A role for Msx genes in mammalian embryonic diapause.

Mammalian embryonic diapause is a reproductive phenomenon defined by the reversible arrest in blastocyst development and metabolic activity within the uterus which synchronously becomes quiescent to implantation. This natural strategy, evident in over 130 species across eight orders, can temporally uncouple conception from delivery until conditions are favorable for the survival of the mother and newborn. While the maternal endocrine milieu has been shown to be important for this process, the local molecular mechanisms by which the uterus and embryo achieve quiescence, maintain blastocyst survival and then resumes blastocyst activation with subsequent implantation in response to endocrine cues remains unclear. Here we review the first evidence that the proximal molecular control of embryonic diapause is conserved in three unrelated mammalian species which employ different endocrine programs to initiate diapause. In particular, uterine expression of muscle segment homeobox (Msx) genes Msx1 or Msx2 persists during diapause, followed by downregulation with blastocyst reactivation and implantation. Mice (Mus musculus) with conditional inactivation of Msx1 and Msx2 in the uterus fail to achieve diapause and reactivation. Remarkably, the mink (Neovison vison) and tammar wallaby (Macropus eugenii) share this pattern of MSX1 or MSX2 expression as in mice during delay - it persists during diapause and is rapidly downregulated upon implantation. Therefore, these findings were the first to provide evidence that there are common conserved molecular regulators in the uterus of unrelated mammals during embryonic diapause.

Trophoblast specialisations during pregnancy in the tammar wallaby, Macropus eugenii: a morphological and lectin histochemical study.

INTRODUCTION: The tammar wallaby has a short gestation (26.5 days) and vascular modifications to expedite transport during that brief pregnancy. Here we examine trophoblast structural attributes that would facilitate materno-fetal exchange. MATERIALS AND METHODS: Four specimens of Macropus eugenii between days 23 and 26 gestation were examined using electron microscopy and 24 lectins to characterise glycosylated secretions and their internalisation. RESULTS: Two trophoblast phenotypes were found, flattened cells generally in contact with the underlying uterine epithelium and giant cells associated with histiotrophe. The latter appeared to penetrate uterine clefts, occasionally detach and become necrotic. Lectin histochemistry and ultrastructure indicated the presence of many lysosomes and residual bodies especially in trophoblast giant cells; these contained glycans, mainly apically, which were also detected in secretions and cell debris. Trophoblast basal membranes bore extensive filopodia. Giant cells were less common in vascular trilaminar areas and here the trophoblast barrier became thinner near term. DISCUSSION: Loss of Maackia amurensis agglutinin binding suggested cleavage of terminal sialic acid residues as an early post-internalisation event in the trophoblast. Lectin staining indicated degradation occurred in an apical-basal direction, and the heavily glycosylated basal membrane appeared specialised for transport out of the cell. CONCLUSION: Granules seen ultrastructurally and histochemically, particularly in giant trophoblast cells of the bilaminar area, suggest that internalised histiotrophe is broken down here and nutrients transferred to the embryo via the specialised basal plasma membrane. The trilaminar vascular area contained mostly flattened trophoblast cells, supporting the suggestion that gaseous exchange is its primary function.

Post-natal imprinting: evidence from marsupials.

Genomic imprinting has been identified in therian (eutherian and marsupial) mammals but not in prototherian (monotreme) mammals. Imprinting has an important role in optimising pre-natal nutrition and growth, and most imprinted genes are expressed and imprinted in the placenta and developing fetus. In marsupials, however, the placental attachment is short-lived, and most growth and development occurs post-natally, supported by a changing milk composition tailor-made for each stage of development. Therefore there is a much greater demand on marsupial females during post-natal lactation than during pre-natal placentation, so there may be greater selection for genomic imprinting in the mammary gland than in the short-lived placenta. Recent studies in the tammar wallaby confirm the presence of genomic imprinting in nutrient-regulatory genes in the adult mammary gland. This suggests that imprinting may influence infant post-natal growth via the mammary gland as it does pre-natally via the placenta. Similarly, an increasing number of imprinted genes have been implicated in regulating feeding and nurturing behaviour in both the adult and the developing neonate/offspring in mice. Together these studies provide evidence that genomic imprinting is critical for regulating growth and subsequently the survival of offspring not only pre-natally but also post-natally.

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.

