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Leydig cell morphology. Distinct Leydig cell types in postnatal mice are shown in testes sections stained for histology as described in methods (Panels A, C, E, G). Immunodetection of 3  -HSD confirms representative LC types at different ages (Panels B, D, F, H). A) Typical FLC cluster (dotted circle) at postnatal day 5 (PND5). B) FLC cluster (dotted circle) was 3  -HSD positive at PND5. C) Newly formed ALC (thin arrows) at PND15, which do not form clusters like FLC. D) Newly formed ALC (arrow) at PND15 were 3  -HSD positive. E) Immature ALC (black arrow head) with larger cytoplasm and numerous lipid droplets at PND30. F) 3  -HSD positive immature ALC. G) Mature ALC (white arrow head) with disappearance of cytoplasmic lipid droplets in adult mice, also showing newly formed ALC (arrow). H) 3  HSD positive immature/mature ALC in adult mice.
Source publication
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 an...
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... of LC types. FLC were identified by rounded nuclei with small nucleoli, darkly stained PAS positive cytoplasm, small-sized cytoplasmic lipid droplets and typical occurrence as cell clusters as previously re- ported (23,49), shown in Figure 1A-B. Mesenchymal pre- cursor and progenitor cells of the ALC lineage were not determined, as these cell types were morphologically in- distinguishable, and while progenitor cells express some steroidogenic enzymes they are not likely to directly pro- duce testosterone, unlike the more advanced ALCs (19,23). ...
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... pre- cursor and progenitor cells of the ALC lineage were not determined, as these cell types were morphologically in- distinguishable, and while progenitor cells express some steroidogenic enzymes they are not likely to directly pro- duce testosterone, unlike the more advanced ALCs (19,23). Newly formed ALC are polygonal compared to spin- dle-shaped precursor-progenitor cells, smaller in size com- pared to immature and mature ALC, exhibit rounded nu- clei featuring a distinct heterochromatin rim and prominent nucleolus, contain no (or very few) cytoplasmic lipid droplets and typically found in the central intersti- tium ( Figure 1C-D). Immature ALCs have a larger cyto- plasm with plentiful lipid droplets that appear larger than those in FLCs ( Figure 1E). ...
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... formed ALC are polygonal compared to spin- dle-shaped precursor-progenitor cells, smaller in size com- pared to immature and mature ALC, exhibit rounded nu- clei featuring a distinct heterochromatin rim and prominent nucleolus, contain no (or very few) cytoplasmic lipid droplets and typically found in the central intersti- tium ( Figure 1C-D). Immature ALCs have a larger cyto- plasm with plentiful lipid droplets that appear larger than those in FLCs ( Figure 1E). The final transition of mature ALCs is characterized by the disappearance of cytoplasmic lipid droplets ( Figure 1G). ...
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... ALCs have a larger cyto- plasm with plentiful lipid droplets that appear larger than those in FLCs ( Figure 1E). The final transition of mature ALCs is characterized by the disappearance of cytoplasmic lipid droplets ( Figure 1G). All fetal and adult Leydig cell types expressed 3-HSD, detected by immunohistochem- istry ( Figure 1B, D, F and H), confirming Leydig cell iden- tification. ...
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... final transition of mature ALCs is characterized by the disappearance of cytoplasmic lipid droplets ( Figure 1G). All fetal and adult Leydig cell types expressed 3-HSD, detected by immunohistochem- istry ( Figure 1B, D, F and H), confirming Leydig cell iden- tification. In addition, the prominent expression of mouse AR normally found in postnatal/adult Leydig and peritu- bular cell populations was also observed in TgSCAR tes- tes, as shown in supplementary data ( Fig S1). ...
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... fetal and adult Leydig cell types expressed 3-HSD, detected by immunohistochem- istry ( Figure 1B, D, F and H), confirming Leydig cell iden- tification. In addition, the prominent expression of mouse AR normally found in postnatal/adult Leydig and peritu- bular cell populations was also observed in TgSCAR tes- tes, as shown in supplementary data ( Fig S1). ...
