Androgen receptor (AR) signaling in Sertoli cells is known to be important for germ-cell progression through meiosis, but the extent to which androgens indirectly regulates specific meiosis stages is not known. Here, we combine synchronization of spermatogenesis, cytological analyses and single-cell RNAseq (scRNAseq) in the S ertoli c ell a ndrogen r eceptor k nock o ut (SCARKO) mutant and control mice, and demonstrate that SCARKO mutant spermatocytes exhibited normal expression and localization of key protein markers of meiotic prophase events, indicating that initiation of meiotic prophase is not androgen dependent. However, spermatocytes from SCARKO testes failed to acquire competence for the meiotic division phase. ScRNAseq analysis of wild type and SCARKO mutant testes revealed a molecular transcriptomic block in an early meiotic prophase state (leptotene/zygotene) in mutant germ cells, and identified several misregulated genes in SCARKO Sertoli cells, many of which have been previously implicated in male infertility. Together, our coordinated cytological and single-cell RNAseq analyses identified germ-cell intrinsic and extrinsic genes responsive to Sertoli-cell androgen signaling that promotes cellular states permissive for the meiotic division phase.

Meiosis defines and dictates much of the success of gametogenesis and is pivotal to our understanding of reproduction and causes of infertility. It is not only a defining event of gametogenesis but also a basic feature of eukaryotic genetics. Meiosis is the engine for genetic diversity, and ensures the genomic integrity and correct number of chromosomes that produce healthy offspring. Genetic analyses have been key to our unfolding knowledge of meiosis, highlighted here in discussions of the intricate chromosomal dynamics of meiotic prophase and the precise reductional and equational segregation of chromosomes in the meiotic division phases. These important aspects of meiosis provide lessons that can be applied to safeguarding human health, fertility, and offspring well-being.

All-trans retinoic acid (atRA), the active metabolite of vitamin A, is a ligand for several nuclear receptors and acts as a critical regulator of many physiologic processes. The cytochrome P450 family 26 (CYP26) enzymes are responsible for atRA clearance, and are potential drug targets to increase concentrations of endogenous atRA in a tissue-specific manner. Talarozole is a potent inhibitor of CYP26A1 and CYP26B1, and has shown some success in clinical trials. However, it is not known what magnitude of change is needed in tissue atRA concentrations to promote atRA signaling changes. The aim of this study was to quantify the increase in endogenous atRA concentrations necessary to alter atRA signaling in target organs, and to establish the relationship between CYP26 inhibition and altered atRA concentrations in tissues. Following a single 2.5-mg/kg dose of talarozole to mice, atRA concentrations increased up to 5.7-, 2.7-, and 2.5-fold in serum, liver, and testis, respectively, resulting in induction of Cyp26a1 in the liver and testis and Rar β and Pgc 1β in liver. The increase in atRA concentrations was well predicted from talarozole pharmacokinetics and in vitro data of CYP26 inhibition. After multiple doses of talarozole, a significant increase in atRA concentrations was observed in serum but not in liver or testis. This lack of increase in atRA concentrations correlated with an increase in CYP26A1 expression in the liver. The increased atRA concentrations in serum without a change in liver suggest that CYP26B1 in extrahepatic sites plays a key role in regulating systemic atRA exposure.

All-trans-retinoic acid (atRA) is the active metabolite of vitamin A. The liver is the main storage organ of vitamin A, but activation of the retinoic acid receptors (RARs) in mouse liver and in human liver cell lines has also been shown. Although atRA treatment improves mitochondrial function in skeletal muscle in rodents, its role in modulating mitochondrial function in the liver is controversial, and little data are available regarding the human liver. The aim of this study was to determine whether atRA regulates hepatic mitochondrial activity atRA treatment increased the mRNA and protein expression of multiple components of mitochondrial beta-oxidation, tricarboxylic acid (TCA) cycle, and respiratory chain. Additionally, atRA increased mitochondrial biogenesis in human hepatocytes and in HepG2 cells with and without lipid loading based on peroxisome proliferator activated receptor gamma coactivator 1 alpha and 1 beta and nuclear respiratory factor 1 mRNA and mitochondrial DNA quantification. atRA also increased beta-oxidation and ATP production in HepG2 cells and in human hepatocytes. Knockdown studies of RAR alpha, RAR beta, and PPAR gamma revealed that the enhancement of mitochondrial biogenesis and beta-oxidation by atRA requires peroxisome proliferator activated receptor delta. In vivo in mice, atRA treatment increased mitochondrial biogenesis markers after an overnight fast. Inhibition of atRA metabolism by talarozole, a cytochrome P450 (CYP) 26 specific inhibitor, increased the effects of atRA on mitochondrial biogenesis markers in HepG2 cells and in vivo in mice. These studies show that atRA regulates mitochondrial function and lipid metabolism and that increasing atRA concentrations in human liver via CYP26 inhibition may increase mitochondrial biogenesis and fatty acid beta-oxidation and provide therapeutic benefit in diseases associated with mitochondrial dysfunction.

