Human Testis Biopsies Research Articles

EXOGENOUS LEPTIN TREATMENT ALTERS THE TESTICULAR IMMUNE MICROIMMUNE-ENVIRONMENT

In men, Leydig cells (LCs) are the primary source of testosterone (T) production. Dysfunction of the LC can lead to T deficiency and male hypogonadism. The development of LCs is influenced by the paracrine factors released by the testicular microenvironment (TME). In our recent study, we demonstrated using cells isolated from human testis biopsies that Leptin, a paracrine factor secreted by TME, is critical for Leydig stem cell (LSCs) differentiation into Adult Leydig Cells (ADL), and subsequent T production.

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

Spermatogonial kinetics in humans.

The human spermatogonial compartment is essential for daily production of millions of sperm. Despite this crucial role, the molecular signature, kinetic behavior and regulation of human spermatogonia are poorly understood. Using human testis biopsies with normal spermatogenesis and by studying marker protein expression, we have identified for the first time different subpopulations of spermatogonia. MAGE-A4 marks all spermatogonia, KIT marks all B spermatogonia and UCLH1 all Apale-dark (Ap-d) spermatogonia. We suggest that at the start of the spermatogenic lineage there are Ap-d spermatogonia that are GFRA1High, likely including the spermatogonial stem cells. Next, UTF1 becomes expressed, cells become quiescent and GFRA1 expression decreases. Finally, GFRA1 expression is lost and subsequently cells differentiate into B spermatogonia, losing UTF1 and acquiring KIT expression. Strikingly, most human Ap-d spermatogonia are out of the cell cycle and even differentiating type B spermatogonial proliferation is restricted. A novel scheme for human spermatogonial development is proposed that will facilitate further research in this field, the understanding of cases of infertility and the development of methods to increase sperm output.

Membrane Transporters for Sulfated Steroids in the Human Testis - Cellular Localization, Expression Pattern and Functional Analysis

Sulfated steroid hormones are commonly considered to be biologically inactive metabolites, but may be reactivated by the steroid sulfatase into biologically active free steroids, thereby having regulatory function via nuclear androgen and estrogen receptors which are widespread in the testis. However, a prerequisite for this mode of action would be a carrier-mediated import of the hydrophilic steroid sulfate molecules into specific target cells in reproductive tissues such as the testis. In the present study we detected predominant expression of the Sodium-dependent Organic Anion Transporter (SOAT), the Organic Anion Transporting Polypeptide 6A1, and the Organic Solute Carrier Partner 1 in human testis biopsies. All of these showed significantly lower or even absent mRNA expression in severe disorders of spermatogenesis (arrest at the level of spermatocytes or spermatogonia, Sertoli cell only syndrome). Only SOAT was significantly lower expressed in biopsies showing hypospermatogenesis. By use of immunohistochemistry SOAT was localized to germ cells at various stages in human testis biopsies showing normal spermatogenesis. SOAT immunoreactivity was detected in zygotene primary spermatocytes of stage V, pachytene spermatocytes of all stages (I–V), secondary spermatocytes of stage VI, and round spermatids (step 1 and step 2) in stages I and II. Furthermore, SOAT transport function for steroid sulfates was analyzed with a novel liquid chromatography tandem mass spectrometry procedure capable of profiling steroid sulfate molecules from cell lysates. With this technique, the cellular inward-directed SOAT transport was verified for the established substrates dehydroepiandrosterone sulfate and estrone-3-sulfate. Additionally, β-estradiol-3-sulfate and androstenediol-3-sulfate were identified as novel SOAT substrates.

Involvement of the prostate and testis expression (PATE)-like proteins in sperm-oocyte interaction

The prostate and testis expression (PATE)-like family of proteins are expressed mainly in the male genital tract. They are localized in the sperm head and are homologous to SP-10, the acrosomal vesicle protein also named ACRV1. Our aim was to characterize the expression and functional role of three PATE-like proteins in the testis and ejaculated sperm. The expression and localization of PATE-like proteins in human testis biopsies (n= 95) and sperm cells were assessed by RT-PCR, immunohistochemistry and immunofluorescence staining (at least 600 sperm cells per specimen). The function of the PATE protein was tested by the hemizona assay and hamster egg penetration test (HEPT). PATE and PATE-M genes and proteins were present almost exclusively in germ cells in the testis: immunoflourescence showed that the percentage of germ cells positive for PATE, PATE-M and PATE-B was 85, 50 and 2%, respectively. PATE and PATE-M proteins were localized in the equatorial segment of the sperm head, while PATE-B protein was localized in the post-acrosomal region. A polyclonal antibody (Ab, at 1:50 and 1:200 dilutions) against the PATE protein did not inhibit sperm-zona binding in the hemizona assay (hemizona index of 89.6 ± 10 and 87 ± 36%, respectively). However, there was inhibition of sperm-oolemma fusion and penetration in the HEPT (penetration index: without Ab 7 ± 3.9; Ab dilution of 1:100, 4 ± 3.5; Ab dilution of 1:20, 0.6 ± 1.2, P < 0.001). Our data suggest that PATE protein is involved in sperm-oolemma fusion and penetration but not sperm-zona binding.

