Differential roles of cyclooxygenase enzymes in the regulation of murine juvenile undifferentiated spermatogonia.

BACKGROUND: Acetaminophen and ibuprofen are widely administered to babies due to their presumed safety as over-the-counter drugs. However, no reports exist on the effects of cyclooxygenase inhibitors on undifferentiated spermatogonia and spermatogonial stem cells. Infancy represents a critical period for spermatogonial stem cell formation and disrupting spermatogonial stem cells or their precursors may be associated with infertility and testicular cancer formation. OBJECTIVES: The goal of this study was to examine the molecular and functional impact of cyclooxygenase inhibition and silencing on early steps of undifferentiated spermatogonia (u spg) and spermatogonial stem cell development, to assess the potential reproductive risk of pharmaceutical cyclooxygenase inhibitors. METHODS: The effects of cyclooxygenase inhibition were assessed using the mouse C18-4 undifferentiated juvenile spermatogonial cell line model, previously shown to include cells with spermatogonial stem cell features, by measuring prostaglandins, cell proliferation, and differentiation, using cyclooxygenase 1- and cyclooxygenase 2-selective inhibitors NS398, celecoxib, and FR122047, acetaminophen, and ibuprofen. Cyclooxygenase 1 gene silencing was achieved using a stable short-hairpin RNA approach and clone selection, then assessing gene and protein expression in RNA sequencing, quantitative real-time polymerase chain reaction, and immunofluorescence studies. RESULTS: Cyclooxygenase 2 inhibitors NS398 and celecoxib, as well as acetaminophen, but not ibuprofen, dose-dependently decreased retinoic acid-induced expression of the spg differentiation gene Stra8, while NS398 decreased the spg differentiation marker Kit, suggesting that cyclooxygenase 2 is positively associated with spg differentiation. In contrast, short-hairpin RNA-based cyclooxygenase 1 silencing in C18-4 cells altered cellular morphology and upregulated Stra8 and Kit, implying that cyclooxygenase 1 prevented spg differentiation. Furthermore, RNA sequencing analysis of cyclooxygenase 1 knockdown cells indicated the activation of several signaling pathways including the TGFb, Wnt, and Notch pathways, compared to control C18-4 cells. Notch pathway genes were upregulated by selective cyclooxygenase inhibitors, acetaminophen and ibuprofen. CONCLUSION: We report that cyclooxygenase 1 and 2 differentially regulate undifferentiated spermatogonia/spermatogonial stem cell differentiation. Cyclooxygenases regulate Notch3 expression, with the Notch pathway targeted by PGD2. These data suggest an interaction between the eicosanoid and Notch signaling pathways that may be critical for the development of spermatogonial stem cells and subsequent spermatogenesis, cautioning about using cyclooxygenase inhibitors in infants.

Do macrophages play a role in the adverse effects of endocrine disrupting chemicals (EDCs) on testicular functions?

During the past decades, several endocrine disrupting chemicals (EDCs) have been confirmed to affect male reproductive function and fertility in animal studies. EDCs are suspected to exert similar effects in humans, based on strong associations between levels of antiandrogenic EDCs in pregnant women and adverse reproductive effects in infants. Testicular macrophages (tMΦ) play a vital role in modulating immunological privilege and maintaining normal testicular homeostasis as well as fetal development. Although tMΦ were not historically studied in the context of endocrine disruption, they have emerged as potential targets to consider due to their critical role in regulating cells such as spermatogonial stem cells (SSCs) and Leydig cells. Few studies have examined the impact of EDCs on the ability of testicular cells to communicate and regulate each other's functions. In this review, we recapitulate what is known about tMΦ functions and interactions with other cell types in the testis that support spermatogenesis and steroidogenesis. We also surveyed the literature for reports on the effects of the EDCs genistein and DEHP on tMΦ, SSCs, Sertoli and Leydig cells. Our goal is to explore the possibility that EDC disruption of tMΦ interactions with other cell types may play a role in their adverse effects on testicular developmental programming and functions. This approach will highlight gaps of knowledge, which, once resolved, should improve the risk assessment of EDC exposure and the development of safeguards to protect male reproductive functions.

