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Germ cell development is an active process in normal testes during the first 4 years after birth, with transformation of the neonatal gonocytes into adult dark spermatogonia and then primary spermatocytes. The hormonal regulation of these changes is not fully understood, with evidence both for and against a role for gonadotrophins and androgens. Early surgical intervention in infancy aims to prevent or reverse germ cell maldevelopment. Although hormonal treatment for maldescent has been shown to be ineffective, there is still controversy over whether it may be useful as an adjunct to surgery to stimulate germ cells. Current evidence suggests that hormonal therapy may not stimulate transformation of neonatal gonocytes but may trigger prepubertal mitosis of primary spermatocytes. Further studies are required to determine the role of hormone treatment on germ cell development.

The stem cell properties of neonatal germ cells have recently been demonstrated by in vivo transplantation. Regulation of proliferation of these cells, however, is not yet understood, and an in vitro system is needed for directly testing the action of differentiation and proliferation-related factors for germ cells. We developed an in vitro model involving micromanipulation and a single-cell clonogenic assay in which results from independent experiments on spermatogonia and gonocytes have been analyzed and compared. Neonatal germ cells can be distinguished by their large size both in vivo and in vitro in a single-cell suspension. These cells are picked up singly using a micropipette and deposited into a 96-well plate precoated with an extracellular matrix component, e.g., collagen IV. The effect of growth factors or cocultured somatic cells was assayed by counting the percentage of wells containing a colony and comparing this percentage with that of control cultures. Addition of platelet-derived growth factor significantly shifted the modal colony size for gonocytes from >16-64 to >64-128 cells/colony (P < 0.001, chi2) but had no effect on spermatogonia-derived colony size and number. For testis somatic cell underlays, there was a profound inhibition of all colony types, and immunohistochemical staining of testis cell underlays showed inhibin/activinbetaA subunit expression. This finding suggests that negative regulation of germ cell proliferation is mediated by inhibin. Addition of activin A to these cultures resulted in significant recovery (P = 0.046) of gonocyte-derived colony numbers but not spermatogonia-derived colonies, which may reflect the functional regulation by these factors observed in vivo. This proliferation assay also highlights many similarities in the regulation of gonocyte and spermatogonia proliferation in vitro, suggesting that proliferation potential is not noticeably affected by the transition of gonocytes to spermatogonia. For example, the average colony cloning efficiency was 80% for gonocytes and 76% for spermatogonia. This technology forms a basis for optimizing growth of neonatal germ cells for applications such as introduction of genetic material into the germ line to produce transgenic mice and to explore gene therapy.

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.