Emeritus Professor John Rodger

Fourteen steps of spermatid development in the tammar wallaby (Macropus eugenii), from the newly formed spermatid to the release of the spermatozoon into the lumen of the seminiferous tubules, were recognised at the ultrastructural level using transmission and scanning electron microscopy. This study confirmed that although the main events are generally similar, the process of the differentiation of the spermatid in marsupials is notably different and relatively more complex than that in most studied eutherian mammals and birds. For example, the sperm head rotated twice in the late stage of spermiogenesis: the shape of the spermatid changed from a T-shape at step 10 into a streamlined shape in step 14, and then back to T-shape in the testicular spermatozoa. Some unique figures occurring during the spermiogenesis in other marsupial species, such as the presence of Sertoli cell spurs, the nuclear ring and the subacrosomal space, were also found in the tammar wallaby. However, an important new finding of this study was the development of the postacrosome complex (PAC), a special structure that was first evident as a line of electron dense material on the nuclear membrane of the step 7 spermatid. Subsequently it became a discontinuous line of electron particles, and migrated from the ventral side of the nucleus to the area just behind the posterior end of the acrosome, which was closely located to the sperm-egg fusion site proposed for Monodelphis domestica. The PAC and its possible role in both American and Australian marsupials requires detailed examination. Distinct immature features were discovered in the wallaby testicular spermatozoa. A scoop shape of the acrosome was found on the testicular spermatozoa of the tammar wallaby, which was completely different to the compact button shape of acrosome in ejaculated spermatozoa. The fibre network found beneath the cytoplasm membrane of the midpiece of the ejaculated sperm also did not occur in the testicular spermatozoa, although the structure of the principle piece was fully formed and had no obvious morphological difference from that of the epididymal and ejaculated spermatozoa. The time frame of the formation of morphologically mature spermatozoa in the epididymis of the tammar wallaby needs to be determined by further studies.

Tammar wallaby spermatozoa undergo maturation during transit through the epididymis. This maturation differs from that seen in eutherian mammals because in addition to biochemical and functional maturation there are also major changes in morphology, in particular formation of the condensed acrosome and reorientation of the sperm head and tail. Of spermatozoa released from the testes, 83% had a large immature acrosome. By the time spermatozoa reached the proximal cauda epididymis 100% of sperm had condensed acrosomes. Similarly 86% of testicular spermatozoa had immature thumb tack or T shape head-tail orientation while only 2% retained this immature morphology in the corpus epididymis. This maturation is very similar to that reported for the common brush tail possum, Trichosurus vulpecula. However, morphological maturation occurred earlier in epididymal transit in the tammar wallaby. By the time spermatozoa had reached the proximal cauda epididymis no spermatozoa had an immature acrosome and thumbtack orientation. Associated with acrosomal maturation was an increase in acrosomal thiols and the formation of disulphides which presumably account for the unusual stability of the wallaby sperm acrosome. The development of motility and progressive motility of tammar wallaby spermatozoa is similar to that of other marsupials and eutherian mammals. Spermatozoa are immotile in the testes and the percentage of motile spermatozoa and the strength of their motility increases during epididymal transit. During passage through the caput and corpus epididymis, spermatozoa first became weakly motile in the proximal caput and then increasingly progressively motile through the corpus epididymis. Tammar wallaby spermatozoa collected from the proximal cauda epididymis had motility not different from ejaculated spermatozoa. Ultrastructural studies indicated that acrosomal condensation involved a complex infolding of the immature acrosome. At spermiation the acrosome of tammar wallaby spermatozoa was a relatively large flat or concave disc which projected laterally and anteriorly beyond the limits of the nucleus. During transit of the epididymal caput and proximal corpus the lateral projections folded inwards to form a cup like structure the sides of which eventually met and fused. The cavity produced by this fusion was lost as the acrosome condensed to its mature form as a small button-like structure contained within the depression on the anterior end of the nucleus. During this process the dorsal surface of the immature acrosome and its outer acrosomal membrane and overlying plasma membrane were engulfed into the acrosomal matrix. This means that the dorsal surface of the acrosomal region of the testicular tammar wallaby sperm. head is a transient structure. The dorsal acrosomal surface of the mature spermatozoon appears ultrastructurally to be the relocated ventral surface of the acrosomal projections which previously extended out beyond the acrosomal depression on the dorsal surface of the nucleus of the immature spermatozoon.

