Synchronizing spermatogenesis in the mouse.

The formation of spermatozoa starts with a germ-line stem cell creating a pool of progenitor cells or undifferentiated spermatogonia. There is a requirement for these progenitor cells to be stimulated by retinoic acid (RA) to enter differentiation and ultimately form spermatocytes, undergo meiosis, form spermatids, and ultimately spermatozoa. After the stimulation by RA, which occurs at sites in the seminiferous tubules, it takes ~35 days to complete this complex process. As a result, the adult testis contains germ cells in all possible states of differentiation, and the isolation of individual cell types or study of functional aspects of the cycle of the seminiferous epithelium is very difficult. We describe the use of WIN 18 446-an inhibitor of RA synthesis followed by injection of RA as a mechanism for the synchronization of spermatogenesis to one to three stages of the cycle of the seminiferous epithelium. The result is that only one to four germ cell types are prevalent during the first wave of spermatogenesis. In the adult only a predictable few stages of the cycle are present throughout the entire testis enriching the targeted cells or stages of the cycle.

Crucial Convolution: Genetic and Molecular Mechanisms of Coiling during Epididymis Formation and Development in Embryogenesis.

As embryonic development proceeds, numerous organs need to coil, bend or fold in order to establish their final shape. Generally, this occurs so as to maximise the surface area for absorption or secretory functions (e.g., in the small and large intestines, kidney or epididymis); however, mechanisms of bending and shaping also occur in other structures, notably the midbrain-hindbrain boundary in some teleost fish models such as zebrafish. In this review, we will examine known genetic and molecular factors that operate to pattern complex, coiled structures, with a primary focus on the epididymis as an excellent model organ to examine coiling. We will also discuss genetic mechanisms involving coiling in the seminiferous tubules and intestine to establish the final form and function of these coiled structures in the mature organism.

Knockout of Cyp26a1 and Cyp26b1 during postnatal life causes reduced lifespan, dermatitis, splenomegaly, and systemic inflammation in mice.

All-trans-retinoic acid (atRA), the active metabolite of vitamin A, is an essential signaling molecule in all chordates. Global knockouts of the atRA clearing enzymes Cyp26a1 or Cyp26b1 are embryonic lethal. In adult rodents, inhibition of Cyp26a1 and Cyp26b1 increases atRA concentrations and signaling. However, postnatal knockout of Cyp26a1 does not cause a severe phenotype. We hypothesized that Cyp26b1 is the main atRA clearing Cyp in postnatal mammals. This hypothesis was tested by generating tamoxifen-inducible knockout mouse models of Cyp26b1 alone or with Cyp26a1. Both mouse models showed dermatitis, blepharitis, and splenomegaly. Histology showed infiltration of inflammatory cells including neutrophils and T lymphocytes into the skin and hyperkeratosis/hyperplasia of the nonglandular stomach. The mice lacking both Cyp26a1 and Cyp26b1 also had a reduced lifespan, failed to gain weight, and showed fat atrophy. There were significant changes in vitamin A homeostasis. Postnatal knockout of Cyp26b1 resulted in increased atRA concentrations in the skin while the postnatal knockout of both Cyp26a1 and Cyp26b1 resulted in increased atRA concentrations in the liver, serum, skin, spleen, and intestines. This study demonstrates the paramount role of Cyp26b1 in regulating retinoid homeostasis in postnatal life.

Dynamic paraspeckle component localisation during spermatogenesis.

Expression profiles and subcellular localisations of core Drosophila behaviour/human splicing (DBHS) proteins (PSPC1, SFPQ and NONO) and NEAT1, a long noncoding RNA (lncRNA), are investigated in developing and adult mouse testes. Core DBHS proteins are markers for the distinct subnuclear domain termed paraspeckles, while a long NEAT1 isoform scaffold facilitates paraspeckle nucleation. Paraspeckles contain many proteins (>40) and are broadly involved in RNA metabolism, including transcriptional regulation by protein sequestration, nuclear retention of A-to-I edited RNA transcripts to regulate translation and promoting survival during cellular stress. Immunohistochemistry reveals cell-specific profiles for core DBHS paraspeckle protein expression, indicating their functional diversity. PSPC1 is an androgen receptor co-activator, and it is detected in differentiating Sertoli cell nuclei from day 15 onwards, as they develop androgen responsiveness. PSPC1 is nuclear in the most mature male germ cell type present at each age, from foetal to adult life. In adult mouse testes, PSPC1 and SFPQ are present in Sertoli cells, spermatocytes and round spermatids, while the NEAT1 lncRNA appears in the punctate nuclear foci delineating paraspeckles only within Leydig cells. Identification of NEAT1 in the cytoplasm of spermatogonia and spermatocytes must reflect non-paraspeckle-related functions. NONO was absent from germ cells but nuclear in Sertoli cells. Reciprocal nuclear profiles of PSPC1 and γ-H2AX in spermatogenic cells suggest that each performs developmentally regulated roles in stress responses. These findings demonstrate paraspeckles and paraspeckle-related proteins contribute to diverse functions during testis development and spermatogenesis.

