Germ Cell Loss Research Articles

Cisplatin and carboplatin result in similar gonadotoxicity in immature human testis with implications for fertility preservation in childhood cancer

BackgroundClinical studies indicate chemotherapy agents used in childhood cancer treatment regimens may impact future fertility. However, effects of individual agents on prepubertal human testis, necessary to identify later risk, have not been determined. The study aimed to investigate the impact of cisplatin, commonly used in childhood cancer, on immature (foetal and prepubertal) human testicular tissues. Comparison was made with carboplatin, which is used as an alternative to cisplatin in order to reduce toxicity in healthy tissues.MethodsWe developed an organotypic culture system combined with xenografting to determine the effect of clinically-relevant exposure to platinum-based chemotherapeutics on human testis. Human foetal and prepubertal testicular tissues were cultured and exposed to cisplatin, carboplatin or vehicle for 24 h, followed by 24–240 h in culture or long-term xenografting. Survival, proliferation and apoptosis of prepubertal germ stem cell populations (gonocytes and spermatogonia), critical for sperm production in adulthood, were quantified.ResultsCisplatin exposure resulted in a significant reduction in the total number of germ cells (− 44%, p < 0.0001) in human foetal testis, which involved an initial loss of gonocytes followed by a significant reduction in spermatogonia. This coincided with a reduction (− 70%, p < 0.05) in germ cell proliferation. Cisplatin exposure resulted in similar effects on total germ cell number (including spermatogonial stem cells) in prepubertal human testicular tissues, demonstrating direct relevance to childhood cancer patients. Xenografting of cisplatin-exposed human foetal testicular tissue demonstrated that germ cell loss (− 42%, p < 0.01) persisted at 12 weeks. Comparison between exposures to human-relevant concentrations of cisplatin and carboplatin revealed a very similar degree of germ cell loss at 240 h post-exposure.ConclusionsThis is the first demonstration of direct effects of chemotherapy exposure on germ cell populations in human foetal and prepubertal testis, demonstrating platinum-induced loss of all germ cell populations, and similar effects of cisplatin or carboplatin. Furthermore, these experimental approaches can be used to determine the effects of established and novel cancer therapies on the developing testis that will inform fertility counselling and development of strategies to preserve fertility in children with cancer.

Homeobox transcription factor Meis1 is crucial to Sertoli cell mediated regulation of male fertility.

Infertility has become a global phenomenon and constantly declining sperm count in males in modern world pose a major threat to procreation of humans. Male fertility is critically dependent on proper functioning of testicular Sertoli cells. Defective Sertoli cell proliferation and/or impaired functional maturation may be one of the underlying causes of idiopathic male infertility. Using high-throughput "omics" approach, wefoundbinding sites for homeobox transcription factor MEIS1 on the promoters of several genes up-regulated in pubertal (mature) Sertoli cells, indicating that MEIS1 may becrucialfor Sertoli cell-mediated regulation of spermatogenesis at and after puberty. To decipher the role of transcription factor MEIS1 in Sertoli cell maturation and spermatogenesis. Sc-specific Meis1 knockdown (KD) transgenic mice were generated using pronuclear microinjection. Morphometric and histological analysis of the testes from transgenic mice was performed to identify defects in spermatogenesis. Epididymal sperm count and litter size were analyzed to determine the effect of Meis1 knockdown on fertility. Sertoli cell (Sc)-specific Meis1 KD led to massive germ cell loss due to apoptosis and impaired spermatogenesis. Unlike normal pubertal Sc, the levels of SOX9 in pubertal Sc of Meis1 KD were significantly high, like immature Sc. A significant reduction in epididymal sperm count was observed in these mice. The mice were found to be infertile or sub-fertile (with reduced litter size), depending on the extent of Meis1 inhibition. The results of this study demonstrated for the first time, a role of Meis1 in Sc maturation and normal spermatogenic progression. Inhibition of Meis1 in Sc was associated with deregulated spermatogenesis and a consequent decline in fertility of the transgenic mice. Our results provided substantial evidence that suboptimal Meis1 expression in Sc may be one of the underlying causes of idiopathic infertility.

