maddison, JW, Rickard, JP, Mooney, E, Bernecic, NC, Soleilhavoup, C, Tsikis, G, Druart, X,
Leahy, T & de Graaf, SP 2016, 'Oestrus synchronisation and superovulation alter the production and biochemical constituents of ovine cervicovaginal mucus', Animal Reproduction
Science, vol. 172, no., pp. 114-122.
Maddison, JW, Rickard, JP, Bernecic, NC, Tsikis, G, Soleilhavoup, C, Labas, V, Combes-
Soia, L, Harichaux, G, Druart, X, Leahy, T & de Graaf, SP 2017, 'Oestrus synchronisation and superovulation alter the cervicovaginal mucus proteome of the ewe', Journal of Proteomics, vol. 155, no., pp. 1-10
Abstracts
Maddison, JW, Rickard, JP, Bathgate, R, Druart, X & de Graaf, SP 2014, 'Changes to ovine cervical mucus induced by the oestrous cycle, oestrus synchronisation and superovulation',
Proceedings of the 18th Annual Conference of the European Society for Domestic Animal
xxiii Reproduction (ESDAR), 11-13th September 2014. Helsinki, Finland. Reproduction in
Domestic Animals, vol. 49, no., pp. 80-80.
Maddison, JW, Rickard, J, Druart, X, Leahy, T & de Graaf, SP 2014, ‘The effect of oestrus synchronisation and superovulation on the migration of spermatozoa through ovine cervical mucus’. 9th Biannual Conference of the Association for Applied Animal Andrology (AAAA),
8-10th August, 2014, Newcastle, Australia.
xxiv Chapter 1 The ovine cervix and its secretions: a review.
1.1. ABSTRACT The advent of controlled breeding practices, artificial insemination (AI) and semen cryopreservation was a turning point for animal production systems worldwide, allowing the dissemination of superior genetics across the globe. The Australian sheep industry has yet to take full advantage of the benefits associated with such practices due to the reduced fertility associated with cryopreserved semen following cervical AI. This is thought to be due to a reduced ability of cryopreserved sperm to traverse the convoluted and tortuous ovine cervix.
While considerable research over the last 30 years has focused on the role that semen plays in this challenge, limited research has examined the involvement of the female environment, specifically the role that the cervix and its mucoid secretions may play. The ovine cervix is a convoluted tortuous lumen, made up of several cartilaginous rings, and acts as a physical barrier to sperm transport. Several studies have examined the use of both physical and chemical cervical dilators on increasing the site of sperm deposition in the female tract, but they have returned widely variable and unsuccessful results on farm. In addition to the physical barrier the ovine cervix poses, its mucosal secretions may also act as physical or chemical barriers to sperm penetration. Mucosal secretions of the reproductive tract are known to be altered by physiological alterations in the hormone profile over the oestrous cycle and through controlled breeding practices that utilise exogenous hormones for oestrus synchronisation and superovulation purposes. However, the effect of altered mucus production and properties on sperm transport and fertility in the Merino ewe has yet to be fully detailed. As such, the aim of this review is to summarise the current literature on the structure of the ovine cervix, the
25 production and composition of reproductive tract mucus and how this changes depending on whether the ewe is in a natural or synchronised oestrus. This thesis will also discuss the interaction between cervical mucus and spermatozoa and what role this may have on sperm transport through the ovine reproductive tract. This information is of importance as it could aid in the improvement of assisted reproductive techniques currently utilised within the Australian sheep industry and abroad.
1.1.1. Key words
Ovine · mucus · oestrus · synchronisation · progesterone · prostaglandin-F2a
1.2. INTRODUCTION The development of semen cryopreservation and artificial insemination (AI) throughout the
20th Century revolutionised animal production systems. However, unlike the dairy industry, the
Australian sheep industry has been unable to fully capitalise on the associated benefits of these techniques. This is principally due to the relatively low pregnancy rates achieved when using frozen thawed spermatozoa for intracervical AI; rarely greater than 40% and often lower
(Salamon and Maxwell 1995a; King et al. 2004a), compared to 60-70% when using fresh spermatozoa (O'Hara et al. 2010). This is due to the reduced ability of frozen thawed spermatozoa to traverse the convoluted and mucus-laden ovine cervix. This is theorised to be a result of the altered membrane structure of spermatozoa that occurs during the processes of cooling, freezing and thawing (Parks and Graham 1992) leading to a ‘cryo-capacitated’ state
(Pérez et al. 1996), however this has yet to be proven. To allow for the continued use of suboptimal cryopreserved semen, research has focused on the development of AI techniques that would bypass the convoluted and tortuous ovine cervix, which acts as a barrier to successful sperm transport. The advent of laparoscopic intrauterine artificial inseminations
26 (Lap AI) in the ewe by Killen & Caffery (1982) has allowed the continued use of frozen-thawed semen. This method facilitates the deposition of semen directly into the uterine horns, thus completely bypassing the cervix. The use of Lap AI increases sperm numbers in the tract and improves fertility rates (60-70 %) when frozen-thawed semen is utilised in comparison to intracervical AI (Maxwell and Hewitt 1986; Yamaki et al. 2003; King et al. 2004a; Anel et al.
2005). Whilst superior in terms of fertility rates, the associated costs, training and equipment required, has precluded the widespread take-up of intrauterine AI. Concerns over the invasiveness of the procedure and the use of sedation are also present within industry and externally. As such, research into methods to increase the fertility rates achieved by intracervical AI and the use of transcervical AI has predominated.
1.3. THE OVINE CERVIX
1.3.1. Anatomy
The ovine cervix is comprised of a highly-convoluted lumen which begins at the cervical os, and connects to the uterine body. The morphology of the external cervical os can be assigned as one of the following: duckbill; with two opposing folds protruding into the vagina with a central horizontal slit, rose or rosette; with a cluster of cervical folds that protrude into the vagina which obscure the external os, papilla; a papilla protruding into the vagina with the external os at the apex, flap; one fold of cervical tissue that protrudes outward, either completely or partially covering the external os, or a slit; no protrusions, just a slit opening into the os [(Dun 1955; Halbert et al. 1990a; Kershaw et al. 2005) Figure 1.1)]. The variation in anatomy of the cervical os can prevent easy access or visualization of the os entry and subsequent entry into the cervical canal by an inseminating pipette, although os type does not provide a clear indication of the complexity of the interdigitating of the cervical rings.
27
Figure 1.1 Classification of common external os types of the ovine cervix; (a) duckbill, (b) slit, (c) rose, (d) papilla and (e) flap (Kershaw et al. 2005).
The lumen of the cervical canal, ranging in length from 2.8 - 9.4 cm (average length 5.5 cm)
(Kershaw et al. 2005), consists of 2-7 annular blind-ended rings or folds, which point caudally and are often not concentrically aligned [(Halbert et al. 1990a; Kershaw et al. 2005; Naqvi et al. 2005) Figure 1.2]. Whilst this may act as a successful barrier to external contamination it is subsequently also the biggest barrier to successful transcervical AI. These rings vary in width and shape as they descend caudally towards the uterine body. The increasing complexity of the convolutions of the canals was used by Kershaw et al. (Kershaw et al. 2005) as a classification tool for grading cervices (1-3, Figure 1.3). Most cervices identified in the study were found to contain a higher proportion of misaligned and interdigitating rings than concentric rings
(Kershaw et al. 2005; Kaabi et al. 2006).
28
cranial caudal Figure 1.2 Silicone cast of the cervical lumen of the lamb cervix. Illustrates the caudally directed rings/folds that comprise the cervix (Naqvi et al. 2005).
Figure 1.3 Classification of cervical grade in the ewe (a) grade 1, (b) grade 2 & (c) grade 3.
Illustrates the variation in misalignment and interdigitating of the cervical rings. Arrow indicates direction and maximum insertion depth of an inseminating pipette (Kershaw et al.
2005)
1.3.2. Influence of cervical anatomy on artificial insemination
Cervical anatomy has been shown to be highly variable, both between individuals (Kershaw et al. 2005) and between sheep breeds (Halbert et al. 1990a), with the Merino having less complex cervices than the Spanish Churra cervix, which is shorter and narrower (Kaabi et al. 2006).
29 Several studies have suggested differences in cervical anatomy between ewe breeds contribute to the differing fertility rates achieved when frozen-thawed semen are inseminated cervically, especially so when comparing fertility rates of Norwegian ewes with other European breeds with the former routinely obtaining fertility rates of over 70%. (A. et al. 1999; Donovan et al.
2004). The age of the ewe has also been shown to result in altered dimensions of the cervix, with older ewes tending to have longer and wider cervical canals with increased ring width depth than younger ewes (Naqvi et al. 2005; Kaabi et al. 2006). This difference in anatomy due to age of the ewe is most likely due to the effects of repeated parturition on cervical morphology
(Dun 1955). This increase in cervix size ultimately allows for deeper insertion of an inseminating pipette, as demonstrated by Eppelston et al. (1994) and Kaabi et al. (2006).
However, this is in conflict with research by Kershaw et al., (2005) who found no relationship between the age of an animal and depth of cervical insemination. Increasing the depth of the cervical insemination pipette can improve fertility rates achieved with cervical AI, although results are not consistent when frozen-thawed semen is utilised (Eppleston et al. 1994; King et al. 2004b; Richardson et al. 2012). These structural studies highlight the challenges associated with designing a transcervical AI method and suggest such a method would not be suitable for use with maiden ewes or indeed the Australian Merino. To overcome the anatomical challenge the ovine cervix poses to transcervical AI (TCAI), chemical and mechanical cervical relaxants have been investigated.
Initially, TCAI techniques utilised tools to help retract the cervix to allow for cervical penetration with an inseminating pipette (Andersen, Aamdal and Fougner 1973). However, this approach was not always successful and was time consuming. The technique, termed the
‘Guelph method’, was further developed in Canada in 1990, to involve the mechanical retraction of the os cervix with forceps. This method attempted to align the rings of the cervical
30 lumen, aiding manipulation and penetration of the inseminating pipette through to the uterine cavity (Halbert et al. 1990b). An early success rate of 82% penetration heralded this method as promising, but permeation and laceration of the cervical wall with the inseminating pipette occurred in one of ten animals, causing gross damage to both the cervical os and canal wall
(Halbert et al. 1990b). Large scale trials of the ‘Guelph’ TCAI technique in Canada showed promising results, with successful cervical penetration in 87.8% of test ewes, yielding pregnancy rates of 50.7% (Buckrell et al. 1994). The application of this technique in Australia using Merino ewes was less successful, only succeeding in penetrating 76% of cervical canals and yielding a pregnancy rate of 32% (Windsor et al. 1994). Whilst still higher than rates achieved by previous cervical AI methods, the uptake of TCAI techniques was poor due to the reports of associated damage caused to the epithelial lining of the tract in all test ewes, and also the rate at which the cervical canal was pierced by the inseminating pipette (Campbell et al. 1996).
Due to the ethical concerns associated with the physical manipulation of the cervix for TCAI, research shifted to focus on chemical methods of cervical dilation. Oxytocin and prostaglandins
(PGE1 and PGE2) were the main candidates tested, as both are highly effective in dilation of the cervix of periparturient women (Tenore 2003) and are often used to induce labour in women
(Atad et al. 1996). Oxytocin has been shown to stimulate myometrial contractility by facilitating increased intracellular calcium concentrations (Arias 2000) whilst prostaglandins effect the extracellular ground substance of the cervix by increasing levels of collagenase, elastase, dermatan sulphate and hyaluronic acid. PGE2 also affects smooth muscle contractility, causing dilation in the cervix and myometrial contractions in the uterus (Arias
2000; Witter 2000). Several studies have investigated the effects of various combinations of
31 both oxytocin and prostaglandins, along with follicle stimulating hormone and oestrogen on the relaxation of the ovine cervix.
Unfortunately, initial reports on lambing rate were not encouraging, with high levels of oxytocin (5 – 10 i.u.) administered at insemination resulting in decreased fertility, and lower levels (0.5 i.u.) yielding similar results to the control. As a result, the authors did not recommend its use in the ewe (Salamon and Lightfoot 1970). Following this, several studies investigating methods of improvement for transcervical embryo transfer (TCET) techniques, reported contradictory findings. With oxytocin use resulting in successful intrauterine entry by a 4 - 8mm stainless steel rod in 77% of treated ewes compared to 0% in controls (Khalifa, Sayre and Lewis 1992). The combination of oestrogen and oxytocin was also investigated with encouraging results, although the rate of successful transcervical penetration was dependent upon timing of treatment application and dose rate of oestrogen (Khalifa, Sayre and Lewis
1992; Wulster-Radcliffe, Costine and Lewis 1999). Despite research by Sayre and Lewis
(1997) indicating that the use of oxytocin had no effect on ovum fertilisation, fertility rates achieved following its use were still low (28%), and continued to be so in more recent studies, with lambing rates of only 10 - 56% achieved using frozen-thawed semen (Stellflug et al. 2001;
King et al. 2004a). Authors attributed this to the TCAI process itself, particularly the manipulation of the pipette through the cervical canal, which even though much more pliable after use of oxytocin, still required to be somewhat manipulated so as to gain entry into the uterine body. Work by Wulster-Radcliffe (2004) suggested that the cervical manipulation required in TCAI techniques could be activating immune responses that result in the early embryonic loss associated with TCAI, which could explain the low fertility rates that were often reported after its use. As a result of the contradictory results obtained with the use of
32 oxytocin, other chemical relaxants were investigated including; prostaglandins, hyaluronan and follicle stimulating hormone (FSH).
The use of alternative chemical cervical dilators has also been investigated. Prostaglandin in the form of the vaginal pessary ‘Cervidil’, was shown to allow cervical penetration and TCAI in 100% of animals in a 2009 study (Candappa et al. 2009). Follow up studies investigating fertility after the Cervidil application provided conflicting results, with successful cervical penetration achieved in 55 - 57% of treated ewes, compared to controls (43%) (Candappa and
Bartlewski 2014; Bartlewski and Candappa 2015). Pregnancy rates were low, with early pregnancy detected in only 35% of animals (2 of 7, Day 25) and no identifiable pregnancies by
Day 55 (Candappa and Bartlewski 2014). The topical application of a prostaglandin E1 analogue (PGE1; misoprostol) or ovine FSH (Ovagen) to the cervix allowed effective penetration of the cervical canal in all treated ewes, in both cases. Furthermore, the study identified that natural cervical relaxation peaked 72 h post sponge removal, which is after acceptable insemination times for cervical AI (Evans and Maxwell 1987). Application of both
PGE1 and FSH enhanced cervical relaxation, allowing successful penetration at 54 h post sponge removal, the optimum time for cervical insemination (Leethongdee et al. 2007). As outlined, TCAI and chemical cervical dilation techniques have resulted in variable, and often low, fertility rates, and as a result have not been taken up by industry. In recent years, research has turned to the investigation of cervical and cervicovaginal mucus, in an effort to better understand how this complex fluid interacts with spermatozoa during cervical transit.
33 1.4. CERVICAL AND CERVICOVAGINAL MUCUS
1.4.1. Composition
Mucus is a complex non-Newtonian biological fluid that is found throughout the body in many organs and systems. Its role as a lubricant allows for normal organ functionality (Wang et al.
2013) and provides both a physical and biochemical barrier to invasion from foreign organisms
(Eggert-Kruse et al. 2000). Mucus is comprised of a semi-solid gel and a low viscosity plasma, with the latter consisting mainly of water (90-98%) and an array of low molecular weight substances, including electrolytes, inorganic ions, carbohydrates, sugars (Tsiligianni et al.
2001), amino acids, various enzymes and bactericidal proteins (Schumacher 1970). In humans, electrolytes, namely sodium chloride, are responsible for the characteristic crystallization or ferning pattern seen in air dried reproductive tract mucus (Igarashi 1954; Schumacher 1970).
Other soluble macromolecules found in mucus plasma include serum transudate proteins, locally produced proteins, peptides, and various polysaccharides. A variety of cell types are present, some which result from the natural development and degradation of the epithelial lining of the reproductive tract, whilst others have specific immune function such as leukocytes
(Schumacher 1970). Mucus of the reproductive tract is produced continuously, creating a level of outward flow, and as such CV mucus is an amalgamation of fluids produced from the oviducts, uterine cavity, cervical canal and the vagina.
The majority of cervical and cervicovaginal mucus compositional studies have been carried out in humans and cattle, with several investigating variation over the reproductive cycle (Wolf et al. 1977b; Eltohamy, Zakaria and Taha 1990). Studies in the ewe have investigated fluids of the oviduct (Perkins and Goode 1966; Restall and Wales 1966) and uterus (Aguilar and Reyley
2005), whilst analyses of ovine cervical and cervicovaginal mucus are currently lacking. Fluid of the oviduct and uterus provide a crucial supportive medium for fertilisation and then
34 implantation of the embryo. As such, these fluids have been thoroughly investigated, especially in humans, with results leading to the development and production of in vitro culture media now used in both research and industry in vitro fertilisation (IVF) settings.
Spermatozoa must navigate this mucus to gain entry into the upper reproductive tract and penetrate the oocyte. As such, detailing the composition of fluid from the cervix and CV regions is central to understanding its role in aiding or hindering semen transport. In the ewe, few studies on the composition of mucus in this region exist. Early work investigated the ability of CV fluid to maintain sperm viability and motility over time (Restall 1969), yet the specific components responsible were not identified. The level of calcium in mucus is of particular interest as calcium has been reported to have roles in sperm maturation, motility, metabolism, motility, capacitation and the acrosome reaction and also hyperactivity (Morton et al. 1974;
Tash and Means 1983; Goh and White 1988; Magnus et al. 1990a; White 1993; Murase et al.
2001b, a). However, the influence of Ca2+ in the female tract on sperm function is not clear.
Levels of Ca2+ in mucus are much higher than seminal plasma, with research suggesting that seminal plasma constituents sequester free Ca2+ in mucus, preventing sperm from being exposed to the higher levels in mucus, and thus from any deleterious effects that it could have on motility or premature activation of the acrosome reaction so early in the female passage
(Magnus et al. 1990b). The composition of calcium, along with other ions has yet to be clearly detailed in the ewe.
1.4.2. Mucus pH
The pH of reproductive tract mucus is known to vary between individual species as well as along different sections of the reproductive tract. Vaginal mucus in women is generally reported to be around pH 4 (Eggertkruse et al. 1993; Olmsted et al. 2000), whereas pH 7.4 is
35 reported in cattle (Lewis and Newman 1984), and 8.5 in the ewe (Singh, Rawal and Kumar
1989). The acidic nature of human reproductive tract mucus, particularly vaginal mucus, is attributed to the high levels of lactic acid produced by the normal vaginal flora, which is predominated by Lactobacillus spp. (Boskey et al. 2001; O'Hanlon, Moench and Cone 2013).
Manes et al. (2010) reported the most common aerobic bacterial species in ewes to be Bacillus spp., Staphylococcus spp. and Corynebacterium spp., with goats also having predominately
Staphylococcus spp. It is likely then that differences in vaginal mucus pH can be, at least partly, attributed to differences in the by-products of differing predominating bacterial species that make up the vaginal biome. As you progress further along the tract, mucus pH is reported to be more basic, with endocervical mucus in cattle reaching 7.4 (Lewis and Newman 1984) and in humans ranging from 5.4 - 8.2 (Eggertkruse et al. 1993), which is a considerable change in environmental conditions for vaginally deposited sperm (such as that from sheep, cattle and human) navigating the tract. Whilst species variation does occur, the range of results could also be heavily influenced by variation in testing method (e.g. Strip tests compared to more accurate pH probes and meters) or the site of testing (in vivo vs in vitro). Method of mucus collection could also both have a strong influence on pH, as it is known that even minimal exposure to air in an in situ environment can alter pH readings by changes in bicarbonate buffering (Correa,
Mattos and Ferrari 2001). As discussed in detail in section 3.4, the pH of mucus is an important determinant of both mucus release and its structure, which can impact on particle translocation.
Thus, any changes in mucus pH could directly impact upon the receptiveness of mucus to migrating spermatozoa and also possibly affect the protective qualities of mucus.
1.4.3. Mucus proteome
Proteomic analysis of mucus began with investigation of total protein concentration and the classification of proteins into functional groups. With the advancement of techniques over the
36 years, analysis of mucus proteins has developed into full proteome identification using a variety of both qualitative and quantitative methods. Proteomic analysis of reproductive tract mucus has been carried out principally in humans, with several studies investigating the proteome of cervical (Andersch-Björkman et al. 2007; Lee et al. 2011; Grande et al. 2015) and cervicovaginal fluids (Dasari et al. 2007a; Shaw, Smith and Diamandis 2007; Tang et al. 2007) during pregnancy and throughout the menstrual cycle. Similarly in cattle, proteomic analysis has also been used to examine and quantify any cyclical changes in mucus proteins (Alavi-
Shoushtari, Asri-Rezai and Abshenas 2006). Analysis of uterine fluids in cattle has identified endometrial proteins and protein secretions that may act as endometrial primers for implantation (Bauersachs et al. 2006) as well as regulators of conceptus survival, growth and development (Forde et al. 2014). Analysis of reproductive tract mucus using mass spectrometry has also been used to identify protein markers of oestrus in sows (Lee et al. 2013) and buffalo cows (Muthukumar et al. 2014), with the aim of optimising current oestrus detection techniques. Proteins involved in cell to cell adhesion, such as oesteopontin and GlyCAM-1 were also identified as key effector proteins in endometrial sections to promote conceptus growth and survival using mass spectrometry (Gray et al. 2002). Whilst currently unavailable, an analysis of the ovine cervicovaginal or cervical mucus proteome could be beneficial in expanding our current understanding of the role mucus plays in successful sperm transport through the sperm restrictive convoluted ovine cervix. Proteomic analysis has also led to identification of mucins; the structural gel-forming proteins of mucus in human cervicovaginal mucus (Andersch-Björkman et al. 2007).
1.4.4. Mucins; their production and release
Mucins constitute the semisolid gel and are the chief determinates of the physicochemical viscoelastic properties of mucus. Mucins are a heterogeneous family of large complex
37 glycoproteins, consisting of a central core protein domain, rich in the amino acids serine, threonine and proline which provide a high number of attachment points for branching oligosaccharide side chains (Lagow, DeSouza and Carson 1999). Unlike the majority of glycoproteins, the oligosaccharides of mucins are predominantly O-linked, resulting in >80% of the molecule being carbohydrate (by weight) (Jentoft 1990; Carraway and Hull 1991;
Andersch-Björkman et al. 2007; Linden et al. 2008). It is this complex arrangement of side chains which give the mucin its filamentous properties and a bottle brush like appearance
(Linden et al. 2008). The carbohydrate side chains can be neutral, sulphated or sialyted
(Andersch-Björkman et al. 2007), with the latter two being partly responsible for conferring a net-negative charge to mucus (Quinton 2010). To date, approximately 20 different mucins have been identified, and these can be divide into three classes; 1. the secreted mucins, 2. membrane associated mucins and 3. small soluble mucins. It should be noted that secreted mucins can be further classified into either gel forming or non-gel forming (Andersch-Björkman et al. 2007).
The mechanisms of mucin production and release have been extensively researched, largely during the investigation of the pathobiology of cystic fibrosis as this disease results in altered mucus production (Quinton 2010). As with other secretory or cell surface associated proteins, the mucin core proteins are produced in the rough endoplasmic reticulum of the cell and are then shuttled to the Golgi apparatus in which O-glycosylation takes place (Pimental et al. 1996;
Ambort et al. 2012). The highly-condensed mucin is then transported to the mucin granule of goblet cells, in which high intergranular levels of calcium and hydrogen ions shield negatively charged sites on mucins from electrostatic repulsion (Verdugo 2012). This allows the highly condensed polyanionic macromolecular mucins to be packaged into mucin granules within goblet cells.
38 − Recent work by several groups has highlighted the intrinsic role of bicarbonate (HCO3 ) in mucus expulsion. Upon release from the granules, the cationic shields are immediately
− 2+ + removed from mucins by extracellular HCO3 via sequestering of Ca and buffering of H , thus allowing rapid expansion of the mucin via electrostatic repulsion (Figure 1.4) (Chen et al. 2010;
Muchekehu and Quinton 2010; Quinton 2010). Work by Kesimer et al. (2010) on the storage and expansion of salivary mucin-5B (MUC5B) highlighted that within the granule, mucins
− - aggregate around a central node, which are mainly composed of NH2 and COOH terminal protein domains. The release of the mucin is controlled by ion exchange between calcium and sodium. This causes increased osmotic pressure, leading to the entry of water into the granule and subsequent exposure of the nodes to enzymatic degradation under optimal pH conditions, resulting in further expansion of the mucin into a more linear structure.
− Figure 1.4 Proposed roles of bicarbonate ( HCO3 ) in mucin release from goblet cell mucin granules, taken from Muchekehu and Quinton (2010).
39 Secreted mucins constitute the bulk of mucins identified in the female reproductive tract; namely in the endocervix and endometrium (Corfield 2015), with cervical mucus reportedly containing around 1.5 % (w/w, wet weight) of mucins (Carlstedt et al. 1983). Mucin content has been shown to be important in particle translocation, with increased mucin content resulting in increased selectivity of mucin hydrogels in an in vitro setting (Lieleg, Vladescu and Ribbeck
2010). In humans, research by Gipson (1997) identified several mucin genes throughout the human female reproductive tract. MUC1 was identified in the fallopian tubes, while small amounts of MUC6 were found in the endometrial epithelium. In the endocervical epithelium
MUC1, MUC4, MUC5AC, MUC5B and MUC6 were identified while MUC1 and MUC4 were also identified in the ectocervical and vaginal epithelia MUC1 and MUC4. Similar results were identified in a recent transcriptome analysis of the bovine cervix, with MUC1, MUC4,
MUC5AC, MUC5B, MUC16 and MUC20 identified (Pluta et al. 2012). Mucin genes of the sheep reproductive tract are still to be fully described, but MUC1 has previously been identified in the ovine endometrium (Raheem et al. 2016). Mucins afford mucus its characteristic physical and rheological qualities which govern its functionality in the female reproductive tract.
