Spermatogonia of rainbow trout: I. Morphological characterization, mitotic activity, and survival in primary cultures of testicular cells

Abstract

Prerequisites of developing in vitro studies for a better understanding of the control mechanisms underlying the proliferation and differentiation of spermatogonia (Go) in the teleost testis are: (1) to be able to identify the different types of Go; (2) to maintain in culture the structural relationships occurring in situ between the various testicular cell types, as intact as possible; and (3) to know how the Go survive and proliferate in culture for several days. After very gentle homogenization of trout testes treated with collagenase, a cell suspension containing mainly spermatocysts (one or several Sertoli cells enclosing one Go or a clone of germ cells) and clusters of myoid cells and Leydig cells was seeded in culture onto a laminin plus fibronectin coating. After 4.5-6 days in culture, then staining with May-Grünwald and Giemsa reagents, the determination of the nuclear and cellular size of the various Go and of the number of Go present in clones has enabled the identification of two types of large Go, in pairs or alone (Go A) and six successive types of smaller Go (Go B). Cell viability determination by staining with Rhodamine 123/propidium iodide (PI)/Hoechst 33342 and with FITC-Annexin V/PI indicated that after 5-7 days in culture, all the somatic cells and most of the Go were viable. Only some of the Go, mainly among the most differentiated ones, underwent apoptosis, as it was the case for a number of spermatocytes and spermatids increasing with the time in culture. Brdu labelling and 3H-Thymidine (3H-Tdr) incorporation indicated that the proliferative activity of Go was at a maximum after 4.5 days in culture and that the response to at least two molecules (QAYL-IGF-I and GTH-I) remained unchanged between 3 and 6 days. As only very scarce somatic cells from immature/spermatogenetic testes synthesized DNA up to 6 days in culture, the measurement of 3H-Tdr incorporation by cells from such testes reliably reflected synthesis of DNA by only the Go (and eventually also by primary spermatocytes when they are present). In conclusion, this study provides information allowing a detailed analysis of the events related with the mitotic phase of spermatogenesis in the trout and it establishes that primary cultures of testicular cells carried out in the reported conditions represent a useful tool to develop an analysis of the mechanisms participating in the control of this phase.