Prepubertal Testis Research Articles

Throughout in vitro first spermatogenic wave: Next-generation sequencing gene expression patterns of fresh and cryopreserved prepubertal mice testicular tissue explants.

Suitable cryopreservation procedures of pre-pubertal testicular tissue associated with efficient culture conditions are crucial in the fields of fertility preservation and restoration. In vitro spermatogenesis remains a challenging technical procedure to undergo a complete spermatogenesis.The number of haploid cells and more specifically the spermatic yield produced in vitro in mice is still extremely low compared to age-matched in vivo controls and this procedure has never yet been successfully transferred to humans. To evaluate the impact of in vitro culture and freezing procedure, pre-pubertal testicular mice testes were directly cultured until day 4 (D4), D16 and D30 or cryopreserved by controlled slow freezing then cultured until D30. Testes composed of a panel of 6.5 dpp (days postpartum), 10.5 dpp, 22.5 dpp, and 36.5 dpp mice were used as in vivo controls. Testicular tissues were assessed by histological (HES) and immunofluorescence (stimulated by retinoic acid gene 8, STRA8) analyses. Moreover, a detailed transcriptome evaluation study has been carried out to study the gene expression patterns throughout the first in vitro spermatogenic wave. Transcriptomic analyses reveal that cultured tissues expression profiles are almost comparable between D16 and D30; highlighting an abnormal kinetic throughout the second half of the first spermatogenesis during in vitro cultures. In addition, testicular explants have shown dysregulation of their transcriptomic profile compared to controls with genes related to inflammation response, insulin-like growth factor and genes involved in steroidogenesis. The present work first shows that cryopreservation had very little impact on gene expression in testicular tissue, either directly after thawing or after 30 days in culture. Transcriptomic analysis of testis tissue samples is highly informative due to the large number of expressed genes and identified isoforms. This study provides a very valuable basis for future studies concerning in vitro spermatogenesis in mice.

Ex vivo effects of 17β-estradiol on the prepubertal rat testis

Although it is well established that testis produces estrogens, their precise effect is not fully documented, particularly during the prepubertal period. In a previous in vivo study, we demonstrated that an exposure of prepubertal rats (15–30 days post-partum (dpp)) to 17β-estradiol (E2) delays the establishment of spermatogenesis. In order to characterize the mechanisms of action and the direct targets of E2 on the immature testis, we developed an organotypic culture model of testicular explants obtained from prepubertal rats (15, 20 and 25 dpp). To determine the involvement of nuclear estrogen receptors (ERs) in the effect of E2, particularly that of ESR1 which is the major ER expressed in the prepubertal testis, a pre-treatment with the full antagonist of this type of ERs (ICI 182.780) was performed. Histological analyses, gene expression studies and hormonal assays were conducted to investigate the effects of E2 on steroidogenesis- and spermatogenesis-related endpoints. Testicular explants from 15 dpp rats were unresponsive to E2 exposure while E2 effects were observed in those obtained from 20 and 25 dpp rats. An E2 exposure of testicular explants obtained from 20 dpp rats seemed to accelerate the establishment of spermatogenesis, whereas an E2 exposure of 25 dpp testicular explants induced a delay of this process. These effects could be related to the E2-induced modulation of steroidogenesis, and involved both ESR1-dependent and -independent mechanisms of action. Overall, this ex vivo study demonstrated differential age- and concentration-related effects of E2 on the testis during the prepubertal period.

Conserved Transcriptome Features Define Prepubertal Primate Spermatogonial Stem Cells as Adark Spermatogonia and Identify Unique Regulators.