The retinoic acid hydroxylase Cyp26a1 has minor effects on postnatal vitamin A homeostasis, but is required for exogenous atRA clearance.

The all-trans-retinoic acid (atRA) hydroxylase Cyp26a1 is essential for embryonic development and may play a key role in regulating atRA clearance also in adults. We hypothesized that loss of Cyp26a1 activity via inducible knockout in juvenile or adult mice would result in decreased atRA clearance and increased tissue atRA concentrations and atRA-related adverse effects. To test these hypotheses, Cyp26a1 was knocked out in juvenile and adult male and female Cyp26a1 floxed mice using standard Cre-Lox technology and tamoxifen injections. Biochemical and histological methods were used to study the effects of global Cyp26a1 knockout. The Cyp26a1 knockout did not result in consistent histopathological changes in any major organs. Cyp26a1-/- mice gained weight normally and exhibited no adverse phenotypes for up to 1 year after loss of Cyp26a1 expression. Similarly, atRA concentrations were not increased in the liver, testes, spleen, or serum of these mice, and the Cyp26a1 knockout did not cause compensatory induction of lecithin:retinol acetyltransferase (Lrat) or retinol dehydrogenase 11 (Rdh11) mRNA or a decrease in aldehyde dehydrogenase 1a1 (Aldh1a1) mRNA in the liver compared with tamoxifen-treated controls. However, the Cyp26a1-/- mice showed increased bone marrow cellularity and decreased frequency of erythroid progenitor cells in the bone marrow consistent with a retinoid-induced myeloid skewing of hematopoiesis. In addition, the Cyp26a1 knockout decreased clearance of exogenous atRA by 70% and increased atRA half-life 6-fold. These findings demonstrate that despite lacking a major impact on endogenous atRA signaling, Cyp26a1 critically contributes as a barrier for exogenous atRA exposure.

Differential localization of histone variant TH2B during the first round compared with subsequent rounds of spermatogenesis.

BACKGROUND: Male germ cells are unique because they express a substantial number of variants of the general DNA binding proteins, known as histones, yet the biological significance of these variants is still unknown. In the present study, we aimed to address the expression pattern of the testis-specific histone H2B variant (TH2B) and the testis-specific histone H2A variant (TH2A) within the neonatal mouse testis. RESULTS: We demonstrate that TH2B and TH2A are present in a testis-enriched for undifferentiated spermatogonia. Co-localization studies with an undifferentiated marker, ZBTB16, revealed that TH2B and ZBTB16 co-localize in the neonatal testis. Upon the appearance of the primary spermatocytes, TH2B no longer co-localized with the ZBTB16 positive spermatogonia but were instead detected within the differentiating spermatogonia. This pattern of expression where TH2B and ZBTB16 no longer co-localize was maintained in the adult testis. CONCLUSION: These findings are in contrast to previous studies, which demonstrated that TH2B and TH2A were found only in adult spermatocytes. Our data are in support of a switch in the expression of these variants following the first round of spermatogonial differentiation. These studies reinforce current understandings that spermatogonia within the neonatal mouse testis are inherently different from those residing within the adult testis.

Does In Vitro Cytochrome P450 Downregulation Translate to In Vivo Drug-Drug Interactions? Preclinical and Clinical Studies With 13-cis-Retinoic Acid.

All-trans-retinoic acid (atRA) downregulates cytochrome P450 (CYP)2D6 in several model systems. The aim of this study was to determine whether all active retinoids downregulate CYP2D6 and whether in vitro downregulation translates to in vivo drug-drug interactions (DDIs). The retinoids atRA, 13cisRA, and 4-oxo-13cisRA all decreased CYP2D6 mRNA in human hepatocytes in a concentration-dependent manner. The in vitro data predicted ~ 50% decrease in CYP2D6 activity in humans after dosing with 13cisRA. However, the geometric mean area under plasma concentration-time curve (AUC) ratio for dextromethorphan between treatment and control was 0.822, indicating a weak induction of dextromethorphan clearance following 13cisRA treatment. Similarly, in mice treatment with 4-oxo-13cisRA-induced mRNA expression of multiple mouse Cyp2d genes. In comparison, a weak induction of CYP3A4 in human hepatocytes translated to a weak in vivo induction of CYP3A4. These data suggest that in vitro CYP downregulation may not translate to in vivo DDIs, and better understanding of the mechanisms of CYP downregulation is needed.

