A dual role for SHH during phallus development in a marsupial.

The mammalian phallus arises from identical primordia in both sexes and is patterned in part by the key morphogen Sonic hedgehog (SHH). We have investigated SHH and other morphogens during phallus development in the tammar wallaby. In this marsupial, testis differentiation and androgen production occurs just after birth, but it takes a further 50-60 days before the phallus becomes sexually dimorphic. One day before birth, SHH was expressed in both sexes in the urethral epithelium. In males, there was a marked upregulation of SHH, GLI2, and AR at day 50 postpartum, a time when testicular androgen production falls. SHH, GLI2, and AR were downregulated in female pouch young treated with androstanediol from days 24-50, but not when treatments were begun at day 29, suggesting an early window of androgen sensitivity. SHH, GLI2, and AR expression in the phallus of males castrated at day 23 did not differ from controls, but there was an increase in SHH and GLI2 and a decrease in FGF8 and BMP4 expression when the animals were castrated at day 29. These results suggest that the early patterning by SHH is androgen-independent followed by an androgen-dependent window of sensitivity and a sharp rise in SHH expression after androgen withdrawal at day 50.

Localization of transforming growth factor beta2 (TGFbeta2) and its receptors, TbetaRI and TbetaRII, in uteri and blastocysts of the stripe-faced dunnart (Sminthopsis macroura) during gastrulation.

This study describes for the first time the spatial and temporal distribution of a growth factor and its receptors in uteri and conceptuses of a marsupial species during the peri-gastrulation period. Uteri (gravid and non-gravid) and blastocysts from 40 female stripe-faced dunnarts (Sminthopsis macroura) were collected over the peri-gastrula period (days 6.0-8.5) and stained immunohistochemically for transforming growth factor beta2 (TGFbeta2) and its receptors, TbetaRI and TbetaRII, to determine possible roles for TGFbeta2 in marsupial embryonic development. The events that occurred during the period examined included the appearance and proliferation of hypoblast and mesoderm, primitive streak and node formation, and early neurulation. Differences in TGFbeta2 quantities between gravid and non-gravid uteri reflect differences in uterine morphology, indicating a role for TGFbeta2 in endometrial remodelling. In blastocysts, large quantities of all three proteins in the trophectoderm during the node stage coincide with both blastocyst expansion before implantation and the appearance of multinucleated cells, indicating that TGFbeta2 plays a role in conceptus elongation and trophectoderm differentiation. In contrast, lack of TbetaRII in blastocysts during hypoblast formation and proliferation negates any role for TGFbeta2 in these processes, as both receptors are required for a response to TGFbeta2. High concentrations of TGFbeta2 but low concentrations of TbetaRII in blastocysts during early primitive streak formation indicate that paracrinal embryo-maternal signalling may be occurring, as blastocysts cannot respond to TGFbeta2 at this stage, but uteri could. A similar situation, but reversed, also occurs during primitive node formation.

Induced ovulation, mating success and embryonic development in the stripe-faced dunnart, Sminthopsis macroura.

Induced ovulation resulting in normal embryos is rare in marsupials. In this study natural and induced ovulations were compared in mature Sminthopsis macroura (n = 122). Comparison of maturation of preovulatory oocytes by ovarian histology and examination of oocytes removed from developing follicles in 12 ovaries of 23 animals receiving 0.058 iu equine serum gonadotrophin (eSG) g(-1) with ovaries of 12 animals undergoing natural cycles showed that oocyte maturation was significantly more irregular when it was induced (P < 0.001). Postovulatory stages were examined by estimating the number of eggs ovulated from ovarian histology, and by counting oviduct and uterine contents recovered after ovulation. S. macroura receiving 0.087 iu eSG g(-1) (n = 34), administered as one (n = 17) or two (n = 17) injections, were significantly (P < 0.05) more likely to ovulate (74%), mate (80%) and have conceptuses (66%) than were animals receiving 0.058 iu eSG g(-1) (12, 53 and 0%, respectively) (n = 17), and the values were similar to those in animals (n = 36) undergoing natural cycles (100, 81 and 56%, respectively). Induced ovulation using 0.087 iu eSG g(-1) yielded significantly (P < 0.05) more oocytes per ovary (20.8 +/- 8.5; combined data) than did ovulation in animals undergoing natural cycles (13.7 +/- 3.2) (ANOVA, t test). The responses of animals induced in different phases of the oestrous cycle with 0.087 iu eSG g(-1) were not significantly different (ANOVA) with respect to the number of corpora lutea per ovary, conceptuses per animal or days to ovulation after injection. However, the proportion of females that responded after receiving 0.058 iu eSG g(-1) in the luteal phase was significantly different from that in animals treated with the same dose in the intermediate phase (P < 0.01) and in non-cyclic females treated with 0.058 iu eSG g(-1) (P < 0.02). The main benefits of the treatment were that normal embryos resulted and that 70-78% of non-cyclic animals could be induced to ovulate.

A staging scheme for assessing development in vitro of organogenesis stage embryos of the stripe-faced dunnart, Sminthopsis macroura (Marsupialia: dasyuridae).

The inaccessibility of mammalian organogenesis stage embryos has precluded their widespread use in embryological and teratological studies. As organogenesis occurs during the last 1.5 days of the 10. 7 days of gestation in the stripe-faced dunnart (Sminthopsis macroura), the aim of the present study was to investigate whether day 9 and day 10 embryos and fetuses could be grown to term in vitro. High glucose Dulbecco's modified Eagle's medium with 10% fetal calf serum (FCS) supported embryonic growth for various periods of time, some to within 5 h of the predicted time of parturition. A roller culture system maintained at 35 degrees C was used to incubate organogenesis stage embryos (n = 43). Nine unincubated (control) embryos were either fixed for microscopic analysis or frozen for microprotein determination. The results of the present study indicate that with some optimization of the culture conditions (increasing oxygen in the gas phase in the culture tubes, replacing FCS with rat serum), it might be possible for organogenesis stage S. macroura embryos to be grown to term. A scoring scheme for assessing morphological development was devised for use as a standard in staging organogenesis stage embryos. This scheme reflects the highly compressed schedule of developmental events that occurs mainly during day 9 of gestation in S. macroura embryos. In comparison, during embryogenesis in Didelphis virginiana these developmental events occur from day 8 to day 10.5 of gestation, and birth occurs on day 13.