Antineoplastic treatments for cancer and other non-malignant disorders can result in long-term or permanent male infertility by ablating spermatogonial stem cells (SSCs). SSC transplantation using testicular tissue harvested before a sterilizing treatment is a promising approach for restoring male fertility in these cases, but a lack of exclusive biomarkers to unequivocally identify prepubertal SSCs limits their therapeutic potential. To address this, we performed single-cell RNA-seq on testis cells from immature baboons and macaques and compared these cells with published data from prepubertal human testis cells and functionally-defined mouse SSCs. While we found discrete groups of human spermatogonia, baboon and rhesus spermatogonia appeared less heterogenous. A cross-species analysis revealed cell types analogous to human SSCs in baboon and rhesus germ cells, but a comparison with mouse SSCs revealed significant differences with primate SSCs. Primate-specific SSC genes were enriched for components and regulators of the actin cytoskeleton and participate in cell-adhesion, which may explain why the culture conditions for rodent SSCs are not appropriate for primate SSCs. Furthermore, correlating the molecular definitions of human SSC, progenitor and differentiating spermatogonia with the histological definitions of Adark/Apale spermatogonia indicates that both SSCs and progenitor spermatogonia are Adark, while Apale spermatogonia appear biased towards differentiation. These results resolve the molecular identity of prepubertal human SSCs, define novel pathways that could be leveraged for advancing their selection and propagation in vitro, and confirm that the human SSC pool resides entirely within Adark spermatogonia.

Testicular Juvenile Granulosa Cell Tumors Demonstrate Recurrent Loss of Chromosome 10 and Absence of Molecular Alterations Described in Ovarian Counterparts

Testicular juvenile granulosa cell tumors (JGCTs) are a rare type of sex cord–stromal tumor, accounting for <5% of all neoplasms of the prepubertal testis. Previous reports have demonstrated sex chromosome anomalies in a small subset of cases, but the molecular alterations associated with JGCTs remain largely undescribed. We evaluated 18 JGCTs using massive parallel DNA and RNA sequencing panels. The median patient age was <1 month (range, newborn to 5 months). The patients presented with scrotal or intra-abdominal masses/enlargement, and all underwent radical orchiectomy (17 unilateral and 1 bilateral). The median tumor size was 1.8 cm (range, 1.3-10.5 cm). Histologically, the tumors were purely cystic/follicular or mixed (ie, solid and cystic/follicular). All cases were predominantly epithelioid, with 2 exhibiting prominent spindle cell components. Nuclear atypia was mild or absent, and the median number of mitoses was 0.4/mm2 (range, 0-10/mm2). Tumors frequently expressed SF-1 (11/12 cases, 92%), inhibin (6/7 cases, 86%), calretinin (3/4 cases, 75%), and keratins (2/4 cases, 50%). Single-nucleotide variant analysis demonstrated the absence of recurrent mutations. RNA sequencing did not detect gene fusions in 3 cases that were sequenced successfully. Recurrent monosomy 10 was identified in 8 of 14 cases (57%) with interpretable copy number variant data, and multiple whole-chromosome gains were present in the 2 cases with significant spindle cell components. This study demonstrated that testicular JGCTs harbor recurrent loss of chromosome 10 and lack the GNAS and AKT1 variants described in their ovarian counterparts.

Comparing the adult and pre-pubertal testis: Metabolic transitions and the change in the spermatogonial stem cell metabolic microenvironment.

Survivors of childhood cancer often suffer from infertility. While sperm cryopreservation is not feasible before puberty, the patient's own spermatogonial stem cells could serve as a germ cell reservoir, enabling these patients to father their own children in adulthood through the isolation, in vitro expansion, and subsequent transplantation of spermatogonial stem cells. However, this approach requires large numbers of stem cells, and methods for successfully propagating spermatogonial stem cells in the laboratory are yet to be established for higher mammals and humans. The improvement of spermatogonial stem cell culture requires deeper understanding of their metabolic requirements and the mechanisms that regulate metabolic homeostasis. This review gives a summary on our knowledge of spermatogonial stem cell metabolism during maintenance and differentiation and highlights the potential influence of Sertoli cell and stem cell niche maturation on spermatogonial stem cell metabolic requirements during development. Fetal human spermatogonial stem cell precursors, or gonocytes, migrate into the seminiferous cords and supposedly mature to adult stem cells within the first year of human development. However, the spermatogonial stem cell niche does not fully differentiate until puberty, when Sertoli cells dramatically rearrange the architecture and microenvironment within the seminiferous epithelium. Consequently, pre-pubertal and adult spermatogonial stem cells experience two distinct niche environments potentially affecting spermatogonial stem cell metabolism and maturation. Indeed, the metabolic requirements of mouse primordial germ cells and pig gonocytes are distinct from their adult counterparts, and novel single-cell RNA sequencing analysis of human and porcine spermatogonial stem cells during development confirms this metabolic transition. Knowledge of the metabolic requirements and their changes and regulation during spermatogonial stem cell maturation is necessary to implement laboratory-based techniques and enable clinical use of spermatogonial stem cells. Based on the advancement in our understanding of germline metabolism circuits and maturation events of niche cells within the testis, we propose a new definition of spermatogonial stem cell maturation and its amendment in the light of metabolic change.