BNC1 Promotes Spermatogenesis by Regulating Transcription of Ybx2 and Papolb via Direct Binding to Their Promotor Elements.

BNC1 is a transcription factor that is crucial for spermatogenesis and male fertility, although the underlying mechanism remains unclear. To study BNC1's specific role in spermatogenesis, we characterized a previously developed mouse model carrying a truncating mutation in Bnc1 (termed Bnc1+/tr for heterozygotes and Bnc1tr/tr for homozygotes) and found that the mutation decreased BNC1 protein levels and resulted in germ cell loss by apoptosis. Given that loss of functional Bnc1 is known to result in decreased expression of the spermatogenesis genes Ybx2 and Papolb, we aimed to explore whether and how BNC1 promotes transcription of Ybx2 and Papolb to mediate its role in spermatogenesis. We confirmed significant reduction in YBX2 and PAPOLB protein levels in testis tissue from Bnc1+/tr and Bnc1tr/tr males compared with wild-type mice (Bnc1+/+). Consistently, knockdown of Bnc1 led to downregulation of Ybx2 and Papolb in CRL-2196 cells in vitro. To investigate if BNC1 directly induces Ybx2 and Papolb gene expression, chromatin immunoprecipitation using mouse testicular tissue and luciferase reporter assays in HEK293 cells were used to identify functional binding of BNC1 to the Ybx2 and Papolb promoters at defined BNC1 binding sites. Taken together, this study reveals a mechanism for BNC1's role in spermatogenesis by directly binding to BNC1 binding elements in the promoter regions of both Ybx2 and Papolb and inducing transcription of these important spermatogenesis genes.

Mutations in non-muscle myosin 2A disrupt the actomyosin cytoskeleton in Sertoli cells and cause male infertility

Mutations in non-muscle myosin 2A (NM2A) encompass a wide spectrum of anomalies collectively known as MYH9-Related Disease (MYH9-RD) in humans that can include macrothrombocytopenia, glomerulosclerosis, deafness, and cataracts. We previously created mouse models of the three mutations most frequently found in humans: R702C, D1424N, and E1841K. While homozygous R702C and D1424N mutations are embryonic lethal, we found homozygous mutant E1841K mice to be viable. However the homozygous male, but not female, mice were infertile. Here, we report that these mice have reduced testis size and defects in actin-associated junctions in Sertoli cells, resulting in inability to form the blood-testis barrier and premature germ cell loss. Moreover, compound double heterozygous (R702C/E1841K and D1424/E1841K) males show the same abnormalities in testes as E1841K homozygous males. Conditional ablation of either NM2A or NM2B alone in Sertoli cells has no effect on fertility and testis size, however deletion of both NM2A and NM2B in Sertoli cells results in infertility. Isolation of mutant E1841K Sertoli cells reveals decreased NM2A and F-actin colocalization and thicker NM2A filaments. Furthermore, AE1841K/AE1841K and double knockout Sertoli cells demonstrate microtubule disorganization and increased tubulin acetylation, suggesting defects in the microtubule cytoskeleton. Together, these results demonstrate that NM2A and 2B paralogs play redundant roles in Sertoli cells and are essential for testes development and normal fertility.

SOX13 gene downregulation in peripheral blood mononuclear cells of patients with Klinefelter syndrome.

Klinefelter syndrome (KS) is the most common sex chromosome disorder in men. It is characterized by germ cell loss and other variable clinical features, including autoimmunity. The sex-determining region of Y (SRY)-box 13 (Sox13) gene is expressed in mouse spermatogonia. In addition, it has been identified as islet cell autoantigen 12 (ICA12), which is involved in the pathogenesis of autoimmune diseases, including type 1 diabetes mellitus (DM) and primary biliary cirrhosis. Sox13 expression has never been investigated in patients with KS. In this age-matched, case–control study performed on ten patients with KS and ten controls, we found that SOX13 is significantly downregulated in peripheral blood mononuclear cells of patients with KS compared to controls. This finding might be consistent with the germ cell loss typical of patients with KS. However, the role of SoX13 in the pathogenesis of germ cell loss and humoral autoimmunity in patients with KS deserves to be further explored.