1.4.5. The function of cervical and cervicovaginal mucus
One of the main functions of mucus is to coat and protect organs from shear force. This allows for the normal functionality of many of the bodies organs and especially so for the female reproductive tract, which undergoes drastic physical transformations over the reproductive cycle and during any ensuing pregnancy. Mucus also acts as a barrier to infection in many organs, especially those with external entry points which increase the likelihood of bacterial invasion and colonisation. The process of mating also introduces foreign material into the body and mucus acts to prevent infection and infiltration in several ways. First, the continuous production of mucus creates a level of outward flow from the upper reproductive tract towards
40 the vagina, where eventually it is discharged, effectively acting to flush foreign bodies from the tract. To maintain this clearance role, mucus biosynthesis, secretion and degradation have to be continuous and balanced. Studies of gastrointestinal mucus propose it takes approximately 60 mins for the inner mucus layer to turnover (Gustafsson et al. 2012), and 24 hours for the whole cycle to occur (production to degradation) (Faure et al. 2002). Secondly, mucus also has the ability to act as a selective permeable barrier, managing the passage of molecules and cells, such as spermatozoa, whilst hindering the entry and colonisation of bacterial, fungal and viral cells. This selective permeability is provided to mucus through complex structural rheological properties, namely the electrostatic, hydrophobic and H- bonding interactions (Harding et al. 1999) of mucins, which afford mucus an ‘adhesive’ quality. The same adhesive properties of mucus are utilised to trap pathogens in the female reproductive tract. In vitro studies using reconstituted mucin hydrogels have described how the electrostatic interactions between diffusing particles in mucus and mucin polymers can be greatly impacted by changes in surface charge of translocating particles, level of mucin density and the pH and ionic strength of utilized buffers (Lieleg, Vladescu and Ribbeck 2010). Whilst these interactions do hamper the growth of bacteria, fungi and viruses in the reproductive tract the former serves as an important part of the microbiological biome of the body which contributes to the management of homeostasis and health of the reproductive tract. As mentioned previously, bacteria of the vagina play an important role in acidifying mucus, which itself acts as a hindrance to infection by other bacterial species, likely those that have pathogenicity, and so prevention of infection rather than just bacterial colonisation is the key.
Mucus is also a dynamic fluid that is adaptive to its environment. An example of this is the increase in viscoelasticity of gastric mucin in response to invasion by Helicobacter pylori bacteria (Markesich et al. 1995; Worku, Sidebotham and Karim 1999). How this change occurs
41 is yet to be fully described but is likely a change in the properties and interactions of mucins.
To combat such an invasion, immune cells must be deployed, but interestingly mucus does not hinder their transport, with studies demonstrating that leukocytes can easily diffuse through mid-cycle cervical mucus in human females (Parkhurst and Saltzman 1994). This ease of passage of leukocytes through mucus is likely due to a complete lack of interaction of such particles with mucin fibres. Work by Li et al (2013), in which peptides diffusing through purified porcine gastric mucin were assessed, suggests this may be due to immune cells such as leukocytes having a spatial charge distribution on their surfaces which favours diffusion.
This is supported by earlier work which showed that soluble proteins densely coated with equal negative and positive surface charges could diffuse freely through mucus. Several studies have investigated particle translocation through native mucus, reporting particles as large as 500 nm can diffuse through mucus gels in an in vitro setting (Olmsted et al. 2001; Lai et al. 2007). The key point is the coating, and thus net surface charge and charge distribution, that these particles had as this governs the type and level of integration that occurred between particles and mucin fibres, ultimately controlling diffusion rates through mucus gels. The low affinity bonds that reportedly form between antibodies and mucin fibres allow them to readily diffuse through mucus and subsequently accumulate to target pathogens, to which they bind tightly and specifically (Cone 2005). However, viruses have adapted so as to have a particular surface charge and charge distribution that aids in diffusion through mucus such as the capsid viruses;
Rhino virus, Norwalk virus and human papilloma virus, which all have a net negative charge
(Wada and Nakamura 1981; Olmsted et al. 2001; Cone 2009b).
As mentioned previously, mucus pH plays an important role in mucus interactions with translocating particles. Work by Lai et al. (2009a) suggests that acidic cervicovaginal mucus effectively traps human immunodeficiency virus (HIV) but when mucus was neutralized to pH
42 6-7, as occurs when semen is deposited in the female tract, HIV easily diffused through the mucus (Lai et al. 2009a). The acidic environment created by bacteria that colonize the human female reproductive tract, lactobacillus pp. (Boskey et al. 1999), reportedly acts to abolish the net negative charge of HIV, which could impact upon its translocation through mucus. Mucus viscosity has also been shown to directly impact on bacterial invasion, with a mucus simulant of 8 Pa·s (a measure of dynamic viscosity) shown to act as an effective bacterial barrier and filter, whilst lower viscosities allowed for raid infiltration and colonisation (Girod et al. 1992;
Lai et al. 2009b). This is in contrast to earlier work that suggested increases in mucin concentration may result in increased mesh size, or the spacing between mucin fibres (Schrank and Verwey 1976).
The immune function of mucus is also crucial as foreign bodies, specifically spermatozoa and any developing conceptus, require acceptance and immune tolerance from the female tract, but the tract concurrently has to defend against pathogenic insults. Mucus of the reproductive tract, and, specifically that produced in the upper reproductive tract and the uterus, is also of importance during fertilisation and any ensuing pregnancy, as the conceptus and amniotic sac are exposed to these secretions during this dynamic period of development.
Mucus is also the transport medium through which sperm are exposed to during their migration of the reproductive tract. As such its components, especially proteins that are expressed throughout the reproductive tract and released into mucus, are in close contact with spermatozoa during this process and can have inhibitory or supportive effects on sperm transport. Research has shown that proteins expressed by the uterovaginal junction (UVJ), namely Heat shock protein-70 (HSPA1A), can support motility in vitro and suggest that it may facilitate the release of sperm from storage tubules in the Japanese quail (Hiyama et al. 2014).
43 Proteins purified from oviductal mucus, namely heat shock proteins (HSPs), have also been shown to aid motility, viability and acrosome integrity of buffalo sperm, and improve its ability to penetrate cervical mucus in vitro, with similar results found in rams (Lloyd et al. 2009), boars and bulls (Elliott et al. 2009) (discussed in detail in section 3.3). HSPA8 has also been demonstrated to reduce polyspermy in porcine spermatozoa and accelerate embryonic development when sperm were exposed to HSPA8 prior to IVF procedures (Elliott et al.
2009).Thus, any changes in mucus composition, especially proteomic changes, could have significant impact on not only mucus structure but also its receptivity to sperm
1.5. VARIATION IN CERVICAL AND CERVICOVAGINAL MUCUS
1.5.1. Across the oestrous cycle
The oestrous cycle is controlled by variation in endogenous hormones levels, and in cases of synchronised or superovulated animals, exogenous hormones are also at play. Mucus properties are also largely under endogenous hormone control and thus naturally fluctuate across the oestrous and menstrual cycles. In women, peri-ovulatory mucus is described as abundant, thin and watery and less viscoelastic than luteal phase mucus, which is scanty, opaque and viscous (Igarashi 1954; Brunelli et al. 2007). The changes in mucus structure over the oestrous cycle are mediated by many factors, including mucus hydration which is largely under the hormonal control of oestrogen and thus peaks during oestrus and ovulation (Igarashi
1954; Chantler and Debruyne 1977). Understandably, as hydration increases, the viscosity of mucus during oestrus and ovulation decreases, and is thus lowest around the time of ovulation
(Schilling and Zust 1968; Wolf et al. 1977b). Changes in mucus characteristics over the menstrual and oestrous cycle are often utilised as a means to determine which stage of the reproductive cycle an animal is at. In humans, the colour and consistency (viscosity) of mucus is often used as a predictor of timing of ovulation in couples trying to conceive. Mucus
44 characteristics are also used in production animals to identify onset of oestrus so as to time breeding or artificial insemination with time of ovulation to improve the efficiency of AI. In the ewe, as with cattle and other production species, increased production of clear, watery mucus is indicative of oestrus onset (Evans and Maxwell 1987).
Mucus pH and electrical conductivity have also been investigated as a means of oestrus detection. In cattle, oestrus onset and ovulation diagnosis are reportedly identified based on vaginal and cervical mucus pH levels, with mucus being most acidic just prior to ovulation
(cervix pH 6.55, vaginal pH 6.97), but the pH range has been shown as quite narrow for both the cervix and vagina (Schilling and Zust 1968). More recent work in cattle utilising pH probes as opposed to pH sensitive strips, identified the same trend, with pH being most acidic at oestrus in cervical (pH 6.2 (Mori et al. 1979)) and vaginal mucus (pH 7.32 (Lewis and Newman 1984)).
Similar research in mares and bitches also identified vaginal mucus pH to be at its lowest at oestrus (Polak and Kammlade 1981; Antonov, Dineva and Greorgiev 2014). Work in cattle has indicated mucus to be closer to neutral pH during the post oestrus and luteal phases (Schilling and Zust 1968; Lewis and Newman 1984). Although in humans cervical mucus pH appears to remain steady across the menstrual cycle (Wolf et al. 1977b). Reports in sheep show that pH also changes over the cycle, trending to be more basic (pH ≥7.5) during the luteal phase; however, data did not identify clearly at which part of the cycle this took place (Singh, Rawal and Kumar 1989). As the onset of oestrus and ovulation is governed by hormone levels, one could assume that as pH varies over the reproductive cycle it too is under hormonal control, but reports on this have been somewhat contradictory. Eggert-Kruse et al. (1993) noted that increasing levels of circulating oestrogen resulted in alkalisation of endocervical mucus in humans, whereas Gorodeski et al. (2005) showed that cultured ectocervical cells treated with oestrogen resulted in an acidified output. The alteration of mucus pH could have flow on effects
45 for both the prevention of microbial colonisation and possible infection of the tract, and for migrating spermatozoa, which maintain optimal functionality at neutral pH (Tampion and
Gibbons 1963).
Changes in mucus pH over the reproductive cycle are important for sperm transit through mucus, as acidic hydrogels in vitro reportedly form tighter barriers and are more selective
(Lieleg, Vladescu and Ribbeck 2010). Brunelli et al.(2007) proposed that pH sensitive domains of the mucin MUC5B, one of the main secretory gel-forming mucins, in human ovulatory cervical mucus drives aggregation of mucin fibres. Studies also report that the network of mucin fibres in mucus of a low pH are more heterogeneous, presumably this altered fibre network and orientation reduces the mesh size of the mucus (Lieleg, Vladescu and Ribbeck
2010). However, a recent study conflicts with this theory, finding that native human cervicovaginal mucus microstructure and bulk rheology was remarkably resistant to changes in pH (Wang et al. 2013). A definitive effect of changes in mucus pH on mucin fibre networks is important as it could be restrictive to successful sperm transport.
The composition of mucus is also altered in a cyclical manner, with oestrous mucus in cattle described to be less proteinaceous than its luteal counterpart (Zaaijer et al. 1993). Proteomic work has highlighted the changes that occur in the cervical mucus proteome of women, with mucin glycosylation suggested as the major alteration of mucus that occurs around the time of ovulation (Andersch-Björkman et al. 2007). Quantitative mass spectrometry of cattle cervical mucus has identified altered abundance of particular proteins during oestrus compared to luteal phase cervical mucus (Pluta et al. 2011). During oestrus the glycoproteins β-galactosidase and sialidase were most abundant 12 h post ovulation whereas β-hexosaminidase and α-fucosidase peaked during the luteal phase (Pluta et al. 2011). In addition to this, mass spectrometry has
46 also identified oestrus specific and luteal specific proteins in humans (Grande et al. 2015) and cattle (Muthukumar et al. 2014). Some studies have investigated certain proteins as putative oestrus detection markers, such as dimethyargenine dimethylaminohydrolase 2 (DDAH2) in sows (Lee et al. 2013) and Heat shock protein (HSP)-70 in buffalo cows (Muthukumar et al.
2014). Of particular interest are the changes in the types and abundance of mucin proteins as they could significantly impact mucus production and structure over the reproductive cycle.
The transcript levels of MUC5B have been shown to be inversely correlated to serum levels of progesterone, with level at oestrus approximately 5-fold higher around the time of ovulation in both women and cows [cow (Pluta et al. 2011), human (Gipson et al. 2001)]. In the human respiratory tract, the MUC5B gene has been shown to be under oestrogenic influence, so it is likely that it, along with other mucins produced in the reproductive tract are directly controlled, if not at least partially influenced by, endogenous hormone levels. Work in the ewe has also identified mucins to be more abundant during oestrus (Soleilhavoup et al. 2015), although further detail in the Merino ewe is yet to be attained. Due to the role mucins have on mucus structure and particle translocation, any changes in mucin content over the oestrous cycle could directly impact sperm migration. From the studies outlined above it is clear that endogenous hormones alter mucus production and its composition but what effect do exogenous hormones have on mucus?
1.5.2. Effect of controlled breeding practices
The use of exogenous hormones for controlled breeding practices has revolutionised breeding systems of domesticated production species across the globe. While their use allows producers to control the timing of oestrus or level of ovulatory output, it also impacts on mucus characteristics and its production. As these programs deliver substantial benefits to animal production systems, the effects they have on mucus production have largely been overlooked.
47 In the ewe, the use of progesterone for oestrus synchronisation has been shown to both increase
(Croker and Shelton 1974; Rexroad and Barb 1977) and decrease (Smith and Allison 1971) mucus production. Despite these results, the use of progesterone for oestrus synchronisation is standard practice on Australian sheep properties. Work on the effects of using prostaglandin-
F2α for oestrus synchronisation are somewhat lacking in the ewe, especially regarding its possible effect on mucus production and how this may impact upon fertility rates. Exogenous oestrogen has also been shown to have a conflicting effect on ovine mucus production, with increased production (Allison 1971; Adams and Tang 1979) reported, as well as decreased production when given in conjunction with progesterone (Croker and Shelton 1974).
Prolonged grazing on oestrogenic clover has been shown to effect mucus production (Lightfoot et al. 1974) and cervical histology, with an initial increase in glandular development, and chronic exposure leading to a reduction in goblet cells and thus mucus production (Lightfoot and Adams 1979). The effects of superovulation practices on mucus production, have also yet to be fully detailed in the ewe.
In addition to the volume of mucus produced, changes in percent dry matter, spinnbarkeit
(Adams and Tang 1979) and protein content (Rexroad and Barb 1977) of mucus have been reported in the ewe following the use of exogenous hormones. As discussed previously, the ionic components of mucus can profoundly influence mucus release and structure (see section
3.4), although to date the ionic components of ovine cervical and cervicovaginal mucus have yet to be fully elucidated. Electrolyte levels are likely to be altered over the natural oestrous cycle, but may also be significantly affected by the use of exogenous hormones for synchronisation and superovulation which could have unintended results on the receptivity of mucus to spermatozoa. The protein component of cervical mucus is also likely to be affected by controlled breeding practices. As mentioned previously, proteins in the secretions of the
48 female reproductive tract have been shown to greatly impact sperm longevity and any changes to the proteome of reproductive fluid may hinder sperm transport through the reproductive tract. The use of both progesterone and prostaglandin-F2α synchronisation methods have resulted in decreased sperm numbers being recovered from throughout the reproductive tract of the ewe [progesterone (Quinlivan and Robinson 1967; Allison and Robinson 1972; Hawk and Cooper 1977), prostaglandin-F2α (Hawk and Cooper 1977)], ultimately resulting in decreased fertility when these practices are adopted on farm. Synchronisation using prostaglandin-F2α reportedly leads to suboptimal follicular development contributing to the reduced fertility seen after this method is utilised (White et al. 1987; Fierro et al. 2011).
1.6. INTERACTION OF SPERMATOZOA WITH THE CERVIX
Numerous studies have demonstrated that the female tract is not a passive conduit in regards to migration of spermatozoa through the tract, but instead is actively involved in restricting sperm numbers and selecting for sperm quality (Mattner 1963a, b; Katz and Overstreet 1982;
Ragni et al. 1985; Chantler, Sharma and Sharman 1989). Mucus of the reproductive tract is key to this sperm-cervix interaction. The initial contact of spermatozoa with the female tract is often one of disparity, with mucus pH often differing from that of semen, depending on the species studied, with the buffering effect of seminal plasma acting to neutralise this. Upon migration into mucus, morphologically abnormal spermatozoa and those with poor motility are filtered out (Katz and Overstreet 1982; Ragni et al. 1985), presumably by becoming lodged in the mucin fibre network. Work by Mullins and Saacke (Mullins and Saacke 1989) also showed that privileged pathways for transport of viable sperm may be present in the cervices of cattle.
These sialomucin laden shallow grooves or channels began in the fornix vagina and extended through the cervical folds and progressed towards the uterus, containing cranial oriented sperm.
Furthermore, it is likely that morphologically abnormal sperm have changes in their surface
49 charge characteristics and distribution, which is known to impact on particle diffusion through mucus (Lieleg, Vladescu and Ribbeck 2010; Li et al. 2013). As such these spermatozoa are captured by mucus through altered electrostatic reactions between the mucin fibres and travelling spermatozoa.
Changes to mucus characteristics that occur both naturally over the oestrous cycle and after exogenous hormone supplementation impact on the interaction between spermatozoa, the cervix and the mucus of the reproductive tract. Of significant interest is the change in mucus hydration over the cycle, with cervical mucus hydration in women shown to be around 90% during the luteal phase (Morales, Roco and Vigil 1993) and increases from 92% to 98% just prior to ovulation (Katz, Slade and Nakajima 1997). Numerous studies have shown that mucus with high water content, like that seen during oestrus under the influence of oestrogen, is more receptive to sperm migration (Morales, Roco and Vigil 1993; Bigelow et al. 2004). Sperm penetrability is substantially increased when mucus hydration is ≥ 97.5%, (Katz, Slade and
Nakajima 1997). Also of interest is the effect that variable mucus protein levels have on the success of sperm migration, with some studies reporting an inverse relationship between protein concentration in cervical mucus of women and its ability to sustain sperm migration
(Morales, Roco and Vigil 1993). Interestingly, mucus protein levels are highest during the luteal phase of the menstrual cycle in women (Morales, Roco and Vigil 1993). As detailed previously, reproductive tract mucus from animals that have undergone superovulation or oestrous synchronisation is significantly altered, and these changes have even been shown to alter sperm survival in cows and ewes in an in vitro setting. In cattle, incubation of sperm in oestrous cervical mucus from synchronised cows leads to reduced motility and forward movement, and a reduced percent of morphologically normal sperm compared to sperm incubated in oestrous mucus of naturally cycling animals (Cal et al. 1973). A recent study also
50 found similar results when ram semen was incubated in vaginal mucus from synchronised ewes compared to mucus from ewes in spontaneous oestrus (Manes et al. 2016). Several fertility trials have also reported lowered fertility rates in animals that have been superovulated compared to rates obtained to naturally cycling animals [sheep (Armstrong and Evans 1983), cattle (Lopez-Gatius 1993)] or when synchronised using either prostaglandin-F2α (Hawk and
Cooper 1977; Fierro et al. 2011) or progesterone. Based upon these findings, it is probable that there is a confounding effect of ‘mucus type’ and ‘sperm type’ which could be contributing to reduced fertility rates reported after use of exogenous hormones for synchronisation, and cooling or cryopreservation of semen. This has yet to be established in the ewe. Furthermore, a recent study looking at the effect of varying concentrations of cervical mucins on sperm motility parameters in humans has identified a dose dependent decrease in motility, and increases in sperm linearity and straight line velocity when incubated in a higher concentration of mucin (Eriksen et al. 1998). Changes in other mucus proteins is also of interest, specifically the level of neuraminidase, also known as sialidase. This enzyme acts to cleave terminal sialic acids from mucins (Wiggins et al. 2001), which could significantly impact the net charge of mucus and thus its interactions with charged spermatozoa. The abundance of neuraminidase in ovine reproductive tract mucus and any effects that changes of neuraminidase levels have on mucins has yet to be detailed in the ewe.
1.7. CONCLUDING REMARKS AND OBJECTIVES OF THE CURRENT STUDY
From the findings summarised above, it is evident that mucus is a complex medium that is crucial for normal functionality of the female reproductive tract and also greatly impacts the likelihood of successful sperm transport and fertilisation. The effect that exogenous hormones have on mucus production, chemical components, mucus characteristics, the mucus proteome, and what effect these factors may have on sperm cervical transport, and therefore fertility, in
51 the Merino ewe requires further investigation. These results could aid in the further development of current artificial insemination (AI) practices in the ewe, with the aim of improving upon current fertility rates achieved when intracervical AI is utilised.
The current hypothesis of this study is that exogenous hormones used for oestrus synchronisation and superovulation significantly impact the production and composition of cervical mucus in the ewe. In particular, these hormones alter the proteomic constituents of cervical mucus and this altered proteome impacts the interaction of spermatozoa (both fresh and processed), mucus and the cervix, ultimately contributing to poor fertility following intracervical AI with frozen-thawed spermatozoa. This hypothesis will be examined by the following objectives:
(I) To characterise and compare the volume, colour, spinnbarkeit, chemical profile
(sodium, potassium, magnesium, calcium and chloride), and total protein
concentration of cervicovaginal mucus collected from naturally cycling,
progesterone synchronised, superovulated and prostaglandin-F2α synchronised
Merino ewes throughout the oestrous cycle
(II) To examine the effect of changes to the structure and composition of mucus
caused by synchronisation (using progesterone or prostaglandin-F2α) and
superovulation methods on the migrating ability of ram spermatozoa
(III) To qualitatively and quantitatively determine the proteomic composition of
ovine cervicovaginal mucus across the oestrous cycle in naturally cycling,
progesterone synchronised and superovulated Merino ewes
(IV) To investigate the effect of changes in mucin and neuraminidase concentration
on the motility, kinematic parameters, membrane viability and acrosome
integrity, and penetrating ability (in vitro) of ram spermatozoa (using a
cervicovaginal mucus simulant to imitate natural mucus)
52 (V) To investigate the influence of synchronisation programs using progesterone or
prostaglandin on the fertility of fresh, chilled and frozen-thawed ram
spermatozoa following intracervical AI
53 Chapter 2. Oestrus synchronisation and superovulation alter the production and biochemical constituents of ovine cervicovaginal mucus
The experiments described herein have been published as:
Maddison, JW, Rickard, JP, Mooney, E, Bernecic, NC, Soleilhavoup, C, Tsikis, G, Druart, X,
Leahy, T & de Graaf, SP 2016, 'Oestrus synchronisation and superovulation alter the production and biochemical constituents of ovine cervicovaginal mucus', Animal Reproduction
Science, vol. 172, no., pp. 114-122.
2.1. ABSTRACT
Controlled breeding programmes utilising exogenous hormones are common in the Australian sheep industry, however the effects of such programs on cervicovaginal mucus properties are lacking. As such, the aim of this study was to investigate cervicovaginal (CV) mucus from naturally cycling (NAT), progesterone synchronised (P4), prostaglandin synchronised (PGF2a), and superovulated (SOV) Merino ewes. Experiment 1; volume, colour, spinnbarkeit, chemical profile and protein concentration of mucus (NAT, P4, PGF2α and SOV; n = 5 ewes/treatment) during the follicular (4 d) and luteal phases (8 d) was investigated. Experiment 2; in vivo mucus pH and in vitro mucus penetration by frozen-thawed spermatozoa (NAT, P4 and SOV; n = 11 ewes/treatment) was investigated over oestrus (2 d) and the mid-luteal phase (pH only, 2 d).
Oestrus mucus was more abundant, clearer in colour and less proteinaceous than luteal phase mucus (p <0.05). Increased mucus production and protein concentration was evident in SOV
(p <0.05) while PGF2a reduced mucus volume (p <0.05). Mucus pH (oestrus 6.2-6.5), chemical
54 profile and mucus penetration by sperm were unchanged (p >0.05) in comparison to naturally cycling animals. Results indicate that exogenous hormones used for controlled breeding affect cervicovaginal mucus production, but few other tested characteristics. Further research is required to explain fertility differences between synchronised and naturally cycling animals following cervical AI.
2.1.1. Key words progesterone ∙ prostaglandin ∙ oestrogen ∙ oestrus ∙ hormone ∙cervicovaginal
2.2. INTRODUCTION
The use of exogenous hormones for oestrus synchronisation and superovulation is commonplace in the Australian sheep industry, allowing farmers a high degree of control over timing of flock mating and insemination, increased reproductive efficiency and widespread dissemination of genetics. However, several studies have reported reduced fertility rates
(Armstrong and Evans 1983) and reduced numbers of spermatozoa in the reproductive tract of the ewe compared to naturally cycling animals, suggesting a failure in maintenance of cervical reservoirs as a possible cause (Quinlivan 1963; Quinlivan and Robinson 1969; Croker and
Shelton 1974; Hawk and Cooper 1977; Salamon and Maxwell 1995a). Progesterone synchronisation has been shown to have a variable effect with reports of increased production
(Croker and Shelton 1974; Rexroad and Barb 1977), decreased production (Smith and Allison
1971) and no change to mucus production (Allison 1971) in the ewe during oestrus. Altered mucus production could result in a less compatible tract for sperm migration and reduce the effectiveness of foreign body clearance from the tract by mucus. These results, while somewhat contradictory, do serve to indicate the marked effect progesterone synchronisation can have on fluids within the female tract relevant to successful reproduction. In humans, it is known that
55 exogenous hormones impact mucus characteristics such as viscosity and protein content
(Chappell et al. 2014) and that these changes negatively impact on sperm penetration in mucus
(Lewis et al. 2010) as, beyond preventing ovulation, these are the principal mechanisms of action of progestagen contraceptives in women. The use of Protaglandin-F2α for synchronisation of oestrus in cattle has also been linked with altered mucus protein content
(Yildiz and Aydin 2005), but similar recent studies on the cervical or cervicovaginal mucus of sheep have not been undertaken. Superovulation treatment has also been linked to reduced fertility (Armstrong and Evans 1983) and lower sperm numbers in the female tract when compared to naturally cycling ewes (Evans and Armstrong 1984). This effect may be caused by reduced sperm transport through the tract possibly due to larger volumes of cervical mucus present as a result of amplified levels of circulating oestrogen (Evans and Armstrong 1983), but this remains unclear. Treatment with exogenous oestrogen, a model for superovulation treatments, has been shown to increase mucus wet weight (Adams and Tang 1979), but its addition in conjunction with exogenous progestagens has resulted in decreased mucus production (Croker and Shelton 1974). In addition to the effects of exogenous hormones, the natural changes in mucus production and composition that result due to circulating endogenous hormones have not been fully defined in the ewe.
While it is clear that controlled breeding practices may impact mucus production within the female tract and even fertility, the means by which these phenomena occur have yet to be fully established. The effect of exogenous hormones on mucus characteristics such as pH, spinnbarkeit, chemical composition and protein concentration, and any correlation these changes have on sperm transport both in vivo and in vitro remains unknown. As such, the aim of this study was to (1) investigate the properties (volume, colour, spinnbarkeit, protein concentration, pH) and chemical profile (sodium, calcium, potassium, magnesium, chloride)
56 of ovine cervicovaginal mucus in naturally cycling, progesterone synchronised, superovulated and prostaglandin-F2α synchronised ewes across the oestrous cycle, and (2) examine the influence of these hormonal treatments on mucus properties, chemical profile and in vitro penetration by spermatozoa.
2.3. MATERIALS AND METHODS
2.3.1. Experimental design
Procedures herein were approved by the University of Sydney Animal Ethics Committee
(protocol number 2013/5999). In experiment 1, 20 mature Merino ewes (housed at the
University of Sydney, Camden campus, Australia) were randomised into four treatment groups: naturally cycling ewes (NAT, n = 5), progesterone sponge synchronised ewes (P4, n =
5), superovulated ewes (SOV, n = 5) and prostaglandin-F2α synchronised ewes (PGF2α, n = 5).
