Fetal Leydig cells: What we know and what we don't.

During male fetal development, testosterone plays an essential role in the differentiation and maturation of the male reproductive system. Deficient fetal testosterone production can result in variations of sex differentiation that may cause infertility and even increased tumor incidence later in life. Fetal Leydig cells in the fetal testis are the major androgen source in mammals. Although fetal and adult Leydig cells are similar in their functions, they are two distinct cell types, and therefore, the knowledge of adult Leydig cells cannot be directly applied to understanding fetal Leydig cells. This review summarizes our current knowledge of fetal Leydig cells regarding their cell biology, developmental biology, and androgen production regulation in rodents and human. Fetal Leydig cells are present in basement membrane-enclosed clusters in between testis cords. They originate from the mesonephros mesenchyme and the coelomic epithelium and start to differentiate upon receiving a Desert Hedgehog signal from Sertoli cells or being released from a NOTCH signal from endothelial cells. Mature fetal Leydig cells produce androgens. Human fetal Leydig cell steroidogenesis is LHCGR (Luteinizing Hormone Chronic Gonadotropin Receptor) dependent, while rodents are not, although other Gαs -protein coupled receptors might be involved in rodent steroidogenesis regulation. Fetal steroidogenesis ceases after sex differentiation is completed, and some fetal Leydig cells dedifferentiate to serve as stem cells for adult testicular cell types. Significant gaps are acknowledged: (1) Why are adult and fetal Leydig cells different? (2) What are bona fide progenitor and fetal Leydig cell markers? (3) Which signaling pathways and transcription factors regulate fetal Leydig cell steroidogenesis? It is critical to discover answers to these questions so that we can understand vulnerable targets in fetal Leydig cells and the mechanisms for androgen production that when disrupted, leads to variations in sex differentiation that range from subtle to complete sex reversal.

Gestational diabetes mellitus suppresses fetal testis development in mice†.

The prevalence of gestational diabetes mellitus (GDM) is increasing rapidly. In addition to the metabolic disease risks, GDM might increase the risks of cryptorchidism in children. However, its mechanism involved in abnormalities of the male reproductive system is still unclear. The purpose of this study was to study the effects of GDM on the development of mouse fetal Leydig cells (FLCs) and Sertoli cells (SCs). Pregnant mice were treated on gestational days 6.5 and 12.5 with streptozotocin (100 mg/kg) or vehicle (sodium citrate buffer). Leydig cell and SC development and functions were evaluated by investigating serum testosterone levels, cell number and distribution, genes, and protein expression. GDM decreased serum testosterone levels, the anogenital distance, and the level of desert hedgehog in SCs of testes of male offspring. FLC number was also decreased in testes of GDM offspring by delaying the commitment of stem Leydig cells into the Leydig cell lineage. RNA-seq showed that FOXL2, RSPO1/ß-catenin signaling was activated and Gsk3ß signaling was inhibited in GDM offspring testis. In conclusion, GDM disrupted reproductive tract and testis development in mouse male offspring via altering genes related to development.

In utero di-(2-ethylhexyl) phthalate-induced testicular dysgenesis syndrome in male newborn rats is rescued by taxifolin through reducing oxidative stress.

Testicular dysgenesis syndrome in male neonates manifests as cryptorchidism and hypospadias, which can be mimicked by in utero phthalate exposure. However, the underlying phthalate mediated mechanism and therapeutic effects of taxifolin remain unclear. Di-(2-ethylhexyl) phthalate (DEHP) is the most abundantly used phthalate and can induce testicular dysgenesis syndrome in male rats. To explore the mechanism of DEHP mediated effects and develop a therapeutic drug, the natural phytomedicine taxifolin was used. Pregnant Sprague-Dawley female rats were daily gavaged with 750 mg/kg/d DEHP or 10 or 20 mg/kg/d taxifolin alone or in combination from gestational day 14 to 21, and male pup's fetal Leydig cell function, testicular MDA, and antioxidants were examined. DEHP significantly reduced serum testosterone levels of male pups, down-regulated the expression of SCARB1, CYP11A1, HSD3B1, HSD17B3, and INSL3, reduced the cell size of fetal Leydig cells, decreased the levels of antioxidant and related signals (SOD2 and CAT, SIRT1, and PGC1α), induced abnormal aggregation of fetal Leydig cells, and stimulated formation of multinucleated gonocytes and MDA levels. Taxifolin alone (10 and 20 mg/kg/d) did not affect these parameters. However, taxifolin significantly rescued DEHP-induced alterations. DEHP exposure in utero can induce testicular dysgenesis syndrome by altering the oxidative balance and SIRT1/PGC1α levels, and taxifolin is an ideal phytomedicine to prevent phthalate induced testicular dysgenesis syndrome.