Cracd Marks the First Wave of Meiosis during Spermatogenesis and Is Mis-Expressed in Azoospermia Mice.

Testicular development starts in utero and maturation continues postnatally, requiring a cascade of gene activation and differentiation into different cell types, with each cell type having its own specific function. As we had previously reported that the Capping protein inhibiting regulator of actin (Cracd) gene was expressed in the adult mouse testis, herein we examine when and where the β-catenin associated Cracd is initially expressed during postnatal testis development. Significantly, Cracd mRNA is present in both the immature postnatal and adult testis in round spermatid cells, with highest level of expression occurring during the first wave of meiosis and spermatogenesis. In the juvenile testes, Cracd is initially expressed within the innermost region but as maturation occurs, Cracd mRNA switches to a more peripheral location. Thereafter, Cracd is downregulated to maintenance levels in the haploid male germ cell lineage. As Cracd mRNA was expressed within developing round spermatids, we tested its effectiveness as a biomarker of non-obstructive azoospermia using transgenic knockout mice models. Meaningfully, Cracd expression was absent in Deleted in azoospermia like (Dazl) null testis, which exhibit a dramatic germ cell loss. Moreover, Cracd was abnormally regulated and ectopically mis-expressed in Polypyrimidine tract binding protein-2 (Ptbp2) conditional germ cell restricted knockout testis, which exhibit a block during spermatid differentiation and a reduction in the number of late stage spermatocytes coincident with reduced β-catenin expression. Combined, these data suggest that Cracd is a useful first wave of spermatogenesis biomarker of azoospermia phenotypes, even prior to an overt phenotype being evident.

Paternal Exposure to Bisphenol-A Transgenerationally Impairs Testis Morphology, Germ Cell Associations, and Stemness Properties of Mouse Spermatogonial Stem Cells.

Bisphenol-A (BPA) exposure in an adult male can affect the reproductive system, which may also adversely affect the next generation. However, there is a lack of comprehensive data on the BPA-induced disruption of the association and functional characteristics of the testicular germ cells, which the present study sought to investigate. Adult male mice were administered BPA doses by gavage for six consecutive weeks and allowed to breed, producing generations F1–F4. Testis samples from each generation were evaluated for several parameters, including abnormal structure, alterations in germ cell proportions, apoptosis, and loss of functional properties of spermatogonial stem cells (SSCs). We observed that at the lowest-observed-adverse-effect level (LOAEL) dose, the testicular abnormalities and alterations in seminiferous epithelium staging persisted in F0–F2 generations, although a reduced total spermatogonia count was found only in F0. However, abnormalities in the proportions of germ cells were observed until F2. Exposure of the male mice (F0) to BPA alters the morphology of the testis along with the association of germ cells and stemness properties of SSCs, with the effects persisting up to F2. Therefore, we conclude that BPA induces physiological and functional disruption in male germ cells, which may lead to reproductive health issues in the next generation.

UXT in Sertoli cells is required for blood-testis barrier integrity†.

Spermatogenesis is a complex process that establishes male fertility and involves proper communication between the germline (spermatozoa) and the somatic tissue (Sertoli cells). Many factors that are important for spermatozoa production are also required for Sertoli cell function. Recently, we showed that the transcriptional cofactor ubiquitously expressed transcript (UXT) encodes a protein that is essential in germ cells for spermatogenesis and fertility. However, the role of UXT within Sertoli cells and how it affects Sertoli cell function was still unclear. Here we describe a novel role for UXT in the Sertoli cell's ability to support spermatogenesis. We find that the conditional deletion of Uxt in Sertoli cells results in smaller testis size and weight, which coincided with a loss of germ cells in a subset of seminiferous tubules. In addition, the deletion of Uxt has no impact on Sertoli cell abundance or maturity, as they express markers of mature Sertoli cells. Gene expression analysis reveals that the deletion of Uxt in Sertoli cells reduces the transcription of genes involved in the tight junctions of the blood-testis barrier (BTB). Furthermore, tracer experiments and electron microscopy reveal that the BTB is permeable in UXT KO animals. These findings broaden our understanding of UXT's role in Sertoli cells and its contribution to the structural integrity of the BTB.

