Peritubular Cells Research Articles

Bisphenol F blocks Leydig cell maturation and steroidogenesis in pubertal male rats through suppressing androgen receptor signaling and activating G-protein coupled estrogen receptor 1 (GPER1) signaling

Bisphenol F (BPF) is a new analog of bisphenol A (BPA). BPA has deleterious effects on the male reproductive system, but the effect of BPF has not been studied in detail. In this study we focus on the effect of BPF on Leydig cell maturation. Male Sprague-Dawley rats were gavaged with 0, 1, 10, or 100 mg/kg BPF from postnatal days 35–56. BPF significantly reduced serum testosterone levels and sperm count in cauda epididymis at dose ≥1 mg/kg. It significantly down-regulated the expression of steroidogenic enzymes, while increasing FSHR and SOX9 levels at 10 and 100 mg/kg. Further studies showed that BPF reduced NR3C4 expression in Leydig and Sertoli cells without affecting its levels in peritubular myoid cells. BPF markedly increased GPER1 in Leydig cells at 100 mg/kg, and it significantly reduced SIRT1 and PGC1α levels in the testes at 100 mg/kg. BPF significantly inhibited testosterone production by immature Leydig cells at 50 μM after 24 h of treatment, which was completely reversed by NR3C4 agonist 7α-methyl-19-nortestosterone and partially reversed by GPER1 antagonist G15 not by ESR1 antagonist ICI 182,780. In conclusion, BPF negatively affects Leydig cell maturation in pubertal male rats through NR3C4 antagonism and GPER1 agonism.

Functions of somatic cells for spermatogenesis in stallions.

Spermatogenesis and testis development are highly structured physiological processes responsible for post-pubertal fertility in stallions. Spermatogenesis comprises spermatocytogenesis, meiosis, and spermiogenesis. Although germ cell degeneration is a continuous process, its effects are more pronounced during spermatocytogenesis and meiosis. The productivity and efficiency of spermatogenesis are directly linked to pubertal development, degenerated germ cell populations, aging, nutrition, and season of the year in stallions. The multiplex interplay of germ cells with somatic cells, endocrine and paracrine factors, growth factors, and signaling molecules contributes to the regulation of spermatogenesis. A cell-to-cell communication within the testes of these factors is a fundamental requirement of normal spermatogenesis. A noteworthy development has been made recently on discovering the effects of different somatic cells including Leydig, Sertoli, and peritubular myoid cells on manipulation the fate of spermatogonial stem cells. In this review, we discuss the self-renewal, differentiation, and apoptotic roles of somatic cells and the relationship between somatic and germ cells during normal spermatogenesis. We also summarize the roles of different growth factors, their paracrine/endocrine/autocrine pathways, and the different cytokines associated with spermatogenesis. Furthermore, we highlight important matters for further studies on the regulation of spermatogenesis. This review presents an insight into the mechanism of spermatogenesis, and helpful in developing better understanding of the functions of somatic cells, particularly in stallions and would offer new research goals for developing curative techniques to address infertility/subfertility in stallions.

O-264 Testosterone potentiates the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the maturation of spermatogonial cells in vitro from prepubertal mice

Abstract Study question Can granulocyte-macrophage colony-stimulating factor (GM-CSF) and testosterone induce spermatogonial cells to develop spermatogenesis in vitro using methylcellulose culture system (MCS)? Summary answer GM-CSF and testosterone induced the development of premeiotic and meiotic cells in vitro. Testosterone also induced and potentiate the development of post-meiotic cells by GM-CSF. What is known already Spermatogenesis is a complicated process of sperm generation. During this process spermatogonial cells proliferate and differentiate to meiotic, postmeiotic stages that continue in spermiogenesis to generate mature sperm. This process is under regulation of autocrine and paracrine factors provided by developed germ cells and somatic cells such as Sertoli, peritubular and Leydig cells. In addition, endocrine factors such as FSH, LH and testosterone are crucial for complete spermatogenesis. Different in vitro culture systems were used to induce the development of complete spermatogenesis in vitro, however this was not yet achieved. Study design, size, duration Sexually immature mice (7-day-old) were used to isolated cells from the seminiferous tubules. These isolated ells were cultured in methylcellulose culture system (MCS) as a 3-dimensinal in vitro system. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and testosterone were added in the beginning of the cultures and after two weeks. The cultures were determined after 4 weeks. Participants/materials, setting, methods Cells were enzymatically isolated from the seminiferous tubules and cultured (2x105/well/0.5ml) in MCS that contained StemPro-34 medium, KSR, rEGF, rGDNF, rLIF, and r-bFGF. The cultures were grown in the presence/absence of GM-CSF (0.1 or 1ng/ml) and/or testosterone (10-7M) and incubated for four weeks in a CO2 incubator at 37°C. The developed cells and colonies/spheroids were examined microscopically, and quantified the developing cells of the different stages of spermatogenesis by immunofluorescence staining (IF) and/or qPCR analyses Main results and the role of chance Our results show the presence of GM-CSF and its receptor (GM-CSFR) in different testicular cells. We demonstrate the development of colonies/spheroids which contain premeiotic, meiotic and post-meiotic cells in MCS, as examined by IF and qPCR analyses. The percentage of pre-meiotic and miotic stage in the cultures with GM-CSF were significantly higher than those in the control group (p < 0.001), and the percentage of cells of the post-meiotic stage were significantly higher in the cultures with GM-CSF and testosterone together compared to each one separately (p < 0.001). In conclusion, testicular cells express GM-CSF/GM-CSFR, and GM-CSF affects only the development of premeiotic and meiotic cells in vitro. In addition, testosterone not only increased the development of cells from the premeiotic, meiotic and post-meiotic stages, but it also potentiated the effect of GM-CSF on the development of post-meiotic cells in vitro. Limitations, reasons for caution no limitation Wider implications of the findings These findings providing evidence that GM-CSF and testosterone could be used to improve the maturation of spermatogonial cells in vitro and that they might play a role in the maturation process of spermatogonial cells in vivo. Trial registration number not applicable