Germ Cell Maintenance and Sustained Testosterone and Precursor Hormone Production in Human Prepubertal Testis Organ Culture with Tissues from Boys 7 Years+ under Conditions from Adult Testicular Tissue.

Human prepubertal testicular tissues are rare, but organ culture conditions to develop a system for human in vitro-spermatogenesis are an essential option for fertility preservation in prepubertal boys subjected to gonadotoxic therapy. To avoid animal testing in line with the 3Rs principle, organ culture conditions initially tested on human adult testis tissue were applied to prepubertal samples (n = 3; patient ages 7, 9, and 12 years). Tissues were investigated by immunostaining and transmission electron microscopy (TEM), and the collected culture medium was profiled for steroid hormones by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Culture conditions proved suitable for prepubertal organ culture since SSCs and germ cell proliferation could be maintained until the end of the 3-week-culture. Leydig cells (LCs) were shown to be competent for steroid hormone production. Three additional testis tissues from boys of the same age were examined for the number of germ cells and undifferentiated spermatogonia (SPG). Using TEM micrographs, eight tissues from patients aged 1.5 to 13 years were examined, with respect to the sizes of mitochondria (MT) in undifferentiated SPG and compared with those from two adult testicular tissues. Mitochondrial sizes were shown to be comparable between adults and prepubertal boys from approximately 7 years of age, which suggests the transition of SSCs from normoxic to hypoxic metabolism at about or before this time period.

Molecular phenotyping of domestic cat (Felis catus) testicular cells across postnatal development – A model for wild felids

Molecular characterisation of testicular cells is a pivotal step towards a profound understanding of spermatogenesis and developing assisted reproductive techniques (ARTs) based on germline preservation. To enable the identification of testicular somatic and spermatogenic cell types in felids, we investigated the expression of five molecular markers at the protein level in testes from domestic cats (Felis catus) at different developmental phases (prepubertal, pubertal I and II, postpubertal I and II) classified by single-cell ploidy analysis. Our findings indicate a prominent co-labelling for two spermatogonial markers, UCHL1 and FOXO1, throughout postnatal testis development. Smaller subsets of UCHL1 or FOXO1 single-positive spermatogonia were also evident, with the FOXO1 single-positive spermatogonia predominantly observed in prepubertal testes. As expected, DDX4+ germ cells increased in numbers beginning in puberty, reaching a maximum at adulthood (post-pubertal phase), corresponding to the sequential appearance of labelled spermatogonia, spermatocytes and spermatids. Furthermore, we identified SOX9+ Sertoli cells and CYP17A1+ Leydig cells in all of the developmental groups. Importantly, testes of African lion (Panthera leo), Sumatran tiger (Panthera tigris sumatrae), Chinese leopard (Panthera pardus japonesis) and Sudan cheetah (Acinonyx jubatus soemmeringii) exhibited conserved labelling for UCHL1, FOXO1, DDX4, SOX9 and CYP17A1. The present study provides fundamental information about the identity of spermatogenic and somatic testicular cell types across felid development that will be useful for developing ART approaches to support endangered felid conservation.

