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C. H. Tyndale-Biscoe and J. C. Rodger

Summary. Ovulation in the tammar wallaby alternates between the ovaries. The genital duct of each side enters the median vaginal culs-de-sac separately. Post-partum oestrus occurred 0·4 days after birth and ovulation 1 day later. After a single copulation spermatozoa were found in both cervical canals at 0·5 h and extended to the oviduct on the non-parturient side only by 8 h. Very few spermatozoa were found in sections of the post-partum uterus or its associated oviduct at any time. Spermatozoa were recovered by flushing from both sides but the numbers were 2–20 times greater in the non-parturient than in the post-partum side: the greatest difference occurred in the cervical canals 2–5 h after copulation. In females which had undergone a previous infertile cycle, spermatozoa were abundant in both cervices and both uteri. It is concluded that the differential distribution of spermatozoa in post-partum animals was (1) due to failure of transport in the recently pregnant side of the tract, rather than attraction of spermatozoa to the ovulation side, and (2) established at the cervix which, on the ovulation side, provides a reservoir of spermatozoa for 24 h after copulation.

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J. C. Rodger and J. M. Bedford

Summary. Oocytes, recovered from the oviduct or from ripe follicles, and spermatozoa flushed from the oviduct of opossums mated under laboratory conditions, were used to study the characteristics of marsupial gamete interaction and sperm incorporation. Opossum oocytes lose all granulosa cells before ovulation and are invested only by a thin zona pellucida when ovulated. The spermatozoa unpair in the oviduct but the motile population flushed at about the time of ovulation includes paired, separating and single spermatozoa. Separation of paired spermatozoa was associated with changes in the acrosome, the amorphous matrix being displaced by membrane-bound vacuoles. Fertilization is normally monospermic. Spermatozoa associated with the zona of unfertilized eggs by the flat acrosomal face of the head, whereas those trapped in mucoid eventually laid down around the zona were orientated at random. In penetrating the zona, the spermatozoon created a relatively large uneven hole that contrasts with the discrete penetration slit seen in the thicker zona of eutherian oocytes. Spermatozoa appeared to associate and fuse with the oolemma by the acrosomal face of the head. Ultrastructurally the sperm head, in oocytes fixed immediately after incorporation into the ooplasm, was devoid of surrounding membranes. This pattern of gamete interaction resembles the mode seen in non-mammalian vertebrates and invertebrates, and not that in eutherian mammals. Sperm penetration by tubal sperm samples occurred readily in vitro within 1 h of placing ripe follicular oocytes with the oviducal flushings containing spermatozoa. None of the oocytes penetrated in vivo or in vitro displayed extra perivitelline spermatozoa. This suggests that the oocyte is able to mount a block to polyspermy at the zona surface, although the mucoid deposited on the zona pellucida may be involved in vivo. The results indicate that the complex mode of sperm incorporation seen in eutherian mammals is unique to the infraclass and not shared by their closest relatives the marsupials.

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J. C. Rodger and J. M. Bedford

Summary. Of 14 lactating opossums maintained in laboratory conditions, 13 mated 4·7–8·5 days after removal of pouch young. The time between this removal and onset of receptive oestrus was negatively correlated with the age of the pouch young. Mating generally occurred between 24:00 and 06:00 h, with ovulation following between 13:00 and 16:00 h. Each animal ovulated a mean of 29·6 eggs (range 19–40), approximately equal numbers coming from both ovaries. Spermatozoa were absent from the uterus and were present only in the oviducts during the periovular period. Those not cleared by flushing (1–160 × 103/oviduct) remained incarcerated in isthmic crypts lined by a simple cuboidal epithelium. Spermatozoa in crypts were paired, separating or single. The progressively motile cells flushed from the oviduct presented a similar pattern to that in the crypts, about 30% of spermatozoa were firmly paired, the others either loosely associated or single. Only single spermatozoa attached to ova. Monospermic fertilization followed shortly after ovulation, and no supplementary spermatozoa were present in the perivitelline space. Deposition of the mucoid layer on the zona pellucida then began, often before incorporation of the fertilizing spermatozoon by the vitellus was complete. The oviducal epithelium was formed throughout by ciliated and secretory cells. In the ampulla and upper isthmus, the secretory cells produced the mucoid material which formed a thick coat over the egg surface. Ovum transit through the oviduct was rapid, in one animal eggs had reached the uterus and acquired a shell within 15–20 h of ovulation.

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J. C. Rodger and K. E. Mate

Summary. Optimal ovarian stimulation with minimal degenerative changes was achieved in brush-tailed possums 3 days after a subcutaneous injection of 10 i.u. PMSG. This treatment alone did not result in ovulation and only rarely in oocyte maturation. Ovulation of 8–24 mature oocytes occurred when on the 3rd day after 10 i.u. PMSG the female received 3 intramuscular injections of 50 μg synthetic gonadotrophin-releasing hormone (GnRH) 90 min apart. Superovulation was achieved in immature, cycling, pregnant and lactating females, but not in animals in the preovulatory phase of a natural oestrous cycle.