Dysregulation of Immature Sertoli Cell Functions by Exposure to Acetaminophen and Genistein in Rodent Cell Models.

Sertoli cells are essential for germ cell development and function. Their disruption by endocrine disrupting chemicals (EDCs) or drugs could jeopardize spermatogenesis, contributing to male infertility. Perinatal exposure to EDCs and acetaminophen (APAP) disrupts male reproductive functions in animals and humans. Infants can be exposed simultaneously to the dietary soy phytoestrogen genistein (GEN) and APAP used for fever or pain relief. Our goal was to determine the effects of 10-100 µM APAP and GEN, alone or mixed, on immature Sertoli cells using mouse TM4 Sertoli cell line and postnatal-day 8 rat Sertoli cells, by measuring cell viability, proliferation, prostaglandins, genes and protein expression, and functional pathways. A value of 50 µM APAP decreased the viability, while 100 µM APAP and GEN decreased the proliferation. Sertoli cell and eicosanoid pathway genes were affected by GEN and mixtures, with downregulation of Sox9, Cox1, Cox2, and genes relevant for Sertoli cell function, while genes involved in inflammation were increased. RNA-seq analysis identified p53 and TNF signaling pathways as common targets of GEN and GEN mixture in both cell types. These results suggest that APAP and GEN dysregulate immature Sertoli cell function and may aid in elucidating novel EDC and drug targets contributing to the etiology of male infertility.

Impact of endocrine disrupting chemicals and pharmaceuticals on Sertoli cell development and functions.

Sertoli cells play essential roles in male reproduction, from supporting fetal testis development to nurturing male germ cells from fetal life to adulthood. Dysregulating Sertoli cell functions can have lifelong adverse effects by jeopardizing early processes such as testis organogenesis, and long-lasting processes such as spermatogenesis. Exposure to endocrine disrupting chemicals (EDCs) is recognized as contributing to the rising incidence of male reproductive disorders and decreasing sperm counts and quality in humans. Some drugs also act as endocrine disruptors by exerting off-target effects on endocrine tissues. However, the mechanisms of toxicity of these compounds on male reproduction at doses compatible with human exposure are still not fully resolved, especially in the case of mixtures, which remain understudied. This review presents first an overview of the mechanisms regulating Sertoli cell development, maintenance, and functions, and then surveys what is known on the impact of EDCs and drugs on immature Sertoli cells, including individual compounds and mixtures, and pinpointing at knowledge gaps. Performing more studies on the impact of mixtures of EDCs and drugs at all ages is crucial to fully understand the adverse outcomes these chemicals may induce on the reproductive system.

Impact of Fetal Exposure to Endocrine Disrupting Chemical Mixtures on FOXA3 Gene and Protein Expression in Adult Rat Testes.

Perinatal exposure to endocrine disrupting chemicals (EDCs) has been shown to affect male reproductive functions. However, the effects on male reproduction of exposure to EDC mixtures at doses relevant to humans have not been fully characterized. In previous studies, we found that in utero exposure to mixtures of the plasticizer di(2-ethylhexyl) phthalate (DEHP) and the soy-based phytoestrogen genistein (Gen) induced abnormal testis development in rats. In the present study, we investigated the molecular basis of these effects in adult testes from the offspring of pregnant SD rats gavaged with corn oil or Gen + DEHP mixtures at 0.1 or 10 mg/kg/day. Testicular transcriptomes were determined by microarray and RNA-seq analyses. A protein analysis was performed on paraffin and frozen testis sections, mainly by immunofluorescence. The transcription factor forkhead box protein 3 (FOXA3), a key regulator of Leydig cell function, was identified as the most significantly downregulated gene in testes from rats exposed in utero to Gen + DEHP mixtures. FOXA3 protein levels were decreased in testicular interstitium at a dose previously found to reduce testosterone levels, suggesting a primary effect of fetal exposure to Gen + DEHP on adult Leydig cells, rather than on spermatids and Sertoli cells, also expressing FOXA3. Thus, FOXA3 downregulation in adult testes following fetal exposure to Gen + DEHP may contribute to adverse male reproductive outcomes.

Role of the Ubiquitin Ligase RNF149 in the Development of Rat Neonatal Gonocytes.

