Expression of Prothymosin alpha during the spermatogenesis of the spotted ray Torpedo marmorata

Abstract

In this study, we show that Prothymosin alpha (Ptma), a small, unfolded, negatively charged protein, is present in the cartilaginous fish Torpedo marmorata. The ptma gene is functional and peculiarly controlled during the male spermatogenesis of T. marmorata, as revealed by in situ hybridization and by immunocytochemistry studies. The data show that the ptma transcript is present in stage-specific germ cells, i.e. spermatocytes II and round spermatids. The Ptma protein is detectable in spermatocytes II, in round and elongated spermatids as well as in spermatozoa before their release from cysts, while it is not evident in spermatozoa located in male genital tracts. The ptma transcript and protein are also evident in some Leydig cells, located among maturing cysts containing meiotic and differentiating male cells. No expression for ptma is observed within Sertoli cells. Furthermore, immunolocalization procedures demonstrate that the protein is preferentially localized in the cytoplasm, whereas a nuclear localization is observed in round and elongated spermatids. The possibility that Ptma is involved in testis activity is discussed.

Introduction

Ptma is a small nuclear protein (109–113 amino acids in length depending on the species) containing more than 50% acidic amino acid residues (glutamic and aspartic). It was originally isolated from rat thymus and is considered to be the most acidic polypeptide present in eukaryotes (isoelectric point 3.55) (Haritos et al., 1984). The extremely acidic character of Ptma and the presence of a nuclear localization signal, strongly suggest that Ptma has an intranuclear site of action (Gómez-Márquez and Segade, 1988). Indeed, the protein has been found to be involved in cell proliferation (Gómez-Márquez et al., 1989, Tsitsiloni et al., 1993), transcriptional regulation (Cotter and Robertson, 2000, Karetsou et al., 2002), chromatin remodeling (Karetsou et al., 1998, Karetsou et al., 2004) and oxidative stress–response (Karapetian et al., 2005). Recent studies demonstrated that Ptma also plays a regulatory role in apoptosis by blocking the formation of apoptosome (Jiang et al., 2003), or via the interaction with an anti-apoptotic protein p8 (Malicet et al., 2006a, Malicet et al., 2006b).

Ptma has been studied in mammals (Pan et al., 1986, Panneerselvam et al., 1988, Schmidt and Werner, 1991, Garcia-Caballero et al., 1994) and nonmammalian vertebrates (Barisone et al., 1998, Aniello et al., 2002, Ferrara et al., 2009). In the mammalian male gonad, in particular in the adult rat testis, ptma expression was reported in spermatogonia and early spermatocytes I (Roson et al., 1990). Furthermore, in the same species Dosil et al. (1990) showed that ptma mRNA is preferentially expressed before puberty, while it significantly decreases in the adult rats. In Rana esculenta testis, the ptma transcript was reported in germ cells (spermatocytes I and II) and Leydig cells (Aniello et al., 2002), and, more recently, the Ptma protein was found in secondary spermatocytes, spermatids, spermatozoa, and Leydig cells (Ferrara et al., 2009). In this regard, it has been proposed that frog Ptma might contribute to the efficiency of spermatogenesis because its expression was found to vary during the spermatogenetic cycle concomitantly with germ cell differentiation (Aniello et al., 2002, Ferrara et al., 2009).

Nothing is known about ptma expression in the testis of other nonmammals. The data so far obtained in vertebrate testis regarding Ptma distribution are limited and new observations concerning Ptma expression in the other vertebrate testis might bring new light on the role of Ptma within spermatogenesis.

We investigated the ptma expression in the testis of a lower vertebrate, the cartilaginous fish Torpedo marmorata, for comparison with the expression pattern of other vertebrates thus far studied before (Roson et al., 1990, Dosil et al., 1990, Aniello et al., 2002, Ferrara et al., 2009). The cyst organization (Prisco et al., 2002) and the peculiar distribution of androgen-producing cells (Prisco et al., 2008) make the Torpedo testis a suitable model to study the role of Ptma in the control of spermatogenesis. A cyst contains about 250 spermatoblasts, which are in turn characterized by a single Sertoli cell associated at the end of the two meiotic divisions to 64 spermatids which will differentiate into spermatozoa (Stanley, 1966). Every cyst during the spermatogenic cycle is characterized by the presence of only one germ cell stage: spermatogonia, spermatocytes I, spermatocytes II, spermatids and spermatozoa (Stanley, 1966, Prisco et al., 2002). After spermiation, the Sertoli cells within a cyst are removed by apoptosis (Prisco et al., 2003). Regarding the organization and the distribution of androgen-producing cells, Torpedo testis is characterized by the presence of true Leydig cells, which are only active when they are located among cysts containing meiotic and differentiating male germ cells. In the testis regions containing cysts with spermatogonia or mature spermatozoa Leydig cells are not active. In such regions, the androgen synthesis is sustained by Sertoli cells as well as by spermatogonia (Prisco et al., 2003, Prisco et al., 2008).

We show that the ptma gene, whose presence is reported for the first time in a cartilaginous fish, is functional and its expression in the testis is controlled during spermatogenesis. Evidences based on in situ hybridization and on immunolocalization experiments show peculiar, cell-specific expression of the ptma gene, suggesting that ptma is involved in spermiogenesis and steroidogenesis.