Keywords: superovulation; marsupial; brush-tailed possum; PMSG; GnRH

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J. C. Rodger and I. G. White

Department of Veterinary Physiology, University of Sydney, N.S.W. 2006, Australia

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J. C. RODGER and I. G. WHITE

Summary.

Total carbohydrate, glucose, fructose and resorcinol-reactive material were estimated in homogenates of the prostates of brush-tailed possum (Trichosurus vulpecula) and red kangaroo (Megaleia rufa). Fructose was virtually absent from the gland in both species. Glucose occurred in quite considerable quantities in the posterior segment of the prostate of M. rufa (92·2 mg/100 g) and to a lesser extent in the small anterior segment of T. vulpecula (22·4 mg/100 g). The posterior prostate of T. vulpecula contained an unidentified material which reacted with the resorcinol reagent as fructose (51·2 mg/100 g) but did not appear to be fructose or a commonly occurring ketose.

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K. E. Mate and J. C. Rodger

Acrosomal loss was induced in marsupial spermatozoa by an intermediate of the phosphoinositide pathway. The diacylglycerol, 1,2-dioctanoyl-sn-glycerol (DiC8; 100 μmol l−1) induced acrosomal loss in 70% of brushtail possum (Trichosurus vulpecula) spermatozoa and in 80% of tammar wallaby (Macropus eugenii) spermatozoa. The DiC8-induced acrosomal loss was not enhanced by co-incubation with calcium ionophore A23187 and occurred in Ca2+-free medium and in the presence of the calcium chelator EGTA (3 mmol l−1). There was no evidence of uptake of 45Ca2+ during the DiC8-induced acrosomal loss. Inhibitors of protein kinase C [1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine] and phospholipase A2 dexamethasone] did not effect DiC8-induced acrosomal loss in wallaby spermatozoa. The phorbol ester, phorbol 12-myristate 13-acetate, at a concentration of 10 μmol l−1 had no effect on possum spermatozoa and induced acrosomal loss in only 6% of wallaby spermatozoa. It appears that the DiC8-induced acrosome reaction is not mediated by activation of the phosphoinositide pathway and that extracellular calcium is not required for the membrane fusion event. As acrosomal loss was seen only at relatively high concentrations of diacylglycerol (> 50 μmol l−1) and there is no evidence of involvement of other phosphoinositide intermediates or analogues, it is likely that its role is as a direct membrane fusogen.

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J. C. RODGER and I. G. WHITE

Appreciable quantities of a compound, now identified as N-acetylglucosamine, have been found in deproteinized, deionized aqueous extracts of the seminal plasma of the red kangaroo (Megaleia rufa), tammar wallaby (Macropus eugenii), grey kangaroo (Macropus giganteus) and the ventral segment of the two segmented prostate gland (Rodger & Hughes, 1973) of the long-nosed bandicoot (Perameles nasuta).

Sperm-free seminal plasma was collected by electroejaculation (J. C. Rodger and I. G. White, in preparation) 5 to 15 min after death from tammar wallabies on Kangaroo Island, South Australia, and red and grey kangaroos near Coonamble, N.S.W. The material was handled and extracts made in much the same manner as that described for prostatic tissue collected in the field (Rodger & White, 1974). Weighed portions (approx. 0·5 to 1·0 g) of coagulated seminal plasma were homogenized in distilled

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J. C. RODGER and I. G. WHITE

Summary.

The seminal plasma of seven eutherian species, including man, was examined for the presence of free N-acetylaminosugar. Only man had appreciable levels of N-acetylaminosugar in the semen (37·1 mg/100 ml), but in all cases this free N-acetylaminosugar was probably the result of breakdown of polysaccharides by semen glycosidases. High levels of free N-acetylaminosugar thus appear to be peculiar to marsupials.

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J. C. RODGER and I. G. WHITE

Summary.

Electroejaculation of a variety of Australian marsupials was attempted in this study. The animals used were conscious, sedated, anaesthetized or recently shot. Electroejaculation proved to be a satisfactory means of obtaining seminal plasma but not spermatozoa. The largest volumes of seminal plasma were collected from animals shortly after death. Anaesthetized animals also provided useful volumes of seminal plasma but only insignificant amounts were obtained from conscious and sedated animals.

Quantitative analyses of N-acetylglucosamine, glucose and anthronereactive material were made of deproteinized, deionized, water extracts of seminal plasma from electroejaculates obtained from wallabies and kangaroos shortly after death. The major seminal sugar of the three macropod species was N-acetylglucosamine and glucose was also present in quite large concentrations. These observations show that the pattern of sugars in the prostate gland of marsupials is reflected in the semen.