Loss of Etv5 decreases proliferation and RET levels in neonatal mouse testicular germ cells and causes an abnormal first wave of spermatogenesis.

Mice that are ets variant gene 5 (ETV5) null (Etv5(-/-)) undergo the first wave of spermatogenesis but lose all spermatogonial stem cells (SSCs) during this time. The SSC loss in Etv5(-/-) mice begins during the neonatal period, suggesting a role for ETV5 in SSC self-renewal during this period. Herein, we show that Etv5 mRNA was present in perinatal mouse testis and that ETV5 was expressed in fetal Sertoli cells and by germ cells and Sertoli cells during the neonatal period. Transplantation of Etv5(-/-) germ cells failed to establish spermatogenesis in W/W(v) mice testes, indicating that germ cell ETV5 has a key role in establishment or self-renewal of transplanted SSCs. The SSC self-renewal is stimulated by glial cell-derived neurotrophic factor (GDNF) acting through the RET/GDNF family receptor alpha 1 (GFRA1) receptor complex in SSCs. Immunohistochemistry, quantitative PCR, and laser capture microdissection revealed decreased RET mRNA and protein expression in spermatogonia of neonatal Etv5(-/-) mice by Postnatal Days 4-8, indicating that disrupted GDNF/RET/GFRA1 signaling may occur before initial spermatogonial stem/progenitor cell decrease. Etv5(-/-) spermatogonia had reduced proliferation in vivo and in vitro. Decreased cell proliferation may cause the observed decreases in the number of type A spermatogonia (Postnatal Day 17) and daily sperm production (Postnatal Day 30) in Etv5(-/-) mice, indicating quantitative impairments in the first wave of spermatogenesis. In conclusion, ETV5 is expressed beginning in fetal Sertoli cells and can potentially have effects on neonatal Sertoli cells and germ cells. In addition, ETV5 has critical effects on neonatal spermatogonial proliferation, which may involve impaired signaling through the RET receptor.

Role of microsomal retinol/sterol dehydrogenase-like short-chain dehydrogenases/reductases in the oxidation and epimerization of 3alpha-hydroxysteroids in human tissues.

Allopregnanolone (ALLO) and androsterone (ADT) are naturally occurring 3alpha-hydroxysteroids that act as positive allosteric regulators of gamma-aminobutyric acid type A receptors. In addition, ADT activates nuclear farnesoid X receptor and ALLO activates pregnane X receptor. At least with respect to gamma-aminobutyric acid type A receptors, the biological activity of ALLO and ADT depends on the 3alpha-hydroxyl group and is lost upon its conversion to either 3-ketosteroid or 3beta-hydroxyl epimer. Such strict structure-activity relationships suggest that the oxidation or epimerization of 3alpha-hydroxysteroids may serve as physiologically relevant mechanisms for the control of the local concentrations of bioactive 3alpha-hydroxysteroids. The exact enzymes responsible for the oxidation and epimerization of 3alpha-hydroxysteroids in vivo have not yet been identified, but our previous studies showed that microsomal nicotinamide adenine dinucleotide-dependent short-chain dehydrogenases/reductases (SDRs) with dual retinol/sterol dehydrogenase substrate specificity (RoDH-like group of SDRs) can oxidize and epimerize 3alpha-hydroxysteroids in vitro. Here, we present the first evidence that microsomal nicotinamide adenine dinucleotide-dependent 3alpha-hydroxysteroid dehydrogenase/epimerase activities are widely distributed in human tissues with the highest activity levels found in liver and testis and lower levels in lung, spleen, brain, kidney, and ovary. We demonstrate that RoDH-like SDRs contribute to the oxidation and epimerization of ALLO and ADT in living cells, and show that RoDH enzymes are expressed in tissues that have microsomal 3alpha-hydroxysteroid dehydrogenase/epimerase activities. Together, these results provide further support for the role of RoDH-like SDRs in human metabolism of 3alpha-hydroxysteroids and offer a new insight into the enzymology of ALLO and ADT inactivation.

Comparative functional analysis of human medium-chain dehydrogenases, short-chain dehydrogenases/reductases and aldo-keto reductases with retinoids.

Retinoic acid biosynthesis in vertebrates occurs in two consecutive steps: the oxidation of retinol to retinaldehyde followed by the oxidation of retinaldehyde to retinoic acid. Enzymes of the MDR (medium-chain dehydrogenase/reductase), SDR (short-chain dehydrogenase/reductase) and AKR (aldo-keto reductase) superfamilies have been reported to catalyse the conversion between retinol and retinaldehyde. Estimation of the relative contribution of enzymes of each type was difficult since kinetics were performed with different methodologies, but SDRs would supposedly play a major role because of their low K(m) values, and because they were found to be active with retinol bound to CRBPI (cellular retinol binding protein type I). In the present study we employed detergent-free assays and HPLC-based methodology to characterize side-by-side the retinoid-converting activities of human MDR [ADH (alcohol dehydrogenase) 1B2 and ADH4), SDR (RoDH (retinol dehydrogenase)-4 and RDH11] and AKR (AKR1B1 and AKR1B10) enzymes. Our results demonstrate that none of the enzymes, including the SDR members, are active with CRBPI-bound retinoids, which questions the previously suggested role of CRBPI as a retinol supplier in the retinoic acid synthesis pathway. The members of all three superfamilies exhibit similar and low K(m) values for retinoids (0.12-1.1 microM), whilst they strongly differ in their kcat values, which range from 0.35 min(-1) for AKR1B1 to 302 min(-1) for ADH4. ADHs appear to be more effective retinol dehydrogenases than SDRs because of their higher kcat values, whereas RDH11 and AKR1B10 are efficient retinaldehyde reductases. Cell culture studies support a role for RoDH-4 as a retinol dehydrogenase and for AKR1B1 as a retinaldehyde reductase in vivo.