Fetal Leydig Cells Research Articles

Simvastatin and dipentyl phthalate lower ex vivo testicular testosterone production and exhibit additive effects on testicular testosterone and gene expression via distinct mechanistic pathways in the fetal rat.

Sex differentiation of the male reproductive tract in mammals is driven, in part, by fetal androgen production. In utero, some phthalate esters (PEs) alter fetal Leydig cell differentiation, reducing the expression of several genes associated with steroid synthesis/transport, and consequently, lowering fetal androgen and Insl3 hormone levels. Simvastatin (SMV) is a cholesterol-lowering drug that directly inhibits HMG-CoA reductase. SMV may also disrupt steroid biosynthesis, but through a different mode of action (MOA) than the PEs. As cholesterol is a precursor of steroid hormone biosynthesis, we hypothesized that in utero exposure to SMV during the critical period of sex differentiation would lower fetal testicular testosterone (T) production without affecting genes involved in cholesterol and androgen synthesis and transport. Secondly, we hypothesized that a mixture of SMV and a PE, which may have different MOAs, would reduce testosterone levels in an additive manner. Pregnant Sprague Dawley rats were dosed orally with SMV, dipentyl phthalate (DPeP), or SMV plus DPeP from gestational days 14-18, and fetuses were evaluated on GD18. On GD18, SMV lowered fetal T production and serum triglycerides, low density lipoprotein, high density lipoprotein, and total cholesterol levels, and downregulated two genes in the fetal testis that were different from those altered by PEs. When SMV and DPeP were administered as a mixture, fetal T production was significantly reduced in an additive manner, thus demonstrating that a mixture of chemicals can induce additive effects on fetal T production even though they display different MOAs.

Exogenous androgens reduce the expression of INSL3, a hormone involved in normal testicular descent, in fetal Leydig cells

Exogenous androgens reduce the expression of INSL3, a hormone involved in normal testicular descent, in fetal Leydig cells

Man is not a big rat: concerns with traditional human risk assessment of phthalates based on their anti-androgenic effects observed in the rat foetus.

Phthalates provide one of the most documented example evidencing how much we must be cautious when using the traditional paradigm based on extrapolation of experimental data from rodent studies for human health risk assessment of endocrine disruptors (EDs). Since foetal testis is known as one of the most sensitive targets of EDs, phthalate risk assessment is routinely based on the capacity of such compounds to decrease testosterone production by the testis or to impair masculinization in the rat during foetal life. In this paper, the well-established inhibiting effects of phthalates of the foetal Leydig cells function in the rat are briefly reviewed. Then, data obtained in humans and other species are carefully analysed. Already in January 2009, using the organotypic culture system named Fetal Testis Assay (FeTA) that we developed, we reported that phthalates might not affect testosterone production in human foetal testes. Several recent experimental studies using xenografts confirm the absence of detectable anti-androgenic effect of phthalates in the human foetal testes. Epidemiological studies led to contradictory results. Altogether, these findings suggest that phthalates effects on foetal Leydig cells are largely species-specific. Consequently, the phthalate threshold doses that disturb foetal steroidogenesis in rat testes and that are presently used to define the acceptable daily intake levels for human health protection must be questioned. This does not mean that phthalates are safe because these compounds have many deleterious effects upon germ cell development that may be common to the different studied species including human. More generally, the identification of common molecular, cellular or/and phenotypic targets in rat and human testes should precede the choice of the toxicological endpoint in rat to accurately assess the safety threshold of any ED in humans.

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.

Expression patterns of DLK1 and INSL3 identify stages of Leydig cell differentiation during normal development and in testicular pathologies, including testicular cancer and Klinefelter syndrome.

