Characterization of the stem cell niche components within the seminiferous tubules in testicular biopsies of Klinefelter patients.

OBJECTIVE: To characterize the tubular environment in testicular biopsy tissues from patients with Klinefelter syndrome (KS). DESIGN: Observational immunohistochemical study. SETTING: Academic research unit. PATIENT(S): Males with KS and controls at different developmental time points: fetal, prepubertal, peripubertal, and adult. INTERVENTION(S): Immunohistochemical analysis of testicular biopsies samples to characterize maturation of Sertoli cells and tubular wall components-peritubular myoid cells (PTMC) and extracellular matrix (ECM) proteins. MAIN OUTCOME MEASURE(S): Intensity of antimüllerian hormone staining; proportion of Sertoli cells expressing androgen receptor (AR); and expression of tubular wall markers as characterized by identifying abnormal staining patterns. RESULT(S): Decreased expression for alpha smooth muscle actin 2 (ACTA2) was observed in peripubertal and adult KS as well as in Sertoli cell only (SCO) patients. Altered expression patterns for all ECM proteins were observed in SCO and KS biopsy tissues compared with controls. Only for collagen I and IV were altered expression patterns observed between KS and SCO patients. In peripubertal samples, no statistically significant differences were observed in the maturation markers, but altered ECM patterns were already present in some samples. CONCLUSION(S): The role of loss of ACTA2 expression in PTMC in the disintegration of tubules in KS patients should be further investigated. Future research is necessary to identify the causes of testicular fibrosis in KS patients. If the mechanism behind this fibrotic process could be identified, this process might be altered toward increasing the chances of fertility in KS patients.

Scaffold protein Lin7 family in membrane skeletal protein complex in mouse seminiferous tubules.

The membrane skeletal complex, protein 4.1G-membrane palmitoylated protein 6 (MPP6), is localized in spermatogonia and early spermatocytes of mouse seminiferous tubules. In this study, we investigated the Lin7 family of scaffolding proteins, which interact with MPP6. By immunohistochemistry, Lin7a and Lin7c were localized in germ cells, and Lin7c had especially strong staining in spermatogonia and early spermatocytes, characterized by staging of seminiferous tubules. By immunoelectron microscopy, Lin7 localization appeared under cell membranes in germ cells. The Lin7 staining pattern in seminiferous tubules was partially similar to that of 4.1G, cell adhesion molecule 1 (CADM1), and melanoma cell adhesion molecule (MCAM). Lin7-positive cells included type A spermatogonia, as revealed by double staining for Lin28a. Lin7 staining became weaker in MPP6-deficient mice by immunohistochemistry and western blotting, indicating that MPP6 transports and maintains Lin7 in germ cells. The histology of seminiferous tubules was unchanged in MPP6-deficient mice compared to that of wild-type mice. In cultured spermatogonial stem cells maintained with glial cell line-derived neurotropic factor (GDNF), Lin7 was clearly expressed and immunolocalized along cell membranes, especially at cell-cell junctions. Thus, Lin7 protein is expressed in germ cells, and Lin7, particularly Lin7c, is a useful marker for early spermatogenesis.

Protein expression of the transcription factors DMRT1, TCLF5, and OCT4 in selected germ cell neoplasms of the testis.

In the present study, we investigated protein expression of the transcription factors mammalian doublesex and mab-3 related transcription factor 1 (DMRT1), basic helix-loop-helix transcription factor-like 5 (TCLF5), and octamer-binding transcription factor 4 (OCT4) in normal human spermatogenesis, testicular mixed germ cell-sex cord stromal tumor (MGC-SCST), spermatocytic tumor, and seminoma. In normal human spermatogenesis, DMRT1 is expressed in the nuclei of spermatogonia but not in those of more mature germ cells. By way of contrast, TCLF5 is expressed in the nuclei of some clusters of primary spermatocytes that have entered meiosis 1, in secondary spermatocytes, and in round (early) spermatids in the seminiferous tubules of adults during the reproductive years. OCT4 is expressed in primordial germ cells but not in the seminiferous tubules of the normal adult testis during the reproductive years. DMRT1 is expressed in the germ cells of both testicular MGC-SCST and spermatocytic tumor, whereas TCLF5 is not expressed in either neoplasm. These low-grade neoplasms, however, differ histologically in that all the germ cell nuclei of testicular MGC-SCST resemble spermatogonia, whereas in spermatocytic tumor, the nuclei of the medium-sized and large cells resemble those of primary spermatocytes. Both neoplasms lack expression of OCT4. By way of contrast, in seminoma, a fully malignant testicular germ cell tumor, the germ cell nuclei express OCT4 but do not express either DMRT1 or TCLF5.