In utero bisphenol AF exposure causes fetal Leydig cell dysfunction and induces multinucleated gonocytes by generating oxidative stress and reducing the SIRT1/PGC1α signals.

Bisphenol AF (BPAF) is one of the primary alternatives of bisphenol A. It has been ubiquitously detected in the environment and is an emerging endocrine disrupting compound. However, the effects of BPAF exposure on fetal Leydig cells and germ cells and the underlying mechanisms remain largely unknown. To this end, pregnant Sprague-Dawley rats were exposed to 10, 50, and 200 mg/kg/d BPAF by gavage from gestational days 14 to 21. The neonatal rats were sacrificed on day 1 at birth. The results showed that serum testosterone levels were significantly decreased at 50 and 200 mg/kg/d, the expression of Scarb1, Star, Cyp17a1, Hsd17b3, and Dhh and their proteins were markedly down-regulated at 50 and 100 mg/kg/d. BPAF exposure also significantly increased the incidence of multinucleated gonocytes at 200 mg/kg/d. We further detected significant increase of testicular malondialdehyde levels and reduction of antioxidants, including SOD1, SOD2, and CAT at 50 and/or 200 mg/kg/d. Furthermore, BPAF markedly reduced the levels of SIRT1 and PGC1α at 200 mg/kg/d while significantly increased AMPK phosphorylation in the testes at 50 and 200 mg/kg/d. In conclusion, our results provide novel in vivo data that BPAF can induce fetal Leydig cell dysfunction by interfering with steroidogenic networks and induce the formation of multinucleated gonocytes after suppressing the antioxidant defense system and reducing SIRT1 and PGC1α signals and increasing the phosphorylation of AMPK, which highlights the potential health risk of environmental exposure to BPAF in inducing male reproductive tract malformation.

Tetramethyl bisphenol A stimulates proliferation but inhibits fetal Leydig cell function in male rats by targeting estrogen receptor α after in utero exposure.

Tetramethyl bisphenol A (TMBPA) is a widely used flame retardant. TMBPA has been a toxic to Leydig cells in puberty, but it remains unclear whether TMBPA has a similar inhibitor effect on fetal Leydig cells (FLCs). This study reported morphological and functional alterations of FLCs in the testes of male offspring at birth after in utero exposure to TMBPA. Pregnant Sprague Dawley rats were dosed via continuous gavage of TMBPA (0, 10, 50, and 200 mg/kg/day) from gestational day 14 to 21. TMBPA markedly raised serum total testosterone level, testicular volume, and FLC number of male offspring at 200 mg/kg dose. The up-regulation of Insl3, Star, and Cyp11a1 mRNAs was observed after 200 mg/kg TMBPA exposure. After normalization to the number of FLCs, TMBPA significantly reduced Lhcgr and Hsd3b1 expressions at 10 mg/kg, and Cyp17a1 at 200 mg/kg paralleling with their protein levels. TMBPA compromised the expression of Esr1, while increased the expression of Cdk2 and Cdk4 as well as their protein levels. TMBPA particularly increased the phosphorylation of AKT1 and AKT2 at 200 mg/kg. In conclusion, the present study suggests that TMBPA may promote FLC proliferation via ESR1-CDK2/4-AKT pathway, while inhibits the function of FLCs by reducing steroidogenic enzyme activity.

Fetal Leydig cells dedifferentiate and serve as adult Leydig stem cells.

Previous studies have established that fetal Leydig cells (FLCs) and adult Leydig cells (ALCs) show distinct functional characteristics. However, the lineage relationship between FLCs and ALCs has not been clarified yet. Here, we reveal that a subset of FLCs dedifferentiate at fetal stages to give rise to ALCs at the pubertal stage. Moreover, the dedifferentiated cells contribute to the peritubular myoid cell and vascular pericyte populations in the neonatal testis, and these non-steroidogenic cells serve as potential ALC stem cells. We generated FLC lineage-specific Nr5a1 (Ad4BP/SF-1) gene-disrupted mice and mice lacking the fetal Leydig enhancer (FLE) of the Nr5a1 gene. Phenotypes of these mice support the conclusion that most of the ALCs arise from dedifferentiated FLCs, and that the FLE of the Nr5a1 gene is essential for both initial FLC differentiation and pubertal ALC redifferentiation.