P-468 Organotypic culture of testicular tissue from infant boys with cryptorchidism

Abstract Study question Can organotypic culture support the survival and maturation of germ cells and niche-related cells within testicular tissue from infant boys with cryptorchidism? Summary answer The testicular structure and the number of germ cells were maintained during organotypic culture, whereas Sertoli cells and peritubular myoid cells (PTMCs) matured. What is known already Testicular tissue cryopreservation (TTC) is a strategy to safeguard the fertility of prepubertal boys who face a risk of infertility. Organotypic culture of immature testicular tissue from mice achieved production of spermatozoa. Similarly, the culture of human fetal gonads resulted in the generation of competent spermatids. However , in vitro spermatogenesis by organotypic culture of human prepubertal testicular tissue has not been achieved. It is also unknown whether germ cells as well as its niche-related cells, in testicular tissue from infant boys with cryptorchidism, can maintain and mature under in vitro conditions. Study design, size, duration Testicular tissue was cryopreserved from four infant boys with bilateral cryptorchidism undergoing orchidopexy (age range: 0.5-1.4 years), as part of a fertility preservation program. Culture media with and without retinoic acid were tested. Testicular fragments were harvested at 30 days and 60 days after culture and evaluated by histological assessment of tissue structure, germ cell development, and immunohistochemical staining for germ cell and somatic cell markers. Participants/materials, setting, methods Cryopreserved-thawed testicular tissue was cut into fragments (1-2 mm3) and placed on top of agarose gel stands and cultured at 34oC with 5% CO2 in Minimum Essential Medium-alpha supplemented with knockout serum replacement, human umbilical cord plasma, Activin A, hormones, growth factors, with or without retinoic acid. Immunohistochemical analyses were performed using germ cell markers (MAGE-A, GAGE, and VASA), Sertoli cell maturation markers (AMH, AR, SOX9), PTMC marker (alpha-SMA). Main results and the role of chance Following the 60-day culture, the lumen of the seminiferous tubules had developed. The number of germ cells per tubule remained stable during this period. However, no further germ cell maturation was observed. Germ cells showed different phenotypes of MAGEA, GAGE, and VASA expression with no significant difference in number. The number of SOX9-positive Sertoli cells was significantly increased from 30 days to 60 days of culture (p <0.0001). No difference in AMH expression was observed, while AR expression in Sertoli cells was induced during culture. Alpha-SMA expression was detected in the PTMCs surrounding the seminiferous tubules. The two different culture conditions, with and without retinoic acid in the culture media, did not affect cell survival or maturation. Limitations, reasons for caution The small number of testicular biopsies available is a limitation. Wider implications of the findings Our organotypic culture conditions support the long-term survival of germ cells in testicular tissue from infant boys with cryptorchidism. Thus, further studies are needed to induce the maturation of germ cells under similar experimental conditions. Trial registration number not applicable

Pleiotropic actions of melatonin in testicular peritubular myoid cells of immature Syrian hamsters