Citations
... By histological examination and stereological analysis, fetal Leydig cell number decline is observed in mouse testes from postnatal Day 7 onward (Hazra et al., 2013;Wen et al., 2014). Ultrastructure examination of human and rat testes identified regression features of poststeroidogenic fetal Leydig cells (Faria et al., 2003;Pelliniemi & Niemi, 1969;Roosen-Runge & Anderson, 1959). ...
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 Gα s ‐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.
... Cell Type Mutation/Activity References ARKO, T-AR −/y Global Ar knock out [3,4] iARKO Global Inducible Ar knock out [6] SCARKO, S-AR −/y Sertoli Ar knock out [7,8] Ar flox(ex1−neo)/Y Sertoli Ar hypomorph [9] TgSCAR Global Ar gain of function [10] SPARKI Global Gr replacement of Ar exon 3 [11] SCGRKO Sertoli Gr knock out [12] Ribotag Leydig, Sertoli Detects translated mRNAs [13] RiboTag-SCARKO Sertoli Detects translated mRNAs in Ar knockout [14] AR-C Sertoli, Global Classical AR activity [15] AR-NC Sertoli, Global Nonclassical AR activity [15] (PTM)-ARKO Smooth Muscle (SM) Ar knock out [16] PM-AR −/y SM Ar knock out [17] L-AR −/Y Leydig Ar knock out [18] G-AR −/y Germ Ar knock out [8] PEARKO Prostate Epithelium Ar knock out [19] Pes-ARKO Prostate Epithelium Ar knock out [20] SM-ARKO SM Ar knock out [21] FSP1-ARKO Fibroblast Ar knock out [22] dARKO Fibroblast + SM Ar knock out [23] ProxE-ARKO Proximal Epididymis Ar knock out [24] CEARKO Principal (Epididymis) ...
... These results suggest that signals downstream of GR in Sertoli cells partially enhance Leydig cell function and reinforce the idea that Sertoli cells communicate with and maximize effectiveness of Leydig cells. Because GR can be detected in post-natal but not adult Sertoli cells, the SCGRKO phenotype may represent developmental defects that lead to lasting effects [12]. ...
... mRNAs encoding members of three Sertoli-Leydig paracrine signaling pathways (Dhh-Ptch1, Pdgfa-Pdgfra, Amh-AMhr2) are induced in TgSCAR mouse testes. Intratesticular testosterone levels per Leydig cell were elevated in TgSCAR mice [12]. ...
Steroid hormones are capable of diffusing through cell membranes to bind with intracellular receptors to regulate numerous physiological processes. Three classes of steroid hormones, namely androgens, estrogens and glucocorticoids, contribute to the development of the reproductive system and the maintenance of fertility. During the past 30 years, mouse models have been produced in which the expression of genes encoding steroid hormone receptors has been enhanced, partially compromised or eliminated. These mouse models have revealed many of the physiological processes regulated by androgens, estrogens and to a more limited extent glucocorticoids in the testis and male accessory organs. In this review, advances provided by mouse models that have facilitated a better understanding of the molecular regulation of testis and reproductive tract processes by steroid hormones are discussed.
... Androgen receptors have been reported in mouse, rat, and human Sertoli cells beginning in the neonatal period and extending to adulthood (Majdic et al., 1995;Rey et al., 2009;Shapiro et al., 2005;You and Sar, 1998) and in fetal and adult Leydig cells (Isomaa et al., 1987;O'Shaughnessy et al., 2019;Shan et al., 1997;Shan et al., 1995;You and Sar, 1998). Genetic ablation of AR signaling in Leydig or Sertoli cells alters Leydig and Sertoli cell function and spermatogenesis in the adult testis (Boukari et al., 2009;De Gendt et al., 2004;Hazra et al., 2013;Kaftanovskaya et al., 2015;Wang et al., 2009). Sertoli cell specific AR ablation alters Leydig cell function (De Gendt et al., 2004;Hazra et al., 2013). ...
... Genetic ablation of AR signaling in Leydig or Sertoli cells alters Leydig and Sertoli cell function and spermatogenesis in the adult testis (Boukari et al., 2009;De Gendt et al., 2004;Hazra et al., 2013;Kaftanovskaya et al., 2015;Wang et al., 2009). Sertoli cell specific AR ablation alters Leydig cell function (De Gendt et al., 2004;Hazra et al., 2013). Androgen receptors were detected in interstitial ovarian stromal cells prenatally and in granulosa cells postnatally. ...
