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Timing of zona pellucida formation in the tammar wallaby (Macropus eugenii) and brushtail possum (Trichosurus vulpecula)
Animal Reproduction Science 53 Ž1998. 237–252
Timing of zona pellucida formation in the tammar wallaby žMacropus eugenii / and brushtail possum žTrichosurus Õulpecula / K.E. Mate
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CooperatiÕe Research Centre for ConserÕation and Management of Marsupials, School of Biological Sciences, Macquarie UniÕersity, New South Wales 2109, Australia
Abstract The zona pellucida ŽZP. is an extracellular coat that surrounds the mammalian egg, and serves as the primary recognition site for fertilizing spermatozoa. The timetable of ZP formation was examined in two marsupials, the tammar wallaby Ž Macropus eugenii . and the brushtail possum ŽTrichosurus Õulpecula. using conventional histological methods, immunofluorescence and electron microscopy. Ovaries from tammar wallaby pouch young less than 80 days of age contained only primordial follicles with a single layer of flattened granulosa cells. There was no evidence of ZP formation until 98 days, when a small number of eggs surrounded by a single layer of cuboidal granulosa cells had a ZP detectable by periodic-acid–schiff staining and rabbit anti-pig ZP polyclonal antibody labelling. Possum ovaries at 108 and 114 days also contained a small number of eggs with a ZP and a single layer of cuboidal granulosa cells. The antibody also labelled the peripheral cytoplasm of oocytes at this stage and, occasionally, the granulosa cells. Antral follicles were first detected at 144 days in the wallaby and 125 days in the possum, and always contained an egg surrounded by a ZP. Ovaries from 147, 158, 165, 181, 184 and 210-day-old tammar wallabies contained a range of follicle types from primordial through early antrum formation. Electron microscopy confirmed observations made at the light microscope level. The ZP was first detectable in small primary follicles with a single layer of cuboidal granulosa cells in areas where microvilli had begun to form on the egg plasma membrane. Immunogold labelling indicated the egg cytoplasm as the origin of the ZP proteins. The ZP completely filled the space between the egg and the adjacent granulosa cells in preantral follicles, so that there was no perivitelline space. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Egg; Follicle; Zona pellucida
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0378-4320r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 Ž 9 8 . 0 0 1 1 6 - X
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1. Introduction The zona pellucida ŽZP. is an extracellular coat that surrounds the mammalian egg, and serves as the primary recognition site for fertilizing spermatozoa. Depending on the species, it is composed of three to four glycoproteins, that have been designated ZP1, ZP2 and ZP3 ŽRinguette et al., 1988; Chamberlin and Dean, 1990; Liang et al., 1990; Harris et al., 1994.. Each of the ZP proteins is modified after translation and secreted from the egg as a glycoprotein that interacts with the other ZP proteins to form the insoluble extracellular zona matrix. The marsupial egg is also surrounded by a ZP made up of at least three glycoproteins, but their functional or structural homology to eutherian ZP proteins has not been examined. Expression of the genes encoding the ZP glycoproteins is restricted to the ovary in eutherian mammals Žreviewed by Dunbar et al., 1994., but occurs in the liver in some species of teleost fish ŽLyons et al., 1993; Murata et al., 1995.. There is some controversy concerning whether synthesis of the ZP proteins within the ovary of eutherian mammals is restricted to the oocyte or whether it also occurs in the granulosa cells. Several studies have indicated that expression of the ZP3 gene is restricted to growing oocytes in the mouse ŽPhilpott et al., 1987; Roller et al., 1989; Epifano et al., 1995.: nongrowing oocytes Ž12–15 mm in diameter. do not contain detectable levels of ZP3, and maximal expression of ZP3 occurs in growing oocytes Ž30–80 mm in diameter. and decreases rapidly by the time of ovulation when up to 98% of ZP3 mRNA is destroyed. The synthesis and secretion of the ZP3 protein follows a similar pattern to gene expression in the mouse ŽBleil and Wassarman, 1980; Salzmann et al., 1983.. Oocyte-specific expression of ZP3 has also been reported in the hamster ŽLeveille et al., 1987. and marmoset ŽThillai-Koothan et al., 1993.. However, studies in other species have indicated that the assembly of the ZP may involve both the oocyte and granulosa cells depending upon the stage of follicular growth and maturation ŽWolgemuth et al., 1984; Lee and Dunbar, 1993.. The granulosa cells surrounding growing oocytes within primary and secondary follicles in the rabbit, mouse, rat, guinea pig, hamster and cat actively synthesise ZP proteins Žreviewed by Dunbar et al., 1991.. The synthesis of ZP proteins by granulosa cells appears strictly limited to certain stages of follicle development, which may explain some of the inconsistencies in the literature. In the rabbit, for example, ZP protein was present in the cytoplasm of the inner layer of granulosa cells in secondary follicles consisting of two to three layers of granulosa cells, but not in the granulosa cells of earlier or later stage follicles ŽWolgemuth et al., 1984.. The timing of ZP formation in marsupials has not been examined in detail, but has been reported to occur during the transition of follicles with a single layer of flattened granulosa cells Žtype 3a. to follicles with a single layer of cuboidal granulosa cells Žtype 3b. in the tammar wallaby ŽAlcorn, 1975.. The same study indicated that most follicles had progressed to at least the type 3b stage of development by 270 days after birth, suggesting that in contrast to eutherian species, the bulk of ZP formation may occur before puberty. A study of oogenesis in the brushtail possum also indicated that the ZP first appeared in follicles with a single layer of cuboidal granulosa cells ŽFrankenberg et al., 1996.. Functional studies of fertilization in marsupials have been severely impeded by the
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inability to achieve fertilization or meaningful sperm–egg interaction in vitro in Australian marsupials. It has been suggested that the marsupial ZP may undergo maturational changes necessary for fertilization based on morphological ŽMate, 1996. and functional studies ŽMoore and Taggart, 1993.. An increased understanding of the structure and function of the marsupial ZP is necessary to overcome the difficulties associated with in vitro fertilization in Australian marsupials. This study has examined the timetable of ZP formation in two marsupials, the tammar wallaby Ž Macropus eugenii . and the brushtail possum ŽTrichosurus Õulpecula. using conventional histological methods, immunofluorescence and electron microscopy.