This study utilised a commercially available monomaleimido-nanogold reagent to directly label cellular thiol groups (SH) of marsupial (tammar wallaby) spermatozoa before and after reduction of disulphides (S-S) with mercaptoethylamine hydrochloride (MEA). The sperm surface, mitochondrial membranes, axoneme and tail fibres were all labelled with gold particles before MEA treatment and the label intensity was increased after S-S reduction. The acrosomal membranes and matrix of spermatozoa contained no detectable SH prior to MEA treatment. However, after moderate MEA treatment (1 mg/ml) gold label was associated with the acrosomal membrane and invaginated acrosomal membrane within the acrosomal matrix. After exposure to 5 and 10 mg/ml MEA, gold particles heavily labelled the acrosomal matrix. Thus, the acrosomal membranes and matrix of tammar wallaby spermatozoa both contain S-S cross-linked structures, and this may contribute to the unusual stability of the marsupial acrosome. Under all treatment conditions the nucleus remained unlabelled. This is consistent with early studies which indicated that cysteine was absent from the nuclear protamines. The study also demonstrated that monomaleimido-nanogold can be used to resolve SH- and S-S-rich cellular structures directly, in addition to its use to label antibodies and Fab fragments for immunochemical localisation. © 1995 Springer-Verlag.

The phagocytic potential of cultured trophoblast from early (ectoplacental cone (EPC); day 7.5 post coitum) and mid-term (placenta; day is to 14 post coitum) pregnancy in the mouse has been examined using a variety of test particles and culture conditions. In suspension, small numbers (< 1 per cent) of large placental trophoblast cells showed limited phagocytic uptake of Staphylococcus aureus but not of opsonized sheep red blood cells (RBCs). In contrast, trophoblast phagocytosis was never seen in monolayer placental cell culture. Placental macrophages consistently exhibited phagocytic uptake of both opsonized sheep RBCs and S. aureus under these conditions. In monolayer culture, EPC trophoblast phagocytosed S. aureus, but there was only limited uptake of RBCs (mouse or sheep) or spermatozoa. When cultured in a three-dimensional matrix (blood and plasma clots), however, EPC trophoblast demonstrated extensive phagocytosis of both RBCs and sperm. These results are discussed with reference to the use of in vitro systems for examining developmental processes, and a possible role for trophoblast phagocytosis in early gestation is proposed. © 1987.

The expression of paternal class I and class II major histocompatibility complex (MHC) antigens in cultures of murine ectoplacental cone trophoblast was examined using immunogold labelled antibodies and electron microscopy. Class I MHC antigens could be induced on ectoplacental cone derived trophoblast following exposure to concanavalin A stimulated T cell supernatants. Class I MHC antigens were not detected in untreated trophoblast cultures. Class II MHC antigens were never detected on trophoblast whether treated or untreated. This is the first report of the experimental induction of Class I MHC antigens on a population of normally MHC-negative trophoblast cells. © 1987.

Active anti-paternal immunization does not compromise pregnancy in eutherian mammals. However, in an earlier study in a marsupial, grafting with paternal skin appeared to have resulted in transient infertility. In the present study, by critically monitoring the breeding efficiency of tammar wallabies sensitized against their mate's transplantation antigens, we aimed to resolve the question of immunologically mediated infertility in marsupials. Eight experimental females received two full-thickness skin grafts from their prospective mate and eight controls grafts of their own skin. The experimental group were monitored for 30 reproductive cycles and produced 24 pouch young (PY), whereas the control animals produced 28 young from 33 cycles. Five of the 11 apparently non-fertile cycles were judged to be normal pregnancies where the young had failed to reach the pouch (cycle length < 28 days: rapid plasma progesterone decline coincident with oestrus). True infertility was thus limited and, although occurring mainly in the male-skin grafted group (5 cycles), this was not significantly different (¿2, P > 0.5) from the controls (1 cycle) and represented the effect of one very poor breeder. We conclude that allogeneic pregnancy in marsupials is not compromised by active anti-paternal immunization. Infertility observed here, and in the earlier study, reflected disturbance of breeding owing to handling of the animals or the poor reproductive efficiency of individual animals in small experimental groups. © 1985.

