Interplay of spermatogonial subpopulations during initial stages of spermatogenesis in adult primates.

The spermatogonial compartment maintains spermatogenesis throughout the reproductive lifespan. Single-cell RNA sequencing (scRNA-seq) has revealed the presence of several spermatogonial clusters characterized by specific molecular signatures. However, it is unknown whether the presence of such clusters can be confirmed in terms of protein expression and whether protein expression in the subsets overlaps. To investigate this, we analyzed the expression profile of spermatogonial markers during the seminiferous epithelial cycle in cynomolgus monkeys and compared the results with human data. We found that in cynomolgus monkeys, as in humans, undifferentiated spermatogonia are largely quiescent, and the few engaged in the cell cycle were immunoreactive to GFRA1 antibodies. Moreover, we showed that PIWIL4+ spermatogonia, considered the most primitive undifferentiated spermatogonia in scRNA-seq studies, are quiescent in primates. We also described a novel subset of early differentiating spermatogonia, detectable from stage III to stage VII of the seminiferous epithelial cycle, that were transitioning from undifferentiated to differentiating spermatogonia, suggesting that the first generation of differentiating spermatogonia arises early during the epithelial cycle. Our study makes key advances in the current understanding of male germline premeiotic expansion in primates.

Identification of two hidden clinical subgroups among men with idiopathic cryptozoospermia.

STUDY QUESTION: Are there subgroups among patients with cryptozoospermia pointing to distinct etiologies? SUMMARY ANSWER: We reveal two distinct subgroups of cryptozoospermic (Crypto) patients based on testicular tissue composition, testicular volume, and FSH levels. WHAT IS KNOWN ALREADY: Cryptozoospermic patients present with a sperm concentration below 0.1 million/ml. While the etiology of the severely impaired spermatogenesis remains largely unknown, alterations of the spermatogonial compartment have been reported including a reduction of the reserve stem cells in these patients. STUDY DESIGN, SIZE, DURATION: To assess whether there are distinct subgroups among cryptozoospermic patients, we applied the statistical method of cluster analysis. For this, we retrospectively selected 132 cryptozoospermic patients from a clinical database who underwent a testicular biopsy in the frame of fertility treatment at a university hospital. As controls (Control), we selected 160 patients with obstructive azoospermia and full spermatogenesis. All 292 patients underwent routine evaluation for endocrine, semen, and histological parameters (i.e. the percentage of tubules with elongated spermatids). Moreover, outcome of medically assisted reproduction (MAR) was assessed for cryptozoospermic (n = 73) and Control patients (n = 87), respectively. For in-depth immunohistochemical and histomorphometrical analyses, representative tissue samples from cryptozoospermic (n = 27) and Control patients (n = 12) were selected based on cluster analysis results and histological parameters. PARTICIPANTS/MATERIALS, SETTING, METHODS: This study included two parts: firstly using clinical parameters of the entire cohort of 292 patients, we performed principal component analysis (PCA) followed by hierarchical clustering on principal components (i.e. considering hormonal values, ejaculate parameters, and histological information). Secondly, for histological analyses seminiferous tubules were categorized according to the most advanced germ cell type present in sections stained with Periodic acid Schif. On the selected cohort of 39 patients (12 Control, 27 cryptozoospermic), we performed immunohistochemistry for spermatogonial markers melanoma-associated antigen 4 (MAGEA4) and piwi like RNA-mediated gene silencing 4 (PIWIL4) followed by quantitative analyses. Moreover, the morphologically defined Adark spermatogonia, which are considered to be the reserve stem cells, were quantified. MAIN RESULTS AND THE ROLE OF CHANCE: The PCA and hierarchical clustering revealed three different clusters, one of them containing all Control samples. The main factors driving the sorting of patients to the clusters were the percentage of tubules with elongated spermatids (Cluster 1, all Control patients and two cryptozoospermic patients), the percentage of tubules with spermatocytes (Cluster 2, cryptozoospermic patients), and tubules showing a Sertoli cells only phenotype (Cluster 3, cryptozoospermic patients). Importantly, the percentage of tubules containing elongated spermatids was comparable between Clusters 2 and 3. Additional differences were higher FSH levels (P < 0.001) and lower testicular volumes (P < 0.001) in Cluster 3 compared to Cluster 2. In the spermatogonial compartment of both cryptozoospermic Clusters, we found lower numbers of MAGEA4+ and Adark spermatogonia but higher proportions of PIWIL4+ spermatogonia, which were significantly correlated with a lower percentage of tubules containing elongated spermatids. In line with this common alteration, the outcome of MAR was comparable between Controls as well as both cryptozoospermic Clusters. LIMITATIONS, REASONS FOR CAUTION: While we have uncovered the existence of subgroups within the cohort of cryptozoospermic patients, comprehensive genetic analyses remain to be performed to unravel potentially distinct etiologies. WIDER IMPLICATIONS OF THE FINDINGS: The novel insight that cryptozoospermic patients can be divided into two subgroups will facilitate the strategic search for underlying genetic etiologies. Moreover, the shared alterations of the spermatogonial stem cell compartment between the two cryptozoospermic subgroups could represent a general response mechanism to the reduced output of sperm, which may be associated with a progressive phenotype. This study therefore offers novel approaches towards the understanding of the etiology underlying the reduced sperm formation in cryptozoospermic patients. STUDY FUNDING/COMPETING INTEREST(S): German research foundation CRU 326 (grants to: SDP, NN). Moreover, we thank the Faculty of Medicine of the University of Münster for the financial support of Lena Charlotte Schülke through the MedK-program. We acknowledge support from the Open Access Publication Fund of the University of Münster. The authors have no potential conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