What is the differentiation stage of human testicular interstitial cells, in particular Leydig cells (LC), within micronodules found in patients with infertility, testicular cancer and Klinefelter syndrome? The Leydig- and peritubular-cell populations in testes with dysgenesis contain an increased proportion of undifferentiated cells when compared with control samples, as demonstrated by increased delta-like homolog 1 (DLK1) and decreased insulin-like peptide 3 (INSL3) expression. Normal LC function is essential for male development and reproduction. Signs of LC failure, including LC micronodules, are often observed in patients with reproductive disorders. In this retrospective study, a panel of markers and factors linked to the differentiation of LCs was investigated in 33 fetal and prepubertal human specimens and in 58 adult testis samples from patients with testicular germ cell tumours, including precursor carcinoma in situ (CIS), infertility or Klinefelter syndrome. The expression patterns of DLK1, INSL3, chicken ovalbumin upstream promoter transcription factor 2 (COUP-TFII), cytochrome P450, family 11, subfamily A, polypeptide 1 (CYP11A1) and smooth muscle actin (SMA) were investigated by immunohistochemistry and quantitative RT-PCR. The percentage of positive LCs was estimated and correlated to total LC numbers and serum levels of reproductive hormones. DLK1, INSL3 and COUP-TFII expression changed during normal development and was linked to different stages of LC differentiation: DLK1 was expressed in all fetal LCs, but only in spindle-shaped progenitor cells and in a small subset of polygonal LCs in the normal adult testis; INSL3 was expressed in a subset of fetal LCs, but in the majority of adult LCs; and COUP-TFII was expressed in peritubular and mesenchymal stroma cells at all ages, in fetal LCs early in gestation and in a subset of adult LCs. CYP11A1 was expressed in the majority of LCs regardless of age and pathology and was the best general LC marker examined here. SMA was weakly expressed in peritubular cells in the fetal and infantile testis, but strongly expressed in the adult testis. In pathological testes, the numbers of DLK1-positive interstitial cells were increased. The proportion of DLK1-positive LCs correlated with total LC numbers (R = 0.53; P < 0.001) and was higher in testis with enlargement of the peritubular layers (P < 0.01), which was also highly associated with DLK1 expression in the peritubular compartment (P < 0.001). INSL3 expression was absent in some, but not all LC micronodules, and in the majority of LCs, it was mutually exclusive of DLK1. The number of samples was relatively small and no true normal adult controls were available. True stereology was not used for LC counting, instead LCs were counted in three fields of 0.5 µm(2) surface for each sample. The population of LCs, especially those clustered in large nodules, are heterogeneous and comprise cells at different stages of differentiation. The study demonstrated that the differentiation and function of LCs, and possibly also peritubular cells, are impaired in adult men with testicular pathologies including testis cancer and Klinefelter syndrome. This work was funded by Rigshospitalet's research funds, the Danish Cancer Society and Kirsten and Freddy Johansen's foundation. The authors have no conflicts of interest.

Loss of function mutation in the palmitoyl-transferase HHAT leads to syndromic 46,XY disorder of sex development by impeding Hedgehog protein palmitoylation and signaling.

The Hedgehog (Hh) family of secreted proteins act as morphogens to control embryonic patterning and development in a variety of organ systems. Post-translational covalent attachment of cholesterol and palmitate to Hh proteins are critical for multimerization and long range signaling potency. However, the biological impact of lipid modifications on Hh ligand distribution and signal reception in humans remains unclear. In the present study, we report a unique case of autosomal recessive syndromic 46,XY Disorder of Sex Development (DSD) with testicular dysgenesis and chondrodysplasia resulting from a homozygous G287V missense mutation in the hedgehog acyl-transferase (HHAT) gene. This mutation occurred in the conserved membrane bound O-acyltransferase (MBOAT) domain and experimentally disrupted the ability of HHAT to palmitoylate Hh proteins such as DHH and SHH. Consistent with the patient phenotype, HHAT was found to be expressed in the somatic cells of both XX and XY gonads at the time of sex determination, and Hhat loss of function in mice recapitulates most of the testicular, skeletal, neuronal and growth defects observed in humans. In the developing testis, HHAT is not required for Sertoli cell commitment but plays a role in proper testis cord formation and the differentiation of fetal Leydig cells. Altogether, these results shed new light on the mechanisms of action of Hh proteins. Furthermore, they provide the first clinical evidence of the essential role played by lipid modification of Hh proteins in human testicular organogenesis and embryonic development.