Male reproductive function depends on the formation of spermatogonial stem cells from their neonatal precursors, the gonocytes. Previously, we identified several UPS enzymes dynamically altered during gonocyte differentiation. The present work focuses on understanding the role of the RING finger protein 149 (RNF149), an E3 ligase that we found to be strongly expressed in gonocytes and downregulated in spermatogonia. The quantification of RNF149 mRNA from postnatal day (PND) 2 to 35 (puberty) in rat testis, brain, liver, kidney, and heart indicated that its highest levels are found in the testis. RNF149 knock-down in PND3 rat gonocytes was performed to better understand its role in gonocyte development. While a proliferative cocktail of PDGF-BB and 17ß-estradiol (P+E) increased both the expression levels of the cell proliferation marker PCNA and RNF149 in mock cells, the effects of P+E on both genes were reduced in cells treated with RNF149 siRNA, suggesting that RNF149 expression is regulated during gonocyte proliferation and that there might be a functional link between RNF149 and PCNA. To examine RNF149 subcellular localization, EGFP-tagged RNF149 vectors were constructed, after determining the rat testis RNF149 mRNA sequence. Surprisingly, two variant transcripts were expressed in rat tissues, predicting truncated proteins, one containing the PA and the other the RING functional domains. Transfection in mouse F9 embryonal carcinoma cells and C18-4 spermatogonial cell lines showed differential subcellular profiles of the two truncated proteins. Overall, the results of this study support a role for RNF149 in gonocyte proliferation and suggest its transcription to variant mRNAs resulting in two proteins with different functional domains. Future studies will examine the respective roles of these variant proteins in the cell lines and isolated gonocytes.

Eicosanoid Biosynthesis in Male Reproductive Development: Effects of Perinatal Exposure to NSAIDs and Analgesic Drugs.

Increasing rates of infertility associated with declining sperm counts and quality, as well as increasing rates of testicular cancer are contemporary issues in the United States and abroad. These conditions are part of the Testicular Dysgenesis Syndrome, which includes a variety of male reproductive disorders hypothesized to share a common origin based on disrupted testicular development during fetal and neonatal stages of life. Male reproductive development is a highly regulated and complex process that relies on an intricate coordination between germ, Leydig, and Sertoli cells as well as other supporting cell types, to ensure proper spermatogenesis, testicular immune privilege, and endocrine function. The eicosanoid system has been reported to be involved in the regulation of fetal and neonatal germ cell development as well as overall testicular homeostasis. Moreover, non-steroidal anti-inflammatory drugs (NSAIDs) and analgesics with abilities to block eicosanoid synthesis by targeting either or both isoforms of cyclooxygenase enzymes, have been found to adversely affect male reproductive development. This review will explore the current body of knowledge on the involvement of the eicosanoid system in male reproductive development, as well as discuss adverse effects of NSAIDs and analgesic drugs administered perinatally, focusing on toxicities reported in the testis and on major testicular cell types. Rodent and epidemiological studies will be corroborated by findings in invertebrate models for a comprehensive report of the state of the field, and to add to our understanding of the potential long-term effects of NSAID and analgesic drug administration in infants.

In vitro impact of genistein and mono(2-ethylhexyl) phthalate (MEHP) on the eicosanoid pathway in spermatogonial stem cells.