2. Materials and methods 2.1. Animals Tammar wallabies Ž M. eugenii . were housed at the Central Animal House at the University of Newcastle or at the Fauna Park at Macquarie University as described previously ŽMate et al., 1997.. Brushtail possums ŽT. Õulpecula. were housed at the Central Animal House at University of Newcastle or the animal facility at Manaaki Whenua Landcare Research in Lincoln, New Zealand. 2.2. Egg collection Two tammar wallabies were treated with 6 mg of porcine follicle-stimulating hormone ŽFSH. ŽFollitropin V; Vetrapharm, Australia. twice daily for four days followed by 4 mg of porcine luteinising hormone ŽLH. ŽLutropin V; Vetrapharm, Australia. ŽMolinia et al., 1998.. Animals were sacrificed by an i.v. injection of 2.5 ml of 350 mgrml sodium pentobarbitone ŽLethobarb; Virbac, NSW. 24 h after the LH injection. Ovaries were removed, rinsed in heparinised phosphate-buffered saline ŽPBS. and eggs Ž n s 16 per category. were collected from primary preantral and antral follicles. Primary follicles were dissected from the ovary using fine needles and examined whole, as it was not possible to remove the tightly adherent granulosa cells. Eggs were recovered from preantral and antral follicles by pricking with a 30-gauge needle attached to a 2.5-ml syringe and flushing the follicle with PBS until the egg was recovered. Granulosa cells were removed from the egg by passing it several times through a pipette with a diameter slightly smaller than that of the egg. 2.3. Pouch young oÕary collection Ovaries from tammar wallaby pouch young that were 59, 74, 98, 104, 144, 147, 158, 165, 181, 184 and 210 days old were collected from the colonies at Macquarie University and the University of Newcastle. Ovaries from brushtail possum pouch young that were 108, 115, 125 and 135 days old were provided by Dr. J. Duckworth from Landcare Research. The age of the pouch young was determined by measuring the
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length of the head from the tip of the snout to the occiput and using age determination charts for the tammar wallaby ŽPoole et al., 1991. and the brushtail possum ŽLyne and Verhagen, 1957.. Adult ovaries from both species were similarly examined. Pouch young were sacrificed by i.p. injection of 350 mgrml sodium pentobarbitone ŽLethobarb; Virbac. and their ovaries were removed. One ovary from each wallaby was divided in two pieces; one piece was processed and embedded in paraffin wax for light microscopy and the other was processed for electron microscopy and immunogold labelling. Paraffin sections were stained with haematoxylin and eosin ŽHE. for routine examination, or period-acid-schiff ŽPAS. or alcian blue which specifically react with glycoproteins, or were labelled with rabbit anti-pig ZP polyclonal antibody Žprovided by Dr. M. Bradley. which cross-reacts with possum and wallaby ZP proteins on Western blot analysis ŽK. Mate, unpublished observations. and paraffin sections Žthis study.. Possum ovaries were examined by light microscopy only. The remaining ovary from each animal was frozen at y808C for use in another study. 2.4. PAS and alcian blue staining Ovaries were fixed in either 4% paraformaldehyde in PBS or Histochoice ŽAustral Scientific, Australia., embedded in paraffin wax and sectioned at a thickness of 5 mm. For PAS staining, the tissue sections were deparaffinized by xylene and rehydrated with 70% ethanol before treatment with periodic acid ŽSigma, St. Louis, MO, USA. for 5 min. Sections were treated with Schiff reagent for 10 min, rinsed in three 30-min changes of sodium bisulfite ŽSigma., dehydrated, and coverslips were attached with DPX mountant ŽBolton, Essex, UK.. Slides for alcian blue staining were rehydrated with water and rinsed in 3% acetic acid. The slides were then stained for 30 min in 1% alcian blue ŽSigma. in 3% acetic acid, washed with water, dehydrated and mounted with DPX. 2.5. Anti-porcine ZP antibody labelling Ovaries were collected and fixed immediately in either 4% paraformaldehyde in PBS or Histochoice. The ovaries were embedded in paraffin wax and sectioned at a thickness of 5 mm. In preparation for antibody labelling, tissue sections were deparaffinized with xylene and rehydrated with ethanol and PBS and then covered in a blocking solution that consisted of PBS containing 2% BSA, 50 mM glycine and 0.02% sodium azide for 1 h. Excess blocking solution was blotted off and the sections were incubated with rabbit anti-pig ZP polyclonal antibody Žprovided by Dr. M. Bradley, Perth Zoo. diluted 1r50 in PBS for 1 h at room temperature ŽRT.. Sections were washed three times in PBS and bound antibody was detected with FITC-conjugated anti-rabbit immunoglobulin diluted 1r100 in PBS ŽAmersham, Australia. for 15 min at RT. After washing three times in PBS, the sections were mounted in Mowiol ŽCalbiochem. containing the anti-fading agent 1,4-diazabicyclo-w2.2.2x-octane ŽDABCO. ŽSigma. and examined by fluorescence microscopy.
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2.6. Electron microscopy Ovaries were fixed immediately in Karnovskys fixative Ž1% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer. overnight at 48C, washed in 0.1 M cacodylate and post-fixed in 1% osmium tetroxide. The ovarian tissue was then dehydrated through an acetone series and embedded in Spurrs resin. Ovarian tissue for immunogold labelling was fixed in Karnovskys fixative as described above, rinsed in 0.1 M cacodylate, dehydrated through an ethanol series and embedded in LR white. 2.7. Immunogold labelling The labelling procedure was carried out in 50-ml drops of solution on a sheet of thermo-plastic film ŽParafilm, American National Can, USA.. Grids were floated on 50 mM glycine in PBS for 15 min and then transferred to incubation buffer which consisted of PBS containing 3% BSA for 1 h at RT. Grids were transferred to rabbit anti-pig ZP polyclonal antibody diluted 1r400 with incubation buffer for 2 h at RT. Grids were washed six times for 5 min each in 50 ml of incubation buffer and transferred to gold-conjugated anti-rabbit antibody ŽICN Biomedicals, Costa Mesa, CA, USA. diluted 1r30 in incubation buffer. Grids were washed six times for 5 min in incubation buffer, washed twice in PBS for 5 min and post-fixed in 2% glutaraldehyde in PBS for 15 min. After fixation, grids were rinsed twice for 5 min in Milli Q water and air-dried.
3. Results 3.1. Adult oÕaries Adult ovaries from the wallaby and possum contained eggs at all stages of development. Eggs from primary follicles in the tammar wallaby were surrounded by a single tightly adherent layer of granulosa cells but it was not possible to detect if a ZP was present in fresh material ŽFig. 1a.. In preantral follicles, the ZP was visible in all eggs examined, but was thin Žmaximum of 4.4 mm. and not evenly distributed around the egg. The ZP was not visible around the entire circumference of all eggs from preantral follicles ŽFig. 1b.. All of the eggs from antral follicles possessed a ZP that was evenly distributed around the circumference of the egg, but varied in thickness between eggs from 4–8.5 mm ŽFig. 1c; Table 1.. Eggs from large antral follicles Ž) 1.5 mm in diameter. generally possessed a thicker ZP than eggs from smaller antral follicles ŽTable 1., but this was not statistically significant. 3.2. Pouch young oÕaries Ovaries from tammar wallaby pouch young 59 and 74 days of age contained only primordial follicles surrounded by a single layer of flattened granulosa cells ŽFig. 2a.; there was weak PAS staining associated with some follicles in 74-day-old pouch young,
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Fig. 1. Eggs from the tammar wallaby Ž M. eugenii . showing progressive thickening of the zona pellucida Žarrows.. Ža. Primary follicle. Žb. Egg from a preantral follicle. Žc. Egg from a 2-mm diameter antral follicle. Barss 25 mm.
but there was no discernible ZP ŽFig. 2b.. By 98 days of age, a small number of primary follicles, consisting of a single layer of cuboidal granulosa cells and a growing oocyte, were present in the inner cortical region of the ovary ŽFig. 2c.. The ZP was detectable in primary follicles as a distinct band of PAS-positive material ŽFig. 2d. and the cytoplasm
Table 1 Width of the zona pellucida during egg development in the adult tammar wallaby Ž M. eugenii . Follicle Type
Egg Diameter Žmm.
Zona Width Žmm.
Primary Preantral Small antral Ž -1.5 mm. Large antral Ž )1.5 mm.
; 50 124.7"3.3 159.6"5.0 175.3"2.3
– 0–4.4 6.5"1.3 7.9"0.4
ns16 eggs per category.
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Fig. 2. Ovarian sections from tammar wallaby Ž M. eugenii . pouch young. Ža. 74-day-old pouch young ovary, stained with haematoxylin and eosin ŽHE. that contains a large number of primordial follicles. Žb. 74-day-old pouch young ovary stained with periodic-acid–schiff ŽPAS.. Several follicles are weakly stained Žarrows., but there is no zona pellucida detectable. Žc. 98-day-old pouch young ovary stained with HE, showing development of primary follicles Žarrows. in the inner ovarian cortex. Žd. 98-day-old pouch young ovary stained with PAS. The zona pellucida Žarrows. is stained in several growing follicles that are present in the inner ovarian cortex. Že. 144-day-old pouch young ovary stained with HE, that contains several growing follicles. Žf. 144-day-old pouch young ovary stained with PAS. The zona pellucida and cytoplasm of two eggs in the same growing follicle Žarrow. are intensely stained. Bars s 50 mm.
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of the egg was also lightly stained in most primary follicles. In some primary follicles, the granulosa cells were also stained by PAS ŽFig. 2d.. Slightly later stage follicles with two to three layers of granulosa cells were present by 104 days in the tammar wallaby. Again, in addition to the ZP and egg cytoplasm, there was also PAS-positive material present in the granulosa cells of some follicles. At this stage, the ZP was also detectable by alcian blue staining Žnot shown.. Follicles around the stage of antrum formation were first detected at 144 days in the tammar wallaby. At this age, the ovary also contained numerous large preantral follicles with growing oocytes surrounded by several layers of granulosa cells ŽFig. 2e.. The eggs in large preantral follicles were intensely stained with PAS, as was the ZP surrounding the egg ŽFig. 2f.. The granulosa cells of large preantral follicles were rarely stained with PAS. Ovaries from 147, 158, 165, 181, 184 and 210-day-old tammar wallabies were similar in appearance; they contained a range of follicle types from primordial through
Fig. 3. Ovarian sections from brushtail possum ŽT. Õulpecula. pouch young. Ža. 108-day-old pouch young ovary stained with haematoxylin and eosin ŽHE.. Primary follicles are present near the boundary of the ovarian cortex and medulla Žarrows.. Žb. 108-day-old pouch young ovary stained with periodic-acid–schiff ŽPAS.. The egg cytoplasm and zona pellucida is darkly stained in growing primary follicles Žarrows.. Žc. 125-day-old pouch young ovary stained with HE, with several growing follicles Žarrows.. Žd. 125-day-old pouch young ovary stained with PAS. The egg cytoplasm and zona pellucida is darkly stained in growing follicles Žarrows.. Barss 50 mm.
