Postnatal development of the reproductive system in the grey short-tailed opossum, Monodelphis domestica.

Postnatal phenotypic sex differentiation has been investigated in a laboratory marsupial, Monodelphis domestica, as part of a larger study to resolve apparent discrepancies between eutherian and marsupial mammals. These include the formation of sex-specific structures in marsupials prior to gonadal differentiation and the retention in both sexes of structures which are sex-specific in eutherians. The time-course and nature of differentiation was investigated in 131 specimens ranging in age from the day of birth to 56 days. Patent wolffian ducts extend to the urogenital sinus in both sexes at birth, while müllerian ducts are identified on day 1 and grow in a cranio-caudal direction to reach the urogenital sinus on day 6. The male müllerian duct shows signs of regression at its cranial end on day 10 and throughout its length on day 12; its lumen has completely disappeared by day 15. By this time the epididymis and vas deferens have developed from the wolffian duct; their histological differentiation occurs between days 26 and 56. Prostatic buds are identifiable in tissue surrounding the male urethra on day 14. In the female, the wolffian duct is larger than the müllerian duct until day 14; thereafter the wolffian duct begins to regress at its cranial end, disappearing by day 17, whereas the müllerian duct begins to enlarge, converging with its fellow at the urogenital sinus by day 19. Lateral vaginae, vaginal culs-de-sac, uteri and oviducts have differentiated from the müllerian ducts by day 25. Gonads of both sexes are elongated in shape at birth, attached along the medial aspect of the large mesonephroi in the abdominal cavity. However, from day 3 onwards the testis becomes more rounded than the ovary. Degeneration of the male mesonephros begins about day 10 and is almost completed by day 19; the female mesonephros is still relatively large at day 14 though it too has almost disappeared by day 19. By postnatal day 13 the abdominal phase of testis descent is underway and the inguinal phase begins at day 15. Testes have reached the scrotal sac by day 24 and achieve their final position at the base of the scrotum by day 28. In summary, postnatal reproductive tract development and gonadal descent has been examined in this important biomedical model, where differentiation of the wolffian and müllerian ducts takes place after gonadal differentiation, according to the normal eutherian pattern.

The role of androgens in development of the scrotum of the grey short-tailed Brazilian opossum ( Monodelphis domestica).

In eutherian mammals, sex differentiation is initiated by expression of the testis-determining gene on the Y chromosome. Subsequent phenotypic development of the reproductive tract and genitalia depends on the production of hormones by the differentiated testis. In marsupials the mechanisms of phenotypic development may vary from this pattern, as differentiation of the scrotal primordia has been shown to occur before that of the gonad. Thus, the development of the scrotum in the marsupial has been regarded as an androgen-independent process. We have sought to clarify the ontogeny of scrotal development and the appearance of androgen receptor immunoreactivity by examining Monodelphis domesticaembryos/pups from 1 day prior to birth until 2 days after birth. We have also used immunocytochemistry to determine the expression of the key steroidogenic enzyme 3beta-hydroxysteroid dehydrogenase as an indicator of when the developing gonad may be capable of synthesizing androgens. Expression of this enzyme was first detected in the gonads and adrenals of both sexes 1 day prior to birth and before the appearance of scrotal bulges. Androgen receptor immunoreactivity was detected in the scrotal anlagen of male opossum pups as early as 1 day following birth. This finding is significantly earlier than previous reports and coincides with the appearance 1 day after birth of distinct scrotal bulges. Androgen receptor immunoreactivity was also observed in the genital tubercles of male pups, but not female pups, 2 days after birth. These results suggest that androgens may play an important role in the development of the male genitalia at a much earlier stage than that indicated by previously published work and that scrotal development in this species may not be androgen-independent.

Species differences in the ovarian distribution of 3beta-hydroxysteroid dehydrogenase/delta5-->4 isomerase (3beta-HSD) in two marsupials: the brushtail possum Trichosurus vulpecula and the grey, short-tailed opossum Monodelphis domestica.