Sources of all-trans retinal oxidation independent of the aldehyde dehydrogenase 1A isozymes exist in the postnatal testis†.

Despite the essential role of the active metabolite of vitamin A, all-trans retinoic acid (atRA) in spermatogenesis, the enzymes, and cellular populations responsible for its synthesis in the postnatal testis remain largely unknown. The aldehyde dehydrogenase 1A (ALDH1A) family of enzymes residing within Sertoli cells is responsible for the synthesis of atRA, driving the first round of spermatogenesis. Those studies also revealed that the atRA required to drive subsequent rounds of spermatogenesis is possibly derived from the ALDH1A enzymes residing within the meiotic and post-meiotic germ cells. Three ALDH1A isozymes (ALDH1A1, ALDH1A2, and ALDH1A3) are present in the testis. Although, ALDH1A1 is expressed in adult Sertoli cells and is suggested to contribute to the atRA required for the pre-meiotic transitions, ALDH1A2 is proposed to be the essential isomer involved in testicular atRA biosynthesis. In this report, we first examine the requirement for ALDH1A2 via the generation and analysis of a conditional Aldh1a2 germ cell knockout and a tamoxifen-induced Aldh1a2 knockout model. We then utilized the pan-ALDH1A inhibitor (WIN 18446) to test the collective contribution of the ALDH1A enzymes to atRA biosynthesis following the first round of spermatogenesis. Collectively, our data provide the first in vivo evidence demonstrating that animals severely deficient in ALDH1A2 postnatally proceed normally through spermatogenesis. Our studies with a pan-ALDH1A inhibitor (WIN 18446) also suggest that an alternative source of atRA biosynthesis independent of the ALDH1A enzymes becomes available to maintain atRA levels for several spermatogenic cycles following an initial atRA injection.

Retinoic acid receptor signaling is necessary in steroidogenic cells for normal spermatogenesis and epididymal function.

Spermatogenesis in mammals is a very complex, highly organized process, regulated in part by testosterone and retinoic acid (RA). Much is known about how RA and testosterone signaling pathways independently regulate this process, but there is almost no information regarding whether these two signaling pathways directly interact and whether RA is crucial for steroidogenic cell function. This study uses a transgenic mouse line that expresses a dominant-negative form of RA receptor α (RAR-DN) and the steroidogenic cell-specific Cre mouse line, Cyp17iCre, to generate male mice with steroidogenic cells unable to perform RA signaling. Testes of mutant mice displayed increased apoptosis of pachytene spermatocytes, an increased number of macrophages in the interstitium and a loss of advanced germ cells. Additionally, blocking RA signaling in Leydig cells resulted in increased permeability of the blood-testis barrier, decreased levels of the steroidogenic enzyme cytochrome P450 17a1 and decreased testosterone levels. Surprisingly, the epididymides of the mutant mice also displayed an abnormal phenotype. This study demonstrates that RA signaling is required in steroidogenic cells for their normal function and, thus, for male fertility.

Beyond stem cells: Commitment of progenitor cells to meiosis.

The first step in established spermatogenesis is the production of progenitor cells by the stem cell population. The progenitor cells (undifferentiated A spermatogonia) expand in number via the formation of syncytial chains by mitosis. The mechanism by which these progenitor cells commit to meiosis and spermatogenesis is tightly controlled and results in complex morphological organization all of which is designed to efficiently achieve large numbers of spermatozoa. The major extrinsic factor that triggers the commitment to meiosis and establishes the structural complexity is retinoic acid (RA). Retinoic acid is produced from retinol via two oxidation steps in low abundance near its site of action. The action of RA on undifferentiated A spermatogonia results in the timed progression of these progenitor cells into the cycle of the seminiferous epithelium. We have utilized a drug WIN 18,446 that inhibits the second oxidation step in RA biosynthesis to block the progression of undifferentiated A spermatogonia in the mouse testis. As a result of this block the undifferentiated progenitor cells accumulate but do not differentiate into A1 spermatogonia. When the block is released and a bolus of RA is simultaneously administered the accumulated spermatogonia progress through the differentiation pathway in complete synchrony and maintain that synchrony with regard to stages of the cycle of the seminiferous epithelium for several months. This procedure allowed us to accumulate sufficient material to measure retinoic acid levels across the cycle and will allow us to isolate and analyze large number of progenitor cells proceeding synchronously down the pathway to meiosis. We have been able to show that the cycle of the seminiferous epithelium is established and maintained by pulses of RA that appear at stages VIII and IX of the cycle.