NLRP3 in somatic non-immune cells of rodent and primate testes.

NLRP3 is part of the NLRP3 inflammasome and a global sensor of cellular damage. It was recently discovered in rodent Sertoli cells. We investigated NLRP3 in mouse, human and non-human primate (marmoset and rhesus macaque) testes, employing immunohistochemistry. Sertoli cells of all species expressed NLRP3, and the expression preceded puberty. In addition, peritubular cells of the adult human testes expressed NLRP3. NLRP3 and associated genes (PYCARD, CASP1, IL1B) were also found in isolated human testicular peritubular cells and the mouse Sertoli cell line TM4. Male infertility due to impairments of spermatogenesis may be related to sterile inflammatory events. We observed that the expression of NLRP3 was altered in the testes of patients suffering from mixed atrophy syndrome, in which tubules with impairments of spermatogenesis showed prominent NLRP3 staining. In order to explore a possible role of NLRP3 in male infertility, associated with sterile testicular inflammation, we studied a mouse model of male infertility. These human aromatase-expressing transgenic mice (AROM+) develop testicular inflammation and impaired spermatogenesis during aging, and the present data show that this is associated with strikingly elevated Nlrp3 expression in the testes compared to WT controls. Interference by aromatase inhibitor treatment significantly reduced increased Nlrp3 levels. Thus, throughout species NLRP3 is expressed by somatic cells of the testis, which are involved in testicular immune surveillance. We conclude that NLRP3 may be a novel player in testicular immune regulation.

Immunocytochemical localization of kisspeptin and kisspeptin receptor in the primate testis.

BACKGROUND: Hypothalamic kisspeptin-kisspeptin receptor signalling in primates ensures the successful progression into puberty during development and maintenance of reproductive capacity during adulthood. Human testis has been shown to express high-to-moderate levels of kisspeptin and kisspeptin receptor gene expression. In this study, we aimed at characterizing the localization of kisspeptin and kisspeptin receptor in adult primate testis tissue. METHODS: Immunocytochemistry was performed on paraffin-embedded testicular sections from adult rhesus monkeys and from common marmoset monkeys. RESULTS: Kisspeptin receptor was detected in Sertoli cells in the periphery of the seminiferous tubules in adult testes of both species. In contrast, kisspeptin was not localized in the seminiferous epithelium and was detected only in the interstitial compartment of the adult rhesus monkey testis. CONCLUSION: Kisspeptin receptor and kisspeptin are localized in the testis of Old World and New World primates.

A Niche for GFRα1-Positive Spermatogonia in the Terminal Segments of the Seminiferous Tubules in Hamster Testes.

In invertebrate species such as flies and nematodes, germline stem cells are maintained in a niche environment, which is restricted to the terminal end of the tubular structure in the gonads. In mice, spermatogonial stem cells (SSCs), a subpopulation of Asingle GFRα1 (glial cell line-derived neurotrophic factor [GDNF] family receptor-α1)-positive spermatogonia, are widely distributed along the longitudinal axis in the convoluted seminiferous tubules, preferentially juxtaposed to the interstitial vasculature. However, whether this area is the only SSC niche is not known. In this study, we identified a valve-like terminal segment of the seminiferous tubules, the Sertoli valve (SV), adjacent to the rete testis as another niche for GFRα1-positive spermatogonia in hamsters. Here, we show that the SV epithelium is composed of the modified Sertoli cells that are still capable of proliferation and missing most spermatogenic activities in the adult stage. The SV epithelium constitutively expresses GDNF, a major niche factor for SSCs, and supports the stable proliferation and selective maintenance of an Asingle subpopulation of GFRα1-positive spermatogonia in hamsters. The SV region of hamster seminiferous tubules has features that are similar to the stem cell niche in invertebrate gonads. Therefore, we propose that the SV may be a novel niche for Asingle GFRá1-positive spermatogonia potentially including a SSC population, at the terminal segments of the seminiferous tubules in hamsters.