The ovarian distribution of the steroidogenic enzyme 3beta-hydroxysteroid dehydrogenase/delta(5-->4) isomerase (3beta-HSD) was investigated by immunocytochemistry in two marsupial species throughout the reproductive cycle, using a rabbit polyclonal antibody raised against human placental 3beta-HSD. In the polyoestrous and polyovular South American opossum Monodelphis domestica, immunostaining was positive for 3beta-HSD in the adrenal cortex, the ovarian interstitial tissue, the corpus luteum and the granulosa cells of antral and atretic follicles. The theca interna was weakly positive for 3beta-HSD, but only in late preantral to early antral stages of follicular development. The adrenal medulla and smaller preantral follicles were completely negative for 3beta-HSD. In contrast, in the polyoestrous and monovular Australian brushtail possum Trichosurus vulpecula, immunostaining showed a strong positive reaction for 3beta-HSD in the theca, whereas the granulosa layer remained predominantly negative for 3beta-HSD except in the largest follicles. The atretic follicles were completely negative for 3beta-HSD. The ovaries of pregnant animals contained grossly enlarged, persistent, antral follicles, which reacted positively for 3beta-HSD. The function of these follicles in T. vulpecula and the 3beta-HSD-positive atretic follicles in M. domestica has not been determined. The differences between the two marsupials represent species variations. The situation in M. domestica does not represent a marsupial-eutherian dichotomy as previously conjectured.

Postnatal development of Leydig cells in the opossum (Monodelphis domestica): an immunocytochemical and endocrinological study.

This study involved characterization of Leydig cells of the opossum Monodelphis domestica, functionally by immunocytochemical identification of the enzyme 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and by measurement of testosterone levels using RIA. Immunostaining for 3 beta-HSD was first detected in a few Leydig cells on Day 16, was increased by Day 24, reached a peak at 4 mo, and was present even in senescent (3 yr) animals. Plasma testosterone was first measurable (0.35 nM) at prepuberty (3.5 mo). Prior to that, plasma testosterone concentrations were uniformly below the level of detection (< 0.3 nM) in both sexes from Day 5 to 2.5 mo. By 4 mo (puberty), plasma testosterone levels in males had risen significantly to 1.53 +/- 0.35 nM, continuing to increase to 1.79 +/- 0.4 nM at 6 mo and peaking at 2.71 +/- 0.29 nM in the adult (1-2 yr). Ovarian testosterone concentrations were consistently lower than those in the testis, as were those of adrenals of both sexes. Thus the testis would appear to be the major source of androgen production throughout life in this species. Our immunocytochemical study suggests that in Monodelphis, puberty is reached at 4 mo, and this was further supported by a rise in circulating testosterone levels at this time.

Migration of primordial germ cells to the developing gonadal ridges in the tammar wallaby Macropus eugenii.

Primordial germ cells (PGCs) of the tammar wallaby Macropus eugenii have a distinctive morphology and stain positively for alkaline phosphatase. PGCs are identifiable in embryos with 12 somites, on about day 17 of the 26.5 day gestation period, when they are located in all three germ layers of the developing embryo and in the endoderm of the bilaminar and vascular (trilaminar) yolk sac membranes. PGCs are positive for alkaline phosphatase (ALP) at least between days 17 and 22 of pregnancy. In whole mounts on day 17, three groups of cells positive for ALP occur: about 40 just caudal to the neural tube, and about 20 distributed on either side of the last three somites. By day 21, there are about 150 PGCs in the newly formed gonadal ridges and 275 in the mesenteries. On days 21-22, there are PGCs in the umbilical mesoderm, the dorsal mesentery and the coelomic angles between the dorsal mesentery and the mesonephroi. On day 22, most ALP-positive PGCs are located in the dorsal mesentery, where they occur in groups. They apparently do not migrate through the hindgut endoderm, but occasional PGCs are seen in sites such as the mesonephros, the adrenals, the blood vessels of the yolk sac and in the vicinity of the dorsal aorta and dorsal nerve cord. Between day 23 and day 25, 1 day before birth, most of the 3200-4000 PGCs complete their migration to the gonadal ridges. Although there are marked differences between embryogenesis of tammars and mice, development and the pattern of migration of PGCs in this marsupial mammal are similar to that of eutherian mammals.

