UN -¿, o I la The Marsupial ZonaPellucida: Its Structure and Glycoconjugate Content Jamie Chapmar BSc (Hons) Department of Anatomical Sciences University of Adelaide Juty 2003 A thesis submitted as fulfïlment of the requirements for the Degree of Doctor of Philosophy Abstract The zona pellucida (ZP) is an extracellular matrix that surrounds the oocytes of all higher vertebrates. Whilst there is considerable data on the ZP of eutherian mammals, the structure and function(s) of theZP of marsupials is poorly known. In this thesis the structure and glycoconjugate composition of the ZP surrounding marsupial oocytes and the changes that occur during ovarian development, following ovulation, and following cortical granule exocytosis was investigated. In addition, the glycoconjugates of the oviduct epithelial lining of the brushtail possum around the time of ovulation were also examined in order to determine if there rwas any contribution of the oviductal secretions to the post-ovulatory ZP. The ultrastructural organisation of ZP and its development during oocyte maturation in five marsupial species was found to differ in the relative thickness around mature oocytes. The thicknesses of the ZP of the marsupials ranged from 3.9¡rm in the fat-tailed dunnart, 4.6¡.lm in the western grey kangaroo, 5.2¡rm in the brushtail possum, 6.6¡rm in the wombat and 8.6¡rm in the koala. The glycoconjugates of the ZP surrounding antral follicular oocytes of seven marsupial species, were determined by differential lectin histochemistry, before and after either removal of sialic acids with neuraminidase, or O-acetyl groups on sialic acids by mild alkali hydrolysis. Interspecific variation in the glycoconjugate composition of the ZP was found, especially that of the mannose-containing glycoconjugates. The presence of terminal sialic acids was indicated since increases in binding of lectins to galactose- and N-acetylgalactosamine- (GalNAc-)-containing glycoconjugates occurred after desialylation. Removal of O-acetyl groups also resulted in the demonstration of the presence of N-acetylglucosamine (GlcNAc) in the ZP of fat-tailed dunnarts, brushtail possums and ringtail possums, and mannose in the ZP of fat-tailed dunnarts, brushtail possums, grey, short-tailed opossums and eastern grey kangaroos. Furthermore, localisation of N-acetylglucosamine residues in all species changed following de-O-acetylation which suggests that a higher proportion of O-acetylated sialic acids occur within the core of the ZP than near the inner and outer surfaces. Ultrastructural changes to the ZP were found to occur following ovulation, with the ZP of ovarian oocytes appearing dense and disorganised, whilst that surrounding ovulated oocytes was highly filamentous and had an ordered structure. A greater abundance of four glycoconjugates (mannose, galactose, N-acetylglucosamine and N-acetylgalactosamine) occurred in the ZP of ovulated, compared to ovarian, oocytes. Artificial induction of cortical granule exocytosis of possum oocytes was carried out to investigate the role of the egg coats following egg activation. Artificial activation showed that N-acetylgalactosamine residues decreased significantly within the ZP, whilst after staining with ruthenium red, the perivitelline space matrix (PVM) appeared to be largely oviductal in origin, and not a "cortical granule envelope" as previously claimed. To determine whether the increase in glycoconjugates of the ZP following ovulation occurs as a result of the incorporation of oviductal secretions into its matrix, the glycoconjugate composition of the oviductal epithelium was also investigated. The epithelium of the oviduct was shown by lectin histochemistry to have an abundance of B-galactose and N- acetylglucosamine residues, with N-acetylgalactosamine being localised to the secretory cells of the ampulla, and absent in the isthmus; a finding that was also supported by lectin immunocytochemistry. Futhermore, lectin immunocytochemistry showed greater N- acetylgalactosamine and mannose residues in the ampulla than in the isthmus, whereas, by contrast, N-acetylglucosamine residues were in higher concentration within the isthmus. Gold labelling of p-galactose residues was equally distributed within the secretory granules between the two regions. Acknowledgments Firstly, I would like to thank my supervisor Bill Breed for many years of patience and persistence. Thankyou for all the encouragement, the constructive criticisms and experience that you have shared. To my other supervisor Ole V/iebkin. Thankyou for helping me see the story of the zona pellucida from another perspective- the carbohydrate's point of view. To Chris Leigh. Your friendship, patience and bottomless depth of practical knowledge have been invaluable to me over the years. To everyone else who played a role in the practical component of my thesis: Nadia Gagliardi and Gail Hermanis for their help and patience in all the facets of light microscopy; Marilyn Henderson and Lyn Waterhouse for help with the cryo-electron microscopy techniques; Craig