In order to understand why sperm pairing has evolved in most American marsupials, the movement parameters of spermatozoa from Monodelphis domestica were analyzed after incubation in capacitating medium for 15 min, 2 h, and 24 h to induce a proportion of sperm pairs to uncouple. Motility characteristics of paired and single spermatozoa were measured in media of differing composition and viscosity by means of computer-aided semen analysis. In minimum essential medium or in RPMI 1640 medium alone, the absolute mean straight-line and curvilinear velocity values of paired spermatozoa (342 +/- 34 and 361 +/- 19 microns/sec, respectively, at 37 degrees C) were significantly greater than those of single spermatozoa (247 +/- 14 and 319 +/- 16 microns/sec), while mean lateral head displacement for paired spermatozoa (5.6 +/- 2.1 microns) was significantly less than for single spermatozoa (11.4 +/- 2.6 microns). However, when medium was made more viscous with polyvinyl pyrrolidone (0.8-82 poise) and sperm motility was calculated as a percentage of maximum attained velocity (in medium alone), there was no significant difference in straight-line or curvilinear velocity for single or paired spermatozoa in medium of the lowest viscosity (0.8 poise). In contrast, paired spermatozoa in medium of higher viscosity (above 1.92 poise) maintained straight-line velocity (e.g., 54 +/- 3% of maximum straight-line velocity in medium of 2.28 poise) while single sperm moved in tight circles and exhibited poor straight-line velocity (5 +/- 1% of maximum velocity). The data show that paired spermatozoa exhibit a significant motility advantage over single spermatozoa in a viscous medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Female brown marsupial mice were mated and changes in the number and distribution of spermatozoa were assessed in several regions of the reproductive tract at 1, 2, 3, 7, 10, 14 and 18 days after mating. Approximately 40 x 10(3) spermatozoa/side were present in the female reproductive tract between Days 1 and 7 after a single mating. This had decreased (to approximately 9 x 10(3) spermatozoa/side) by Days 10 and 14 after mating; by Day 18 no spermatozoa were recovered. The maximum number of spermatozoa recorded in a female tract was approximately 72 x 10(3) spermatozoa/side (Day 5 female, death in laboratory) and the minimum recorded was approximately 2 x 10(3) spermatozoa/side on Day 2 after mating. Between Days 1 and 7 after mating most spermatozoa were located in the uterus and lower isthmus (isthmus 1) and spermatozoa were rarely found in the lateral vaginae. By 24 h after mating most spermatozoa (approximately 60%) were found in isthmus 1, but approximately 35% were still present in the uterus. Histological observations of the lower isthmus at this time showed that large numbers of spermatozoa were present in both the lumen of the duct and the sperm storage crypts which are located in this region. By Day 7 after mating approximately 91% of all spermatozoa in the female tract were in isthmus 1, most of these being confined to the sperm storage crypts. On Days 10 and 14 after mating almost all spermatozoa in the tract were in the crypt regions of isthmus 1 and on Day 18 degenerating spermatozoa were observed. No special orientation or association of spermatozoa in relation to crypt cells was observed. These results show that, although the number of spermatozoa inseminated is low by mammalian standards sperm transport in this species is extremely efficient and a large proportion of spermatozoa reaches the isthmus before ovulation (approximately 1 in 1 to 1 in 7). Several observations may explain the remarkable success of these low numbers of spermatozoa, including specializations of the reproductive tract which may have a directing effect on sperm movement and the special relationship which exists between spermatozoa and the oviducal environment which results in viable sperm storage. Recent observations suggest that an unusual sinusoidal mode of progressive motility observed in this species, may also influence the success of the low numbers of ejaculated spermatozoa.

