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At birth, the mouse gonocyte does not resume mitotic activity for several days in vivo but, in an in vitro clonogenic system, cell division commences soon after culture. Somatic testis cell underlays had potent inhibitory activity on gonocyte-derived colony formation (23 +/- 15% compared with 84 +/- 1% in controls; P = 0.0001) when added to cultures of gonocytes in vitro. A Sertoli cell line, TM4B, had an even more pronounced effect on gonocyte clonogenic capacity, with 1 +/- 1% compared with 72 +/- 17% colony formation in controls (P = 0.0003). Testis cells appeared to have a direct inhibitory effect since testis-conditioned medium did not show a significant reduction in the number of colonies. The observed reduction in colony formation with the testis cell underlay was not accounted for by decreased attachment of gonocytes as simultaneous addition of a single cell suspension of testis cells was still effective in significantly reducing colony number when compared with controls (P = 0.01). Therefore, the observed inhibition exerted by testis cells appears to be a consequence of decreased proliferation of gonocytes. Growth factors belonging to the transforming growth factor beta superfamily which are known to be expressed in testis, such as transforming growth factor beta and epidermal growth factor, did not exert any inhibitory action on gonocyte-derived colony formation when added together or alone. However, a shift to a smaller colony size occurred in the presence of transforming growth factor beta and transforming growth factor beta plus epidermal growth factor, indicating a reduction in colony cell proliferation. Evidence for the expression of the Müllerian inhibiting substance receptor on newborn gonocytes using in situ hybridization was inconclusive. This finding was in agreement with the lack of a direct action of Müllerian inhibiting substance on the formation of gonocyte-derived colonies in vitro. Leukaemia inhibitory factor, alone or in combination with forskolin, had neither an inhibitory nor an enhancing effect on gonocyte-derived colony formation. An in vitro clonogenic method to assay for the proliferation of gonocytes in the presence of specific growth factors, cell lines, testis cell underlays and cell suspensions was used to identify a somatic cell-mediated inhibitor which may be responsible for the inhibitory action on gonocyte proliferation in vivo shortly after birth.

Survival and proliferation of mouse gonocytes was studied using a single cell clonogenic assay in vitro. The effect of growth factors and extracellular matrix on clonogenic development was quantitated. Fundamental requirements for growth of neonatal gonocytes included addition of fetal calf serum and coating culture wells with collagen IV alone or with added fibronectin. After 4-5 days, colonies ranged in size from four to > 128 cells, and some contained very elongated cells indicating migratory behaviour. Soluble stem cell factor did not have any effect on clonogenicity, although STO (subline of SIM mouse fibroblasts) cells, which produce membrane-bound stem cell factor, reduced colony formation from 79 +/- 5.9% to 20 +/- 3.3% without added growth factor. The majority of gonocytes and type A spermatogonia express the c-kit receptor according to in situ hybridization studies. However, the results indicate that the receptor may not be functional in neonatal gonocytes and their immediate progeny. The current assay for gonocytes can be extended to test new growth factors or proliferation-inducing agents directly, as well as to study cell-cell interactions. This assay and long-term propagation of neonatal germ cells will provide the much needed resources to enable greater understanding of the early development of germ cells.

Sexual dimorphism of the genitofemoral nerve spinal nucleus has been demonstrated in normal rodents. Calcitonin gene-related peptide is a neurotransmitter, present in the genitofemoral nerve, that has been implicated in the regulation of gubernacular migration and inguinoscrotal testicular descent. A combination of retrograde fluorescent labelling of the genitofemoral nerve and immunohistochemistry for calcitonin gene-related peptide was used in 1\p=n-\3-day-oldmutant TS rats with 85% incidence of congenital cryptorchidism and an absence of the normal sexual dimorphism of the genitofemoral nerve spinal nucleus was demonstrated. There was no significant difference between male and female nuclei with respect to fluorescent-labelled neurones as well as those immunoreactive for calcitonin gene-related peptide, in contrast to an obvious sexual dimorphism present in normal control animals. This lack of normal sexual dimorphism of the genitofemoral nerve nucleus is likely to be important in the pathogenesis of cryptorchidism in this animal model.