BackgroundPeritubular myoid cells are emerging as key regulators of testicular function in adulthood. However, little is known about the role of testicular peritubular myoid cells (TPMCs) in the development of the male gonad. We found that, compared to testes of young adult hamsters, gonads of 21 day-old animals show increased melatonin concentration, seminiferous tubular wall thickening and a heterogeneous packaging of its collagen fibers thus raising the question whether melatonin may be involved in the regulation of TPMCs. MethodsWe established primary cultures of TPMCs from immature hamsters (ihaTPMCs), which we found express melatonergic receptors. ResultsExogeneous melatonin decreased the levels of inflammatory markers (NLRP3 inflammasome, IL1β) but increased the expression of cyclooxygenase 2 (COX2, key enzyme mediating prostaglandin synthesis) and of the glial cell line-derived neurotrophic factor (GDNF) in ihaTPMCs. Melatonin also stimulated ihaTPMCs proliferation and the expression of extracellular matrix proteins such as collagen type I and IV. Furthermore, collagen gel contraction assays revealed an enhanced ability of ihaTPMCs to contract in the presence of melatonin. ConclusionMelatonin regulates immune and inflammatory functions as well as contractile phenotype of the peritubular wall in the hamster testis. General significanceIf transferable to the in vivo situation, melatonin-dependent induction of ihaTPMCs to produce factors known to exert paracrine effects in other somatic cell populations of the gonad suggests that the influence of melatonin may go beyond the peritubular wall and indicates its contribution to testicular development and the establishment of a normal and sustainable spermatogenesis.

Deciphering the origins and fates of steroidogenic lineages in the mouse testis.

Leydig cells (LCs) are the major androgen-producing cells in the testis. They arise from steroidogenic progenitors (SPs), whose origins, maintenance, and differentiation dynamics remain largely unknown. Single-cell transcriptomics reveal that the mouse steroidogenic lineage is specified as early as embryonic day 12.5 (E12.5) and has a dual mesonephric and coelomic origin. SPs specifically express the Wnt5a gene and evolve rapidly. At E12.5 and E13.5, they give rise first to an intermediate population of pre-LCs, and finally to fetal LCs. At E16.5, SPs possess the characteristics of the dormant progenitors at the origin of adult LCs and are also transcriptionally closely related to peritubular myoid cells (PMCs). In agreement with our in silico analysis, invivo lineage tracing indicates that Wnt5a-expressing cells are bona fide progenitors of PMCs as well as fetal and adult LCs, contributing to most of the LCs present in the fetal and adult testis.

Establishment of a pure culture of immature Sertoli cells by PDGFRA staining and cell sorting.

Sertoli cells are key somatic cells in the testis that form seminiferous tubules and support spermatogenesis. The isolation of pure Sertoli cells is important for their study. However, it is a difficult effort because of the close association of Sertoli cells with peritubular myoid cells surrounding seminiferous tubules. Here, we propose a novel approach to the establishment of a pure Sertoli cell culture from immature mouse testes. It is based on the staining of testicular cells for platelet-derived growth factor receptor alpha (PDGFRA), followed by fluorescence-activated cell sorting and culturing of a PDGFRA-negative cell population. Cells positive for a Sertoli cell marker WT1 accounted for more than 96% of cells in cultures from 6 to 12 days postpartum (dpp) mice. The numbers of peritubular myoid cells identified by ACTA2 staining did not exceed 4%. Cells in the cultures were also positive for Sertoli cell proteins SOX9 and DMRT1. Amh and Hsd17b3 expression decreased and Ar and Gata1 expression increased in 12 dpp cultures compared to 6 dpp cultures, which suggests that cultured Sertoli cells at least partially retained their differentiation status. This method can be employed in various applications including the analysis of differential gene expression and functional studies.

Single-cell analysis of human testis aging and correlation with elevated body mass index.

Aging men display reduced reproductive health; however, testis aging is poorly understood at the molecular and genomic levels. Here, we utilized single-cell RNA-seq to profile over 44,000 cells from both young and older men and examined age-related changes in germline development and in the testicular somatic cells. Age-related changes in spermatogonial stem cells appeared modest, whereas age-related dysregulation of spermatogenesis and somatic cells ranged from moderate to severe. Altered pathways included signaling and inflammation in multiple cell types, metabolic signaling in Sertoli cells, hedgehog signaling and testosterone production in Leydig cells, cell death and growth in testicular peritubular cells, and possible developmental regression in both Leydig and peritubular cells. Remarkably, the extent of dysregulation correlated with body mass index in older but not in younger men. Collectively, we reveal candidate molecular mechanisms underlying the complex testicular changes conferred by aging and their possible exacerbation by concurrent chronic conditions such as obesity.

