Quantification of stem cell factor mRNA levels in the rat testis: usefulness of clusterin mRNA as a marker of the amount of mRNA of sertoli cell origin in post pubertal rats

Abstract

Spermatogenesis is a complex cellular process regulated by gonadotrophins and local cell-cell interactions. Stem cell factor (SCF) is one of the paracrine factors, produced by the Sertoli cells, involved in the local regulation of spermatogenesis. Measurement of its testicular level is important for addressing its role in testis physiopathology. However, the relative cell composition of experimental and pathological testis samples may lead to misinterpretation in relating SCF mRNA levels to the amount of RNA extracted from the whole tissue sample. Taking into account the relative RNA content of Sertoli cell origin should provide more significant data. In the present study, three sets of experiments were intended for modifying the proportion of RNA of Sertoli cell origin in RNA extracted from whole testis tissue samples: during postnatal development; following methoxy-acetic acid (MAA) administration; and after injecting a long-acting gonadotrophin-releasing hormone agonist (GnRHa). In a first step, we demonstrated clusterin mRNA level stability in purified Sertoli cell preparations between 20 days and adulthood, and following MAA or GnRHa treatment. In a second step, we used a competitive RT-PCR assay to measure SCF and clusterin mRNA levels and expressed the amount of SCF mRNA relative to the amount of clusterin mRNA under the above experimental conditions. The SCF/clusterin mRNA level ratio was found to remain roughly stable from 20 days post-partum to adulthood; i.e. during the development of spermatogenesis. MAA administration led to an overall increase in the SCF/clusterin mRNA level ratio between 7 and 14 days after administration, consistent with the replenishment of the testis with pachytene spermatocytes and round spermatids. Conversely, after long-acting GnRHa injection, the SCF/clusterin mRNA level ratio decreased only slightly from day 21 onward. Hence, the present studies indicate that, under physiopathological conditions, the amount of clusterin mRNA is a good marker of the amount of RNA of Sertoli cell origin in testis samples at day 20 or later; different experimental alterations of spermatogenesis are associated with different patterns of SCF mRNA levels; the relationship between FSH and SCF in vivo is not as simple as that described in vitro.

Propylthiouracil-induced hypothyroidism delays apoptosis during the first wave of spermatogenesis

“…Here we have confirmed previous results showing that neonatal PTU-treated rats have a lower body weight, along with smaller testes and reduced diameter of seminiferous tubules, along with retardation of germ cell differentiation (Cook et al, 2005;De Franca et al, 1995;Holsberger and Cooke, 2005;Plotton et al, 2005). In addition, we have confi rmed that there is a delay in the appearance of Sertoli cell differentiation markers, such as clusterin, related to retardation in Sertoli cell differentiation.…”

“…In mice and rats, Sertoli cells begin to proliferate at birth, but stop dividing around postnatal day 15, which is accompanied by expression of specifi c markers, such as clusterin (McKinnell and Sharpe, 1997;Plotton et al, 2005). This differentiation involves the induction of the cyclin-dependent kinase inhibitor p27 kip1 by the thyroid hormone (T3) (Holsberger et al, 2005a;Holsberger et al, 2003).…”

The RNA-binding protein Mex3b regulates the spatial organization of the Rap1 pathway

“…For that purpose, we determined by quantitative RT-PCR the level of the mRNA coding for clusterin, the major protein synthesized by differentiated Sertoli cells that is deposited on sperm membranes (Plotton et al, 2005). As shown in Fig.…”

Expression Pattern of Sulfated Glycoprotein-2 (SGP-2) mRNA in Rat Testes Exposed to Endocrine Disruptors

“…Plotton et al (2005) reported that the amount of SGP-2 (clusterin) mRNA is a good marker of the amount of mRNA of Sertoli cell origin in the testes of post pubertal rats [26]. In the present study, we examined the effects of various endocrine disruptors on spermatogenesis using SGP-2 mRNA expression as a biomarker for spermatogenesis in the Sertoli cells of rat testes.…”

“…The Sertoli cell is the only cell type shown to have a significant level of SGP-2 mRNA in the testes by in situ hybridization [25]. Plotton et al (2005) reported that the amount of SGP-2 (clusterin) mRNA is a good marker of the amount of mRNA of Sertoli cell origin in the testes of post pubertal rats [26].…”