H3K9 Demethylases JMJD1A and JMJD1B Control Prospermatogonia to Spermatogonia Transition in Mouse Germline.

SummaryHistone H3 lysine 9 (H3K9) methylation is dynamically regulated by methyltransferases and demethylases. In spermatogenesis, prospermatogonia differentiate into differentiating or undifferentiated spermatogonia after birth. However, the epigenetic regulation of prospermatogonia to spermatogonia transition is largely unknown. We found that perinatal prospermatogonia have extremely low levels of di-methylated H3K9 (H3K9me2) and that H3K9 demethylases, JMJD1A and JMJD1B, catalyze H3K9me2 demethylation in perinatal prospermatogonia. Depletion of JMJD1A and JMJD1B in the embryonic germline resulted in complete loss of male germ cells after puberty, indicating that H3K9me2 demethylation is essential for male germline maintenance. JMJD1A/JMJD1B-depleted germ cells were unable to differentiate into functional spermatogonia. JMJD1 isozymes contributed to activation of several spermatogonial stem cell maintenance genes through H3K9 demethylation during the prospermatogonia to spermatogonia transition, which we propose is key for spermatogonia development. In summary, JMJD1A/JMJD1B-mediated H3K9me2 demethylation promotes prospermatogonia to differentiate into functional spermatogonia by establishing proper gene expression profiles.

Testicular immune cells and vasculature in Klinefelter syndrome from childhood up to adulthood.

Is the distribution of immune cells and the testicular vasculature altered in testicular biopsies from patients with Klinefelter syndrome (KS)? Increased numbers of macrophages and mast cells, an increased expression of decorin and an increased blood vessel density were found in KS samples compared to controls. Most KS patients are infertile due to an early germ cell loss. From puberty onwards, testicular fibrosis can be detected. How this fibrotic process is initiated remains unknown. In this study, the number of macrophages, mast cells and their secretory products were evaluated in KS, Sertoli cell only (SCO) and control patient samples. The association between immune cell numbers and level of fibrosis in KS tissue was examined. In addition, the vascularization within these testicular tissue biopsies was studied. For immunohistochemical evaluation, KS patients at different stages of testicular development were included: prepubertal (aged 4-7 years; n = 4), peripubertal (aged 11-17 years; n = 21) and adult (aged >18 years; n = 37) patients. In addition, testicular tissue biopsies of adult SCO (n = 33) and control samples for the three KS age groups (prepubertal n = 9; peripubertal n = 5; adult n = 25) were analysed. Gene expression analysis was performed on adult testicular tissue from KS (n = 5), SCO (n = 5) and control (n = 5) patients. Adult (>18 years) KS, SCO and control testicular tissue biopsies were obtained during a testicular sperm extraction procedure. KS peripubertal (11-18 years), prepubertal (<11 years) and age-matched control biopsies were obtained from the biobank of the university hospital. Immunohistochemistry was used to determine the tubular structure (H/PAS), the number of spermatogonia (MAGE-A4), macrophages (CD68) and mast cells (tryptase) and the blood vessel density (Von Willebrand factor). In addition, quantitative real-time polymerase chain reaction was used to determine the expression of secretory products of macrophages and mast cells (tryptase, tumour necrosis factor alpha and decorin). A significant increase in the number of macrophages (P < 0.0001) and mast cells (P = 0.0008) was found in the peritubular compartment of testes of adult KS patients compared to control samples. However, no association between the number of immune cells and the degree of fibrosis was observed. In adult SCO samples, a significant increase was seen for peritubular macrophage (P < 0.0001) and mast cell (P < 0.0001) numbers compared to control samples. In the interstitial compartment, a significant increase in mast cell number was found in adult SCO samples compared to KS (P < 0.0001) and control (P < 0.0001) tissue. A significant difference (P = 0.0431) in decorin expression could be detected in adult KS compared to control patients. Decorin expression was mostly seen in the walls of the seminiferous tubules. When comparing the vascularization between KS patients and age-matched controls, a significant increase (P = 0.0081) in blood vessel density could be observed only in prepubertal KS testicular tissue. N/A. As controls for this study, testicular tissue biopsies of men who underwent a vasectomy reversal or orchiectomy were used, but these men may not represent fertile controls. In addition, a high variability in immune cell numbers, secretory products expression and number of blood vessels could be observed amongst all patient samples. Increased numbers of macrophages and mast cells have previously been described in non-KS infertile men. Our results show that these increased numbers can also be detected in KS testicular tissue. However, no association between the number of macrophages or mast cells and the degree of fibrosis in KS samples could be detected. Decorin has previously been described in relation to fibrosis, but it has not yet been associated with testicular fibrosis in KS. Our results suggest a role for this proteoglycan in the fibrotic process since an increased expression was observed in adult KS tissue compared to controls. Impaired vascularization in KS men was suggested to be responsible for the KS-related disturbed hormone levels. Our results show a significant difference in blood vessel density, especially for the smallest blood vessels, between prepubertal KS samples and age-matched controls. This is the first study to report differences between KS and control testicular tissue at prepubertal age. The project was funded by grants from the Vrije Universiteit Brussel (E.G.) and the scientific Fund Willy Gepts from the UZ Brussel (D.V.S.). D.V.S. is a post-doctoral fellow of the Fonds voor Wetenschappelijk Onderzoek (FWO; 12M2819N). No conflict of interest is declared for this research project.