Sex-linked and autosomal microsatellites provide new insights into island populations of the tammar wallaby.

The emerging availability of microsatellite markers from mammalian sex chromosomes provides opportunities to investigate both male- and female-mediated gene flow in wild populations, identifying patterns not apparent from the analysis of autosomal markers alone. Tammar wallabies (Macropus eugenii), once spread over the southern mainland, have been isolated on several islands off the Western Australian and South Australian coastlines for between 10,000 and 13,000 years. Here, we combine analyses of autosomal, Y-linked and X-linked microsatellite loci to investigate genetic variation in populations of this species on two islands (Kangaroo Island, South Australia and Garden Island, Western Australia). All measures of diversity were higher for the larger Kangaroo Island population, in which genetic variation was lowest at Y-linked markers and highest at autosomal markers (θ=3.291, 1.208 and 0.627 for autosomal, X-linked and Y-linked data, respectively). Greater relatedness among females than males provides evidence for male-biased dispersal in this population, while sex-linked markers identified genetic lineages not apparent from autosomal data alone. Overall genetic diversity in the Garden Island population was low, especially on the Y chromosome where most males shared a common haplotype, and we observed high levels of inbreeding and relatedness among individuals. Our findings highlight the utility of this approach for management actions, such as the selection of animals for translocation or captive breeding, and the ecological insights that may be gained by combining analyses of microsatellite markers on sex chromosomes with those derived from autosomes.

The fetomaternal interface shows vascular hypoglycosylation in the tammar wallaby Macropus eugenii: comparison with a range of non-mammalian and eutherian placentae.

INTRODUCTION: Blood vessel glycosylation at the fetomaternal interface of four near-term specimens of tammar wallaby, Macropus eugenii, has been examined at days 23-26 of the 26.5 day pregnancy and compared with that of other species. METHODS: A panel of 23 lectins was used to compare vasculature in tammar with non-mammalian (shark, skink) and eutherian species at early and late gestation (camel, horse and alpaca), and term/near-term (cat, lion, dog, mink and elephant). RESULTS: Strikingly low levels of all the glycans tested, apart from sialic acids, were found in capillary endothelium of both the trilaminar omphalopleure and underlying surface endometrium of the tammar, though deeper endometrial vessels showed normally high levels of glycosylation. Only maternal vasculature of the mink placenta showed a comparable lack of expression. DISCUSSION: One reason for a reduced endothelial glycocalyx may be to facilitate diffusion of gases and nutrients as the tammar trophoblast lacks the indentation by overlying vessels that is seen in the other near-term placentae. Early epitheliochorial placentae of other species with equal diffusion distances to the tammar, showed normal vascular glycosylation. However, their pregnancies are much longer. CONCLUSION: The hypoglycosylation of tammar vessels at the fetomaternal interface may allow continued transfer of nutrients and gaseous exchange during the extremely rapid period of organogenesis which occurs during the short 26.5 day pregnancy of this marsupial. Given the short gestation period of the tammar, we suggest that a thinner endothelial glycocalyx has evolved to facilitate diffusion of gases and nutrients between the maternal and fetal compartments.

Development of the penile urethra in the tammar wallaby.

Hypospadias is increasingly common, and requires surgery to repair, but its aetiology is poorly understood. The marsupial tammar wallaby provides a unique opportunity to study hypospadias because penile differentiation occurs postnatally. Androgens are responsible for penile development in the tammar, but the majority of differentiation, in particular formation and closure of the urethral groove forming the penile urethra in males, occurs when there is no measurable sex difference in the concentrations of testosterone or dihydrotestosterone in either the gonads or the circulation [corrected]. Phalluses were examined morphologically from the sexually indifferent period (when androgens are high) to well after the time that the phallus becomes sexually dimorphic. We show that penile development and critical changes in the positioning of the urethra occur in the male phallus begin during an early window of time when androgens are high. Remodelling of the urethra in the male occurs between days 20-60. The critical period of time for the establishment urethral closure occurs during the earliest phases of penile development. This study suggests that there is an early window of time before day 60 when androgen imprinting must occur for normal penile development and closure of the urethral groove.

Fibroblast growth factor-9 in marsupial testicular development.

