Antagonistic Effects of Testosterone and the Endocrine Disruptor Mono-(2-Ethylhexyl) Phthalate on INSL3 Transcription in Leydig Cells

Abstract

Insulin-like 3 (INSL3) is a small peptide produced by testicular Leydig cells throughout embryonic and postnatal life and by theca and luteal cells of the adult ovary. During fetal life, INSL3 regulates testicular descent in males, whereas in adults, it acts as an antiapoptotic factor for germ cells in males and as a follicle selection and survival factor in females. Despite its considerable roles in the reproductive system, the mechanisms that regulate Insl3 expression remain poorly understood. There is accumulating evidence suggesting that androgens might regulate Insl3 expression in Leydig cells, but transcriptional data are still lacking. We now report that testosterone does increase Insl3 mRNA levels in a Leydig cell line and primary Leydig cells. We also show that testosterone activates the activity of the Insl3 promoter from different species. In addition, the testosterone-stimulating effects on Insl3 mRNA levels and promoter activity require the androgen receptor. We have mapped the testosterone-responsive element to the proximal Insl3 promoter region. This region, however, lacks a consensus androgen response element, suggesting an indirect mechanism of action. Finally we show that mono-(2-ethylhexyl) phthalate, a widely distributed endocrine disruptor with antiandrogenic activity previously shown to inhibit Insl3 expression in vivo, represses Insl3 transcription, at least in part, by antagonizing testosterone/androgen receptor action. All together our data provide important new insights into the regulation of Insl3 transcription in Leydig cells and the mode of action of phthalates.

The INSL3 gene is a direct target for the orphan nuclear receptor, COUP-TFII, in Leydig cells

“…For instance, Insl3 expression is known to be repressed by estradiol in vivo in mice and rats (Nef et al 2000, Cederroth et al 2007, Strauss et al 2009, Zhou et al 2011) and this repression was also observed in MA-10 Leydig cells ). Insl3 expression is upregulated by androgens in vivo (Zhou et al 2010) as well as in primary Leydig cells and in MA-10 Leydig cells (Laguë & Tremblay 2008). Therefore, the MA-10 Leydig cell line is an appropriate model to study Insl3 gene regulation in the adult Leydig cell population.…”

“…The K1087 bp Insl3 reporter construct harbouring mutations in both DR0-like and DR3 elements was generated via the same two-step process using the K1087 bp Insl3 DR3 mutated construct as template and the sense and antisense oligonucleotides containing the mutations for the both half-site of the DR0-like element described above. The human K1137 bp INSL3 and rat K508 bp Insl3 promoter constructs have been described previously (Robert et al 2006, Laguë & Tremblay 2008). The mouse COUP-TFII expression vector (Pereira et al 1999) was obtained from Dr Ming Tsai (Baylor College of Medicine, Houston, TX, USA).…”

Antiandrogenic and Estrogenic Compounds: Effect on Development and Function of Male Reproductive System

“…Phthalates were also shown to decrease the expression of Insl-3, a factor produced by fetal Leydig cells. Insl-3 is an important regulator of testicular descent and phthalateinduced reduction of Insl-3 is consistent with the high incidence of cryptorchidism (Gray et al, 2006;Laguë & Tremblay, 2008;Wilson et al, 2004). In contrast to phthalates, in utero exposure to prochloraz decreases testosterone production by direct inhibition of the activity of steroidogenic enzymes without affecting the mRNA expression of these enzymes Wilson et al, 2008).…”

Structural Validation and Homology Modeling of Differentially Expressed Proteins in Rattus Norvergicus Induced by Bisphenol a and Probiotic Treatement.

“…In humans, circulating INSL3 increases through puberty, to reach a maximum in early adulthood, and subsequently appears to decline to significantly lower plasma levels in aging men [16,17]. Recently, it has been suggested [18] that androgens and phthalates at high concentration may modulate Insl3 gene expression in cultured Leydig cells. Caudal enlargement of the gubernaculum during relative transabdominal movement of the testis is known as the " gubernacular swelling reaction" or "gubernacular outgrowth" and is caused by cell division and an increase in glycosaminoglycans and hyaluronic acid (19).…”