Mammary glands in male marsupials: I. Primordia in neonatal opossums Didelphis virginiana and Monodelphis domestica

Neonates of the American didelphid marsupials Didelphis virginiana and Monodelphis domestica were sexed by karyotype and histologically examined on the day of birth. Mammary anlagen were found in both sexes of both species, but the neonatal males had less than one-third of the full female complement of mammary glands. Male neonates of both species also had paired scrotal bulges anterior to the genital tubercle but these were never present in females, once again raising the question of whether the pouch and scrotum are homologous structures. Mammary anlagen are not found in male neonates of the Australian marsupial species so far studied, which suggests a dichotomy in the control of some aspects of sexual differentiation in the two marsupial lineages.     

Proffered papers 9.30–10.00 Monday 15 September 2003

Both the original Bethesda system and the current UK classifications of cervical cytology have proved robust but each has a major weakness in the area of abnormalities of uncertain significance. Cytologists recognize that sometimes it is simply impossible to differentiate between reactive and dyskaryotic material. For this reason, the Australian version of the Bethesda system introduced a new category of 'high grade inconclusive' with a recommendation for referral to colposcopy. Approximately 60% of such cases are found to have high grade lesions at colposcopy (Schoolland M, Sterrett G, Knowles S et al .). The present UK system even with the proposed changes requires of the pathologist, a decision as to whether such cases are probably high grade (=a report of moderate dyskaryosis) or not (= a report of borderline). This continues to ignore the fact that sometimes you just cannot tell, even on review. We have taken a consecutive series of 50 referral smears, reported as moderate dyskaryosis, where the histological outcome (by loop cone) is known. These cases were rescreened and then reviewed blind by a pathologist with extensive experience of the Australian NH & MRC modified Bethesda system. On review, the material was reclassified along NH & MRC lines. The results were compared with the biopsy findings in order to determine whether the category of 'inconclusive' might be of value in the context of the NHSCSP.     

Differential expression of WNT4 in testicular and ovarian development in a marsupial

Background  

WNT4 is a key regulator of gonadal differentiation in humans and mice, playing a pivotal role in early embryogenesis. Using a marsupial, the tammar wallaby, in which most gonadal differentiation occurs after birth whilst the young is in the pouch, we show by quantitative PCR during early testicular and ovarian development that WNT4 is differentially expressed ingonads.     

Identification of a Novel PNMA-MS1 Gene in Marsupials Suggests the LTR Retrotransposon-Derived PNMA Genes Evolved Differently in Marsupials and Eutherians

Two major gene families derived from Ty3/Gypsy long terminal repeat (LTR) retrotransposons were recently identified in mammals. The sushi-ichi retrotransposon homologue (SIRH) family comprises 12 genes: 11 in eutherians including Peg10 and Peg11/Rtl1 that have essential roles in the eutherian placenta and 1 that is marsupial specific. Fifteen and 12 genes were reported in the second gene family, para-neoplastic antigen MA (PNMA), in humans and mice, respectively, although their biological functions and evolutionary history remain largely unknown. Here, we identified two novel candidate PNMA genes, PNMA-MS1 and -MS2 in marsupials. Like all eutherian-specific PNMA genes, they exhibit the highest homology to a Gypsy12_DR (DR, Danio rerio) Gag protein. PNMA-MS1 is conserved in both Australian and South American marsupial species, the tammar wallaby and grey short-tailed opossum. However, no PNMA-MS1 orthologue was found in eutherians, monotremes or non-mammalian vertebrates. PNMA-MS1 was expressed in the ovary, mammary gland and brain during development and growth in the tammar, suggesting that PNMA-MS1 may have acquired a marsupial-specific function. However, PNMA-MS2 seems to be a pseudogene. The absence of marsupial orthologues of eutherian PNMA genes suggests that the retrotransposition events of the Gypsy12_DR-related retrotransposons that gave rise to the PNMA family occurred after the divergence of marsupials and eutherians.     

