Activin maintains the condensed type of mitochondria in germ cells

Abstract

Development of germ cells during spermatogenesis is characterized by a complex series of differentiation events finally leading to the production of spermatozoa. Beside the main hormonal regulators, paracrine interactions are thought to play a major role in this process. Mitochondria in germ cells pass through unique alterations ranging from the ‘typical’ cristae-rich mitochondria found in spermatogonia to the ‘condensed’ form in pachytene spermatocytes. This study provides further support that paracrine factors produced by Sertoli cells, most likely activin A, are involved in germ cell differentiation as monitored by the maintenance of the physiological ‘condensed’ mitochondrial phenotype in primary spennatocytes. Culture of primary spermatocytes in Sertoli cell conditioned medium (SCCM) for 18 h resulted in the maintenance of a high percentage of ‘condensed-type’ mitochondria in comparison to cells cultured in Dulbecco's minimum essential medium (DMEM). Activin A, a product of Sertoli cells, showed at subnanogram concentrations a similar ability to SCCM to maintain a high percentage of spematocyte mitochondria in the ‘condensed’ state, while inhibin had no effect. The addition of an antiserum specific for activin A resulted in a neutralization of the effect caused by activin A or SCCM. This strongly suggested that the active substance in SCCM was activin A. Taken together these data indicate that activin A is the first Sertoli cell product that has been identified to influence differentiation of male meiotic germ cells.

Introduction

It is well recognized that the mitochondria of germ cells modify their morphological organisation, number and location during the processes leading to sperm production (Andre, 1962, Fawcett, 1970, Machado de Domenech et al., 1972). Mitochondria gradually develop from the usual cristae rich type (‘orthodox-type’) found in the various types of spermatogonia via an intermediate form located in very early primary spermatocytes (leptotene and zygotene spermatocytes) to a so-called ‘condensed type’ in pachytene spermatocytes and early spermatids according to the terminology of Hackenbrock (Hackenbrock, 1966a, Hackenbrock, 1968b). In leptotene and zygotene spermatocytes, the space delimited between the two membranes forming the mitochondrial cristae, increases in size giving rise to the ‘intermediate-type’ (De Martino et al., 1979). In pachytene spermatocytes, the mitochondria are grouped together in small clusters characterized by an electron-dense material, the nuage, between them. The space between the membranes composing the cristae is greatly enlarged, with the membrane pushed to the periphery leaving almost no cristae. The matrix is condensed to a small ring-like structure beneath the outer membrane leaving the interior of these mitochondria looking ‘empty’. This type is regarded as the final stage of condensation (De Martino et al., 1979, Meinhardt et al., 1995a, Paranko et al., 1996, Meinhardt et al., 1999b). In early round spermatids, the mitochondria retain the ‘empty’ central regions but later in spermiogenesis more cristae become evident. These organelles elongate in the stages of the spermatid maturation phase. The fate of such organelles is species-dependent. In the rat, a subpopulation, characterized by sulphydryl oxidase (SOx) immunoreactivity aggregate and are retained in the residual body, while SOx-negative mitochondria finally fuse to form the mitochondrial sheath wrapped around the sperm midpiece (Kumari et al., 1990).

Very little is known about the factors that regulate or trigger the various steps of mitochondrial differentiation during spermatogenesis. Recent studies from Seitz et al. (1995) indicate the involvement of factors secreted by Sertoli cells which act in a paracrine fashion on primary spermatocytes. The physiological condensed shape of mitochondria was maintained when germ cells were co-cultured in dual compartment chambers with hormone stimulated Sertoli cells. The same result was achieved in germ cell only cultures treated with Sertoli cell conditioned medium (SCCM). The absence of SCCM or the degradation of the proteinaceous components of SCCM by heat denaturation or protease treatment resulted in a dedifferentiation back to the intermediate mitochondrial isotype (Meinhardt et al., 1999a; Seitz et al., 1995). The nature of this proteinaceous substance originally termed paracrine mitochondrial maturation factor (PMMF) remained unknown, but may represent one or more of the known products of the Sertoli cell (Seitz et al., 1995).

The activins are homo- and heterodimeric proteins consisting of a combination of the β_A and β_B subunits of inhibin and give rise to activin A (β_Aβ_A) activin B (β_Bβ_B) and activin AB (β_Aβ_B), each with the capacity to stimulate FSH secretion by the pituitary gland. In addition to their widespread biological effects in many tissues, the local production of activins in the testis may cause paracrine effects since activin A is a product of the Sertoli cell (De Winter et al., 1993b) and activin type IIB receptors are found on many germ cell types (De Winter et al., 1992a, Kaipia et al., 1993). Further, Krummen et al. (Krummen et al., 1994) showed binding of ¹²⁵I-labeled activin to spermatocytes and round spermatids, but no activin action on meiotic or post-meiotic cell types has been reported thus far.

In view of some similarities in the biochemical characteristics of the paracrine factor(s) in Sertoli cell culture media and the activins, we have assessed the effects of activin A on the mitochondria in primary spermatocytes maintained in vitro. These studies demonstrated that activin A could maintain the physiological ‘condensed’ type of mitochondrial morphology in subnanogram doses and significantly expands the role of this growth factor in the paracrine regulation of testicular function. The present study for the first time demonstrates the action of activin A on meiotic germ cells in influencing the physiological state of differentiation monitored by the type of mitochondria.