Pleiotropic Activities of HGF/c-Met System in Testicular Physiology: Paracrine and Endocrine Implications.

In the last decades, a growing body of evidence has been reported concerning the expression and functional role of hepatocyte growth factor (HGF) on different aspects of testicular physiology. This review has the aim to summarize what is currently known regarding this topic. From early embryonic development to adult age, HGF and its receptor c-Met appeared to be clearly detectable in the testis. These molecules acquire different distribution patterns and roles depending on the developmental stage or the post-natal age considered. HGF acts as a paracrine modulator of testicular functions promoting the epithelium–mesenchyme cross-talk as described even in other organs. Interestingly, it has been reported that testicular HGF acts even as an autocrine factor and that its receptor might be modulated by endocrine signals that change at puberty: HGF receptor expressed by Sertoli cells, in fact, is up-regulated by FSH administration. HGF is in turn able to modify endocrine state of the organism being able to increase testosterone secretion of both fetal and adult Leydig cells. Moreover, c-Met is expressed in mitotic and meiotic male germ cells as well as in spermatozoa. The distribution pattern of c-Met on sperm cell membrane changes in the caput and cauda epididymal sperms and HGF is able to maintain epididymal sperm motility in vitro suggesting a physiological role of this growth factor in the acquisition of sperm motility. Noteworthy changes in HGF concentration in seminal plasma have been reported in different andrological diseases. All together these data indicate that HGF has a role in the control of spermatogenesis and sperm quality either directly, acting on male germ cells, or indirectly acting on tubular and interstitial somatic cells of the testis.

Effects of prenatal and lactation nicotine exposure on rat testicular interstitial tissue

Nicotine is largely consumed as a component of cigarettes. It induces apoptosis, interferes with endocrine function by changing the sex hormones secretion and leads to male infertility. Testosterone is produced from cholesterol by Leydig cells (LC), with the participation of testicular macrophages (MO). Thus, to investigate whether nicotine administration to pregnant and lactating rats changes cholesterol and sexual hormone levels and LC and MO populations of offspring, female rats received nicotine (2 mg/kg/day) through osmotic minipumps from the first day of pregnancy up to the end of weaning. At 1, 30, 60 and 90 days post-partum (dpp) the plasma cholesterol and testosterone levels were obtained, as well as the biometric, histopathological and stereological testicular parameters. Nicotine reduced the body weight, cholesterol levels and lipid droplet number in foetal LC at 1 dpp. The number of apoptotic LC did not change in the offspring of nicotine group at any age studied. No alterations in the numerical densities of MO and LC occurred at 60 and 90 dpp. Hypertrophy of mature LC and increase in cholesterol and testosterone levels were noted at 90 dpp. In conclusion, nicotine when administered to rats throughout pregnancy and lactation induces morphofunctional alterations of foetal and mature LC and affects cholesterol and testosterone levels.

Exposure to Perfluorooctane Sulfonate In Utero Reduces Testosterone Production in Rat Fetal Leydig Cells

BackgroundPerfluorooctane sulfonate (PFOS) is a synthetic material that has been widely used in industrial applications for decades. Exposure to PFOS has been associated with decreased adult testosterone level, and Leydig cell impairment during the time of adulthood. However, little is known about PFOS effects in utero on fetal Leydig cells (FLC).Methods and ResultsThe present study investigated effects of PFOS on FLC function. Pregnant Sprague Dawley female rats received vehicle (0.05% Tween20) or PFOS (5, 20 mg/kg) by oral gavage from gestational day (GD) 11–19. At GD20, testosterone (T) production, FLC numbers and ultrastructure, testicular gene and protein expression levels were examined. The results indicate that exposures to PFOS have affected FLC function as evidenced by decreased T production, impaired FLC, reduced FLC number, and decreased steroidogenic capacity and cholesterol level in utero.ConclusionThe present study shows that PFOS is an endocrine disruptor of male reproductive system as it causes reduction of T production and impairment of rat fetal Leydig cells.