Development of the ovary in the brushtail possum Trichosurus vulpecula (Marsupialia).

The urogenital region of 25 fetuses and 75 pouch young, ranging in age from newborn to 103 days (d) in development, was examined in serial histological sections. The rete ridges formed the anterior extensions of the gonadal ridges and gave rise to the rete system and gonads respectively. Sexual differentiation of the ovary commenced 2.5 d after birth, when 2 cell types appeared: the larger of these then clumped together to form the medullary cords, while the smaller cells gave rise to the stroma. Primordial germ cells were still migrating, dividing and populating the peripheral gonadal regions on d 8. Cortex and medulla were distinguishable by d 12, when a thick fibrous zone separated them. The cortex was augmented by cells from the mesothelium. The rete ovarii developed from cell condensations within the rete ridges, made secondary contact with the mesonephroi and penetrated the ovaries but did not contribute to the granulosa cells. It is concluded that, contrary to the situation in most eutherians, in Trichosurus, as in other marsupials examined, the mesonephros does not contribute to rete formation, or to the granulosa cells, which appear to arise from the medullary cords.

Testis development in the opossum Monodelphis domestica.

Testis development in the grey short-tailed opossum, Monodelphis domestica, was investigated by light and electron microscopy in 180 animals. On the day of birth, half the karyotyped males were found to have histologically differentiated testes. By day (d) 1 testicular cords were clearly distinguished in all XY gonads and the tunica albuginea was fully developed. At this stage the large and pale primordial germ cells could be differentiated from dark pre-Sertoli cells. From d 3 the testis became progressively rounded and testicular cords were surrounded by peritubular cells. Leydig cells were then distinguishable by the expected ultrastructural features of steroidogenically active cells, showing abundant vesicles of SER, extensive mitochondria with tubular cristae and numerous lipid inclusions. Subsequently these cells formed clusters and were surrounded by envelope cells until wk 12. Testes were located in the abdomen, attached to the large mesonephroi, until d 24 after birth when they began their descent to the scrotal sac. From 7 wk the interstitial tissue became less cellular. At the prepubertal stage (12 wk), the seminiferous tubules lacked lumina. Leydig cell cytoplasm was electron-dense with increased amounts of SER forming parallel profiles. By 4 mo (pubertal stage), seminiferous tubules were patent and various spermatogenic stages, including spermatozoa, were seen for the first time. Leydig cells then greatly outnumbered other interstitial tissue cells and were closely-packed around blood vessels but no longer clustered by envelope cells; their SER was very highly organised into masses of parallel arrays and lipid inclusions were reduced. In the adult (1 y) Leydig cells reached their greatest size; their morphological features resembled those seen at 4 mo except that lipid inclusions were sparse. In ageing Leydig cells (2-3 y), large amounts of SER were present but disorganised.

Mammalian oocyte organelles with special reference to pleomorphic mitochondria and vacuole formation in marsupials.

Marsupial oocytes are larger and have a thinner zona than eutherian oocytes; and the ooplasm becomes almost completely filled with empty-looking vacuoles the contents of which have, so far, defied histochemical analysis. In the opossum, Monodelphis domestica, apart from orthodox mitochondria, a 'hooded' form is found occasionally in young primary oocytes, and a novel 'spiked' form-which is very elongate and has longitudinally-running filaments attached to the outer membrane--is found in mature oocytes. On the genesis of the ooplasmic vacuoles in mammals, information is available only for two marsupials. In Monodelphis, the vacuoles originate from endoplasmic, endocytotic and Golgi vesicles which generate multivesicular bodies; these give rise to the vacuoles. For the bandicoot, Isoodon macrourus, evidence is presented for the formation of the vacuoles from enlarged, transformed mitochondria which undergo a complex evolution during development. Primordial oocytes of Isoodon contain three ooplasmic localizations--a paranuclear complex, a vesicle microtubule complex and an aggregate of tubular cistenae-which have not been described for other mammalian oocytes. The origin, fate and function of these organelle localizations is unknown. In this paper, problems with respect to the definition of 'yolk' are described and the extent of our ignorance concerning oocyte organelles is discussed.