WWC2 expression in the testis: Implications for spermatogenesis and male fertility.

The family of WWC proteins is known to regulate cell proliferation and organ growth control via the Hippo signaling pathway. As WWC proteins share a similar domain structure and a common set of interacting proteins, they are supposed to fulfill compensatory functions in cells and tissues. While all three WWC family members WWC1, WWC2, and WWC3 are found co-expressed in most human organs including lung, brain, kidney, and liver, in the testis only WWC2 displays a relatively high expression. In this study, we investigated the testicular WWC2 expression in spermatogenesis and male fertility. We show that the Wwc2 mRNA expression level in mouse testes is increased during development in parallel with germ cell proliferation and differentiation. The cellular expression of each individual WWC family member was evaluated in published single-cell mRNA datasets of murine and human testes demonstrating a high WWC2 expression predominantly in early spermatocytes. In line with this, immunohistochemistry revealed cytosolic WWC2 protein expression in primary spermatocytes from human testes displaying full spermatogenesis. In accordance with these findings, markedly lower WWC2 expression levels were detected in testicular tissues from mice and men lacking germ cells. Finally, analysis of whole-exome sequencing data of male patients affected by infertility and unexplained severe spermatogenic failure revealed several heterozygous, rare WWC2 gene variants with a proposed damaging function and putative impact on WWC2 protein structure. Taken together, our findings provide novel insights into the testicular expression of WWC2 and show its cell-specific expression in spermatocytes. As rare WWC2 variants were identified in the background of disturbed spermatogenesis, WWC2 may be a novel candidate gene for male infertility.

Bi-allelic Mutations in M1AP Are a Frequent Cause of Meiotic Arrest and Severely Impaired Spermatogenesis Leading to Male Infertility.

Male infertility affects ∼7% of men, but its causes remain poorly understood. The most severe form is non-obstructive azoospermia (NOA), which is, in part, caused by an arrest at meiosis. So far, only a few validated disease-associated genes have been reported. To address this gap, we performed whole-exome sequencing in 58 men with unexplained meiotic arrest and identified the same homozygous frameshift variant c.676dup (p.Trp226LeufsTer4) in M1AP, encoding meiosis 1 associated protein, in three unrelated men. This variant most likely results in a truncated protein as shown in vitro by heterologous expression of mutant M1AP. Next, we screened four large cohorts of infertile men and identified three additional individuals carrying homozygous c.676dup and three carrying combinations of this and other likely causal variants in M1AP. Moreover, a homozygous missense variant, c.1166C>T (p.Pro389Leu), segregated with infertility in five men from a consanguineous Turkish family. The common phenotype between all affected men was NOA, but occasionally spermatids and rarely a few spermatozoa in the semen were observed. A similar phenotype has been described for mice with disruption of M1ap. Collectively, these findings demonstrate that mutations in M1AP are a relatively frequent cause of autosomal recessive severe spermatogenic failure and male infertility with strong clinical validity.

Male minipuberty in human and non-human primates: planting the seeds of future fertility.