Bone morphogenetic protein 4 inhibits rat stem/progenitor Leydig cell development and regeneration via SMAD-dependent and SMAD-independent signaling

Bone morphogenetic protein 4 (BMP4) is an important member of the transforming growth factor-β superfamily. BMP4 is expressed in the Leydig cell lineage. We hypothesized that BMP4 might regulate the development of stem/progenitor Leydig cells. The BMP4 receptors, BMPR1A, BMPR1B, and BMPR2 were found to be expressed in progenitor Leydig cells of prepubertal testis and isolated cells. BMP4 at 1 and 10 ng/mL significantly reduced androgen production and down-regulated steroidogenesis-related gene and protein expression possibly by activating the SMAD signaling pathway (increasing SMAD1/5 phosphorylation and SMAD4) at 24 h treatment. BMP4 at 0.1 ng/mL and higher concentrations markedly reduced the EdU labeling index of CD90+ stem Leydig cells after 24 h treatment and significantly reduced the number of EdU+ stem Leydig cells on the surface of seminiferous tubules after 7 days of culture. BMP4 at 0.01 ng/mL and higher concentrations significantly blocked the differentiation of stem Leydig cells into adult cells, as shown by the reduction of testosterone secretion and the downregulation of Lhcgr, Scarb1, Cyp11a1, Hsd11b1, and Insl3 and their function after 3D seminiferous tubule culture for 3 weeks, and this effect was reversed by co-treatment with the BMP4 antagonists noggin and doxomorphine. In addition, BMP4 also blocked stem Leydig cell differentiation through SMAD-independent signaling pathways (ERK1/2 and AMPK). Ethanedimethane sulfonate (EDS) single injection can result in reduction of testosterone, restoration can happen post treatment. In an in vivo model of Leydig cell regeneration following EDS treatment, intratesticular injection of BMP4 from day 14 to day 28 post-elimination significantly reduced serum testosterone levels and down-regulated the expression of Scarb1, Star, Hsd11b1, and Insl3 and its proteins, possibly through SMAD-dependent and SMAD-independent (ERK1/2 and AMPK) signaling pathways. In conclusion, BMP4 is expressed in cells of the Leydig cell lineage and blocks entry of stem/progenitor Leydig cells into adult Leydig cells through SMAD-dependent and SMAD-independent signaling pathways.

Single-cell RNA sequencing uncovers dynamic roadmap and cell-cell communication during buffalo spermatogenesis

Spermatogenesis carries the task of precise intergenerational transmission of genetic information from the paternal genome and involves complex developmental processes regulated by the testicular microenvironment. Studies performed mainly in mouse models have established the theoretical basis for spermatogenesis, yet the wide interspecies differences preclude direct translation of the findings, and farm animal studies are progressing slowly. More than 32,000 cells from prepubertal (3-month-old) and pubertal (24-month-old) buffalo testes were analyzed by using single-cell RNA sequencing (scRNA-seq), and dynamic gene expression roadmaps of germ and somatic cell development were generated. In addition to identifying the dynamic processes of sequential cell fate transitions, the global cell-cell communication essential to maintain regular spermatogenesis in the buffalo testicular microenvironment was uncovered. The findings provide the theoretical basis for establishing buffalo germline stem cells invitro or culturing organoids and facilitating the expansion of superior livestock breeding.

ODP373 A Tale of Two Syndromes: Coexistence of Russell-Silver and Klinefelter Syndrome

Abstract Background/Aims Klinefelter syndrome is a common condition characterized by an extra X chromosome, tall stature, gynecomastia, and hypogonadism. Russell-Silver syndrome is a rare disorder characterized by intrauterine growth restriction, poor growth after birth, and triangular facies. The coexistence of Russell-Silver and Klinefelter syndrome has so far not been reported in the literature. This study aimed to characterize the phenotype of such an individual. Methods Clinical and endocrinal investigations were performed and followed by pathogenic variant screening of candidate genes. Results An 8-year-old boy was evaluated for short stature. He was a former 27-week preemie who had postnatal growth failure. His mean parental height was5'6" (66 inches). His growth chart showed that he was below the 0. 01% percentile in both height and weight for age. He had behavioral issues. On physical exam, he had high riding prepubertal testes and proportional limbs. He was evaluated by both genetics and endocrine. His bone age was delayed by 4 years. He passed his growth hormone stimulation test. He had a nondiagnostic bone survey. His chromosomal microarray showed an XXY karyotype. Further testing was collected because it did not explain his short stature. He was found to have maternal uniparental disomy of 7q32.2. He was started on growth hormone and his most recent growth velocity was 4 cm a year. He continues to be below the 0. 01% percentile in both height and weight for age Conclusion: This is the first report on a patient with the coexistence of Russell-Silver and Klinefelter syndrome. It shows that the short stature and postnatal growth of Russell-Silver syndrome is the prevailing phenotype in this rare combination. Presentation: No date and time listed