FGF9 is a member of the fibroblast growth factor (FGF) family and is critical for early testicular development and germ cell survival in the mouse. Fgf9 reinforces the testis determinant Sox9 and antagonizes Wnt4, an ovarian factor. To determine whether FGF9 has a conserved role in the mammalian gonad, we examined its expression in the gonads of a marsupial, the tammar wallaby Macropus eugenii, and compared it to WNT4 expression. Marsupial FGF9 is highly conserved with orthologues from eutherian mammals, including humans. FGF9 protein was detected in both the testis and ovary before sexual differentiation, but it subsequently became sexually dimorphic during the period of testicular differentiation. The protein was specifically enriched in the seminiferous cords of the developing testis in the Sertoli and germ cells. FGF9 mRNA expression was upregulated in the tammar testis at the time of seminiferous cord formation and downregulated in the developing ovary in an opposite profile to that of marsupial WNT4. These observations suggest that FGF9 promotes male fate in the early gonad of marsupials through an antagonistic relationship with WNT4 as it does in eutherian mammals.

Review: Marsupials: placental mammals with a difference.

The placenta is the most varied organ within the Mammalia. There are many similarities, as well as some differences, between the marsupial embryo and those of eutherian mammals. The most striking difference is the lack of the inner cell mass in the blastocyst which consists solely of a single layer of trophoblast cells. The trophoblast expands and eventually becomes part of the definitive chorio-vitelline placenta. The degree of functional differentiation between the vascular and non-vascular parts of the yolk sac placenta differs between species in the relative surface area that is attached to the endometrium, in trophoblast thickness, in yolk sac fusion with the luminal epithelium and most markedly in the degree of invasiveness. In marsupials, placental physiology has been best studied in the tammar wallaby. Despite the lack of invasion in the tammar, there is nevertheless maternal recognition of pregnancy in response to trophoblast formation. Contrary to popular opinion, the tammar placenta also elaborates hormones: at term it secretes prostaglandin F2alpha and accumulates cortisol, and it expresses genes for hormones such as growth hormone, IGF2 and relaxin. As in eutherian mammals, genomic imprinting is important for placental function. Despite the relatively short period of placentation, it is clear that the trophoblast and the placenta it forms are as important for successful pregnancy in marsupial as in eutherian mammals. Marsupials are certainly placental mammals. However marsupials have an additional trick in their pouches, with the physiologically sophisticated and extended lactation that has allowed them to exchange the umbilical cord for the teat.

Early expression of the androgen receptor in the Sertoli cells of a marsupial coincides with downregulation of anti-Müllerian hormone at the time of urogenital virilization.

Anti-Müllerian hormone (AMH), responsible for the regression of Müllerian ducts, is strongly expressed by eutherian fetal and postnatal Sertoli cells. Both AMH and testosterone levels are high during the period of fetal reproductive tract virilization which occurs largely in utero in eutherian mammals. Taking advantage of the fact that differentiation of the urogenital tract occurs after birth in marsupials, we studied the ontogeny and regulation of AMH in the tammar wallaby testis and related it to the expression of the androgen receptor in Sertoli cells. Testicular AMH expression was high between days 10-30 post partum, then fell to basal levels by day 60 and remained low until day 90, the oldest age examined. AMH expression was repressed by treatment of male pouch young with the potent androgen androstanediol. Thus, in the tammar, AMH expression decreases in response to androgen at the time of initial urogenital masculinization, in contrast to the situation in humans in which AMH is repressed by testosterone only at the time of puberty. The difference might be explained by the timing of androgen receptor expression which appears in tammar Sertoli cells at around day 40 of pouch life but only at a later developmental stage in eutherians.

Marsupial WT1 has a novel isoform and is expressed in both somatic and germ cells in the developing ovary and testis.

The Wilms' tumour 1 gene is essential for the formation of the mouse and human urogenital systems. We characterised this gene and examined its expression throughout gonadal development in a marsupial, the tammar wallaby. WT1 protein was detected in the Sertoli and granulosa cells of the developing testis and ovary, respectively. There was also strong immunostaining in the germ cells of both males and females at all stages of gonadal development. In the adult gonads WT1 appears to be dynamically regulated during spermatogenesis and oogenesis. Tammar WT1 has a novel isoform in which a portion of exon 1 is removed, partially deleting the RNA recognition motif (RRM). Despite its removal, WT1 still localised to RNA rich regions of the oocyte including speckled bodies within the nucleus, in the nucleolus and the perinucleolar compartment. This suggests that the RRM is not required for WT1 co-localisation with RNA. This is also the first report of WT1 in association with the perinucleolar compartment, important for RNA metabolism. Our data suggest that WT1 has a conserved function in both the somatic and germ cell lineages of the gonads of marsupials.

