Sertoli Cell Development and Function in an Animal Model of Testicular Dysgenesis Syndrome1

Abstract

Pregnancy exposure to di(n-butyl) phthalate (DBP) in rats induces a testicular dysgenesislike syndrome (TDS) in male offspring. Earlier studies suggested altered Sertoli cell development/maturation may result, especially in testes that become cryptorchid. This study quantitatively assessed Sertoli cell numerical and functional development in DBP-exposed rats and compared (unilaterally) cryptorchid and scrotal testes. Pregnant rats were gavaged with 500 mg/kg/day DBP or corn oil from embryonic (E) Days 13.5 to 21.5. Male offspring were sampled on E21.5 or Postnatal Day 6, 10, 15, 25, or 90. Sertoli cell number in DBP-exposed males was reduced by approximately 50% at E21.5 but recovered to normal by Days 25-90, accompanied by significant changes in plasma inhibin B and testosterone levels. Sertoli cell maturational development in DBP-exposed males, assessed using five protein markers (anti-müllerian hormone, cytokeratin, androgen receptor, CDKN1B, and Nestin), was largely normal, with some evidence of delayed maturation. However, in adulthood, Sertoli cells (SC) in areas lacking germ cells (Sertoli cell-only [SCO] tubules) often exhibited immature features, especially in cryptorchid testes. Sertoli cells in DBP-exposed animals supported fewer germ cells during puberty, but this normalized in scrotal testes by adulthood. Scrotal and especially cryptorchid testes from DBP-exposed animals exhibited abnormalities (SCO tubules, focal dysgenetic areas) at all postnatal ages. Cryptorchid testes from DBP-exposed animals exhibited more Sertoli cell abnormalities at Day 25 compared with scrotal testes, perhaps indicating more severe underlying Sertoli cell malfunction in these testes. Our findings support the concept of altered Sertoli cell development in TDS, especially in cryptorchid testes, but show that maturational defects in Sertoli cells in adulthood most commonly reflect secondary dedifferentiation in absence of germ cells.

Experimentally induced testicular dysgenesis syndrome originates in the masculinization programming window

“…To visualize germ cells and thus to identify SCO tubules and tubules with incomplete spermatogenesis in adult rat testes, sections were immunostained with an antibody to detect the germ cell marker DAZL (gift from H. Cooke, University of Edinburgh; 1:1,000), using standard immunohistochemistry techniques as described elsewhere (73). Two complete cross sections of each adult testis were then systematically evaluated by a person blinded to the treatment group who identified any tubules with incomplete spermatogenesis (i.e., tubules lacking specific germ cell types that should be present, tubules with clearly reduced germ cell numbers, or tubules with no germ cells [SCO]); note that SCO tubules located at the testis periphery were ignored, as these are likely to be a normal part of the rete testis.…”

Fetal programming of adult Leydig cell function by androgenic effects on stem/progenitor cells

“…Because we used scrotal and cryptorchid testes from DBP-exposed animals for these studies, we used a control gene (Sox9) expressed specifically in Sertoli cells to correct expression of the target genes, because Leydig cell mRNAs would be overrepresented in cryptorchid vs. scrotal testes because of massive germ cell loss in the former. Sox9 was chosen, because adult Sertoli cell number is unchanged in DBP-exposed animals, irrespective of whether testes are scrotal or cryptorchid (41). In DBP-exposed animals, expression of Lhcgr, Cyp11a1, Cyp17a1, and 17β-hsd3 were unchanged, whereas expression of StAR and 3β-hsd were both significantly reduced compared with controls (Fig.…”

“…Immunostaining was for two to three proteins to delineate cell types for analysis using methods and antibodies validated previously (33,41). Slides were dewaxed and rehydrated and underwent heat-induced antigen retrieval (33).…”

“…Numbers per testis of COUP-TFII + adult Leydig stem cells and adult Leydig cells were determined by stereology (33,41). A Zeiss Axio-Imager microscope (Carl Zeiss) fitted with a Hitachi HVC20 camera (Hitachi Denshi Europe) and a Prior automatic stage (Prior Scientific Instruments Ltd.) was used plus Image-Pro Plus v7.0 with Stereologer Analyzer Pro (Media Cybernetics).…”

“…A Zeiss Axio-Imager microscope (Carl Zeiss) fitted with a Hitachi HVC20 camera (Hitachi Denshi Europe) and a Prior automatic stage (Prior Scientific Instruments Ltd.) was used plus Image-Pro Plus v7.0 with Stereologer Analyzer Pro (Media Cybernetics). Using random fields, testis cross-sections were analyzed for COUP-TFII + /3β-HSD neg adult Leydig stem cells and adult Leydig cells (3β-HSD + ) and expressed as relative volumes per testis before conversion to absolute volumes using testis weight; then, they were converted to cell number per testis using the average nuclear diameter (∼150 nuclei) measured for each sample, group, and age (33,41).…”