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.

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.

Mammals generally ejaculate many more spermatozoa than seem to be needed for fertilization. This apparent profligacy has not been explained, but observations made in marsupials may shed light on it. The Virginia opossum, Didelphis virginiana, inseminates only about three million spermatozoa, a very low number. As a corollary, relatively few (ca. 13 x 106) are stored in each cauda epididymidis. However, some 5% of the spermatozoa that the opossum ejaculates populate the oviduct about 12 h later when ovulation can be anticipated - a success rate in the female orders of magnitude greater than in eutherian mammals. I t is not certain what determines the unusually efficient transport to and the high survival rate of spermatozoa in the oviduct of Didelphis, but two unusual features suggest themselves as possible contributors. Didelphis (and all other American marsupial) spermatozoa undergo a head-to-head pairing in the epididymis by the acrosomal face; this serves to isolate the acrosome of ejaculated spermatozoa from the female milieu until the pairs separate in the oviduct. Secondly, spermatozoa are housed in special crypts in the isthmus of the oviduct. Australian marsupials, which usually lack such features, store spermatozoa in the epididymis in numbers more close to those in comparably sized eutheriam mammals. Exceptions which store very low sperm numbers there can be seen in one Australian Family, the Dasyuridae. The spermatozoa of dasyurids are not paired, but the species examined possess distinctive sperm storage crypts in the oviducal isthmus similar to those in the opossum. The present findings suggest that where mechanisms exist that could protect the acrosome and, or, the whole spermatozoon in the female tract, a much lower level of sperm production can be maintained without compromising fertility. While the number ejaculated typically by any one species is probably determined ultimately by several interacting factors, it therefore seems likely that a most important one in this respect relates to conditions spermatozoa face in the female tract.

The phosphorylation of tyrosine residues in cellular proteins is a major signal transduction event during sperm capacitation. In this study protein phosphorylation was monitored using a fluorescein isothiocyanate (FITC)-labeled antiphosphotyrosine monoclonal antibody and a flow cytometric procedure optimized for sperm. Using this technique, the correlation between tyrosine phosphorylation and sperm capacitation was examined in two marsupial species, the brushtail possum and the tammar wallaby and compared with that of ram spermatozoa. The levels of tyrosine phosphorylation in sperm from all three species were increased by the addition of cyclic AMP (cAMP) and vandate, a phosphotyrosine phosphatase inhibitor and were decreased by the addition of the phosphotyrosine kinase inhibitor, staurosporine. Oviductal conditioned media (CM) induced a progressive increase in tyrosine phosphorylation in both marsupial species and also induced morphological transition from a streamlined to a ‘T’-shape configuration in brushtail possum spermatozoa but not in tammar wallaby spermatozoa. Transition to the ‘T’-shape orientation associated with capacitation in marsupial spermatozoa was observed by 2 h of incubation in both species when tyrosine phosphorylation was increased by higher levels of cAMP i.e. 5 mM dibutyryl cAMP plus 3 mM pentoxyphylline. Thus the tyrosine phosphorylation trigger with CM may differ in these two marsupial species. Ram sperm tyrosine phosphorylation could be increased by addition of lower levels of cAMP (1 mM). These results support the finding that tyrosine phosphorylation is associated with sperm capacitation in marsupials. Similar results were obtained by using SDS PAGE/Western blot analysis of tyrosine phosphorylation in the brushtail possum spermatozoa. The specificity, efficiency and sensitivity of the procedure described here make it applicable for routine assessment of capacitation in large numbers of samples and in other species.