Fetal Testis Dysgenesis and Compromised Leydig Cell Function in Tgfbr3 (Betaglycan) Knockout Mice1

Abstract

Betaglycan (Tgfbr3) is a coreceptor for transforming growth factor-beta (TGFB) superfamily ligands. In the current study, a defect in seminiferous cord formation was detected in 12.5-13.5 days postcoitum (dpc) beta glycan null murine testis. Immunohistochemistry with antibodies against cell-specific markers revealed defects in somatic cell populations. To confirm these data, quantitative real-time PCR was performed to determine changes in the expression levels of genes involved in fetal testis cell differentiation and function. The expression levels of the Leydig cell markers Insl3, Cyp17a1, Cyp11a1, Star, and Hsd3b1 were reduced in knockout testis compared to wild-type testis, beginning at 12.5 dpc. Whole mount in situ hybridization confirmed that Cyp11a1 expression was reduced in the null testis, but its distribution pattern was unchanged. Apoptosis was not affected by the loss of beta glycan, but proliferation within the interstitium was reduced at 14.5 dpc. However, morphometric analysis showed no changes in Leydig cell counts between the wild-type and the knockout testes at 14.5 dpc, indicating that fetal Leydig function, rather than number, was affected by the loss of beta glycan. The expression levels of Sertoli cell markers Dhh, Sox9, and Amh were also reduced in the knockout testis at 14.5 dpc. However, the expression of fetal germ cell markers Pou5f1 and DDX4 were not changed across the genotypes at any age examined. Our data show that the presence of beta glycan is required for normal cord formation, normal fetal Leydig cell development, and the establishment of fetal testis endocrine function, thus implicating TGFB superfamily members as regulators of early fetal testis structure and function.

Insulin-like factor 3 as a monitor of endocrine disruption

“…If its expression is similarly regulated in the fetus, then this would imply that INSL3 in fetal Leydig cells should become detectable soonest after about day e12.5 in the mouse, or by weeks 8-10 in the human. Indeed, the earliest evidence for Insl3 mRNA detection in the mouse is on day e12.5 (Sarraj et al 2010) and, for the human, INSL3 peptide has been measured in the amniotic fluid at gestational week 11 (R Anand-Ivell, unpublished observations). There is less evidence available for other species, although sporadic measurements of INSL3 in the bovine (Anand-Ivell et al 2011), porcine, or rat systems (Anand-Ivell, Vernunft, Barthol & Ivell, unpublished observations) confirm that high levels of INSL3 are detectable in fetal blood, or in fetal fluids, at the time of the first transabdominal phase of testicular descent (Fig.…”

Endocrinology of the mammalian fetal testis

“…It is likely that Leydig cell activity is regulated during this period by local growth factors secreted within the testis or that constitutive activity within the Leydig cells is sufficient to ensure adequate androgen production. Local factors that may be involved include DHH, PDGFA, insulin-like growth factor 1, vasoactive intestinal polypeptide, pituitary adenylate cyclase-activating polypeptide, natriuretic peptides, and the transforming growth factor-b superfamily (El Gehani et al 1998a, 2001, Griswold & Behringer 2009, Scott et al 2009, Sarraj et al 2010. Realistically, the role that each of these factors plays in fetal Leydig cell activity is only likely to be determined once a fetal Leydig cell-specific Cre mouse model is developed.…”

Research on the Isolation of Mouse Leydig Cells Using Differential Digestion with a Low Concentration of Collagenase

“…After Leydig cells were seeded and had completely adhered to the culture plate, an inverted microscope was used to observe the morphology and growth. Immunofluorescence was used to determine the expression of Leydig biomarkers [19] at different stages of the primary culture. Histochemical staining for 3β-HSD was conducted again to determine the postculture purity on the seventh day of primary culture.…”