Testis

Clonogenic Culture of Normal Spermatogonia: In Vitro Regulation of Postnatal Germ Cell Proliferation

Abstract

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. This report describes an in vitro model involving micromanipulation and a single cell clonogenic assay in which results from independent experiments on spermatogonia and gonocytes have been analysed 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 which is pre-coated with an extracellular matrix component eg. collagen IV. The effect of growth factors or co-cultured somatic cells was assayed by counting the percentage of wells containing a colony and comparing this to control cultures. Addition of platelet-derived growth factor (PDGF) shifted the modal colony size for gonocytes from >16-64 to > 64-128 cells/colony which was significant (P<0.001) using chi-square analysis, but had an immeasurable effect on spermatogonial-derived colony size and number. When the effect of testis somatic cell underlays were studied a profound inhibition of all colony types occurred and immunohistochemical staining of testis cell underlays showed inhibin/activin _A subunit expression. This suggests that negative regulation of germ cell proliferation is mediated by inhibin. Addition of activin A to these cultures resulted in significant (P=0.046) recovery 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 between 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 optimising 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.