Phthalate-induced pathology in the foetal testis involves more than decreased testosterone production

Foetal exposure to phthalates is known to adversely impact male reproductive development and function. Developmental anomalies of reproductive tract have been attributed to impaired testosterone synthesis. However, species differences in the ability to produce testosterone have been noted; e.g., following foetal exposure, abnormal clustering of Leydig cells or decreased production of testosterone that is manifested in rats does not occur in mice or humans. Nonetheless, other facets of testicular dysgenesis occur in both rats and mice as well as in some other species tested. We recently published a comprehensive evaluation of the foetal rat testis proteome, following in utero exposure to diethylhexyl phthalate (DEHP), which revealed changes in individual proteins that are known to be factors in cellular differentiation and migration or related to the capacity of the foetal Leydig cell to produce testosterone and fit a pathway network in which each is regulated directly or indirectly by oestradiol. Plasma oestradiol indeed was found to be elevated approximately twofold in 19-day-old DEHP-exposed foetal male rats. In this brief review, we discuss our new findings vis-à-vis 'oestrogen hypothesis' as a cause for testicular dysgenesis syndrome.

Endocrine disrupter effects on the gonads and pubertY

Evidence accumulating the past decades indicates that human populations are exposed to environmental chemicals interfering with endocrine systems. There is growing concern of the potential adverse impact of exposure to such endocrine disrupting chemicals (EDCs) on human health, based on observations in wildlife, animal model systems and reports of yet unexplained increased incidences of hormone related disorders in humans. Exposure in fetal and neonatal age may be associated with developmental and reproductive disturbances due to interference with the programming of normal hormone signaling and metabolic pathways. Boys are conceived to be more vulnerable than girls due to their more complicated and androgen driven prenatal sex differentiation. Male prenatal sex differentiation is crucially dependent on the functioning of fetal Leydig cells. Exposure of these cells to EDCs at critical time windows of development may result in undermasculinization and disorder (s) of sex development (DSD), such as hypospadias, cryptorchidism and ambiguous genitalia. Furthermore, a novel trend of earlier start of puberty has been found during the past few decades, at least in some regions and mostly affecting girls. Among plausible causes behind this phenomenon it has been suggested that exposure to EDCs affecting the programming or triggering of the pubertal clock could result in premature activation of gonadotropin secretion. Another hypothesis is that gonadotropin independent effects of EDCs are associated with an earlier start of puberty. Proof of principle of such potential roles of EDCs has been obtained from a multitude of studies in experimental animals and gained some support by case studies, exposure data and epidemiological investigations in humans. This presentation will review recent data and ongoing studies on the mechanism(s) of action of EDCs, including effects of mixtures, on the hypothalamuspituitary-gonadal axis in experimental animals and humans. The impact of genetic susceptibility on the degree of disrupting activity caused by certain EDCs will be discussed on the basis of ongoing animal studies.

Prenatal phthalate exposure is associated with human fetal Leydig cell function but not with cryptorchidism and hypospadias

Background: Studies on rats indicate that prenatal exposure to certain phthalates may cause reproductive tract disorders by reducing fetal insulin like factor 3 (INSL3) and testosterone production....

Developmental Expression of Translocator Protein/Peripheral Benzodiazepine Receptor in Reproductive Tissues

