Sertoli Cell Maturation Research Articles

Puberty induction in boys with congenital isolated hypogonadotropic hypogonadism

Gonadotropin therapy in boys with congenital isolated hypogonadotropic hypogonadism helps to increase testes volume and induce spermatogenesis in comparison with testosterone therapy. However, difficulties with dose titration, partial therapy success, absence of generally accepted regimen protocols don't allow to use this therapy in order to induce puberty in adolescents with Kallmann syndrome or normosmic hypogonadotropic hypogonadism. To assess the effectiveness of combination hormonal replacement therapy via human chorionic gonadotropin and recombinant follicle stimulation hormone in adolescents with congenital isolated normosmic hypogonadotropic hypogonadism and with Kallmann syndromeMATERIALS AND METHODS: This is an open single-center prospective non-controlled study. Boys with hypogonadotropic hypogonadism were receiving hormonal replacement therapy for 12 months. Initial dose of human chorionic gonadotropin was 500 IU per week. Initial dose of recombinant follicle stimulation hormone was 37.5 IU per week. Doses were doubled in 6 months. Antropometric data, Tanner stage, testes volumes, inhibin B and anti-Mullerian hormone (AMH) levels were evaluated in all the patients before the treatment, after 6 and 12 months of the therapy. 8 boys with hypogonadotropic hypogonadism were included into the study. Median age before therapy initiation was 15.7 years [15.33; 16.41]. In 12 months after the therapy initiation puberty development, testosterone increase from 0.44 [0.34;0.62] to 4.39 [0.88;10.51] nmol/l (p=0.012), AMH decrease from 35.70 [18.00;59.00] to 14.41 [11.60;16.65] ng/ml were noted in all the patients (p=0.017). Testes volumes increase and inhibin B level increase were not statistically significant. Gonadotropin therapy is effective in order to puberty initiation in adolescents with congenital hypogonadotropic hypogonadism. In helps to achieve not only androgenization, but also to Sertoli cells maturation.

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.

Multiomics approach to profiling Sertoli cell maturation during development of the spermatogonial stem cell niche.

Spermatogonial stem cells (SSCs) are the basis of spermatogenesis, a complex process supported by a specialized microenvironment, called the SSC niche. Postnatal development of SSCs is characterized by distinct metabolic transitions from prepubertal to adult stages. An understanding of the niche factors that regulate these maturational events is critical for the clinical application of SSCs in fertility preservation. To investigate the niche maturation events that take place during SSC maturation, we combined different '-omics' technologies. Serial single cell RNA sequencing analysis revealed changes in the transcriptomes indicative of niche maturation that was initiated at 11 years of age in humans and at 8 weeks of age in pigs, as evident by Monocle analysis of Sertoli cells and peritubular myoid cell (PMC) development in humans and Sertoli cell analysis in pigs. Morphological niche maturation was associated with lipid droplet accumulation, a characteristic that was conserved between species. Lipidomic profiling revealed an increase in triglycerides and a decrease in sphingolipids with Sertoli cell maturation in the pig model. Quantitative (phospho-) proteomics analysis detected the activation of distinct pathways with porcine Sertoli cell maturation. We show here that the main aspects of niche maturation coincide with the morphological maturation of SSCs, which is followed by their metabolic maturation. The main aspects are also conserved between the species and can be predicted by changes in the niche lipidome. Overall, this knowledge is pivotal to establishing cell/tissue-based biomarkers that could gauge stem cell maturation to facilitate laboratory techniques that allow for SSC transplantation for restoration of fertility.

The role of steroidogenic factor 1 (SF-1) in steroidogenic cell function of the testes and ovaries of mature mice.

The nuclear receptor steroidogenic factor 1 (SF-1) is essential for mature mouse gonad steroidogenic gene expression, for Leydig and Sertoli cell function, and depletion of SF-1 in steroidogenic cells of the testis compromises steroidogenesis, spermatogenesis and male fertility. Steroidogenic factor 1 (SF-1 or NR5A1) plays an essential role in the development of fetal gonads and regulates genes involved in steroid biosynthesis. Since SF-1 is expressed in multiple cell types in mouse gonads, we developed three novel conditional knockout (cKO) mouse models employing Cre-recombinase and floxed alleles of SF-1 (Nr5a1f/f) to identify its role in testes and ovaries of mature mice: Cytochrome P450 17α-hydroxylase (Cyp17Cre/+;Nr5a1f/f, Leydig and theca cell-specific), aromatase (Cyp19Cre/+;Nr5a1f/f, Sertoli and granulosa cell-specific), as well as a combination of both (Cyp17+Cyp19-Cre;Nr5a1f/f). Compared to control animals, Cyp19-Cre;Nr5a1f/f cKO males showed normal fertility and testicular function. The Cyp17Cre/+;Nr5a1f/f cKO males had smaller testis, with drastically reduced Leydig cell volumes and impaired steroidogenesis, though their reproductive performance remained comparable to controls. Some 50% of Cyp17Cre/++Cyp19Cre/+;Nr5a1f/f double-cKO (dKO) males were infertile, while the remaining 50% showed significantly reduced fertility. These dKO males also had smaller testis with degenerative seminiferous tubules, abnormal Leydig cell morphology and lower levels of intra-testicular testosterone. Abnormal Sertoli cell localization was noted in dKO testes, with increased Sox9, p27 and inhibin subunit ßb and decreased androgen receptor expression. Female mice from all genotypes showed normal reproductive capacity, though steroidogenic gene expression levels were significantly decreased in both Cyp17Cre/+;Nr5a1f/f cKO and dKO females. These results show the essential role of SF-1 in mature mouse gonad steroidogenic gene expression, for Leydig and Sertoli cell function, and that depletion SF-1 in all steroidogenic cells of the testis compromises steroidogenesis, spermatogenesis and male fertility.