In utero exposure to dipentyl phthalate disrupts fetal and adult Leydig cell development.

Phthalates as plasticizers are widely used in many consumer products. Dipentyl phthalate (DPeP) is one of phthalates. However, there are currently few data on whether DPeP exposure affects rat Leydig cell development. In this study, we investigated the effects of in utero DPeP exposure on Leydig cell development in the testes of male newborn and adult rats. From gestational days 14 to 21, Sprague-Dawley pregnant rats were gavaged vehicle (corn oil, control) or DPeP (10, 50, 100, and 500 mg/kg body weight/day). Testosterone and the expression of Leydig cell genes and proteins in the testis at birth and at postnatal day 56 were examined. DPeP dose-dependently reduced serum testosterone levels of male offspring at birth and at postnatal day 56 at 100 and 500 mg/kg and lowered serum luteinizing hormone levels at adult males at ≥10 mg/kg when compared with the control. In addition, DPeP increased number of fetal Leydig cells by inducing their proliferation but down-regulated the expression of Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, and Insl3 in fetal Leydig cells per se. DPeP reduced number of adult Leydig cells by inducing cell apoptosis and down-regulated the expression of Lhcgr and Star in adult Leydig cells at postnatal day 56. DPeP lowered SIRT1 and BCL2 levels in the testis of adult rats. In conclusion, DPeP adversely affects both fetal and adult Leydig cell development after in utero exposure.

Triadimefon increases fetal Leydig cell proliferation but inhibits its differentiation of male fetuses after gestational exposure.

Triadimefon is a broad-spectrum fungicide widely applied in the agriculture. It is believed to be an endocrine disruptor. Whether triadimefon can inhibit the development of fetal Leydig cells and the underlying mechanisms are unknown. Thirty-two female pregnant Sprague-Dawley rats were randomly assigned into four groups and were dosed via gavage of triadimefon (0, 25, 50, and 100 mg/kg/day) for 9 days from gestational day (GD) 12-20. Triadimefon significantly reduced serum testosterone level in male fetuses at 100 mg/kg. The double immunofluorescence staining of proliferating cell nuclear antigen (PCNA) and cytochrome P450 cholesterol side-chain cleavage (a biomarker for fetal Leydig cells) was used to measure PCNA-labeling in fetal Leydig cells. It markedly increased fetal Leydig cell number primarily via increasing single cell population and elevated the PCNA-labeling of fetal Leydig cells in male fetuses at 100 mg/kg while it induced abnormal aggregation of fetal Leydig cells. The expression levels of fetal Leydig cell genes, Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Insl3 and Nr5a1, were determined to explore its effects on fetal Leydig cell development. We found that triadimefon markedly down-regulated the expression of Leydig cell genes, Hsd17b3, Insl3, and Nr5a1 as low as 25 mg/kg and Scarb1 and Cyp11a1 at 100 mg/kg. It did not affect Sertoli cell number but markedly down-regulated the expression of Sertoli cell gene Amh at 50 and 100 mg/kg. Triadimefon significantly down-regulated the expression of antioxidant genes Sod1, Gpx1, and Cat at 25-100 mg/kg, suggesting that it can induce oxidative stress in fetal testis, and it reduced the phosphorylation of ERK1/2 and AKT2 at 100 mg/kg, indicating that it can inhibit the development of fetal Leydig cells. In conclusion, gestational exposure to triadimefon inhibits the development of fetal Leydig cells in male fetuses by inhibiting its differentiation.

An exploration of the role of Sertoli cells on fetal testis development using cell ablation strategy.