Low-dose every-second-day LHRH treatment following bilateral orchidopexy in children with bilateral cryptorchidism may improve their fertility outcome

Currently the standard treatment for bilateral cryptorchidism is bilateral surgical orchidopexy. Whether a hormonal treatment should be routinely administered postoperatively to increase fertility is debatable. Low-dose postoperative luteinizing hormone releasing hormone (LHRH) can increase spermatogonial numbers, but the effect of native LHRH (Kryptocur®) on adult fertility is unclear. To determine if low-dose every-second-day postoperative LHRH administration in children with bilateral cryptorchidism improves fertility in adulthood and if Nistal testicular histological grading could guide the decision to administer LHRH. All patients, actually at least 16yr of age, that underwent a bilateral orchidolysis and orchidopexy for bilateral cryptorchidism (surgery between 1997 and 2018) were contacted and offered a clinical exam, hormone levels, sperm analysis, and a scrotal ultrasound. At the original surgery, testicular biopsy was performed (if 60% of the tubuli contain >1 spermatogonia, this is normal=Nistal-1, if 30-60% filled=Nistal-2, if <30%=Nistal-3 and if Sertoli only=Nistal-4) and if in at least one testis impaired. A low dose native LHRH treatment was offered to the patients, as this treatment is known to increase the number of spermatogonia in a short term. Kryptocur® (LHRH, Gonadorelin, Hoechst®) was prescribed and dosed at 200μg (one spray in one nostril) every other day for 6-8 months. Forty-two men were eligible for this study. 20/42 accepted the invitation for a clinical and hormonal evaluation. 16/20 men accepted the invitation for an additional sperm analysis. Fourteen of 20 men received low-dose LHRH postoperatively in a nonrandomized manner. Three men had Nistal grade 1, eight grade 2, seven grade 3, and two had grade 4. Inhibin B levels were higher in men with Nistal 1 and 2 compared with Nistal 3 and 4P≤0.037). Severe oligospermia/azoospermia (<1×106/ejaculate) was observed in 33% of the treated group vs 67% of the untreated group (P≤0.036.) DISCUSSION AND CONCLUSIONS: Low-dose every-second-day postoperative LHRH treatment improves fertility outcome in bilateral cryptorchidism. Histological analysis of prepubertal testes according to Nistal grading cannot be used as a predictive diagnostic test for LHRH treatment.

Localization and characterization of SSCs from pre-pubertal bovine testes

Self renewal and proliferation ability of spermatogonial stem cells (SSCs) support spermatogenesis during adult life. Theoretically, these stem cells can be utilized for transmission of genetic information to descendants via testicular transplantation. However, lack of knowledge in methodologies for identification of SSCs limits the application of SSCs transplantation in domestic animals. Accumulated studies have shown that SSCs specific markers (DBA, UCHL1) and stem cell marker (Sox2, Oct4) are useful to screen SSCs that able to be used for transplantation. However, in cattle, less information is available on the expression status of these markers till date. Therefore, a study was carried out in 2019 at Tsukuba University, Japan where testes from 3, 5 and 7 months old calves were utilized to examine testicular localization and in vitro propogation of stem cell markers. SSCs were isolated by enzymatic digestion combined with centrifugal separation on discontinuous Percoll density gradient. Cell propagation and SSCs marker expression were determined at 5, 10 and 15 days post-culture. Immunostaining in conjunction with Western Blot analysis of cultured cells showed that stem cell markers (UCHL1, Oct4 and Sox2) were expressed in SSCs suggesting that differentiation of gonocyte started by 3 months and SSCs differentiation begins after 5 months of age. Taken together, these results demonstrated marker expression and localization of bull SSCs and showed that in vitro culturing of bull SSCs is implementable.