Efficiency of immunocastration with an anti-gonadotropin-releasing hormone vaccine on cryptorchid bulls.

Cryptorchid bulls have low economic value owing to the effects of masculinization. Moreover, surgical removal of an ectopic testis is difficult in certain clinical cases. Recently, immunocastration has garnered popularity as a nonsurgical castration method in pig farming; however, the effects of immunocastration on cryptorchid bulls are yet to be yet. Herein, we investigated endocrine changes due to immunocastration in cryptorchid bulls and studied its effectiveness. This study included 13 Holstein bulls diagnosed with cryptorchidism and classified into two groups based on pubertal period: <8 months of age (pregroup) and ≥8 months of age (postgroup). Antigonadotropin-releasing hormone (GnRH) vaccine was used for immunocastration, and two vaccine doses were administered. Blood testosterone and anti-Müllerian hormone (AMH) levels were measured and analyzed for endocrine evaluation. The testosterone levels significantly decreased following the start of immunocastration in both groups, thereby confirming the efficacy of antiGnRH vaccination in cryptorchid bulls. The AMH levels significantly increased in the pregroup with two antiGnRH vaccination, suggesting a compensatory response via the neutralization of GnRH antibodies. The AMH levels did not significantly change in the postgroup, indicating the partial suppression of AMH secretion in Sertoli cells during sexual maturation and failure of Sertoli cell maturation. Thus, we successfully restrained the serum testosterone levels in cryptorchid bulls using antiGnRH vaccine. The testosterone levels are a useful indicator of the immunocastration effect on cryptorchid bulls. Hereafter, a vaccine program that can sustain the castration effect on cryptorchid bulls is necessary.

Expression of Intermediate Filaments in the Developing Testis and Testicular Germ Cell Cancer.

Cytokeratin and desmin expression have been associated with Sertoli cell maturity and the development of testicular germ cell cancer (TGCC). Thus, the present study aimed to characterize the expression of these intermediate filaments in normal testis development and TGCC. Cytokeratin and desmin were determined by immunohistochemistry and immunofluorescence in human fetal, and adult testis and tissue from patients with pre-invasive germ cell neoplasia in-situ (GCNIS) or invasive TGCC. Desmin was expressed in Sertoli cells of the human fetal testis, and the proportion of desmin expressing Sertoli cells was significantly reduced in the second trimester, compared with the first trimester (31.14% vs. 6.74%, p = 0.0016). Additionally, Desmin was expressed in the majority of Sertoli cells in the adult testis and TGCC samples. Cytokeratin was detected in Sertoli cells of human fetal testis but was not expressed in Sertoli cells of human adult testis. In patients with TGCC, cytokeratin was not expressed in Sertoli cells in tubules with active spermatogenesis but was detected in Sertoli cells in tubules containing GCNIS cells in patients with both pre-invasive and invasive TGCC. In conclusion, desmin was not associated with Sertoli cell maturation or progression to TGCC. However, cytokeratin appeared to be an indicator of impaired Sertoli cell maturation.

Two complementary approaches for efficient isolation of Sertoli cells for transcriptomic analysis.

Sertoli cells (SCs) are the only somatic cells that reside in seminiferous tubules of testis. They directly interact with and support the development of germ cells, thus have an indispensable role in the process of spermatogenesis. SCs first appear in a proliferative state and then, with the initiation of the first wave of spermatogenesis, progress to a mature “nurturing” state which supports lifelong continuous sperm production. During this development, the SC transcriptome must adapt rapidly as obstacles in SC maturation often result in deficiencies in male fertility. Due to its importance in spermatogenesis, a reliable, rapid, and precise method for the isolation of high purity, viable and unadulterated SC has been largely missing. We have developed an improved method for the preparation of a testicular single cell suspension comprised of two alternative protocols to separate SCs from the rest of the testicular cells by FACS. The first sorting scheme is based on their co-expression of surface specific markers, FSHr and Occludin-1, while the second focuses on the co-staining of SCs with FSHr-specific antibody and Hoechst 33342, which discriminates DNA content of testicular cells. The entire procedure can be completed in less than 3 h which permits the analysis of the development-related transcriptional profile of these cells. Notably, our comparative study showed that this method resulted in a SC transcriptome that is largely comparable to SCs which were briskly isolated due to their cell-specific expression of fluorescent protein. Interestingly, we also show that SCs sorted as FSHr+Occludin+ cells contained a tangible portion of transcripts from all types of testicular germ cells. Sorting of SCs according to their 2C DNA content significantly reduced the presence of these transcripts, thus seems to be the most suitable approach for accurate determination of the SC transcriptome. We believe that these novel approaches for the isolation of SCs will assist researchers in the elucidation of their function as well as their role in spermatogenesis and disorders related to male infertility.