Histological and Biochemical Studies of Germ Cell Toxicity in Male Rats Exposed to Sodium Benzoate

Background: One of the consequences of increasing urbanization is that many people eat processed foods that contain various chemical substances applied as additives. Some of them may have the ability to suppress male fertility.&#x0D; Aims: To determine the effects of sodium benzoate (NaB) on the histology of the testis, and biochemical and semen parameters in rats. The potential of Vitamin E to protect the testis was also studied.&#x0D; Methodology: Six groups of 8 rats each were treated with these substances: Group A (Control) had olive oil. Groups B and C had 200 mg/kg of NaB with Vitamin E added to group C. Groups D and E had 400 mg/kg of NaB with Vitamin E added to group E. Groups F and G were given NaB only at 200 mgkg and 400 mg/kg respectively but were left for an extra 4 weeks after the last treatment dose. All groups were treated daily for 8weeks. Outcome measures were testosterone assays and cell counts and morphometry in the testis. We also examined biochemical parameters such as catalase, glutathione and malondialdehyde levels.&#x0D; Results: This study showed that NaB impaired reproduction. Total sperm count in some treated groups were reduced to half (50%) of the count in control animals. There was reduction in anti-oxidant levels and elevation in markers of lipid peroxidation suggesting oxidative stress. There was histologic evidence of impaired spermatogenesis and considerable testicular damage with micrographs showing widespread germ cell loss and sloughing of germinal epithelium in many places. Vitamin E offered significant protection from testicular damage in the groups given the vitamin.&#x0D; Conclusion: We conclude that NaB has the potential to impair fertility in rats and more studies are needed to determine its safety level in the male reproductive system.

In vitro administration of sodium arsenite in mouse prepubertal testis induces germ cell loss and apoptosis

High levels of arsenic contamination in drinking water pose serious health risks in numerous countries. The documentation reporting arsenic toxicity on reproduction and development is increasing, with evidence of arsenic inducing fertility and developmental issues. Nonetheless, the impact of arsenic exposure on the development of the male reproductive system is not fully elucidated. In the present study, we have investigated the direct effects of arsenic on prepubertal mouse testis using an in vitro testicular organ culture system. Culture medium was supplemented with a range of concentrations of sodium arsenite, examining effects of low (0.5 and 1 μM) and high (10, 50, 100 μM) concentrations, in cultures of post-natal day 5 CD1 mouse testis. In vitro exposure of low arsenic concentrations (0.5 or 1 μM) for 6 days did not cause any change in the testicular morphology, germ cells density, or apoptotic marker cleaved caspase 3 (CC3) expression. In contrast, exposure of prepubertal testis to high arsenic concentrations (10, 50 or 100 μM) induced drastic changes: severe destruction of testicular morphology, with loss of seminiferous tubule integrity; a dose-dependent decrease in germ cell density, and a hundred-fold increase in CC3 expression after 50 μM arsenic exposure. In conclusion, high arsenic treatment induced a dose-dependent induction of apoptosis and germ cell loss in prepubertal mouse testis.

