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Observations were made of the early differentiation of the gonad in the grey short-tailed opossum, Monodelphis domestica, using light and electron microscopy. An investigation was made also of the effects of neonatal treatment with oestradiol benzoate on sexual differentiation.

Neonates were removed from the nipple at various times after birth (0-14 days), killed and karyotyped to determine the genetic sex. Some individuals were then fixed in buffered formalin, embedded in paraffin and prepared for light microscopy. Alternatively the mesonephri and gonadal anlagen were dissected free of tissue, fixed in buffered 2.5% glutaraldehyde and prepared for electron microscopy. Twenty-two offspring from nine litters were examined.

At birth, pups were sexually undifferentiated and had rudimentary indifferent gonadal anlagen. By three days after birth, the gonad rudiments of males exhibited the first formation of sex cords with the coalescing of presumptive Sertoli cells. The sex cords of males were well defined by six days. By contrast, the gonads of female pups failed to display defined sex cords up to two weeks after birth.

An unusual feature was the initial appearance of a scrotal bulge in males as early as two days after birth, thus preceding any obvious morphological gonadal differentiation.

In all 34 pups were coated with oestradiol (1-2 μg per application) for various lengths of time. Some individuals were allowed to reach maturity so that growth rate and sexual behaviour could be studied.

Oestradiol treatment had a profound effect on male neonates, but no apparent effect on female neonates. Testis development was inhibited with the failure of the rudimentary gonad to form sex cords. With the exception of near-normal scrotum development, external and internal genitalia were completely feminized. Treated males allowed to reach maturity had growth rates similar to control females and exhibited oestrous behaviour in the presence of a control male.

The results suggest that gonadal differentiation in marsupials can be profoundly influenced by exogenous hormone. The early development of a scrotal bulge, preceding morphological differentiation, and the lack of an effect of oestradiol on scrotal formation would indicate that the differentiation of this somatic tissue may not be dependent on gonadal determination.