A definition of normal human fetal and early postnatal ovarian development is critical to the ability to accurately diagnose the presence or absence of functional ovarian tissue in clinical specimens. Through assembling an extensive histologic and immunohistochemical developmental ontogeny of human ovarian specimens from 8 weeks of gestation through 16 years of postnatal, we present a comprehensive immunohistochemical mapping of normal protein expression patterns in the early fetal through post-pubertal human ovary and detail a specific expression-based definition of the early stages of follicular development. Normal fetal and postnatal ovarian tissue is defined by the presence of follicular structures and characteristic immunohistochemical staining patterns, including granulosa cells expressing Forkhead Box Protein L2 (FOXL2). However, the current standard array of immunohistochemical markers poorly defines ovarian stromal tissue, and additional work is needed to identify new markers to advance our ability to accurately identify ovarian stromal components in gonadal specimens from patients with disorders of sexual differentiation.
... In cases of cryptorchidism in humans, the maintenance of germ cells at body temperature induces the appearance of mutations, leading to germ cell alterations, testicular germ cell tumor formation, and progressive germ cell depletion [42]. Similarly, disruption of the androgen signaling pathway has been shown to lead to germ cell loss [43,44] or to azoospermia [45][46][47]. ...
In vitro spermatogenesis appears to be a promising approach to restore the fertility of childhood cancer survivors. The rat model has proven to be challenging, since germ cell maturation is arrested in organotypic cultures. Here, we report that, despite a meiotic entry, abnormal synaptonemal complexes were found in spermatocytes, and in vitro matured rat prepubertal testicular tissues displayed an immature phenotype. RNA-sequencing analyses highlighted up to 600 differentially expressed genes between in vitro and in vivo conditions, including genes involved in blood-testis barrier (BTB) formation and steroidogenesis. BTB integrity, the expression of two steroidogenic enzymes, and androgen receptors were indeed altered in vitro. Moreover, most of the top 10 predicted upstream regulators of deregulated genes were involved in inflammatory processes or immune cell recruitment. However, none of the three anti-inflammatory molecules tested in this study promoted meiotic progression. By analysing for the first time in vitro matured rat prepubertal testicular tissues at the molecular level, we uncovered the deregulation of several genes and revealed that defective BTB function, altered steroidogenic pathway, and probably inflammation, could be at the origin of meiotic arrest.
... Perhaps as a compensatory mechanism, there was Leydig cell hypertrophy. These findings indicated that AR actions in Sertoli cells regulate the proliferation and differentiation of Leydig cells [66,67]. ...
... A gain of function mouse model in which AR levels were increased by an AR transgene expressed only in Sertoli cells resulted in lower numbers of Sertoli cells due to precocious maturation that limited the proliferation window for Sertoli cell proliferation due to the premature expression of AR that was detected as early as PND 2. As a result, testes were smaller because the fewer Sertoli cells present could support only smaller populations of germ cells but, the development of the germ cells was accelerated [67]. The same mouse model having the transgenic AR expressed at even higher levels were infertile (or severely subfertile) with a 70% reduction of Sertoli cells and normal ratios of spermatogonia and spermatocytes to Sertoli cells but with elevated ratios of spermatids to Sertoli cells. ...
Testosterone is essential for spermatogenesis and male fertility. In this review, topics related to testosterone control of spermatogenesis are covered including testosterone production and levels in the testis, classical and nonclassical testosterone signaling pathways, cell- and temporal-specific expression of the androgen receptor in the testis and autocrine and paracrine signaling of testis cells in the testis. Also discussed are the contributions of testosterone to testis descent, the blood-testis barrier, control of gonocyte numbers and spermatogonia expansion, completion of meiosis and attachment and release of elongaed spermatids. Testosterone-regulated genes identified in various mouse models of idsrupted Androgen receptor expression are discussed. Finally, examples of synergism and antagonism between androgen and follicle-stimulating hormone signaling pathways are summarized.