In brief: Minipuberty is a transient activity period of the hypothalamic-pituitary-gonadal axis in the postnatal and infant period including surging serum concentrations of reproductive hormones. Increasing evidence points to an important role of this period for maturation of the testes and thereby for male reproductive function. Abstract: Minipuberty is a transient activity period of the hypothalamic-pituitary-gonadal (HPG) axis in the postnatal and infant period in humans and non-human primates. Hallmarks of this period are surging serum concentrations of reproductive hormones. While in females, the role of minipuberty seems to be dispensable for future fertility, in males, it is significantly associated with reproductive function in later life. In males, this activity period promotes further masculinization, including testicular and penile growth, as well as completion of testicular descent if not already achieved at birth. At the testicular level, both, somatic and germ cells undergo proliferation and partial maturation during this period. Minipuberty is thought to prime male gonadal tissue for subsequent growth and maturation. Notably, perturbed or absent minipuberty is associated with reduced male reproductive function in adulthood. While the sustained HPG axis activity during adulthood is known to control reproductive function, minipuberty appears to be a prerequisite for obtaining full male reproductive function in later life, thereby determining future fertility potential, i.e. the ability to father a child. This review maps the role of male minipuberty for reproductive function and presents suitable animal models to study minipuberty. Also, it describes the development and maturation of testicular cell types, discusses short- and long-term effects of minipuberty and highlights future research perspectives.

Healthy ageing and spermatogenesis.

Delayed family planning and increased parental age increase the risk for infertility and impaired offspring health. While the impact of ageing on oogenesis is well studied, this is less understood on spermatogenesis. Assessing ageing effects on the male germline presents a challenge in differentiating between the effects of ageing-associated morbidities, infertility and 'pure' ageing. However, understanding the impact of ageing on male germ cells requires the separation of age from other factors. In this review, we therefore discuss the current knowledge on healthy ageing and spermatogenesis. Male ageing has been previously associated with declining sperm parameters, disrupted hormone secretion and increased time-to-pregnancy, among others. However, recent data show that healthy ageing does not deteriorate testicular function in terms of hormone production and spermatogenic output. In addition, intrinsic, age-dependent, highly specific processes occur in ageing germ cells that are clearly distinct from somatic ageing. Changes in spermatogonial stem cell populations indicate compensation for stem cell exhaustion. Alterations in the stem cell niche and molecular ageing signatures in sperm can be observed in ageing fertile men. DNA fragmentation rates as well as changes in DNA methylation patterns and increased telomere length are hallmarks of ageing sperm. Taken together, we propose a putative link between the re-activation of quiescent Adark spermatogonia and molecular changes in aged sperm descending from these activated spermatogonia. We suggest a baseline of 'pure' age effects in male germ cells which can be used for subsequent studies in which the impact of infertility or co-morbidities will be studied.

New Insights Into Extended Steroid Hormone Profiles in Transwomen in a Multi-Center Setting in Germany.

BACKGROUND: Little information is available on steroid hormone profiles in transwomen on the day of gender affirming surgery (GAS) after gender affirming hormone therapy (GAHT). AIM: We compared extended serum steroid hormone profiles of 77 transwomen with 3 different treatment regimens in order to get more insight on how GAHT changes the hormone system. METHODS: Samples were obtained from 3 independent clinics. Individuals in clinic A (n = 13) and B (n = 51) discontinued GAHT 4-6 weeks and 2 weeks before GAS, individuals in clinic C (n = 13) continued treatment. Testicular tissue, blood samples and questionnaires on age, weight, height, and medication use were received from each patient. Steroid hormones were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS), 6 sex hormones were determined by immunofluorometric assays, and ELISA. Spermatogenesis was scored using the Bergman/Kliesch score. OUTCOMES: Participants were not different with regard to age, BMI, treatment duration, and dosage. Feminized blood serum levels with low LH, low FSH and low testosterone, however, were achieved in persons taking GAHT until GAS. Significantly reduced cortisone levels were seen after stopping GAHT before GAS. RESULTS: GAHT had marked effects on the sex-steroid profile in each person. Factor analysis provided a model explaining 78% of the variance and interdependency of sex steroid levels. Stopping treatment was inversely associated with intactness of the corticosteroid-axis with adrenal steroidogenesis as well as it was inversely associated with pituitary-gonadal hormone production. CLINICAL IMPLICATIONS: Transwomen generally did not have elevated cortisone levels but differed significantly depending on and when GAHT was stopped. STRENGTHS & LIMITATIONS: This is the first study examining the steroid hormone profiles of transgender persons on the day of GAS in a multi-center setting. Additional studies (including follow ups before and after GAS and stress questionnaires) will be necessary to assess these conflicting results about the possible psychological impact on persons undergoing GAS to improve care. CONCLUSION: Concerning feminized blood serum levels, continued GAHT seems the better alternative, however stopping treatment 4-6 weeks prior to surgery was associated with reduced cortisone levels. Schneider F, Wistuba J, Holterhus P-M, et al. New Insights Into Extended Steroid Hormone Profiles in Transwomen in a Multi-Center Setting in Germany. J Sex Med 2021;18:1807-1817.