In Children with 46, XY DSD; HCG Testing is not always the Best Answer for Testicular Function Assessment

Hormonal levels are the hallmark for the assessment of testicular function in XY DSD (Disorders of sex development). Traditionally, it has relied on testosterone level increment after hCG (human Chorionic Gonadotropin) stimulation testing. More recently role of Sertoli cell hormones is more emphasized. Objectives: Evaluating the role of serum anti-mullerian hormone and inhibin B on function of the pre-pubertal testis without the need for hCG stimulation test. Method: The study was conducted in the Endocrinology Clinic in Alexandria University Children’s Hospital. All patients who present with XY DSD were tested for Testosterone (T), Dihydrotestosterone (DHT), Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH), Anti-Mullerian Hormone (AMH), inhibin B. All cases had hCG stimulation test. Results: The hCG stimulation test was done for 32 cases. There was significant positive correlation between serum testosterone levels before and after hCG stimulation test (p &lt;0.001). Similarly, significant correlation was identified between basal AMH and testosterone increment after hCG stimulation (p &lt;0.001) and between basal levels of AMH and inhibin (MCp= 0.025). Conclusion: Single measurement of basal AMH and/or inhibin B can detect the presence and function of testes by a reliable non-invasive way. Basal AMH assessment is an important tool to distinguish between cryptorchidism and anorchia. hCG test is needed in the work-up of patients with inconclusive results.

Testicular Expression of Antioxidant Enzymes and Changes in Response to a Slow-Release Deslorelin Implant (Suprelorin® 4.7 mg) in the Dog.

Simple SummaryWe investigated the expression of the testicular antioxidant enzyme system, i.e., superoxide dismutase (SOD1 and 2), catalase (CAT), glutathione peroxidase (GPx1), and glutathione disulfide reductase (GSR), in healthy adult and prepubertal dogs and after slow-release GnRH superagonist deslorelin (Suprelorin® 4.7 mg subcutaneous implant, Virbac, France) treatment in adult dogs. qPCR and immunohistochemistry were used to detect gene expression and protein expression and localization, respectively. These antioxidant enzymes were variably expressed within cellular compartments in the seminiferous epithelium and the interstitium. SOD1, CAT, and GSR protein expression were also dependent on the stage of the seminiferous epithelium cycle in normal adult dogs. Deslorelin treatment and prepubertal status affected the expression and localization of the investigated antioxidant enzymes. Our results confirmed the presence of a functional antioxidant enzyme system in the canine testis, which was also observed during deslorelin-induced spermatogenic and steroidogenic arrest, as well as in prepubertal dogs. These results improve our current understanding of the local antioxidant protective mechanisms within the testis in the dog. Spermatogenesis takes place in a hypoxic environment, and antioxidant enzymes protect germ and somatic cells from free radical-mediated damage. Expression of the antioxidant enzyme system in the canine testis has not yet been investigated. We hypothesized that the slow-release GnRH superagonist deslorelin 4.7 mg implant, which induces temporary reversible suppression of endocrine and germinative testicular function, would affect the testicular expression of antioxidant enzymes compared to untreated adult and prepubertal dogs. The goal of this study was to investigate and compare gene (by qPCR, in whole-tissue homogenates) and protein expression (by immunohistochemistry) of superoxide dismutase (SOD1, SOD2), catalase (CAT), glutathione peroxidase (GPx1), and glutathione disulfide reductase (GSR) in the testes of untreated adult (CON, n = 7), prepubertal (PRE, n = 8), and deslorelin-treated (DES, n = 5, 16 weeks after implantation) dogs. We found that in DES dogs, the gene expression of SOD1 was significantly (p < 0.05) lower and GPx1 was higher than in CON, and SOD2 was higher than in PRE. Expression of all, except for the SOD2 mRNA, differed between the CON and PRE dogs. Immunohistochemistry showed distinct cell-specific localization and expression patterns for the antioxidant enzymes in each experimental group. Additionally, in the CON animals, cell-specific SOD1, CAT, and GSR expression was dependent on the stage of the seminiferous epithelium cycle. These findings confirm that members of the antioxidant enzyme system are present in normal adult and prepubertal testis as well as in the deslorelin-treated downregulated adult canine testis, and that this local antioxidant system protects developing germ cells and somatic cells from oxidative damage. Different expression patterns of antioxidant enzymes in various germ cell populations and stages of the seminiferous epithelium cycle may indicate differences in their susceptibility to oxidative stress depending on their developmental and maturation stage. The continued presence of the antioxidant enzymes in the testis of DES dogs offers protection to spermatogonia as well as Sertoli and Leydig cells from oxidative stress during temporary infertility, potentially contributing to ensure the reversibility of suppression and the return of normal spermatogenesis and steroidogenesis after the end of deslorelin treatment.