Sertoli cells (SCs) are presumed to be the center of testis differentiation because they provide both structural support and biological regulation for spermatogenesis. Previous studies suggest that SCs control germ cell (GC) count and Leydig cell (LC) development in mouse testes. However, the regulatory role of SCs on peritubular myoid (PTM) cell fate in fetal testis has not been clearly reported. Here, we employed Amh-Cre; diphtheria toxin fragment A (DTA) mouse model to selectively ablate SCs from embryonic day (E) 14.5. Results found that SC ablation in the fetal stage caused the disruption of testis cords and the massive loss of GCs. Furthermore, the number of α-smooth muscle actin-labeled PTM cells was gradually decreased from E14.5 and almost lost at E18.5 in SC ablation testis. Interestingly, some Ki67 and 3ß-HSD double-positive fetal LCs could be observed in Amh-Cre; DTA testes at E16.5 and E18.5. Consistent with this phenomenon, the messenger RNA levels of Hsd3b1, Cyp11a1, Lhr, Star and the protein levels of 3ß-HSD and P450Scc were significantly elevated by SC ablation. SC ablation appears to induce ectopic proliferation of fetal LCs although the total LC number appeared reduced. Together, these findings bring us a better understanding of SCs' central role in fetal testis development.

Gestational vinclozolin exposure suppresses fetal testis development in rats.

Vinclozolin is a common dicarboximide fungicide used to protect crops from diseases. It is also an endocrine disruptor and is thought to be related to abnormalities of the reproductive tract. However, its mechanism of inducing abnormalities of the male reproductive tract is still unclear. The purpose of this study was to study the effect of gestational vinclozolin exposure on the development of rat fetal Leydig cells. Female pregnant Sprague-Dawley rats were exposed to vinclozolin (0, 25, 50, and 100 mg/kg body weight/day) by gavage from gestational day 14-21. Vinclozolin dose-dependently reduced serum testosterone levels at doses of 50 and 100 mg/kg and the anogenital distance at 100 mg/kg. RNA-seq, qPCR, and Western blotting showed that vinclozolin down-regulated the expression of Nr5a1, Sox9, Lhcgr, Cyp11a1, Hsd3b1, Hsd17b3, Amh, Pdgfa, and Dhh and their encoded proteins. Vinclozolin reduced the number of NR2F2-positive stem Leydig cells at a dose of 100 mg/kg and enhanced autophagy in the testes. In conclusion, vinclozolin disrupts reproductive tract development and testis development in male fetal rats via several pathways.

Development of fetal and adult Leydig cells.

BACKGROUND: In mammals, two distinct Leydig cell populations, fetal Leydig cells (FLCs) and adult Leydig cells (ALCs), appear in the prenatal and postnatal testis, respectively. Although the functional differences between these cell types have been well described, the developmental relationship between FLCs and ALCs has not been fully understood. In this review, I focus on the cellular origins of FLCs and ALCs as well as the developmental and functional links between them. METHODS: I surveyed previous reports about FLC and/or ALC development and summarized the findings. MAIN FINDINGS: Fetal Leydig cells and ALCs were identified to have separate origins in the fetal and neonatal testis, respectively. However, several studies suggested that FLCs and ALCs share a common progenitor pool. Moreover, perturbation of FLC development at the fetal stage induces ALC dysfunction in adults, suggesting a functional link between FLCs and ALCs. Although the lineage relationship between FLCs and ALCs remains controversial, a recent study suggested that some FLCs dedifferentiate at the fetal stage, and that these cells serve as ALC stem cells. CONCLUSION: Findings obtained from animal studies might provide clues to the causative mechanisms of male reproductive dysfunctions such as testicular dysgenesis syndrome in humans.

Steroidogenic factor 1 differentially regulates fetal and adult leydig cell development in male mice.

The nuclear receptor steroidogenic factor 1 (SF-1, AD4BP, NR5A1) is a key regulator of the endocrine axes and is essential for adrenal and gonad development. Partial rescue of Nr5a1(-/-) mice with an SF-1-expressing transgene caused a hypomorphic phenotype that revealed its roles in Leydig cell development. In contrast to controls, all male rescue mice (Nr5a1(-/-);tg(+/0)) showed varying signs of androgen deficiency, including spermatogenic arrest, cryptorchidism, and poor virilization. Expression of various Leydig cell markers measured by immunohistochemistry, Western blot analysis, and RT-PCR indicated fetal and adult Leydig cell development were differentially impaired. Whereas fetal Leydig cell development was delayed in Nr5a1(-/-);tg(+/0) embryos, it recovered to control levels by birth. In contrast, Sult1e1, Vcam1, and Hsd3b6 transcript levels in adult rescue testes indicated complete blockage in adult Leydig cell development. In addition, between Postnatal Days 8 and 12, peritubular cells expressing PTCH1, SF-1, and CYP11A1 were observed in control testes but not in rescue testes, indicating SF-1 is needed for either survival or differentiation of adult Leydig cell progenitors. Cultured prepubertal rat peritubular cells also expressed SF-1 and PTCH1, but Cyp11a1 was expressed only after treatment with cAMP and retinoic acid. Together, data show SF-1 is needed for proper development of fetal and adult Leydig cells but with distinct primary functions; in fetal Leydig cells, it regulates differentiation, whereas in adult Leydig cells it regulates progenitor cell formation and/or survival.