41,XXY * male mice: An animal model for Klinefelter syndrome.

Klinefelter syndrome (KS, 47,XXY) is the most frequent male chromosomal aneuploidy resulting in a highly heterogeneous clinical phenotype associated with hormonal dysbalance, increased rate of co-morbidities, and reduced lifespan. Two hallmarks of KS-affecting testicular functions are consistently observed: Hypergonadotropic hypogonadism and germ cell (GC) loss resulting in infertility. Although KS is being studied for decades, the underlying mechanisms for the observed pathophysiology are still unclear. Due to ethical restrictions, studies in humans are limited, and consequently, suitable animal models are needed to address the consequences of a supernumerary X chromosome. Mouse strains with comparable aneuploidies have been generated and yielded highly relevant insights into KS. We briefly describe the establishment of the KS mouse models, summarize the knowledge gained by their use, compare findings from the mouse models to those obtained in clinical studies, and also reflect on limitations of the currently used models derived from the B6Ei.Lt-Y* mouse strain, in which the Y chromosome is altered and its centromere position changed into a more distal location provoking meiotic non-disjunction. Breeding such as XY* males to XX females, the target 41,XXY *, and 41,XXY males are generated. Here, we summarize features of both models but report in particular findings from our 41,XXY * mice including some novel data on Sertoli cell characteristics.

Age-related distribution and potential role of SNCB in topographically different retinal areas of the common marmoset Callithrix jacchus, including the macula.

Evidence of an age-related increase of ß-synuclein (SNCB) in several parts of the visual system including the retina has been reported. SNCB is thought to function as an antagonist of α-synuclein in neurodegenerative diseases, but the exact role of SNCB remains unclear. The presented work studies two different aspects of the onset and role of SNCB in the retinal pigment epithelium (RPE). First, the topographical and intracellular distributions of SNCB in the RPE of non-human marmoset monkey (Callithrix jacchus) were evaluated in paraffin-embedded eyes and RPE whole mounts from different developmental stages (neonatal, adolescent, and adult). Thus, revealed distinct lifetime-related alterations of the topographical and intracellular distributions of SNCB in the primate macula compared to the retinal periphery. Furthermore, the function and influences of SNCB on ARPE-19 cells and primary porcine RPE (ppRPE) cells were characterized by exposing these cells with recombinant SNCB (rSNCB) at different concentrations. Moreover, apoptosis, protein- and mRNA-expression levels of factors of the p53/MDM2 signaling cascade and inflammation- and oxidation-related genes were investigated. The observed dose-depended decreased apoptosis rates together with the PLD2 mediated activation of the p53 pathway promotes senescence-related processes in SNCB exposed common ARPE-19 cells from human origin. Further, increased HMOX1 and NOX4 levels indicate increased oxidative stress and inflammatory responses triggered by SNCB. The obtained differences in the distribution of SNCB in primate RPE together with alterations of cellular functions in rSNCB-exposed RPE cells (e.g., ARPE-19, ppRPE) support SNCB-related effects like inflammatory response and stress-related properties on RPE over lifetime. The possible functional relevance of SNCB in physiological aging converting into a pathophysiological condition should be investigated in further studies.

Follicle activation is a significant and immediate cause of follicle loss after ovarian tissue transplantation.