Mass spectrometry imaging with laser-induced postionization.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can simultaneously record the lateral distribution of numerous biomolecules in tissue slices, but its sensitivity is restricted by limited ionization. We used a wavelength-tunable postionization laser to initiate secondary MALDI-like ionization processes in the gas phase. In this way, we could increase the ion yields for numerous lipid classes, liposoluble vitamins, and saccharides, imaged in animal and plant tissue with a 5-micrometer-wide laser spot, by up to two orders of magnitude. Critical parameters for initiation of the secondary ionization processes are pressure of the cooling gas in the ion source, laser wavelength, pulse energy, and delay between the two laser pulses. The technology could enable sensitive MALDI-MS imaging with a lateral resolution in the low micrometer range.

Expression of steroid hormone receptors in the genital structures of a true hermaphrodite pug dog.

Hermaphroditism is a rare and a not well-understood disordered sexual development (DSD) in dogs. The objective of the study was to analyse the sex steroid hormone receptor (STHR) expression patterns in the internal genital structures, because the responsiveness of the different tissue types to the steroid hormones may have a key role in pathological alterations based on DSDs. Furthermore, the adhesion molecule ß-catenin was investigated by means of immunohistochemistry because of its important role in development, tissue integrity and disease. Molecular sexing was performed via PCR targeting DBX/DBY genes to identify the pug dog as a true XX hermaphrodite. The portions of uterine tissue revealed comparable expression patterns for STHRs as investigated in normal female reproductive tissue. In the male parts, ß-catenin showed strong expression in the Sertoli cells of the seminiferous tubules; this was in contrast to normal testicular tissue. Likewise, the layers of smooth muscle actin-positive cells surrounding the seminiferous tubules were reduced in the hermaphrodite. The results of this study deepen the knowledge of tissue characteristics in a hermaphrodite dog and highlight the importance of early diagnosis because the STH responsiveness in maldeveloped reproductive tissue might lead to serious problems for the dog.

Differential expression and localization of ADAM10 and ADAM17 during rat spermatogenesis suggest a role in germ cell differentiation.

BACKGROUND: Extracellular metolloproteases have been implied in different process such as cell death, differentiation and migration. Membrane-bound metalloproteases of the ADAM family shed the extracellular domain of many cytokines and receptor controlling auto and para/juxtacrine cell signaling in different tissues. ADAM17 and ADAM10 are two members of this family surface metalloproteases involved in germ cell apoptosis during the first wave of spermatogenesis in the rat, but they have other signaling functions in somatic tissues. RESULTS: In an attempt to further study these two enzymes, we describe the presence and localization in adult male rats. Results showed that both enzymes are detected in germ and Sertoli cells during all the stages of spermatogenesis. Interestingly their protein levels and cell surface localization in adult rats were stage-specific, suggesting activation of these enzymes at particular events of rat spermatogenesis. CONCLUSIONS: Therefore, these results show that ADAM10 and ADAM17 protein levels and subcellular (cell surface) localization are regulated during rat spermatogenesis.

Raman spectroscopy as an ex vivo noninvasive approach to distinguish complete and incomplete spermatogenesis within human seminiferous tubules.

OBJECTIVE: To evaluate the potential clinical application of Raman spectroscopy (RS) as a tool that may identify spermatogenesis within human seminiferous tubules. DESIGN: RS scanning of human testicular tissue at different maturational stages; immunohistochemistry study and metabolomic analysis of nonobstructive azoospermic/obstructive azoospermic testes. SETTING: State-owned hospital. PATIENT(S): Fifty-two patients with clinical indications of nonobstructive azoospermia (NOA) and obstructive azoospermia (OA) who underwent infertility evaluation and treatment. INTERVENTION(S): None. MAIN OUTCOME MEASUREMENT(S): Raman spectra of seminiferous tubules, thickness of lamina propria (LP), immunohistochemistry of type I, III, and IV collagens and laminin, metabolites of human testes. RESULT(S): Tubules of OA patients had spectral intensities below 2,000 (au), while tubules of NOA patients had higher intensities, depending on the degree of spermatogenesis. RS was able to separate samples of NOA and OA testicular tissue with a sensitivity of 90% and specificity of 85.71%. The LP of NOA tubules were thickened and had increased deposition of type I and type III collagens. Gas chromatography-mass spectrometer (GC-MS) detected 12 metabolites that showed significant differences between NOA and OA testes. CONCLUSION(S): RS can noninvasively distinguish seminiferous tubules with complete and incomplete spermatogenesis and may serve as a novel and potentially useful tool to guide surgeons performing micro-testicular sperm extraction to improve sperm retrieval.