Translocator protein (TSPO) present in the outer mitochondrial membrane has been suggested to be critical for cholesterol import, a rate-limiting step for steroid hormone biosynthesis. Despite the importance of steroidogenesis in regulating reproductive functions, the developmental profile of TSPO expression in the gonads and accessory sex organs has not been completely characterized. As a first step towards understanding the function of TSPO, we studied its expression in male and female murine reproductive organs. We examined testes and ovaries at embryonic days 14.5 and 18.5, and postnatal days 0, 7, 14, 21 and 56 of development. In the adult testis, TSPO was expressed in both Leydig cells and Sertoli cells. In the developing testes TSPO expression was seen in immature Sertoli cells, fetal Leydig cells and gonocytes. In the ovary, TSPO was expressed in the ovarian surface epithelium, interstitial cells granulosa cells and luteal cells. Corpora lutea of ovaries from pregnant mice showed strong expression of TSPO. In the developing ovary, TSPO expression was seen in the squamous pregranulosa cells associated with germ line cysts, together with progressively increasing expression in interstitial cells and the ovarian surface epithelium. In adult mice, the epithelia of other reproductive tissues like the epididymis, prostate, seminal vesicle, oviduct and uterus also showed distinct patterns of TSPO expression. In summary, TSPO expression in both male and female reproductive tissues was not only restricted to steroidogenic cells. Expression in Sertoli cells, ovarian surface epithelium, efferent ductal epithelium, prostatic epithelium, seminal vesiclular epithelium, uterine epithelium and oviductal epithelium suggest either previously unknown sites for de novo steroidogenesis or functions for TSPO distinct from its well-studied role in steroid hormone production.

Sertoli cell dedifferentiation in human cryptorchidism and gender reassignment shows similarities between fetal environmental and adult medical treatment estrogen and antiandrogen exposure

Studies over the last years show an increase in testicular cancer, hypospadias and cryptorchidism in industrial countries, leading to the concept of testicular dysgenesis syndrome (TDS). It is hypothesized that TDS is caused by estrogen and antiandrogen exposure during fetal life, accompanied by incomplete maturation of testicular Sertoli cells (SC). However, it is not known if SC disruption is a primary cause or a response to fetal Leydig cell testosterone production changes.To determine if SC differentiation is directly affected by estrogens, we compared SC maturation between adult gender reassignment cases exposed to estrogen and antiandrogen therapy, and those of typical TDS in adult cryptorchidism.We found similar expression of immature SC markers M2A antigen, inhibin bodies and Anti Mullerian Hormone, and the absence of maturation marker androgen receptor in SC of both types of patients. These data supports the occurrence of true SC dedifferentiation caused by estrogen exposure in adult humans. Our data also suggests that SC maturation is directly disrupted in TDS.

Aristaless Related Homeobox Gene, Arx, Is Implicated in Mouse Fetal Leydig Cell Differentiation Possibly through Expressing in the Progenitor Cells

Development of the testis begins with the expression of the SRY gene in pre-Sertoli cells. Soon after, testis cords containing Sertoli and germ cells are formed and fetal Leydig cells subsequently develop in the interstitial space. Studies using knockout mice have indicated that multiple genes encoding growth factors and transcription factors are implicated in fetal Leydig cell differentiation. Previously, we demonstrated that the Arx gene is implicated in this process. However, how ARX regulates Leydig cell differentiation remained unknown. In this study, we examined Arx KO testes and revealed that fetal Leydig cell numbers largely decrease throughout the fetal life. Since our study shows that fetal Leydig cells rarely proliferate, this decrease in the KO testes is thought to be due to defects of fetal Leydig progenitor cells. In sexually indifferent fetal gonads of wild type, ARX was expressed in the coelomic epithelial cells and cells underneath the epithelium as well as cells at the gonad-mesonephros border, both of which have been described to contain progenitors of fetal Leydig cells. After testis differentiation, ARX was expressed in a large population of the interstitial cells but not in fetal Leydig cells, raising the possibility that ARX-positive cells contain fetal Leydig progenitor cells. When examining marker gene expression, we observed cells as if they were differentiating into fetal Leydig cells from the progenitor cells. Based on these results, we propose that ARX acts as a positive factor for differentiation of fetal Leydig cells through functioning at the progenitor stage.