PURPOSE: Extensive follicle loss has been demonstrated in ovarian grafts post transplantation, reducing their productivity and lifespan. Several mechanisms for this loss have been proposed, and this study aims to clarify when and how the massive follicle loss associated with transplantation of ovarian tissue graft occurs. An understanding of the mechanisms of follicle loss will pinpoint potential new targets for optimization and improvement of this important fertility preservation technique. METHODS: Frozen-thawed marmoset (n = 15), bovine (n = 37), and human (n = 46) ovarian cortical tissue strips were transplanted subcutaneously into immunodeficient castrated male mice for 3 or 7 days. Histological (H&E, Masson's trichrome) analysis and immunostaining (Ki-67, GDF9, cleaved caspase-3) were conducted to assess transplantation-associated follicle dynamics, with untransplanted frozen-thawed tissue serving as a negative control. RESULTS: Evidence of extensive primordial follicle (PMF) activation and loss was observed already 3 days post transplantation in marmoset, bovine, and human tissue grafts, compared to frozen-thawed untransplanted controls (p < 0.001). No significant additional PMF loss was observed 7 days post transplantation. Recovered grafts of all species showed markedly higher rates of proliferative activity and progression from dormant to growing follicles (Ki-67 and GDF9 staining) as well as higher growing/primordial (GF/PMF) ratio (p < 0.02) and higher collagen levels compared with untransplanted controls. CONCLUSIONS: This multi-species study demonstrates that follicle activation plays an important role in transplantation-induced follicle loss, and that it occurs within a very short time frame after grafting. These results underline the need to prevent this activation at the time of transplantation in order to retain the maximal possible follicle reserve and extend graft lifespan.

Epigenetic germline mosaicism in infertile men.

Imprinted genes are expressed either from the paternal or the maternal allele, because the other allele has been silenced in the mother's or father's germline. Imprints are characterized by DNA methylation at cytosine phosphate guanine sites. Recently, abnormal sperm parameters and male infertility have been linked to aberrant methylation patterns of imprinted genes in sperm DNA. However, these studies did not account for possible epigenetic heterogeneity in sperm. We have investigated whether spermatozoa are a homogeneous cell population regarding DNA methylation of imprinted genes. Swim-up sperm was obtained from 45 men with normal (n = 19) and abnormal (n = 26) sperm parameters. DNA methylation of the imprinted gene KCNQ1OT1 was measured in multiple pools of 10 spermatozoa by a highly sensitive pyrosequencing-based oligo-sperm methylation assay (OSMA). DNA methylation of four imprinted genes (KCNQ1OT1, MEST, H19 and MEG3) was further analysed by deep bisulfite sequencing, which allows analysis at the single-cell level. Using OSMA, we found a significantly increased variation in the DNA methylation values of the maternally methylated gene KCNQ1OT1 in samples with abnormal sperm parameters. DBS showed that normozoospermic samples had a homogenous pattern of DNA methylation, whereas oligoasthenozoospermic samples contained discrete populations of spermatozoa with either normal or abnormal methylation patterns. Aberrant methylation of H19 appears to occur preferentially on the maternally inherited allele. Our results demonstrate the presence of epigenetic mosaicism in the semen of oligoasthenozoospermic men, which probably results from errors in imprint erasure.

Quantitative analysis of retinal perfusion in mice using optical coherence tomography angiography.

PURPOSE: To evaluate repeatability of the quantitative analysis of vessel density in the retinas of healthy mice using optical coherence tomography angiography (OCT-A). METHODS: Seventeen eyes of seventeen healthy mice aged 10-15 weeks (young) and 75-95 weeks (old) were included in this study. OCT-A was performed using RTVue XR Avanti (Optovue Inc., Fremont, California, USA) under general anaesthesia. The retina was imaged twice using a 3 × 3 mm2 scan. Retinal thickness and flow density data in the superficial and deep retinal OCT angiograms were extracted and analysed. RESULTS: The differences between the flow density values (whole en face) in the first and second sessions were non-significant (superficial retinal OCT angiogram: first session: 45.4 ± 4.1% (39.1-55.3%); second session: 46.1 ± 4.7% (39.1-59.1%); p = 0.14; deep retinal OCT angiogram: first session: 47.1 ± 3.8% (39.4-53.4%); second session: 47.3 ± 3.7% (39.4-53.8%); p = 0.50). The repeatability assessment of retinal thickness yielded intraclass correlation coefficient (ICC) values ranging between (0.86-0.99) while the ICCs for the flow density measurements ranged from 0.87 to 0.92 for the superficial retinal OCT angiogram and 0.68 to 0.93 for the deep retinal OCT angiogram. CONCLUSIONS: Repeated OCT-A measurement of mice retinal vessel density (VD) revealed valid repeatability, indicating that this non-invasive technology is sufficient for longitudinal assessment of vascular changes in various mouse models and thereby opening the way to in-depth, experimental analysis of the vascular aspects of different retinal diseases and monitoring of disease progression and the effects of treatments.