Placental function in two distantly related marsupials.

The biochemical composition of uterine and fetal fluids during pregnancy of the grey short-tailed opossum was compared with new and published data on the tammar wallaby. In the grey short-tailed opossum, there are three main phases of embryonic nourishment. During the first phase, the embryo obtains nutrients from uterine secretion transferred into the yolk sac. The amount of uterine secretion declines during the second phase up to the time of shell coat rupture. As a result, the protein concentration in yolk sac fluid also declines. During phase three, which begins with shell coat rupture, nutrients are predominantly available from the maternal blood. In the grey short-tailed opossum that lacks a vesicular, fluid-filled allantois, waste products such as urea are apparently stored in the yolk sac and from there pass into the maternal circulation across the invasive yolk sac placenta. In contrast, in the tammar wallaby, the main source of nutrients available to the late term fetus is glandular secretion that is complemented by substances from the maternal circulation via the chorio-vitelline placenta, and waste products are stored in the large, fluid-filled allantois.

Changes in semen quality and morphology of the reproductive tract of the male tammar wallaby parallel seasonal breeding activity in the female.

Changes in semen quality and morphology of the male reproductive tract were studied throughout the year in the highly promiscuous tammar wallaby. Body size, semen quality and gross morphology of the reproductive organs were assessed in adult males each month from January to November. The mean weight of males was similar in most periods sampled, but males were slightly heavier in the minor (P < 0.05) than the non-breeding season. Since body weight was correlated with weights of the testes, epididymides and accessory sex glands, organ weights were adjusted for body weight in subsequent analyses. In the major breeding season (late January/early February), when most females go through a brief, highly synchronized oestrus, the testes, prostate, Cowper's glands, crus penis and urethral bulb were heaviest, volume and coagulation of ejaculates were greatest, and sperm motility had increased. Semen samples collected by electroejaculation at this time contained low numbers of spermatozoa, possibly as a result of dilution and entrapment by the seminal coagulum or depletion of epididymal stores during intense multiple mating activity. In the non-breeding season (late May-July), when mating does not normally occur in the wild, there was a significant decrease in the relative weight of nearly all male reproductive organs and a decline in most semen parameters. In the minor breeding season (September-November), when pubertal females undergo their first oestrus and mating, the weights of testes, epididymides and most accessory sex glands had significantly increased similar to those of males in the major breeding season. The total number and motility of ejaculated spermatozoa were highest during this period, but the volume and coagulation of ejaculates and weight of the prostate had only increased to levels that were intermediate between the major and non-breeding seasons. Ejaculate volume was strongly correlated with prostate weight, and % motile spermatozoa was strongly correlated with epididymis weight. Semen quality thus varied seasonally with changes in androgen-dependent reproductive organs in the male tammar wallaby and appeared to be influenced by the seasonal timing of oestrus in females. Semen quality may also improve in response to an increase in the number of available oestrous females.

Long-term effects of deslorelin implants on reproduction in the female tammar wallaby (Macropus eugenii).

The contraceptive and endocrine effects of long-term treatment with implants containing the GnRH agonist deslorelin were investigated in female tammar wallabies (Macropus eugenii). Fertility was successfully inhibited for 515 +/- 87 days after treatment with a 5 mg deslorelin implant (n = 7), while control animals gave birth to their first young 159 +/- 47 days after placebo implant administration (n = 8). The duration of contraception was highly variable, ranging from 344 to 761 days. The strict reproductive seasonality in the tammar wallaby was maintained once the implant had expired. This inhibition of reproduction was associated with a significant reduction in basal LH concentrations and a cessation of oestrous cycles, as evidenced by low progesterone concentrations. There was evidence to suggest that some aspect of either blastocyst survival, luteal reactivation, pregnancy or birth may be affected by deslorelin treatment in some animals. These results show that long-term inhibition of fertility in the female tammar wallaby is possible using slow-release deslorelin implants. The effects of deslorelin treatment were fully reversible and there was no evidence of negative side effects. Slow-release GnRH agonist implants may represent a practicable method for reproductive management of captive and semi-wild populations of marsupials.