Primary cultures of murine trophoblast (ectoplacental cone and mid-term placenta) and their supernatants were found to inhibit in vitro lymphocyte proliferative responses to concanavalin A (77-87%) and allo-antigen (52-84%). However, cultures and cell-conditioned media from non-trophoblastic tissues (embryonic sac, adult lung and liver, and B16 melanoma line) produced similar results. In all cases, the inhibitory effects were not due to reduced cell viability. Addition of anti-progesterone serum to the ectoplacental cone-lymphocyte co-cultures, at a concentration known to bind the available trophoblast-derived progesterone, did not overcome the observed suppression. The results clearly demonstrate that a range of cultured cell types, and their conditioned media, will suppress immune reponses in vitro. We conclude that cultured trophoblast is not an appropriate model for studies of placental immunoregulation.

In general, breeding pairs are not major histocompatibility complex (MHC)-compatible, and therefore the fetoplacental unit can be considered as a natural allograft. In many mammals pregnancy leads to the production of nonlytic antibodies of antipaternal MHC specificity. It has been suggested that these protect the semiallogeneic fetus from rejection by acting as blocking or enhancing factors¿or, alternatively, that they are part of a humoral response involved in the establishment of normal pregnancy. These hypotheses were tested in allomated mice made B cell deficient by continuous treatment with aIgMµ antiserum. The status of the maternal immune system was assessed by in vivo antibody production, in vitro mitogen responses, and allograft rejection. By these criteria B cell function could not be demonstrated in aIgMµ treated female mice, but T cell responses were unaffected. Allogeneic pregnancy, however, was not compromized by this humoral immune system dysfunction¿litter size and neonatal survival being the same in the agMµ and control serum-treated groups. These results indicate that a maternal humoral immune response is not essential for the establishment of pregnancy or the survival of the semiallogeneic: fetus. © 1985 by The Williams & Wilkins Co.

The present study examines and compares the structure of the testis and its excurrent ducts in a caenolestid and four didelphid marsupials. Of particular interest was the site of sperm pairing in the epididymis and whether this feature, shared by both American marsupial families but not by any Australian marsupial, was associated with changes in the morphology of the duct. In contrast to the testes of most Australian marsupials, except the peramelids (bandicoots), the intertubular space in the American marsupials was filled by Leydig cells (around 20% of testis volume). The opossum testes were unusual compared with those of eutherian mammals in that histological sections of individual seminiferous tubules contained only a single cellular association irrespective of the length of the tubule sectioned. The rete testis, as in the Australian dasyurids (devil, quoll, etc.), was a simple branching duct system that arose deep within the testis and emerged as a single duct at the testicular hilus. This arrangement is completely different from that in kangaroos and Australian possums, indicating a diversity of rete form in the marsupials similar to that seen in eutherian mammals. The rete emptied into a single, essentially straight, efferent duct that became convoluted towards the epididymis, where it formed a distinct structure adjacent to the caput epididymidis. The efferent ducts were highly variable in diameter and epithelial height, suggesting that the duct was not of uniform character along its length, or that the initial single duct had divided to form ducts of different characters. Sperm pairs were first seen in the proximal cauda epididymidis, and their appearance was correlated with changes in the character of the duct and its epithelium. The distal ductus deferens of Caenolestes, in contrast to those of the didelphids and indeed all other marsupials, was a convoluted ampulla-like structure adjacent to the prostate gland. In the other marsupials the only accessory sex glands are a segmented prostate and bulbourethral glands. Copyright © 1982 Wiley-Liss, Inc.

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 x 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 oviductal 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.

