Sertoli cell modulates MAA‐induced apoptosis of germ cells throughout voltage‐operated calcium channels

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Abstract
SPECIFIC AIMS The aim of our study is to demonstrate that in the mammalian testis, Sertoli cells, which regulate germ cell proliferation and differentiation, are also involved in the control of germ cell apoptosis throughout voltage-operated calcium channels (VOCCs) and that the contacts between Sertoli cells and germ cells are necessary for such a modulation. As a marker of germ cell apoptosis, we chose clusterin, a ubiquitous glycoprotein synthesized by Sertoli cells, since it has been shown that Sertoli cell-derived clusterin is present in large amounts in dying germ cells, i.e., pachytene spermatocytes. We used methoxyacetic acid (MAA) as an inductor of in vitro germ cell apoptosis. PRINCIPAL FINDINGS 1. In cultured rat Sertoli cells, clusterin expression is modulated by MAA; Sertoli cell VOCCs are involved in such a modulation 2. In cultured seminiferous tubules, Sertoli cell VOCC block prevents accumulation of clusterin in germ cells As a consequence, the spermatocytes, although exposed to MAA, do not undergo apoptosis (Fig. 1 ⤻ ). Figure 1. Quantitative analysis by flow cytometry of testicular cell population derived from seminiferous tubules cultured for 12 h. Data are expressed as mean ± se of fluorescence values for n = 3 independent experiments. White bar represents clusterin amount in cells immunostained with antibodies raised against clusterin (448±36, 218±23, 252±13, 516±28: white histograms 1–4). Black bar represents apoptosis levels determined by in situ end-labeling (TUNEL) (1032±73, 349±65, 312±34, 3726±34: black histograms 1–4). 1: Control; 2: MAA plus ω-CTX; 3: MAA plus nifedipine; 4: MAA. Download figure Download PowerPoint 3. Effects of VOCC blockers are strictly correlated to the integrity of seminiferous epithelium In a mixed population of Sertoli and germ cells cultured in the presence of MAA and with MAA + VOCC inhibitors, clusterin accumulation, which is visible in seminiferous tubules in the germ cells 6 h post-MAA treatment, is not observable in spermatocytes under any of the experimental conditions (Fig. 2 ⤻ A, black histograms 1–4). As expected, spermatocytes do not undergo apoptosis (Fig. 2B⤻ , black histograms). The peculiar localization of VOCCs in the seminiferous epithelium and their ability to quantitatively and qualitatively modulate Sertoli cell protein secretion fit well with their involvement in controlling germ cell apoptosis. In fact, the block of Sertoli cell VOCCs prevents pachytenespermatocytes undergoing apoptosis and inhibits the accumulation of Sertoli cell-produced clusterin in the cytoplasm of germ cells. This is the first time a role for VOCCs in germ cell death is demonstrated in the testis while evidence of the involvement of voltage-operated calcium channels in apoptosis is accumulating in other systems. Figure 2. Quantitative analysis by flow cytometry of clusterin accumulation and apoptotic cell levels in mixed cell populations represented by spermatogonia and Sertoli cells (2C) and primary spermatocytes (4C) cultured for 6 (A) and 12 (B) h. Data are expressed as mean ± se of fluorescence values for n = 3 independent experiments. 1: Control; 2: MAA plus ω-CTX; 3: MAA plus nifedipine; 4: MAA. A) Clusterin content in 2C cell population cultured for 6 h. Clusterin content in 4C cell population cultured for 6 h. B)Apoptotic levels in 2C cell population cultured for 12 h. Apoptotic levels in 4C cell population cultured for 12 h. Clusterin content in 2C cell population (mainly Sertoli cells) is increased in the presence of MAA (874±49: histogram 4) and reduced to control level (563±18: histogram 1) by VOCC blockers (576±5, 473±12: histograms 2 and 3). Clusterin content in 4C population (pachytene spermatocytes) is not observable under these experimental conditions (6±1, 15±2, 6±1, 18±2: histograms 1–4). The apoptotic level in 2C cell population (274±12, 235±15, 243±8, 263±2: white histograms 1–4) and in 4C cell population (480±13, 396±7, 337±9, 460±12: black histograms 1–4) cultured for 12 h is not influenced by MAA and is not reduced by VOCC blockers. Download figure Download PowerPoint Our results demonstrate that Sertoli cells and not germ cells are the major target for the apoptosis, so that cells showing the damage (i.e., germ cells) are not the ones actually hit by the toxic substance. Thus, the Sertoli cell not only is the mediating agent in the hormonal control of spermatogenesis, being the target of FSH and testosterone; it is also the mediator in response to injuries that could damage germ cell differentiation. Our data suggest that Sertoli cells could “select” germ cells at risk of damage by allowing the accumulation of clusterin in their cytoplasm. Finally, our results showing that the methoxyacetic acid is ineffective on germ cells not in contact with Sertoli cells indicate that the junctions existing in the seminiferous epithelium between Sertoli cells and between Sertoli cells and germ cells are essential not only for allowing correct spermatogenesis, but also for the control of germ cell viability. 1To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0347fje Figure 3. Schematic diagram. Sertoli cells can modulate germ cell apoptosis through their voltage-operated calcium channels (VOCCs). The block of Sertoli cell VOCCs inhibits the accumulation of Sertoli cell-produced clusterin in the cytoplasm of germ cells and prevents pachytene spermatocytes undergoing apoptosis, induced by MAA treatment. Download figure Download PowerPoint
Funding Information
  • Ministero dell’Istruzione, dell’Università e della Ricerca

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