MeDIP-seq and RNA-seq analysis during porcine testis development reveals functional DMR at the promoter of LDHC

Testis development requires tight regulation of gene expression programmed by epigenetic modifiers. However, their mechanism remains to be elucidated. Here, we investigated the genome-wide DNA methylation landscape in the Duroc and Meishan boar testes using methylated DNA immunoprecipitation sequencing (MeDIP-seq). We identified over 1100 promoter differential methylation genes (DMGs) before and after puberty, most of which are associated with testis development. Furthermore, we discovered that the expression of lactate dehydrogenase C (LDHC) gene during testis development is regulated by DNA methylation. The promoter of LDHC in pre-pubertal testes is substantially methylated, whereas considerably demethylated in post-pubertal testes. Artificial demethylation with the demethylating agent 5-Aza-CdR induced LDHC expression in immature Sertoli cells (SCs). Mechanistically, we confirmed the transcription factor SP1 was recruited to bind in hypomethylated differentially methylated regions (DMRs) in LDHC promoter, which upregulated the expression of LDHC. Functionally, we demonstrated that LDHC was activated in mature SCs (mSCs) and its overexpression significantly increases lactate secretion in SCs. In conclusion, our results highlight the function and regulation of dynamic DNA methylation in testis development.

Mesenchymal stem cells promote spermatogonial stem/progenitor cell pool and spermatogenesis in neonatal mice in vitro

Prepubertal cancer treatment leads to irreversible infertility in half of the male patients. Current in vitro spermatogenesis protocols and cryopreservation techniques are inadequate to expand spermatogonial stem/progenitor cells (SSPC) from testicles. Bone marrow derived mesenchymal stem cells (BM-MSC) bearing a close resemblance to Sertoli cells, improved spermatogenesis in animal models. We asked if a co-culture setup supported by syngeneic BM-MSC that contributes to the air–liquid interphase (ALI) could lead to survival, expansion and differentiation of SSPCs in vitro. We generated an ALI platform able to provide a real-time cellular paracrine contribution consisting of syngeneic BM-MSCs to neonatal C57BL/6 mice testes. We aimed to evaluate the efficacy of this culture system on SSPC pool expansion and spermatogenesis throughout a complete spermatogenic cycle by measuring the number of total germ cells (GC), the undifferentiated and differentiating spermatogonia, the spermatocytes and the spermatids. Furthermore, we evaluated the testicular cell cycle phases, the tubular and luminal areas using histochemical, immunohistochemical and flow cytometric techniques. Cultures in present of BM-MSCs displayed survival of ID4(+) spermatogonial stem cells (SSC), expansion of SALL4(+) and OCT4(+) SSPCs, VASA(+) total GCs and Ki67(+) proliferative cells at 42 days and an increased number of SCP3(+) spermatocytes and Acrosin(+) spermatids at 28 days. BM-MSCs increased the percentage of mitotic cells within the G2-M phase of the total testicular cell cycle increased for 7 days, preserved the cell viability for 42 days and induced testicular maturation by enlargement of the tubular and luminal area for 42 days in comparison to the control. The percentage of PLZF(+) SSPCs increased within the first 28 days of culture, after which the pool started to get smaller while the number of spermatocytes and spermatids increased simultaneously. Our findings established the efficacy of syngeneic BM-MSCs on the survival and expansion of the SSPC pool and differentiation of spermatogonia to round spermatids during in vitro culture of prepubertal mice testes for 42 days. This method may be helpful in providing alternative cures for male fertility by supporting in vitro differentiated spermatids that can be used for round spermatid injection (ROSI) to female oocyte in animal models. These findings can be further exploited for personalized cellular therapy strategies to cure male infertility of prepubertal cancer survivors in clinics.