Effects of a gonadotropin-releasing hormone agonist implant on reproduction in a male marsupial, Macropus eugenii.

This study evaluated the potential of slow-release GnRH agonist (deslorelin) implants to inhibit reproductive function in the male tammar wallaby. The specific aim was to measure the effects of graded dosages of deslorelin on testes size and plasma LH and testosterone concentrations. Adult male tammar wallabies were assigned to four groups (n = 6 per group) and received the following treatment: control, placebo implant; low dose, 5 mg deslorelin; medium dose, 10 mg; high dose, 20 mg. All dosages of deslorelin induced acute increases (P < 0.001) in plasma LH and testosterone concentrations within 2 h, with concentrations remaining elevated during the first 24 h but returning to pretreatment levels by Day 7. Thereafter, there was no evidence of a treatment-induced decline in plasma testosterone concentrations. There was no detectable difference in basal LH concentrations between treated and control animals, nor was there a significant change in testes width or length (P > 0.05). These results suggest that the male tammar wallaby is resistant to the contraceptive effects of chronic GnRH agonist treatment. Despite the maintenance of testosterone secretion, the majority of male tammars (10 of 17) failed to respond to a GnRH challenge with a release of LH between Days 186 and 197 of treatment. The failure of animals to respond to exogenous GnRH suggests a direct effect of deslorelin on the pituitary, resulting in a level of desensitization that was sufficient to inhibit a LH surge but insufficient to inhibit basal LH secretion. The variation between animals is believed to result from earlier recovery of some individuals, in particular those that received a lower dose, or individual resistance to the desensitization process.

Functional analysis of the cooled rat testis.

Direct cooling of the testis results in the depletion of most germ cells in vivo. Germ cell-depleted testes are now commonly used to investigate spermatogenic regeneration and can serve as recipients for germ cell transplantation. The present study explored the effects of cooling rat testes on the depletion of endogenous germ cells, spermatogenic regeneration, and Sertoli cell function. Adult rat testes were cooled with iced Ringer's solution for 60 minutes, which results in the initiation of apoptotic germ cell loss within 8 hours. Pachytene spermatocytes at stages XII-I were the cells most sensitive to cooling. In 46%-67% of seminiferous tubule cross-sections, only Sertoli cells remained in the cooled testes 3-10 weeks after treatment. Germ cell loss was accompanied by a significant decrease in circulating inhibin B and an increase in follicle-stimulating hormone concentrations, which indicated a change in Sertoli cell function. Quantitative analysis of mRNA expression associated with apoptotic signals showed no significant uniform changes among the cooled testes, although some individuals had a distinct up-regulation of FAS mRNA at 24 hours. Attempts to use the cooled testes as recipient testes for mouse-to-rat germ cell transplantation were undertaken, but none of the mouse germ cells transplanted into the testes 15-34 days after cooling appeared to have undergone spermatogenesis 64-92 days after transplantation. These data suggest that modifications to Sertoli cell function resulting from testicular cooling create an environment that is unable to support spermatogenesis by donor germ cells.

Expression of DMRT1 in the mammalian ovary and testis--from marsupials to mice.

Doublesex and mab3 related transcript (DMRT1) was identified as a candidate gene for human 9p24.3 associated sex reversal. DMRT1 orthologues have highly conserved roles in sexual differentiation from flies and worms to humans. A DMRT1 orthologue was isolated from a marsupial, the tammar wallaby Macropus eugenii. The wallaby gene is highly conserved with other vertebrate DMRT1 genes, especially within the P/S and DM domains. It is expressed in the differentiating testis from the late fetus, during pouch life and in the adult. As in eutherian mammals, DMRT1 protein was localized in the germ cells and the Sertoli cells of the testis, but in addition it was detected in the Leydig cells, peri-tubular myoid cells and within the acrosome of the sperm heads. DMRT1 protein was also detected in the fetal and adult ovary pre-granulosa, granulosa and germ cells. Similarly, we also detected DMRT1 in the granulosa cells of all developing follicles in the adult mouse ovary. This is the first report of DMRT1 expression in the adult mammalian ovary, and suggests a wider role for this gene in mammals, in both the testis and ovarian function.

Long-term fertility control in the kangaroo and the wallaby using levonorgestrel implants.