Sertoli Cell Androgen Receptor Expression Regulates Temporal Fetal and Adult Leydig Cell Differentiation, Function, and Population Size

We recently created a mouse model displaying precocious Sertoli cell (SC) and spermatogenic development induced by SC-specific transgenic androgen receptor expression (TgSCAR). Here we reveal that TgSCAR regulates the development, function, and absolute number of Leydig cells (LCs). Total fetal and adult type LC numbers were reduced in postnatal and adult TgSCAR vs control testes, despite normal circulating LH levels. Normal LC to SC ratios found in TgSCAR testes indicate that SC androgen receptor (SCAR)-mediated activity confers a quorum-dependent relationship between total SC and LC numbers. TgSCAR enhanced LC differentiation, shown by elevated ratios of advanced to immature LC types, and reduced LC proliferation in postnatal TgSCAR vs control testes. Postnatal TgSCAR testes displayed up-regulated expression of coupled ligand-receptor transcripts (Amh-Amhr2, Dhh-Ptch1, Pdgfa-Pdgfra) for potential SCAR-stimulated paracrine pathways, which may coordinate LC differentiation. Neonatal TgSCAR testes displayed normal T and dihydrotestosterone levels despite differential changes to steroidogenic gene expression, with down-regulated Star, Cyp11a1, and Cyp17a1 expression contrasting with up-regulated Hsd3b1, Hsd17b3, and Srd5a1 expression. TgSCAR males also displayed elevated postnatal and normal adult serum testosterone levels, despite reduced LC numbers. Enhanced adult-type LC steroidogenic output was revealed by increased pubertal testicular T, dihydrotestosterone, 3α-diol and 3β-diol levels per LC and up-regulated steroidogenic gene (Nr5a1, Lhr, Cyp11a1, Cyp17a1, Hsd3b6, Srd5a1) expression in pubertal or adult TgSCAR vs control males, suggesting regulatory mechanisms maintain androgen levels independently of absolute LC numbers. Our unique gain-of-function TgSCAR model has revealed that SCAR activity controls temporal LC differentiation, steroidogenic function, and population size.

Diethylstilboestrol Exposure Does Not Reduce Testosterone Production in Human Fetal Testis Xenografts

In rodents, in utero exposure to exogenous estrogens including diethylstilboestrol (DES) results in major suppression of steroidogenesis in fetal testes. Whether similar effects occur in the human fetal testis is equivocal. Based on the results of the rodent studies, we hypothesised that exposure of human fetal testes to DES would result in a reduction in testosterone production. We show, using a xenograft approach, that testosterone production is not reduced in human fetal testis following DES exposure. Human fetal testes (15–19 weeks’ gestation, n = 6) were xenografted into castrate male nude mice which were then treated for 35 days with vehicle or 100 µg/kg DES three times a week. For comparison, similar treatment was applied to pregnant rats from e13.5–e20.5 and effects on fetal testes evaluated at e21.5. Xenograft testosterone production was assessed by measuring host seminal vesicle (SV) weights as an indirect measure over the entire grafting period, and single measurement of serum testosterone at termination. Human fetal testis xenografts showed similar survival in DES and vehicle-exposed hosts. SV weight (44.3 v 26.6 mg, p = 0.01) was significantly increased in DES compared to vehicle-exposed hosts, respectively, indicating an overall increase in xenograft testosterone production over the grafting period, whilst serum testosterone at termination was unchanged. In contrast intra-testicular testosterone levels were reduced by 89%, in fetal rats exposed to DES. In rats, DES effects are mediated via Estrogen Receptor α (ESR1). We determined ESR1 protein and mRNA expression in human and rat fetal testis. ESR1 was expressed in rat, but not in human, fetal Leydig cells. We conclude that human fetal testis exposure to DES does not impair testosterone production as it does in rats, probably because ESR1 is not expressed in human fetal Leydig cells. This indicates that DES exposure is likely to pose minimal risk to masculinization of the human fetus.