Perinatal exposures to endocrine disrupting chemicals (EDCs) alter the male reproductive system. Infants are exposed to genistein (GEN) through soy-based formula, and to Mono(2-ethylhexyl) Phthalate (MEHP), metabolite of the plasticizer DEHP. Spermatogonial stem cells (SSCs) are formed in infancy and their integrity is essential for spermatogenesis. Thus, understanding the impact of EDCs on SSCs is critical. Prostaglandins (PGs) are inflammatory mediators synthesized via the eicosanoid pathway starting with cyclooxygenases (Coxs), that regulate physiological and pathological processes. Our goal was to study the eicosanoid pathway in SSCs and examine whether it was disrupted by GEN and MEHP, potentially contributing to their adverse effects. The mouse C18-4 cell line used as SSC model expressed high levels of Cox1 and Cox2 genes and proteins, and eicosanoid pathway genes similarly to levels measured in primary rat spermatogonia. Treatments with GEN and MEHP at 10 and 100 µM decreased Cox1 gene and protein expression, whereas Cox2, phospholipase A2, prostaglandin synthases transcripts, PGE2, PGF2a and PGD2 were upregulated. Simultaneously, the transcript levels of spermatogonia progenitor markers Foxo1 and Mcam and differentiated spermatogonial markers cKit and Stra8 were increased. Foxo1 was also increased by EDCs in primary rat spermatogonia. This study shows that the eicosanoid pathway is altered during SSC differentiation and that exposure to GEN and MEHP disrupts this process, mainly driven by GEN effects on Cox2 pathway, while MEHP acts through an alternative mechanism. Thus, understanding the role of Cox enzymes in SSCs and how GEN and MEHP exposures alter their differentiation warrants further studies.

Cholesterol-binding translocator protein TSPO regulates steatosis and bile acid synthesis in nonalcoholic fatty liver disease.

Translocator protein (TSPO, 18 kDa) levels increase in parallel with the evolution of simple steatosis (SS) to nonalcoholic steatohepatitis (NASH) in nonalcoholic fatty liver disease (NAFLD). However, TSPO function in SS and NASH is unknown. Loss of TSPO in hepatocytes in vitro downregulated acetyl-CoA acetyltransferase 2 and increased free cholesterol (FC). FC accumulation induced endoplasmic reticulum stress via IRE1A and protein kinase RNA-like ER kinase/ATF4/CCAAT-enhancer-binding protein homologous protein pathways and autophagy. TSPO deficiency activated cellular adaptive antioxidant protection; this adaptation was lost upon excessive FC accumulation. A TSPO ligand 19-Atriol blocked cholesterol binding and recapitulated many of the alterations seen in TSPO-deficient cells. These data suggest that TSPO deficiency accelerated the progression of SS. In NASH, however, loss of TSPO ameliorated liver fibrosis through downregulation of bile acid synthesis by reducing CYP7A1 and CYP27A1 levels and increasing farnesoid X receptor expression. These studies indicate a dynamic and complex role for TSPO in the evolution of NAFLD.

Impact of endocrine-disrupting chemicals on steroidogenesis and consequences on testicular function.

Testicular steroidogenesis is a tightly regulated process that produces the androgens important for the development, maintenance and function of the male reproductive system. These androgens are also essential for overall health, and well-being. Disruptions in the ability of the testis to form steroids can result in developmental abnormalities, dysfunction, and infertility. Endocrine-disrupting chemicals (EDCs) can interfere with the intricate signaling and metabolizing networks that produce androgens and promote their dysfunction. These chemicals are found ubiquitously in our environment, as they are integral components of products that are used every day. The effects of EDCs, such as bisphenols, phthalates, and alkyl chemicals, have been studied independently, revealing deleterious effects; but the combined influence of these structures on steroidogenesis has yet to be completely elucidated. This manuscript presents an updated review on EDC mixtures and their impact on testicular function and fertility, highlighting new findings that illustrate the anti-androgenic capabilities of EDC mixtures.

In utero exposure to low doses of genistein and di-(2-ethylhexyl) phthalate (DEHP) alters innate immune cells in neonatal and adult rat testes.