Mucus was collected from each ewe every 6 hours for 4 days over the follicular phase (with oestrus occurring in last two days of the follicular phase for all ewes), then once per day for 8 days (collected every second day) over the luteal phase. Circulating progesterone concentrations and androgenised wethers were used to ascertain precise onset of oestrus and timing of follicular and luteal phases (data not shown). Initial assessments of volume, spinnbarkeit and colour were made on samples at time of collection whilst chemical profile and protein concentration were determined at a later date.
In experiment 2, 30 mature Merino ewes were randomised into three treatment groups: naturally cycling ewes (NAT, n = 10), progesterone sponge synchronised ewes (P4, n = 11) and superovulated ewes (SOV, n = 11). Treatments were applied so oestrus occurred at approximately the same point for all treatment groups. Measurement of circulating progesterone and oestrogen concentrations, as well as marking by androgenised wethers was
57 used to ascertain precise onset of oestrus and timing of follicular and luteal phases (data not shown). Mucus was collected daily for 2 days over the follicular phase (oestrus) then for 2 days during the mid-luteal phase (d 7-8 of cycle). The experiment was replicated twice in the breeding season (March and June 2014), using the same animals. pH measurements were taken in vivo prior to mucus sample collection and sperm migration tests carried out following sample collection.
2.3.2. Hormone administration
Hormone schedules were the same for both experiments, with treatments applied so that expected onset of oestrus occurred at the same time for all treatment groups (although this was also confirmed by circulating hormone concentrations and markings by androgenised wethers).
To ensure all ewes in the NAT treatment group cycled at approximately the same time, they were treated in the cycle prior to the start of sample collection with intra vaginal progesterone sponges (30 mg Flugestone acetate; Vetoquinol, Lure cedex, France) for 14 days and injected with equine chorionic gonadotropin (400 IU; Pregnocol, Vetoquinol) at sponge removal (Evans and Maxwell 1987). Mucus was collected from the second oestrous cycle following synchronisation, at which time mucus was determined not to be directly affected by hormonal administration. P4 ewes were treated with intra vaginal progesterone sponges (30 mg
Flugestone acetate; Vetoquinol) for 14 days and injected with equine chorionic gonadotropin
(400 IU, Pregnocol; Vetoquinol) at sponge removal (Evans and Maxwell 1987). SOV ewes were treated with intra vaginal progesterone sponges (30 mg Flugestone acetate; Vetoquinol) for 12 days. Ewes received injections of follicle stimulating hormone (Foltropin; Vetoquinol)
2 days prior to sponge removal (22 mg x 2/ewe/day, am & pm), at sponge removal (22 mg x
2/ewe, am & pm) and the following day (22 mg/ewe, am) (de Graaf 2010). Ewes also received an injection of equine chorionic gonadotropin (400 IU, Pregnocol; Vetoquinol,) 2 days prior to
58 sponge removal. PGF2α ewes were given two injections of prostaglandin-F2α (125 mg, cloprostenol sodium, ‘Estrumate’; Merck animal health, Bendigo, VIC, Australia), ten days apart (Evans and Maxwell 1987).
2.3.3. Oestrus detection
Oestrus was detected by the use of testosterone treated wethers (n = 3) fitted with ram harnesses and crayons. Wethers were injected once/week over three weeks with testosterone (150 mg;
Testoprop, Jurox, Rutherford, NSW, Australia) prior to introduction to the flock. Ewes were checked at each collection point for new wether markings, indicating onset of oestrus (Evans and Maxwell 1987). Timing of oestrus was also checked with circulating concentrations of progesterone (experiment 1 and 2) and oestrogen (experiment 2) in ewe blood plasma, determined by commercially available radio immunoassay kits for progesterone (Coat-a-Count
Progesterone; Siemens Medical Solutions Diagnostics, Los Angeles, CA, USA) and oestrogen
(Ultrasensitive Estradiol RIA, Beckman Coulter, Brea, CA, USA). Blood collection was carried out by jugular venipuncture using lithium heparinized collection tubes daily for 7 days then every second day for 5 days. Samples were centrifuged (1228 × g, 10 min, room temperature) and the plasma frozen (-20ᵒC) for later analysis. For experiments 1 and 2 the progesterone assay sensitivity was 0.02 ng/mL, the intra assay coefficients of variation (CV) were 4.9%, 3.9% for experiments 1 and 2 1% for low and high controls, respectively. For oestrogen the intra assay CV was 19.2%, 16.6%, 16.4% and 6.9% for low and high controls, and assay 1 and 2, respectively. The inter assay CV for oestrogen was 2.23%, 6.40% for low and high controls, respectively. Data for progesterone and oestrogen not shown.
59 2.3.4. Cervical mucus collection and handling
Mucus was collected from the cervicovaginal region via aspiration using a modified cervical insemination pipette attached to a disposable 20 ml syringe. Prior to aspiration the vulva was cleaned with water and mild disinfectant to prevent contamination; insemination pipettes were thoroughly cleaned with alcohol and sterile water between collections to prevent sample contamination. For experiment 1, mucus collections began a day prior to the onset of oestrus and continued for 13 days in total. Collections occurred at 6 hour intervals (5am, 11am, 5pm and 11pm) for 4 days (follicular phase), then every second day (8am) for 8 days (luteal phase).
For experiment 2, mucus was collected once daily for two days at oestrus and then for two days at the mid-luteal phase. After aspiration mucus was deposited into sterile tubes and centrifuged
(1228 × g, 15 min, room temperature) to remove cellular debris with the supernatant retained.
After initial assessments were carried out directly after aspiration, samples destined for chemical or proteomic analysis were stored at -80ᵒC and thawed on ice prior to examination, whilst those intended for sperm migration tests were briefly stored at room temperature (≤
20°C) until testing was carried out. For proteomic analysis, samples from all ewes within a given treatment were pooled for each time point (5am, 11am, 5pm and 11pm), for follicular and luteal phases so as to provide sufficient volume for analysis. Biochemical samples were pooled across time point and ewe (using equal volumes where possible) per day, over oestrus
(2 days) for each treatment so as to provide sufficient volume for analysis. Luteal samples for biochemical analysis were pooled across ewes, as only a single time point of collection was used during this period.
60 2.3.5. Assessment of cervical mucus characteristics
Samples from each animal at each time point were assessed for volume (ml), colour and spinnbarkeit immediately after aspiration. Colour was determined via a scoring system (1-7) based on observations (1: clear, 2: clear-cloudy, 3: cloudy; 4: cloudy-milky, 5: milky, 6: milky- creamy, 7: creamy). Spinnbarkeit of the mucus was measured when enough sample was recovered, briefly a droplet of mucus was placed between two slides which were slowly separated until the strand between them broke. The length of this strand was measured (cm)
(Rexroad and Barb 1977) and given a score (1 < 0.1cm, 2 > 0.1cm, 3 > 0.5cm, 4 > 1cm, 5 >
1.5cm, 6 > 2cm, 7 > 2.5cm), with more ‘viscous’ or ‘thicker’ mucus having a shorter strand length.
2.3.6. Chemical assessments
A small aliquot of mucus was used to assess the chemical profile (mmol/L); sodium (Na), calcium (Ca), potassium (K), chloride (Cl) and magnesium (Mg). These ions were selected based on their suggested roles relating to reproduction (Casslen and Nilsson 1984; Magnus et al. 1990a). Samples were assessed by Regional Laboratory services (NATA accredited;
Benalla, Victoria, Australia), with technique varying depending on element tested. Chloride,
Magnesium and Calcium were all assessed using specific reagents, namely Mercury/Iron thiocyanate (95% level of confidence ± 2.2mM at 99mM), CPZ (Roche) (95% level of confidence ± 0.148mM at 1.29mM) and Arsenazo III (95% level of confidence ± 0.1mM at
2.3mM), respectively, coupled with spectroscopy techniques. Both potassium and sodium were assessed via flame photometry techniques (potassium; 95% level of confidence ± 0.18mM at
4.2mM, sodium; 95% level of confidence ± 2.8mM at 138mM).
61 2.3.7. Protein concentration
The protein concentration (µg/µl) of sample pools was determined using a Pierce bicinchoninic acid assay (BCA; Thermo Scientific, Rockford, IL, USA; range of detection 20-2000µg/ml) as per manufacturer’s instructions.
2.3.8. pH
The pH of samples was determined in vivo using a portable probe and sensor (Sentron, VD
Leek, The Netherlands, limits of detection pH 0-14, ± 0.01) with measurements taken from the cervicovaginal mucus pool just prior to sample collection. To ensure pH readings were accurate, measurements were taken only when the sensor chip could be visualized to be within a pool of mucus. The sensor was calibrated periodically as per manufacturer’s instructions.
2.3.9. Sperm migration
The ability of spermatozoa to penetrate cervicovaginal mucus was determined using a cervical mucus migration test (CMMT) (Katz, Overstreet and Hanson 1980). After collection, follicular phase mucus was aspirated into flat glass capillary tubes (3 replicates/animal, Vitrocom Inc.
Mtn. Lakes, NJ, USA; 0.3х3.0mm I.D.) and sealed at one end (Cristaseal; Cope laboratories,
Lancing, Sussex, UK). Capillaries were incubated (1 hour, 37ᵒC) vertically in beem capsules
(ProScitech, Thuringowa, QLD, Australia) with 50µl frozen thawed merino ram semen (20 x
106 motile sperm/ml; stained 1:1 with 20 µg/ml Hoechst 33342; IDENT, Hamilton Thorne,
Beverly, MA, USA). Following incubation, capillaries were heat fixed to render sperm immotile. Briefly, capillaries were removed from beem capsules, wiped dry, immediately sealed before being placed on a heating block (30 sec, 50ᵒC) and then placed in a freezer (60 sec, -20ᵒ) on a Styrofoam mat. The number of spermatozoa to reach 1cm in each cervical mucus sample was determined using a fluorescent microscope (400 × magnification, Olympus BHS,
62 Tokyo, Japan) comprising a 270-380 nm band pass filter. Emissions were observed through a
380 nm dichroic mirror.
2.3.10. Statistical analysis
Analysis of volume, pH, chemical analysis and protein concentration data was carried out using a restricted maximum likelihood method (REML) in Genstat (version 15, VSN International,
Hemel Hempstead, Hertfordshire, UK) with treatment (NAT, P4, SOV and PGF2α) and phase of oestrous cycle (follicular/oestrus and luteal) set as the fixed model whilst ewe (experiment
1; n = 20, experiment 2; n = 30) was set as the random model for volume, pH and protein, and replicate was used for chemical analysis. Means are reported as ± standard error of the means
(SEM) (µl, pH, mmol/L or µg/µl, respectively) with p <0.05 considered significant.
Analysis of colour and spinnbarkeit was carried out using an ordinal logistic regression model in the program R (version 3.1.2, R Foundation for Statistical Computing, Vienna, Austria). The fixed effects of treatment (NAT, P4, SOV and PGF2α), and phase of cycle (follicular and luteal) were assessed, with ewe set as the random model. Values reported are range values that denote a score for colour and spinnbarkeit.
Analysis of sperm count data was carried out using a generalized linear mixed model (GLMM) with Poisson distribution in Genstat (version 15, VSN International, Hemel Hempstead,
Hertfordshire, UK). Treatment (NAT, P4 and SOV) and observer (capillaries counted by two observers to ensure result reliability) were set as fixed effects, whilst collection period (March and June), ewe (n = 30) and replicate (n = 3) were set as the random model. Means are reported as ± SEM (sperm count) with p <0.05 considered significant.
63 2.4. RESULTS
2.4.1. Volume
The average volume of mucus produced per ewe (n =5 ewes/treatment) was greater at oestrus than during the luteal phase for naturally cycling, P4 and SOV ewes (p <0.05, Figure 2.1 A).
Levels of mucus produced by PGF2α ewes during the follicular phase were similar to luteal levels (p >0.05). During the follicular phase, treatment with exogenous hormones greatly affected the average volume of mucus produced per ewe, with SOV ewes producing significantly more mucus than NAT ewes, and PGF2α ewes having significantly less mucus than NAT ewes (p <0.05). P4 ewes produced similar volumes of mucus compared to NAT ewes during the follicular phase (p >0.05). Volumes of mucus produced during the luteal phase were comparable to NAT, and all other treatments (p >0.05).
64 A B
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Figure 2.1 Average volume per ewe (A), colour (B), spinnbarkeit (C) and protein
concentration (D) of cervicovaginal mucus from naturally cycling (NAT; white bars),
progesterone synchronised (P4; light grey bars), superovulated (SOV; dark grey bars) and
prostaglandin-F2α synchronised (PGF2α; black bars) ewes (n = 5 ewes/treatment) during the
follicular (N = 24 aspirations/ewe) and luteal phases (N = 8 aspirations/ewe) of the oestrous
cycle. Values without common superscripts differ significantly (p <0.05).
2.4.2. Colour
Luteal phase mucus of NAT, P4 and SOV ewes was milkier (p <0.05) in appearance than mucus produced during the follicular phase (Figure 2.1 B), however mucus colour of PGF2α ewes was comparable across the cycle (p >0.05). Exogenous hormones also affected mucus colour during the follicular phase, with P4 and SOV animals having cloudier mucus than that of NAT animals during the follicular phase (p <0.05).
65
2.4.3. Spinnbarkeit
Mucus viscosity, measured by spinnbarkeit, did not differ significantly between the follicular and luteal phases of the oestrous cycle for NAT, P4, SOV or PGF2α ewes (p >0.05). The use of exogenous hormones for oestrous synchronisation and superovulation did not have a marked effect on the mucus spinnbarkeit either (p >0.05, Figure 2.1 C).
2.4.4. Chemical assessment
Exogenous hormones had a varied effect on levels of sodium, potassium, calcium and magnesium in oestrus mucus (Table 2.1). Levels of sodium in mucus of NAT and P4 ewes were comparable (p >0.05, Table 1). Levels of potassium, magnesium and calcium were all similar to NAT, and all other treatments (p >0.05). Whilst levels of chloride in SOV animals were significantly higher than those found in mucus of P4 and PGF2α (p <0.05), levels of all three treatments compared to those of NAT mucus were alike (p >0.05, Table 2.1). Low sample volumes obtained during the luteal phase prevented sufficient replicates of samples for chemical analysis to be obtained, as such, results for luteal phase mucus were not included.
66 Table 2.1 Levels of sodium (Na), magnesium (Mg), calcium (Ca), potassium (K) and chloride
(Cl) in the cervicovaginal mucus of naturally cycling (NAT), progesterone synchronised (P4), superovulated (SOV) and prostaglandin-F2α synchronised (PGF2α) ewes during oestrus (n = 5 ewes/treatment, N = 8 aspirations/ewe).
Mg Ca Treatment Na (mmol/L) K (mmol/L) Cl (mmol/L) (mmol/L) (mmol/L)
Naturally cycling (NAT) 102.00 ±14.32 a,b 0.24 ± 0.09 a 0.75 ± 0.08 a 17.75 ± 3.10 a 129.00 ± 3.54 a,b
a,b a a a a P₄ Syncronised (P4) 98.50 ± 5.67 0.26 ± 0.04 0.8 ± 0.18 13.73 ± 1.56 111.00 ± 6.36 Superovulated (SOV) 129.00 ± 6.94 a 0.24 ± 0.06 a 0.67 ± 0.1 a 15.43 ± 0.80 a 131.67 ± 5.90 b
PGF₂α Synchronised (PGF₂α) 69.50 ± 12.96 b 0.29 ± 0.03 a 0.64 ± 0.15 a 20.03 ± 2.79 a <100 a
Only partial results were available for chloride as testing method used was not able to detect levels of <100 mmol/L. Within column, values without common superscripts differ significantly (p <0.05)
2.4.5. Protein concentration
The protein concentration of mucus (µg/µl) from NAT ewes varied markedly between the phases of the oestrous cycle, with significantly more protein present in luteal mucus than follicular phase mucus (p <0.05, Figure 2.1 D). However, this trend was not evident in the mucus of P4, SOV or PGF2α ewes, which each had comparable protein levels between follicular and luteal phases (p >0.05). Superovulation (SOV) did however result in increased protein during the follicular phase, in comparison to NAT mucus (p <0.05).
2.4.6. pH
In vivo mucus pH values, were comparable between NAT, P4 and SOV during oestrus, only ranging from 6.2-6.5 (p >0.05, Figure 2.2). Mucus was significantly more basic during the mid- luteal phase for SOV animals when compared to values during oestrus (p <0.05), however pH
67 values between the phases of the cycle were comparable for P4 ewes (p >0.05). Data for NAT animals during the mid-luteal phase could not be obtained due to insufficient sample volume, as such comparisons across the oestrous cycle cannot be made.
Figure 2.2 pH of cervicovaginal mucus from naturally cycling (NAT; white bars), progesterone synchronised (P4; light grey bars) and superovulated (SOV; dark grey bars) ewes
(n = 11 ewes/treatment) during oestrus and the mid-luteal phase of the oestrous cycle. Values without common superscripts differ significantly (p <0.05).
2.4.7. Sperm migration in CV mucus
Comparable numbers (p >0.05) of frozen-thawed spermatozoa penetrated 1cm into the follicular phase mucus of NAT, P4 and SOV ewes (average number of sperm; 13.2 ± 3.6, 14.6
± 3.0, 18.1 ± 1.2, respectively).
2.5. DISCUSSION
This study has highlighted the changes in cervicovaginal mucus that occur naturally across the oestrous cycle, and as a result of exogenous hormones used for synchronisation and
68 superovulation in sheep. During the follicular phase mucus from naturally cycling ewes was more abundant, clearer in colour and less proteinaceous than that produced during the luteal phase. The most pronounced effects of exogenous hormones were seen after either superovulation or synchronisation with PGF2α, with the former resulting in increased mucus production and protein concentration, whilst the latter significantly reduced mucus production.
2.5.1. Mucus volume increases during the follicular phase and is altered by superovulation and prostaglandin-F2α synchronisation
Research has shown that both mucus production and hydration are largely under hormonal control (Bigelow et al. 2004) and as such vary across the reproductive cycle, with peak hydration and mucus volume during the ovulatory period in humans (Igarashi 1954; Chantler and Debruyne 1977). This would explain the increased volume of mucus produced in the present study during the follicular phase, a period during which oestrogen concentrations are highest. Supplementation studies have highlighted the role of oestrogen in mucus production and hydration, as it results in increases in both production and hydration (Allison 1971; Adams and Tang 1979) in a dose sensitive manner (Allison 1971). The increase in mucus production may in part be due to increased para-cellular permeability of ectocervical cells, which has been shown to occur after oestrogen supplementation in human ectocervical cell cultures (Gorodeski
2000).
In addition to changes over the oestrous cycle due to natural variation in endogenous hormone concentrations, the effects of exogenous hormone use on mucus hydration and production have also been reported. Synchronisation by injection of prostaglandin-F2α had a marked effect on mucus production with PGF2α ewes producing approximately half the mucus produced by naturally cycling animals. Altered mucus volumes can impact on tract health, with lowered
69 volumes resulting in a decreased ability to eliminate foreign bodies from the tract and a less compatible tract for sperm migration. Indeed, reports have shown that various PGF2α synchronisation methods in sheep result in lower sperm numbers within the tract (Hawk and
Cooper 1977; Boland, Gordon and Kelleher 1978), altered ovulation rates (Fierro et al. 2011) and concentrations of progesterone (White et al. 1987; Fierro et al. 2011) and decreased fertility
(Boland, Gordon and Kelleher 1978; Menchaca et al. 2004). This is the first report to suggest this may be due to significant reduction in cervicovaginal mucus in the ewe. The deleterious effects of low mucus volumes in the tract could possibly be overcome on farm through the use of laparoscopic insemination as opposed to cervical insemination in artificial insemination programmes.
In the present study progesterone synchronisation did not significantly impact mucus volume when compared to naturally cycling animals. Previous reports are inconsistent, with increases
(Croker and Shelton 1974; Rexroad and Barb 1977) and decreases (Smith and Allison 1971) in levels of mucus produced at oestrus following progesterone synchronisation in addition to no changes to mucus production (Allison 1971). Some have suggested that delayed oestrus onset and length of oestrus could in part explain this discrepancy amongst reports (Allison
1971), although oestrus length has been shown not to be effected by progesterone synchronisation (Croker and Shelton 1974; Hawk and Cooper 1977). The variation in effect of progesterone synchronisation on mucus production in the literature could be due to differences in the type of progesterone used (progesterone/ 6-α-methyl-17α-acetoxyprogesterone: MAP/ several variants of 17α-acetoxy-9α-fluoro-11β-hydroxypregn-4-ene-3, 20-dion; Cronolone/
Flugestone acetate), dose (10/30/60/90 mg) and method of application (intravaginal sponge/ intramuscular injection). The results of the present study, in which progesterone
70 synchronisation did not alter mucus production, provide a positive outlook for the continued use of this controlled breeding practice on farm.
The higher volumes of mucus reported in the present study in SOV animals have not been directly supported within the literature. However, the result was expected as oestrogen concentrations are clearly linked to increased mucus production, evident in the higher mucus volumes produced during the follicular phase, and widespread anecdotal evidence of increased mucus volumes after superovulation. Oestradiol supplementation studies can serve as a model for superovulation as they mirror the high levels of oestrogen that result from increased follicular growth. Such supplementation studies are conflicting, showing both an increase
(Allison 1971; Adams and Tang 1979) and decrease in mucus production (Croker and Shelton
1974) along with no change in production (Rexroad and Barb 1977). Excessive mucus volumes may also negatively impact sperm transit in the tract by preventing establishment of sperm reservoirs in the cervix, possibly via excess mucus acting as a wave and effectively flushing the tract of sperm. Although as the sperm reservoirs are likely within the grooves of the convoluted ewe cervix, the most likely site at which a wave of mucus could effectively flush spermatozoa would be in the lumen, during periods of sperm transit into these ‘privileged pathways’ (Mullins and Saacke 1989). Previous work has shown that laparoscopic insemination as opposed to cervical insemination can overcome the reduced fertility associated with this controlled breeding technique. As a result, laparoscopic intrauterine insemination is widely used around the world for insemination of superovulated ewes.
2.5.2. Protein concentration peaks during the luteal phase and after superovulation
Protein concentration of mucus reported in the present study varied between phases of the oestrous cycle, with levels increasing from the follicular to the luteal phase. This is supported
71 by previous research (Hamana, Elbanna and Hafez 1971), with protein content reported to be inversely proportional to the level of hydration of mucus (Wolf et al. 1977b; Morales, Roco and Vigil 1993), with peak mucus hydration occurring during the follicular period during which protein levels are lowest. Cyclical changes occur in 3 major groupings of human cervical mucus proteins with albumin and a mucoid fraction both peaking around ovulation, suggestive of a link with higher oestrogen levels at this time (Moghissi and Neuhaus 1966). Recent quantitative proteomic analysis of human cervical mucus supports this as phase (pre-ovulatory, ovulatory
& post-ovulatory) specific proteins were identified (Grande et al. 2015). Protein concentration in the present study was altered by exogenous hormones, with mucus from superovulated ewes having higher average protein content than mucus from naturally cycling ewes during the follicular phase. As discussed, a link between oestrogen and protein concentration is evident in the literature (Moghissi and Neuhaus 1966; Rexroad and Barb 1977). This supports our finding of higher protein concentration in SOV mucus but contradicts the levels in NAT animals, which were lowest during the follicular phase. The diluting effect that increased mucus volume during the follicular phase has on protein content (Wolf et al. 1977a; Morales, Roco and Vigil 1993) could partially explain this result. Interestingly, mucus volume was also highest for SOV animals compared to NAT, so whilst mucus volume increased it was not simply a result of increased hydration, but was also accompanied by an increase in production of mucus proteins.
Perhaps superovulation also affects the composition of proteins produced in addition to increased mucus volumes, which could have flow on effects for interactions between sperm and mucus. Mucus synchronisation with progesterone or PGF2α has resulted in 5 fold increases in protein concentration of oestrus cervical mucus compared to levels in mucus of cows in a spontaneous oestrus (Tsiligianni et al. 2001). However, these results do not support our findings in regards to P4 or PGF2α ewes, which had comparable mucus protein levels to naturally cycling animals. This difference is not explained by variation in protocols as both
72 were similar; perhaps variation in site of mucus collection is the cause or perhaps different parts of the female reproductive tract have varied protein secreting abilities. Whilst total protein concentration does give an overall idea of protein changes as a result of exogenous hormone use, a more detailed proteomic analysis of cervical mucus could illustrate specific proteomic changes in cervical mucus and even allow the potential correlation of individual proteins to mucus characteristics which influence sperm function and successful fertilisation.
2.5.3. Mucus colour, spinnbarkeit and penetrability; over the oestrous cycles and after synchronisation and superovulation
The marked differences in mucus volumes, hydration and protein concentration over the oestrous cycle and after exogenous hormone use directly affect mucus colour, its spinnbarkeit value and penetrability. In the present study, mucus from the follicular phase was significantly less cloudy than that of the luteal phase for NAT, P4 and SOV animals. This cyclical nature of mucus colour over the oestrous cycle is supported by previous work in humans (Hamana,
Elbanna and Hafez 1971; Bigelow et al. 2004) and in the ewe (Evans and Maxwell 1987) and is in part due to changes in protein content over the cycle, with increased protein content resulting in cloudier more opaque mucus. Accordingly, we found that mucus colour variation was very similar to protein differences in NAT, P4, SOV and PGF2α animals during the follicular phase. The spinnbarkeit of mucus is also largely dictated by the level of hydration and protein concentration, specifically of mucins (Wolf et al. 1977a; Wolf, Sokoloski and Litt
1980), over the oestrous cycle. Follicular phase mucus is generally accepted to be more hydrated and less viscous (Wolf et al. 1977b; Morales, Roco and Vigil 1993; Bigelow et al.
2004), whilst luteal mucus is thicker or more viscous (Schumacher 1970; Hamana, Elbanna and Hafez 1971; Chantler and Debruyne 1977) and is inhibitory towards sperm during this period (Bigelow et al. 2004; Grande et al. 2015). This was not the case in the present study as
73 spinnbarkeit was similar between the oestrus and luteal phases. However, comparisons with some studies on this mucus characteristic are difficult due to the complex structural behaviour of mucus as it is a non-Newtonian fluid (Cone 2009a; Lai et al. 2009b) and variation in the parameter tested; viscosity, viscoelasticity, spinnbarkeit and thickness. Reports also vary in regards to the effect of synchronisation on mucus spinnbarkeit, with progesterone and prostaglandin-F2α synchronisation resulting in unaltered (Rexroad and Barb 1977) and decreased (Tsiligianni et al. 2000) spinnbarkeit values in comparison to mucus of naturally cycling animals. Research has also indicated that calcium plays a significant role in mucus structure through its involvement in mucin charge, bonding, release and expansion, which effects mucus swelling, hydration, structure and therefore also ‘viscosity’ (Chen et al. 2010;
Muchekehu and Quinton 2010) and as such any alteration to available extracellular, free or mucus bound forms might alter mucus structure. In this study, we found similar levels of calcium ions in mucus from NAT, P4, SOV and PGF2α ewes, which might partially explain the similarity in spinnbarkeit values between these groups.