Reduced retinoic acid synthesis accelerates prophase I and follicle activation.

In female mammals, reproductive potential is determined during fetal life by the formation of a non-renewable pool of primordial follicles. Initiation of meiosis is one of the defining features of germ cell differentiation and is well established to commence in response to retinoic acid. WIN 18,446 inhibits the conversion of retinol to retinoic acid, and therefore it was used to explore the impact of reduced retinoic acid synthesis on meiotic progression and thus germ cell development and subsequent primordial follicle formation. e13.5 mouse fetal ovaries were cultured in vitro and treated with WIN 18,446 for the first 3 days of a total of up to 12 days. Doses as low as 0.01 µM reduced transcript levels of the retinoic acid response genes Stra8 and Rarβ without affecting germ cell number. Higher doses resulted in germ cell loss, rescued with the addition of retinoic acid. WIN 18,446 significantly accelerated the progression of prophase I; this was seen as early as 48 h post treatment using meiotic chromosome spreads and was still evident after 12 days of culture using Tra98/Msy2 immunostaining. Furthermore, ovaries treated with WIN 18,446 at e13.5 but not at P0 had a higher proportion of growing follicles compared to vehicle controls, thus showing evidence of increased follicle activation. These data therefore indicate that retinoic acid is not necessary for meiotic progression but may have a role in the regulation of its progression and germ cell survival at that time and provide evidence for a link between meiotic arrest and follicle growth initiation.

A dual death/survival role of autophagy in the adult ovary of Lagostomus maximus (Mammalia- Rodentia).

Follicular atresia is a cell death event that occurs in the great majority of follicles before ovulation in the mature mammalian ovary. Germ cell loss has been mainly associated to apoptosis although autophagy also seems to be at play. Aimed to increase our understanding on the possible cooperating role of autophagy and apoptosis in follicular atresia and/or follicular survival, we analyzed both programmed cell death mechanisms in a rodent model, the South American plains vizcacha, Lagostomus maximus. Female vizcacha shows highly suppressed apoptosis-dependent follicular atresia in the adult ovary, with continuous folliculogenesis and massive polyovulation. This strategy of massive ovulation requires a permanent remodeling of the ovarian architecture to maintain the availability of quiescent primordial follicles throughout the individual's reproductive lifespan. We report here our analysis of autophagy (BECN1, LAMP1 and LC3B-I/II) and apoptosis (BCL2 and ACTIVE CASPASE-3) markers which revealed interactive behaviors between both processes, with autophagy promoting survival or cell death depending on the ovarian structure. Strong BECN1, LC3B-II and LAMP1 staining was observed in atretic follicles and degenerating corpora lutea that also expressed nuclear ACTIVE CASPASE-3. Healthy follicles showed a slight expression of autophagy proteins but a strong expression of BCL2 and no detectable ACTIVE CASPASE-3. Transmission electron microscopy revealed a high formation of autophagosomes, autolysosomes and lysosomes in atretic follicles and degenerating corpora lutea and a low number of autophagic vesicles in normal follicles. The co-expression of LC3B-BECN1, LC3B-LAMP1 and LC3B-ACTIVE CASPASE-3 was only detected in atretic follicles and degenerating corpora lutea, while co-expression of BCL2-BECN1 was only observed in normal follicles. We propose that autophagy could act as a mechanism to eliminate altered follicles and remnant corpora lutea providing the necessary space for maturation of primordial follicles that continuously enter the growing follicular pool to sustain massive ovulation.

Integration and reanalysis of transcriptomics and methylomics data derived from blood and testis tissue of men with 47,XXY Klinefelter syndrome indicates the primary involvement of Sertoli cells in the testicular pathogenesis.