Secretome analysis of testicular peritubular cells: a window into the human testicular microenvironment and the spermatogonial stem cell niche in man.

Spermatogonial stem cells (SSCs) are vital for lifelong spermatogenesis in man. In their niches, a special growth factor milieu and structural support by surrounding cells are thought to ensure their maintenance. In man, the cells of the wall of seminiferous tubules, human testicular peritubular cells (HTPCs), are considered to contribute to this microenvironment and the overall testicular microenvironment via secreted proteins. Therefore, the secretome of cultured HTPCs from five individual men was analyzed by LC-MS/MS. Quantification and comparison to the proteome of HTPC lysates revealed 263 out of 660 identified secretome proteins to be at least 5-fold enriched in the culture media. To obtain additional evidence for secretion, signal peptide and gene ontology (GO) enrichment analyses were applied. The latter revealed--besides extracellular matrix (ECM) components--a significant over-representation of chemokines and growth factors acting in signaling pathways that appear critical for SSC maintenance. Immunohistochemistry, performed with human testicular sections, depicted expression of selected proteins in vivo. The significant enrichment of proteins related to cell adhesion and migration may indicate their involvement in SSC regulation. Our data strongly support the hypothesis of a crucial role of HTPCs in the composition of SSC niches in man.

Role of P-glycoprotein in the distribution of the HIV protease inhibitor atazanavir in the brain and male genital tract.

The blood-testis barrier and blood-brain barrier are responsible for protecting the male genital tract and central nervous system from xenobiotic exposure. In HIV-infected patients, low concentrations of antiretroviral drugs in cerebrospinal fluid and seminal fluid have been reported. One mechanism that may contribute to reduced concentrations is the expression of ATP-binding cassette drug efflux transporters, such as P-glycoprotein (P-gp). The objective of this study was to investigate in vivo the tissue distribution of the HIV protease inhibitor atazanavir in wild-type (WT) mice, P-gp/breast cancer resistance protein (Bcrp)-knockout (Mdr1a-/-, Mdr1b-/-, and Abcg2-/- triple-knockout [TKO]) mice, and Cyp3a-/- (Cyp) mice. WT mice and Cyp mice were pretreated with a P-gp/Bcrp inhibitor, elacridar (5 mg/kg of body weight), and the HIV protease inhibitor and boosting agent ritonavir (2 mg/kg intravenously [i.v.]), respectively. Atazanavir (10 mg/kg) was administered i.v. Atazanavir concentrations in plasma (Cplasma), brain (Cbrain), and testes (Ctestes) were quantified at various times by liquid chromatography-tandem mass spectrometry. In TKO mice, we demonstrated a significant increase in atazanavir Cbrain/Cplasma (5.4-fold) and Ctestes/Cplasma (4.6-fold) ratios compared to those in WT mice (P<0.05). Elacridar-treated WT mice showed a significant increase in atazanavir Cbrain/Cplasma (12.3-fold) and Ctestes/Cplasma (13.5-fold) ratios compared to those in vehicle-treated WT mice. In Cyp mice pretreated with ritonavir, significant (P<0.05) increases in atazanavir Cbrain/Cplasma (1.8-fold) and Ctestes/Cplasma (9.5-fold) ratios compared to those in vehicle-treated WT mice were observed. These data suggest that drug efflux transporters, i.e., P-gp, are involved in limiting the ability of atazanavir to permeate the rodent brain and genital tract. Since these transporters are known to be expressed in humans, they could contribute to the low cerebrospinal and seminal fluid antiretroviral concentrations reported in the clinic.