BACKGROUND: Although humans are exposed to mixtures of endocrine disruptor chemicals, few studies have examined their toxicity on male reproduction. We previously found that fetal exposure to a mixture of the phytoestrogen genistein (GEN) and the plasticizer di(2-ethylhexyl) phthalate (DEHP) altered gene expression in adult rat testes. OBJECTIVES: Our goal was to investigate the effects of fetal exposure to GEN-DEHP mixtures at two doses relevant to humans on testicular function and transcriptome in neonatal and adult rats. MATERIALS AND METHODS: Pregnant SD rats were gavaged with vehicle, GEN or DEHP, alone or mixed at 0.1 and 10 mg/kg/day, from gestation day 14 to birth. Fertility, steroid levels, and testis morphology were examined in neonatal and adult rats. Testicular transcriptomes were examined by gene array and functional pathway analyses. Cell-specific genes/proteins were determined by quantitative real-time PCR and immunohistochemistry. RESULTS: GEN-DEHP mixtures increased the rates of infertility and abnormal testes in adult rats. Gene array analysis identified more genes exclusively altered by the mixtures than individual compounds. Altered top canonical pathways included urogenital/reproductive developmental and inflammatory processes. GEN-DEHP mixtures increased innate immune cells and macrophages markers at both doses and ages, more strongly and consistently than DEHP or GEN alone. Genes exclusively increased by the mixture in adult testis related to innate immune cells and macrophages included Kitlg, Rps6ka3 (Rsk2), Nr3c1, Nqo1, Lif, Fyn, Ptprj (Dep-1), Gpr116, Pfn2, and Ptgr1. DISCUSSION AND CONCLUSION: These findings demonstrate that GEN-DEHP mixtures at doses relevant to human induce adverse testicular phenotypes, concurrent with age-dependent and non-monotonic changes in testicular transcriptomes. The involvement of innate immune cells such as macrophages suggests immediate and delayed inflammatory responses which may contribute to testicular dysfunction. Moreover, these effects are complex and likely involve multiple interactions between immune and non-immune testicular cell types that will entail further studies.

Cyclooxygenase 2 (COX2) expression and prostaglandin synthesis in neonatal rat testicular germ cells: Effects of acetaminophen and ibuprofen.

BACKGROUND: In infants, fever is often treated with acetaminophen or ibuprofen, two antipyretic and analgesic drugs inhibiting cyclooxygenases (COXs), enzymes catalyzing prostaglandin production. Infancy represents a critical developmental period when neonatal germ cells/gonocytes differentiate to spermatogonial stem cells required for spermatogenesis. OBJECTIVES: (a) Determine the expression of Cox2 and associated genes in postnatal day (PND)3 rat gonocytes compared to spermatogonia. (b) Examine whether acetaminophen or ibuprofen disrupts neonatal gonocyte functions. (c) Determine whether neonatal gonocytes produce prostaglandins and whether this process is altered by acetaminophen and ibuprofen. MATERIALS AND METHODS: The expression of Cox2 and related genes was determined by gene arrays and qPCR. Cox2 protein levels were determined by immunocyto/histochemistry and immunoblots. The effects of acetaminophen and ibuprofen on PND3 gonocyte viability, apoptosis, proliferation, and differentiation were examined alone and with a proliferation cocktail or differentiation factor. Prostaglandins were examined by immunocyto/histochemistry and LC-MS. RESULTS: Cox2 and related genes are highly expressed in gonocytes and spermatogonia. Acetaminophen and ibuprofen did not affect gonocyte survival or apoptosis, but they increased gonocyte proliferation. Ibuprofen significantly reduced RA-induced Stra8 expression, indicating an inhibitory effect on differentiation. Ibuprofen combined with RA decreased Cox2 mRNA and protein expression. PGE2 and PGF2α were produced by neonatal gonocytes and decreased by acetaminophen and ibuprofen. DISCUSSION: The concomitant decrease of Stra8 expression, Cox2 expression, and PGE2 and PGF2a production in gonocytes co-treated with RA suggests that Cox2 plays a role in PND3 gonocyte differentiation. The effects of acetaminophen and ibuprofen on proliferation suggest a negative relationship between Cox2 and proliferation. Treating neonates with acetaminophen or ibuprofen could disrupt gonocyte development, leading to adverse reproductive effects. CONCLUSION: Understanding COX2 role in neonatal gonocytes and the potential risk of acetaminophen and ibuprofen treatment of infants may help prevent male reproductive pathologies.

Directing differentiation of human induced pluripotent stem cells toward androgen-producing Leydig cells rather than adrenal cells.