Influence of progesterone and oestradiol-17 beta on blastocysts of the tammar wallaby (Macropus eugenii) during seasonal diapause

Forty tammar wallabies, presumed to be carrying quiescent blastocysts, were injected with progesterone and oestradiol alone, or in combination, during seasonal quiescence when the corpus luteum is inactive. Plasma progesterone concentrations were increased to values equivalent to those of late pregnancy for the duration of the treatment in progesterone-treated groups but otherwise remained at values equivalent to seasonal quiescence. Tammars treated with low doses of oestradiol showed no measurable increase in plasma oestradiol concentrations but in those treated with high doses plasma concentrations were increased to oestrous levels. At autopsy on Day 18 after the start of treatment the embryos and reproductive tracts were assessed. While progesterone alone caused reactivation of about 50% of the embryos, blastocysts in tammars treated with oestradiol alone remained in diapause (low dose) or disappeared from the uterus (high dose): 2 blastocysts collapsed after some slight expansion. No synergistic effect on pregnancy was noted in tammars receiving both oestradiol and progesterone. We conclude that oestrogen alone is not capable of stimulating normal growth of blastocysts, and its role during early pregnancy in tammars remains unclear.     

Experimental manipulation of sexual differentiation in wallaby pouch young treated with exogenous steroids

We have investigated the effects of androgen or oestrogen treatment of female or male tammar wallabies from the day of birth, when the gonads are histologically undifferentiated, to day 25 of pouch life, when the gonads and the Wolffian and Müllerian ducts have differentiated and the testes have migrated through the inguinal canal. Female tammars treated with testosterone propionate (24-50 mg kg-1 day-1) orally for 25 days had enlarged Wolffian and Müllerian ducts. Mammary and pouch development, however, was indistinguishable from that of control females. The treatment had no apparent effect on ovarian development, or on ovarian position in the abdomen. The phallus of males and females was similar in size, and neither experimental treatment had a significant effect on its size at day 25. Male tammars treated with oestradiol benzoate (1.2-2.5 mg kg-1 day-1) orally for 25 days had gross hypertrophy of the urogenital sinus. Testicular morphology was abnormal; many of the germ cells appeared necrotic, the seminiferous tubules were of reduced diameter, and there were few Leydig cells and increased amounts of fibrous tissue between the tubules. The cortex of these gonads contained some areas which had an ovarian appearance, lacking tubules and containing numerous germ cells. The Müllerian ducts of control males had regressed, but this was prevented by oestrogen treatment, suggesting an inhibition of either Müllerian Inhibiting Substance (MIS) production or its action. Normal testicular migration was inhibited in treated males; the testes remained high in the abdomen, similar in position to the ovaries of control females, whilst control males all had testes in the inguinal region. The gubernaculum and processus vaginalis of control males extended into the scrotum, but in treated males they terminated outside it. Oestrogen treatment had no effect on the size of the scrotum and did not induce mammary or pouch development. These experiments show that marsupials, like eutherians, have a dual hormonal control of Wolffian and Müllerian development. By contrast, the initial development of the mammary glands, pouch, gubernaculum and scrotum does not appear to be under hormonal control and is therefore likely to be autonomous and dependent on genotype.     

The effects of corpus luteum removal during gestation on parturition in the tammar wallaby (Macorpus eugenii).

Removal of the corpus luteum of pregnancy of the tammar wallaby interfered with successful parturition if carried out before Day 17 of the 27-day pregnancy. After removal at Days 17 and 21, 40% of animals gave birth but pouch young died with 24 h; if performed at Days 23 or 25, pouch young survived. However, surgery also affected sham-operated controls if performed between Days 15 and 21. In control animals, sodium pentobarbitone followed by halothane anaesthesia was the least disruptive anaesthetic procedure. This study shows that the corpus luteum has an essential role in parturition and subsequent survival of the neonate in the pouch.