Blood Count Under the Effect of Erythropoietin (EPO) in Patients with Chronic Hemodialysis

One of the most common associated diseases in dialysis patients is anemia. The number of erythrocytes, hemoglobin and hematocrit is reduced in patients that suffer from anemia. These three measurement parameters represent the most important "red part" of the blood count. Due to renal insufficiency, patients on hemodialysis do not have enough erythropoietin, which healthy kidneys excreted, and is necessary for hematopoiesis. Erythropoietin (EPO) is the main regulator of the daily production of red blood cells. EPO that is execrated by peritubular capillary membrane cells in the kidneys circulates within plasma in order to interact with target cells in the bone marrow to maintain or stimulate erythropoiesis. The main purpose of the action of EPO is the formation of erythroid colonies. During observation of differences in the ordination of EPO in each group between men and women, there is no statistical significance evident in the incidence of anemia before and after therapy, as in p=0.70. The target concentration of hemoglobin, which must be constantly maintained in people with chronic renal anemia, is 110-120 g/l, while the target value of the number of red blood cells is 4-5x10 12/l and the hematocrit value is 0.35-4.5 l/l, with a slight outflow depending on the gender of the patient. The final values of the participants in our study treated with epoetin alpha show higher values of hemoglobin, hematocrit and erythrocytes, while the average values recorded in participants treated with darbepoetin decreased.

Transmigration of macrophages through primary adult rat Sertoli cells

ABSTRACT The blood testis barrier (BTB) is often studied with isolated immature Sertoli cells (SCs), transepithelial resistance (TER) measurements and FITC dextran diffusion assays. Recently, it was found that even in the absence of SCs, only few immune cells enter the seminiferous tubules. Thus, in this study, we evaluated the testicular immunological barrier (TIB) in vitro by transmigration of macrophages through SCs with and without peritubular cells (PCs) and with or without matrigel (MG). Primary PCs were isolated from adult rat testis and kept in mono- or co-cultures with the conditionally reprogrammed primary adult Sertoli cell line (PASC1) from rat that has been recently generated by our group. Rat monocytes isolated from fresh blood were differentiated into M0 macrophages, and after polarization to M1 or M2 macrophages characterized by gene expression of CXCL11 and TNF-α for M1, or CCL17 and CCL22 for M2. Transmigration of LeukoTracker-labeled M0, M1, and M2 macrophages through mono- and co-cultures of PCs/SCs with and without MG demonstrated that SCs are the main constituent of the TIB in vitro with only a negligible contribution of PCs or MG. Moreover, M2 macrophages showed less migration activity compared to M0 or M1. Treatment of SCs with testosterone (T) showed positive effects on the barrier in contrast to negative effects by interleukin-6 (IL-6) or tumor necrosis factor-α (TNF-α). The new transmigration model is suitable to evaluate transmigration of macrophages through a barrier consisting of testicular cells and can be applied to study the integrity of testicular barriers with respect to immunological aspects.

Testis on a chip—a microfluidic three-dimensional culture system for the development of spermatogenesis in-vitro

This research presents a novel testis-on-a-chip (ToC) platform. Testicular cells are enzymatically isolated from the seminiferous tubules of sexually immature mice, seeded in a methylcellulose gel and cultured in a microfluidic chip. The unique design sandwiches the soft methylcellulose between stiffer agar support gels. The cells develop into spheroids continuing to proliferate and differentiate. After seven weeks of culture the cells have over 95% viability. Confocal microscopy of the developed spheroids reveals a structure containing the various stages of spermatogenesis up to and including meiosis II: premeiotic, meiotic and post-meiotic germ cells. The spheroid structure also contains the supporting Sertoli and peritubular cells. The responsiveness of the system to the addition of testosterone and retinoic acid to the culture medium during the experiment was also investigated. As a benchmark, the ToC is compared to a conventional three-dimensional methylcellulose cell culture system in a well plate. Analysis via fluorescence-activated cell sorting shows more haploid cells in the chip as compared to the plates. Immunofluorescence staining after seven weeks of culture shows more differentiated cells in the chip as compared to the well plate. This demonstrates the feasibility of our platform as well as its advantages. This research opens new horizons for the study and realization of spermatogenesis in-vitro. It can also enable the implementation of microfluidic technologies in future therapeutic strategies for pre-pubertal male fertility preservation and adults with maturation arrest. Lastly, it can serve as a platform for drug and toxin testing.

A Role for Exchange of Extracellular Vesicles in Porcine Spermatogonial Co-Culture.