Reduced serum testosterone (T), or hypogonadism, affects millions of men and is associated with many pathologies, including infertility, cardiovascular diseases, metabolic syndrome, and decreased libido and sexual function. Administering T-replacement therapy (TRT) reverses many of the symptoms associated with low T levels. However, TRT is linked to side effects such as infertility and increased risk of prostate cancer and cardiovascular diseases. Thus, there is a need to obtain T-producing cells that could be used to treat hypogonadism via transplantation and reestablishment of T-producing cell lineages in the body. T is synthesized by Leydig cells (LCs), proposed to derive from mesenchymal cells of mesonephric origin. Although mesenchymal cells have been successfully induced into LCs, the limited source and possible trauma to donors hinders their application to clinical therapies. Alternatively, human induced pluripotent stem cells (hiPSCs), which are expandable in culture and have the potential to differentiate into all somatic cell types, have become the emerging source of autologous cell therapies. We have successfully induced the differentiation of hiPSCs into either human Leydig-like (hLLCs) or adrenal-like cells (hALCs) using chemically defined culture conditions. Factors critical for the development of LCs were added to both culture systems. hLLCs expressed all steroidogenic genes and proteins important for T biosynthesis, synthesized T rather than cortisol, secreted steroid hormones in response to dibutyryl-cAMP and 22(R)-hydroxycholesterol, and displayed ultrastructural features resembling LCs. By contrast, hALCs synthesized cortisol rather than T. The success in generating hiPSC-derived hLLCs with broad human LC (hLC) features supports the potential for hiPSC-based hLC regeneration.

Protective Role of Peroxiredoxins against Reactive Oxygen Species in Neonatal Rat Testicular Gonocytes.

Peroxiredoxins (PRDXs) are antioxidant enzymes that protect cells from oxidative stress and play a role in reactive oxygen species (ROS)-mediated signaling. We reported that PRDXs are critical for human fertility by maintaining sperm viability and regulating ROS levels during capacitation. Moreover, studies on Prdx6-/- mice revealed the essential role of PRDX6 in the viability, motility, and fertility competence of spermatozoa. Although PRDXs are abundant in the testis and spermatozoa, their potential role at different phases of spermatogenesis and in perinatal germ cells is unknown. Here, we examined the expression and role of PRDXs in isolated rat neonatal gonocytes, the precursors of spermatogonia, including spermatogonial stem cells. Gene array, qPCR analyses showed that PRDX1, 2, 3, 5, and 6 transcripts are among the most abundant antioxidant genes in postnatal day (PND) 3 gonocytes, while immunofluorescence confirmed the expression of PRDX1, 2, and 6 proteins. The role of PRDXs in gonocyte viability was examined using PRDX inhibitors, revealing that the 2-Cys PRDXs and PRDX6 peroxidases activities are critical for gonocytes viability in basal condition, likely preventing an excessive accumulation of endogenous ROS in the cells. In contrast to its crucial role in spermatozoa, PRDX6 independent phospholipase A2 (iPLA2) activity was not critical in gonocytes in basal conditions. However, under conditions of H2O2-induced oxidative stress, all these enzymatic activities were critical to maintain gonocyte viability. The inhibition of PRDXs promoted a two-fold increase in lipid peroxidation and prevented gonocyte differentiation. These results suggest that ROS are produced in neonatal gonocytes, where they are maintained by PRDXs at levels that are non-toxic and permissive for cell differentiation. These findings show that PRDXs play a major role in the antioxidant machinery of gonocytes, to maintain cell viability and allow for differentiation.

Ubiquitin Ligase Huwe1 Modulates Spermatogenesis by Regulating Spermatogonial Differentiation and Entry into Meiosis.

Spermatogenesis consists of a series of highly regulated processes that include mitotic proliferation, meiosis and cellular remodeling. Although alterations in gene expression are well known to modulate spermatogenesis, posttranscriptional mechanisms are less well defined. The ubiquitin proteasome system plays a significant role in protein turnover and may be involved in these posttranscriptional mechanisms. We previously identified ubiquitin ligase Huwe1 in the testis and showed that it can ubiquitinate histones. Since modulation of histones is important at many steps in spermatogenesis, we performed a complete characterization of the functions of Huwe1 in this process by examining the effects of its inactivation in the differentiating spermatogonia, spermatocytes and spermatids. Inactivation of Huwe1 in differentiating spermatogonia led to their depletion and formation of fewer pre-leptotene spermatocytes. The cell degeneration was associated with an accumulation of DNA damage response protein γH2AX, impaired downstream signalling and apoptosis. Inactivation of Huwe1 in spermatocytes indicated that Huwe1 is not essential for meiosis and spermiogenesis, but can result in accumulation of γH2AX. Collectively, these results provide a comprehensive survey of the functions of Huwe1 in spermatogenesis and reveal Huwe1's critical role as a modulator of the DNA damage response pathway in the earliest steps of spermatogonial differentiation.