Cellular microenvironment dictates androgen production by murine fetal Leydig cells in primary culture.

Despite the fact that fetal Leydig cells are recognized as the primary source of androgens in male embryos, the mechanisms by which steroidogenesis occurs within the developing testis remain unclear. A genetic approach was used to visualize and isolate fetal Leydig cells from remaining cells within developing mouse testes. Cyp11a1-Cre mice were bred to mT/mG dual reporter mice to target membrane-tagged enhanced green fluorescent protein (GFP) within steroidogenic cells, whereas other cells expressed membrane-tagged tandem-dimer tomato red. Fetal Leydig cell identity was validated using double-labeled immunohistochemistry against GFP and the steroidogenic enzyme 3beta-HSD, and cells were successfully isolated as indicated by qPCR results from sorted cell populations. Because fetal Leydig cells must collaborate with neighboring cells to synthesize testosterone, we hypothesized that the fetal Leydig cell microenvironment defined their capacity for androgen production. Microfluidic culture devices were used to measure androstenedione and testosterone production of fetal Leydig cells that were cultured in cell-cell contact within a mixed population, were isolated but remained in medium contact via compartmentalized co-culture with other testicular cells, or were isolated and cultured alone. Results showed that fetal Leydig cells maintained their identity and steroidogenic activity for 3-5 days in primary culture. Microenvironment dictated proficiency of testosterone production. As expected, fetal Leydig cells produced androstenedione but not testosterone when cultured in isolation. More testosterone accumulated in medium from mixed cultures than from compartmentalized co-cultures initially; however, co-cultures maintained testosterone synthesis for a longer time. These data suggest that a combination of cell-cell contact and soluble factors constitute the ideal microenvironment for fetal Leydig cell activity in primary culture.

Emerging concepts on Leydig cell development in fetal and adult testis.

Leydig cells (Lc) reside in the interstitial compartment of the testis and are the target of Luteinising hormone (LH) for Testosterone (T) production, thus critically regulates male fertility. Classical histological studies have identified two morphologically different populations of Lc during testicular development [fetal (FLc) and adult (ALc)]. Recent progress in ex vivo cell/organ culture, genome-wide analysis, genetically manipulated mouse models, lineage tracing, and single-cell RNA-seq experiments have revealed the diverse cellular origins with differential transcriptomic and distinct steroidogenic outputs of these populations. FLc originates from both coelomic epithelium and notch-active Nestin-positive perivascular cells located at the gonad-mesonephros borders, and get specified as Nr5a1 (previously known as Ad4BP/SF-1) expressing cells by embryonic age (E) 12.5 days in fetal mouse testes. These cells produce androstenedione (precursor of T, due to lack of HSD17ß3 enzyme) and play critical a role in initial virilization and patterning of the male external genitalia. However, in neonatal testis, FLc undergoes massive regression/dedifferentiation and gradually gets replaced by T-producing ALc. Very recent studies suggest a small fraction (5-20%) of FLc still persists in adult testis. Both Nestin-positive perivascular cells and FLc are considered to be the progenitor populations for ALc. This minireview article summarizes the current understanding of Lc development in fetal and adult testes highlighting their common or diverse cellular (progenitor/stem) origins with respective functional significance in both rodents and primates. (227 words).

Exposure to 4-bromodiphenyl ether during pregnancy blocks testis development in male rat fetuses.