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Fig. 4. Phase contrast Ža,c,e. and fluorescent micrographs Žb,d,f. of tammar wallaby Ž M. eugenii . and brushtail possum ŽT. Õulpecula. ovaries labelled with rabbit anti-pig zona pellucida polyclonal antibody. Although there was variation in the intensity of labelling between different sections in both the wallaby and possum, there was no consistent difference in the intensity of labelling between the two species. Ža,b. Adult tammar wallaby ovary. Žc,d. Adult brushtail possum ovary. Že,f. 144-day-old tammar wallaby ovary. Barss 50 mm.
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to early antrum formation. Each of the follicle types had a characteristic staining pattern: primordial follicles were not PAS stained; the ZP, egg cytoplasm and, occasionally, granulosa cells were PAS-stained in follicles with one to two layers of granulosa cells; the ZP and egg cytoplasm only were stained in large antral follicles with three or more layers of granulosa cells. The 181, 184 and 210-day-old tammar wallabies, generally, had a greater number of medium and large preantral follicles than younger animals. Although antral follicles were present in wallabies aged 144 days and older, they were present only in limited numbers and the follicle did not grow significantly after antrum formation. A similar pattern of ZP formation was observed in the brushtail possum. The youngest brushtail possum examined was 108 days of age ŽFig. 3a.. At this stage of development, there were numerous primary follicles near the boundary of the ovarian cortex and medulla, consisting of a growing oocyte surrounded by PAS-stained ZP and a single layer of cuboidal granulosa cells. The oocyte cytoplasm was also stained by PAS in primary follicles ŽFig. 3b.. The ovaries from a 115-day-old possum were at a similar
Fig. 5. Electron micrographs of tammar wallaby Ž M. eugenii . eggs during the process of zona pellucida deposition. Ža. Egg from a primordial follicle which has no microvilli on the plasma membrane and no zona pellucida. Bar s 3 mm. Žb. Egg from a primary follicle at an early stage of zona pellucida formation; a small amount of zona material is visible in areas where microvilli are present on the egg plasma membrane. Bar s1 mm. Žc. Egg from a primary follicle with a thin and incomplete zona pellucida. Bar s1 mm. Žd. Egg from a primary follicle showing deposition of zona pellucida material between granulosa cells. Bar s 0.5 mm. Egg cytoplasm Žegg.; egg plasma membrane Žarrow.; zona pellucida Žzp.; granulosa cell Žgc..
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stage of development. The older possums examined, 125 and 135 days old, did not contain any antral follicles ŽFig. 3c., but did contain numerous large preantral follicles with PAS-stained oocytes and ZP ŽFig. 3d.. The several layers of granulosa cells in the large preantral follicles were not stained by PAS. 3.3. Anti-porcine ZP antibody labelling The pattern of ZP formation in the tammar wallaby and brushtail possum detected by antibody labelling was consistent with that described above using PAS staining. The ZP and peripheral egg cytoplasm were labelled in large preantral follicles with three or more layers of granulosa cells and in antral follicles in adult tammar wallaby ŽFig. 4a,b. and brushtail possum ŽFig. 4c,d. ovaries. The antibody did not bind to primordial follicles in the adult ovary, confirming the absence of a ZP at this stage. The ZP was first detectable by antibody labelling at 98 days of age in the wallaby and 108 days in the possum, in follicles with one to two layers of granulosa cells ŽFig. 4e,f.. Granulosa cells were also labelled occasionally in follicles with one to two layers of granulosa cells. 3.4. Electron microscopy Electron microscopy confirmed observations made at the light microscopic level. The plasma membrane of the oocyte and the adjacent granulosa cells were closely apposed in
Fig. 6. Electron micrograph of a tammar wallaby Ž M. eugenii . egg from a preantral follicle fully surrounded by a zona pellucida. Oocyte and granulosa cell microvilli traverse the zona pellucida to maintain contact. Bar s1 mm. Egg cytoplasm Žegg.; egg plasma membrane Žarrow.; zona pellucida Žzp.; granulosa cell Žgc..
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primordial follicles with no sign of ZP material, regardless of the age of the animal. There were no microvilli on the egg plasma membrane in primordial follicles ŽFig. 5a.. Primary follicles with a single layer of cuboidal granulosa cells contained eggs in the process of ZP formation; small patches of microvilli formed on the surface of the egg, creating an intercellular space between the egg and granulosa cell plasma membranes ŽFig. 5b.. The ZP was deposited in the newly created intercellular space very soon after formation of the egg microvilli. During its formation, the ZP completely filled the space between the egg and the adjacent granulosa cells and there was no perivitelline space ŽFig. 5c.. Numerous Golgi and membrane bound vesicles are present in the peripheral cytoplasm of eggs at this time. The ZP matrix material often appeared between granulosa cells during its formation process ŽFig. 5d.. When the ZP was fully formed,
Fig. 7. Electron micrographs of tammar wallaby Ž M. eugenii . eggs labelled with rabbit anti-pig zona pellucida polyclonal antibody. Ža. Egg from a primary follicle at an early stage of zona pellucida formation, showing localisation of zona proteins in the perivitelline space and egg cytoplasm Žarrows.. Bar s1 mm. The inset shows an enlarged view of the antibody labelling in the egg cytoplasm. Bar s 0.3 mm. Žb. Egg from a primary follicle with a partially formed zona pellucida. Note the absence of label in the granulosa cells. Bar s 2 mm. Egg cytoplasm Žegg.; egg plasma membrane Ždouble arrow.; zona pellucida Žzp.; granulosa cell Žgc..
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oocyte and granulosa cell microvilli traversed the ZP in order to maintain contact ŽFig. 6.. Immunogold labelling confirmed the presence of the ZP in the intercellular space between the egg and granulosa cells often before any matrix was visible using conventional electron microscopy ŽFig. 7a.. Small patches of ZP protein were detected within the cytoplasm of the egg at the time of ZP formation ŽFig. 7a, inset.. There was no ZP-reactive material detected in the granulosa cells of any follicles examined Že.g. Fig. 7b..