Spermatogonial stem cells (SSCs) provide the basis for lifelong male fertility through self-renewal and differentiation. Prepubertal male cancer patients may be rendered infertile by gonadotoxic chemotherapy and, unlike sexually mature men, cannot store sperm. Alternatively, testicular biopsies taken prior to treatment may be used to restore fertility in adulthood. Testicular SSC populations are limited, and in vitro culture systems are required to increase numbers of SSCs for treatment, demanding culture systems for SSC propagation. Using the pig as a non-rodent model, we developed culture systems to expand spermatogonia from immature testis tissue, comparing different feeders (Sertoli cells, peritubular myoid cells (PMCs) and pig fetal fibroblasts (PFFs)). Spermatogonia co-cultured with Sertoli cells, PMCs and PFFs had comparable rates of proliferation and apoptosis. To elucidate the mechanism behind the beneficial nature of feeder layers, we investigated the role of extracellular vesicles in crosstalk between spermatogonia and feeder cells. Sertoli cell-released exosomes are incorporated by spermatogonia, and inhibition of exosomal release reduces spermatogonial proliferation. Together, these results show that PMCs, PFFs and Sertoli cells promote spermatogonial proliferation in co-culture, with exosomal exchange representing one possible mechanism. Further characterization of exosomal cargo may ultimately allow the development of feeder-free culture systems for clinical use.

Single-cell RNA-seq analysis of testicular somatic cell development in pigs

Spermatogenesis is the process by which diploid male germ cells propagate and differentiate into haploid flagellated spermatozoa. This highly complicated process is dependent on testicular somatic cells maturation. While the role of these somatic cells in spermatogenesis is relatively well established, knowledge about their development and maturation, particularly at the molecular level, is limited. In this study, we profiled the testicular single-cell transcriptomes of Guanzhong black pigs at the age of 7, 30, 60, 90, and 150 days. Five types of Sertoli cells, five types of Leydig cells, and four types of peritubular myoid cells were identified. Histological analysis revealed the changes in proliferation levels and marker gene expressions, and the prion-like protein gene (PRND) was identified as a novel marker for Sertoli cells. Additionally, integrated analyses of porcine and human datasets revealed similarities between human and pig testicular somatic cells. Overall, the data obtained in this study contribute to the understanding of testicular development in pigs as a model species.

Characteristics of alpha smooth muscle actin-positive peritubular cells in prepubertal bovine testes

Characteristics of peritubular myoid cells (PMCs) in sexually immature cattle remain largely unknown. Here, we report the character and behavior of peritubular cells expressing alpha smooth muscle actin (αSMA), a marker of PMCs in adult testes, in prepubertal testes procured from 5-months-old bulls. Elastin distribution around αSMA + PMCs was dim and discontinuous in prepubertal testes, but strong and continuous in adult testes. Fibroblast-specific protein 1 (FSP1) was rarely expressed in αSMA + PMCs of prepubertal testes, while in adult testes, majority of αSMA + PMCs were FSP1+. Moreover, αSMA + PMCs in prepubertal testes proliferated more actively than those in adult testes. In vitro culture of isolated seminiferous tubules from prepubertal testes revealed that αSMA + PMCs migrate from peritubular to interstitial area. Hence, in prepubertal bovine testes, (1) PMCs rarely portray fibroblast-like properties, (2) PMCs exhibit heterogeneity in FSP1 expression, (3) PMCs proliferate more actively than those in adult testes, and (4) PMCs have a potential to migrate to the interstitium. Our observations help to understand the maturation of PMCs and their involvement in bovine testicular function.

Endogenous Effects of Leptin on Leydig Stem Cell Differentiation through Desert Hedgehog Signaling Pathway

