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. Thus masculinisation of the phenotype is controlled by androgens and anti-Mƒllerian hormone (AMH) produced by the testes, feminisation being essentially the default mode. In marsupials it appears that the mechanisms of phenotypic development may vary from the eutherian pattern, as differentiation of the mammary and scrotal primordia occur before that of the gonad. This suggests that the development of at least some structures in the urogenital tract of the male marsupial may be independent of testicular signals.

Monodelphis domestica was used as a model to investigate the development of the marsupial urogenital tract as, besides having a short gestation (14 days), it has a large litter size (up to nine). Immunocytochemistry was used to determine the expression of the key steroidogenic enzyme 3β-HSD as an indication as to when the developing gonad may be capable of synthesising androgens. As a complementary test, an anti-androgen receptor antibody was also used to determine when the urogenital tract first became receptive to androgen stimulation.

Male and female opossum pups from the first to the third day of postnatal age (PN0 = day of birth) were humanely killed by decapitation, fixed in 4 % paraformaldehyde and embedded in paraffin wax. 6 µm sections were cut and mounted on APES (3-aminopropyltriethoxysilane)-coated slides. Immunocytochemistry was performed using primary antibodies to 3β-HSD (S683) and androgen receptor (NCL-ARp). Antibody binding was visualised using a biotinylated secondary antibody followed by standard immunoperoxidase staining.

3β-HSD expression was detected in the gonads and adrenals of both male and female PN0 pups. Androgen receptor expression was first observed in the genital tubercles and scrotal primordia of PN1 male pups. No similar staining was observed in female pups. An earlier investigation by Sonea et al. (1997) failed to detect androgen receptor immunoreactivity until PN4, but these authors only sampled PN0 and PN4 pups. Our results show steroidogenic capability of the gonads, as manifested by expression of 3β-HSD, as early as PN0 and the presence of androgen receptor in the developing male genitalia from PN1.

These findings indicate that androgens may play a role in the development of the marsupial male genitalia at a much earlier stage than previously thought.This work was supported by the BBSRC.

Sonea, I.M., Iqbal, J., Prins, G.S. & Jacobson, C.D. (1997). Biol. Reprod. 56, 852-860.