4-Bromodiphenyl ether (BDE3) is a photodegradation product of higher polybrominated diphenyl ether flame retardants and is known as an endocrine disruptor. However, it is unclear whether and how BDE3 affects the development of fetal testes. This study aimed to investigate the effect of in utero exposure to BDE3 on fetal testicular development in rats. From gestational day (GD) 12-21, BDE3 (0, 50, 100, and 200 mg/kg) was daily gavaged to female pregnant Sprague Dawley rats. BDE3 significantly reduced serum testosterone levels of male pups starting at 50 mg/kg. BDE3 reduced fetal Leydig cell number at a dose of 200 mg/kg without affecting fetal Leydig cell cluster frequency and Sertoli cell number. In addition, BDE3 down-regulated the expression of fetal Leydig cell genes (Cyp11a1, Hsd3b1, Cyp17a1, and Hsd17b3) and their proteins at 100 and/or 200 mg/kg. RNA-seq analysis showed that genes responsive to cAMP (Ass1, Gpd1, Rpl13a) were down-regulated and hypoxia-related genes (Egln3 and P4ha1) were up-regulated at 200 mg/kg. In utero exposure to BDE3 can promote autophagy and apoptosis of fetal Leydig cells via increasing the levels of Beclin1, LC3-II, BAX, and by decreasing the levels of p62 and BCL2. In conclusion, in utero exposure to BDE3 blocks the development of fetal rat testes.

Effects of bis(2-butoxyethyl) phthalate exposure in utero on the development of fetal Leydig cells in rats.

Phthalates are plasticizers widely found in the environment. They are potential endocrine disruptors. Bis(2-butoxyethyl) phthalate (BBOP) is a unique phthalate that contains oxygen atoms in the carbon backbone. Little is known about its reproductive and developmental toxicity. The objective of this study was to determine the effect of BBOP on fetal Leydig cell development after in utero exposure to rats. Sprague Dawley pregnant dams were randomly allocated into 6 groups, and were gavaged with BBOP (0, 10, 100, 250, 500, and 1000 mg/kg body weight/day) from gestational day (GD) 14-21. Seven of the 8 dams in the 1000 mg/kg BBOP group died before giving birth. Twelve of the 20 dams in the 500 mg/kg BBOP group had whole litter loss. BBOP significantly reduced the body weight of dams and male offspring and serum testosterone level and anogenital distance of male fetus on GD 21 at 500 mg/kg. BBOP markedly increased fetal Leydig cell proliferation and number at 500 mg/kg while inducing their abnormal aggregation at 250 and 500 mg/kg. BBOP down-regulated the expression of Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Insl3, and Nr5a1 at various doses while up-regulating the expression of Sertoli cell gene Fshr and Sox9. The phosphorylation of AKT1, AKT2, and ERK1/2 was also markedly reduced by BBOP. In conclusion, BBOP in utero exposure can disrupt fetal Leydig cell development, possibly via the mechanism that may include inhibiting the phosphorylation of AKT1, AKT2, and ERK1/2.

The Production of Testosterone and Gene Expression in Neonatal Testes of Rats Exposed to Diisoheptyl Phthalate During Pregnancy is Inhibited.

Background: Diisoheptyl phthalate (DIHP) is a phthalate plasticizer, which is a branched phthalate. Here, we reported the effects of gestational exposure to DIHP on testis development in male rats. Methods: Pregnant Sprague-Dawley rats were orally fed with vehicle (corn oil, control) or DIHP (10, 100, 500, and 1,000 mg/kg) from gestational day (GD) 12-21. At GD21, serum testosterone levels, the number and distribution of fetal Leydig cells, and testicular mRNA and protein levels, the incidence of multinucleated gonocytes, and focal testicular hypoplasia in the neonatal testis were measured. Results: DIHP increased the fetal Leydig cell cluster size and decreased the fetal Leydig cell size with LOAEL of 10 mg/kg. DIHP did not affect the fetal Leydig cell number. DIHP significantly lowered serum testosterone levels, down-regulated the expression of steroidogenesis-related genes (Lhcgr, Star, Cyp11a1, Hsd3b1, Cyp17a1, and Hsd17b3) and testis descent-related gene (Insl3) as well as protein levels of cholesterol side-chain cleavage enzyme (CYP11A1) and insulin-like 3 (INSL3). DIHP dose-dependently increased the percentage of multinucleated gonocytes with the low observed adverse-effect level (LOAEL) of 100 mg/kg. DIHP induced focal testicular hypoplasia. Conclusion: Gestational exposure to DIHP causes testis dysgenesis in rats.