ABSTRACT Introduction Although testosterone deficiency (TD) may be present in 1 out of 5 men 40 years or older, the factors responsible for TD remain largely unknown. Leydig stem cells (LSCs) differentiate into adult Leydig cells (ALC) and produce testosterone in the testes under the pulsatile control of luteinizing hormone (LH) from the pituitary gland. However, our recent studies have suggested that the testicular microenvironment (TME), which is comprised of Sertoli and peritubular myoid cells (PMC), plays an instrumental role in LSC differentiation and testosterone production under the regulation of the desert hedgehog signaling pathway (DHH). In the present study, we hypothesized that testicular TME releases paracrine factors that modulate LSC differentiation. Objective To further examine the TME its see if its release of paracrine factors modulates LSC differentiation. Methods A total of 13 men with testicular failure underwent testis biopsies for sperm retrieval. Using an IRB approved protocol, about 10mg of testicular tissue from each of these men were processed for Leydig stem cell isolation, culture and characterized. Cytokine antibody array was performed to identify the paracrine factors released by TME. The cells were treated with hedgehog signaling agonist and antagonist in the presence or absence of identified paracrine factors from the conditioned media. Two independent biopsies were treated with siRNA against LEPR (25pmol) for 48hrs. This was followed by evaluating the extent of knockdown on LEPR and B3HSD. Further, to evaluate the functional aspects (cell proliferation) modulated by Leptin-DHH signalling by first sorting the Sertoli cells, Peritubular myoid cells, Leydig stem cells and Adult Leydig cells using surface markers. These cells were then treated with Hedgehog agonist (SAG) and antagonist (Vismodigib), in the presence or absence of increasing doses of Leptin (0, 1, 10 ng/ml respectively) 48 hours post treatment, these cells were used for checking the impacts of Leptin-DHH treatments on cell proliferation by MTT assay. GraphPad Prism (GraphPad Software) was used for statistical analysis. All data were presented as the means ± SEM. The statistical significance between two groups was estimated by unpaired two-tailed t-test. Results We successfully isolated and cultured up to 5 × 106million LSC's / biopsy from all 13 human testis biopsies. These cells were characterized for the presence or absence of different cell types in testis such as adult Leydig cells, Leydig stem cells, Sertoli cells, peritublar myoid cells and germ cells. Results showed that, of the cells cultured, up to 70% of the cells were Leydig stem cells and 10% of them were Sertoli-cell in origin on day 14. IF and qPCR data showed as the majority of cell population was undifferentiated (PDGFR-α). Upon stimulation by LH, the expression of 3βHSD (mature Leydig cells) was increased and that of PDGFR-α was decreased. Results from siRNA against LEPR has shown the down-regulated LEPR and B3HSD expression levels. The results from cell proliferation showed that there is minimal to no impact of leptin treatment on upon DHH agonist (SAG) or antagonist (Vismodigib) treatment in either cell types therefore suggesting that Leptin is not involving in the proliferation mechanism. Furthermore, the results demonstrated that TME secretes leptin which induces LSC differentiation and increases T production. Leptin's effects on LSC differentiation and T production, however, are inversely concentration-dependent: positive at low doses and negative at higher doses. Mechanistically, leptin acts on LSCs upstream of DHH; leptin-DHH regulation functions unidirectionally insofar as DHH gain or loss of function has no effects on leptin levels. Conclusions Taken together, results from this study demonstrate the instrumental role of leptin as a paracrine factor secreted by TME on human LSC function and differentiation. Moreover, this study established the molecular events (DHH signaling) that underlie the leptin-modulated effects on LSCs. Further studies are ongoing to validate the implications of these Leptin in terms of its role in LSCs function, differentiation and survival Disclosure No

Leptin Effects on Leydig Stem Cell Differentiation are Specific to Patients BMI

ABSTRACT Introduction Adult Leydig cells in the testes are essential for testosterone production. The growth and differentiation of Leydig cells could be affected by paracrine factors released by testicular microenvironment (TME) (Sertoli and peritubular myoid cells). In our recent study, we demonstrated that Leptin, a paracrine factor secreted by TME, is critical for Leydig stem cells (LSCs) differentiation and subsequent testosterone production via its interaction with Leptin receptor on LSCs, followed by downstream regulation of desert hedgehog (DHH) signaling. Although it is well-known that obesity adversely affects male fertility and testosterone production, the endogenous effects of Leptin on Leydig stem cell differentiation and transcription factors genes (TFs) binding to leptin receptor (LEPR) and the mechanism that are specific to patient's BMI (normal, obese and lean men) are understudied. Additionally, the underlying mechanism behind the differential effects of Leptin (if any) is unknown. Therefore, In the present study, we evaluated the impacts and mechanism of Leptin as a paracrine factor on LSC differentiation in patients with different BMI's. Objective To uncover the mechanism of Leptin as a paracrine factor on LSC differentiation in patients with different BMI's. Methods A total of 13 men with testicular failure that were subcategorized as obese (BMI >35), normal (BMI 25-30), and lean (BMI <25) underwent testis biopsies. Using an IRB approved protocol, about 10mg of testicular tissue from each of these men were processed for LSC isolation, culture and characterization. After characterization using Flow cytometry and Immunostaining, cells were treated for dose dependent (0, 1, 10 ng/mL respectively) and time dependent (24, 48, 72, and 96 hours) effects of Leptin in the presence or absence of testicular microenvironment. To validate the effects of leptin on DHH, - LSCs were treated with Leptin in the presence of DHH agonists (SAG) and antagonists (Vismodigib). For the functional studies, UCSC genome browser was used to identify the TFs binding the LEPR promoter. The list of TFs was subjected for Enrichr for enrichment analysis. To evaluate the effects of shortlisted TFs on leptin-LEPR, qPCR and western blotting were performed. Furthermore, to validate the specificity of interaction between Leptin-Leptin receptor on LSC differentiation, sorted LSCs were exposed to siRNA against LEPR in the presence of increased dose of Leptin, followed by evaluating the percentage shift in LSCs to ALCs. After treatment, qPCR, western blot and flow cytometry analysis were performed to evaluate the effects of treatment with respective to the BMI levels. RNA sequencing analysis was performed on cells extracted from men with different BMI's, treated with 0, 0.25, 1 and 10ng/ml Leptin to study the signatures specific effects of leptin with respect to patients BMI. For statistical analysis GraphPad Prism (GraphPad Software) were used. All data were presented as the means ± SEM. The statistical significance between two groups were estimated by unpaired two-tailed t test. Results Results showed that a strong relationship exists between BMI and leptin levels. Immunostaining, flow cytometry and RNA sequencing results showed that in the men with normal BMI, in the presence of low doses of leptin, there was a shift of the testicular cell population towards adult Leydig cells (increased population of cells staining positive for 3BHSD, LHR) and increased DHH signaling (GLI and SMO). On the contrary, at higher concentrations, the positive effects of leptin on cells from men with normal BMI were neutralized. Furthermore, in cells from lean patients, leptin treatment had a consistent, linear and positive impact on LSC differentiation markers and DHH signaling. Moreover, in cells from obese patients, leptin had a consistent, linear and negative impact on LSC differentiation and markers of DHH signaling. Enrichment analysis identified 50 TFs, of which SOX2, SOX8 and SOX9 were enriched for abnormal pituitary function and therefore for hypothalamic pituitary gonadal axis. The role of these TFs on LSC differentiation remained under-explored. RNA sequencing data from cells exposed to different concentrations of Leptin showed an inducing effect of Leptin on SOX2, SOX8 Sox9 respectively. Consequently, with increasing leptin doses SOX2, SOX8 Sox9 protein and expression has been upregulated. Mechanistically, Leptin-LEPR-TFs induced DHH signaling regulates LSCs differentiation in a different manner which is specific to patient's BMI. Conclusions Our results demonstrate the influence, BMI has on Leptin induced LSC differentiation. These findings suggest the future potential use of Leptin as a personalized therapy for inducing LSC differentiation and overcoming low Testosterone levels. Further studies are necessary to identify potential therapeutic effects of leptin treatment in improving fertility in the setting of leptin resistance and obesity. Financial Support: Supported by the American Urological Association Research Scholar Award to H.A. J.M.H. is supported by NIH grants 1R01 HL137355, 1R01 HL107110, 1R01 HL134558, 5R01 CA136387, 5UM1 HL113460 and Soffer Family Foundation. Disclosure No