Penetration of spermatozoa through all three mucus types (NAT, P4 and SOV) was found to be comparable in the current study. This result was expected given the similarity in mucus spinnbarkeit between NAT, P4 and SOV ewes, although unexpected given the reported link between exogenous hormone use and impaired sperm transport in vivo (Quinlivan and
Robinson 1969; Hawk and Cooper 1977) as well as altered motility in vitro (Cal et al. 1973).
A possible explanation for this disparity is the static nature of mucus within the CMMT in vitro, in addition, structural alteration of mucus could be taking place when loading mucus into the CMMT capillary tubes. Furthermore, spermatozoa appeared to migrate through channels in the mucus, these channels are comprised of the liquid phase or ‘mucus plasma’ of mucus that fills any residual spaces that are present within the mucus gel phase. Large variation both
74 between capillaries for the same ewe and ewes of the same treatment were evident, highlighting the degree of variability that is seen when using natural CV mucus for sperm migration tests.
However, as the aim was to test if exogenous hormone use affected mucus in such a way that sperm migration was altered, the use of synthetic mucus was not an option.
2.5.4. Mucus pH is unaltered by exogenous hormones
Previous research has found that increasing circulating oestrogen concentrations results in more basic vaginal mucus (Eggertkruse et al. 1993; Olmsted et al. 2000; Gorodeski et al. 2005).
Unfortunately, the low sample volumes obtained in the present study prevented measurements of naturally cycling ewes during the luteal phase so this theory has remained untested.
Nonetheless, it is worth noting that reports in this area are somewhat contradictory. In dairy cattle, pH is reported to be lowest during oestrus in vaginal (Lewis and Newman 1984) and cervical mucus (Mori et al. 1979), whilst human cervical mucus did not change over the reproductive cycle (Wolf et al. 1977b). In the present study, mucus pH was not altered by hormonal synchronisation or superovulation. Similar work in cattle supports this finding, in which mucus from cattle in spontaneous oestrus had similar pH to mucus from P4 and PGF2α synchronised animals (Tsiligianni et al. 2000). The opposite has also been reported, with increasing circulating oestrogen levels resulting in more basic vaginal mucus (Eggertkruse et al. 1993; Olmsted et al. 2000). Cervicovaginal mucus pH for NAT, P4 and SOV ranged from
6.2 to 6.5, which is more alkaline than the generally reported vaginal pH of 4 in humans
(Eggertkruse et al. 1993; Olmsted et al. 2000) but more acidic than the average pH of 7.4 in dairy cattle (Lewis and Newman 1984). This discrepancy could be in part be related to air exposure of mucus between speculum insertion, pH probe insertion and meter reading.
Previous work has highlighted the necessity of timely pH readings when taking in situ mucus pH, as air exposure leads to alkalization of the mucus (Olmsted et al. 2000; Correa, Mattos and
75 Ferrari 2001). While this could explain the more alkaline pH reported here, readings within this study were taken immediately after visualisation of the mucus pool so as to reduce the effects of air exposure so this is unlikely. The precise in vivo analysis of mucus pH used in the present study was very accurate (± 0.01) and so raises the question of why results vary. Perhaps this variation in vaginal pH is due to differences in normal vaginal microflora between humans and ruminants; humans having high levels of lactic acid produced by Lactobacillus spp.
(Boskey et al. 2001; O'Hanlon, Moench and Cone 2013) whereas ruminants may have less, or perhaps a different dominate species of microflora or by-product produced. Differences may also be due to variation in sampling site and what is considered within literature as an ‘ecto- cervical’, ‘vaginal’ or ‘cervicovaginal’ mucus sample, as not all of these areas are precisely demarcated. All are considered external to the cervical os, although if part of this sample had just been released from the reportedly more alkaline os or intentionally sampled from ‘around’ the os, then perhaps the pH would be higher. In any case, results reported here can be taken as the current pH of cervicovaginal mucus in the merino ewe.
In conclusion, the present study has demonstrated that mucus varies considerably across the cycle and following the use of exogenous hormones to enable oestrus synchronisation and superovulation in the ewe. Oestrus mucus was more abundant and less proteinaceous than luteal phase mucus, whilst superovulation and synchronisation with PGF2α, significantly altered mucus properties; the former resulted in increased mucus production and protein concentration, whilst the latter significantly reduced mucus production. Further studies into the effect of pH and chemical changes on mucus structure, sperm migration and survival are needed, as is the effect of exogenous hormones on the proteomic composition of cervical mucus. Additional research could assist in the improvement of sperm transport within the
76 female tract (especially through the cervix) following cervical artificial insemination and ultimately improve fertility.
2.6. Acknowledgements
Research was supported by the NSW Stud Merino Breeders Association Trust and Australian
Wool Innovation. The authors would like to thank Mr Byron Biffin, Mr Keith Tribe, Miss
Taylor Pini and Mr Bradley James King for their on farm assistance, Miss Evelyn Hall and Mr
Peter Thomson for their statistical consultation and Miss Kim Heasman for laboratory assistance. The authors would also like to acknowledge Bioniche Animal Health Australasia
(now Vetoquinol) for the generous donation of Ovagest sponges, eCG (Pregnocol) and FSH
(Foltropin-V).
77 Chapter 3. Oestrus synchronisation and superovulation alter the cervicovaginal mucus proteome of the ewe
The experiments described herein have been published as:
Maddison, JW, Rickard, JP, Bernecic, NC, Tsikis, G, Soleilhavoup, C, Labas, V, Combes-Soia,
L, Harichaux, G, Druart, X, Leahy, T & de Graaf, SP 2017, 'Oestrus synchronisation and superovulation alter the cervicovaginal mucus proteome of the ewe', Journal of Proteomics, vol. 155, no., pp. 1-10.
3.1. ABSTRACT
Although essential for artificial insemination and MOET (multiple ovulation and embryo transfer), oestrus synchronisation and superovulation are associated with increased female reproductive tract mucus production and altered sperm transport. The effects of such breeding practices on the ovine cervicovaginal mucus proteome have not been detailed. The aim of this study was to qualitatively and quantitatively investigate the Merino CV mucus proteome in naturally cycling ewes at oestrus and mid-luteal phase, and quantitatively compare CV oestrus mucus proteomes of NAT, progesterone synchronised and superovulated ewes. Quantitative analysis revealed 60 proteins were more abundant during oestrus and 127 were more abundant during the luteal phase, with 27 oestrus specific and 40 luteal specific proteins identified. The oestrus proteins most disparate in abundance compared to mid-luteal phase were ceruloplasmin
(CP), chitinase-3-like protein 1 (CHI3L1), clusterin (CLU), alkaline phosphatase (ALPL) and mucin-16 (MUC16). Exogenous hormones greatly altered the proteome with 51 and 32 proteins
78 more abundant and 98 and 53 proteins less abundant, in P4 and SOV mucus, respectively when compared to NAT mucus. Investigation of the impact of these proteomic changes on sperm motility and longevity within mucus may help improve sperm transport and fertility following cervical AI.
3.2. INTRODUCTION
Mucus is a complex non-Newtonian (Lai et al. 2009b) biological fluid found in many of the body’s organs, which plays a vital role in their maintenance, protection and overall function.
Mucus is found throughout the reproductive tract and plays a dynamic role as a lubricant for normal organ function (Wang et al. 2013), as a protective barrier to infection (Eggert-Kruse et al. 2000) and also plays a critical role in mediating sperm trasnport through the female repordcutive tract. Female cervicovaginal mucus consists of an amalgamation of fluids from the oviducts, uterus (endometrial), cervical canal and vagina. In humans, cervicovaginal (CV) mucus is largely comprised of water (90-99 %) (Katz, Slade and Nakajima 1997), cellular material (cell debris from the natural growth and degradation of the reproductive tract or cells with an immune function), various electrolytes, carbohydrates, amino acids, lipids, peptides and proteins, which includes mucins; large filamentous glycoproteins secreted from goblet cells within the endometrium, oviduct and cervical tissues (Gipson et al. 1997; Lagow,
DeSouza and Carson 1999). Mucus is continuously produced in the female and ovine reproductive tract and varies in response to hormonal (endogenous and exogenous) fluctuations
(Schumacher 1970; Allison 1971; Smith and Allison 1971; Evans and Maxwell 1987). During oestrus, mucus is generally more profuse, watery, clear and favourable for sperm penetration, in comparison to the scant, thick, opaque mucus that is unfavourable to sperm penetration during the luteal phase (Schumacher 1970).
79 Mucus plays a pivotal role in sperm migration through the tract, especially cervicovaginal mucus in vaginal depositors such as sheep (Evans and Maxwell 1987). Several studies have highlighted changes in the production and composition of mucus from the application of exogenous hormones for controlled breeding purposes (Allison 1971; Smith and Allison 1971).
The administration of progesterone to synchronise oestrus has resulted in contradictory reports of both increased (Allison 1971; Croker and Shelton 1974; Rexroad and Barb 1977) and decreased (Smith and Allison 1971) mucus production. Furthermore, its use in conjunction with oestradiol has also resulted in decreased mucus production (Croker and Shelton 1974).
Oestradiol has also been shown to increase the protein concentration of mucus (Rexroad and
Barb 1977) and wet weight of mucus, (Adams and Tang 1979) whilst prolonged grazing on oestrogenic clover caused morphological changes to the cervix (Lightfoot et al. 1974; Lightfoot and Adams 1979) which consequently resulted in decreased mucus production. Superovulation has also been shown to significantly increase mucus production and protein concentration during oestrus in the Merino ewe (Maddison et al. 2016). These modifications to the production and composition of mucus have been linked to impaired sperm transport and reduced fertility
(Quinlivan and Robinson 1967; Croker and Shelton 1974). An understanding of the proteomic composition of ovine CV mucus and the effects of such controlled breeding practices could help elucidate the environment that spermatozoa are exposed to on their journey through the female tract, especially the cervix, a known barrier to sperm transport. Such proteomic information could also shed light on the acceptability of oestrus mucus to spermatozoa in comparison to its luteal counterpart and also the mechanisms behind reduced sperm transport in hormonally treated ewes, as reported previously (Quinlivan and Robinson 1969; Lightfoot et al. 1974; Hawk and Cooper 1977). As such, the aim of this study was to qualitatively and quantitatively determine the protein composition of ovine cervicovaginal mucus across the
80 oestrous cycle and after use of exogenous hormones commonly used for synchronisation and superovulation.
3.3. MATERIALS AND METHODS
Procedures herein where approved by the University of Sydney Animal Ethics Committee
(protocol number 2013/5999) with sheep (Ovis aries) housed at the University of Sydney
Camden Farms.
3.3.1. Hormone administration
Hormone administration was carried out so that oestrus occurred at the same time for each treatment group. Naturally cycling ewes (NAT; n = 12) were treated with intra vaginal progesterone sponges (30 mg Flugestone acetate, Vetoquinol Australia, Brisbane, Australia) for 14 days, to ensure synchrony of their cycles and correct timing of oestrus comparable to other treatments, then injected with equine chorionic gonadotropin (400IU, Pregnocol,
Vetoquinol) at sponge removal. Mucus was collected from the second oestrous cycle following synchronisation, at which time mucus was determined not to be directly affected by hormonal administration.
Progesterone synchronised ewes (P4; n = 12) were treated with intravaginal progesterone sponges (30 mg Flugestone acetate, Vetoquinol) for 14 days, then injected with equine chorionic gonadotropin (400IU, Pregnocol, Vetoquinol) at sponge removal. Samples from P4 synchronised ewes were collected directly following sponge removal at the subsequent oestrus.
81 Superovulated ewes (SOV; n = 12) were treated with intravaginal progesterone sponges (30 mg Flugestone acetate; Vetoquinol) for 12 days. Ewes received injections of follicle stimulating hormone (Foltropin; Vetoquinol) 2 days prior to sponge removal (22mg x
2/ewe/day, am & pm), at sponge removal (22mg x 2/ewe, am & pm) and the following day
(22mg/ewe, am) (de Graaf 2010). Ewes also received an injection of equine chorionic gonadotropin (400IU, Pregnocol; Vetoquinol) 2 days prior to sponge removal (de Graaf 2010).
The entire experiment was repeated during the same breeding season, producing samples from two collection periods (Period 1; P1 and Period 2; P2).
3.3.2. Oestrus detection
The onset of oestrus was determined by the use of testosterone treated wethers fitted with ram harnesses with crayons. Ewes were checked for markings at regular intervals in conjunction with expected onset of oestrus determined through previous experiments (data not shown).
Wethers were treated with three weekly injections of testosterone (150 mg, Testoprop, Jurox,
Rutherford, NSW, Australia) prior to introduction into the ewe flock (Evans and Maxwell
1987). Onset of oestrus was also determined using circulating concentrations of progesterone and oestrogen in ewe blood plasma, determined by commercially available radio-labelled immunoassay kits for progesterone (Coat-a-Count Progesterone; Siemens Medical Solutions
Diagnostics, Los Angeles, CA, USA) and oestrogen (Ultrasensitive Estradiol RIA, Beckman
Coulter, Brea, CA, USA) (supplementary figure S-1). Blood collection was carried out by jugular venepuncture using lithium heparinised collection tubes 2 days prior to expected onset of oestrus until 1 day after mid luteal phase mucus collections, totalling 11 days. Samples were centrifuged (1228 × g, 10 min, 25°C) and plasma frozen (-20°C) until analysis. The progesterone assay sensitivity was 0.02 ng/mL and the intra assay coefficients of variation were
82 4.9% and 3.9% for low and high controls, respectively. For oestrogen assays, the intra assay coefficients of variation were 19.2%, 16.6%, 16.4% and 6.9% for low and high controls, and assay 1 and 2, respectively. The inter assay coefficients of variation for oestrogen were 2.23%,
6.40% for low and high controls, respectively.
3.3.3. Cervicovaginal mucus collection
Mucus was collected from the cervicovaginal region of the ewe reproductive tract by aspiration using a modified cervical insemination pipette attached to a disposable 20 ml syringe.
Collections were carried out daily for two days over the oestrus period and daily for two days over the midpoint of the luteal phase, for all treatments. After collection, mucus was deposited into sterile tubes then centrifuged (1228 × g, 15 min, 25°C) to remove cellular debris with the supernatant retained. Samples were immediately stored at -80°C until assessments were carried out.
3.3.4. Sample preparation
To qualitatively assess the protein composition of cervicovaginal mucus across the entire oestrous cycle, equal volumes (10 µl/ewe) of CV mucus from naturally cycling ewes at oestrus and mid-luteal phase were pooled (n = 3 ewes) using positive displacement pipettes to ensure volumes were accurate. To quantitatively assess the protein composition of CV mucus at oestrus and mid-luteal phase, CV mucus from naturally cycling ewes (n = 3 ewes) at oestrus (2 days) and mid-luteal points (2 days) over the course of one oestrous cycle was used (P1 and P2 included equally). For comparison of protein composition across treatments (NAT, P4 and
SOV), samples of CV mucus (n = 6 ewes/treatment) during oestrus were pooled according to treatment group, separately for each collection period (P1 and P2).
83 All samples were thawed on ice and treated with acetonitrile (1:1) to reduce viscosity. Samples were then sonicated with an ultrasonic bath sonicator (Fisher Scientific, Bremen, Germany) to solubilize proteins (30 min, 25°C) and centrifuged (15000 × g, 10 min, 4°C) with the supernatant retained. Mucus protein concentration was determined using a BC Protein Assay
(Interchim, Montlucon Cedex, France), using bovine serum albumin (Sigma-Aldrich, Saint
Quentin Fallavier, France) as the standard.
3.3.5. 1D-SDS PAGE of qualitative samples
SDS-PAGE was carried out to separate the proteins within the whole cycle (qualitative) sample using an 8-16% gradient gel (180V, 60 min) with a protein load of 30 µg total protein per lane.
The gel was then stained with Coomassie blue (overnight, room temp. (RT) with agitation) then the whole lane was excised into 30 individual bands ready for digestion and analysis.
3.3.6. Protein digestion
3.3.6.1 In-gel digestion of qualitative samples
Gel pieces were washed separately in water: acetonitrile solution (1:1, 5 min) followed by
100% acetonitrile (10 min). Reduction and cysteine alkylation was performed by successive incubation with 10 mM dithiothreitol in 50 mM NH4HCO3 (30 min, 56°C), then 55 mM iodoacetamide in 50 mM NH4HCO3 (20 min, RT, in dark). Pieces were then incubated with 50 mM NH4HCO3 and acetonitrile (1:1, 10min) followed by acetonitrile (15 min). Proteolytic digestion was carried out overnight using 25 mM NH4HCO3 with 12.5 ng/µl Trypsin
(Sequencing grade, Roche diagnostics, Paris, France). Resultant peptides were extracted by incubation in 5% formic acid (sonicated) with the supernatant removed and saved, followed by incubation in acetonitrile and 1% formic acid (1:1, 10 min) and a final incubation with acetonitrile (5 min), again supernatant was removed and saved. These two peptide extractions
84 were pooled and dried using a SPD1010 speedvac system (Thermosavant, Thermofisher
Scientific, Bremen, Germany) and the resultant peptide mixture was analysed by nanoflow liquid chromatography tandem mass spectrometry (Nano LC-MS/MS).
3.3.6.1 In-solution digestion of quantitative samples
Mucus proteins in all other samples bar the whole cycle qualitative samples were solubilized by adding 0.5% ProteaseMAX TM (Promega biosciences, San Luis Obipso, CA, USA).
Reduction and cysteine alkylation was performed by successive incubation in 10 mM dithiothreitol in 50 mM NH4HCO3 (30 min, 56°C), then 55 mM iodoacetamide in 50 mM
NH4HCO3 (30 min, RT, in the dark). Proteolytic digestion was carried out overnight using 1
µg Trypsin (Sequencing grade, Roche diagnostics, Paris, France) and acidified with 0.1%
Formic Acid (30 min, 60°C) so as to degrade ProteaseMAX TM surfactant (Promega biosciences, San Luis Obipso, CA, USA). The resultant peptide mixture was analysed by nanoLC-MS/MS.
3.3.7. NanoLC- MS/MS
All experiments were performed on a LTQ Orbitrap Velos Mass Spectrometer (Thermo Fisher
Scientific, Bremen, Germany) coupled to an Ultimate® 3000 RSLC chromatographer (Dionex,
Amsterdam, The Netherlands). Samples were loaded on a trap column (Acclaim PepMap 100
C18, 100 mm i.d. x 2 cm long, 3 mm particles) and desalted for 10 min at 5 mL/min with 4% solvent B. Mobile phases consisted of (A) 0.1% formic acid, 97.9% water, 2% acetonitrile
(v/v/v) and (B) 0.1% formic acid, 15.9% water, 84% acetonitrile (v/v/v). Separation was conducted using a nano-column (Acclaim PepMap C18, 75 mm i.d x 50 cm long, 3 mm particles) at 300 nl/min by applying gradient consisted of 4–55% B during 180 min for liquid mucus products or 90 min for gel piece products. The mass spectrometer was operated in data
85 dependent scan mode. Survey full scan MS spectra (from 300–1800 m/z) were acquired with a resolution set at 60 000. The 20 most intense ions with charge states ≥2 were sequentially isolated (isolation width: 2 m/z; 1 micro scan) and fragmented using CID mode (normalized collision energy of 35% and wideband-activation enabled). Dynamic exclusion was active during 30 s with a repeat count of 1. Polydimethylcyclosiloxane (m/z, 445.1200025) ions were used for internal calibration.
3.3.8. Protein identification and validation
MS/MS ion searches were performed using Mascot search engine v 2.2 (Matrix Science,
London, UK) via Proteome Discoverer 1.4 software (ThermoFisher Scientific, Bremen,
Germany) against a local database (128 674 entries). From NCBI database (download
10/01/14), a sub-database was generated using Proteome Discoverer 1.4 software from keywords targeting mammalian taxonomy. The search parameters included trypsin as a protease with two allowed missed cleavages and carbamidomethylcysteine, methionine oxidation and acetylation of N-term protein as variable modifications. The tolerance of the ions was set to 5 ppm for parent and 0.8 Da for-fragment ion matches. Mascot results obtained from the target and decoy databases searches were subjected to Scaffold 4 software (version 4.4,
Proteome Software, Portland, USA). Peptide and proteins identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm and by the Protein Prophet algorithm (Keller et al. 2002), respectively. Protein identifications were accepted if they contained at least two identified peptides.
3.3.9. Label-free protein quantification using spectral counting
For comparative analysis, we employed Scaffold 4 software (version 4.4, Proteome Software,
Portland, USA) using the protein cluster analysis option with spectral counting quantitative
86 module. Quantification was performed with “Weighed Spectra” method and carried out on protein clusters. Thus, numbers of Normalized Weighed spectra (NWS) were tabulated using experiment-wide protein clusters. The reproducibility linked directly to the nanoLC-MS methodology was evaluated by the quantitative variance for each biological condition (luteal vs oestrus, NAT vs P4 vs SOV) considering 12 technical replicates and for each protein group.
Significance between phases of the cycle and treatments was determined using statistical tests within Scaffold software; t-test (phase of cycle comparison) and an ANOVA (treatment comparison), respectively, where p <0.05 was considered significant. Limits of an average
NWS of ≥5 and fold change/ratio of ≥1.5 were included to increase validity of any comparisons made.
3.3.10. Molecular function analysis
Proteins were assessed for molecular function and biological process using the Protein Analysis
Through Evolutionary Relationships (PANTHER; version 9.0; http://pantherdb.org/) classification system web server, which uses Gene Ontology (GO) annotations for functional categorization using Homo sapiens as the reference species. Default settings were used and results displayed are represented as percent of functional hits.
3.4. RESULTS
3.4.1. Qualitative whole oestrous cycle analysis of mucus
A total of 950 proteins in 622 clusters were identified in CV mucus samples pooled throughout the entire oestrous cycle in naturally cycling animals (Supplementary table S-1). Molecular process analysis using PANTHER revealed the majority of proteins identified had catalytic activity, closely followed by those with binding activity (40.2% and 28.0% of functional hits,
87 respectively). Several proteins identified also had structural or enzymatic regulation activity
(8.9% and 8.3% of functional hits, respectively; Figure 3.1).
Figure 3.1 Molecular functions and GO annotations of proteins identified in ovine cervicovaginal mucus pooled from the entire oestrous cycle (follicular and luteal phases).
Molecular function and GO annotations determined by PANTHER analysis.
As the main structural component of mucus, we identified numerous mucins (MUC1, MUC5B,
MUC16 and MUC21), along with the enzyme sialidase (NEU1). Immune based proteins were also abundant including several heat shock family proteins (HSPA4, HSPA4L, HSPA5,
HSPA8, HAP90AB1, HSP90B1, HASPA1A, HSPB1, HSPD1 and HSPE1), a number of complement cascade components (C3, C4A, C7, C9, CFB, CFH and CFI), several cytokine proteins (A2ML1, SPP1, DDT, CD109, A2M, and TF), those with antigen processing and presentation function (B2M, RNH1 and CTSS) along with numerous macrophage activation proteins (BPIFA2, S100A2, BPIFB1, ALOX15B, S100A11, S100A7, S100A9, PRTN3,
S100A12, S100A14, COL18A1, LBP, DMBT1, LGALS3BP, S100A4, DDT).
88
3.4.2. Quantitative comparison of oestrus and mid-luteal phase mucus
A total of 436 protein clusters were found within ovine mucus by a quantitative method
(Supplementary table S-2). Comparison of average NWS between the two phases of the oestrous cycle indicates that 87 identified proteins were found to be relatively more abundant during the oestrus period than the mid-luteal phase, whilst 194 were more abundant during the mid-luteal period than during oestrus. The remaining 155 proteins identified had similar abundances during oestrus and the mid-luteal phase (p >0.05). After limits (NWS ≥5, fold change ≥1.5, p <0.05) were applied, a total of 187 protein clusters remained (Supplementary table S-3), 60 of these were significantly more abundant during oestrus (Table 3.1) whilst 127 were most abundant during the mid-luteal phase (p <0.05), when compared to mid-luteal and oestrus phases, respectively. There were 10 proteins that did not significantly differ (p >0.05) between the two phases of the oestrus cycle. The variation in abundance of proteins in mucus at oestrus and the mid-luteal phase is depicted in figure 2, which displays the proteins identified in oestrus and luteal mucus that were the most disparate in abundance between the two phases, based on fold change (a total of 70 proteins with FC >5 displayed, Figure 3.2).
89 Table 3.1 Proteins (NWS >5, fold change between oestrus and luteal NWS ≥1.5) identified within ovine cervicovaginal mucus found to be significantly (p <0.05) more abundant during oestrus when compared to the mid-luteal phase.
GI Accession Molecular Oestrus Luteal Fold Protein Gene Symbol Number Weight NWS NWS Change Cluster of ceruloplasmin precursor gi|57619174 CP 119 kDa 535.3 0.7 769.7 Cluster of chitinase-3-like protein 1 gi|426239361 CHI3L1 44 kDa 28.2 0.1 192.1 clusterin isoform 1 gi|426220555 CLU 51 kDa 163.4 1.7 96.5 Cluster of alkaline phosphatase, tissue- gi|426222804 ALPL 56 kDa 127.5 3.9 33.1 nonspecific isozyme Cluster of mucin-16-like gi|548472212 MUC16 239 kDa 86.4 3.1 27.8 Cluster of TPA: mucin-5B gi|326806946 MUC5B 690 kDa 142.5 5.3 27.0 Cluster of olfactomedin-4 gi|426236681 OLFM4 58 kDa 55.0 2.4 22.9 Cluster of desmocollin-2 gi|426253649 DSC2 101 kDa 21.3 1.0 21.7 Cluster of IgGFc-binding protein gi|426243806 FCGBP 331 kDa 39.8 1.9 21.4 Cluster of inhibitor of carbonic gi|426218286 LOC101117129 73 kDa 32.0 1.6 19.8 anhydrase-like Cluster of sulfhydryl oxidase 1 gi|426240519 QSOX1 87 kDa 43.0 2.7 16.1 pancreatic secretory granule membrane gi|426254385 GP2 59 kDa 16.8 1.6 10.8 major glycoprotein GP2 isoform 2 programmed cell death 6-interacting gi|426249775 PDCD6IP 100 kDa 16.7 2.0 8.3 protein BPI fold-containing family B member 1 gi|426241325 BPIFB1 52 kDa 10.7 1.3 8.0 Cluster of CD109 antigen gi|426235067 CD109 169 kDa 205.9 27.1 7.6 Cluster of aminopeptidase N gi|426248094 ANPEP 109 kDa 10.8 1.6 7.0 mesothelin gi|548519629 MSLN 45 kDa 14.8 2.9 5.1 desmoglein-3 gi|426253641 DSG3 107 kDa 14.2 2.9 5.0 zinc-alpha-2-glycoprotein-like gi|426255398 AZGP1 38 kDa 22.5 5.0 4.5 thioredoxin gi|27806783 TXNL1 12 kDa 8.8 2.6 3.4 Cluster of deleted in malignant brain gi|556726702 DMBT1 151 kDa 100.5 31.1 3.2 tumors 1 protein WAP four-disulfide core domain protein gi|426242091 WFDC2 20 kDa 42.1 14.0 3.0 2 Cluster of complement C3 gi|548472985 C3 136 kDa 182.5 61.6 3.0 cystatin-M gi|426252062 CST6 17 kDa 58.9 20.6 2.9 Cluster of transcobalamin-1 gi|426245518 TCN1 48 kDa 20.8 8.4 2.5 complement component C3 gi|12649541 C3 40 kDa 7.0 2.9 2.5 trefoil factor 2 gi|426218349 TFF2 15 kDa 7.1 2.9 2.4 alpha-1-antitrypsin transcript variant 1 gi|385682605 SERPINA1 46 kDa 26.0 11.2 2.3 transthyretin precursor gi|57526651 TTR 16 kDa 6.4 3.1 2.1 Cluster of vascular non-inflammatory gi|556744205 VNN2 62 kDa 82.9 40.9 2.0 molecule 2 isoform X1 trefoil factor 3 gi|426218347 TFF3 9 kDa 31.2 15.5 2.0 Cluster of protein S100-A2 isoform X1 gi|149751286 S100A2 11 kDa 5.8 3.0 2.0 Cluster of immunoglobulin alpha heavy gi|2582411 CD79A 50 kDa 75.2 40.3 1.9 chain Cluster of polymeric immunoglobulin gi|426239425 PIGR 82 kDa 128.8 71.5 1.8 receptor isoform 1 complement factor B gi|426250509 CFB 85 kDa 32.0 18.8 1.7 polyubiquitin-C-like gi|511937197 UBC-like 28 kDa 20.2 12.8 1.6 double-headed protease inhibitor, gi|548475514 LOC102171242 14 kDa 7.5 5.1 1.5 submandibular gland-like
Values ranked in decreasing order of fold change value.