Non-lethal management techniques are needed for overabundant captive and wild populations of macropodid marsupials for which lethal techniques are considered inappropriate and may be hazardous. The tammar wallaby, Macropus eugenii, was used as a model species to investigate the effect of s.c. levonorgestrel implants on reproduction during breeding and non-breeding seasons. Implants were tested on captive and wild populations of eastern grey kangaroos, Macropus giganteus. In both species, levonorgestrel successfully stopped reproduction by inhibiting oestrus. However, levonorgestrel did not affect the reactivation and subsequent development of blastocysts in diapause that had been conceived before treatment and it did not impair lactation, as young were reared to weaning in both species. The contraceptive effects of the implant were reversible, as removal of the implants from tammar wallabies was followed by a rapid return of fertility. Levonorgestrel implants had no apparent adverse effects on body condition in either species, although in the wild kangaroos a small increase in body condition did occur. Levonorgestrel implants are long-acting and so far have provided 48 months of contraception in the tammar wallaby and 27 months of contraception in the kangaroo, although the implants should provide contraception for more than 5 years in the kangaroo. Thus, levonorgestrel implants provide a safe, highly effective and long-term method of fertility control for macropodid marsupials and should be applicable for the management of overabundant captive and selected wild populations.

Effects of oestrogen treatment on testicular descent, inguinal closure and prostatic development in a male marsupial, Macropus eugenii.

This study reports the effect of oestrogen treatment on the development of the genital ducts, prostate gland, testicular descent and inguinal canal closure in male tammar wallaby young treated with oestrogen over four time spans during the first 25 days of pouch life (days 0-10, 10-15, 15-25 and 0-25) and sampled at day 50. In control males, the Müllerian ducts had regressed and the Wolffian ducts had developed into the vas deferens and epididymis. The prostate gland had formed epithelial buds extending from the ventral, lateral and posterior walls of the urethra. The testes were in the neck of the scrotum and the gubernaculum and processus vaginalis were present at the base of the scrotum. In most males treated with oestradiol from day 0 to day 25, the testes had failed to descend by day 50. The gubernaculae were long and thin. The retained Müllerian ducts formed a lateral vaginal expansion like that of normal day 50 females. The Wolffian ducts of the males treated on days 0-25 were regressed, but were present in males in the other three treatment groups. The prostate glands were hyperplastic and epithelial budding was highly invasive. Some treated males from the day 10-25 and 0-25 groups had inguinal hernias. These results demonstrate that oestrogen treatment has profound effects on the development of the internal genitalia of a male marsupial, preventing inguinal closure and interfering with testicular descent. Therefore, the tammar wallaby may provide a useful experimental model animal in which to investigate the hormonal control of testicular migration and closure of the inguinal canal.

Effect of an anti-androgen on testicular descent and inguinal closure in a marsupial, the tammar wallaby (Macropus eugenii).

Androgens are essential for testicular descent in eutherian mammals, but little is known about its hormonal control in marsupials. This study reports the effects of daily treatment with the anti-androgen flutamide (10 mg kg(-1)) from day 9 to day 75 after birth on the descent of the testis and inguinal closure in tammar wallabies. By day 75 after birth, the testes of control males had descended and the prostate gland was well developed. The testes of all flutamide-treated males had passed through the inguinal canal and were situated in the base of the scrotum. Three of the nine flutamide-treated males had unilateral inguinal hernias. The size of the inguinal canal, regardless of whether a hernia was present, was significantly wider than that of control males. Development of the prostate gland was significantly inhibited. By day 75 after birth, the phallus was significantly longer in control males than in females, whereas the phallus of flutamide-treated males was similar to that of control females. In flutamide-treated males, the lumbar 1 dorsal root ganglia was feminized and significantly fewer cell bodies expressed calcitonin gene- related peptide. As the anti-androgen treatment resulted in a reduction in the number of calcitonin gene-related peptide-positive cell bodies in the dorsal root ganglion supplying the genitofemoral nerve, the process of inguinal closure in tammar wallabies may be mediated by calcitonin gene-related peptide via the genitofemoral nerve, as indicated in humans. Flutamide treatment inhibited development of the prostate gland and phallus, which are both androgen-dependent structures, but it did not affect the normal descent of the testis, indicating that testicular descent can proceed when the action of androgens is blocked.