The active metabolite of vitamin A, all‐trans‐retinoic acid (atRA), regulates many physiological processes including lipid homeostasis, adiposity and spermatogenesis. Clinically atRA is used in the treatment of acute promyelocytic leukemia but use is associated with high relapse rates likely due to autoinduction of CYP26s, the major enzymes responsible for atRA clearance. To overcome autoinduction and increase efficacy, retinoic acid metabolism blocking agents (RAMBAs) were developed. Subsequently, inhibition of CYP26s has become a drug development strategy with the aim to increase endogenous levels of atRA in other diseases.The RAMBA talarozole (TLZ) is a selective and potent CYP26 inhibitor. The objective of this work was to determine whether TLZ treatment increases endogenous atRA concentrations in retinoid target tissues as predicted from in vitro CYP26 inhibition data and TLZ disposition in mice, and to establish whether TLZ treatment alters retinoid signaling related to fatty acid oxidation in the liver and spermatogonial differentiation in the testesTalarozole was administered to mice as a single dose (2.5 mg/kg) or as multiple doses for three days. Serum TLZ concentrations and serum, liver and testes atRA concentrations were measured by LC‐MS/MS. Inhibition of CYP26 and changes in atRA concentrations in each tissue were predicted based on CYP26 activity in vitro and TLZ disposition. Markers of fatty acid oxidation in the liver and spermatogonial differentiation in the testes were measured following TLZ treatment.A maximum 84% inhibition of CYP26 activity at 0.5 hours post‐dose was predicted based on TLZ Cmax of 80 nM and a Ki of 1 nM following a single dose of TLZ. Due to the short TLZ half‐life (2.2 hrs) CYP26 activity was predicted to return to 100% by 12 hours. In agreement with the predictions, atRA concentrations were increased by 82, 63 and 60% at 4 hours post‐dose in the serum, liver and testes, respectively, and concentrations returned to baseline by 24 hours. Following multiple doses of TLZ, liver CYP26 mRNA and activity were increased suggesting autoinduction of CYP26 due to increased atRA concentrations. In agreement, atRA concentrations were elevated in serum and liver at all timepoints measured. This increase in atRA concentrations was associated with increased mRNA of the mitochondrial biogenesis markers PGC‐1β and NRF‐1 in comparison to control mice. atRA signaling in the testes was increased following multiple doses of TLZ based on Stra8 expression, a marker of atRA signaling, and spermatogonial differentiation, but the increase in testes atRA concentrations was not significant.This study shows that administration of a RAMBA to inhibit the enzymes primarily responsible for atRA elimination (CYP26s) increases atRA concentrations in the serum, liver and testes of mice and increases the expression of genes that are markers of retinoid signaling in the target tissues. These studies suggest that blocking atRA metabolism in vivo via CYP26 inhibition may be a viable drug target in select tissues.Support or Funding InformationThis work is support by NIH grants R01GM111772 and T32GM007750.

All-trans-retinoic acid (atRA) is the active metabolite of vitamin A. The liver is the main storage organ of vitamin A, but activation of the retinoic acid receptors (RARs) in mouse liver and in human liver cell lines has also been shown. AlthoughatRA treatment improves mitochondrial function in skeletal muscle in rodents, its role in modulating mitochondrial function in the liver is controversial, and little data are available regarding the human liver. The aim of this study was to determine whetheratRA regulates hepatic mitochondrial activity.atRA treatment increased the mRNA and protein expression of multiple components of mitochondrialβ-oxidation, tricarboxylic acid (TCA) cycle, and respiratory chain. Additionally,atRA increased mitochondrial biogenesis in human hepatocytes and in HepG2 cells with and without lipid loading based on peroxisome proliferator activated receptor gamma coactivator 1αand 1βand nuclear respiratory factor 1 mRNA and mitochondrial DNA quantification.atRA also increasedβ-oxidation and ATP production in HepG2 cells and in human hepatocytes. Knockdown studies of RARα, RARβ, and PPARδrevealed that the enhancement of mitochondrial biogenesis andβ-oxidation byatRA requires peroxisome proliferator activated receptor delta. In vivo in mice,atRA treatment increased mitochondrial biogenesis markers after an overnight fast. Inhibition ofatRA metabolism by talarozole, a cytochrome P450 (CYP) 26 specific inhibitor, increased the effects ofatRA on mitochondrial biogenesis markers in HepG2 cells and in vivo in mice. These studies show thatatRA regulates mitochondrial function and lipid metabolism and that increasingatRA concentrations in human liver via CYP26 inhibition may increase mitochondrial biogenesis and fatty acidβ-oxidation and provide therapeutic benefit in diseases associated with mitochondrial dysfunction.