Specific Transcriptomic Signatures and Dual Regulation of Steroidogenesis Between Fetal and Adult Mouse Leydig Cells.

Leydig cells (LC) are the main testicular androgen-producing cells. In eutherian mammals, two types of LCs emerge successively during testicular development, fetal Leydig cells (FLCs) and adult Leydig cells (ALCs). Both display significant differences in androgen production and regulation. Using bulk RNA sequencing, we compared the transcriptomes of both LC populations to characterize their specific transcriptional and functional features. Despite similar transcriptomic profiles, a quarter of the genes show significant variations in expression between FLCs and ALCs. Non-transcriptional events, such as alternative splicing was also observed, including a high rate of intron retention in FLCs compared to ALCs. The use of single-cell RNA sequencing data also allowed the identification of nine FLC-specific genes and 50 ALC-specific genes. Expression of the corticotropin-releasing hormone 1 (Crhr1) receptor and the ACTH receptor melanocortin type 2 receptor (Mc2r) specifically in FLCs suggests a dual regulation of steroidogenesis. The androstenedione synthesis by FLCs is stimulated by luteinizing hormone (LH), corticotrophin-releasing hormone (CRH), and adrenocorticotropic hormone (ACTH) whereas the testosterone synthesis by ALCs is dependent exclusively on LH. Overall, our study provides a useful database to explore LC development and functions.

Taxifolin attenuates the developmental testicular toxicity induced by di-n-butyl phthalate in fetal male rats.

Di-n-butyl phthalate (DBP) is widely used in consumer products as a plasticizer. Here, we report a natural product taxifolin that can attenuate developmental and reproductive toxicity of DBP. Pregnant rats were daily gavaged with 500 mg/kg DBP alone or together with taxifolin (10 and 20 mg/kg) from gestational day (GD) 12-21. At GD21, sera and testes of male fetus were collected. DBP significantly lowered serum testosterone level at 500 mg/kg and taxifolin can completely reverse its action. DBP caused abnormal aggregation of fetal Leydig cells and taxifolin can reverse it. DBP down-regulated the expression of the genes of cholesterol side-chain cleavage enzyme (Cyp11a1), 17ß-hydroxysteroid dehydrogenase 3 (Hsd17b3), and insulin-like 3 (Insl3) and taxifolin can reverse its action. DBP increased malondialdehyde levels and decreased superoxide dismutase and glutathione peroxidase expression and taxifolin can reverse it. DBP increased incidence of multinucleated gonocytes and taxifolin can prevent it. Moreover, DBP lowered sirtuin 1 (SIRT1)/peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) and phosphorylated AMP-activated protein kinase (pAMPK) signalling and taxifolin antagonized DBP. In conclusion, in utero exposure to DBP caused developmental/reproductive toxicity of male offspring via increasing reactive oxygen species and taxifolin is an effective food component that completely reverses DBP-mediated action.

Maternal exposure to zearalenone in masculinization window affects the fetal Leydig cell development in rat male fetus.

Zearalenone is a phenolic Fusarium mycotoxin, which is ubiquitous in human and animal feedstuff and often co-occurs with other mycotoxins. ZEA has been reported to disturb Leydig cell function and even cause the apoptosis to the Leydig cells. However, the effects of gestational exposure to zearalenone on fetal Leydig cells and the underlying mechanism remain unknown. Sprague Dawley dams were daily gavaged with 0, 2.5, 5, 10, and 20 mg/kg body weight ZEA from gestational day 14-21. On gestational day 21, rats were euthanized and serum testosterone levels were measured, and testes were collected for further evaluation of Leydig cell number, cell size, gene, and protein expression. Zearalenone significantly decreased anogenital distance and its index of male fetus, serum testosterone levels, Leydig cell proteins (SCARB1, STAR, CYP11A1, CYP17A1, and INSL3), and fetal Leydig cell number at 10 and/or 20 mg/kg by delaying the commitment of stem Leydig cells into the Leydig cell lineage and proliferation. Further study found that Notch signaling (RFNG, PSEN1, NOTCH1, and NOTCH3) was up-regulated by zearalenone. In conclusion, gestational exposure to high doses of zearalenone (10 and 20 mg/kg) blocks fetal Leydig cell development, thus possibly causing the anomalies of the male reproductive tract.