Differential expression and localization of ADAM10 and ADAM17 during rat spermatogenesis suggest a role in germ cell differentiation

BACKGROUND: Extracellular metolloproteases have been implied in different process such as cell death, differentiation and migration. Membrane-bound metalloproteases of the ADAM family shed the extracellular domain of many cytokines and receptor controlling auto and para/juxtacrine cell signaling in different tissues. ADAM17 and ADAM10 are two members of this family surface metalloproteases involved in germ cell apoptosis during the first wave of spermatogenesis in the rat, but they have other signaling functions in somatic tissues. RESULTS: In an attempt to further study these two enzymes, we describe the presence and localization in adult male rats. Results showed that both enzymes are detected in germ and Sertoli cells during all the stages of spermatogenesis. Interestingly their protein levels and cell surface localization in adult rats were stage-specific, suggesting activation of these enzymes at particular events of rat spermatogenesis. CONCLUSIONS: Therefore, these results show that ADAM10 and ADAM17 protein levels and subcellular (cell surface) localization are regulated during rat spermatogenesis.

Germ cell migration across Sertoli cell tight junctions.

The blood-testis barrier includes strands of tight junctions between somatic Sertoli cells that restricts solutes from crossing the paracellular space, creating a microenvironment within seminiferous tubules and providing immune privilege to meiotic and postmeiotic cells. Large cysts of germ cells transit the Sertoli cell tight junctions (SCTJs) without compromising their integrity. We used confocal microscopy to visualize SCTJ components during germ cell cyst migration across the SCTJs. Cysts become enclosed within a network of transient compartments fully bounded by old and new tight junctions. Dissolution of the old tight junctions releases the germ cells into the adluminal compartment, thus completing transit across the blood-testis barrier. Claudin 3, a tight junction protein, is transiently incorporated into new tight junctions and then replaced by claudin 11.

Expression of basigin in reproductive tissues of estrogen receptor-{alpha} or -{beta} null mice.

Basigin plays important roles in both male and female reproduction because basigin (Bsg) null male and female mice are infertile. The aim of the present study was to determine whether basigin expression in reproductive organs requires estrogen receptor-alpha (ESR1, ERalpha) or -beta (ESR2, ERbeta). Expression of basigin protein in the testis, ovary, and male and female reproductive tracts was studied in adult wild-type (WT), Esr1-null (alphaERKO), and Esr2-null (betaERKO) mice by immunohistochemistry and immunoblotting. Basigin mRNA levels in ovary and uterus were examined by quantitative RT-PCR. In females, basigin protein expression was observed mainly in granulosa and interstitial cells of the ovary and epithelial cells of the proximal oviduct in all genotypes. Basigin protein was also expressed in the uterine epithelium at proestrus and estrus in WT and betaERKO mice but not in alphaERKO mice. However, a higher level of basigin mRNA was observed in uteri of alphaERKO mice compared with WT and betaERKO mice. In males, basigin was expressed in Leydig cells and all germ cells except spermatogonia in all genotypes. Basigin was present in epithelial cells lining the efferent ductules in WT and betaERKO mice, but expression was greatly reduced in alphaERKO mice. In epididymal ducts, basigin expression was observed in epithelial cells in the caput and cauda in all genotypes. These data suggest that expression of basigin protein requires ESR1, but not ESR2, in the uterus and efferent ductules, but is independent of estrogen receptor in the ovary, oviduct, testis, and epididymis.

EPAS1 Is Required for Spermatogenesis in the Postnatal Mouse Testis.

Spermatogenesis, a process involving the differentiation of spermatogonial stem cells into mature spermatozoa, takes place throughout masculine life. A complex system in the testis, including endocrine signaling, physical interactions between germ and somatic cells, spermatocyte meiosis, and timely release of spermatozoa, controls this cycle. We demonstrate herein that decreased O(2) levels and Epas1 activation are critical components of spermatogenesis. Postnatal Epas1 ablation leads to male infertility, with reduced testis size and weight. While immature spermatogonia and spermatocytes are present in Epas1(Delta/Delta) testes, spermatid and spermatozoan numbers are dramatically reduced. This is not due to germ cell-intrinsic defects. Rather, Epas(Delta/Delta) Sertoli cells exhibit decreased ability to form tight junctions, thereby disrupting the blood-testis barrier necessary for proper spermatogenesis. Reduced numbers of tight junction complexes are due to decreased expression of multiple genes encoding tight junction proteins, including TJP1 (ZO1), TJP2 (ZO2), and occludin. Furthermore, Epas1(Delta/Delta) testes exhibit disrupted basement membranes surrounding the seminiferous tubules, causing the premature release of incompletely differentiated germ cells. We conclude that low O(2) levels in the male gonad regulate germ cell homeostasis in this organ via EPAS1.