The active metabolite of vitamin A, retinoic acid (RA), is known to be essential for spermatogenesis. Changes to RA levels within the seminiferous epithelium can alter the development of male germ cells, including blocking their differentiation completely. Excess RA has been shown to cause germ cell death in both neonatal and adult animals, yet the cells capable of degrading RA within the testis have yet to be investigated. One previous study alluded to a requirement for one of the RA degrading enzymes, CYP26B1, in Sertoli cells but no data exist to determine whether germ cells possess the ability to degrade RA. To bridge this gap, the roles of CYP26A1 and CYP26B1 within the seminiferous epithelium were investigated by creating single and dual conditional knockouts of these enzymes in either Sertoli or germ cells. Analysis of these knockout models revealed that deletion of both Cyp26a1 and Cyp26b1 in either cell type resulted in increased vacuolization within the seminiferous tubules, delayed spermatid release, and an increase in the number of STRA8-positive spermatogonia, but spermatozoa were still produced and the animals were found to be fertile. However, elimination of CYP26B1 activity within both germ and Sertoli cells resulted in severe male subfertility, with a loss of advanced germ cells from the seminiferous epithelium. These data indicate that CYP26 activity within either Sertoli or germ cells is essential for the normal progression of spermatogenesis and that its loss can result in reduced male fertility.

Perturbations in the vitamin A metabolism pathway could be a significant cause of male infertility, as well as a target toward the development of a male contraceptive, necessitating the need for a better understanding of how testicular retinoic acid (RA) concentrations are regulated. Quantitative analyses have recently demonstrated that RA is present in a pulsatile manner along testis tubules. However, it is unclear if the aldehyde dehydrogenase (ALDH) enzymes, which are responsible for RA synthesis, contribute to the regulation of these RA concentration gradients. Previous studies have alluded to fluctuations in ALDH enzymes across the spermatogenic cycle, but these inferences have been based primarily on qualitative transcript localization experiments. Here, we show via various quantitative methods that the three well-known ALDH enzymes (ALDH1A1, ALDH1A2, and ALDH1A3), and an ALDH enzyme previously unreported in the murine testis (ALDH8A1), are not expressed in a stage-specific manner in the adult testis, but do fluctuate throughout juvenile development in perfect agreement with the first appearance of each advancing germ cell type. We also show, via treatments with a known ALDH inhibitor, that lowered testicular RA levels result in an increase in blood-testis barrier permeability, meiotic recombination, and meiotic defects. Taken together, these data further our understanding of the complex regulatory actions of RA on various spermatogenic events and, in contrast with previous studies, also suggest that the ALDH enzymes are not responsible for regulating the recently measured RA pulse.

The asynchronous cyclic nature of spermatogenesis is essential for continual sperm production and is one of the hallmarks of mammalian male fertility. While various mRNA and protein localization studies have indirectly implicated changing retinoid levels along testis tubules, no quantitative evidence for these changes across the cycle of the seminiferous epithelium currently exists. This study utilized a unique mouse model of induced synchronous spermatogenesis, localization of the retinoid-signaling marker STRA8, and sensitive quantification of retinoic acid concentrations to determine whether there are fluctuations in retinoid levels at each of the individual stages of germ cell differentiation and maturation to sperm. These data show that processive pulses of retinoic acid are generated during spermatogonial differentiation and are the likely trigger for cyclic spermatogenesis and allow us, for the first time, to understand how the cycle of the seminiferous epithelium is generated and maintained. In addition, this study represents the first direct quantification of a retinoid gradient controlling cellular differentiation in a postnatal tissue.