... Androgen receptor (AR) has fundamental roles in spermatogenesis and male fertility. Testosterone acts directly on both Sertoli cells and spermatogenic cells to enhance spermatogenesis via AR that requires normal spermatid differentiation and liberation from seminiferous epithelium (Hazra et al. 2013). Moreover, testosterone supports Leydig cell development and functions via AR and motivates growth and function of male reproductive system. ...
The current research was constructed to throw the light on the protective possibility of Chlorella vulgaris (C. vulgaris) and Spirulina platensis (S. platensis) against lead acetate-promoted testicular dysfunction in male rats. Forty rats were classified into four groups: (i) control, (ii) rats received lead acetate (30 mg/kg bw), (iii) rats concomitantly received lead acetate and C. vulgaris (300 mg/kg bw), (vi) rats were simultaneously treated with lead acetate and S. platensis (300 mg/kg bw) via oral gavage for 8 weeks. Lead acetate promoted testicular injury as expressed with fall in reproductive organ weights and gonadosomatic index (GSI). Lead acetate disrupted spermatogenesis as indicated by sperm cell count reduction and increased sperm malformation percentage. Lead acetate-deteriorated steroidogenesis is evoked by minimized serum testosterone along with maximized follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels. Testicular oxidative, inflammatory, and apoptotic cascades are revealed by elevated acid phosphatase (ACP) and sorbitol dehydrogenase (SDH) serum leakage, declined testicular total antioxidative capacity (TAC) with elevated total oxidative capacity (TOC), tumor necrosis factor alpha (TNF-α), caspase-3 levels, lessened androgen receptor (AR) expression, and histopathological lesions against control. Our research highlights that C. vulgaris or S. platensis therapy can modulate lead acetate-promoted testicular dysfunction via their antioxidant activity as expressed by elevated TAC and reduced TOC, immunomodulatory effect as indicated by lessened TNF-α level, and anti-apoptotic potential that was revealed by minimized caspase-3 levels. As well as restoration of testicular histoarchitecture, androgen receptor, steroidogenesis, and spermatogenesis were detected with better impacts to S. platensis comparing with C. vulgaris. Therefore, further clinical trials are needed to test S. platensis and C. vulgaris as a promising candidate in treating male infertility.
... Increased FSH with downregulation of FSHR in experimental rats of the present study may attest SC resistance to FSH. AR expression in SCs has been shown to influence LC number and differentiation [83], by acting synergistically with FSH [84]. Therefore, decreased expression of FSHR and AR might have reduced paracrine factors secreted by SCs that could regulate steroidogenesis in LCs [85] of F 1 rats exposed to Cr(VI). ...
We have reported sub-fertility in F1 progeny rats with gestational exposure to hexavalent chromium [Cr(VI)], which had disrupted Sertoli cell (SC) structure and function, and decreased testosterone (T). However, the underlying mechanism for reduced T remains to be understood. We tested the hypothesis “transient prenatal exposure to Cr(VI) affects testicular steroidogenesis by altering hormone receptors and steroidogenic enzyme proteins in Leydig cells (LCs).” Pregnant Wistar rats were given drinking water containing 50, 100, and 200 mg/L potassium dichromate during gestational days 9 to 14, encompassing fetal differentiation window of the testis from the bipotential gonad. F1 male rats were euthanized on postnatal day 60 (peripubertal rats with adult-type LCs alone). Results showed that prenatal exposure to Cr(VI): (i) increased accumulation of Cr(III) in the testis of F1 rats; (ii) increased serum levels of luteinizing and follicle stimulating hormones (LH and FSH), and 17β estradiol, and decreased prolactin and T; (iii) decreased steroidogenic acute regulatory protein, cytochrome P450 11A1, cytochrome P450 17A1, 3β- and 17β-hydroxysteroid dehydrogenases, cytochrome P450 aromatase and 5α reductase proteins, (iv) decreased specific activities of 3β and 17β hydroxysteroid dehydrogenases; (v) decreased receptors of LH, androgen and estrogen in LCs; (vi) decreased 5α reductase and receptor proteins of FSH, androgen, and estrogen in SCs. The current study concludes that prenatal exposure to Cr(VI) disrupts testicular steroidogenesis in F1 progeny by repressing hormone receptors and key proteins of the steroidogenic pathway in LCs and SCs.