Opossum and rabbit sperm sonicates dispersed the mouse cumulus oophorus in vitro at the same rate on a per sperm basis, despite much higher activities of the glycosidic acrosomal enzymes N-acetylhexosaminidase (350 ×) and arylsulphatase (40 ×) in the opossum preparation. Activities of another glycosidase, hyaluronidase, and the protease acrosin where similar in sperm extracts from both species. However, specific inhibitors of N-acetylhexosaminidase (iodoacetate) and arylsulphatase (SO42-, PO43-) markedly reduced the rate of cumulus dispersal by rabbit sperm sonicates. These findings suggest that, while hyaluronidase action probably represents the rate-limiting (slow) step, several glycosidases act together to disperse the cumulus and in the passage of the fertilizing spermatozoon through it in eutherian mammals. The likely role of these acrosomal enzymes in the marsupial, where the ovulated oocytes lack a cumulus oophorus, is more uncertain. Copyright © 1981 Alan R. Liss, Inc.

1. 1. The semen of the brush tailed possum Trichosurus culpecula and the prostates of other marsupials were found to contain prostaglandins identified positively in possum semen as predominantly 19-OH PGF1a, and 19-OH PGF2a. 2. 2. The 19-OH prostaglandins can no longer be regarded as characteristic of primate semen. The 19-OH nature of the PGFs in possum semen may be of some evolutionary interest in view of the restricted occurrence of the 19-OH-PGs in the semen of eutherian species so far examined. © 1981.

1. 1. The major carbohydrate of the prostate of dasyurid and didelphid marsupials was found to be glycogen. 2. 2. Neither N-acetylglucosamine, the characteristic seminal and prostatic sugar of all but one previously examined marsupial species, nor fructose were detected. 3. 3. Glycogen was only a minor constituent of the phalangerid and peramelid, prostate gland (high N-acetylglucosamine species). 4. 4. The prostatic secretion of the dasyurid, Sarcophilus, had a very high concentration of glycogen, suggesting that the polysaccharide is the seminal sugar in this species. 5. 5. These and earlier data indicate that there are at least three prostatic carbohydrate patterns in marsupials. © 1980.

The origin of glycerylphosphorylcholine (GPC), N-acetylaminosugar, inositol, and prostaglandins in human seminal plasma was investigated by correlating the concentration of these components in split ejaculates with known marker constituents. Fructose and acid phosphatase were selected as markers of the secretory activity of the seminal vesicles and prostate gland, respectively, and spermatozoa indicated epididymal origin. The concentration of fructose was lowest in the first fraction of the semen and increased to a maximum in the final portion. Prostaglandins E and F and N-acetylaminosugar values closely followed this pattern, indicating that these components originate in the seminal vesicles. The concentration of spermatozoa was high in the first 2 fractions, decreasing to a minimum in the final fraction. The distribution of GPC was similar to that of the spermatozoa, indicating that the epididymis secretes this compound. Inositol levels were similar in all fractions, indicating that it is probably present in epididymal, vesicular, and prostatic fluid. Human spermatozoa were unable to utilize N-acetylglucosamine or inositol. High concentrations of some prostaglandins (100 µg/ml of PGF1(a), 15S 15 met. F2(a), PGA1, and PGA2) depressed the endogenous oxygen uptake of human spermatozoa.

The origin of glycerylphosphorylcholine (GPC), N-acetylaminosugar, inositol, and prostaglandins in human seminal plasma was investigated by correlating the concentration of these components in split ejaculates with known marker constituents. Fructose and acid phosphatase were selected as markers of the secretory activity of the seminal vesicles and prostate gland, respectively, and spermatozoa indicated epididymal origin. The concentration of fructose was lowest in the first fraction of the semen and increased to a maximum in the final portion. Prostaglandins E and F and N-acetylaminosugar values closely followed this pattern, indicating that these components originate in the seminal vesicles. The concentration of spermatozoa was high in the first two fractions, decreasing to a minimum in the final fraction. The distribution of GPC was similar to that of the spermatozoa, indicating that the epididymis secretes this compound. Inositol levels were similar in all fractions, indicating that it is probably present in epididymal, vesicular, and prostatic fluid. Human spermatozoa were unable to utilize N-acetylglucosamine or inositol. High concentrations of some prostaglandins (100 µg/ml of PGF1a, 15S 15 met. F2a, PGA1, and PGA2) depressed the endogenous oxygen uptake of human spermatozoa. © 1980 Informa UK Ltd.

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 nonparturient 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.