Klinefelter syndrome (KS; 47,XXY) is the most common sex chromosomal anomaly and causes a multitude of symptoms. Often the most noticeable symptom is infertility caused by azoospermia with testicular histology showing hyalinization of tubules, germ cells loss, and Leydig cell hyperplasia. The germ cell loss begins early in life leading to partial hyalinization of the testis at puberty, but the mechanistic drivers behind this remain poorly understood. In this systematic review, we summarize the current knowledge on developmental changes in the cellularity of KS gonads supplemented by a comparative analysis of the fetal and adult gonadal transcriptome, and blood transcriptome and methylome of men with KS. We identified a high fraction of upregulated genes that escape X-chromosome inactivation, thus supporting previous hypotheses that these are the main drivers of the testicular phenotype in KS. Enrichment analysis showed overrepresentation of genes from the X- and Y-chromosome and testicular transcription factors. Furthermore, by re-evaluation of recent single cell RNA-sequencing data originating from adult KS testis, we found novel evidence that the Sertoli cell is the most affected cell type. Our results are consistent with disturbed cross-talk between somatic and germ cells in the KS testis, and with X-escapee genes acting as mediators.

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.

Germ cell loss in Klinefelter syndrome: When and why?

Klinefelter syndrome (KS) is a quite common disorder with an incidence of 1-2 in 1,000 new-born males. Most patients are diagnosed in the light of a clinical checkup when consulting a fertility clinic with an unfulfilled child wish. Infertility in KS patients is caused by a massive germ cell loss, leading to azoospermia in more than 90% of the adult patients. Most seminiferous tubules in the adult KS testis are degenerated or hyalinized and testicular fibrosis can be observed, starting from puberty. However, focal spermatogenesis can be found in the testis of some patients. This offers the opportunity to extract spermatozoa from the testis by testicular sperm extraction (TESE). Nevertheless, TESE is only successful in about half of the KS adults seeking to father children. The reason for the germ cell loss remains unclear. To date, it is still debated whether the testicular tissue changes and the germ cell loss seen in KS is directly caused by an altered X-linked gene expression, the altered somatic environment, or a deficiency in the germ cells. In this review, we provide an overview of the current knowledge about the germ cell loss in KS patients.

A multi-organ-chip co-culture of liver and testis equivalents: a first step toward a systemic male reprotoxicity model.

Is it possible to co-culture and functionally link human liver and testis equivalents in the combined medium circuit of a multi-organ chip? Multi-organ-chip co-cultures of human liver and testis equivalents were maintained at a steady-state for at least 1week and the co-cultures reproduced specific natural and drug-induced liver-testis systemic interactions. Current benchtop reprotoxicity models typically do not include hepatic metabolism and interactions of the liver-testis axis. However, these are important to study the biotransformation of substances. Testicular organoids derived from primary adult testicular cells and liver spheroids consisting of cultured HepaRG cells and hepatic stellate cells were loaded into separate culture compartments of each multi-organ-chip circuit for co-culture in liver spheroid-specific medium, testicular organoid-specific medium or a combined medium over a week. Additional multi-organ-chips (single) and well plates (static) were loaded only with testicular organoids or liver spheroids for comparison. Subsequently, the selected type of medium was supplemented with cyclophosphamide, an alkylating anti-neoplastic prodrug that has demonstrated germ cell toxicity after its bioactivation in the liver, and added to chip-based co-cultures to replicate a human liver-testis systemic interaction in vitro. Single chip-based testicular organoids were used as a control. Experiments were performed with three biological replicates unless otherwise stated. The metabolic activity was determined as glucose consumption and lactate production. The cell viability was measured as lactate dehydrogenase activity in the medium. Additionally, immunohistochemical and real-time quantitative PCR end-point analyses were performed for apoptosis, proliferation and cell-specific phenotypical and functional markers. The functionality of Sertoli and Leydig cells in testicular spheroids was specifically evaluated by measuring daily inhibin B and testosterone release, respectively. Co-culture in multi-organ chips with liver spheroid-specific medium better supported the metabolic activity of the cultured tissues compared to other media tested. The liver spheroids did not show significantly different behaviour during co-culture compared to that in single culture on multi-organ-chips. The testicular organoids also developed accordingly and produced higher inhibin B but lower testosterone levels than the static culture in plates with testicular organoid-specific medium. By comparison, testosterone secretion by testicular organoids cultured individually on multi-organ-chips reached a similar level as the static culture at Day 7. This suggests that the liver spheroids have metabolised the steroids in the co-cultures, a naturally occurring phenomenon. The addition of cyclophosphamide led to upregulation of specific cytochromes in liver spheroids and loss of germ cells in testicular organoids in the multi-organ-chip co-cultures but not in single-testis culture. N/A. The number of biological replicates included in this study was relatively small due to the limited availability of individual donor testes and the labour-intensive nature of multi-organ-chip co-cultures. Moreover, testicular organoids and liver spheroids are miniaturised organ equivalents that capture key features, but are still simplified versions of the native tissues. Also, it should be noted that only the prodrug cyclophosphamide was administered. The final concentration of the active metabolite was not measured. This co-culture model responds to the request of setting up a specific tool that enables the testing of candidate reprotoxic substances with the possibility of human biotransformation. It further allows the inclusion of other human tissue equivalents for chemical risk assessment on the systemic level. This work was supported by research grants from the Scientific Research Foundation Flanders (FWO), Universitair Ziekenhuis Brussel (scientific fund Willy Gepts) and the Vrije Universiteit Brussel. Y.B. is a postdoctoral fellow of the FWO. U.M. is founder, shareholder and CEO of TissUse GmbH, Berlin, Germany, a company commercializing the Multi-Organ-Chip platform systems used in the study. The other authors have no conflict of interest to declare.