Development of the human fetal testis: Morphology and expression of cellular differentiation markers

A comprehensive immunohistochemical ontogeny of the developing human fetal testis has remained incomplete in the literature to date. We collected human fetal testes from 8 to 21 weeks of fetal age, as well as postnatal human testes at minipuberty, pre-pubertal, and pubertal stages. Immunohistochemistry was performed with a comprehensive panel of antigens targeting gonadocytes, Sertoli cells, fetal Leydig cells, peritubular myoid cells, and other hormonal and developmental targets.Testicular cords, precursor structures to seminiferous tubules, developed from 8 to 14 weeks of fetal age, separating the testis into the interstitial and intracordal compartments. Fetal gonadocytes were localized within the testicular cords and evaluated for Testis-Specific Protein Y, Octamer-binding transcription factor 4, Sal-like protein 4, and placental alkaline phosphatase expression. Fetal Sertoli cells were also localized in the testicular cords and evaluated for SRY-box Transcription Factor 9, inhibin, and anti-Mullerian hormone expression. Fetal Leydig cells were present in the interstitium and stained for cytochrome p450c17 and calretinin, while interstitial peritubular myoid cells were examined using smooth muscle α-actin staining. Androgen receptor expression was localized close to the testicular medulla at 8 weeks and then around the testicular cords in the interstitium as they matured in structure.Postnatal staining showed that Testis-Specific Protein Y remained positive of male gonadocytes throughout adulthood. Anti-Mullerian hormone, SRY-box Transcription Factor 9, and Steroidogenic factor 1 are expressed by the postnatal Sertoli cells at all ages examined. Leydig cell markers cytochrome p450c17 and calretinin are expressed during mini-puberty and puberty, but not expressed during the pre-pubertal period. Smooth muscle α-actin and androgen receptor were not expressed during mini-puberty or pre-puberty, but again expressed during the pubertal period.The ontogenic map of the human fetal and postnatal testicular structure and expression patterns described here will serve as a reference for future investigations into normal and abnormal testicular development.

Localization of Xenobiotic Transporters Expressed at the Human Blood-Testis Barrier.