Huwe1 Regulates the Establishment and Maintenance of Spermatogonia by Suppressing DNA Damage Response.

Spermatogenesis is sustained by a heterogeneous population of spermatogonia that includes the spermatogonial stem cells. However, the mechanisms underlying their establishment from gonocyte embryonic precursors and their maintenance thereafter remain largely unknown. In this study, we report that inactivation of the ubiquitin ligase Huwe1 in male germ cells in mice led to the degeneration of spermatogonia in neonates and resulted in a Sertoli cell-only phenotype in the adult. Huwe1 knockout gonocytes showed a decrease in mitotic re-entry, which inhibited their transition to spermatogonia. Inactivation of Huwe1 in primary spermatogonial culture or the C18-4 cell line resulted in cell degeneration. Degeneration of Huwe1 knockout spermatogonia was associated with an increased level of histone H2AX and an elevated DNA damage response that led to apparent mitotic catastrophe but not apoptosis or senescence. Blocking this increase in H2AX prevented the degeneration of Huwe1-depleted cells. Taken together, these results reveal a previously undefined role of Huwe1 in orchestrating the physiological DNA damage response in the male germline that contributes to the establishment and maintenance of spermatogonia.

In Utero and Lactational Exposure Study in Rats to Identify Replacements for Di(2-ethylhexyl) Phthalate.

Di(2-ethylhexyl) phthalate (DEHP) and other phthalates are ubiquitous environmental contaminants with endocrine disrupting properties. Two novel plasticizers, 1,4 butanediol dibenzoate (BDB) and dioctyl succinate (DOS), have been proposed as potential replacements. Both have desirable properties as plasticizers and minimal in vitro biological effects. Herein, we present an in utero and lactational exposure study comparing DEHP with BDB, DOS, and 1,2-cyclohexane dicarboxylic acid diisononyl ester (DINCH), a commercial alternative. Timed-pregnant Sprague-Dawley rats were gavaged with vehicle or one of these chemicals at 30 or 300 mg/kg/day from gestational day 8 until postnatal day (PND) 21. The offspring were examined for effects on developmental and endocrine markers until PND 46. DEHP treatment (300 mg/kg) decreased heart weights in dams and induced a significant decrease in anogenital index and an increase in hemorrhagic testes and multinucleated gonocytes in PND 3 male pups. An increase in the incidence of hemorrhagic testes was also observed on PND 8 after exposure to DINCH (30 and 300 mg/kg). The only other effects observed were decreases in serum alanine transaminase and magnesium in BDB 30 exposed dams. These data suggest that both BDB and DOS are viable alternative plasticizers.

Regulation of Translocator Protein 18 kDa (TSPO) Expression in Rat and Human Male Germ Cells.

Translocator protein 18 kDa (TSPO) is a high affinity cholesterol- and drug-binding protein highly expressed in steroidogenic cells, such as Leydig cells, where it plays a role in cholesterol mitochondrial transport. We have previously shown that TSPO is expressed in postnatal day 3 rat gonocytes, precursors of spermatogonial stem cells. Gonocytes undergo regulated phases of proliferation and migration, followed by retinoic acid (RA)-induced differentiation. Understanding these processes is important since their disruption may lead to the formation of carcinoma in situ, a precursor of testicular germ cell tumors (TGCTs). Previously, we showed that TSPO ligands do not regulate gonocyte proliferation. In the present study, we found that TSPO expression is downregulated in differentiating gonocytes. Similarly, in F9 embryonal carcinoma cells, a mouse TGCT cell line with embryonic stem cell properties, there is a significant decrease in TSPO expression during RA-induced differentiation. Silencing TSPO expression in gonocytes increased the stimulatory effect of RA on the expression of the differentiation marker Stra8, suggesting that TSPO exerts a repressive role on differentiation. Furthermore, in normal human testes, TSPO was located not only in Leydig cells, but also in discrete spermatogenic phases such as the forming acrosome of round spermatids. By contrast, seminomas, the most common type of TGCT, presented high levels of TSPO mRNA. TSPO protein was expressed in the cytoplasmic compartment of seminoma cells, identified by their nuclear expression of the transcription factors OCT4 and AP2G. Thus, TSPO appears to be tightly regulated during germ cell differentiation, and to be deregulated in seminomas, suggesting a role in germ cell development and pathology.