Gene conversion of the major histocompatibility complex class I Caja-G in common marmosets (Callithrix jacchus).

Currently, the amount of sequenced and classified MHC class I genes of the common marmoset is limited, in spite of the wide use of this species as an animal model for biomedical research. In this study, 480 clones of MHC class I G locus (Caja-G) cDNA sequences were obtained from 21 common marmosets. Up to 10 different alleles were detected in each common marmoset, leading to the assumption that the Caja-G loci duplicated in the marmoset genome. In the investigated population, four alleles occurred more often, giving evidence for higher immunological advantage of these alleles. In contrast to the human non-classical MHC class I genes, Caja-G shows high rates of polymorphism at the relevant peptide-binding sites, despite its phylogenetic relationship to the non-classical HLA-G. Our results provide information for better understanding of the immunological properties of the common marmoset and confirm the theory of a gene conversion of the Caja-G due to its detected plasticity and the absence of any known HLA-A equivalent.

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 Sertoli cell-specific connexin43 knockout leads to altered interstitial connexin expression and increased Leydig cell numbers.

The Sertoli cell (SC)-specific knockout (KO) of connexin43 (Cx43) results in spermatogenic arrest at the level of spermatogonia and/or SC-only syndrome. Histology of the interstitial compartment suggests Leydig cell (LC) hyperplasia. Our aim has been to investigate possible effects of the SC-specific KO of Cx43 (SCCx43KO) on interstitial LC. We therefore counted LC via the optical dissector method (per microliter of testicular tissue and per testis) and found LC to be significantly increased in SCCx43KO(-/-) compared with wild-type mice. Semiquantitative western blot together with Cx43 and 3ß-hydroxysteroid dehydrogenase immunohistochemistry showed that Cx43 protein was significantly reduced and barely detectable in LC in adult SCCx43KO(-/-) mice. This reduction of Cx43 protein was accompanied by a reduction of Cx43 mRNA as analyzed by laser-assisted microdissection of interstitial cells and subsequent quantitative real-time polymerase chain reaction (PCR). Interestingly, Cx45, another recently detected connexin in LC, was also downregulated. Preliminary qualitative data of LC differentiation markers (Thb2, Hsd3b6) and a steroidogenic marker (Hsd17b3) obtained by reverse transcription plus PCR revealed no obvious differences. Thus, the loss of Cx43 in SC also provokes the downregulation of connexins in interstitial LC at the transcriptional and translational levels. Moreover, SCCx43KO leads to alterations in LC numbers. Despite these alterations, steroidogenesis seems not to be impaired. Further studies, including ultrastructural analysis of the tissue as well as quantitative examination of additional LC markers and testosterone, and functional in vitro experiments, should provide more information about LC differentiation and function in SCCx43KO(-/-) mice.

Developmental expression patterns of chemokines CXCL11, CXCL12 and their receptor CXCR7 in testes of common marmoset and human.

The chemokine receptor CXCR7 interacts with the chemokines CXCL11 and CXCL12. During development, this ligand receptor system (C-X-C) provokes cell-type-specific responses in terms of migration, adhesion or ligand sequestration. It is active in zebrafish and rodents but no data are available for its presence or function in primate testes. Real-time quantitative polymerase chain reaction was performed in monkeys to detect CXCL11, CXCL12 and CXCR7. At the protein level, CXCL12 and CXCR7 were localized in the testes of the marmoset (Callitrix jacchus) whereas CXCR7 patterns were determined for various stages in human testes. Morphometry and flow cytometry were applied to quantify CXCR7-positive cells in monkeys. Transcript levels and protein expression of CXCR7 were detectable throughout testicular development. In both species, CXCR7 protein expression was restricted to premeiotic germ cells. In immature marmoset testes, 69.9% ± 9% of the total germ cell population were labelled for CXCR7, whereas in the adult, 4.7% ± 2.7% were positive for CXCR7. CXCL12 mRNA was detectable in all developmental stages in marmosets. The CXCL12 protein was exclusively localized to Sertoli cells. This pattern of CXCL12/CXCR7 indicates their involvement in regulatory processes that possibly orchestrate the interaction between undifferentiated germ cells and Sertoli cells.