The blood-testis barrier (BTB) is formed by basal tight junctions between adjacent Sertoli cells (SCs) of the seminiferous tubules and acts as a physical barrier to protect developing germ cells in the adluminal compartment from reproductive toxicants. Xenobiotics, including antivirals, male contraceptives, and cancer chemotherapeutics, are known to cross the BTB, although the mechanisms that permit barrier circumvention are generally unknown. This study used immunohistological staining of human testicular tissue to determine the site of expression for xenobiotic transporters that facilitate transport across the BTB. Organic anion transporter (OAT) 1, OAT2, and organic cation transporter, novel (OCTN) 1 primarily localized to the basal membrane of SCs, whereas OCTN2, multidrug resistance protein (MRP) 3, MRP6, and MRP7 localized to SC basal membranes and peritubular myoid cells (PMCs) surrounding the seminiferous tubules. Concentrative nucleoside transporter (CNT) 2 localized to Leydig cells (LCs), PMCs, and SC apicolateral membranes. Organic cation transporter (OCT) 1, OCT2, and OCT3 mostly localized to PMCs and LCs, although there was minor staining in developing germ cells for OCT3. Organic anion transporting polypeptide (OATP) 1A2, OATP1B1, OATP1B3, OATP2A1, OATP2B1, and OATP3A1-v2 localized to SC basal membranes with diffuse staining for some transporters. Notably, OATP1C1 and OATP4A1 primarily localized to LCs. Positive staining for multidrug and toxin extrusion protein (MATE) 1 was only observed throughout the adluminal compartment. Definitive staining for CNT1, OAT3, MATE2, and OATP6A1 was not observed. The location of these transporters is consistent with their involvement in the movement of xenobiotics across the BTB. Altogether, the localization of these transporters provides insight into the mechanisms of drug disposition across the BTB and will be useful in developing tools to overcome the pharmacokinetic and pharmacodynamic difficulties presented by the BTB. SIGNIFICANCE STATEMENT: Although the total mRNA and protein expression of drug transporters in the testes has been explored, the localization of many transporters at the blood-testis barrier (BTB) has not been determined. This study applied immunohistological staining in human testicular tissues to identify the cellular localization of drug transporters in the testes. The observations made in this study have implications for the development of drugs that can effectively use transporters expressed at the basal membranes of Sertoli cells to bypass the BTB.

Ontogeny of mouse Sertoli, Leydig and peritubular myoid cells from embryonic day 10 to adulthood

We present a comprehensive description of the differentiating somatic cell types (Sertoli, Leydig, and peritubular myoid cells) of the mouse testis from embryonic day 10.5 (E10.5) to adulthood, postnatal day 60 (P60). Immunohistochemistry was used to analyze expression of: Sox9 (a Sertoli cell marker), 3βHSD-1 (a fetal Leydig cell marker), 3βHSD-6 (an adult Leydig cell marker), α-actin (a peritubular myoid cell marker), and androgen receptor (a marker of all three somatic cell types). The temporal-spatial expression of these markers was used to interrogate findings of earlier experimental studies on the origin of Sertoli, Leydig and peritubular myoid cells, as well as extend previous descriptive studies across a broader developmental period (E10.5-P60). Such comparisons demonstrate inconsistencies that require further examination and raise questions regarding conservation of developmental mechanisms across higher vertebrate species.

Using 2-Photon Microscopy to Quantify the Effects of Chronic Unilateral Ureteral Obstruction on Glomerular Processes.

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface glomeruli provide a unique opportunity to investigate physiological and pathophysiological processes using intravital 2-photon microscopy. Quantification of glomerular capillary blood flow and vasoconstriction and dilatation in response to drugs, permeability, and inflammation are just some of the processes that can be studied. In addition, transgenic rats, i.e., podocytes labeled with fluorescent dyes and other molecular biomarker approaches, provide increased resolution to directly monitor and quantify protein-protein interactions and the effects of specific molecular alterations. In mice, which lack surface glomeruli after four weeks of age, unilateral ureteral obstruction (UUO) for several weeks has been used to induce surface glomeruli. As this induction model does not allow for baseline studies, we quantified the effects of UUO on glomerular processes in the UUO model in Munich Wistar Frömter (MWF) rats, which have surface glomeruli under physiologic conditions. The UUO model for five weeks or more induced significant alterations to gross renal morphology, the peritubular and glomerular microvasculature, as well as the structure and function of tubular epithelia. Glomerular and peritubular red blood cell (RBC) flow decreased significantly (p < 0.01), probably due to the significant increase in the adherence of white blood cells (WBCs) within glomerular and peritubular capillaries. The glomerular sieving coefficient of albumin increased from 0.015 ± 0.002 in untreated MWFs to 0.045 ± 0.05 in 5-week-old UUO MWF rats. Twelve weeks of UUO resulted in further increases in surface glomerular density and glomerular sieving coefficient (GSC) for albumin. Fluorescent albumin filtered across the glomeruli was not reabsorbed by the proximal tubules. These data suggest that using UUO to induce surface glomeruli limits the ability to study and interpret normal glomerular processes and disease alterations.