The ductus epididymidis of the marsupial mouse Antechinus stuartii was divided into caput, corpus, and caudal regions using several constant morphological landmarks. Tubule diameter and epithelial height increased gradually from caput to cauda. In contrast, the surface area of the lumen of the ductus epididymidis increased to a maximum in the distal caput region, but decreased markedly in the distal cauda in association with characteristic changes in lumen shape (from circular to slit-shaped) and epithelial height. Epithelial cells of the ductus epididymidis were generally similar in structure to those described in other mammalian species. Principal and basal cells were common throughout the epithelium. Clear and mitochondria-rich cells were also identified, but occurred less frequently. Regional variations in cell ultrastructure were observed only in principal cells. Numerous vesicular inclusions occurred in the apical cytoplasm of cells in caput segments, membrane-bounded, electron-dense bodies were common in distal corpus regions, and a brush border of microvilli characterized the luminal surface of principal cells in caudal segments. Sperm index increased in the proximal caput, declined to basal levels in the distal caput and proximal corpus, and then increased to a maximum in segment 9 of the distal corpus and remained at about this level throughout the cauda epididymidis. Nuclear rotation, loss of cytoplasmic droplets, and other sperm maturational changes were observed along the epididymis. Discarded cytoplasmic droplets collected in large masses interspersed between aggregates of spermatozoa throughout the distal regions of the duct. There was no evidence of phagocytosis by principal cells of cytoplasmic droplets. The epididymis of A. stuartii differs from that of other mammals.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in the number and distribution of spermatozoa in the epididymis of the adult brown marsupial mouse were examined during July/August in mated and unmated males. The effects of mating on epididymal sperm populations were studied in 2 groups of males each mated 3 times and compared with the number and distribution of spermatozoa in the epididymides of 4 unmated control groups. One testis and epididymis were removed from each animal (hemicastration) either before or early in the mating season to provide information on initial sperm content and distribution. The contralateral side was removed later in the mating season to examine the effects of mating or sexual abstinence on epididymal sperm distribution. Epididymal sperm number peaked in both the distal caput and distal corpus/proximal cauda epididymidis in late July. The total number of spermatozoa, including those remaining in the testis, available to each male at the beginning of the mating season in early August was approximately 4.4 x 10(6)/side. Although recruitment of spermatozoa into the epididymis from the testis continued until mid-August, sperm content of the epididymis reached a peak of about 3.5 x 10(6)/epididymis in early August. At this time approximately 0.9 x 10(6) spermatozoa remained in the testis which had ceased spermatogenic activity. Throughout the mating season, epididymal spermatozoa were concentrated in the distal corpus/proximal cauda regions of the epididymis and were replenished by spermatozoa from upper regions of the duct. Relatively few spermatozoa were found in the distal cauda epididymidis, confirming a low sperm storage capacity in this region. A constant loss of spermatozoa from the epididymis, probably via spermatorrhoea, occurred throughout the mating season and very few spermatozoa remained in unmated males in late August before the annual male die-off. Mating studies showed that an average of 0.23 x 10(6) spermatozoa/epididymis were delivered per mating in this species, but the number of spermatozoa released at each ejaculation may be as few as 0.04 x 10(6)/epididymis when sperm loss via spermatorrhoea is taken into account. We suggest that the unusual structure of the cauda epididymidis, which has a very restricted sperm storage capacity, may function to limit the numbers of spermatozoa available at each ejaculation and thus conserve the dwindling epididymal sperm reserves in order to maximize the number of successful matings which are possible during the mating season.

To establish the mode of fertilization in a marsupial, a morphological investigation was made of the gametes of the South American grey short-tailed opossum. Monodelphis domestica, at the time of fertilization in vivo and in vitro. Oestrus was induced in females by the introduction of an unfamiliar male. To obtain oocytes recently fertilized in vivo, females were killed 18-24 hours after the first mating and the region of the oviduct containing eggs excised and fixed. Unfertilized mature oocytes were recovered from ovarian follicles 15-18 hours after first mating and fertilized in vitro with cauda epididymal spermatozoa in a modified MEM medium supplemented with bovine serum albumin at 37 degrees C in 5% CO2 in air. Following sperm-egg binding and fertilization, oocytes were fixed and prepared for light and electron microscopy. Spermatozoa unpaired prior to fertilization in vivo and in vitro and single spermatozoa bound to the zona surface by their plasmalemma overlying the acrosome on the dorsal face of the sperm head. The acrosome reaction was only observed at the zona surface (suggesting that it may be induced by zona components) and involved a vesiculation of sperm plasma and acrosomal membranes over the main body of the acrosome but not over the narrow, marginal region which persisted after the acrosome reaction was complete. Sperm penetration of the zona pellucida caused a large breach in the zona and the dispersal of perivitelline material. The fusion of the spermatozoon with the oolemma occurred first over the marginal acrosomal region and was accompanied by a fertilization cone which protruded through the zona penetration hole. Activation of the egg was characterized by the release of material from vesicles in the peripheral cytoplasm and extrusion of the second polar body. The mode of fertilization in Monodelphis was compared with what is known in other marsupials (New World and Australian) and eutherian (placental) mammals. It was concluded that the general features of the acrosome reaction and sperm-egg fusion may be essentially similar in both groups and that an evolutionary schism did not occur following the development of the eutherian mode of fertilization.