90
Figure 3.2 Proteins (gene names listed) in the cervicovaginal mucus of ewes which differed markedly in abundance between oestrus and the mid-luteal phase of the oestrous cycle (based on fold change (FC) comparison of average NWS, FC 0 = green, 5 = black, 35 ≥ red, 70 most disparate proteins displayed).
91 The five most abundant proteins overall during oestrus, based on average normalised weighted spectra (NWS) after limits were applied, were ceruloplasmin (CP; NWS 535), CD109 antigen
(CD109; NWS 206), complement C3 (C3; NWS 183), protein S100-A12 (S100A12; NWS
177) and clusterin (CLU; NWS 163) (Table 3.1). When comparing abundance between phases of the cycle (fold change), the top 3 proteins all had remarkably higher abundance during oestrus than mid-luteal phase, demonstrated by high fold change values. Ceruloplasmin (CP; fold change 770) was the most different, with a fold change more than four times higher than that of chitinase-3-like protein (CHI3L1; fold change 192), whist CHI3L1 had a fold change almost double that of clusterin (CLU; fold change 97) (Figure 3.2). Abundance of structural mucus glycoproteins mucin-16 (MUC16; fold change 28) and mucin-5B (MUC5B; fold change
27) was also significantly higher in oestrus mucus than luteal phase mucus, with NWS of 128 vs. 4 and 86 vs. 3 for oestrus and luteal phase, for MUC16 and MUC5B respectively.
Of the 27 proteins present in oestrus phase mucus only, the five most copious were folate receptor alpha-like isoform 1 (FOLR1), matrilysin (MMP7), oviductin (OVN), complement factor I (CFI) and sialadase-1 (NEU1), having NWS values of 117, 47, 45, 44 and 41, respectively (Table 3.2). Several immune related proteins identified in the qualitative analysis were found to be significantly more abundant during oestrus than the mid-luteal phase, including complement cascade proteins complement component 3 (C3), which was the third most abundant protein during oestrus, complement factor I (CFI) found in oestrus mucus only, and complement factor B (CFB). Others were more abundant during the mid-luteal phase, namely heat shock proteins HSPA8, HSPB1, HSP90AA1, HSP90AB1, with the latter found in mid-luteal phase samples only.
92 Table 3.2 Proteins identified within ovine cervicovaginal mucus found only during oestrus.
GI Accession Gene MW Oestrus Luteal Protein Number Symbol (kda) NWS NWS
Cluster of folate receptor alpha-like isoform 1 gi|426245067 FOLR1 30 kDa 117.3 0.0 matrix metallopeptidase 7 gi|211063451 MMP7 30 kDa 46.9 0.0 oviductal glycoprotein gi|550821891 OVGP1 60 kDa 44.8 0.0 Cluster of complement factor I gi|426231287 CFI 69 kDa 44.5 0.0 sialidase-1 gi|426250521 NEU1 45 kDa 40.7 0.0 Cluster of low-density lipoprotein receptor- related protein 2 gi|426222491 LRP2 520 kDa 39.7 0.0 fibronectin isoform 1 gi|426221515 FN1 262 kDa 37.0 0.0 ceruloplasmin gi|531998806 CP 122 kDa 28.8 0.0 Cluster of cornulin gi|403302821 CRNN 51 kDa 25.7 0.0 galectin-3-binding protein gi|426239209 LGALS3BP 61 kDa 20.9 0.0 Cluster of beta-hexosaminidase subunit beta gi|426246297 HEXB 61 kDa 19.8 0.0 Cluster of SPARC-like protein 1 gi|426232015 SPARCL1 74 kDa 18.0 0.0 retinoic acid receptor responder protein 1 isoform 1 gi|426218046 RARRES1 33 kDa 16.0 0.0 Cluster of serum amyloid A3.2 precursor LOC100135684 protein gi|165940902 11 kDa 14.9 0.0 Cluster of lysozyme 1a precursor, partial gi|165964 LYZ 14 kDa 13.6 0.0 Cluster of extracellular superoxide dismutase [Cu-Zn] isoform 1 gi|426231525 SOD3 26 kDa 11.5 0.0 Cluster of uroplakin-3b-like protein gi|426255354 UPK3BL 27 kDa 10.5 0.0 cystatin-A gi|426217560 CSTA 11 kDa 9.2 0.0 factor XIIa inhibitor-like gi|426246147 LOC101121454 67 kDa 8.9 0.0 prolactin-inducible protein homolog gi|426228154 PIP-like 16 kDa 8.7 0.0 cysteine-rich secretory protein 3 precursor gi|297591805 CRISP3 27 kDa 8.6 0.0 disintegrin and metalloproteinase domain- containing protein 28 gi|426222231 ADAM28 82 kDa 8.0 0.0 Cluster of choline transporter-like protein 4 isoform 1 gi|426250517 SLC44A4 79 kDa 7.8 0.0 phosphatidylethanolamine-binding protein 4 gi|426220060 PEBP4 25 kDa 6.8 0.0 desmoplakin gi|426251400 DSP 331 kDa 6.7 0.0 coagulation factor V gi|426239635 F5 229 kDa 5.8 0.0 sodium-dependent phosphate transport protein 2B gi|426231543 SLC34A2 75 kDa 5.4 0.0
Values ranked in decreasing order of average normalised weighted spectra (NWS).
The five most abundant proteins during the mid-luteal phase, based on average NWS, were protein S100-A12 (S100A12; NWS 260), immunoglobin heavy chain C region (SLC7A4;
NWS 238), 14-3-3 protein sigma (SFN; NWS 207), immunoglobulin lambda light chain constant region segment 1 (IGL; NWS 116) and cathelicidin-1-like isoform X1 (CATHL1B;
93 NWS 171). Normalised weighted spectra of the five most abundant proteins in mid-luteal mucus (NWS range 260-171) were lower than that of oestrus mucus normalised weighted spectra counts (NWS range 535-163). In regards to fold change (FC), tubulin beta 2C
(TUBB4B; FC 140), elongation factor 1-alpha 1-like isoform 4 (EEF1A1; FC 86), phosphoglycerate kinase 1 (PGK1; FC 69), leukotriene A-4 hydrolase isoform 1 (LTA4H; FC
54) and tropomyosin alpha-4 chain (TPM4; FC 52) were the top 5 (Figure 3.2). Of the 40 proteins that were present in mid-luteal samples only, the 5 proteins that had the highest NWS were heat shock protein HSP 90-alpha (HSP90AA1; NWS), cathelin-related peptide SC5-like
(CAMP-like; NWS 35), non-specific cytotoxic cell receptor protein 1 homolog (NCCRP1;
NWS 30), rho GDP-dissociation inhibitor 2 isoform 1 (ARHGDIB; NWS 23) and glycogen phosphorylase (PYGL; NWS 22).
3.4.3. Quantitative comparison of NAT, P4 and SOV mucus produced during oestrus
A total of 347 proteins were identified within the three treatments; NAT, P4 and SOV
(Supplementary table S-4). Following the application of limits 149 proteins remained
(Supplementary table S-5). Comparison of the normalised average weighted (NWS) spectra values of proteins across the three oestrus mucus types clearly indicates that exogenous hormones affect the cervicovaginal mucus proteome and that many protein families are affected by fluctuations in circulating endogenous and exogenous oestrogen and progesterone concentrations in the ewe. Figure 3.3 depicts the top 20 proteins most significantly affected by progesterone synchronisation and superovulation practices, showing an increase or decrease in abundance when compared to NAT mucus. Of the proteins most affected by exogenous hormones, 51 and 32 were found to be significantly more abundant in P4 and SOV samples, respectively, when compared to NAT (Supplementary table S-6). Furthermore, 98 and 53 proteins were identified to be significantly less abundant in P4 and SOV samples, respectively,
94 again in comparison to NAT (Supplementary table S-6). Both myeloperoxidase (MPO) and bcl-2-like protein 15 (BCL2L15) were found only in NAT samples, indicating the most significant effect of treatment. Furthermore, following P4 treatment, several proteins were completely removed from the proteome, namely L-serine dehydratase/L-threonine deaminase
(SDS), UPF0762 protein C6orf58 (C6orf58), mammaglobin-A (SCGB2A2) and binder of sperm 5 precursor (BSP5) (Figure 3.3).
Figure 3.3 Proteins identified (gene names listed) in ovine cervicovaginal mucus of progesterone synchronised (P4), and progesterone synchronised then superovulated (SOV) ewes that were most disparate in abundance (based on fold change (FC) comparison of average
NWS, FC value; 0 = blue, red ≥ 15, grey = present only in corresponding sample, 20 most disparate proteins displayed) when compared to levels in mucus of naturally cycling ewes
(NAT).
Proteins that differed significantly in abundance when compared to that of NAT mucus after progesterone synchronisation and superovulation were analysed for differences in molecular function (according to PANTHER analysis). The 51 proteins that were significantly more abundant in P4 mucus compared to NAT had predominantly catalytic molecular activity, followed by binding, enzyme regulation or receptor molecular activity (37%, 22%, 17% and
95 13% of functional hits, respectively). The 98 proteins found to be significantly less abundant in P4 mucus compared to NAT also had predominately catalytic or binding molecular activity
(38% and 32% of functional hits, respectively). Of the 32 proteins that were markedly more abundant in SOV than NAT mucus, the most common molecular functions were again mainly catalytic, binding activity or enzyme regulation molecular activity (35%, 27% and 21% of functional hits, respectively). The 53 proteins found to be significantly decreased in abundance in SOV mucus compared to levels in NAT mucus had similar molecular functions also (33% and 35% of functional hits for catalytic and binding activity, respectively, Figure 3.4).
Figure 3.4 Differences in molecular function (MF) of the proteins identified in ovine cervicovaginal mucus significantly affected (p <0.05, Fold change ≤1.5) by progesterone synchronisation (A; P4) and superovulation (B; SOV) in comparison to a natural cycle (NAT).
Proteins that were more abundant (red bar for each MF category) and less abundant (blue bar for each MF category) in comparison to levels in NAT mucus are shown. Molecular function determined by PANTHER analysis.
Supplementary data for this chapter can be found online at http://dx.doi.org/10.1016/j.jprot.2017.01.007.
3.5. DISCUSSION
The results of this study indicate that the cervicovaginal mucus proteome of the ewe undergoes natural variation across the oestrous cycle, and is significantly altered by progesterone
96 synchronisation and superovulation practices (Figure 3.2 and 3.3). The variation in protein abundance across the cycle and between hormonal treatments (particularly with high abundance of proteins in biological conditions during which oestrogen dominates e.g. following superovulation) suggests a key role of endogenous hormone levels in the control of protein expression and secretion into the female reproductive tract of the ewe. This variation relates to the varied roles that these proteins have at the two stages of the cycle. Namely that during oestrus, cervicovaginal mucus needs to be receptive to a possible influx of spermatozoa and potential pathogens, whereas luteal mucus is more likely to contain proteins involved in tract modification and readiness for possible implantation and pregnancy.
Results in the present study show that mucin 5B (MUC5B), one of the main gel forming mucins responsible for the viscoelastic properties of lower reproductive tract mucus (Gipson et al.
1999) and saliva (Raynal et al. 2003), varied over the cycle and was highest at oestrus (top ten in abundance). MUC5B is expressed in human oviducal (Ma et al. 2012) and endocervical epithelium (Gipson et al. 1999), and excreted into cervical mucus (Andersch-Björkman et al.
2007). In the present study levels were highest at oestrus, during which progesterone levels would have been at their lowest. This result agrees with previous work indicating MUC5B levels in mucus are inversely related to serum progesterone levels (Gipson et al. 1999) and as such peak at mid cycle in humans (Gipson et al. 2001). Furthermore, previous work has shown
MUC5B gene expression in the human respiratory tract to be under control of oestrogen (Choi et al. 2009). Due to its gel forming nature, high levels of MUC5B might be indicative of highly thick or viscous mucus. However, as MUC5B is highly hydrophilic (Gerken 1993), increased viscosity as a result of increased MUC5B content would be negated. At oestrus, mucus needs to allow for foreign spermatozoa to migrate through the reproductive tract but also prevent bacterial invasion after semen deposition, thus its viscoelastic properties are paramount. A
97 suggested function of the increased water in the tract due to higher levels of MUC5B is to keep the tract patent to support sperm motility (Gipson 2001). Sialidase (NEU1) was also present during oestrus, in fact it was identified only in oestrus samples. This enzymatic protein is responsible for the degradation of mucins by cleaving terminal sialic acid residues (Wiggins et al. 2001), leading to a reduction in viscoelastic properties. Thus, high levels of NEU1 may also contribute to a reduction in mucus viscosity during oestrus, allowing spermatozoa successful transit through the tract. The absence of NEU1 in luteal phase samples might contribute to increased mucus viscosity during this period, perhaps resulting in an increased ability of mucus to act as a barrier to infection and spermatozoa. Detection of NEU1 solely in oestrus mucus may suggest its expression is under oestrogenic influence, and thus we might expect levels to be highest in mucus of SOV ewes, which have amplified circulating oestrogen levels as a result of the large number of follicles present in these animals. Interestingly this was not the case; with NEU1 levels significantly lower in SOV mucus compared to NAT. Low levels of NEU1 in SOV could result in altered mucus viscosity and ultimately mucus receptivity to spermatozoa.
As mucus is produced continuously from all parts of the female tract, a level of outward flow from the oviduct to the vagina takes place. While this likely aids in the prevention of infiltration by foreign bodies, upward diffusion of molecules also occurs (Kunz et al. 1996; Barnhart et al.
2001) and as such, immune regulation of mucus remains important to prevent infection. There is an increased risk of foreign body infiltration of the lower reproductive tract after mating or insemination, with immune cells increasing during this period (Kunz et al. 1996; Kaushic et al.
1998; Scott, Ketheesan and Summers 2009), so it is interesting to note that several proteins related to immune function and non-immune defence were found in higher levels (or only) during oestrus. Indeed, several of the most abundant proteins during oestrus were serum
98 proteins with known protective functionality such as clusterin (CLU), which is related to damaging oxidative reactions and stress induced protein precipitation (Poon et al. 2000;
Trougakos 2013). Protein S100-A12 (S100A12) is also directly involved in immune function through its role as an antimicrobial and anti-inflammatory reagent released from the cytoplasm of neutrophils (calgranulin) (Foell et al. 2007; Hsu et al. 2009; Cunden, Gaillard and Nolan
2016) and also has mast cell chemoattractant abilities (Yan et al. 2008) (Yang et al. 2007; Yan et al. 2008). Polymeric immunoglobulin receptor (PIGR), the seventh most abundant protein during oestrus, is also directly involved in the immune response as it mediates transcytosis of
IgA across the epithelium and its subsequent excretion into the mucus of the female productive tract (Wira and Sullivan 1985). As a secretory product of PIGR is also endocytosed and secreted across the membrane with IgA (Delves, Martin and Burton 2011), PIGR levels within mucus could be representative of levels of secretory IgA within reproductive tract mucus.
Complement proteins C3, CFB and CFI were all highest at oestrus, the latter found only in oestrus mucus, and play a central role in both the classical and alternate complement pathways.
The high levels of C3 and CFB reported here at oestrus are supported by previous work which found production, from endometrial epithelial cells in mice (Li, Huang and Chen 2002) and the oviducts of women (Lee et al. 2004), to be under oestrogenic control (Li, Huang and Chen
2002; Lee et al. 2004). Galectin-3-binding protein (LGALS3BP), found only during oestrus in the present study, also has anti-inflammatory roles, is involved in T-cell mediated immunity
(Radosavljevic et al. 2012) and has been localised to the luminal epithelium of the bovine uterus (Bauersachs et al. 2006). The influence of proteins within seminal plasma and spermatozoa can also affect the output of reproductive tract mucus, with seminal fluid affecting the inflammatory response as well as the influx of cytokines and neutrophils to the tract, especially so during the oestrus phase (Scott, Ketheesan and Summers 2006; Sharkey et al.
2007; Scott, Ketheesan and Summers 2009). Research has shown that immunoglobulin and
99 antibody levels vary across the menstrual cycle in response to hormonal profile changes
(Sharkey et al. 2012). Thus, changes to hormonal levels through exogenous hormone use might also result in altered inflammatory and immune responses.
The abundance of several immune related proteins was also found to differ between mucus of
NAT, P4 and SOV ewes. Several proteins involved in antibacterial defence such as lactoperoxidase precursor (LPO), BPI fold-containing protein (BPIFB1) and complement factor H (CFH) were more abundant in the mucus of both progesterone synchronised (P4) and superovulated ewes (SOV). Interleukin-8 (IL8) was also more abundant in CV mucus of both
P4 and SOV animals and has previously been described to be hormonally regulated and have neutrophil chemotactic activities in does (Lü et al. 1999), and ewes (Scott, Ketheesan and
Summers 2009). Conversely, myeloperoxidase (MPO), which contributes to the microbicide effect of neutrophils, was completely absent in mucus of P4 and SOV ewes, when compared to levels in NAT. In addition, the role of many proteins in immune tolerance of the conceptus should also be considered, such as PAEP, having many immune altering effects in the human female tract and has been shown to increase directly after ovulation and continues to do so throughout pregnancy (Dalton et al. 1995; Sonoda et al. 1998). The effects of these numerous changes to the efficacy of immune responses are as yet unknown, and show no apparent trend in abundance relative to synchronisation or superovulation. As immune processes and protective functions of the female reproductive tract are predominately a collection of complex cascades and multistep reactions as opposed to one step responses, the effects of any alteration to immune protein abundance could be far reaching. As such, the outcome of these protein alterations is dependent on the functionality of each protein affected.
100 The reproductive tract also undergoes drastic remodelling over the reproductive cycle and throughout pregnancy as a result of protein modification over the cycle. Several proteins more abundant in CV mucus during oestrus than the mid-luteal phase, such as matrilysin (MMP7), a matrix metalloproteinase identified in oestrus samples only, are involved in breakdown of the extra cellular matrix (Curry and Osteen 2003; Clark and Schust 2013), which occurs during tract remodelling. Cysteine-rich secretory protein 3 (CRISP3), also identified only in oestrus phase mucus, has been localised in the human and mouse endometrium and enhances adhesion and proliferation of human endometrial epithelial cells, suggesting a role in endometrial repair and regeneration (Feng, Woessner and Zhu 1998). Changes to the tract also occur throughout the luteal phase, with proteins again playing a significant role in these changes. Indeed, several proteins most abundant in luteal mucus compared to oestrus such as actin-β (ACTB) and coronin (CORO1A), a major cytoskeletal structural protein and actin binding protein, respectively, could be by-products of increased tissue remodelling during the luteal phase, ensuring tract readiness for possible implantation and maintenance of pregnancy. Matrix metallopeptidase 7 (MMP7), found only in luteal phase mucus, aids extracellular matrix remodelling via degradation of several extracellular matrix (ECM) components including proteoglycans, fibronectin, collagens and gelatins’ (Nagase, Visse and Murphy 2006; Evans et al. 2015). Aplha-2-Macroglobulin (A2M) is a proteinase inhibitor of many proteinases including collagenase, and was 3 times more abundant in luteal phase mucus than oestrus phase mucus, thus perhaps levels indicate a halt on tissue alteration in the tract until implantation of a conceptus.
Apart from the aforementioned functions, proteins produced within the female reproductive tract are also involved in several reproductive processes. Heat shock family proteins, several of which were more abundant in mid-luteal phase mucus (HSPA1A, HSPB1, HSPA4,
101 HSPA5, HSPA8, HSP90AA1 and HSP90B1), have been shown to have supportive effects on spermatozoa, with HSPA4 aiding sperm migration within the chicken oviduct (Hulboy,
Rudolph and Matrisian 1997) and HSPA8 shown to improve viability of ram semen (Lloyd et al. 2009; Hiyama et al. 2014) although as they were identified during the mid-luteal phase in the present study they likely do not have similar function in ovine cervicovaginal mucus or this action is species specific. HSPs are also involved in protein synthesis and folding, and have immunogenic and protective functions (Lloyd et al. 2012). HSPA8 and HSPB1 expression in ovarian, cervical and uterine tissues has been reported in humans (Mangurten et al. 1997; Schmitt et al. 2007), thus supporting our finding of these proteins within cervicovaginal mucus of the ewe. Several mucus proteins found in higher abundance or only during oestrus have also been linked specifically to sperm function and fertilisation, such as sialidase-1 (NEU1), with involvement in capacitation (Ma et al. 2012), and along with oviducal glycoprotein 1 (OVGP1) is reportedly involved in zona-pellucida binding
(Schlesinger 1986; McCauley et al. 2003; Pang et al. 2011; Ma et al. 2012), both of which have been identified in the oviduct (Guillermo Velasquez et al. 2007; Ma et al. 2012) of several species with NEU1 also identified in the endometrium and uterine secretions of humans (Buhi 2002). Ceruloplasmin (CP), which was identified in oestrus phase mucus only in the present study, has previously been shown to be impacted by oestrogen concentrations
(Evans, Cornatzer and Cornatzer 1970; Ganguly, Sarkar and Ghosh 1976) and is a serum copper binding protein with antioxidant abilities, both of which may be beneficial for sperm function and transport. Clusterin (CLU), a serum transudate protein has also been identified within seminal plasma (Trougakos 2013) and is involved in agglutination of abnormal spermatozoa (Carruthers, Hobbs and Warren 1966). Perhaps the high levels of clusterin found in oestrus mucus, almost 100 times more than levels in luteal phase mucus, are indicative of a similar role for this protein, thus aiding in transport of only normal spermatozoa to the ova.
102 Development of the embryo and growing foetus may also be influenced by proteins found within the female reproductive tract, as they contribute to the milieu that surrounds the developing conceptus. An example of such a protein could be ubiquitin like modifier activating enzyme 1 (UBA1), an enzyme found only in luteal samples and involved in DNA damage response processes that mediate genomic integrity, crucial for correct embryo, and foetal growth and development (Yeung et al. 2009). Several proteins more abundant at oestrus, for example derivatives of C3, also have roles in early embryonic development
(Moudry et al. 2012) through effects on blastocyst size and hatching rate (Lee et al. 2004).
NEU1 was also identified in quantitative analysis of NAT, P4 and SOV mucus; however, it occurred in lower abundance in SOV and P4 samples when compared to NAT. As such, any of the described roles that such a protein may have in supporting sperm function in the female could be negated due to decreased abundance. Whilst the exact mechanism of action of proteins identified in the cervicovaginal mucus of the ewe is unknown, alterations of abundance might adversely affect fertility and could in part explain the reportedly lower fertility rates achieved in progesterone synchronised and superovulated ewes (McCauley et al. 2003).
It should be noted that mucus samples were taken from the lumen of the cervical canal and not specifically the bases of cervical crypts or folds. However, luminal mucus would likely consist of a combination of mucus produced from the apices and bases of cervical folds (crypts).
Histological studies have indicated mucus varies between the regions of cervical folds, with sialomucins (mucins with terminal sialic groups) staining more abundantly at the bases of cervical crypts compared to sulfomucins (mucins with terminal sulfate groups) (Heydon and
Adams 1979; Pluta et al. 2011). The importance of such a glycosylation change is not fully understood. It has been theorised that sialomucins are favoured by spermatozoa and that their
103 presence in the base of the cervical folds guides spermatozoa into these areas, which have been described as ‘preferred pathways’ (Mullins and Saacke 1989), but this theory has not been well established experimentally. Whilst it is likely that mucus of varying composition is produced from these varying regions of cervical folds, analysis of apical and basal cervical crypt is mucus would not be feasible due to logistical issues with differentiation of mucus from each area.
This study has highlighted the marked changes that occur in the cervicovaginal mucus proteome throughout the oestrous cycle of the ewe and during oestrus synchronisation and/or superovulation. Many proteins were found in higher abundance during the oestrus or luteal phases, with several appearing to be phase specific. Exogenous hormones also significantly impacted the mucus proteome, with many proteins having either an increased or decreased abundance in comparison to levels in NAT mucus. These results clearly show that the mucus proteome in the ewe is greatly impacted by circulating endogenous hormone concentrations as well as controlled breeding programs which utilise exogenous hormones. Such changes could negatively impact upon tract health, immune tolerance and function, sperm transport through the tract, and ultimately successful fertilisation and maintenance of pregnancy. Further research is required to define the exact effects of these protein changes and how this knowledge could be used to refine the current practices of artificial breeding and ultimately improving fertility rates achieved when cervical insemination programs are used on farm.
3.6. Acknowledgements
Research was supported by the NSW Stud Merino Breeders Association Trust and Australian
Wool Innovation. The authors would like to thank Mr. Byron Biffin and Mr. Keith Tribe for their on-farm assistance, Miss Evelyn Hall and Mr. Peter Thomson for their statistical consultation and Miss Kim Heasman for laboratory assistance. The authors would also like to
104 acknowledge Vetoquinol for the generous donation of Ovagest sponges, eCG (Pregnecol) and
FSH (Folltropin-V). The high-resolution mass spectrometer was financed (SMHART project) by the European Regional Development Fund (ERDF), the Conseil Régional du Centre, the
French National Institute for Agricultural Research (INRA) and the French National Institute of Health and Medical Research (INSERM). Authors would like to thank Guilluame Tsikis and
Clement Soleilhavoup for assistance in sample preparation for mass spectrometer and Lucie
Combes-Soia and Greg Harichaux for running of mass spectrometry samples.