Isolation and Characterization of Pluripotent Human Spermatogonial Stem Cell-Derived Cells

Several reports have documented the derivation of pluripotent cells (multipotent germline stem cells) from spermatogonial stem cells obtained from the adult mouse testis. These spermatogonia-derived stem cells express embryonic stem cell markers and differentiate to the three primary germ layers, as well as the germline. Data indicate that derivation may involve reprogramming of endogenous spermatogonia in culture. Here, we report the derivation of human multipotent germline stem cells (hMGSCs) from a testis biopsy. The cells express distinct markers of pluripotency, form embryoid bodies that contain derivatives of all three germ layers, maintain a normal XY karyotype, are hypomethylated at the H19 locus, and express high levels of telomerase. Teratoma assays indicate the presence of human cells 8 weeks post-transplantation but limited teratoma formation. Thus, these data suggest the potential to derive pluripotent cells from human testis biopsies but indicate a need for novel strategies to optimize hMGSC culture conditions and reprogramming.

Increased achondroplasia mutation frequency with advanced age and evidence for G1138A mosaicism in human testis biopsies

To evaluate the influence of aging on the achondroplasia mutation rate in the male germline. Studies in sperm and testis biopsy DNA according to donor's age. University teaching hospital. Seventeen donors aged 30 to 65 years for sperm collection and 14 deceased donors aged 53 to 95 years for testis biopsies, all with normal stature. Testes were obtained from 14 deceased donors, and sperm was obtained from 17 patients who requested ART. Real-time polymerase chain reaction quantification of the G1138A mutation in sperm and testis biopsies. The rate of G1138A mutation did not significantly vary with age in sperm, whereas in testis biopsies it increased markedly past the age of 70 years. Moreover, and for the first time, a mosaic for this mutation was detected in the testis of three subjects who were >80 years of age. These findings could contribute to providing a molecular explanation for the increased incidence of achondroplastic offspring with advanced paternal age.

Deletions in L-type calcium channel α1 subunit testicular transcripts correlate with testicular cadmium and apoptosis in infertile men with varicoceles

To identify and understand predictors of successful varicocelectomy. Examination of testicular L-type voltage-dependent calcium channel (L-VDCC) mRNAs and proteins in testis biopsies and comparison of presence and absence of various mRNAs with testicular cadmium levels, with apoptosis, and with sperm count change after varicocelectomy. University clinical urology practice and research laboratories. Infertile men with varicocele (left varicocele only, n = 18; bilateral varicoceles, n = 26) and controls (men with obstructive azoospermia undergoing testicular sperm extraction before intracytoplasmic sperm injection; n = 7). Left testis biopsies by percutaneous needle aspiration biopsy. Varicocele repair by subinguinal approach. Calcium channel mRNA sequence by reverse transcription-polymerase chain reaction and amplicon analysis; calcium channel protein distribution by immunocytochemistry; cadmium levels by atomic absorption and apoptosis by deoxynucleotidyl transferase labeling; and sperm counts in the ejaculate before and after varicocelectomy. Calcium channel mRNAs are polymorphic in human testis biopsies from different men. Proteins from sequence-deleted exons 7 and/or 8 localize to germ cell membranes. Expression of undeleted L-type calcium channel mRNAs correlates with normal testes cadmium and increased sperm count after varicocelectomy. Apoptosis is lower in such cases. Expression of normal testicular L-VDCC sequence in exons 7-8 predicts postvaricocelectomy sperm count increase. Deletions may alter calcium channel function and affect testicular cadmium and apoptosis.