This paper reports the first metabolic study of marsupial spermatozoa. The oxidative metabolism of the spermatozoa of the Australian brush-tailed possum (Trichosurus vulpecula) was examined using a micro Warburg system. Semen was collected by electro-ejaculation and washed twice in Ca2+ free Krebs-Ringer-phosphate buffer containing antibiotics (KRPA). Washed spermatozoa suspended in fresh KRPA, were then incubated for 3 hours at 37° C in the presence and absence of added substrates (4 mM). The exogenous substrates tested were N-acetylglucosamine, glucosamine, and glucose. Small quantities of radioactively labeled [14C] substrates were included in the incubation media to allow measurement of substrate oxidation. Although the respiratory rate varied considerably between semen pools (replicate experiments), the relationship between total oxygen consumption (measured manometrically), and oxygen consumption accounted for by exogenous substrate utilization (calcuated from radioactivity recovered in the respiratory CO2) was remarkably consistent. Oxidation of exogenous substrate accounted for 49¿54% of the oxygen consumption, depending on the substrate used. There was, however, no evidence that addition of these substrates stimulated the respiratory rate over that found when no substrate was added. Lactate formation accounted for the greater part of exogenous substrate consumed. Copyright © 1978 Alan R. Liss, Inc.

1. 1. Despite large gaps in the literature of semen biochemistry, especially of wild species, recent studies of the carbohydrate biochemistry of marsupial semen allow an approach to the question of the origin and evolution of mammalian seminal plasma. 2. 2. The marsupials are a very important part of an overall picture of seminal plasma evolution because they are the end product of an alternative line of mammalian evolution genetically separated from the eutherians for over 100 million years. 3. 3. The present paper discusses the accessory sex glands and seminal sugars of mammals from a comparative point of view in the light of present knowledge of evolutionary relationships and suggests possible schemes of evolution of mammalian seminal plasma. © 1976.

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 3 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.

Despite considerable activity in the field of semen biochemistry over the last thirty years no generalization about the function of the myriad of seminal plasma constituents has emerged. As a result, some have commented, if not in print, that the seminal plasma has no essential function at all. The present paper examines the literature in the light of the premise that the answer to the question of the role of seminal plasma constituents will require an overall view of each species' reproductive physiology as it relates to such events and processes as gamete production, copulation, gamete transport and the environment within the female tract. Guidelines are suggested that should be followed when attempting to suggest a role or roles for seminal constituents. © 1975.

The reproductive tracts of males from eight species of Australian marsupial were examined (Macropus eugenii, Potorous tridactylus, Sminthopsis crassicaudata, Antechinus stuartii, Pseudocheirus peregrinus, Trichosurus vulpécula, Isoodon macrourus, and Perameles nasuta). The prostate glands of these species were found to be of two shapes, carrot-like or heart-like. From one to three pairs of Cowper's glands were observed; these were mostly bulbous in shape but some were kidney-shaped. Both prostate and Cowper's glands were tubular in structure with the glandular tubules lined by a simple columnar epithelium. The glandular tubules of Cowper's glands were of much larger diameter than those of the prostate. The prostate glands were segmented, and this segmentation was usually shown by variations in the height and staining reactions of the tubular epithelium and in the volume of connective tissue between glandular tubules. Differences in microanatomy between pairs of Cowper's glands were far less than those between prostate segments. Mucosubstance appeared to be the major contribution of the prostate to the seminal plasma. This mucosubstance was mainly neutral, with glycogen largely absent. The present results indicate that the Cowper's glands secrete mucus but that various glands also contributed lipid and glycogen. © 1973 CSIRO. All Rights Reserved.

There is a growing need to manage marsupial populations as a means to mitigate economic and environmental damage and resolve animal welfare problems. In Australia, the problems of population management are highly specific and localized. In contrast, in New Zealand the problem is the control of the many millions of widely-distributed brushtail possums which are the country's major vertebrate pest. The needs of the two countries are thus very different but immunocontraception may provide an effective and humane alternative to current lethal control strategies. This paper discusses the features of marsupial reproduction and development that offer potential as targets for immunocontraceptive interference, including: (1) sperm production and maturation in the male; (2) sperm transport and maturation in the female; and (3) sperm and egg antigens and the early embryo. Some of these antigen targets are shared with eutherian mammals but others are likely to be unique to marsupials.