Three-dimensional analysis of connexin 43 gap junction in the ex vivo rat seminiferous tubules: short-term effects of hormonal effectors.

Cx43 gap junctions are essential for proliferation, differentiation, and apoptosis of germ cells during spermatogenesis. However, only few and indirect observations have been reported on the distribution of Cx43, the predominant Cx within the seminiferous tubules. In the present study, we developed an innovative method that allows visualization of the three- dimensional localization of Cx43 associated with gap junctions and their functionality in isolated spermatogenic stage-specific seminiferous tubules. Cx43 gap junctions were present between myoid cells, between Sertoli cells, and between Sertoli and germ cells. Cx43 levels and coupling were stage-dependent with higher values at stages VI-VIII of spermatogenesis and markedly reduced at stages IX-X. Short-term exposure of seminiferous tubule fragments at stages VI-VIII and of the 42GPA9 Sertoli cell line transfected with a Cx43-GFP vector, to FSH, cAMP, DHT, and 17beta-E(2) significantly altered Cx43 distribution as well as gap junction coupling. These observations highlight a nongenomic effect of these testicular effectors on Cx43 gap junction.

Testicular annexin A5 expression augmented by experimental cryptorchidism and could affect germ cell apoptosis in rats.

OBJECTIVES: To examine the expression of annexin A5, a recently emerged multifunctional protein, in the testis after experimental cryptorchidism. METHODS: The hemilateral testes of rats were surgically relocated from the scrotum into the abdomen. The annexin A5 content was evaluated by Western blot analysis, and its distribution was determined by immunohistochemistry. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) was performed to detect the apoptotic cells. RESULTS: Although annexin A5 content was not changed in the scrotal testis, it was dramatically increased in the abdominal testis during the 4-week observation period. TUNEL-positive apoptotic spermatogonia appeared in some of the seminiferous tubules only 1 day after the surgery. The appearance of TUNEL-positive cells was accompanied by augmented expression of annexin A5. Spermatocytes disappeared by 1 week after cryptorchidism, and annexin A5 expression was significantly increased inside the seminiferous tubules. In the cryptorchid testis, annexin A5 of the interstitial cells disappeared, as did 3beta-hydroxy steroid dehydrogenase. Annexin A5 on endothelial cells was not changed by the cryptorchidism. CONCLUSIONS: These data demonstrate first that the expression of annexin A5 is increased in the seminiferous tubules of cryptorchid testes and suggest that it is related to the degenerative response of germ cells.

PCNA indexing as a preclinical immunohistochemical biomarker for testicular toxicity.

It is well known that toxicants such as cyclophosphamide and ethanol can have deleterious effects on normal spermatogenesis. End points such as testis weight and sperm counts have been used widely to assess gross structural and functional changes in testes resulting from toxicant exposure. Histopathological assessments are more sensitive measures of testicular health, but generally they are neither quantitative nor sensitive enough to detect early toxicity. Recently, immunolabeling cells with proliferating cell nuclear antigen (PCNA) has been used to identify proliferating spermatogonia; however, there have been no systematic attempts to quantify these changes. We have developed a sensitive, reliable and quantitative assay using immunohistochemistry on formalin fixed, paraffin embedded rat testes to assess the degree of proliferation-related toxicity. An indexing scheme was derived based on the determination of radially positioned PCNA-positive cells within similarly staged seminiferous tubules presenting a single layer of PCNA-positive cells along the basement membrane of the basal tubular compartment. An average of 60 tubules in the testes were counted per animal. Our results show significant decreases in the PCNA index in rats treated with an experimental compound that has been shown to produce testicular histopathology. The analysis provides a quick, reliable, sensitive, and quantitative means for assessing early testicular toxicity. The assay has potential utility as an in vivo biomarker for detecting early testicular toxicity of experimental compounds in preclinical development as well as for refining follow-up compounds with reduced testicular toxicity.