all-trans retinoic acid (atRA), the active metabolite of vitamin A, is an essential signaling molecule. Specifically the concentrations of atRA are spatiotemporally controlled in target tissues such as the liver and the testes. While the enzymes of the aldehyde dehydrogenase 1A family (ALDH1A) are believed to control the synthesis of atRA, a direct relationship between altered ALDH1A activity and tissue atRA concentrations has never been shown. To test whether inhibition of ALDH1A enzymes decreases atRA concentrations in a tissue specific manner, the potent ALDH1A inhibitor WIN 18,446 was used to inhibit ALDH1A activity in mice. The ALDH1A expression, atRA formation kinetics, ALDH1A inhibition by WIN 18,446 and WIN 18,446 disposition were used to predict the time course and extent of inhibition of atRA formation in the testis and liver. The effect of WIN 18,446 on atRA concentrations in testis, liver and serum were measured following single and multiple doses of WIN 18,446. ALDH1A1 and ALDH1A2 were responsible for the majority of atRA formation in the testis while ALDH1A1 and aldehyde oxidase contributed to atRA formation in the liver. Due to the different complement of enzymes contributing to atRA formation in different tissues and different inhibition of ALDH1A1 and ALDH1A2 by WIN 18,446, WIN 18,446 caused only a 50% decrease in liver atRA but testicular atRA decreased over 90%. Serum atRA concentrations were also reduced. These data demonstrate that inhibition of ALDH1A enzymes will decrease atRA concentrations in a tissue specific manner and selective ALDH1A inhibition could be used to alter atRA concentrations in select target tissues.

Retinoic acid (RA), the active metabolite of vitamin A, is required for spermatogenesis and many other biological processes. RA formation requires irreversible oxidation of retinal to RA by aldehyde dehydrogenase enzymes of the 1A family (ALDH1A). While ALDH1A1, ALDH1A2, and ALDH1A3 all form RA, the expression pattern and relative contribution of these enzymes to RA formation in the testis is unknown. In this study, novel methods to measure ALDH1A protein levels and intrinsic RA formation were used to accurately predict RA formation velocities in individual human testis samples and an association between RA formation and intratesticular RA concentrations was observed. The distinct localization of ALDH1A in the testis suggests a specific role for each enzyme in controlling RA formation. ALDH1A1 was found in Sertoli cells, while only ALDH1A2 was found in spermatogonia, spermatids, and spermatocytes. In the absence of cellular retinol binding protein (CRBP)1, ALDH1A1 was predicted to be the main contributor to intratesticular RA formation, but when CRBP1 was present, ALDH1A2 was predicted to be equally important in RA formation as ALDH1A1. This study provides a comprehensive novel methodology to evaluate RA homeostasis in human tissues and provides insight to how the individual ALDH1A enzymes mediate RA concentrations in specific cell types.

Retinoic acid (RA), the active form of vitamin A (retinol), is indispensable for maintaining many essential biological processes. RA formation depends on a complex network of enzymes. The rate limiting step is irreversible formation of RA by aldehyde dehydrogenase1A (ALDH1A1‐1A3). However, the specific roles, tissue expression, and localization of these enzymes are not well understood.While RA is necessary for spermatogenesis in animal models, the source of intratesticular RA is unknown. Sertoli cell specific ALDH1A knockouts have demonstrated intratesticular RA formation is required for male fertility. Due to the requirement for intratesticular RA formation and presence of mRNA for all three ALDH1A enzymes in the human testis, the human testis was chosen as a model of the biochemistry of RA formation.In order to model RA formation, a novel LC‐MS/MS based peptide quantification method was developed to quantify ALDH1A in a cohort of 18 men. Each ALDH1A concentration was used to predict the velocity of RA formation in each donor. The accuracy of our predictions was confirmed by measuring intrinsic RA formation and RA concentrations in the same samples. Additionally, weidentified a distinct localization pattern for ALDH1A enzymes in the human testis using immunohistochemistry.In conclusion, these novel techniques were successfully used to model RA formation in human testicular tissue. In the future, RA formation in other tissues can be investigated using the assays developed.Grant Funding Source: Supported by NIH/NICHD grant U54 HD042454 and TL1TR000422

The asynchronous cyclic nature of spermatogenesis is essential for continual sperm production and is one of the hallmarks of mammalian male fertility. While various mRNA and protein localization studies have indirectly implicated changing retinoid levels along testis tubules, no quantitative evidence for these changes across the cycle of the seminiferous epithelium currently exists. This study utilized a unique mouse model of induced synchronous spermatogenesis, localization of the retinoid signaling marker STRA8 and sensitive quantification of retinoic acid concentrations to determine whether there are fluctuations in retinoid levels at each of the individual stages of germ cell differentiation and maturation to sperm. These data show that processive pulses of retinoic acid are generated during spermatogonial differentiation, and are the likely trigger for cyclic spermatogenesis and allow us, for the first time, to understand how the cycle of the seminiferous epithelium is generated and maintained. In addition, this study represents the first direct quantification of a retinoid gradient controlling cellular differentiation in a postnatal tissue.

