The publication data currently available has been vetted by Vanderbilt faculty, staff, administrators and trainees. The data itself is retrieved directly from NCBI's PubMed and is automatically updated on a weekly basis to ensure accuracy and completeness.
If you have any questions or comments, please contact us.
Phthalate exposure impairs testis development and function; however, whether phthalates affect nonreproductive functions is not well understood. To investigate this, C57BL/6J mice were fed 1-500 mg di-n-butyl phthalate (DBP) in corn oil, or vehicle only, daily from 4 to 14 days, after which tissues were collected (prepubertal study). Another group was fed 1-500 mg/kg·d DBP from 4 to 21 days and then maintained untreated until 8 weeks for determination of adult consequences of prepubertal exposure. Bones were assessed by microcomputed tomography and dual-energy X-ray absorptiometry and T by RIA. DBP exposure decreased prepubertal femur length, marrow volume, and mean moment of inertia. Adult animals exposed prepubertally to low DBP doses had lower bone mineral content and bone mineral density and less lean tissue mass than vehicle-treated animals. Altered dynamics of the emerging Leydig population were found in 14-day-old animals fed 100-500 mg/kg·d DBP. Adult mice had variable testicular T and serum T and LH concentrations after prepubertal exposure and a dose-dependent reduction in cytochrome p450, family 11, subfamily A, polypeptide 1. Insulin-like 3 was detected in Sertoli cells of adult mice administered the highest dose of 500 mg/kg·d DBP prepubertally, a finding supported by the induction of insulin-like 3 expression in TM4 cells exposed to 50 μM, but not 5 μM, DBP. We propose that low-dose DBP exposure is detrimental to bone but that normal bone mineral density/bone mineral content after high-dose DBP exposure reflects changes in testicular somatic cells that confer protection to bones. These findings will fuel concerns that low-dose DBP exposure impacts health beyond the reproductive axis.
The physiological role of multidrug resistance protein 4 (Mrp4, Abcc4) in the testes is unknown. We found that Mrp4 is expressed primarily in mouse and human Leydig cells; however, there is no current evidence that Mrp4 regulates testosterone production. We investigated its role in Leydig cells, where testosterone production is regulated by cAMP, an intracellular messenger formed when the luteinizing hormone (LH) receptor is activated. Because Mrp4 regulates cAMP, we compared testosterone levels in Mrp4(-/-) and Mrp4(+/+) mice. Young Mrp4(-/-) mice had significantly impaired gametogenesis, reduced testicular testosterone, and disruption of Leydig cell cAMP homeostasis. Both young and adult mice had impaired testosterone production. In Mrp4(-/-) primary Leydig cells treated with LH, intracellular cAMP production was impaired and cAMP-response element-binding protein (CREB) phosphorylation was strongly attenuated. Notably, expression of CREB target genes that regulate testosterone biosynthesis was reduced in Mrp4(-/-) Leydig cells in vivo. Therefore, Mrp4 is required for normal Leydig cell testosterone production. However, adult Mrp4(-/-) mice are fertile, with a normal circulating testosterone concentration. The difference is that in 3-week-old Mrp4(-/-) mice, disruption of gonadal testosterone production up-regulates hepatic Cyp2b10, a known testosterone-metabolizing enzyme. Therefore, defective testicular testosterone production de-regulates hepatic Cyp-mediated testosterone metabolism to disrupt gametogenesis. These findings have important implications for understanding the side effects of therapeutics that disrupt Mrp4 function and are reported to alter androgen production.
Proper cell fate determination in mammalian gonads is critical for the establishment of sexual identity. The Hedgehog (Hh) pathway has been implicated in cell fate decision for various organs, including gonads. Desert Hedgehog (Dhh), one of the three mammalian Hh genes, has been implicated with other genes in the establishment of mouse fetal Leydig cells. To investigate whether Hh alone is sufficient to induce fetal Leydig cell differentiation, we ectopically activated the Hh pathway in Steroidogenic factor 1 (SF1)-positive somatic cell precursors of fetal ovaries. Hh activation transformed SF1-positive somatic ovarian cells into functional fetal Leydig cells. These ectopic fetal Leydig cells produced androgens and insulin-like growth factor 3 (INLS3) that cause virilization of female embryos and ovarian descent. However, the female reproductive system remained intact, indicating a typical example of female pseudohermaphroditism. The appearance of fetal Leydig cells was a direct consequence of Hh activation as evident by the absence of other testicular components in the affected ovary. This study provides not only insights into mechanisms of cell lineage specification in gonads, but also a model to understand defects in sexual differentiation.
Lanosterol 14alpha-demethylase (CYP51) is a cytochrome P450 enzyme involved primarily in cholesterol biosynthesis. CYP51 in the presence of NADPH-cytochrome P450 reductase converts lanosterol to follicular fluid meiosis activating sterol (FF-MAS), an intermediate of cholesterol biosynthesis which accumulates in gonads and has an additional function as oocyte meiosis-activating substance. This work shows for the first time that cholesterogenic enzymes are highly expressed only in distinct stages of spermatogenesis. CYP51, NADPH-P450 reductase (the electron transferring enzyme needed for CYP51 activity) and squalene synthase (an enzyme preceding CYP51 in the pathway) proteins have been studied. CYP51 was detected in step 3-19 spermatids, with large amounts in the cytoplasm/residual bodies of step 19 spermatids, where P450 reductase was also observed. Squalene synthase was immunodetected in step 2-15 spermatids of the rat, indicating that squalene synthase and CYP51 proteins are not equally expressed in same stages of spermatogenesis. Discordant expression of cholesterogenic genes may be a more general mechanism leading to transient accumulation of pathway intermediates in spermatogenesis. This study provides the first evidence that step 19 spermatids and residual bodies of the rat testis have the capacity to produce MAS sterols in situ.
We have isolated and characterized a P-45017 alpha cDNA fragment from a rat testis library. The partial length rat P-45017 alpha cDNA (1Kb) has high overall nucleotide and deduced amino acid similarity with human and bovine P-45017 alpha cDNA's and contains the conserved tridecapeptide and heme regions, the termination codon and polyadenylation site. Using this rat testis cDNA probe we measured P-45017 alpha mRNA levels of rat Leydig cells from animals treated with hCG. Temporal studies with a low hCG dose showed an early increase in mRNA levels returning to control values at later times, while a higher desensitizing dose caused a marked reduction in the mRNA (24 h) and a small recovery at 48 h. Fetal rat Leydig cells maintained in the presence of LH treated with estradiol showed a 70% decrease in P-45017 alpha mRNA levels and testosterone production followed closely the changes in P-45017 alpha mRNA. These studies suggest that gonadotropin stimulation and desensitization of P-45017 alpha dependent enzymes in the adult rat testis as well as estradiol induced desensitization in fetal Leydig cells are related to levels P-45017 alpha mRNA.