Testicular Functions and Clinical Characterization of Patients with Gender Dysphoria (GD) Undergoing Sex Reassignment Surgery (SRS).

INTRODUCTION: Cross-sex hormone treatment of gender dysphoria (GD) patients changing from male to female a prerequisite for sex reassignment. For initial physical adaptation, a combined treatment of anti-androgens and estrogens is used. Provided that patients fulfill specific criteria, sex reassignment surgery (SRS) presents the final step toward physical adaptation. However, systematic studies analyzing effects of hormone treatment regimens are lacking. AIM: The aim of this study was to compare the effects of three different hormonal treatment strategies regarding endocrinological parameters and testicular histology. METHODS: Testicular tissues were obtained in a multicenter study from 108 patients on the day of SRS from three clinics following different treatment strategies. Patients either discontinued treatment 6 weeks (clinic A) or 2 weeks (clinic B) prior to SRS or not at all (clinic C). Testicular tissues, ethylenediaminetetraacetic acid blood and questionnaires were obtained on the day of SRS. MAIN OUTCOME MEASURES: Blood hormone and intratesticular testosterone (ITT) levels were measured. Testicular weight and histology were evaluated and the percentage of luteinizing hormone/choriogonadotropin receptor (LHCGR) positive cells was determined. RESULTS: According to the questionnaires, patients showed desired phenotypical changes including breast growth (75%) and smooth skin (32%). While patients from clinics A and B presented with rather virilized hormonal levels, patients from clinic C showed generally feminized blood serum levels. Histological evaluation revealed highly heterogeneous results with about 24% of patients presenting with qualitatively normal spermatogenesis. In accordance with serum endocrine profile, ITT levels were lowest in clinic C and correlated with testosterone and free testosterone, but not with the spermatogenic state. The percentage of LHCGR-positive cells and ITT levels did not correlate. CONCLUSION: Only patients that did not discontinue hormonal treatment showed feminized blood levels on the day of SRS. The ones who stopped re-virilized quickly. Interestingly, testicular histology was highly heterogeneous irrespective of the treatment strategy, a phenomenon that requires further investigation.

Sperm competition and the evolution of spermatogenesis.

Spermatogenesis is a long and complex process that, despite the shared overall goal of producing the male gamete, displays striking amounts of interspecific diversity. In this review, we argue that sperm competition has been an important selection pressure acting on multiple aspects of spermatogenesis, causing variation in the number and morphology of sperm produced, and in the molecular and cellular processes by which this happens. We begin by reviewing the basic biology of spermatogenesis in some of the main animal model systems to illustrate this diversity, and then ask to what extent this variation arises from the evolutionary forces acting on spermatogenesis, most notably sperm competition. We explore five specific aspects of spermatogenesis from an evolutionary perspective, namely: (i) interspecific diversity in the number and morphology of sperm produced; (ii) the testicular organizations and stem cell systems used to produce them; (iii) the large number and high evolutionary rate of genes underpinning spermatogenesis; (iv) the repression of transcription during spermiogenesis and its link to the potential for haploid selection; and (v) the phenomenon of selection acting at the level of the germline. Overall we conclude that adopting an evolutionary perspective can shed light on many otherwise opaque features of spermatogenesis, and help to explain the diversity of ways in which males of different species perform this fundamentally important process.

Separation of somatic and germ cells is required to establish primate spermatogonial cultures.

