Basic features of bovine spermatogonial culture and effects of glial cell line-derived neurotrophic factor

Abstract

Spermatogonial stem cells (SSC) are a small self-renewing subpopulation of type A spermatogonia, which for the rest are composed of differentiating cells with a very similar morphology. We studied the development of primary co-cultures of prepubertal bovine Sertoli cells and A spermatogonia and the effect of glial cell line-derived neurotropic factor (GDNF) on the numbers and types of spermatogonia, the formation of spermatogonial colonies and the capacity of the cultured SSC to colonize a recipient mouse testis. During the first week of culture many, probably differentiating, A spermatogonia entered apoptosis while others formed pairs and chains of A spermatogonia. After 1 week colonies started to appear that increased in size with time. Numbers of single (A_s) and paired (A_pr) spermatogonia were significantly higher in GDNF treated cultures at Days 15 and 25 (P < 0.01 and 0.05, respectively), and the ratio of A_s to A_pr and spermatogonial chains (A_al) was also higher indicating enhanced self-renewal of the SSC. Furthermore, spermatogonial outgrowths in the periphery of the colonies showed a significantly higher number of A spermatogonia with a more primitive morphology under the influence of GDNF (P < 0.05). Spermatogonial stem cell transplantation experiments revealed a 2-fold increase in stem cell activity in GDNF treated spermatogonial cultures (P < 0.01). We conclude that GDNF rather than inducing proliferation, enhances self-renewal and increases survival rates of SSC in the bovine spermatogonial culture system.

Introduction

Stem cells are capable of both self-renewal and the production of differentiating cells and are present in almost every tissue in the body. Starting from spermatogonial stem cells (SSC) that reside on the basal membrane of the seminiferous tubules in the testis, spermatogenesis takes place as a highly coordinated and efficient process, which produces millions of terminally differentiated spermatozoa a day. SSC, as other stem cells, are found in very low numbers. For example, in the mouse testis, only 0.03% of all germ cells are stem cells [1]. Furthermore, the morphological appearance of SSC is very similar to that of early spermatogonia that already are committed to differentiation and have lost stem cell properties. To make progress in understanding the regulation of self-renewal and differentiation of SSC and to characterize these cells at the molecular level it is essential to develop methods for their culture and purification. In cell culture systems, stem cells can be exposed to selected growth factors, so that the complexity of the regulatory mechanisms that govern spermatogenesis in vivo, involving a myriad of different signals, can be simplified.

In this respect, progress has been made in other stem cell systems like hemopoiesis in which several cytokines and growth factors were found that induce hemopoietic stem cells to proliferate in vitro without undergoing differentiation [2]. Within the testis, the renewal mechanism of SSC is still not completely understood. One important factor, which enhances SSC renewal, is glial cell line-derived neurotrophic factor (GDNF) [3] that originally was found in nervous tissue where it can promote the survival of neurons [4]. GDNF is also involved in non-neural tissues like the kidney, where it plays an essential role in early developmental stages [5].

During the past years, GDNF has been associated with SSC renewal in vivo [3], [6] and in vitro situations [7], [8]. Normally, in the seminiferous epithelium Sertoli cells secrete GDNF and the receptors for this growth factor, GFR-alfa 1 and c-ret, are present on SSC [3], [9], [10]. Furthermore, FSH was found to stimulate GDNF production by Sertoli cells [10].

We now have studied the development of primary co-cultures of bovine Sertoli cells and A spermatogonia and we quantified the effects of GDNF on the numbers of SSC and spermatogonial clones, the formation of spermatogonial colonies and stem cell activity by way of transplantation techniques. Our system uses a monolayer of autologous Sertoli cells to provide germ cells an environment that resembles that in vivo as closely as possible [11], [12], [13]. We used 4.5–5-month-old calf testes, an age at which type A spermatogonia are the predominant type of germ cells, with low numbers of type In and B spermatogonia and preleptotene spermatocytes [14], [15]. We hypothesized that GDNF increases the number of SSC in vitro, by stimulating their proliferation.