Culture of preantral ovarian follicles in the grey, short-tailed opossum, Monodelphis domestica.

A culture system designed to support the development of individual preantral mouse ovarian follicles has been employed to study follicle growth in the New World marsupial species Monodelphis domestica. Preantral follicles were isolated mechanically and cultured individually in microdrops under oil. Preliminary results indicate that follicle growth was positively correlated to the concentration of follicle-stimulating hormone (FSH) provided, with 1.0-1.5 IU FSH mL-1 producing the best results. Incubation at the body temperature of M. domestica (33 degrees C) was found to be preferable to that at 37 degrees C. The culture system was able to support follicle growth; however, despite follicles exceeding the size when antrum formation occurs in vivo, they remained preantral.

Gonadal development in the opossum, Monodelphis domestica: the rete ovarii does not contribute to the steroidogenic tissues.

The gonads of 273 animals, ranging in age from newborn to adult, were examined in serial histological sections. Primordial germ cells were located in the hindgut, dorsal mesentery and gonadal primordia of neonates but were alkaline phosphatase negative. The testis differentiated between d 13 of gestation and birth, about half a day later. Testis cords, composed of pre-Sertoli cells and containing germ cells, were distributed peripherally in the gonad. Ovarian differentiation began on d 6, when an ill defined cortex and medulla became discernible. Meiosis commenced on d 14, medullary cords formed from blastema cells by d 26 and gave rise to granulosa cells around d 29. The rete ovarii was first observed in the hilar region of the gonad primordium. It penetrated maximally two thirds of the ovarian length between d 26 and d 29. It is concluded that, at least postnatally, the mesonephros does not contribute to the rete ovarii and that the granulosa cells are derived not from the rete but from the medullary cords.

Differentiation of the gonads and initiation of mammary gland and scrotum development in the brushtail possum Trichosurus vulpecula (Marsupialia).

The first appearance of the mammary and scrotal primordia and the sexual differentiation of the gonads of the brushtail possum, Trichosurus vulpecula, are described. Primordial germ cells were first observed, in fetuses of 7.5 mm crown-rump length, in the gonadal ridges and migrating up the dorsal mesentery. Mammary primordia were first observed in fetuses of 11 mm, and scrotal primordia in those of 12 mm crown-rump length. These structures were diagnostic of female and male brushtail possums respectively. Processus vaginales and gubernacula showed sexual dimorphism, being better developed and appearing earlier in males than in females. Sexual differentiation of the gonads occurred after the appearance of mammary and scrotal primordia, the testes being first recognisable in a 14.5-mm fetus and the ovaries postnatally. Birth occurred between the stages of 14 and 15 mm crown-rump length. These observations appear to indicate that the development of mammary and scrotal primordia are not under gonadal hormonal control, but under direct genetic control, as suggested for the tammar wallaby by previous authors.

Early differentiation of the testis in the native cat, Dasyurus viverrinus (Marsupialia).

The development of the testis in the native cat Dasyurus viverrinus (Marsupialia) is described, from Day 3 to 2.5 months post partum. The gonad rudiment consists of a mass of undifferentiated blastema cells of mesenchymal origin. The primordial germ cells populate the rudiment around the neonatal period. On Day 3 two cell types autodifferentiate from the gonadal blastema (pre-Sertoli and stroma cells) and the tunica albuginea begins to form. Pre-Sertoli cells are large, pale, cells which encompass the primordial germ cells and come to form a zone just beneath the tunica albuginea. By the end of the second week the cell complex has become transformed into a series of sex cords which encircle the gonad rudiment peripherally and converge on the rete cord in the hilar region. The stroma cells are fibroblast-like cells and occupy the central portion of the rudiment; by the eighth week, Leydig cells are appearing among them. No evidence was found for mesothelial invaginations or mesonephric contributions to the gonad. The classically recognised regions of 'cortex' and 'medulla' cannot be recognised and the seminiferous tubules differ in arrangement from that in eutherians.