4. Discussion The ZP of two Australian marsupials, the brushtail possum and tammar wallaby first appears in growing primary follicles with a single cuboidal layer of granulosa cells. The proteins of the possum and wallaby ZP appear to be secreted by growing oocytes which are present from 98 days of pouch life through to adulthood in the tammar wallaby, and at least from 108 days in the brushtail possum. The detection of a ZP in primary follicles at 108 days of age in the possum is consistent with a previous study that reported the appearance of primary follicles at 103 days of age ŽFrankenberg et al., 1996.. The time that ZP protein was first detected in the tammar wallaby in this study Ž; 100 days after birth. coincides with commencement of the follicular stage of ovarian development, which occupies 110 days of pouch life to puberty ŽAlcorn, 1975; Alcorn and Robinson, 1983.. The number of growing follicles present within the tammar wallaby ovary peaks at about 240 days after birth ŽAlcorn, 1975.. Shortly after 240 days, a wave of follicle atresia follows and the population of growing follicles then remains low through the pre-pubertal period ŽAlcorn, 1975.. A similar phenomenon also occurs in eutherian species, such as the rabbit where the number of growing follicles peaks at six weeks of age, followed by follicular atresia ŽDunbar et al., 1991.. This period of ovarian development in the rabbit also represents the time of maximal expression of the ZP genes; there is a 600-fold decrease in the levels of ZP transcripts in the adult rabbit ovary compared to the immature six-week old ovary ŽDunbar et al., 1991.. It is therefore predicted that maximal expression of the tammar wallaby ZP genes would occur around 240 days of age. Large antral follicles were observed only in adult tammar wallaby and possum ovaries. Follicle development in the pouch young ovaries did not significantly progress past the stage of antrum formation. Similarly, Alcorn Ž1975. reported the absence of follicles greater than 500 mm in diameter in animals less than 270 days of age. This is consistent with findings in eutherian mammals that the continued development of antral follicles is dependent upon the gonadotrophins, FSH and LH Žreviewed by Greewald and Roy, 1994.. Immunofluorescence studies of the ZP in several eutherian species detected a heterogeneity in its structure characterised by more intense labelling at the inner and outer boundaries of the zona ŽWolgemuth et al., 1984; Leveille et al., 1987.. There was no such heterogeneity detectable in the ZP of the tammar wallaby or brushtail possum at the light microscope level when stained with PAS or labelled with rabbit anti-pig ZP
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polyclonal antibody. This result may indicate that the marsupial ZP is a more homogeneous structure than that of eutherian mammals or, alternatively, may be due to insufficient resolution to discern distinct regions in the much thinner marsupial ZP. Electron microscopy has also indicated that the tammar wallaby ZP is a homogeneous structure, which has also been reported in several other marsupial species ŽKress, 1996; Mate, 1996.. Labelling with the anti-pig ZP polyclonal and PAS staining indicated that although the bulk of ZP material appeared to be derived from the egg, the granulosa cells of small preantral follicles in both the tammar wallaby and brushtail possum may also synthesise ZP proteins. The pattern of ZP deposition, when examined by electron microscopy, was also suggestive of a contribution from granulosa cells as patches of zona material appeared between granulosa cells quite a distance from the egg plasma membrane. Analysis of eggs in the process of ZP formation using immunogold labelling confirmed synthesis of ZP proteins in the cytoplasm of the egg, but did not detect any synthesis in the granulosa cells. Molecular studies are required to determine unequivocally whether granulosa cells contribute to the formation of the marsupial ZP as they do in the rabbit Žreviewed by Dunbar et al., 1994. or whether the immunoreactive zona or PAS-positive material detected in the granulosa cells is part of a degradative process as described in the hamster ŽDelgado and Zoller, 1987; Leveille et al., 1987.. The fully formed ZP in the tammar wallaby was approximately 8 mm thick in this study, which is slightly larger than previously reported ŽRenfree and Tyndale-Biscoe, 1978.. Secretion of the ZP appears to continue throughout follicular development in the wallaby as it was thicker in eggs recovered from large Ž) 1.5 mm. antral follicles ŽZP s 8 mm. than those from small Ž- 1.5 mm. antral follicles ŽZP s 6.5 mm.. The alteration andror addition of material to the marsupial ZP in the later stages of follicle development is consistent with functional studies carried out in an American marsupial, grey short-tailed opossum Ž Monodelphis domestica.. In order to be penetrated and fertilized in vitro, eggs from the opossum must have completed the process of meiotic maturation ŽMoore and Taggart, 1993.. Ultrastructural differences between the ZP of follicular and ovulated eggs from the brushtail possum and grey short-tailed opossum ŽMate, 1996. further suggest that marsupial ZP may undergo late maturational changes. The process of ZP formation described here is similar to that reported in the grey short-tailed opossum, Mon. domestica, which begins in eggs surrounded by a complete follicular epithelium ŽKress, 1996.. In the tammar wallaby, ZP material first accumulated in areas where small patches of microvilli appeared on the egg surface. The perivitelline space in the tammar wallaby must develop during the antral stage of follicular development as it is not present in preantral follicles with two to three layers of granulosa cells. Similar observations were also made in the opossum ŽKress, 1996..
5. Conclusion The process of ZP formation in the tammar wallaby and brushtail possum is similar to that described for a variety of eutherian species based on the histological, immunofluorescence and electron microscopy findings reported in this study. Further studies are
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required to investigate whether subtle changes to marsupial ZP occur during the latter stages of oocyte development. Acknowledgements This work was supported by the Cooperative Research Centre for Conservation and Management of Marsupials. Thanks to Amanda Harman and Janine Buist for their skilled technical assistance, Ron Claassens and Steve McLeod for assistance in collecting tammar wallaby pouch young, Dr. Janine Duckworth for supplying possum pouch young ovaries and Dr. Mark Bradley for supplying antibody. References Alcorn, G.T., 1975. Development of the ovary and urogenital ducts in the tammar wallaby Macropus eugenii ŽDesmarest, 1817., PhD Thesis. Macquarie University, Australia. Alcorn, G.T., Robinson, E.S., 1983. Germ cell development in female pouch young of the tammar wallaby Ž Macropus eugenii .. J. Reprod. Fertil. 67, 319–325. Bleil, J.D., Wassarman, P.M., 1980. Synthesis of zona pellucida proteins by denuded and follicle-enclosed mouse oocytes during culture in vitro. Proc. Natl. Acad. Sci. USA 77, 1029–1033. Chamberlin, M.E., Dean, J., 1990. Human homolog of the mouse sperm receptor. Proc. Natl. Acad. Sci. USA 87, 6014–6018. Delgado, M.V., Zoller, L.C., 1987. A quantitative and qualitative cytochemical analysis of glycosaminoglycan content of hamster ovarian follicles. Histochemistry 87, 279–287. Dunbar, B.S., Prasad, S.V., Timmons, T.M., 1991. Comparative structure and function of mammalian zonae pellucidae. In: Dunbar, B.S., O’Rand, M.G. ŽEds.., A Comparative Overview of Mammalian Fertilization. Plenum, New York, pp. 97–116. Dunbar, B.S., Avery, S., Lee, V., Prasad, S., Schwahn, D., Schwoebel, E., Skinner, S., Wilkins, B., 1994. The mammalian zona pellucida: its biochemistry, immunochemistry, molecular biology and developmental expression. Reprod. Fertil. Dev. 6, 331–347. Epifano, O., Liang, F.-F., Dean, J., 1995. Coordinate expression of the three zona pellucida genes during mouse oogenesis. Development 121, 1947–1956. Frankenberg, S., Newell, G., Selwood, L., 1996. A light microscopic study of oogenesis in the brushtail possum Trichosurus Õulpecula. Reprod. Fertil. Dev. 8, 541–546. Greewald, G.S., Roy, S.K., 1994. Follicular development and its control. In: Knobil, E., Neill, J.D. ŽEds.., The Physiology of Reproduction. Raven Press, New York. pp. 629–724. Harris, J.D., Hibler, D.W., Fontenant, G.K., Hsu, K.T., Yurewicz, E.C., Sacco, A.G., 1994. Cloning and characterization of zona pellucida genes and cDNAs from a variety of mammalian species: ZPA, ZPB and ZPC gene families. DNA Sequence 4, 361–393. Kress, A., 1996. A comparison of oocyte organelles in Monodelphis domestica with those of other marsupials and eutherians. Reprod. Fertil. Dev. 8, 521–533. Lee, V., Dunbar, B., 1993. Developmental expression of the rabbit 55-kDa zona pellucida protein and mRNA in ovarian follicles. Dev. Biol. 155, 371–382. Leveille, M.C., Roberts, K.D., Chevalier, S., Chapdelaine, A., Bleau, G., 1987. Formation of the hamster zona pellucida in relation to ovarian differentiation and follicular growth. J. Reprod. Fertil. 79, 173–183. Liang, L.F., Chamow, S.M., Dean, J., 1990. Oocyte-specific expression of mouse Zp-2: developmental regulation of the zona pellucida genes. Mol. Cell. Biol. 10, 1507–1515. Lyne, A.G., Verhagen, A.M.W., 1957. Growth of the marsupial Trichosurus Õulpecula and a comparison with some higher mammals. Growth 21, 167–195. Lyons, C.E., Payette, K.L., Price, J., Huang, R., 1993. Expression and structural analysis of a teleost homolog of a mammalian zona pellucida gene. J. Biol. Chem. 268, 21351–21358.