105 Chapter 4. Mucin content and neuraminidase impacts cervicovaginal mucus viscosity and ram sperm motility, migrating ability and viability.
4.1. ABSTRACT
Proteins that affect the physicochemical and viscoelastic properties of mucus vary over the oestrous cycle of the ewe and after the application of exogenous hormones. It has been hypothesised that these changes could influence sperm transport in cervical mucus. As such, the aim of this study was to investigate the effect of variable mucin and neuraminidase-1
(NEU1; an enzyme which alters the structure of mucins proteins) content on the viscosity
(centipoise; cp) of a cervicovaginal mucus simulant (CMS) and how this influences the characteristics of fresh and frozen-thawed ram spermatozoa. An increase in the concentration of mucins resulted in a significant increase in CMS viscosity (p <0.05). However, the treatment of CMS with NEU1 did not significantly affect CMS viscosity (p >0.05) alone but did reduce
CMS viscosity following incubation with semen (1:1) (p <0.05). The migrating ability of fresh spermatozoa was unaffected by the addition of mucins (p>0.05) but the migrating ability of frozen-thawed spermatozoa increased following the addition of 2% and 4% mucins (p <0.05).
The migrating ability of spermatozoa (fresh or frozen-thawed) was unaffected by the inclusion of NEU1 into CMS (p >0.05). The inclusion of mucin into CMS resulted in spermatozoa (fresh and frozen-thawed) with an overall reduced straight line velocity (VSL) and increased average path and curvilinear velocity (VAP and VCL, respectively, p <0.05) whereas, the treatment of
CMS with NEU1 had no effect on resultant kinematic parameters. Spermatozoa had reduced membrane damage and reacted acrosomes when incubated in CMS with 2% or 4% mucins, or
106 CMS pre-treated with NEU1 (p <0.05). These results show that variation in mucin and neuraminidase concentrations alters the viscosity of a cervicovaginal mucus simulant, the motility and viability of spermatozoa, and the mucus migration ability of frozen-thawed ram spermatozoa.
4.1.1. Keywords cervicovaginal mucus ∙ mucins ∙ neuraminidase ∙ mucus viscosity ∙ ovine ∙ spermatozoa · cryopreservation
4.2. INTRODUCTION
The mucus of the female reproductive tract undergoes hormone-mediated changes throughout the oestrous cycle, resulting in altered rheological properties (humans; (Igarashi 1954; Brunelli et al. 2007), ewe; (Evans and Maxwell 1987; Maddison et al. 2016)). These changes have been suggested to impede or support sperm transit (Lewis et al. 2010; Chappell et al. 2014). The use of exogenous hormones during controlled breeding practices such as oestrus synchronisation and superovulation has also been shown to impact the quantity of mucus produced by the ewe
(Smith and Allison 1971; Croker and Shelton 1974), as well as sperm transit through the reproductive tract, ultimately resulting in reduced fertility (Maxwell et al. 1993). In women, changes in mucus characteristics after exogenous hormone use are utilised as a mode of action in contraceptive solutions. They have been shown to affect mucus viscosity, pH and protein composition (Chappell et al. 2014), ultimately resulting in reduced mucus quality according to
World Health Organisation (WHO) standards. These changes impair the successful penetration of sperm through mucus, thus aiding in the contraceptive effects of “the pill” (Lewis et al.
2010). Recent work in the ewe has shown that exogenous hormones alter the volume, protein concentration and composition of cervicovaginal mucus (Maddison et al. 2016; Maddison et
107 al. 2017), yet the effect of these alterations on mucus viscosity and sperm transit have yet to be tested.
One of the protein families previously identified in reproductive tract mucus and shown to vary significantly in abundance over the oestrous cycle are mucins (Maddison et al. 2016). Mucins are large filamentous glycoproteins secreted from goblet cells within the endometrium, oviduct and cervical tissues (Gipson et al. 1997; Lagow, DeSouza and Carson 1999). They constitute the semisolid gel portion of cervicovaginal mucus and are the chief determinates of its physicochemical viscoelastic properties, particularly gel forming mucins. Previous work by
(Wolf et al. 1977a, b) suggests that variation in rheological properties of cervical mucus across the menstrual cycle in women is due in part to variation in mucin concentration, with human cervical mucus reportedly containing around 1.5 % (w/w, wet weight) of mucins (Carlstedt et al. 1983). More recent work has intimated that even small changes in mucin concentration are sufficient to elicit significant alteration to mucus viscoelasticity. An example of this is non- ovulatory cervicovaginal mucus in women, which is roughly 100 fold more viscous than ovulatory mucus, although there is only a moderate difference in mucin concentration between the two (Lai et al. 2009b). Work published within our research group has identified significant changes occur in the level of mucins in cervicovaginal mucus of the ewe, with increased abundance of MUC5B, one of the main gel forming mucins, identified during oestrus
(Maddison et al. 2017) and this has also been reported in humans (Gipson et al. 2001). It is probable then that changes in mucin concentration could significantly affect mucus viscoelasticity, which could ultimately influence the motility and migration of spermatozoa through cervical mucus. Work by Katz and Berger (1980) suggested that the spermatozoon is sufficiently small to experience mucus ‘not as a whole continuous gel but as a discrete network of macromolecules suspended within a simple fluid’. The interaction of spermatozoa with
108 mucin is key to allow for the successful transit of spermatozoa through the tract. Any changes to this relationship could negatively impact upon sperm migration and successful fertilisation.
Variation in mucin levels has been shown to affect motility and kinematic parameters of human spermatozoa in vitro (Eriksen et al. 1998), with the inclusion of mucins resulting in increased motility, linearity and straight line velocity parameters and higher mucin content resulting in a more linear sperm trajectory. In the present study lyophilized porcine gastric mucin was utilised to allow for ease of experimentation. Furthermore, its main structural component, MUC2 mucin, is one of the gel forming secreted mucins. Previous work has highlighted that gel forming mucins predominate the female reproductive tract (Andersch-Björkman et al. 2007), with work by one group suggesting MUC2 can also be found within the female endocervical region (Gipson 2001).
Another way to alter the viscosity of mucus is to vary the production of the enzyme neuraminidase. Neuraminidase cleaves terminal sialic acid residues from mucin proteins
(Wiggins et al. 2001). This removes the electrostatic repulsion between mucin protein which, in part, affords mucins their structural conformity and prevents them from re-aggregating after expulsion into the tract lumen (Andersch-Björkman et al. 2007). Alteration in levels of neuraminidase could result in modified mucus structure and viscosity which could greatly impact sperm motility and migration in mucus. To date the effect of variable mucin and neuraminidase levels on mucus viscosity, and how this may affect sperm motility and migration, have not been described in the ewe. As such, our aim was to investigate the effects of varying mucin content and neuraminidase treatment of CMS on the motility, viability, acrosome integrity and migration ability of ram spermatozoa following incubation in CMS.
109 4.3. MATERIALS AND METHODS
4.3.1. Experimental Design
Procedures herein were approved by the University of Sydney Animal Ethics Committee
(protocol number 2013/5854). Unless otherwise stated, all chemicals were supplied by Sigma
Aldrich (St Louis, MO, USA). Two experiments were conducted to assess the effect of mucin content (Experiment 1) and neuraminidase concentration (Experiment 2) on the viscosity of mucus and sperm transport. The motility, viability, acrosome integrity and mucus penetration ability of fresh (FRESH) and frozen-thawed (FROZEN) ram spermatozoa was also assessed over time (0 h and 3 h). In experiment 1, spermatozoa were incubated 1:1 (v/v) with CMS (see
4.3.2 for components) containing 0%, 1%, 2% or 4% mucins (3 h, 37°C) and assessments made at 0 h and 3 h after diluting semen 1:1 with CMS. For experiment 2, spermatozoa were incubated 1:1 (v/v) with CMS containing no mucins (no MUCINS) or CMS containing 2% mucins and was pre-treated with either no neuraminidase (#N2876) enzyme (no NEU1), 1 unit
NEU1/ml CMS (NEU1-1) or 2 units NEU1/ml CMS (NEU1-2). Briefly, NEU1 was added to
CMS and incubated in a water bath (1 h, 37°C) to allow the enzyme to function, then the reaction was halted by immersing the samples in boiling water for 3 minutes (Nasir ud et al.
2003), samples were then left to return to 37°C before 1:1 (v/v) incubation with spermatozoa.
Assessments were made at 0 h and 3 h post 1:1 of semen and CMS. Three technical replicates for each experiment were carried out.
4.3.2. Artificial mucus
For both experiments, an artificial mucus media, combined with varying levels of purified lyophilized porcine gastric mucins, was used as an ovine cervicovaginal mucus simulant in place of actual cervicovaginal mucus. The subsequent CMS recipe was adapted from compositional studies and mucus simulant recipes of human cervicovaginal and vaginal mucus
110 (Dorr et al. 1982; Owen and Katz 1999), and also ovine fallopian tube fluid (Restall B.J. and
Wales 1966). Concentrations of ingredients for 50 ml of CMS are as follows; sodium chloride
0.176g (60.23mM), calcium hydroxide 0.011g (2.97mM), potassium hydroxide 0.07g
(24.95mM), lactic acid 0.1g (9.17mM), acetic acid 47.65µl, glycerol 6.4µl, urea 0.02g
(6.66mM), glucose 0.25g (27.75mM), sodium bicarbonate 0.063g (15.00mM), bovine serum albumin (BSA) 0.001g, non-essential amino acids (2%) 1ml, streptomycin 0.0003g (8.23mM) and Penicillin 0.0029g (155.71mM). Lyophilized porcine gastric mucins (#M1778) mucins were added to CMS media in a flat bottom jar at 0%, 1%, 2% or 4% (w/v) for experiment 1 and at 0% or 2% for experiment 2. Solutions were gently mixed via an orbital shaker (5°C, 95 rpm) overnight and warmed to 37C before use.
4.3.3. Semen collection and freezing
Animals were housed at the University of Sydney, Camperdown campus, during the Australian breeding season (2016). Ejaculates were collected by artificial vagina from mature rams (n =
3) in the presence of a teaser ewe, with only ejaculates of good quality (wave motion ≥ 3, thick and creamy appearance) retained. Sperm concentration was determined using an SDM1 photometer (Minitube Australia, Smythesdale, VIC) calibrated for ram semen, after which they were split into fresh and frozen aliquots. Aliquots destined for fresh assessment were diluted to 200×106 spermatozoa mL-1 with Tyrode’s medium supplemented with 0.3% BSA (w/v), lactate and pyruvate (TALP) (Parrish et al. 1988). TALP was adjusted to pH 7.4 and supplemented with penicillamine (PEN; 1mM) to prevent sperm agglutination (Leahy et al.
2016). Aliquots destined for freezing were slowly diluted with a Tris-citrate-glucose diluent
(Evans and Maxwell 1987) supplemented with 15% egg yolk and 5% glycerol to 200×106 spermatozoa mL-1, chilled to 5°C over 2 h and frozen via the pellet method (Evans and Maxwell
1987). Pellets were then thawed in a water bath at 37°C for 2-3 min with agitation. Aliquots of
111 fresh (FRESH) and frozen-thawed (FROZEN) spermatozoa were then further diluted 1:1 (v/v) with CMS and maintained at 37°C. Aliquots were then taken at 0 h and 3 h for assessment.
Additional aliquots of each sample were kept separate for mucus migration tests at 0 h and 3 h.
4.3.4. Viscosity of CMS
The viscosity of all CMS types used in experiment 1 (0%, 1%, 2% and 4% mucin) and experiment 2 (no MUCINS, no NEU1, NEU1-1 and NEU1-2) was assessed prior to incubation with spermatozoa to determine the effect of variable mucin content and NEU1 on mucus viscosity. The viscosity of neat CMS and after 1:1 dilution with spermatozoa was also assessed during each experiment to evaluate any changes in viscosity due to treatment differences. A 4 chamber Leja slide (20µm×21mm×6mm;, Niuew-Vennep, The Netherlands) was used to assess the viscosity of mucus as per the method described by Rijnders et al.(2007). The filling time (sec) of a capillary-filling lamellar slide was determined to be correlated to the viscosity of seminal plasma, and thus can be utilised as a diagnostic measure. Briefly, 5µl of sample was placed directly at the opening of a Leja side chamber without touching the entrance. The pipette was depressed at the same time as a stop watch, with the time taken to completely fill the chamber recorded. Using the regression coefficient determined by Rijnders et al. (2007) (y
= 0.34x + 1.34, where y = viscosity in cp, x = filling time in sec), these values were converted to centipoise (cp), a common measure of viscosity of fluids. Two technical replicates were conducted at each time point for each sample.
4.3.5. Sperm migration in compositionally altered mucus
The ability of spermatozoa to migrate through mucus was assessed using a modified swim up method (Garcia-Lopez et al. 1996). Fresh spermatozoa were mixed with Dextran sulphate
112 (#67578, final concentration 30mg/ml) to allow for correct layering, whilst frozen-thawed spermatozoa were thawed and immediately used without further dilution. Both semen types
(200µl; FRESH; 160×106 spermatozoa mL-1, FROZEN; 200×106 spermatozoa mL-1) were then layered under CMS (400µl) in a warmed tube before being incubated at 37°C for 1 hr. After incubation, an aliquot (100µl) was taken from the uppermost section of the CMS layer and diluted 1:2 (v/v) with sodium chloride solution (3%; w/v) to render sperm immotile.
Concentration counts were then performed using a Haemocytometer (Neubauer Improved;
Pericolour HBG, Giessen-Lützellinden, Germany) to determine the concentration of sperm that had migrated through CMS.
4.3.6. Motility and kinematic parameters
At each time point, a small aliquot of each 1:1 sample (semen: CMS, 1:1), was further diluted from 100×106 spermatozoa mL-1 (concentration post 1:1) to 20×106 spermatozoa mL-1 with
TALP which had been adjusted to pH 7.4 and supplemented with BSA (0.3% w/v) and PEN
(1mM). Samples were further diluted in this manner so as to attain suitable concentrations for
CASA assessment. Samples were then assessed for motility and other kinematic parameters using computer-assisted sperm analysis (CASA; HTM-IVOS II Animal Breeder Software version 1.4, Hamilton-Thorne, Beverly, MA, USA). Samples (6µl) were placed on pre-warmed
(37°C) slides (Cell Vu; Milllenium Sciences, Mulgrave, VIC, AUS) and enclosed using a
22×22cm coverslip and immediately viewed on CASA. Motility and kinematic parameters
(total motility, progressive motility, average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), beat cross frequency (BCF), straightness (STR) and linearity (LIN) were determined by assessment of several fields (200-300 total cells per sample) using factory settings for ram.
113 4.3.7. Membrane viability and acrosome integrity
At each time point, a small aliquot of each 1:1 sample (semen: CMS, 1:1), was further diluted from 100×106 spermatozoa mL-1 (concentration post 1:1) to 20×106 spermatozoa mL-1 with
TALP which had been adjusted to pH 7.4 and supplemented with BSA (0.3% w/v) and PEN
(1mM). Samples were further diluted in this manner so as to attain suitable concentrations for membrane viability and acrosome integrity assessment. This was carried out by staining fixed
(0.1% formalin) fresh and frozen-thawed spermatozoa with propidium iodide (PI; final staining concentration 24µM) and fluorescein isothiocyanate conjugated peanut agglutinin (FITC/PNA; final staining concentration 0.4µg/ml). Following incubation (10 mins, dark, 37°C), spermatozoa were counted (200 cells) using a fluorescent microscope (400 × magnification,
Olympus BHS, Tokyo, Japan) comprising a 270-380 nm band pass filter. Emissions were observed through a 380nm dichroic mirror. Cells were classified as membrane viable-acrosome intact cells (PI-, FITC/PNA-), non-viable membranes-acrosome intact cells (PI+, FITC/PNA-) or non-viable membrane-reacted acrosome cells (PI+, FITC/PNA+).
4.3.8. Statistical analysis
Viscosity, sperm migration in mucus, kinematic parameters, membrane viability and acrosome integrity of spermatozoa following incubation with CMS were analysed using a REML in
GENSTAT (Version 15; VSN International, Hemel Hempstead, UK). Fixed effects for all parameters analysed were treatment (experiment 1; 0%, 1%, 2% or 4% mucin content, experiment 2; no MUCINS, no NEU1, NEU1-1 or NEU1-2), time (0 h and 3 h) and semen type
(fresh semen; FRESH or frozen-thawed semen; FROZEN), with all interactions also considered. Random effects for all parameters were defined as ram (n = 3), rep (n = 3), semen type (FRESH or FROZEN) and treatment (experiment 1; 0%, 1%, 2% or 4% mucin content,
114 experiment 2; no MUCINS, no NEU1, NEU1-1 or NEU1-2). Means are reported as ± SEM with p <0.05 considered significant.
4.4. RESULTS
4.4.1. Viscosity
In experiment 1, the viscosity of CMS significantly increased with increasing levels of mucin content, in a dose dependent manner (p <0.05, Figure 4.1 A). In experiment 2, while the addition of mucins to CMS media significantly increased viscosity (p <0.05), there was no effect of neuraminidase on reducing viscosity (p >0.05, Figure 4.1 B).
A B
1 2 6
d
1 0 b b b
)
)
P p
c 8 c
( 4 a
(
y
y
t
t i
6 c i
s
s
o
o c
b c
s 4 s i 2
a i
v v
2
0 0 C M S C M S + 1 % C M S + 2 % C M S + 4 % C M S C M S + 2 % C M S + 2 % C M S + 2 % M U C I N S M U C I N S M U C I N S M U C I N S M U C I N S M U C I N S
+ N E U 1 - 1 + N E U 1 - 2
M u c u s T y p e M u c u s T y p e
Figure 4.1 Viscosity of cervicovaginal mucus simulant (CMS) containing (A) varying percent of mucins (black bar; CMS, dark grey bar; CMS with 1% mucins, light grey bar; CMS with
2% mucins, lighter grey bar; CMS with 4% mucins), and (B) CMS pre-treated with varying levels of Neuraminidase (NEU1) (black bar; CMS with no mucins or NEU1, dark grey bar;
CMS with 2% mucins and no NEU1, light grey bar; CMS with 2% mucins and 1 unit NEU1/ml
CMS, and lighter grey bar; CMS with 2% mucins and 2 units NEU1/ml CMS). Significant differences between viscosity of CMS is denoted by differing superscripts. Data are means ± sem. Six replicates per treatment for A and B respectively.
115
In experiment 1, the viscosity of CMS following incubation with spermatozoa also increased with increasing mucin content, in a dose dependent manner (p <0.05, Figure 4.2 A). In experiment 2, the inclusion of NEU1 at 1 or 2 units/ml (NEU1-1 and NEU1-2, respectively) significantly reduced the viscosity of CMS following incubation with spermatozoa when compared to CMS containing no NEU1 (no NEU1) (p <0.05, Figure 4.2 B). There was no significant difference between the viscosity of CMS treated with 1 or 2 units of NEU1 (NEU1-
1 and NEU1-2, respectively, p >0.05, Figure 4.2 B).
A B
8 6
d )
6 ) P
p b
c c
c 4 c
(
(
c y
y a t
b t i
4 i s
a s
o
o
c
c s
s 2
i
i v 2 v
0 0
C M S + C M S + 1 % C M S + 2 % C M S + 4 % C M S + C M S + 2 % C M S + 2 % C M S + 2 %
s p e r m M U C I N S M U C I N S M U C I N S s p e r m M U C I N S M U C I N S M U C I N S
+ s p e r m + s p e r m + s p e r m + s p e r m + N E U 1 - 1 + N E U 1 - 2
+ s p e r m + s p e r m
M u c u s T y p e M u c u s T y p e
Figure 4.2 Viscosity of cervicovaginal mucus simulant (CMS) following incubation (1:1; v/v) with spermatozoa (fresh and frozen-thawed results pooled) when CMS contains (A) varying percent of mucins (black bar; CMS, dark grey bar; CMS with 1% mucins, light grey bar; CMS with 2% mucins, lighter grey bar; CMS with 4% mucins), and (B) CMS pre-treated with varying levels of Neuraminidase (NEU1) (black bar; CMS with no mucins or NEU1, dark grey bar; CMS with 2% mucins and no NEU1, light grey bar; CMS with 2% mucins and 1 unit
NEU1/ml CMS, and lighter grey bar; CMS with 2% mucins and 2 units NEU1/ml CMS).
Significant differences between the viscosity of CMS following incubation with spermatozoa
116 is denoted by differing superscripts. Data are means ± sem. Twelve replicates per treatment for
A and B respectively.
4.4.2. Sperm migration in mucus
In experiment 1, while there was no effect of mucins on the migrating ability of fresh spermatozoa (p > 0.05, Figure 4.3 A), frozen-thawed spermatozoa migrated further in CMS with increasing concentrations of mucins (p <0.05, Figure 4.3 A). Treatment of CMS with
NEU1 did not affect sperm migration, with comparable numbers of sperm migrating through no MUCINS, NO NEU1, NEU1-1 and NEU1-2 (p >0.05, Figure 4.3 B).
A B
7 7
5 1 0 c 5 1 0
n
n o
7 o 7 i 4 1 0 i t 4 1 0
t
a b
a
r
r
t
t n 7 n 7 3 1 0 3 1 0
e
e a
c
c n
n a
a o 7 o 7
2 1 0 a 2 1 0 a c
c
a a a a a
a a
m
m
r r
7 7 e
1 1 0 e 1 1 0
p
p
s s
0 0
f r e s h s e m e n f r o z e n - t h a w e d f r e s h s e m e n f r o z e n - t h a w e d
s e m e n s e m e n
S e m e n T y p e S e m e n T y p e
Figure 4.3 Concentration of fresh and frozen-thawed sperm that migrated into cervicovaginal mucus simulant (CMS) containing (A) varying percent of mucins (black bar; 0% mucins, dark grey bar; 1% mucins, light grey bar; 2% mucins, lighter grey bar; 4% mucins), and (B) CMS pre-treated with varying levels of Neuraminidase (NEU1) (black bar; no MUCINS: artificial mucus media with no mucins or NEU1, dark grey bar; no NEU1: CMS with 2% mucins and no NEU1, light grey bar; NEU1-1: CMS with 2% mucins and 1 unit NEU1/ml CMS, and lighter grey bar; NEU1-2: CMS with 2% mucins and 2 units NEU1/ml CMS). Significant difference in sperm concentration between treatments denoted by differing superscripts for each graph.
Data are means ± sem. Six replicates per treatment for A and B respectively.
117
4.4.3. Motility parameters
In experiment 1, the motility of sperm incubated in CMS containing no mucins (0%) was significantly higher in comparison to CMS containing 1%, 2% and 4% mucins (p <0.05, Figure
4.4 A). Variation in percent content of mucins in CMS had no effect on sperm motility, with sperm incubated in CMS containing 1%, 2% or 4% mucins having comparable motility values
(p >0.05, Figure 4.4 A). Both the process of cryopreservation and incubation of samples for 3 hrs resulted in significantly reduced motility (p <0.05).
In experiment 2, the inclusion of 2% mucins into CMS (no NEU1) significantly increased sperm motility in comparison to CMS containing no mucins (no MUCINS, p <0.05, Figure 4.4
B). Pre-treatment of CMS with NEU1 had no effect on motility, with sperm incubated in no
NEU1, NEU1-1 and NEU1-2 having similar motility values (p >0.05, Figure 4.4 B). The process of cryopreservation and incubation of samples for 3 hours resulted in significantly reduced motility overall (p <0.05).
118 A B
a 1 0 0 1 0 0
b b 8 0 b 8 0 b
b b
y
y
t
t
i
i l
6 0 l 6 0
i
i
t
t
o
o m
m a
4 0 4 0
% %
2 0 2 0
0 0
C M S + C M S + 1 % C M S + 2 % C M S + 4 % C M S + C M S + 2 % C M S + 2 % C M S + 2 %
s p e r m M U C I N S M U C I N S M U C I N S s p e r m M U C I N S M U C I N S M U C I N S
+ s p e r m + s p e r m + s p e r m + s p e r m + N E U 1 - 1 + N E U 1 - 2
+ s p e r m + s p e r m
M u c u s T y p e M u c u s T y p e
Figure 4.4 Motility of sperm (fresh and frozen-thawed pooled) following incubation (1:1; v/v) with cervicovaginal mucus simulant (CMS) containing (A) varying percent of mucins (black bar; CMS, dark grey bar; CMS with 1% mucins, light grey bar; CMS with 2% mucins, lighter grey bar; CMS with 4% mucins), and (B) CMS pre-treated with varying levels of
Neuraminidase (NEU1) (black bar; CMS with no mucins or NEU1, dark grey bar; CMS with
2% mucins and no NEU1, light grey bar; CMS with 2% mucins and 1 unit NEU1/ml CMS, and lighter grey bar; CMS with 2% mucins and 2 units NEU1/ml CMS). Significant difference between motility of sperm in CMS denoted by differing superscripts. Data are means ± sem.
Twelve replicates per sample for A and B respectively.
In experiment 1 the addition of mucins into CMS significantly affected sperm kinematic parameters, with increased sperm straightness (STR), linearity (LIN), beat cross frequency
(BCF) and decreased amplitude of lateral head movement (ALH), however not in a dose dependent manner, with results similar between 1%, 2% and 4% (p <0.05). After freeze- thawing, spermatozoa had significantly increased LIN but significantly decreased STR and
BCF (p <0.05). The inclusion of mucins into CMS significantly affected velocity parameters,
119 with increased curvilinear velocity (VCL) and average path velocity (VAP), but decreased straight line velocity (p <0.05, Figure 4.5 A). VSL and VAP were further reduced in CMS containing 4% mucins compared to those with 1% or 2% mucin content (p <0.05, Figure 4.5
A).
A B
3 0 0 3 0 0
2 5 0 2 5 0 )
* )
s s
/ *
* * / m
2 0 0 m 2 0 0
(
(
y 1 5 0
1 5 0 y
t
t
i
i
c
c
o o
l 1 0 0 1 0 0
l
e
e
v
v
5 0 5 0
0 0
C M S C M S + 1 % C M S + 2 % C M S + 4 % C M S C M S + C M S + 2 % C M S + 2 %
M U C IN S M U C IN S M U C IN S 2 % M U C I N S M U C I N S M U C I N S
+ N E U 1 - 1 + N E U 1 - 2
M u c u s T y p e M u c u s T y p e
Figure 4.5 Velocity parameters (average path velocity; solid line, straight line velocity; dotted line and curvilinear velocity; dashed line) of sperm (fresh and frozen-thawed pooled) incubated
1:1 (v/v) with cervicovaginal mucus simulant (CMS) containing (A) varying percent content of mucins; CMS with no mucins (CMS), CMS with 1% mucins (CMS +1% MUCINS), CMS with 2% mucins (CMS + 2% MUCINS) or CMS with 4% mucins (CMS + 4% MUCINS), and
(B) CMS pre-treated with or without varying levels of Neuraminidase; CMS with no mucins or NEU1 (CMS), CMS with 2% mucins but no NEU1 (CMS + 2% MUCINS), CMS with 2% mucins and 1 unit NEU1/ml CMS (CMS + 2% MUCINS + NEU1-1), or CMS with 2% mucins and 2 units NEU1/ml CMS (CMS + 2% MUCINS + NEU1-2). Significant differences between types of CMS denoted by asterix superscript. Data are means ± sem. Twelve replicates per sample for A and B respectively.