Leydig cell genes change their expression and association with polysomes in a stage-specific manner in the adult mouse testis.

Spermatogenesis in mammals occurs in a very highly organized manner within the seminiferous epithelium regulated by different cell types in the testis. Testosterone produced by Leydig cells regulates blood-testis barrier formation, meiosis, spermiogenesis, and spermiation. However, it is unknown whether Leydig cell function changes with the different stages of the seminiferous epithelium. This study utilized the WIN 18,446 and retinoic acid (RA) treatment regime combined with the RiboTag mouse methodology to synchronize male germ cell development and allow for the in vivo mapping of the Leydig cell translatome across the different stages of one cycle of the seminiferous epithelium. Using microarrays analysis, we identified 11 Leydig cell-enriched genes that were expressed in stage-specific manner such as the glucocorticoid synthesis and transport genes, Cyp21a1 and Serpina6. In addition, there were nine Leydig cell transcripts that change their association with polysomes in correlation with the different stages of the spermatogenic cycle including Egr1. Interestingly, the signal intensity of EGR1 and CYP21 varied among Leydig cells in the adult asynchronous testis. However, testosterone levels across the different stages of germ cell development did not cycle. These data show, for the first time, that Leydig cell gene expression changes in a stage-specific manner during the cycle of the seminiferous epithelium and indicate that a heterogeneous Leydig cell population exists in the adult mouse testis.

Retinoic acid deficiency leads to an increase in spermatogonial stem number in the neonatal mouse testis, but excess retinoic acid results in no change.

The onset of spermatogenesis occurs in response to retinoic acid (RA), the active metabolite of vitamin A. However, whether RA plays any role during establishment of the spermatogonial stem cell (SSC) pool is unknown. Because designation of the SSC population and the onset of RA signaling in the testis that induces differentiation have similar timing, this study asked whether RA influenced SSC establishment. Whole mount immunofluorescence and flow cytometric analysis using the Id4-eGfp transgenic reporter mouse line revealed an enrichment for ID4-EGFP+ cells within the testis following inhibition of RA synthesis by WIN 18,446 treatment. Transplantation analyses confirmed a significant increase in the number of SSCs in testes from RA-deficient animals. Conversely, no difference in the ID4-EGFP+ population or change in SSC number were detected following exposure to an excess of RA. Collectively, reduced RA altered the number of SSCs present in the neonatal testis but precocious RA exposure in the neonatal testis did not, suggesting that RA deficiency causes a greater proportion of progenitor undifferentiated spermatogonia to retain their SSC state past the age when the pool is thought to be determined.

Intact piRNA pathway prevents L1 mobilization in male meiosis.

The PIWI-interacting RNA (piRNA) pathway is essential for retrotransposon silencing. In piRNA-deficient mice, L1-overexpressing male germ cells exhibit excessive DNA damage and meiotic defects. It remains unknown whether L1 expression simply highlights piRNA deficiency or actually drives the germ-cell demise. Specifically, the sheer abundance of genomic L1 copies prevents reliable quantification of new insertions. Here, we developed a codon-optimized L1 transgene that is controlled by an endogenous mouse L1 promoter. Importantly, DNA methylation dynamics of a single-copy transgene were indistinguishable from those of endogenous L1s. Analysis of Mov10l1-/- testes established that de novo methylation of the L1 transgene required the intact piRNA pathway. Consistent with loss of DNA methylation and programmed reduction of H3K9me2 at meiotic onset, the transgene showed 1,400-fold increase in RNA expression and consequently 70-fold increase in retrotransposition in postnatal day 14 Mov10l1-/- germ cells compared with the wild-type. Analysis of adult Mov10l1-/- germ-cell fractions indicated a stage-specific increase of retrotransposition in the early meiotic prophase. However, extrapolation of the transgene data to endogenous L1s suggests that it is unlikely insertional mutagenesis alone accounts for the Mov10l1-/- phenotype. Indeed, pharmacological inhibition of reverse transcription did not rescue the meiotic defect. Cumulatively, these results establish the occurrence of productive L1 mobilization in the absence of an intact piRNA pathway but leave open the possibility of processes preceding L1 integration in triggering meiotic checkpoints and germ-cell death. Additionally, our data suggest that many heritable L1 insertions originate from individuals with partially compromised piRNA defense.