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Mate, K.E., 1996. Cytoplasmic maturation of the marsupial oocyte during the periovulatory period. J. Reprod. Fertil. 8, 509–519. Mate, K.E., Gentle, R.L., Fletcher, T.P., Rodger, J.C., 1997. Australian marsupials. In: Poole, T.B. ŽEd.., UFAW Handbook on the Care and Management of Laboratory Animals, 7th edn. Blackwells Press, in press. Molinia, F.C., Gibson, R.J., Smedley, M.A., Rodger, J.C., 1998. Further observations of the ovarian response of tammar wallaby, Macropus eugenii, to exogenous gonadotrophin treatment and an improved method for superovulation using FSHrLH, Anim. Reprod. Sci. Žthis issue.. Moore, H.D.M., Taggart, D.A., 1993. In vitro fertilization and embryo culture in the grey short-tailed opossum, Monodelphis domestica. J. Reprod. Fertil. 98, 267–274. Murata, K., Sasaki, T., Yasumasu, S., Iuchi, I., Enami, J., Yasumasu, I., Yamagami, K., 1995. Cloning of cDNAs for the precursor protein of a low-molecular weight subunit of the inner layer of the egg envelope Žchorion. of the fish Oryzias latipes. Dev. Biol. 167, 9–17. Philpott, C.C., Ringuette, M.J., Dean, J., 1987. Oocyte specific expression and developmental regulation of ZP3, the sperm receptor of the mouse zona pellucida. Dev. Biol. 121, 568–575. Poole, W.E., Simms, N.G., Wood, J.T., Lubulwa, M., 1991. Table for age determination of the Kangaroo Island wallaby ŽTammar., Macropus eugenii, from body measurements, CSIRO Division of Wildlife and Ecology Technical Memorandum, 32, 1–37. Renfree and Tyndale-Biscoe, 1978. Ringuette, M.J., Chamberlin, M., Baur, A., Sobieski, D., Dean, J., 1988. Molecular analysis of cDNA coding for ZP3, a sperm-binding protein of the mouse zona pellucida. Dev. Biol. 127, 287–295. Roller, R.J., Kinloch, R.A., Hiraoka, B.Y., Li, S.S., Wassarman, P.M., 1989. Gene expression during mammalian oogenesis and early embryogenesis: quantification of three messenger RNAs abundant in fully grown mouse oocytes. Development 106, 251–261. Salzmann, G.S., Greve, J.M., Roller, R.J., Wassarman, P.M., 1983. Biosynthesis of the sperm receptor during oogenesis in the mouse. EMBO J. 2, 1451–1456. Thillai-Koothan, P., van Duin, M., Aitken, R.J., 1993. Cloning, sequencing and oocyte-specific expression of the marmoset sperm receptor protein, ZP3. Zygote 1, 93–101. Wolgemuth, D., Celenza, J., Dunbar, B.S., 1984. Formation of the rabbit zona pellucida and its relationship to ovarian follicular development. Dev. Biol. 106, 1–14.
Timing of zona pellucida formation in the tammar wallaby žMacropus eugenii / and brushtail possum žTrichosurus Õulpecula / K.E. Mate
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CooperatiÕe Research Centre for ConserÕation and Management of Marsupials, School of Biological Sciences, Macquarie UniÕersity, New South Wales 2109, Australia
Abstract The zona pellucida ŽZP. is an extracellular coat that surrounds the mammalian egg, and serves as the primary recognition site for fertilizing spermatozoa. The timetable of ZP formation was examined in two marsupials, the tammar wallaby Ž Macropus eugenii . and the brushtail possum ŽTrichosurus Õulpecula. using conventional histological methods, immunofluorescence and electron microscopy. Ovaries from tammar wallaby pouch young less than 80 days of age contained only primordial follicles with a single layer of flattened granulosa cells. There was no evidence of ZP formation until 98 days, when a small number of eggs surrounded by a single layer of cuboidal granulosa cells had a ZP detectable by periodic-acid–schiff staining and rabbit anti-pig ZP polyclonal antibody labelling. Possum ovaries at 108 and 114 days also contained a small number of eggs with a ZP and a single layer of cuboidal granulosa cells. The antibody also labelled the peripheral cytoplasm of oocytes at this stage and, occasionally, the granulosa cells. Antral follicles were first detected at 144 days in the wallaby and 125 days in the possum, and always contained an egg surrounded by a ZP. Ovaries from 147, 158, 165, 181, 184 and 210-day-old tammar wallabies contained a range of follicle types from primordial through early antrum formation. Electron microscopy confirmed observations made at the light microscope level. The ZP was first detectable in small primary follicles with a single layer of cuboidal granulosa cells in areas where microvilli had begun to form on the egg plasma membrane. Immunogold labelling indicated the egg cytoplasm as the origin of the ZP proteins. The ZP completely filled the space between the egg and the adjacent granulosa cells in preantral follicles, so that there was no perivitelline space. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Egg; Follicle; Zona pellucida
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Tel.: q61 2 9850 9257; fax: q61 2 9850 9686; e-mail: [email protected]
0378-4320r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 Ž 9 8 . 0 0 1 1 6 - X
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1. Introduction The zona pellucida ŽZP. is an extracellular coat that surrounds the mammalian egg, and serves as the primary recognition site for fertilizing spermatozoa. Depending on the species, it is composed of three to four glycoproteins, that have been designated ZP1, ZP2 and ZP3 ŽRinguette et al., 1988; Chamberlin and Dean, 1990; Liang et al., 1990; Harris et al., 1994.. Each of the ZP proteins is modified after translation and secreted from the egg as a glycoprotein that interacts with the other ZP proteins to form the insoluble extracellular zona matrix. The marsupial egg is also surrounded by a ZP made up of at least three glycoproteins, but their functional or structural homology to eutherian ZP proteins has not been examined. Expression of the genes encoding the ZP glycoproteins is restricted to the ovary in eutherian mammals Žreviewed by Dunbar et al., 1994., but occurs in the liver in some species of teleost fish ŽLyons et al., 1993; Murata et al., 1995.. There is some controversy concerning whether synthesis of the ZP proteins within the ovary of eutherian mammals is restricted to the oocyte or whether it also occurs in the granulosa cells. Several studies have indicated that expression of the ZP3 gene is restricted to growing oocytes in the mouse ŽPhilpott et al., 1987; Roller et al., 1989; Epifano et al., 1995.: nongrowing oocytes Ž12–15 mm in diameter. do not contain detectable levels of ZP3, and maximal expression of ZP3 occurs in growing oocytes Ž30–80 mm in diameter. and decreases rapidly by the time of ovulation when up to 98% of ZP3 mRNA is destroyed. The synthesis and secretion of the ZP3 protein follows a similar pattern to gene expression in the mouse ŽBleil and Wassarman, 1980; Salzmann et al., 1983.. Oocyte-specific expression of ZP3 has also been reported in the hamster ŽLeveille et al., 1987. and marmoset ŽThillai-Koothan et al., 1993.. However, studies in other species have indicated that the assembly of the ZP may involve both the oocyte and granulosa cells depending upon the stage of follicular growth and maturation ŽWolgemuth et al., 1984; Lee and Dunbar, 1993.. The granulosa cells surrounding growing oocytes within primary and secondary follicles in the rabbit, mouse, rat, guinea pig, hamster and cat actively synthesise ZP proteins Žreviewed by Dunbar et al., 1991.. The synthesis of ZP proteins by granulosa cells appears strictly limited to certain stages of follicle development, which may explain some of the inconsistencies in the literature. In the rabbit, for example, ZP protein was present in the cytoplasm of the inner layer of granulosa cells in secondary follicles consisting of two to three layers of granulosa cells, but not in the granulosa cells of earlier or later stage follicles ŽWolgemuth et al., 1984.. The timing of ZP formation in marsupials has not been examined in detail, but has been reported to occur during the transition of follicles with a single layer of flattened granulosa cells Žtype 3a. to follicles with a single layer of cuboidal granulosa cells Žtype 3b. in the tammar wallaby ŽAlcorn, 1975.. The same study indicated that most follicles had progressed to at least the type 3b stage of development by 270 days after birth, suggesting that in contrast to eutherian species, the bulk of ZP formation may occur before puberty. A study of oogenesis in the brushtail possum also indicated that the ZP first appeared in follicles with a single layer of cuboidal granulosa cells ŽFrankenberg et al., 1996.. Functional studies of fertilization in marsupials have been severely impeded by the
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inability to achieve fertilization or meaningful sperm–egg interaction in vitro in Australian marsupials. It has been suggested that the marsupial ZP may undergo maturational changes necessary for fertilization based on morphological ŽMate, 1996. and functional studies ŽMoore and Taggart, 1993.. An increased understanding of the structure and function of the marsupial ZP is necessary to overcome the difficulties associated with in vitro fertilization in Australian marsupials. This study has examined the timetable of ZP formation in two marsupials, the tammar wallaby Ž Macropus eugenii . and the brushtail possum ŽTrichosurus Õulpecula. using conventional histological methods, immunofluorescence and electron microscopy.