120 In experiment 2, neither the inclusion of mucins nor the pre-treatment of CMS with NEU1 significantly affected sperm motion kinematics, with ALH, LIN, STR and BCF being comparable between no MUCINS, no NEU1, NEU1-1 and NEU1-2 (p >0.05). After freeze- thawing, sperm had significantly decreased BCF, LIN and STR values but significantly increased ALH (p <0.05). Velocity parameters were not significantly affected by the addition of mucins to CMS or pre-treatment of CMS with NEU1 at 1 or 2 units/ml CMS, with VAP,
VSL and VCL being comparable between no MUCINS, no NEU1, NEU1-1 and NEU1-2 (p
>0.05, Figure 4.5 B). Freeze-thawing resulted in significant decreases to VSL and VAP, but significant increases to VCL (p <0.05, Figure 4.5 B).
4.4.4. Membrane viability and acrosome integrity
The addition of mucins to CMS significantly improved sperm membrane status and acrosome integrity. Spermatozoa incubated in CMS containing 1%, 2% or 4% mucins (experiment 1) had a significantly higher percentage of membrane viable and acrosome intact cells (PI+,
FITC/PNA-), and non-viable membranes and acrosome intact cells (PI+, FITC/PNA-) than those incubated in CMS alone (p <0.05, Figure 4.6 A). The amount of non-viable membranes and acrosome reacted cells (PI+, FITC/PNA+) was significantly decreased by the inclusion of mucins in CMS (p <0.05, Figure 4.6 A). The effects of mucins were not consistently dose dependent with comparable levels of viable and acrosome intact spermatozoa present in CMS containing 1%, 2% or 4% mucins (p >0.05). Increasing mucin content of CMS from 1% to 2% or 4% resulted in significantly increased levels of spermatozoa with non-viable membranes and acrosome intact spermatozoa (PI+, FITC/PNA-), and non-viable membrane and acrosome reacted cells (PI+, FITC/PNA+) (p <0.05, Figure 4.6 A).
121 In experiment 2, the inclusion of NEU1 in CMS at either 1 or 2 units NEU1/ml CMS resulted in significantly increased levels of spermatozoa with non-viable membranes and intact acrosome. The inverse was also true, with NEU1 inclusion in CMS (NEU1-1 and NEU1-2) resulting in significantly decreased amounts of sperm with non-viable membranes and reacted acrosomes (p <0.05, Figure 4.6 B). There was no significant effect of concentration of NEU1
(1 or 2 units/ml CMS) on the percentage of membrane viable and acrosome intact spermatozoa
(p > 0.005).
For both experiments 1 and 2, the process of freeze-thawing significantly reduced the amount of viable and acrosome intact spermatozoa, irrespective of the type of CMS incubated with spermatozoa (p <0.05), however no treatment by spermatozoa status (fresh or frozen-thawed) effect was evident (p >0.05)
122 A B
6 0 7 0
* 5 0 6 0 * * * * 5 0
4 0 * *
s
s
l
l l
l 4 0 * e
e 3 0
c
c
3 0
% % 2 0 2 0
1 0 1 0
0 0
C M S C M S + 1 % C M S + 2 % C M S + 4 % C M S C M S + C M S + 2 % C M S + 2 %
M U C IN S M U C IN S M U C IN S 2 % M U C I N S M U C I N S M U C I N S
+ N E U 1 - 1 + N E U 1 - 2
M u c u s T y p e M u c u s T y p e
Figure 4.6 Percent of membrane viable/acrosome intact (solid line; PI-, FITC/PNA-), non- viable membranes/acrosome intact (dashed line; PI+, FITC/PNA-) and non-viable membranes/acrosome reacted (dotted line; PI+, FITC/PNA+) spermatozoa (pooled data for fresh and frozen-thawed) incubated 1:1 (v/v) in cervicovaginal mucus simulant (CMS) containing (A) varying percent content of mucins; CMS with no mucins (CMS), CMS with 1% mucins (CMS +1% MUCINS), CMS with 2% mucins (CMS + 2% MUCINS) or CMS with
4% mucins (CMS + 4% MUCINS), and (B) CMS pre-treated with or without varying levels of
Neuraminidase; CMS with no mucins or NEU1 (CMS), CMS with 2% mucins but no NEU1
(CMS + 2% MUCINS), CMS with 2% mucins and 1 unit NEU1/ml CMS (CMS + 2%
MUCINS + NEU1-1), or CMS with 2% mucins and 2 units NEU1/ml CMS (CMS + 2%
MUCINS + NEU1-2). Significant difference in viability between types of CMS denoted by asterix superscript for each experiment. Data are means ± sem. Twelve replicates per sample for A and B respectively.
4.5. DISCUSSION
We have shown that varying the concentration of mucins can significantly alter the viscosity of a CMS, leading to an improvement in mucus penetration ability for frozen-thawed
123 spermatozoa. Following incubation in CMS, spermatozoa had decreased motility, increased velocity, and an increase in the percentage of membrane viable, acrosome intact cells. Results show that pre-treatment of a cervicovaginal mucus simulant with the enzyme neuraminidase does not affect neat viscosity, nor does it impact on the mucus penetration ability of fresh or frozen-thawed spermatozoa. Following incubation in CMS, spermatozoa had unchanged motility and velocity, but an increase in the percentage of membrane viable and acrosome intact cells. As the ability of spermatozoa to migrate through mucus is crucial for successful transit of the female tract, any unexpected changes due to altered abundance of proteins, such as mucins and neuraminidase, could have deleterious effects. Results reported herein suggest that increases in mucin content may be affecting the migration of frozen-thawed sperm. If the changes reported herein are in fact occurring in vivo, the success of migrating spermatozoa in reaching the ova may well be impacted upon when frozen-thawed semen are utilised, which may be impacting on fertility.
A variety of mucus migration or swim up tests are used to assess, in an in vitro setting, semen and their in vivo capability to migrate through mucus and the female tract, ultimately reaching the ova. Previous work in goats supports this, with mucus migration in vitro being related to the in vivo ability of sperm to transit the tract and reach the utero-tubal junction (Cox et al.
2002). Work by Robayo et al. (2008) highlighted the link between sperm kinematic parameters and ability of sperm to migrate in mucus in vitro. They found the curvilinear velocity (VCL) and average path velocity (VAP) measures of ram sperm had a moderate positive correlation with the ability to migrate through homologous cervical mucus. Migration of ram sperm in caprine mucus also showed positive correlations between VCL and VAP as well as straight line velocity (VSL) and linearity (LIN) measures. Cox et al. (2006) also reported that increased velocity parameters, particularly VAP and VCL, of buck ejaculates resulted in increased
124 migrating abilities. Applying this logic to the present data one might propose that the sperm: mucus samples with the highest velocities would have increased sperm migration, however this was not necessarily the case. Whilst sperm incubated with CMS containing mucins did have higher velocities than sperm incubated in CMS alone, velocity measures of CMS were either comparable with increasing mucin content (VCL), or significantly lower in CMS with 4% mucins (VAP and VSL), compared to that containing 1% or 2% mucin content. The migrating ability of sperm however, increased with increasing levels of mucins, although this was only the case for frozen-thawed semen. A possible explanation is that the increasing mucin content which resulted in increased viscosity led to either similar or reduced velocities, due to perhaps increased friction between travelling sperm and its surrounding CMS.
Previous studies utilising purified human cervical mucins from cervical mucus plugs from pregnant women have highlighted the effects that mucins can have on human sperm motility and kinematic parameters in vitro. Eriksen et al.(1998) showed that increased mucin content caused increased sperm linearity and straight line velocity in human sperm, and whilst inclusion of mucins in CMS did cause increased LIN in the current study, effects were not dose dependant. Furthermore, increasing levels of mucins in CMS resulted in decreased straight line velocity (VSL) when compared to CMS with no mucins. The concentration of mucins used may be a contributing factor in result disparity between the present study and work by Eriksen et. al. (1998), with the current study using up to 3 times more mucins than Eriksen et al. (1998), however this is unlikely to produce opposing results but rather a weaker or stronger result. The level of mucin utilised in the present study was based upon work by Carlstedt et. al. (1983) which identified cervical mucus in humans to contain 1.5 % mucin content (w/w).
125 Perhaps the differences seen in sperm linearity and velocity between Eriksens works and the present study were in part due to variation in the type of mucins utilised within each respective study. Purified mucins from cervical mucus plugs of pregnant women were used in Eriksens’ works, whilst reconstituted porcine gastric mucins were utilised in the present study. Several mucins have been identified in the reproductive tract, including MUC1, 4, 5AC, 5B, 6 and 8
(Gipson et al. 1997; Lagow, DeSouza and Carson 1999), so perhaps a different mucin type dominates in the cervical mucus plug in pregnant women compared to porcine gastric mucins, the type of mucin utilised in the present study. Porcine gastric mucin coats the intestinal epithelium and has Mucin-2 (MUC2) as its central molecule (Smit et al. 1988), this disparity in mucus type might explain any opposing results.
Whilst treatment of CMS with NEU1 did not significantly reduce viscosity of CMS alone, its inclusion in CMS at 1 or 2 units/ml CMS (NEU1-1 and NEU1-2, respectively) did significantly reduce the viscosity of 1:1 samples and result in more motile sperm, it did not affect sperm migration in mucus, suggesting that a reduction in abundance of the protein neuramindase-1 in ovine cervicovaginal mucus after superovulation, as previously reported by this group
(Maddison et al. 2017), may not greatly impact on sperm transit and successful fertilisation. It is interesting that decreased viscosity of 1:1 samples after treatment with NEU1 did not translate into changes in motility and altered sperm migration, although whilst significantly different, the viscosity values of 1:1 samples did not differ immensely. Perhaps morphology of the tested spermatozoa is key here; we know that mucus in vivo acts to filter out morphologically abnormal spermatozoa (Ragni et al. 1985) so it is possible that whilst motile they could have possessed abnormal morphology and thus were retained in lower sections of the CMS layer in the swim up device. Morphology was not tested in either experiment and as such this theory cannot currently be verified. It is also very likely that whilst useful, the in vitro
126 assay utilised to assess the effects of NEU1 are unable to fully replicate in vivo effects of this protein.
Also, worthy to note is the disparity in motility for CMS only control treatments between the two experiments, experiment 1 (mucin content) sample having motility of 93% and experiment
2 (neuraminidase content) having motility of 32%. Viscosity of both mucus alone and 1:1 samples were similar between the two experiments, and thus likely cannot explain the disparity in motility observed. One could assume that as experiments were performed at different times that the quality of original semen samples may have differed between the two experiments, however initial semen sample assessments of wave motion and motility ensured only optimal samples were utilised and as such could not be the cause either. All experimentation was carried out using the same batch of CMS, so variances in CMS can also not explain this. Unfortunately, at present this variation remains unexplained.
In conclusion, we have shown that alteration of mucin levels can greatly impact the viscosity of a mucus simulant and impacts the motility, membrane viability and acrosome integrity, and migrating ability of sperm incubated with a cervicovaginal mucus simulant.
4.6. Acknowledgements
Research was supported by the NSW Stud Merino Breeders Association Trust and Australian
Wool Innovation. The authors would like to thank Miss Naomi Bernecic, Miss Taylor Pini,
Mrs Angela Crean, Miss Jessica Ricakrd and Miss Tamara Leahy for their laboratory assistance. The authors would also like to thank Miss Evelyn Hall and Mr. Peter Thomson for their statistical consultation.
127 Chapter 5. Oestrus synchronisation using prostaglandin-F2α decreases fertility in the Merino ewe
5.1. ABSTRACT
Oestrus synchronisation techniques are commonly used in the sheep (Ovis aries) industry to facilitate fixed time artificial insemination of chilled or frozen semen. Semen storage procedures have previously been shown to have deleterious effects on fertility especially when intracervical artificial insemination is employed. Such effects may be exacerbated by the use of synchronisation protocols which alter conditions inside the female tract at oestrus. As such, the aim of this study was to compare the pregnancy rates of naturally cycling (NAT; n = 111), progesterone synchronised (P4; n = 135) and prostaglandin-F2α (PGF2α; n = 133) synchronised
Merino ewes following intracervical artificial insemination with fresh, chilled or cryopreserved ram semen. Ewes synchronised using prostaglandin-F2α had significantly lower pregnancy rates compared to both naturally cycling ewes and progesterone synchronised ewes (6%, 17% and 25%, respectively, p <0.001), whereas progesterone synchronised ewes had comparable pregnancy rates to naturally cycling ewes (p >0.05). While ewes inseminated with cryopreserved semen had lower pregnancy rates than those inseminated with chilled or fresh semen, differences were not significant (10%, 20% and 17%, respectively, p >0.05). No significant interactions between semen type (fresh, chilled or cryopreserved semen) and synchronisation method (NAT, P4 or PGF2α) were identified (p >0.05). These results indicate that oestrus synchronisation using prostaglandin-F2α limits fertility in the Merino ewe following intracervical artificial insemination with fresh, chilled and cryopreserved sperm.
128
5.2. INTRODUCTION
Oestrus synchronisation is an essential tool used to facilitate artificial breeding programs around the world. In the southern hemisphere, synchronisation of ewes by progestagen impregnated intravaginal pessaries (either sponges or controlled intravaginal drug release devices; CIDRs) is favoured due to their ease of application and ability to synchronise and induce oestrus outside the breeding season. Prostaglandin analogues are more commonly utilised by sheep industries in the northern hemisphere due to regional restrictions on the use of progesterone for synchronisation purposes in food-producing animals (Lane, Austin and
Crowe 2008). While both methods successfully achieve flock synchrony, questions remain over their relative impact on the physiology of the female reproductive tract at oestrus and the subsequent consequences for ewe fertility. Indeed, progesterone synchronisation has been shown to modify the volume (Allison 1971; Rexroad and Barb 1977), protein concentration
(Croker and Shelton 1974; Rexroad and Barb 1977) and protein composition (Maddison et al.
2016) of cervical mucus, while also reducing the number of spermatozoa retrieved from the female tract (Quinlivan and Robinson 1967, 1969; Hawk and Cooper 1977). PGF2α use has had similar effects in the ewe, with altered mucus composition (Prasad et al. 1981) and volume
(Maddison et al. 2016), reduced sperm in the tract after natural mating (Hawk and Cooper
1977), decreased fertilisation (Boland, Gordon and Kelleher 1978) and pregnancy rates
(Boland, Lemainque and Gordon 1978) all reported. While it is clear that oestrus synchronisation may have negative effects on ewe fertility, to date no study has simultaneously investigated the impact of both methods on the transit of spermatozoa across the cervix.
Studies have demonstrated that liquid and particularly frozen storage of ram semen dramatically reduces the ability of spermatozoa to cross the cervix (Salamon and Maxwell
129 1995a; Druart et al. 2009). While easily explained for frozen spermatozoa given the widespread changes which occur to this population (e.g. reduced motility and viability (Barrios et al. 2000), and destabilised plasma membranes (Bailey, Bilodeau and Cormier 2000)) it is less expected for liquid stored spermatozoa as they display similar motility and velocity to fresh spermatozoa
(Druart 2009). In any event, the majority of experiments that investigate the interaction between spermatozoa and the cervix have been undertaken with ewes that have been synchronised for oestrus (Richardson et al. 2012). The few studies that involve insemination of ewes at a natural oestrus still report a negative effects of semen storage (Salamon and
Maxwell 1995b) but seemingly to a reduced degree. The prospect of the method of oestrus synchrony exacerbating the impact of semen storage on sperm transport has not been considered and the potential interaction between type of spermatozoa and cervical environment has not been investigated. As such, the aim of this experiment was to investigate the effect of oestrus synchronisation (none, progesterone or prostaglandin synchronised) and semen storage method (fresh, chilled or cryopreserved) on the ability of ram spermatozoa to navigate the cervix as assessed by pregnancy following cervical AI.
5.3. MATERIALS AND METHODS
Procedures herein were approved by the University of Sydney Animal Ethics Committee
(protocol number 2013/5999). One experiment was repeated over two weeks to assess the interaction of semen type (fresh; FRESH, chilled; CHILLED and cryopreserved; FROZEN) and synchronisation protocol (naturally cycling ewes, progesterone synchronised ewes and prostaglandin-F2α synchronised ewes) on the field fertility of Merino ewes following intracervical AI. A sub-group of P4 ewes (n = 31) were inseminated with cryopreserved semen
(FROZEN) by intrauterine laparoscopic intrauterine AI to act as the control and confirm flock fertility.
130
5.3.1. Animals
The trial was carried out during the Southern hemisphere breeding season (April-May) at
‘Arthursleigh’ (Marulan, NSW, Australia). Mature Merino ewes (n = 379; 2-4 years old, body condition score (BCS) 2-3) were kept on a pasture based diet supplemented with barley grain.
Mature rams (n = 3; BCS 2-4) were kept on a chaff-based diet (Oaten: Lucerne chaff, 1:1) supplemented with lupin grain and housed in an animal house at the Faulty of Veterinary science, University of Sydney, Camperdown, NSW, Australia during the cryopreservation of semen and at ‘Arthursleigh’ during the insemination. Androgenised wethers (n = 33; BCS 2-
4) were kept on a pasture based diet supplemented with barley grain at ‘Arthursleigh, NSW
Australia’.
5.3.2. Hormone administration
Hormone administration was carried out so that oestrus occurred at approximately the same time for each treatment group, for NAT this occurred at the ‘natural’ oestrus following a synchronised oestrous cycle, and for P4 and PGF2α ewes at the synchronised oestrus immediately following hormone administration. Naturally cycling ewes (N = 111, week 1; n =
64, week 2; n = 47) were treated with intra vaginal progesterone sponges (30 mg Flugestone acetate; Vetoquinol, Lure cedex, France) for 12 days, to ensure synchrony of their cycles and correct timing of oestrus comparable to other treatments. Post sponge removal, they were injected with 400IU equine chorionic gonadotropin (Pregnocol, Vetoquinol). Two days prior to expected onset of ‘natural oestrus’ of the second oestrous cycle, ewes were exposed to harnessed androgenised wethers at a flock percentage of approximately 16 % during week 1 and 19 % during week 2. NAT ewes were inseminated at the second oestrus following synchronisation (‘natural cycle’) only if marked by an androgenised wether, thus insemination
131 occurred at 12 h (minimum) - 24 h (maximum) post onset of oestrus (i.e. marking). Ewes not marked by wethers were returned to the mob until marking occurred.
Progesterone synchronised ewes destined for either intracervical AI (N = 135, week 1; n = 7 0, week 2; n = 65) or laparoscopic intrauterine AI (n = 31, week 1) were treated with intravaginal progesterone sponges (30 mg Flugestone acetate, Vetoquinol) for 12 days. At sponge removal, ewes were injected with equine chorionic gonadotropin (400IU, Pregnocol, Vetoquinol) and then exposed to harnessed androgenised wethers at a flock percentage of approximately 16 % during week 1 and 19 % during week 2. P4 ewes destined for intracervical AI were inseminated at the synchronised oestrus if marked by an androgenised wether, thus insemination occurred at 12 h (minimum) - 24 h (maximum) post onset of oestrus (i.e. marking). Ewes not marked by wethers were returned to the mob until marking occurred. All P4 LAP AI ewes were removed from the main mob to fast 24 h prior to insemination, which occurred 60 h post sponge removal
(Evans and Maxwell 1987).
Prostaglandin-F2α synchronised ewes (N = 133, week 1; n = 71, week 2; n = 62) were given two injections of prostaglandin-F2α (125 mg, cloprostenol sodium, ‘Estrumate’; Merck animal health, Bendigo, VIC, Australia), ten days apart (Evans and Maxwell 1987). After the second injection, ewes were exposed to harnessed androgenised wethers at a flock percentage of approximately 16 % during week 1 and 19 % during week 2. PGF2α ewes were inseminated at the synchronised oestrus only if marked by an androgenised wether, thus insemination occurred at 12 h (minimum) - 24 h (maximum) post onset of oestrus (i.e. marking). Ewes not marked by wethers were returned to the mob until marking occurred.
132 5.3.3. Oestrus detection
The onset of oestrus was determined by the use of androgenised wethers fitted with harnesses with crayons. A double draft method was employed through which ewes were checked for markings on their hind quarters twice daily (6 am, 6 pm) in conjunction with expected onset of oestrus (Evans and Maxwell 1987), and inseminated after the next marking assessment (12 h).
Ewes not marked, along with wethers, were returned to paddock until the next draft. Wethers were treated with one injection of testosterone enanthate (450 mg, Ropel, Jurox, Rutherford,
NSW, Australia) one week prior to introduction into the week 1 ewe flock, followed by a single injection one week later (225 mg) after introduction into the week 2 flock. LAP AI ewes were not drafted based on oestrus onset but rather removed from the main mob so as to fast 24 h prior to timed insemination at 60 h post sponge removal.
5.3.4. Semen collection and processing
All semen was collected from mature Merino rams (n = 3) using an artificial vagina in the presence of a teaser ewe. Each ejaculate was immediately assessed for wave motion (with samples ≥4 being accepted for further study; data not shown) and sperm concentration, determined using a photometer (SDM1; Minitube, Tiefenbach, Germany). Ejaculates destined for cryopreservation were collected, frozen and banked prior to the trial during February-April,
2016. Ejaculates used for fresh (FRESH) or chilled (CHILLED) samples were collected twice daily (6 am & 6 pm) during the trial period. All ejaculates were kept separate per ram.
Ejaculates used for FRESH and CHILLED processing were slowly diluted 1+3 (semen: diluent, v/v) in warmed (30ᵒC) Tris-citrate-fructose diluent containing 15 % egg yolk (Evans and Maxwell 1987). Here the sample volume was split and CHILLED samples were slowly cooled to 5ᵒC for a minimum of 12 h, whereby they were warmed to 30ᵒC ready for
133 insemination. FRESH samples were held at 30ᵒC ready for insemination. Ejaculates used for
FROZEN processing were slowly diluted 1+3 (semen: diluent, v/v) with a warmed Tris-citrate- glucose diluent containing 15 % egg yolk and 5 % glycerol (Evans and Maxwell 1987).
Samples were then slowly chilled to 5ᵒC over 2 h and frozen via the pellet method (Evans and
Maxwell 1987). Briefly, 250µl of sample was placed into small indentations on a block of dry ice for 2-3 mins, before being plunged into liquid nitrogen where they were stored until required. On the day of insemination, appropriate amounts of pellets were thawed by vigorous agitation in a glass tube (max 2 per tube) for a minimum of 2 minutes in a 37ᵒC water bath, then cooled to 30ᵒC ready for insemination. Subjective motility was assessed for each FRESH,
CHILLED and FROZEN sample per ram to ascertain total motile dose for each sample.
5.3.5. Insemination
After drafting, all marked ewes, for each synchronisation method (NAT, P4, PGF2α), were divided evenly (were possible) amongst semen types (FRESH, CHILLED or FROZEN) for each ram (n = 3), to eliminate bias. Ewes inseminated by intracervical AI (NAT; n = 111, P4;
6 n = 135, PGF2α; n = 133, 150 x 10 motile spermatozoa/ewe) were fasted for 12 h prior to insemination after identification of marks by an androgenised wether. Thus, insemination of intracervical AI ewes occurred at 12 h (minimum) - 24 h (maximum) post onset of oestrus (i.e. marking). Ewes not marked by wethers were returned to the mob until marking occurred.
The sub-group of P4 LAP AI ewes were inseminated by laparoscopic intrauterine AI (n = 31,
50 x 106 motile spermatozoa/ewe) at 60 h after sponge removal. LAP AI ewes were fasted for
24 h prior to insemination and given pre-operative analgesia, sedation and a muscle relaxant via an i.m. injection of Xylaxin (3.75mg; Troy Laboratories, Glendenning, NSW, Australia).
LAP AI ewes were monitored for 3 h post insemination for any adverse effects to sedation. All
134 ewes left undisturbed for 12 h post insemination and the use of dogs throughout the trial period was prohibited so as to prevent the animals from experiencing any undue stress. Pregnancy diagnosis of all ewes was determined at Day 84-91 post insemination using real-time cutaneous ultrasonography.
5.3.6. Statistical analysis
Pregnancy data was analysed using a GLMM in Genstat (Version 15, VSN International,
Hemel Hempstead, UK) for both the intracervical AI and laparoscopic intrauterine AI groups.
For intracervical AI ewes, synchronisation method (NAT, P4 and PGF2α) and sperm type
(FRESH, CHILLED and FROZEN) and any interactions between the two were specified as fixed effects and ewe number and ram specified as random effects. Pregnancy was reported as
% pregnancy and p <0.05 was considered statistically significant.
For analysis of fertilizing capability of cryopreserved semen, a subset of data (to ensure balance between the two treatment groups) of P4 ewes (n = 46) inseminated with FROZEN semen by intracervical AI was analysed with data for laparoscopic intrauterine AI (n = 31), for which
FROZEN sperm was inseminated. Analysis was carried out using a GLMM in Genstat (Version
15, VSN International, Hemel Hempstead, UK), with insemination method (intracervical AI or laparoscopic AI) specified as fixed effects, while ewe and ram were specified as the random effects. Pregnancy was reported as % pregnancy and p <0.05 was considered statistically significant.
5.4. RESULTS
The pre-insemination subjective motility of frozen-thawed semen (FROZEN) (51 ± 1.1 %) was significantly lower than FRESH and CHILLED (86 ± 1.0 % and 82 ± 0.8 %, respectively, p
135 <0.001; Figure 5.1). Whilst the difference in subjective motility between FRESH and
CHILLED semen was only 4 %, this was also significant (p <0.001, Figure 5.1).
Figure 5.1 Subjective Motility of fresh ram semen (FRESH; n = 32), ram semen chilled for 12 h at 5°C (CHILLED; n = 40) and frozen-thawed ram semen (FROZEN; n = 37) used for intracervical and laparoscopic intrauterine artificial insemination. Values without common superscripts differ significantly (p <0.05).
There was no significant interaction between oestrus synchronisation method and sperm type on pregnancy rate (p = 0.202). Method of oestrus synchronisation had a significant impact on percent pregnancy rate, with ewes synchronised using PGF2α having a lower (p <0.001) pregnancy rate (6 ± 0.04 %) than both naturally cycling ewes (17 ± 0.02 %) and ewes synchronised with progesterone (25 ± 0.06 %, Table 5.1, Figure 5.2). There was no significant difference between the pregnancy rate of P4 and NAT ewes (p >0.05, Figure 5.2). While ewes inseminated with FROZEN semen had lower pregnancy rates than both CHILLED and
FRESH, differences were not significant (10 ± 0.04 %, 20 ± 0.04 % and 17 ± 0.10 %, respectively, p = 0.07, Figure 5.3).