Inhibition of the all-trans Retinoic Acid (atRA) Hydroxylases CYP26A1 and CYP26B1 Results in Dynamic, Tissue-Specific Changes in Endogenous atRA Signaling.

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 ß and Pgc 1ß 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.

Characterizing the Spermatogonial Response to Retinoic Acid During the Onset of Spermatogenesis and Following Synchronization in the Neonatal Mouse Testis.

Retinoic acid (RA), the active metabolite of vitamin A, is known to be required for the differentiation of spermatogonia. The first round of spermatogenesis initiates in response to RA and occurs in patches along the length of the seminiferous tubule. However, very little is known about the individual differentiating spermatogonial populations and their progression through the cell cycle due to the heterogeneous nature of the onset of spermatogenesis. In this study, we utilized WIN 18,446 and RA as tools to generate testes enriched with different populations of spermatogonia to further investigate 1) the undifferentiated to differentiating spermatogonial transition, 2) the progression of the differentiating spermatogonia through the cell cycle, and 3) Sertoli cell number in response to altered RA levels. WIN 18,446/RA-treated neonatal mice were used to determine when synchronous S phases occurred in the differentiating spermatogonial population following treatment. Five differentiating spermatogonial S phase windows were identified between spermatogonial differentiation and formation of preleptotene spermatocytes. In addition, a slight increase in Sertoli cell number was observed following RA treatment, possibly implicating a role for RA in Sertoli cell cycle progression. This study has enhanced our understanding of the spermatogonial populations present in the neonatal testis during the onset of spermatogenesis by mapping the cell cycle kinetics of both the undifferentiated and the differentiating spermatogonial populations and identifying the precise timing of when specific individual differentiating spermatogonial populations are enriched within the testis following synchrony, thus providing an essential tool for further study of the differentiating spermatogonia.

Germ Cell Commitment to Oogenic Versus Spermatogenic Pathway: The Role of Retinoic Acid.

The core of the decision to commit to either oogenesis or spermatogenesis lies in the timing of meiotic entry. Primordial germ cells within the fetal ovary become committed to the female pathway prior to birth and enter meiosis during embryonic development. In the fetal testis, however, the germ cells are protected from this signal before birth and instead receive this trigger postnatally. There is a growing body of evidence to indicate that RA is the meiosis-inducing factor in both sexes, with the gender-specific timing of meiotic entry controlled via degradation of this molecule only within the fetal testis. This chapter will review our current understanding of how RA controls germ cell fate in both the embryonic ovary and postnatal testis, highlighting the key studies that have led to the hypothesis that RA can drive the commitment to meiosis in both sexes and discussing the current debate over whether RA truly is the meiosis-inducing factor in the fetal ovary.

Retinoid signaling controls spermatogonial differentiation by regulating expression of replication-dependent core histone genes.

Retinoic acid (RA) signaling is crucial for spermatogonial differentiation, which is a key step for spermatogenesis. We explored the mechanisms underlying spermatogonial differentiation by targeting expression of a dominant-negative mutant of retinoic acid receptor α (RARα) specifically to the germ cells of transgenic mice to subvert the activity of endogenous receptors. Here we show that: (1) inhibition of retinoid signaling in germ cells completely blocked spermatogonial differentiation identical to vitamin A-deficient (VAD) mice; (2) the blockage of spermatogonial differentiation by impaired retinoid signaling resulted from an arrest of entry of the undifferentiated spermatogonia into S phase; and (3) retinoid signaling regulated spermatogonial differentiation through controlling expression of its direct target genes, including replication-dependent core histone genes. Taken together, our results demonstrate that the action of retinoid signaling on spermatogonial differentiation in vivo is direct through the spermatogonia itself, and provide the first evidence that this is mediated by regulation of expression of replication-dependent core histone genes.

All-Trans-Retinoic Acid Enhances Mitochondrial Function in Models of Human Liver.

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 mitochondrialß-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 1ßand nuclear respiratory factor 1 mRNA and mitochondrial DNA quantification.atRA also increasedß-oxidation and ATP production in HepG2 cells and in human hepatocytes. Knockdown studies of RARα, RARß, and PPARδrevealed that the enhancement of mitochondrial biogenesis andß-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 acidß-oxidation and provide therapeutic benefit in diseases associated with mitochondrial dysfunction.

ALDH Enzyme Expression Is Independent of the Spermatogenic Cycle, and Their Inhibition Causes Misregulation of Murine Spermatogenic Processes.

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.

CYP26 Enzymes Are Necessary Within the Postnatal Seminiferous Epithelium for Normal Murine Spermatogenesis.

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.