2. Materials and methods 2.1. Animals Tammar wallabies Ž M. eugenii . were housed at the Central Animal House at the University of Newcastle or at the Fauna Park at Macquarie University as described previously ŽMate et al., 1997.. Brushtail possums ŽT. Õulpecula. were housed at the Central Animal House at University of Newcastle or the animal facility at Manaaki Whenua Landcare Research in Lincoln, New Zealand. 2.2. Egg collection Two tammar wallabies were treated with 6 mg of porcine follicle-stimulating hormone ŽFSH. ŽFollitropin V; Vetrapharm, Australia. twice daily for four days followed by 4 mg of porcine luteinising hormone ŽLH. ŽLutropin V; Vetrapharm, Australia. ŽMolinia et al., 1998.. Animals were sacrificed by an i.v. injection of 2.5 ml of 350 mgrml sodium pentobarbitone ŽLethobarb; Virbac, NSW. 24 h after the LH injection. Ovaries were removed, rinsed in heparinised phosphate-buffered saline ŽPBS. and eggs Ž n s 16 per category. were collected from primary preantral and antral follicles. Primary follicles were dissected from the ovary using fine needles and examined whole, as it was not possible to remove the tightly adherent granulosa cells. Eggs were recovered from preantral and antral follicles by pricking with a 30-gauge needle attached to a 2.5-ml syringe and flushing the follicle with PBS until the egg was recovered. Granulosa cells were removed from the egg by passing it several times through a pipette with a diameter slightly smaller than that of the egg. 2.3. Pouch young oÕary collection Ovaries from tammar wallaby pouch young that were 59, 74, 98, 104, 144, 147, 158, 165, 181, 184 and 210 days old were collected from the colonies at Macquarie University and the University of Newcastle. Ovaries from brushtail possum pouch young that were 108, 115, 125 and 135 days old were provided by Dr. J. Duckworth from Landcare Research. The age of the pouch young was determined by measuring the
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length of the head from the tip of the snout to the occiput and using age determination charts for the tammar wallaby ŽPoole et al., 1991. and the brushtail possum ŽLyne and Verhagen, 1957.. Adult ovaries from both species were similarly examined. Pouch young were sacrificed by i.p. injection of 350 mgrml sodium pentobarbitone ŽLethobarb; Virbac. and their ovaries were removed. One ovary from each wallaby was divided in two pieces; one piece was processed and embedded in paraffin wax for light microscopy and the other was processed for electron microscopy and immunogold labelling. Paraffin sections were stained with haematoxylin and eosin ŽHE. for routine examination, or period-acid-schiff ŽPAS. or alcian blue which specifically react with glycoproteins, or were labelled with rabbit anti-pig ZP polyclonal antibody Žprovided by Dr. M. Bradley. which cross-reacts with possum and wallaby ZP proteins on Western blot analysis ŽK. Mate, unpublished observations. and paraffin sections Žthis study.. Possum ovaries were examined by light microscopy only. The remaining ovary from each animal was frozen at y808C for use in another study. 2.4. PAS and alcian blue staining Ovaries were fixed in either 4% paraformaldehyde in PBS or Histochoice ŽAustral Scientific, Australia., embedded in paraffin wax and sectioned at a thickness of 5 mm. For PAS staining, the tissue sections were deparaffinized by xylene and rehydrated with 70% ethanol before treatment with periodic acid ŽSigma, St. Louis, MO, USA. for 5 min. Sections were treated with Schiff reagent for 10 min, rinsed in three 30-min changes of sodium bisulfite ŽSigma., dehydrated, and coverslips were attached with DPX mountant ŽBolton, Essex, UK.. Slides for alcian blue staining were rehydrated with water and rinsed in 3% acetic acid. The slides were then stained for 30 min in 1% alcian blue ŽSigma. in 3% acetic acid, washed with water, dehydrated and mounted with DPX. 2.5. Anti-porcine ZP antibody labelling Ovaries were collected and fixed immediately in either 4% paraformaldehyde in PBS or Histochoice. The ovaries were embedded in paraffin wax and sectioned at a thickness of 5 mm. In preparation for antibody labelling, tissue sections were deparaffinized with xylene and rehydrated with ethanol and PBS and then covered in a blocking solution that consisted of PBS containing 2% BSA, 50 mM glycine and 0.02% sodium azide for 1 h. Excess blocking solution was blotted off and the sections were incubated with rabbit anti-pig ZP polyclonal antibody Žprovided by Dr. M. Bradley, Perth Zoo. diluted 1r50 in PBS for 1 h at room temperature ŽRT.. Sections were washed three times in PBS and bound antibody was detected with FITC-conjugated anti-rabbit immunoglobulin diluted 1r100 in PBS ŽAmersham, Australia. for 15 min at RT. After washing three times in PBS, the sections were mounted in Mowiol ŽCalbiochem. containing the anti-fading agent 1,4-diazabicyclo-w2.2.2x-octane ŽDABCO. ŽSigma. and examined by fluorescence microscopy.
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2.6. Electron microscopy Ovaries were fixed immediately in Karnovskys fixative Ž1% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer. overnight at 48C, washed in 0.1 M cacodylate and post-fixed in 1% osmium tetroxide. The ovarian tissue was then dehydrated through an acetone series and embedded in Spurrs resin. Ovarian tissue for immunogold labelling was fixed in Karnovskys fixative as described above, rinsed in 0.1 M cacodylate, dehydrated through an ethanol series and embedded in LR white. 2.7. Immunogold labelling The labelling procedure was carried out in 50-ml drops of solution on a sheet of thermo-plastic film ŽParafilm, American National Can, USA.. Grids were floated on 50 mM glycine in PBS for 15 min and then transferred to incubation buffer which consisted of PBS containing 3% BSA for 1 h at RT. Grids were transferred to rabbit anti-pig ZP polyclonal antibody diluted 1r400 with incubation buffer for 2 h at RT. Grids were washed six times for 5 min each in 50 ml of incubation buffer and transferred to gold-conjugated anti-rabbit antibody ŽICN Biomedicals, Costa Mesa, CA, USA. diluted 1r30 in incubation buffer. Grids were washed six times for 5 min in incubation buffer, washed twice in PBS for 5 min and post-fixed in 2% glutaraldehyde in PBS for 15 min. After fixation, grids were rinsed twice for 5 min in Milli Q water and air-dried.
3. Results 3.1. Adult oÕaries Adult ovaries from the wallaby and possum contained eggs at all stages of development. Eggs from primary follicles in the tammar wallaby were surrounded by a single tightly adherent layer of granulosa cells but it was not possible to detect if a ZP was present in fresh material ŽFig. 1a.. In preantral follicles, the ZP was visible in all eggs examined, but was thin Žmaximum of 4.4 mm. and not evenly distributed around the egg. The ZP was not visible around the entire circumference of all eggs from preantral follicles ŽFig. 1b.. All of the eggs from antral follicles possessed a ZP that was evenly distributed around the circumference of the egg, but varied in thickness between eggs from 4–8.5 mm ŽFig. 1c; Table 1.. Eggs from large antral follicles Ž) 1.5 mm in diameter. generally possessed a thicker ZP than eggs from smaller antral follicles ŽTable 1., but this was not statistically significant. 3.2. Pouch young oÕaries Ovaries from tammar wallaby pouch young 59 and 74 days of age contained only primordial follicles surrounded by a single layer of flattened granulosa cells ŽFig. 2a.; there was weak PAS staining associated with some follicles in 74-day-old pouch young,
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Fig. 1. Eggs from the tammar wallaby Ž M. eugenii . showing progressive thickening of the zona pellucida Žarrows.. Ža. Primary follicle. Žb. Egg from a preantral follicle. Žc. Egg from a 2-mm diameter antral follicle. Barss 25 mm.
but there was no discernible ZP ŽFig. 2b.. By 98 days of age, a small number of primary follicles, consisting of a single layer of cuboidal granulosa cells and a growing oocyte, were present in the inner cortical region of the ovary ŽFig. 2c.. The ZP was detectable in primary follicles as a distinct band of PAS-positive material ŽFig. 2d. and the cytoplasm
Table 1 Width of the zona pellucida during egg development in the adult tammar wallaby Ž M. eugenii . Follicle Type
Egg Diameter Žmm.
Zona Width Žmm.
Primary Preantral Small antral Ž -1.5 mm. Large antral Ž )1.5 mm.
; 50 124.7"3.3 159.6"5.0 175.3"2.3
– 0–4.4 6.5"1.3 7.9"0.4
ns16 eggs per category.
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Fig. 2. Ovarian sections from tammar wallaby Ž M. eugenii . pouch young. Ža. 74-day-old pouch young ovary, stained with haematoxylin and eosin ŽHE. that contains a large number of primordial follicles. Žb. 74-day-old pouch young ovary stained with periodic-acid–schiff ŽPAS.. Several follicles are weakly stained Žarrows., but there is no zona pellucida detectable. Žc. 98-day-old pouch young ovary stained with HE, showing development of primary follicles Žarrows. in the inner ovarian cortex. Žd. 98-day-old pouch young ovary stained with PAS. The zona pellucida Žarrows. is stained in several growing follicles that are present in the inner ovarian cortex. Že. 144-day-old pouch young ovary stained with HE, that contains several growing follicles. Žf. 144-day-old pouch young ovary stained with PAS. The zona pellucida and cytoplasm of two eggs in the same growing follicle Žarrow. are intensely stained. Bars s 50 mm.