Delayed Consequences of the Toxic Effect of Paclitaxel on the Testes of Prepubertal Rats and Their Correction with p-Tyrosol.

Delayed gonadotoxic effects were revealed in outbred male sexually mature rats (SD) after exposure to paclitaxel in the prepubertal period, and the possibility of their correction with p-tyrosol was shown. It was found, that administration of paclitaxel does not inhibit the ability of animals to conceive, but impairs the reserve capacity of the testicular tissue. In intact female rats crossed with male rats receiving paclitaxel, increased post-implantation fetal death was observed. Combined administration of paclitaxel and p-tyrosol alleviated the delayed effects of the cytostatic treatment on the prepubertal testis.

Achievement of complete in vitro spermatogenesis in testicular tissues from prepubertal mice exposed to mono- or polychemotherapy

The assessment of the impact of chemotherapies on in vitro spermatogenesis in experimental models is required before considering the application of this fertility restoration strategy to prepubertal boys who received these treatments before testicular tissue cryopreservation. The present work investigated the effects of exposure of prepubertal mice to mono- (vincristine or cyclophosphamide) and polychemotherapy (a combination of vincristine and cyclophosphamide) on the first wave of in vitro spermatogenesis. When testicular tissue exposed to monochemotherapy was preserved, polychemotherapy led to severe alterations of the seminiferous epithelium and increased apoptosis in prepubertal testes prior in vitro maturation, suggesting a potential additive gonadotoxic effect. These alterations were also found in the testicular tissues of polychemotherapy-treated mice after 30 days of organotypic culture and were associated with a reduction in the germ cell/Sertoli cell ratio. The different treatments neither altered the ability of spermatogonia to differentiate in vitro into spermatozoa nor the yield of in vitro spermatogenesis. However, more spermatozoa with morphological abnormalities and fragmented DNA were produced after administration of polychemotherapy. This work therefore shows for the first time the possibility to achieve a complete in vitro spermatogenesis after an in vivo exposure of mice to a mono- or polychemotherapy before meiotic entry.

Comparative Efficiency for in vitro Transfection of Goat Undifferentiated Spermatogonia Using Lipofectamine Reagents and Electroporation.

IntroductionSpermatogonial stem cells (SSC), also referred to as undifferentiated spermatogonia, are the germline stem cells responsible for continuous spermatogenesis throughout a male’s life. They are, therefore, an ideal target for gene editing. Previously, SSC from animal testis have been isolated and transplanted to homologous recipients resulting in the successful reestablishment of donor-derived spermatogenesis.MethodsEnhanced green fluorescent protein (eGFP) gene transfection into goat SSC was evaluated using liposomal carriers and electroporation. The cells were isolated from the prepubertal Galla goats testis cultured in serum-free defined media and transfected with the eGFP gene. Green fluorescing of SSC colonies indicated transfection.ResultsThe use of lipofectamineTM stem reagent and lipofectamineTM 2000 carriers resulted in more SSC colonies expressing the eGFP gene (25.25% and 22.25%, respectively). Electroporation resulted in 15% ± 0.54 eGFP expressing SSC colonies. Furthermore, cell viability was higher in lipofectamine transfection (55% ± 0.21) as compared to electroporation (38% ± 0.14).ConclusionThese results indicated that lipofectamine was more effective in eGFP gene transfer into SSC. The successful transient transfection points to a possibility of transfecting transgenes into male germ cells in genetic engineering programs.

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

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