Gene expression patterns associated with infertility in humans and rodent models

Modern genomic technologies such as DNA arrays provide the means to investigate molecular interactions at an unprecedented level, and arrays have been used to carry out gene expression profiling as a means of identifying candidate genes involved in molecular mechanisms underlying a variety of phenotypes. By comparing gene expression profiles from normal and abnormal human testes with those from comparable infertile mouse models, we endeavored to identify genes and gene networks critical for male fertility. We used commercially available filter-based DNA arrays to analyze testicular gene expression from eight human testis biopsies and three different infertile mouse models (atrichosis mutation, ataxia telangiectasia knockout and CREMtau knockout). Forty-seven mouse genes exhibited differential testicular gene expression ( P<0.01) associated with male infertility. These included genes involved in DNA repair (Vim, Rad23A, Rad23B), glutathione metabolism (Gsr, Gstp 1, Mgst1), proteolysis (Ace, Casp1, Ctsd), spermatogenesis (Prlr, Tmsb4 and Zfp-37) and stress response (Hsp 1, Osp94). The expression of 19 human genes was different ( P<0.05) between normal and abnormal samples, including those associated with apoptosis (GADD45), gonad development (SOX9), proteolysis (PSMC3, SPINK2, TIMP3, UBE213) and signal transduction (DLK1, NAP4, S100A10). Direct comparison of differentially expressed human and mouse genes identified glucose phosphate isomerase, and the highly similar human tissue inhibitor of metalloproteinase 3 (TIMP3) and mouse Timp2. Using DNA microarrays to profile gene expression in testes from infertile animal models and humans will be useful for understanding congenital infertility, and also infertility caused by environmental exposures where the same genes and molecular mechanisms are involved.

The proliferation of spermatogonia in normal and pathological human seminiferous epithelium: an immunohistochemical study using monoclonal antibodies against Ki-67 protein and proliferating cell nuclear antigen.

The quantitative distribution pattern of Ki-67 protein and proliferating cell nuclear antigen (PCNA) immunoreactivity was studied in human testis biopsies. In normal seminiferous epithelium Ki-67 is expressed in nuclei of spermatogonia, while PCNA additionally occurs in nuclei of primary spermatocytes. The staining pattern of spermatogonia is as follows (Ki-67-positive/PCNA-positive): 26.6 +/- 12.4%/46.3 +/- 9.5%. No stage-dependent differences were found. Biopsies with mixed atrophy (score < or =7) showed a significant (P < 0.05) decrease of immunopositive spermatogonia to 19.9 +/- 3.0%/31.4 +/- 5.7% (score 1) with minimal variation between different samples (score 7 to 1). Associated with defined histological defects such as hypospermatogenesis (hyp), spermatogenic arrest at the level of spermatids (sda), spermatocytes (sca) or spermatogonia (sga), however, there was a significant (P < 0.05) decrease of Ki-67 staining in tubules showing hyp (28.6 +/- 8.8%), sda (25.6 +/- 9.3%), sca (23.7 +/- 9.3%) and sga (16.2 +/- 6.0%) and of PCNA staining in sca (32.2 +/- 11.8%) and sga (20.0 +/- 9.5%), respectively. The decrease of immunoreactive spermatogonia did not correspond to elevation of follicle stimulating hormone (FSH). These data demonstrate that the low spermatogenic efficiency in infertile men is not only due to postmeiotic events, but also to a decrease in the meiotic activity of spermatogonia, and is not related to serum FSH.

Distribution of vimentin-type intermediate filaments in Sertoli cells of the human testis, normal and pathologic.

The presence, distribution and spatial arrangement of vimentin-type intermediate filaments in Sertoli cells from human testis biopsies, were studied in semithin and ultrathin sections using a polyclonal rabbit antiserum. At the ultrastructural level, vimentin immunoreactivity was seen concentrated around the nuclei, along fibrillary material within the cytoplasm and at the ectoplasmic specializations of the Sertoli cell junctions, as well as throughout the periphery of the Sertoli cell processes. It is therefore well suited as a marker for Sertoli cell configuration. In computer-aided 3D reconstructions of 20 serial sections, Sertoli cells displayed particular configurations of intermediate filaments in the different stages of spermatogenesis. Two basic configurations, named AS (before spermiation, stages V, VI, I and II), and PS (after spermiation, stages III and IV) respectively, could be differentiated. In addition to the reconstruction and morphological analysis of vimentin filaments in Sertoli cells from patients with unaltered spermatogenesis (obstructive azoospermia), pathological specimens (spermatogenetic arrest, Sertoli cells only-syndrome) were studied with respect to vimentin immunohistochemistry. The results indicate that vimentin filaments play an important role in the adaptation of Sertoli cells to the varying configurations of neighbouring cells during spermatogenesis as well as under pathological conditions.

In vitro autoradiographic studies of DNA synthesis in human testis with spermatogenic arrest.

The uptake of 3H‐thymidine by spermatogonia and resting spermatocytes was studied by autoradiography in human testis biopsies incubated in vitro from four groups of subjects: normal, prepuberal (hypogonadotropic hypogonadism), with spermatocytic arrest and with spermatidic arrest. In the prepuberal group there was a very low DNA synthesis rate. In the spermatidic arrest group the number of DNA synthesizing cells was higher than that of the normal control group while in the spermatocytic arrest group it was lower. These results indicate a possible different pathogenesis for the two forms of spermatogenic arrest. In the spermatocytic arrest there is a primary deficiency of germ cells synthesizing DNA, while in the spermatidic arrest, synthesis is active and the arrest is probably due to an enzymatic (?) metabolic block.