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of the egg was also lightly stained in most primary follicles. In some primary follicles, the granulosa cells were also stained by PAS ŽFig. 2d.. Slightly later stage follicles with two to three layers of granulosa cells were present by 104 days in the tammar wallaby. Again, in addition to the ZP and egg cytoplasm, there was also PAS-positive material present in the granulosa cells of some follicles. At this stage, the ZP was also detectable by alcian blue staining Žnot shown.. Follicles around the stage of antrum formation were first detected at 144 days in the tammar wallaby. At this age, the ovary also contained numerous large preantral follicles with growing oocytes surrounded by several layers of granulosa cells ŽFig. 2e.. The eggs in large preantral follicles were intensely stained with PAS, as was the ZP surrounding the egg ŽFig. 2f.. The granulosa cells of large preantral follicles were rarely stained with PAS. Ovaries from 147, 158, 165, 181, 184 and 210-day-old tammar wallabies were similar in appearance; they contained a range of follicle types from primordial through
Fig. 3. Ovarian sections from brushtail possum ŽT. Õulpecula. pouch young. Ža. 108-day-old pouch young ovary stained with haematoxylin and eosin ŽHE.. Primary follicles are present near the boundary of the ovarian cortex and medulla Žarrows.. Žb. 108-day-old pouch young ovary stained with periodic-acid–schiff ŽPAS.. The egg cytoplasm and zona pellucida is darkly stained in growing primary follicles Žarrows.. Žc. 125-day-old pouch young ovary stained with HE, with several growing follicles Žarrows.. Žd. 125-day-old pouch young ovary stained with PAS. The egg cytoplasm and zona pellucida is darkly stained in growing follicles Žarrows.. Barss 50 mm.
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Fig. 4. Phase contrast Ža,c,e. and fluorescent micrographs Žb,d,f. of tammar wallaby Ž M. eugenii . and brushtail possum ŽT. Õulpecula. ovaries labelled with rabbit anti-pig zona pellucida polyclonal antibody. Although there was variation in the intensity of labelling between different sections in both the wallaby and possum, there was no consistent difference in the intensity of labelling between the two species. Ža,b. Adult tammar wallaby ovary. Žc,d. Adult brushtail possum ovary. Že,f. 144-day-old tammar wallaby ovary. Barss 50 mm.
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to early antrum formation. Each of the follicle types had a characteristic staining pattern: primordial follicles were not PAS stained; the ZP, egg cytoplasm and, occasionally, granulosa cells were PAS-stained in follicles with one to two layers of granulosa cells; the ZP and egg cytoplasm only were stained in large antral follicles with three or more layers of granulosa cells. The 181, 184 and 210-day-old tammar wallabies, generally, had a greater number of medium and large preantral follicles than younger animals. Although antral follicles were present in wallabies aged 144 days and older, they were present only in limited numbers and the follicle did not grow significantly after antrum formation. A similar pattern of ZP formation was observed in the brushtail possum. The youngest brushtail possum examined was 108 days of age ŽFig. 3a.. At this stage of development, there were numerous primary follicles near the boundary of the ovarian cortex and medulla, consisting of a growing oocyte surrounded by PAS-stained ZP and a single layer of cuboidal granulosa cells. The oocyte cytoplasm was also stained by PAS in primary follicles ŽFig. 3b.. The ovaries from a 115-day-old possum were at a similar
Fig. 5. Electron micrographs of tammar wallaby Ž M. eugenii . eggs during the process of zona pellucida deposition. Ža. Egg from a primordial follicle which has no microvilli on the plasma membrane and no zona pellucida. Bar s 3 mm. Žb. Egg from a primary follicle at an early stage of zona pellucida formation; a small amount of zona material is visible in areas where microvilli are present on the egg plasma membrane. Bar s1 mm. Žc. Egg from a primary follicle with a thin and incomplete zona pellucida. Bar s1 mm. Žd. Egg from a primary follicle showing deposition of zona pellucida material between granulosa cells. Bar s 0.5 mm. Egg cytoplasm Žegg.; egg plasma membrane Žarrow.; zona pellucida Žzp.; granulosa cell Žgc..
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stage of development. The older possums examined, 125 and 135 days old, did not contain any antral follicles ŽFig. 3c., but did contain numerous large preantral follicles with PAS-stained oocytes and ZP ŽFig. 3d.. The several layers of granulosa cells in the large preantral follicles were not stained by PAS. 3.3. Anti-porcine ZP antibody labelling The pattern of ZP formation in the tammar wallaby and brushtail possum detected by antibody labelling was consistent with that described above using PAS staining. The ZP and peripheral egg cytoplasm were labelled in large preantral follicles with three or more layers of granulosa cells and in antral follicles in adult tammar wallaby ŽFig. 4a,b. and brushtail possum ŽFig. 4c,d. ovaries. The antibody did not bind to primordial follicles in the adult ovary, confirming the absence of a ZP at this stage. The ZP was first detectable by antibody labelling at 98 days of age in the wallaby and 108 days in the possum, in follicles with one to two layers of granulosa cells ŽFig. 4e,f.. Granulosa cells were also labelled occasionally in follicles with one to two layers of granulosa cells. 3.4. Electron microscopy Electron microscopy confirmed observations made at the light microscopic level. The plasma membrane of the oocyte and the adjacent granulosa cells were closely apposed in
Fig. 6. Electron micrograph of a tammar wallaby Ž M. eugenii . egg from a preantral follicle fully surrounded by a zona pellucida. Oocyte and granulosa cell microvilli traverse the zona pellucida to maintain contact. Bar s1 mm. Egg cytoplasm Žegg.; egg plasma membrane Žarrow.; zona pellucida Žzp.; granulosa cell Žgc..
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primordial follicles with no sign of ZP material, regardless of the age of the animal. There were no microvilli on the egg plasma membrane in primordial follicles ŽFig. 5a.. Primary follicles with a single layer of cuboidal granulosa cells contained eggs in the process of ZP formation; small patches of microvilli formed on the surface of the egg, creating an intercellular space between the egg and granulosa cell plasma membranes ŽFig. 5b.. The ZP was deposited in the newly created intercellular space very soon after formation of the egg microvilli. During its formation, the ZP completely filled the space between the egg and the adjacent granulosa cells and there was no perivitelline space ŽFig. 5c.. Numerous Golgi and membrane bound vesicles are present in the peripheral cytoplasm of eggs at this time. The ZP matrix material often appeared between granulosa cells during its formation process ŽFig. 5d.. When the ZP was fully formed,
Fig. 7. Electron micrographs of tammar wallaby Ž M. eugenii . eggs labelled with rabbit anti-pig zona pellucida polyclonal antibody. Ža. Egg from a primary follicle at an early stage of zona pellucida formation, showing localisation of zona proteins in the perivitelline space and egg cytoplasm Žarrows.. Bar s1 mm. The inset shows an enlarged view of the antibody labelling in the egg cytoplasm. Bar s 0.3 mm. Žb. Egg from a primary follicle with a partially formed zona pellucida. Note the absence of label in the granulosa cells. Bar s 2 mm. Egg cytoplasm Žegg.; egg plasma membrane Ždouble arrow.; zona pellucida Žzp.; granulosa cell Žgc..
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oocyte and granulosa cell microvilli traversed the ZP in order to maintain contact ŽFig. 6.. Immunogold labelling confirmed the presence of the ZP in the intercellular space between the egg and granulosa cells often before any matrix was visible using conventional electron microscopy ŽFig. 7a.. Small patches of ZP protein were detected within the cytoplasm of the egg at the time of ZP formation ŽFig. 7a, inset.. There was no ZP-reactive material detected in the granulosa cells of any follicles examined Že.g. Fig. 7b..