Noble for his help with analySiS; the rest of the staff at CEMMSA (now Adelaide Microscopy) for their help and friendship over the years; Tavik Morgenstern for his help in the printing of the plates. Thanks to Mario Ricci for his friendship over the years of our PhDs: I'm very fortunate to have gone through both Honours and a PhD with such a great mate.; Thanks also to all of the other members (past and present) of the Reproductive Biology group for your friendship and constructive criticism. To my family, Kerry, Roger and Stacy Chapman. Thankyou for your love and patience while I went off and did whatever it was I did at Uni. And lastly, to my beautiful wife Michelle. Your love, commitment, patience and understanding have been amazing and I couldn't have finished without you. You kept me sane when I thought my head would explode and you helped me more than you will ever know. Thankyou so much for everything, I love you very much. Research arising from this thesis has been published and/or presented at conferences as follows, examples of which can be found at the rear of this thesis: Publications: Breed, W. G., Hope, R. M.,'Wiebkin, O. W., Spargo, S. C. and Chapman, J. A. (2002).The marsupial zona pellucida: its structural organízation and evolution. Reproduction l23z 13-21. Chapman, J. 4., Wiebkin, O. W. and Breed, W. G. (2000). Interspecific variation of zona pellucida glycoconjugates in several species of marsupial. Journal of Reproduction and Fertility 11.9: I ll-120. Chapman, J. A. and Breed, Vy'. G. (in press). The egg coats of the brushtail possum (Trichosurus vulpeculø) following ovulation and artificial activation with particular reference to the origin of the Perivitelline Space Matrix. In 'The Biology of Australian Possums and Gliders' (Eds. Goldingay, R. L. & Jackson, S.M.) Surrey Beatty & Sons: Chipping Norton. C o nfere nc e P re s e ntations : Chapman, J. A. and Breed, W. G. (2003). The oviduct of the brushtail possum, Trichosurus vulpecula: the glycoconjugate environment at ovulation and its probable contribution to the post-ovulatory zona pellucida. 21" New Zealand Conference on Microscopy, Wellington, February 7-12. 'W., Chapman, J. 4., Wiebkin, O. and Breed, W. G. (2002). Structure of the zona pellucida of a marsupial, the brushtail possum Trichosurus vulpeculø, following ovulation and egg activation. The l7h Australian Conference for Electron Microscopy (ACEM17), Adelaide Convention Centre, South Australia, February 4-8. Chapman, J. 4., and Breed, W. G. (2001). What prevents polyspermy in a marsupial egg? A study in the brushtail possum. The 47h Scientific Meeting and Possum and Glider Symposium of the Australian Mammal Society, Queensland Museum, Brisbane, July 2-6. Hope, R. M., Chapman, J. 4., Wiebkin, O. V/. and Breed, W. G. (2000). How similar is the molecular structural organization of the marsupial zona pellucida to that of ettherians? The Third International Symposium on the Molecular and Cellular Biology of the Egg and Embryo Extracellular Matrix, Grandlibakken Resort and Conference Center, Lake Tahoe, Caliþrnia, August 27-September l. 'Wiebkin, Chapman, J. 4., O. W., and Breed, W. G. (2000). Structure of the zona pellucida of a model marsupial, the brushtail possum (Trichosurus vulpecula: Famlly, Phalangeridae). Fertility 2000: EICC Edinburgh, 2nd Joint Meeting of BAS|BFSISSF, July 3l-August 2. 'W. Chapman, J. 4., Wiebkin, O. W., and Breed, G. (1999). Interspecific variation in glycoconjugates of the zonae pellucidae of several marsupial species. 31" Annual Conference of the Australian Society for Reproductive Biology, Melbourne, September 26-29. Contents Chapter 1 Literature Review ........ ............1 1.1 Introduction .... ........ 1 1.2 Evolution of the Oocyte Vestments ............2 1.3 The Metatherian and EutherianZonaPellucida (ZP) ............ ............9 1.3.1 Functions of the ZP ............ ............9 1.3.2 Morphology of the ZP ........ ..........12 1.3.3 Glycoproteins of theZP ..........15 1.3.4 Carbohydrate composition of the ZP ... ..........21 1.3.5 Development of the ZP ........... 24 1.4 The Oviduct ....... 27 1.4.1 Overview 27 1.4.2 General morphology of the Oviduct 28 1.4.3 Functional Histology of the Oviduct 29 1.4.4 Biosynthetic activity - Oviductal-specific proteins ....... 32 1.4.5 Functions of Oviductal-Specific Glycoproteins (OGPs) 39 1.5 Summary and Project Proposal ...... 42 Chapter 2 Research Project 41 2.1 Animals . 47 2.1.1 Brushtail possums (Trichosurus vulpecula) .. 47 2. | .2 F at-tailed dunnart s (S mintho p s i s cr as s i c audat a) 48 2.1 .3 Koalas (Phas c olar cto s c iner e us) ....... 48 2. 1 .4 S outhern hairy-nosed w omb ats (I'a s i o rhinu s I atifr o n s) .49 2.1.5 Eastern Grey Kangaroos (Macropus giganteus) 49 2.1.6 Western Grey Kangaroos (Macropus fuliginosus fuliginosus) 50 2. 1 .7 Ringtail Possum s (P s eudo cheirus p er e grinus) 50 2.1.8 Southern Brown Bandicoot (Isoodon obesulus) 50 2.1.9 Grey Short-tailed Opossum (Monodelphis domestica) ........'.'. 51 2.2 Superovulation by priming with Gonadotrophins .