Dynamic changes in fetal Leydig cell populations influence adult Leydig cell populations in mice

Testes contain two distinct Leydig cell populations during development: fetal and adult Leydig cells (FLCs and ALCs, respectively). ALCs are not derived from FLCs, and it is unknown whether these two populations share common progenitors. We discovered that hedgehog (Hh) signaling is responsible for transforming steroidogenic factor 1-positive (SF1(+)) progenitors into FLCs. However, not all SF1(+) progenitors become FLCs, and some remain undifferentiated through fetal development. We therefore hypothesized that if FLCs and ALCs share SF1(+) progenitors, increased Hh pathway activation in SF1(+) progenitor cells could change the dynamics and distribution of SF1(+) progenitors, FLCs, and ALCs. Using a genetic model involving constitutive activation of Hh pathway in SF1(+) cells, we observed reduced numbers of SF1(+) progenitor cells and increased FLCs. Conversely, increased Hh activation led to decreased ALC populations prepubertally, while adult ALC numbers were comparable to control testes. Hence, reduction in SF1(+) progenitors temporarily affects ALC numbers, suggesting that SF1(+) progenitors in fetal testes are a potential source of both FLCs and ALCs. Besides transient ALC defects, adult animals with Hh activation in SF1(+) progenitors had reduced testicular weight, oligospermia, and decreased sperm mobility. These defects highlight the importance of properly regulated Hh signaling in Leydig cell development and testicular functions.

Testosterone Levels Influence Mouse Fetal Leydig Cell Progenitors Through Notch Signaling1

Leydig cells are the steroidogenic lineage of the mammalian testis that produces testosterone, a key hormone required throughout male fetal and adult life for virilization and spermatogenesis. Both fetal and adult Leydig cells arise from a progenitor population in the testis interstitium but are thought to be lineage-independent of one another. Genetic evidence indicates that Notch signaling is required during fetal life to maintain a balance between differentiated Leydig cells and their progenitors, but the elusive progenitor cell type and ligands involved have not been identified. In this study, we show that the Notch pathway signals through the ligand JAG1 in perivascular interstitial cells during fetal life. In the early postnatal testis, we show that circulating levels of testosterone directly affect Notch signaling, implicating a feedback role for systemic circulating factors in the regulation of progenitor cells. Between Postnatal Days 3 and 21, as fetal Leydig cells disappear from the testis and are replaced by adult Leydig cells, the perivascular population of interstitial cells active for Notch signaling declines, consistent with distinct regulation of adult Leydig progenitors.

Rat Gnrhr promoter directs species-specific gene expression in the pituitary and testes of transgenic mice

The GnRH receptor (GnRHR) is expressed in several non-pituitary tissues, notably in gonads. However, mechanisms underlying the gonad-specific expression of Gnrhr are not well understood. Here, Gnrhr expression was analysed in the developing testes and pituitaries of rats and transgenic mice bearing the human placental alkaline phosphatase reporter gene (ALPP) under the control of the rat Gnrhr promoter. We showed that the 3.3 kb, but not the pituitary-specific 1.1 kb promoter, directs ALPP expression exclusively to testis Leydig cells from embryonic day 12 onwards. Real-time PCR analysis revealed that promoter activity displayed the same biphasic profile as marker genes in Leydig cells, i.e. abrupt declines after birth followed by progressive rises after a latency phase, in coherence with the differentiation and evolution of foetal and adult Leydig cell lineages. Interestingly, the developmental profile of transgene expression showed high similarity with the endogenous Gnrhr profile in the rat testis, while mouse Gnrhr was only poorly expressed in the mouse testis. In the pituitary, both transgene and Gnrhr were co-expressed at measurable levels with similar ontogenetic profiles, which were markedly distinct from those in the testis. Castration that induced pituitary Gnrhr up-regulation in rats did not affect the mouse Gnrhr. However, it duly up-regulated the transgene. In addition, in LβT2 cells, the rat, but not mouse, Gnrhr promoter was sensitive to GnRH agonist stimulation. Collectively, our data highlight inter-species variations in the expression and regulation of Gnrhr in two different organs and reveal that the rat promoter sequence contains relevant genetic information that dictates rat-specific gene expression in the mouse context.