4. Discussion The ZP of two Australian marsupials, the brushtail possum and tammar wallaby first appears in growing primary follicles with a single cuboidal layer of granulosa cells. The proteins of the possum and wallaby ZP appear to be secreted by growing oocytes which are present from 98 days of pouch life through to adulthood in the tammar wallaby, and at least from 108 days in the brushtail possum. The detection of a ZP in primary follicles at 108 days of age in the possum is consistent with a previous study that reported the appearance of primary follicles at 103 days of age ŽFrankenberg et al., 1996.. The time that ZP protein was first detected in the tammar wallaby in this study Ž; 100 days after birth. coincides with commencement of the follicular stage of ovarian development, which occupies 110 days of pouch life to puberty ŽAlcorn, 1975; Alcorn and Robinson, 1983.. The number of growing follicles present within the tammar wallaby ovary peaks at about 240 days after birth ŽAlcorn, 1975.. Shortly after 240 days, a wave of follicle atresia follows and the population of growing follicles then remains low through the pre-pubertal period ŽAlcorn, 1975.. A similar phenomenon also occurs in eutherian species, such as the rabbit where the number of growing follicles peaks at six weeks of age, followed by follicular atresia ŽDunbar et al., 1991.. This period of ovarian development in the rabbit also represents the time of maximal expression of the ZP genes; there is a 600-fold decrease in the levels of ZP transcripts in the adult rabbit ovary compared to the immature six-week old ovary ŽDunbar et al., 1991.. It is therefore predicted that maximal expression of the tammar wallaby ZP genes would occur around 240 days of age. Large antral follicles were observed only in adult tammar wallaby and possum ovaries. Follicle development in the pouch young ovaries did not significantly progress past the stage of antrum formation. Similarly, Alcorn Ž1975. reported the absence of follicles greater than 500 mm in diameter in animals less than 270 days of age. This is consistent with findings in eutherian mammals that the continued development of antral follicles is dependent upon the gonadotrophins, FSH and LH Žreviewed by Greewald and Roy, 1994.. Immunofluorescence studies of the ZP in several eutherian species detected a heterogeneity in its structure characterised by more intense labelling at the inner and outer boundaries of the zona ŽWolgemuth et al., 1984; Leveille et al., 1987.. There was no such heterogeneity detectable in the ZP of the tammar wallaby or brushtail possum at the light microscope level when stained with PAS or labelled with rabbit anti-pig ZP
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polyclonal antibody. This result may indicate that the marsupial ZP is a more homogeneous structure than that of eutherian mammals or, alternatively, may be due to insufficient resolution to discern distinct regions in the much thinner marsupial ZP. Electron microscopy has also indicated that the tammar wallaby ZP is a homogeneous structure, which has also been reported in several other marsupial species ŽKress, 1996; Mate, 1996.. Labelling with the anti-pig ZP polyclonal and PAS staining indicated that although the bulk of ZP material appeared to be derived from the egg, the granulosa cells of small preantral follicles in both the tammar wallaby and brushtail possum may also synthesise ZP proteins. The pattern of ZP deposition, when examined by electron microscopy, was also suggestive of a contribution from granulosa cells as patches of zona material appeared between granulosa cells quite a distance from the egg plasma membrane. Analysis of eggs in the process of ZP formation using immunogold labelling confirmed synthesis of ZP proteins in the cytoplasm of the egg, but did not detect any synthesis in the granulosa cells. Molecular studies are required to determine unequivocally whether granulosa cells contribute to the formation of the marsupial ZP as they do in the rabbit Žreviewed by Dunbar et al., 1994. or whether the immunoreactive zona or PAS-positive material detected in the granulosa cells is part of a degradative process as described in the hamster ŽDelgado and Zoller, 1987; Leveille et al., 1987.. The fully formed ZP in the tammar wallaby was approximately 8 mm thick in this study, which is slightly larger than previously reported ŽRenfree and Tyndale-Biscoe, 1978.. Secretion of the ZP appears to continue throughout follicular development in the wallaby as it was thicker in eggs recovered from large Ž) 1.5 mm. antral follicles ŽZP s 8 mm. than those from small Ž- 1.5 mm. antral follicles ŽZP s 6.5 mm.. The alteration andror addition of material to the marsupial ZP in the later stages of follicle development is consistent with functional studies carried out in an American marsupial, grey short-tailed opossum Ž Monodelphis domestica.. In order to be penetrated and fertilized in vitro, eggs from the opossum must have completed the process of meiotic maturation ŽMoore and Taggart, 1993.. Ultrastructural differences between the ZP of follicular and ovulated eggs from the brushtail possum and grey short-tailed opossum ŽMate, 1996. further suggest that marsupial ZP may undergo late maturational changes. The process of ZP formation described here is similar to that reported in the grey short-tailed opossum, Mon. domestica, which begins in eggs surrounded by a complete follicular epithelium ŽKress, 1996.. In the tammar wallaby, ZP material first accumulated in areas where small patches of microvilli appeared on the egg surface. The perivitelline space in the tammar wallaby must develop during the antral stage of follicular development as it is not present in preantral follicles with two to three layers of granulosa cells. Similar observations were also made in the opossum ŽKress, 1996..
5. Conclusion The process of ZP formation in the tammar wallaby and brushtail possum is similar to that described for a variety of eutherian species based on the histological, immunofluorescence and electron microscopy findings reported in this study. Further studies are
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required to investigate whether subtle changes to marsupial ZP occur during the latter stages of oocyte development. Acknowledgements This work was supported by the Cooperative Research Centre for Conservation and Management of Marsupials. Thanks to Amanda Harman and Janine Buist for their skilled technical assistance, Ron Claassens and Steve McLeod for assistance in collecting tammar wallaby pouch young, Dr. Janine Duckworth for supplying possum pouch young ovaries and Dr. Mark Bradley for supplying antibody. References Alcorn, G.T., 1975. Development of the ovary and urogenital ducts in the tammar wallaby Macropus eugenii ŽDesmarest, 1817., PhD Thesis. Macquarie University, Australia. Alcorn, G.T., Robinson, E.S., 1983. Germ cell development in female pouch young of the tammar wallaby Ž Macropus eugenii .. J. Reprod. Fertil. 67, 319–325. Bleil, J.D., Wassarman, P.M., 1980. Synthesis of zona pellucida proteins by denuded and follicle-enclosed mouse oocytes during culture in vitro. Proc. Natl. Acad. Sci. USA 77, 1029–1033. Chamberlin, M.E., Dean, J., 1990. Human homolog of the mouse sperm receptor. Proc. Natl. Acad. Sci. USA 87, 6014–6018. Delgado, M.V., Zoller, L.C., 1987. A quantitative and qualitative cytochemical analysis of glycosaminoglycan content of hamster ovarian follicles. Histochemistry 87, 279–287. Dunbar, B.S., Prasad, S.V., Timmons, T.M., 1991. Comparative structure and function of mammalian zonae pellucidae. In: Dunbar, B.S., O’Rand, M.G. ŽEds.., A Comparative Overview of Mammalian Fertilization. Plenum, New York, pp. 97–116. Dunbar, B.S., Avery, S., Lee, V., Prasad, S., Schwahn, D., Schwoebel, E., Skinner, S., Wilkins, B., 1994. The mammalian zona pellucida: its biochemistry, immunochemistry, molecular biology and developmental expression. Reprod. Fertil. Dev. 6, 331–347. Epifano, O., Liang, F.-F., Dean, J., 1995. Coordinate expression of the three zona pellucida genes during mouse oogenesis. Development 121, 1947–1956. Frankenberg, S., Newell, G., Selwood, L., 1996. A light microscopic study of oogenesis in the brushtail possum Trichosurus Õulpecula. Reprod. Fertil. Dev. 8, 541–546. Greewald, G.S., Roy, S.K., 1994. Follicular development and its control. In: Knobil, E., Neill, J.D. ŽEds.., The Physiology of Reproduction. Raven Press, New York. pp. 629–724. Harris, J.D., Hibler, D.W., Fontenant, G.K., Hsu, K.T., Yurewicz, E.C., Sacco, A.G., 1994. Cloning and characterization of zona pellucida genes and cDNAs from a variety of mammalian species: ZPA, ZPB and ZPC gene families. DNA Sequence 4, 361–393. Kress, A., 1996. A comparison of oocyte organelles in Monodelphis domestica with those of other marsupials and eutherians. Reprod. Fertil. Dev. 8, 521–533. Lee, V., Dunbar, B., 1993. Developmental expression of the rabbit 55-kDa zona pellucida protein and mRNA in ovarian follicles. Dev. Biol. 155, 371–382. Leveille, M.C., Roberts, K.D., Chevalier, S., Chapdelaine, A., Bleau, G., 1987. Formation of the hamster zona pellucida in relation to ovarian differentiation and follicular growth. J. Reprod. Fertil. 79, 173–183. Liang, L.F., Chamow, S.M., Dean, J., 1990. Oocyte-specific expression of mouse Zp-2: developmental regulation of the zona pellucida genes. Mol. Cell. Biol. 10, 1507–1515. Lyne, A.G., Verhagen, A.M.W., 1957. Growth of the marsupial Trichosurus Õulpecula and a comparison with some higher mammals. Growth 21, 167–195. Lyons, C.E., Payette, K.L., Price, J., Huang, R., 1993. Expression and structural analysis of a teleost homolog of a mammalian zona pellucida gene. J. Biol. Chem. 268, 21351–21358.
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