Further observations on the potoroo (Potorous tridactylus) in captivity.

The maintenance and breeding performance of potoroos in captivity over a 7-year period is described. By removing offspring from the pouch and allowing 29 days for completion of embryonic development and birth to occur, pouch young of known ages can be obtained.

Observations on the primordial germ cells of bandicoots (Peramelidae, Marsupialia).

Characteristically, bandicoot primordial germ cells are large, lightly staining cells with vesicular nuclei in which the chromatin is clumped beneath the nuclear envelope. Rounded, lightly staining cells in the yolk sac wall of 9 1/2 days old embryos are tentatively identified as primordial germ cells; as they lack other primordial germ cell attributes it is suggested that they may be prospective primordial germ cells in the process of differentiation. The primordial germ cells reach the prominent gonadal rudiments by migration through the hindgut and dorsal mesentery, as in eutherian mammals. In 9 1/2 days old embryos the primordial germ cells are found in the hindgut endoderm and mesoderm. By day 11 they are migrating in the dorsal mesentery. During migration the primordial germ cells undergo both division and attrition. Primordial germ cells reach the gonadal ridges around the perinatal period. No sexual differences are evident at this stage. As the gonadal ridges develop before the primordial germ cells reach their vicinity their development is clearly independent of the primordial germ cells.

Observations on the primordial oocyte of the bandicoot Isoodon macrourus (Peramelidae, Marsupialia).

An ultrastructural study of bandicoot primordial follicles and oocytes was undertaken, as information on this subject is lacking in marsupials. Conspiculous features of the ooplasm are a paranuclear complex (PNC), a vesicle-microtubule complex (VMC) and an aggregate of tubular cisternae (ATC). The PNCappears as one or, more rarely, several homogeneous eosinophil bodies at the light microscope level. Ultrastructurally it is particulate, consisting of five distinct types of bodies, most of which are composed of concentric fibrillar whorls, but others appear homogeneous, granular or crystalline. Embedded among the particles is a group of Golgi-like vesicles. The bandicoot PNC-unlike similar structures found in the ooplasm of a variety of vertebrates, and known variously as "Balbiani body", "yolk nucleus", etc.-totally lacks nitochondria. The VMC consists of vesicle-like organelles which may be drawn out into tubular extensions, while the bounding membrane may be decorated with granules. Bundles of microtubules ramify between the vesicles, from which they appear to originate. The vesicles contain a matrix similar to the ooplasm. The ATC contains a homogeneous substance more electron-dense than the surrounding ooplasm. 'Dense bodies' occur in the cytoplasm of both the follicle cells and the oocytes. These are elongate membrane-bound organelles, circular in cross section. An electron-dense core is separated from the membrane by a narrow, less dense zone. The genesis and morphogenetic significance of these various organelles is unknown.

Anomalous litters in hybrid mice and the retention of spermatozoa in the female tract.

Suspected superfetation was investigated in a Glasgow hybrid stock of mice. The male was removed either (i) a few days before parturition, or (ii) immediately after mating and on 23 and 25 occasions, respectively, a second litter was born. Members of the anomalous litters were inbred for 10 generations, but the incidence of supernumerary litters did not increase beyond 2-5%. The anterior part of over 500 reproductive tracts, at various stages of pregnancy and after parturition, were serially sectioned but a second set of embryos was not found. The second gestation was of normal length and superfetation was not therefore considered to be the cause of the anomalous litters. In two females, one non-pregnant and one pregnant, spermatozoa were found in the uterus and oviducts 8 days after mating and in distended uterine glands 15 days after mating respectively. It is concluded that the anomalous litters were derived from the fertilization of eggs ovulated at the post-partum oestrus by spermatozoa which had been retained in the female tract for at least 23 days.