STUDY QUESTION: Can primate spermatogonial cultures be optimized by application of separation steps and well defined culture conditions? SUMMARY ANSWER: We identified the cell fraction which provides the best source for primate spermatogonia when prolonged culture is desired. WHAT IS KNOWN ALREADY: Man and marmoset show similar characteristics in regard to germ cell development and function. Several protocols for isolation and culture of human testis-derived germline stem cells have been described. Subsequent analysis revealed doubts on the germline origin of these cells and characterized them as mesenchymal stem cells or fibroblasts. Studies using marmosets as preclinical model confirmed that the published isolation protocols did not lead to propagation of germline cells. STUDY DESIGN, SIZE, DURATION: Testicular cells derived from nine adult marmoset monkeys (Callithrix jacchus) were cultured for 1, 3, 6 and 11 days and consecutively analyzed for the presence of spermatogonia, differentiating germ cells and testicular somatic cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: Testicular tissue of nine adult marmoset monkeys was enzymatically dissociated and subjected to two different cell culture approaches. In the first approach all cells were kept in the same dish (non-separate culture, n = 5). In the second approach the supernatant cells were transferred into a new dish 24 h after seeding and subsequently supernatant and attached cells were cultured separately (separate culture, n = 4). Real-time quantitative PCR and immunofluorescence were used to analyze the expression of reliable germ cell and somatic markers throughout the culture period. Germ cell transplantation assays and subsequent wholemount analyses were performed to functionally evaluate the colonization of spermatogonial cells. MAIN RESULTS AND THE ROLE OF CHANCE: This is the first report revealing an efficient isolation and culture of putative marmoset spermatogonial stem cells with colonization ability. Our results indicate that a separation of spermatogonia from testicular somatic cells is a crucial step during cell preparation. We identified the overgrowth of more rapidly expanding somatic cells to be a major problem when establishing spermatogonial cultures. Initiating germ cell cultures from the supernatant and maintaining germ cells in suspension cultures minimized the somatic cell contamination and provided enriched germ cell fractions which displayed after 11 days of culture a significantly higher expression of germ cell markers genes (DDX-4, MAGE A-4; P < 0.05) compared with separately cultured attached cells. Additionally, germ cell transplantation experiments demonstrated a significantly higher absolute number of cells with colonization ability (P < 0.001) in supernatant cells after 11 days of separate culture. LIMITATIONS, REASONS FOR CAUTION: This study presents a relevant aspect for the successful setup of spermatogonial cultures but provides limited data regarding the question of whether the long-term maintenance of spermatogonia can be achieved. Transfer of these preclinical data to man may require modifications of the protocol. WIDER IMPLICATIONS OF THE FINDINGS: Spermatogonial cultures from rodents have become important and innovative tools for basic and applied research in reproductive biology and veterinary medicine. It is expected that spermatogonia-based strategies will be transformed into clinical applications for the treatment of male infertility. Our data in the marmoset monkey may be highly relevant to establish spermatogonial cultures of human testes. STUDY FUNDING/COMPETING INTERESTS: Funding was provided by the DFG-Research Unit FOR 1041 Germ Cell Potential (SCHL394/11-2) and by the Graduate Program Cell Dynamics and Disease (CEDAD) together with the International Max Planck Research School - Molecular Biomedicine (IMPRS-MBM). The authors declare that there is no conflict of interest. TRIAL REGISTRATION NUMBER: Not applicable.

Germ cell loss is associated with fading Lin28a expression in a mouse model for Klinefelter's syndrome.

Klinefelter's syndrome is a male sex-chromosomal disorder (47,XXY), causing hypogonadism, cognitive and metabolic deficits. The majority of patients are infertile due to complete germ cell loss after puberty. As the depletion occurs during development, the possibilities to study the underlying causes in humans are limited. In this study, we used the 41,XX(Y*) mouse model to characterise the germ line postnatally. We examined marker expression of testicular cells focusing on the spermatogonial stem cells (SSCs) and found that the number of germ cells was approximately reduced fivefold at day 1pp in the 41,XX(Y*) mice, indicating the loss to start prenatally. Concurrently, immunohistochemical SSC markers LIN28A and PGP9.5 also showed decreased expression on day 1pp in the 41,XX(Y*) mice (48.5 and 38.9% of all germ cells were positive), which dropped to 7.8 and 7.3% on 3dpp, and were no longer detectable on days 5 and 10pp respectively. The differences in PCNA-positive proliferating cells in XY* and XX(Y*) mice dramatically increased towards day 10pp. The mRNA expression of the germ cell markers Lin28a (Lin28), Pou5f1 (Oct4), Utf1, Ddx4 (Vasa), Dazl, and Fapb1 (Sycp3) was reduced and the Lin28a regulating miRNAs were deregulated in the 41,XX(Y*) mice. We suggest a model for the course of germ cell loss starting during the intrauterine period. Neonatally, SSC marker expression by the already lowered number of spermatogonia is reduced and continues fading during the first postnatal week, indicating the surviving cells of the SSC population to be disturbed in their stem cell characteristics. Subsequently, the entire germ line is then generally lost when entering meiosis.