... These results are in keeping with earlier work in mice, which showed that when AR in Sertoli cells was knocked out, AMH downregulation occurred at the same time as it did in the control [31]. Further evidence for AR-independent AMH downregulation has been demonstrated in a gain of function transgenic Sertoli cell-specific AR (TgSCAR) mouse model [37]. ...
The future fertility of prepubertal boys with cancer may be irreversibly compromised by chemotherapy and/or radiotherapy. Successful spermatogenesis has not been achieved following the xenotransplantation of prepubertal human testis tissue, which is likely due to the failure of somatic cell maturation and function. We used a validated xenograft model to identify the factors required for Leydig and Sertoli cell development and function in immature human testis. Importantly, we compared the maturation status of Sertoli cells in xenografts with that of human testis tissues (n = 9, 1 year-adult). Human fetal testis (n = 6; 14–21 gestational weeks) tissue, which models many aspects of prepubertal testicular development, was transplanted subcutaneously into castrated immunocompromised mice for ~12 months. The mice received exogenous human chorionic gonadotropin (hCG; 20IU, 3×/week). In xenografts exposed continuously to hCG, we demonstrate the maintenance of Leydig cell steroidogenesis, the acquisition of features of Sertoli cell maturation (androgen receptor, lumen development), and the formation of the blood–testis barrier (connexin 43), none of which were present prior to the transplantation or in xenografts in which hCG was withdrawn after 7 months. These studies provide evidence that hCG plays a role in Sertoli cell maturation, which is relevant for future investigations, helping them generate functional gametes from immature testis tissue for clinical application.
... Hazra and colleagues have created a mouse model displaying precocious SC and spermatogenic development induced by SC-specific transgenic androgen receptor expression (TgSCAR). Here they reveal that TgSCAR regulates the development, function, and absolute number of LCs without reflex changes in serum LH, indicating a local intratesticular regulatory mechanism [21]. ...
Introduction
A reduction of testicular volume (TV) represents an important clinical sign, which may hide sperm abnormalities and predispose to hypogonadism.
Aim
The primary purpose of this study was to evaluate the serum levels of total testosterone after treatment with urofollitropin in selected patients with male infertility and idiopathic mild reduction of testicular volume.
Methods
In this 1-year-long prospective design, patients with abnormal sperm parameters, mild reduction in TV (8–12 mL) and normal gonadotropin, and total testosterone (TT) serum levels were recruited in this study. Patients treated for 4 months with urofollitropin were included in group A, those treated with intracytoplasmatic sperm injection due to a female-factor infertility were included in group B. Hormone values, sperm parameters, and TV were detected at baseline (T0), after 4 (T1) and 12 months (T2) in group A and at T0 and T2 in group B.
Results
Group A (n = 80) showed increased follicle-stimulating hormone (FSH) at T1 and sperm morphology at T1 and T2 compared to T0 (all p < 0.05). Group B (n = 50) had lower TT and higher FSH levels at T2 compared to T0 (all p < 0.05). At T2, TT, VT, total sperm count, progressive motility, total motility, and sperm morphology were higher in group A compared to group B (all p < 0.05).
Conclusion
Reduced TV may predispose to infertility and hypogonadism. FSH treatment may improve Sertoli and Leydig cell function and prevent the development of hypogonadism.
... Levels of LH are normal in adult animals following neonatal SC ablation suggesting that the LC hyperactivity is due either to altered development of the cells around puberty or to increased activity in the mature cells because of SC loss. A number of factors have been postulated to mediate SC-LC communication [17][18][19][20][21][22][23] but definitive studies remain to be carried out. ...
Objectives:
The current study aims to identify markers that would reflect the number of Leydig cells present in the testis, to help determine whether labour-intensive methods such as stereology are necessary. We used our well-characterised Sertoli cell ablation model in which we have empirically established the size of the Leydig cell population, to try to identify transcriptional biomarkers indicative of population size.
Results:
Following characterisation of the Leydig cell population after Sertoli cell ablation in neonatal life or adulthood, we identified Hsd3b1 transcript levels as a potential indicator of Leydig cell number with utility for informing decision-making on whether to engage in time-consuming stereological cell counting analysis.