136
Table 5.1 Pregnancy rates of naturally cycling (NAT), progesterone synchronised (P4) and prostaglandin-F2α (PGF2α) synchronised ewes after intracervical artificial insemination with fresh ram semen (FRESH), ram semen chilled for 12 h at 5°C (CHILLED) and frozen-thawed ram semen (FROZEN).
Oestrus Synchronisation Method
NAT P4 PGF2α Ewes Ewes Ewes Ewes Ewes Ewes Semen Type inseminated pregnant inseminated pregnant inseminated pregnant (%)a (%)a (%)a FRESH 30 4 (13) 43 15 (35) 39 1 (3) CHILLED 37 8 (22) 43 11 (26) 46 6 (13) FROZEN 39 6 (15) 46 6 (13) 41 1 (2) a Pregnancy was determined by real time cutaneous ultrasound
Figure 5.2 Pregnancy rates of naturally cycling (NAT; n = 106), progesterone synchronised
(P4; n =132) and prostaglandin-F2α synchronised (PGF2α; n = 126) merino ewes after intracervical artificial insemination. Semen type data pooled. Values without common superscripts differ significantly (p <0.05).
137
Figure 5.3 Pregnancy rates of ewes inseminated by intracervical artificial insemination with fresh ram semen (FRESH; n = 112), ram semen chilled for 12 h at 5°C (CHILLED; n = 126) and frozen-thawed ram semen (FROZEN; n = 126). Data pooled for all oestrus synchronisation methods. Values without common superscripts differ significantly (p <0.05).
The pregnancy rate achieved when utilising FROZEN ram semen was significantly reduced (p
<0.001) when inseminated by intracervical AI (FROZEN; 13 %), compared to laparoscopic AI
(58 %, Figure 5.4). This indicates that whilst frozen-thawed semen inseminated by intracervical
AI could result in pregnancy, significant reductions in pregnancy rate resulted when this method was utilised, when compared to pregnancy rates achieved when laparoscopic AI was utilised.
138
Figure 5.4 Pregnancy rates of ewes inseminated by intracervical artificial insemination (Intra- cervical AI; n = 46) or laparoscopic intrauterine artificial insemination (Laparoscopic
Intrauterine AI; n = 31) methods with frozen-thawed (FROZEN) ram semen at progesterone synchronised oestrus. Values without common superscripts differ significantly (p <0.05).
5.5. DISCUSSION
The current study has illustrated the negative impact of oestrus synchronisation with prostaglandins on the fertility of Merino ewes. Oestrus synchronisation using PGF2α resulted in a pregnancy rate of only 6 %, compared to the 17 % achieved in NAT ewes. This detrimental effect of oestrus synchronisation was not evident when ewes were synchronised using progesterone (P4), which had a comparable pregnancy rate to NAT ewes (25 % P4 v 17 % NAT;
Figure 5.2). The lack of interaction between oestrus synchronisation method (NAT, P4, PGF2α) and sperm type (FRESH, CHILLED or FROZEN-THAWED) suggest the negative impacts of sperm processing are not exacerbated by synchronisation method.
Results reported here are in agreeance with several previous studies regarding the negative impact of PGF2α oestrus synchronisation on fertility rates in the ewe (Boland, Gordon and
139 Kelleher 1978; Menchaca et al. 2004). Several reports have attributed the low fertility rates to a reduction in sperm numbers in the upper reproductive tract, with sperm numbers retrieved from the ovine cervix and oviducts after natural mating representing only a small fraction of those retrieved from untreated naturally cycling ewes (Hawk 1973; Hawk and Cooper 1977).
Low sperm numbers at the site of fertilisation could partially explain the low fertility results reported here. Successful migration of sperm through the female tract requires the ‘active’ movement of sperm and the ‘passive’ involvement of uterine contractions. Previous work in the ewe has suggested that decreased sperm transport through the female tract could partially be due to reductions of in vivo uterine contractility after synchronisation protocols. Work by
Hawk (1973) highlighted that controlled oestrus practices, specifically oestrus synchronisation using PGF2α, resulted in ewes having significantly fewer uterine contractions moving towards the oviducts of the ewe compared to NAT ewes.
The decreased fertility of ewes synchronised with PGF2α observed in the current trial may also be due to the significant reductions in cervicovaginal mucus volume observed in Merino ewes and a subsequent negative effect on sperm transport (Maddison et al. 2016). Along with mucus volume, the composition and consistency of reproductive tract mucus also impacts on sperm migration. Reports clearly highlight the significant negative impact that PGF2α synchronisation protocols can have on fertility in the ewe by altering the female tract and its mucoid secretions.
PGF2α synchronisation has been shown to alter the total protein content of cervical mucus, however reports are conflicting with decreases reported in cervical mucus of buffalo cows
(Prasad et al. 1981), whilst significant increases were described in cervical mucus of cows
(Yildiz and Aydin 2005). Several groups have indicated that altered steroidogenic function of follicles after PGF2α synchronisation are factors in reportedly lowered fertility rates. White et al. (1987) concluded that ovulatory follicles with lowered steroidogenic activity become
140 corpora lutea that secrete below normal levels of progesterone. More recent work in corriedale ewes reported that PGF2α use resulted in larger ovulatory follicles which grew faster but ultimately resulted in a lowered ovulation rate, culminating in lowered conception rates, prolificacy and fecundity (Fierro et al. 2011). The reported variation in the effect of PGF2α synchrony on pregnancy rates can be attributed to differences in the dose schedule and dose rate, number of doses (single or double), interval between doses (7,10 or 11 days apart) and start of treatment in relation to ovulation (3, 5, 7, 10 or 16 days after ovulation), with the end goal of causing luteolysis and cycle synchrony. Whilst hormonal levels and ovarian activity were not monitored in this trial, suboptimal ovarian activity resulting from PGF2α use is a possible contributor to the low pregnancy rates reported herein.
The use of progesterone for synchronisation is more commonplace in the Australian sheep industry than the use of prostaglandin synchronisation, which predominates in some parts of
Europe and the Americas. While largely advantageous, on farm usage can result in slightly lower pregnancy rates when compared to naturally cycling animals. Contrary to these on farm reports, in the current study, no significant differences in pregnancy rate were found between ewes inseminated at a progesterone synchronised oestrus and ewes inseminated at a naturally cycling oestrus. This confirms work by Donovan et al. (2004) which found comparable results between synchronised and naturally cycling crossbred ewes in Ireland. Studies which have reported negative impacts of P4 sync have attributed reduced fertility to; lower sperm numbers traversing the cervix and therefore migrating further (Allison and Robinson 1972; Hawk and
Cooper 1977) as well as altered mucus production. However, reports are contradictory with both increased (Rexroad and Barb 1977) and decreased mucus production reported following
P4 synchronisation (Smith and Allison 1971). In the current study, perhaps the time between ovulation and the onset of oestrus in NAT ewes was not as precise as the P4 ewes, but there is
141 no direct evidence for this. Whatever the case, the lack of evidence of a negative impact of progesterone synchronisation on fertility in the present study is encouraging as its use is commonplace in the Australian sheep industry.
An interaction between sperm type (FRESH, CHILLED or FROZEN-THAWED) and oestrus synchronisation method (NAT, P4, PGF2α) was not identified in the present study. Whilst the number of ewes included in the trial was determined to provide enough power to ensure statistical significance the lower than expected fertility rates of each treatment reduced statistical power. This likely also contributed to the unexpected finding that pregnancy rates following AI with frozen-thawed semen, while lower, were not significantly so when compared with fresh semen. Future experiments would be well served by an increase in the number of ewes within each treatment in addition to utilising animals on multiple properties to reduce the chances of low fertility to cervical AI that is occasionally reported at the property where this study was conducted (Ell-Hajj Ghaoui et al. 2007).
A clear effect of deposition site on fertility was identified by this study, with LAP AI resulting in significantly higher pregnancy rates when compared to cervical AI. This clearly demonstrates that the cervix, and very likely its secretions are key determinants of the reduced fertility associated with cervical AI. Furthermore, it highlights the role that cervical and cervicovaginal mucus, not mucus of the upper tract, have on successful fertility in the ewe.
In conclusion, results reported in the current study indicate that synchronisation with prostaglandin-F2α reduces pregnancy rates in the Merino ewe, whilst ewes synchronised with
P4 achieved comparable pregnancy rates to ewes inseminated at natural oestrus (inseminated
12 – 24 h after oestrus onset). However, these synchronisation methods to not exacerbate any
142 of the deleterious effects of semen cryopreservation on sperm-cervix interactions and fertility following cervical AI. Nonetheless, the use of prostaglandin-F2α in cervical AI programs of any sperm type is not recommended unless further research identifies the causative agents and methods of amelioration. Additional research into the factors that reduce fertility of some synchronised animals could benefit the development of more effective cervical AI in sheep.
5.6. Acknowledgements
Research was supported by the NSW Stud Merino Breeders Association Trust and Australian
Wool Innovation. The authors would like to thank Miss Taylor Pini, Miss Naomi Bernecic,
Mr. Max Lloyd, Mr. Cameron Negus, Miss Dannielle Glencourse, Mr. Cameron Sharpe and
Mr. Keith Tribe for on farm assistance. The authors would also like to thank Miss Evelyn Hall and Mr Peter Thomson for their statistical consultation. The authors thank Mr. Steve Burgun and the staff at ‘Arthursleigh’ for their help, donation of animals and provision of accommodation during the trial. The authors would also like to acknowledge Bioniche Animal
Health Australasia (now Vetoquinol) for the generous donation of Ovagest sponges, eCG
(Pregnocol) and FSH (Foltropin-V).
143 Chapter 6. General discussion and conclusions
The vast genetic, animal health and production gains made by animal industries through artificial insemination and embryo transfer would not have been possible without the development of oestrus synchronisation and superovulation. However, whilst facilitating controlled breeding programs, oestrus synchronisation and superovulation require the use of exogenous hormones which impact upon the mucus within the female reproductive tract. Such mucus is crucial for maintenance of tract health and vital for sperm transit so any changes in production or composition are likely to have direct impacts on fertility. Until recently, only limited information was available on the modifications that occur in mucus production, its properties and composition across the oestrous cycle, and after controlled breeding practices have been employed. Both increased (Croker and Shelton 1974; Rexroad and Barb 1977) and decreased mucus production (Smith and Allison 1971) have been reported following oestrus synchronisation. In addition to this, modern proteomic tools have only recently been utilised to investigate ovine reproductive tract mucus (Soleilhavoup et al. 2015), with the majority of previous studies on the mucus proteome carried out in humans (Dasari et al. 2007b; Grande et al. 2015). Whilst very early studies indicated oestrus synchronisation reduced the number of spermatozoa recovered from the reproductive tract of ewes (Quinlivan and Robinson 1967;
Hawk and Cooper 1977), the effect that such changes to mucus has on the motility, viability, and migration of spermatozoa has yet to be fully elucidated in the ewe. Based on the findings reported herein we have enhanced the understanding of the impact controlled breeding practices have on mucus of the reproductive tract in the ewe. Studies within this thesis have detailed the fluctuations in ovine cervicovaginal mucus production and composition that occur during the oestrous cycle in naturally cycling, synchronised and superovulated ewes. Results
144 clearly indicate that controlled breeding practices significantly influence mucus production, especially so for superovulated ewes. These ewes were shown to produce significantly higher volumes of mucus, and animals synchronised using prostaglandin-F2α were shown to produce dramatically lower volumes of mucus. Subsequent qualitative and quantitative proteomic analysis brought attention to the effects of superovulation and synchronisation of oestrus on the occurrence and abundance or proteins within mucus of the female reproductive tract.
Furthermore, fertility was confirmed to be significantly affected by prostaglandin-F2α synchronisation, compared to fertility of naturally cycling animals, as has been reported previously (Menchaca et al. 2004; Fierro et al. 2011).
Even with these accomplishments, some ambiguity remains as to the precise effect that progesterone synchronisation has on mucus production. Results obtained in this thesis unfortunately add to the conflicting reports of past studies, whereby progesterone synchronisation resulted in both increased and decreased mucus production (Smith and Allison
1971; Rexroad and Barb 1977). Studies within this thesis did not clearly show that controlled breeding practices affect mucus composition, with no evident trend of hormone level on the tested characteristics. In addition to this, the impact that exogenous hormones have on reproductive tract mucus and how this contributes to the reduced sperm transport previously reported in synchronised animals (Quinlivan and Robinson 1969; Boland, Gordon and Kelleher
1978; Menchaca et al. 2004) remains unclear. Perhaps a more significant focus needs to be placed on the variation that exists between ewes in regards to their response to synchronisation protocols. Animals are assumed to respond in a similar manner to various synchronisation protocols, and whilst a large majority of them will, a portion often do not. This complicates results and contributes to the lack of clear outcomes being obtained by research. Consideration also needs to be given to the suitability of tests used and the reproducibility of results. It may
145 be the case that some questions asked by researchers are unable to be entirely resolved given the confines of in vitro testing methods available at present.
Repeated sampling of mucus throughout oestrus, as was carried out in Chapter 2, clearly demonstrated the integral role that circulating concentrations of oestrogen have on mucus production over the cycle. This work confirmed the disparity in mucus production between the two phases of the oestrous cycle, namely that mucus production during oestrus is significantly more abundant than during the luteal phase, during which time oestrogen is at its lowest. This theory of oestrogen concentrations mirroring mucus output was supported by comparisons of mucus production in naturally cycling and superovulated ewes, with the latter producing almost double the volume of mucus during oestrus and the luteal phase. These results attest to the impact that exogenous hormonal inputs can have on endogenous hormone concentrations, and thus those processes usually governed by endogenous hormone concentrations, such as mucus production. Whilst a clear link between circulating concentrations of oestrogen and mucus production has been elucidated (Maddison et al. 2016), no clear evidence was found for similar changes to the composition and characteristics of mucus. Mucus ph was found to vary over only a small range of 6.2-6.5 and was not greatly affected by increased oestrogen during oestrus, despite work in humans that suggests oestrogen alkalises endocervical mucus of humans (Eggertkruse et al. 1993). Whilst not evident in our results, alkalisation of mucus at oestrus is theoretically sound as spermatozoa are susceptible to acidic environments.
Compositional changes to chemical profile also did not show a clear trend in relation to exogenous hormone use, although protein content did, with total protein peaking during the luteal phase and after superovulation during the follicular phase, when compared to naturally cycling animals.
146 Proteomic analysis of cervicovaginal mucus indicated the significant fluctuations in occurrence and abundance of proteins throughout a natural oestrous cycle and also detailed the impacts that controlled breeding can have on the mucus proteome. A total of 436 proteins were identified across the cycle, with 87 found to be more abundant during the oestrus phase, whereas 194 were more abundant during the luteal phase. This variation in abundance of proteins between the two phases of the oestrous cycle is suggestive of the varied roles that proteins might play during each of the phases. Specifically, that during oestrus, proteins within mucus should be receptive to a possible influx of spermatozoa through the tract whilst also preventing infection from any introduced pathogens during the fertile window of oestrus.
During the luteal phase however, it is more likely that proteins are involved in tract modification, preparation for possible implantation or beginning of a new cycle, and protection of the tract against invasion by pathogens.
Proteomic analysis in chapter 3 identified several proteins key to the structure of mucus, namely mucins, which were identified to vary greatly in abundance over the oestrous cycle, particularly mucin-5B and mucin-16 which were more abundant during oestrus than the luteal phase. Also of interest is the enzymatic protein NEU1, identified only in oestrus mucus by proteomic analysis. NEU1 cleaves the terminal sialic acids of mucins, ultimately causing changes to conformity in mucin fibres and thus its elastic and structural properties. Chapter 4 aimed to investigate these two important proteins further in an in vitro setting with results regarding mucin content of mucus being informative. They showed that increased mucin content led to increased viscosity, but this did not translate to reduced sperm motility in a dose dependant manner, as was expected (as reported in Chapter 4). Previous work in vitro in humans showed that variation in mucin levels has been shown to affect motility and kinematic parameters of human spermatozoa. It could be hypothesized that the level of mucin increase
147 utilised in chapter 3 was too small to elicit an effect on sperm motility. As described in chapter
3, the type and source of mucin (lyophilised versus fresh), and its preparation could have also impacted results reported. Regrettably though, no clear evidence was found in this thesis to support the theory that changes in abundance of the enzyme NEU1 alters mucus viscosity, and thus impacts the motility, viability and migration of spermatozoa. Unfortunately, due to logistical restraints, the effects of prostaglandin-F2α used for oestrus synchronisation on mucus proteomics was not investigated. It is likely though, given the extreme effect it had on mucus production, that proteomic variation in mucus would have also occurred after its use, as was the case for superovulated ewes and ewes synchronised using progesterone.
Chapter 4 aimed to investigate the effects that changes to mucus composition have on sperm migration effects in vitro, no clear relationship between changes to mucus composition or proteome as a result of oestrus synchronisation or superovulation, could be linked to reduced sperm transport. With results in Chapter 2 and Chapter 4 finding no consistently significant effect of oestrus synchronisation on in vitro sperm migration. This is surprising given that several previous studies have highlighted the reduced capability of spermatozoa to traverse the ovine female reproductive tract of synchronised ewes (Quinlivan and Robinson 1969; Allison and Robinson 1972; Hawk and Cooper 1977). Further to this, studies in cattle have also showed that mucus of synchronised animals caused decreased motility and forward movement of spermatozoa incubated within it. Clearly the main effectors in mucus of superovulated or synchronised animals that impact sperm migration in the female tract requires further clarification. While disappointing that no clear causal effects of oestrus synchronisation and reduced sperm migration through the tract were identified, chapter 2 and 3 did provide abundant information on mucus properties and the mucus proteome both over the oestrous cycle and after exogenous hormone use. In vitro testing methods are often the only solution
148 when, due to logistical, ethical and cost related issues, in vivo methods are non-viable. Whilst crucial to reveal many experimental queries, in vitro techniques often fail to fully replicate in vivo conditions, resulting in an experimental method conundrum. For instance, a multitude of sperm migration methods have been utilised by previous studies, but they often lack the ability to take into account the complex nature of the female reproductive tract. The reproductive tract is much more dynamic than the static vessels, such as tubes and glass capillary filled with mucus that are often employed for in vitro testing. The tract has muscular contractions, mucus is constantly produced and thus there is an outward flow through the tract, protein secretions are added along the way, which are all very difficult to reproduce in a laboratory setting. So, whilst current in vitro sperm migration methods can provide us with useful information, they may not always provide the entire story. This could, at least in part, explain why in chapter 2 there was no effect on migration of spermatozoa through mucus from ewes synchronised using
PGF2α, despite clear in vivo effects detailed in chapter 5 which suggest such a synchronisation method does have an effect.
As highlighted in chapter 2, synchronisation with prostaglandin-F2α significantly reduced mucus production in comparison to naturally cycling and progesterone synchronised ewes.
Reduced mucus production and quality is known to have contraceptive effects, as this is one of the modes of action of the contraceptive pill (Lewis et al. 2010; Chappell et al. 2014). This results in mucus with properties more akin to that produced during the luteal phase; scant and viscous (Igarashi 1954; Brunelli et al. 2007). Based on this it is likely that the low pregnancy rates reported in chapter 5, are at least somewhat related to reduced mucus production and quality. Work published as a result of this thesis (Chapter 2), confirms that reduced mucus volume as a result of prostaglandin-F2α used for synchronisation purposes could be responsible for the reduced fertility often reported after its use in the ewe (Boland, Gordon and Kelleher
149 1978; Menchaca et al. 2004). This theory has been further supported by subsequent work reported herein (chapter 5), which clearly demonstrates the negative impact synchronisation using prostaglandin-F2α can have on pregnancy rates. Its use resulted in pregnancy rates of only
6%, significantly lower than the 17% obtained by naturally cycling ewes and the 25% reached by ewes synchronised using progesterone.
The hypothesis that exogenous hormones used for oestrus synchronisation and superovulation significantly affect the production and composition of cervical mucus in the ewe was confirmed by the studies completed within this thesis. Sampling of cervicovaginal mucus throughout the oestrous cycle in naturally cycling, progesterone synchronised, superovulated, and prostaglandin-F2α synchronised Merino ewes was conducted and elucidated the changes in production and composition that occur naturally, and as a consequence of controlled breeding practices. The first detailed proteomic analysis of ovine cervicovaginal mucus collected throughout the oestrous cycle of naturally cycling, progesterone synchronised and superovulated ewes highlighted the natural fluctuations in occurrence and abundance of key protein groups that occur throughout the oestrous cycle. It also elucidated the effects that controlled breeding programs have on the mucus proteome, with large numbers of proteins having altered incidence and abundance, compared to the natural state. Whilst it is still not clear if and how exogenous hormones used for controlled breeding affect sperm migration through the tract, it is evident that changes in mucus caused by such breeding practices do alter mucus properties and ultimately impacts on sperm migration through mucus in the reproductive tract. The results reported herein, coupled with further studies could perhaps unravel the causative agents that often cause lowered fertility of cervically inseminated Merino ewes after controlled breeding practices have been employed. The inclusion of oestrus synchronisation methods into breeding programmes would benefit from consideration of the changes in timing
150 of onset of oestrus and volume of mucus produced. Future studies investigating additive agents to semen diluents utilised for cervical AI should take into consideration the challenges associated with the cervical and cervicovaginal mucus barrier.
151 References
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175 Appendix 1: Conference Proceedings
I. Maddison, JW, Rickard, JP, Bathgate, R, Druart, X & de Graaf, SP 2014, 'Changes to
ovine cervical mucus induced by the oestrous cycle, oestrus synchronisation and
superovulation', Proceedings of the 18th Annual Conference of the European Society for
Domestic Animal Reproduction (ESDAR), 11-13th September 2014. Helsinki, Finland.
Reproduction in Domestic Animals, vol. 49, no., pp. 80-80.
II. Maddison, JW, Rickard, J, Druart, X, Leahy, T & de Graaf, SP 2014, ‘The effect of oestrus
synchronisation and superovulation on the migration of spermatozoa through ovine cervical
mucus’. 9th Biannual Conference of the Association for Applied Animal Andrology
(AAAA), 8-10th August, 2014, Newcastle, Australia.
176 I Changes to ovine cervical mucus induced by the oestrous cycle, oestrus synchronisation and superovulation
Jessie W. Maddison1, Jessica P. Rickard1, Roslyn Bathgate1, Xavier Druart2, Simon P. de Graaf1 1Faculty of Veterinary Science, University of Sydney NSW 2006, Australia 2Institut National de la Recherché Agronomique (INRA), 37380 Nouzilly, France
The variation in the properties of cervical mucus over the course of the oestrous cycle or as a result of exogenous hormone use for oestrus synchronisation and superovulation are poorly described in the ewe. As such, this study aimed to determine the changes in volume, pH and viscosity of cervical mucus throughout the oestrous cycle and after administration of exogenous hormones. Mucus was collected every 6h for 4d (follicular phase), then daily for 12d (luteal phase) from naturally cycling (NAT), progesterone synchronised (P4), superovulated (SOV) and prostaglandin synchronised (PGF2a) merino ewes (n = 5/group). A greater (p <0.05) volume of mucus was produced in the follicular phase when compared to the luteal phase of NAT, P4 and SOV ewes. In the follicular phase, SOV animals produced more mucus (p <0.05) than all other treatments, NAT and P4 ewes produced similar volumes (p >0.05), while PGF2α ewes produced significantly less mucus than P4, but not NAT ewes. Luteal phase mucus was found to be more alkaline than follicular phase mucus, while mucus from SOV ewes was more likely to be alkaline than mucus from NAT or P4 ewes. Luteal phase mucus was found to be more viscous than that from the follicular phase (p <0.05). Results indicate that the properties of mucus vary across the oestrous cycle and as a result of controlled breeding. Further research is required into the possible implications of these effects on sperm transport following cervical AI.
177 II The effect of oestrus synchronisation and superovulation on the migration of spermatozoa through ovine cervical mucus
Jessie W. Maddison1, Jessica P. Rickard1, Roslyn Bathgate1, Xavier Druart2, Simon P. de Graaf1 1Faculty of Veterinary Science, University of Sydney NSW 2006, Australia 2Institut National de la Recherché Agronomique (INRA), 37380 Nouzilly, France
Exogenous hormones used for controlled breeding purposes are commonplace in the Australian sheep industry. However, their use has been suggested to negatively impact successful fertility. Research and on farm reports have indicated exogenous hormone use can result in increased cervicovaginal mucus production, lowered sperm concentrations in the female tract and reduced fertility rates. Little research has focused on the possible effects of controlled breeding on sperm penetrability in mucus. As such, this study aimed to investigate what effects exogenous hormones have on the ability of cryopreserved spermatozoa to penetrate ovine cervicovaginal mucus through the use of the cervical mucus penetration test. Mucus was collected from naturally cycling (Nat), progesterone synchronised (P4) and superovulated (SOV) mature merino ewes (n = 22/treatment) via aspiration from the cervicovaginal region twice during oestrous; determined by circulating progesterone and oestrogen levels and allowing testosterone treated wethers to mount. Mucus samples were aspirated into flat glass capillary tubes, incubated with frozen merino spermatozoa (20 x 106 motile sperm/ml) for 1 hour, with the concentration of sperm assessed at 1cm. Whilst results show that sperm travelled further in the mucus of SOV ewes compared to Nat and P4, differences were not significant (p >0.05). Significant variation within and between samples was observed. Previous research in cattle and humans has indicated the penetrability of mucus to vary greatly, suggesting variation in mucus properties as a possible cause for this. Further investigation into the proteomic and structural properties of mucus across the oestrous cycle and after exogenous hormone use is needed as it may explain the negative impacts on fertility reported after use of exogenous hormone use.
178 Appendix 2: Supplementary Files
Chapter 3: Oestrus synchronisation and superovulation alter the cervicovaginal mucus proteome of the ewe
Supplementary data for this chapter can be found online at http://dx.doi.org/10.1016/j.jprot.2017.01.007.
Supplementary Table 1: Proteins identified by qualitative whole oestrous cycle analysis
(Located in Table S-1).
Supplementary Table 2: Proteins identified by quantitative comparison of oestrus and mid- luteal phase mucus (Located in Table S-2).
Supplementary Table 3: Proteins identified by quantitative comparison of oestrus and mid- luteal phase mucus with limits (SPC ≥ 5, fold change ≥ 1.5) applied (Located in Table S-3).
Supplementary Table 4: Proteins identified by quantitative comparison of mucus from naturally cycling (NAT), progesterone synchronised (P4), and progesterone synchronised and superovulated (SOV) ewes (Located in Table S-3).
Supplementary Table 5: Proteins identified by quantitative comparison of mucus from naturally cycling (NAT), progesterone synchronised (P4), and progesterone synchronised and superovulated (SOV) ewes with limits (SPC ≥ 5, fold change ≥ 1.5 in comparison to NAT) applied (Located in Table S-5).
179 Supplementary Table 6: Proteins in mucus most altered by progesterone synchronisation (P4), and progesterone synchronised and superovulation (SOV), in comparison to levels found in mucus of naturally cycling (NAT) ewes (Located in Table S-6).
180