In vitro functional screening as a means to identify new plasticizers devoid of reproductive toxicity.

Plasticizers are indispensable additives providing flexibility and malleability to plastics. Among them, several phthalates, including di (2-ethylhexyl) phthalate (DEHP), have emerged as endocrine disruptors, leading to their restriction in consumer products and creating a need for new, safer plasticizers. The goal of this project was to use in vitro functional screening tools to select novel non-toxic plasticizers suitable for further in vivo evaluation. A panel of novel compounds with satisfactory plasticizer properties and biodegradability were tested, along with several commercial plasticizers, such as diisononyl-cyclohexane-1,2-dicarboxylate (DINCH®). MEHP, the monoester metabolite of DEHP was also included as reference compound. Because phthalates target mainly testicular function, including androgen production and spermatogenesis, we used the mouse MA-10 Leydig and C18-4 spermatogonial cell lines as surrogates to examine cell survival, proliferation, steroidogenesis and mitochondrial integrity. The most promising compounds were further assessed on organ cultures of rat fetal and neonatal testes, corresponding to sensitive developmental windows. Dose-response studies revealed the toxicity of most maleates and fumarates, while identifying several dibenzoate and succinate plasticizers as innocuous on Leydig and germ cells. Interestingly, DINCH®, a plasticizer marketed as a safe alternative to phthalates, exerted a biphasic effect on steroid production in MA-10 and fetal Leydig cells. MEHP was the only plasticizer inducing the formation of multinucleated germ cells (MNG) in organ culture. Overall, organ cultures corroborated the cell line data, identifying one dibenzoate and one succinate as the most promising candidates. The adoption of such collaborative approaches for developing new chemicals should help prevent the development of compounds potentially harmful to human health.

ACBD2/ECI2-Mediated Peroxisome-Mitochondria Interactions in Leydig Cell Steroid Biosynthesis.

Fatty acid metabolism and steroid biosynthesis are 2 major pathways shared by peroxisomes and mitochondria. Both organelles are in close apposition to the endoplasmic reticulum, with which they communicate via interorganelle membrane contact sites to promote cellular signaling and the exchange of ions and lipids. To date, no convincing evidence of the direct contact between peroxisomes and mitochondria was reported in mammalian cells. Hormone-induced, tightly controlled steroid hormone biosynthesis requires interorganelle interactions. Using immunofluorescent staining and live-cell imaging, we found that dibutyryl-cAMP treatment of MA-10 mouse tumor Leydig cells rapidly induces peroxisomes to approach mitochondria and form peroxisome-mitochondrial contact sites/fusion, revealed by the subcellular distribution of the endogenous acyl-coenzyme A-binding domain (ACBD)2/ECI2 isoform A generated by alternative splicing, and further validated using a proximity ligation assay. This event occurs likely via a peroxisome-like structure, which is mediated by peroxisomal and mitochondrial matrix protein import complexes: peroxisomal import receptor peroxisomal biogenesis factor 5 (PEX5), and the mitochondrial import receptor subunit translocase of outer mitochondrial membrane 20 homolog (yeast) protein. Similar results were obtained using the mLTC-1 mouse tumor Leydig cells. Ectopic expression of the ACBD2/ECI2 isoform A in MA-10 cells led to increased basal and hormone-stimulated steroid formation, indicating that ACBD2/ECI2-mediated peroxisomes-mitochondria interactions favor in the exchange of metabolites and/or macromolecules between these 2 organelles in support of steroid biosynthesis. Considering the widespread occurrence of the ACBD2/ECI2 protein, we propose that this protein might serve as a tool to assist in understanding the contact between peroxisomes and mitochondria.