Vitamin A has been shown to be essential for a multitude of biological processes vital for mammalian development and homeostasis. Its active metabolite, retinoic acid (RA), is important for establishing and maintaining proper germ cell development. During spermatogenesis, the germ cells orient themselves in very distinct patterns, which have been organized into stages. There is evidence to show that, in the mouse, RA is needed for many steps during germ cell development. Interestingly, RA has been implicated as playing a role within the same two Stages: VII and VIII, where meiosis is initiated and spermiation occurs. The goal of this review is to outline this evidence, exploring the relevant players in retinoid metabolism, storage, transport, and signaling. Finally, this review will provide a potential model for how RA activity is organized across the murine stages of the spermatogenic cycle.

The BDADs (bis-[dichloroacetyl]-diamines) are compounds that can inhibit spermatogenesis via blocking the metabolism of vitamin A. We utilized one specific BDAD, WIN 18,446, to manipulate the endogenous production of retinoic acid (RA) in the testis to further investigate the action of this compound on mammalian sperm production. Transient treatment of adult male mice with WIN 18,446 blocked spermatogonial differentiation and induced significant changes in the cycle of the seminiferous epithelium. WIN 18,446 treatment of neonatal mice also blocked spermatogonial differentiation and, followed by injection of RA, induced synchronous spermatogenesis in adulthood. The net result was pulsatile, rather than normal continuous, release of sperm from the seminiferous epithelium. This study describes a novel technique that can enrich for specific germ cell populations within the testis, representing a valuable new tool for studying spermatogenesis.

Bis-(dichloroacetyl)-diamines (BDADs) are compounds that inhibit spermatogenesis and function as male contraceptives in many species; however, their mechanism of action has yet to be fully investigated. It has been proposed that BDADs may function via inhibition of testicular retinoic acid (RA) biosynthesis. We employed an organ culture technique and the expression of a marker for RA activity, Stra8 (stimulated by retinoic acid gene 8), to investigate if the BDAD WIN 18,446 inhibited the biosynthesis of RA from retinol (ROL) in neonatal and adult murine testis and in the embryonic murine gonad. After culturing either whole testes or germ cells isolated from mice at 2 days postpartum (dpp) with WIN 18,446 or with WIN 18,446 plus ROL, Stra8 expression was suppressed, demonstrating that WIN 18,446 inhibited the conversion of ROL to RA in both systems. We also utilized a transgenic mouse containing an RA-responsive LacZ reporter gene to demonstrate limited RA induction of LacZ expression in 2-dpp testes cultured with WIN 18,446 plus ROL. The expression of Stra8 was downregulated in adult mouse testis tubules cultured with WIN 18,446 when compared to tubules cultured with the vehicle control. WIN 18,446 also inhibited the conversion of ROL to RA in embryonic ovaries and testes cultured for 48 h. These murine results provide critical insights regarding how the BDADs can inhibit spermatogenesis by blocking the ability of vitamin A to drive germ cell development. In addition, these techniques will be useful for screening novel inhibitors of RA biosynthesis as potential male contraceptives.

BDADs (Bis-(dichloroacetyl)-diamines) are a group of compounds that inhibit the conversion of vitamin A (retinol, (ROL)) to its active metabolite, retinoic acid (RA), which is essential for mammalian sperm production. This study investigates the feasibility of using BDADs to inhibit spermatogenesis leading to their potential use as male contraceptives. A novel testis organ culture technique and the expression of a marker of RA activity, Stra8, were utilized to demonstrate that a specific BDAD, WIN 18,446, can inhibit the metabolism of ROL to RA in the neonatal and adult murine testis, and the embryonic murine gonad. After culturing either whole testes or germ cells isolated from 2 days post partum (dpp) mice with BDAD or BDAD plus ROL, Stra8 expression was suppressed, demonstrating that BDAD inhibited the conversion of ROL to RA in both systems. We also utilized a transgenic mouse containing an RA-activated LacZ reporter gene to demonstrate that there was limited RA activation of LacZ in 2 dpp testes cultured with BDAD or BDAD plus ROL. The expression of Stra8 was also downregulated in adult mouse testis tubules cultured with BDAD when compared to tubules cultured with the vehicle control. BDAD also inhibited the conversion of ROL to RA in embryonic ovaries and testes cultured for 48 hours. These murine results, combined with the significant reduction in sperm count also observed in rabbits treated with WIN 18,446, provide critical insights into how the BDADs can inhibit spermatogenesis through blocking the ability of vitamin A to drive germ cell development. This work was supported by the NIH Grants HD 10808 to MDG and U01 HD060408 to JA. (platform)