An exploration of the role of Sertoli cells on fetal testis development using cell ablation strategy.

Sertoli cells (SCs) are presumed to be the center of testis differentiation because they provide both structural support and biological regulation for spermatogenesis. Previous studies suggest that SCs control germ cell (GC) count and Leydig cell (LC) development in mouse testes. However, the regulatory role of SCs on peritubular myoid (PTM) cell fate in fetal testis has not been clearly reported. Here, we employed Amh-Cre; diphtheria toxin fragment A (DTA) mouse model to selectively ablate SCs from embryonic day (E) 14.5. Results found that SC ablation in the fetal stage caused the disruption of testis cords and the massive loss of GCs. Furthermore, the number of α-smooth muscle actin-labeled PTM cells was gradually decreased from E14.5 and almost lost at E18.5 in SC ablation testis. Interestingly, some Ki67 and 3β-HSD double-positive fetal LCs could be observed in Amh-Cre; DTA testes at E16.5 and E18.5. Consistent with this phenomenon, the messenger RNA levels of Hsd3b1, Cyp11a1, Lhr, Star and the protein levels of 3β-HSD and P450Scc were significantly elevated by SC ablation. SC ablation appears to induce ectopic proliferation of fetal LCs although the total LC number appeared reduced. Together, these findings bring us a better understanding of SCs' central role in fetal testis development.

Functional analysis of vasa/PL10-like genes in the ovary of Schistosoma mansoni

The RNA helicase Vasa plays a pivotal role in the development of the germ line. To decipher the functional roles of vasa/PL10-like genes in the human blood fluke Schistosoma mansoni, we performed RNA interference followed by the analysis of the ovary in the adult female. Double-stranded RNA targeting the schistosome vasa-like gene Smvlg1 reduced the volume of the ovary. Changes in morphology of the ovary were analysed using carmine red-staining of the parasites followed by a novel confocal laser scanning microscopy (CLSM)-based approach to control for natural autofluorescence in female schistosome tissues. The reduction in the ovary volume may have been promoted by the loss of germ cells. By contrast, significant differences were not apparent in the number of eggs produced or hatching rate of eggs laid by the female schistosomes transfected with Smvlg1-specific dsRNA. The findings suggested a role for S. mansoni vasa/PL10-like gene -1 in germ cell development within the schistosome ovary that might impact in the pathogenesis and disease transmission by this neglected tropical disease pathogen.