Role of p38 MAPK Signalling in Testis Development and Male Fertility.

The testis is an important male reproductive organ, which ensures reproductive function via the secretion of testosterone and the generation of spermatozoa. Testis development begins in the embryonic period, continues after birth, and generally reaches functional maturation at puberty. The stress-activated kinase, p38 mitogen-activated protein kinase (MAPK), regulates multiple cell processes including proliferation, differentiation, apoptosis, and cellular stress responses. p38 MAPK signalling plays a crucial role in testis development by regulating spermatogenesis, the fate determination of pre-Sertoli, and primordial germ cells during embryogenesis, the proliferation of testicular cells in the postnatal period, and the functions of mature Sertoli and Leydig cells. In addition, p38 MAPK signalling is involved in decreased male fertility when exposed to various harmful stimuli. This review will describe in detail the biological functions of p38 MAPK signalling in testis development and male reproduction, together with its pathological role in male infertility.

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.

Connexin43 represents an important regulator for Sertoli cell morphology, Sertoli cell nuclear ultrastructure, and Sertoli cell maturation

The Sertoli cell (SC)-specific knockout (KO) of connexin43 (Cx43) was shown to be an effector of multiple histological changes in tubular morphology, resulting in germ cell loss through to a Sertoli-cell-only (SCO) phenotype and vacuolated seminiferous tubules containing SC-clusters. Our present study focused on the effects of Cx43 loss on SC ultrastructure. Using serial block-face scanning electron microscopy (SBF-SEM), we could confirm previous results. Ultrastructural analysis of Sertoli cell nuclei (SCN) revealed that these appear in clusters with a phenotype resembling immature/proliferating SCs in KO mice. Surprisingly, SCs of fertile wild type (WT) mice contained SCN with a predominantly smooth surface instead of deep indentations of the nuclear envelope, suggesting that these indentations do not correlate with germ cell support or spermatogenesis. SBF-SEM facilitated the precise examination of clustered SCs. Even if the exact maturation state of mutant SCs remained unclear, our study could detect indications of cellular senescence as well as immaturity, emphasising that Cx43 affects SC maturation. Moreover, Sudan III staining and transmission electron microscopy (TEM) demonstrated an altered lipid metabolism in SCs of Cx43 deficient mice.

Organotypic Culture of Testicular Tissue from Infant Boys with Cryptorchidism.

Organotypic culture of human fetal testis has achieved fertilization-competent spermatids followed by blastocysts development. This study focuses on whether the organotypic culture of testicular tissue from infant boys with cryptorchidism could support the development of spermatogonia and somatic cells. Frozen-thawed tissues were cultured in two different media, with or without retinoic acid (RA), for 60 days and evaluated by tissue morphology and immunostaining using germ and somatic cell markers. During the 60-day culture, spermatocytes stained by boule-like RNA-binding protein (BOLL) were induced in biopsies cultured with RA. Increased AR expression (p < 0.001) and decreased AMH expression (p < 0.001) in Sertoli cells indicated advancement of Sertoli cell maturity. An increased number of SOX9-positive Sertoli cells (p < 0.05) was observed, while the percentage of tubules with spermatogonia was reduced (p < 0.001). More tubules with alpha-smooth muscle actin (ACTA, peritubular myoid cells (PTMCs) marker) were observed in an RA-absent medium (p = 0.02). CYP17A1/STAR-positive Leydig cells demonstrated sustained steroidogenic function. Our culture conditions support the initiation of spermatocytes and enhanced maturation of Sertoli cells and PTMCs within infant testicular tissues. This study may be a basis for future studies focusing on maintaining and increasing the number of spermatogonia and identifying different factors and hormones, further advancing in vitro spermatogenesis.

The PI3K/AKT signaling pathway: How does it regulate development of Sertoli cells and spermatogenic cells?

The PI3K/AKT signaling pathway is one of the most crucial regulatory mechanisms in animal cells, which can mainly regulate proliferation, survival and anti-apoptosis in cell lines. In the seminiferous epithelium, most studies were concentrated on the role of PI3K/AKT signaling in immature Sertoli cells (SCs) and spermatogonia stem cells (SSCs). PI3K/AKT signaling can facilitate the proliferation and anti-apoptosis of immature Sertoli cells and spermatogenic cells. Besides, in mature Sertoli cells, this pathway can disintegrate the structure of the blood-testis barrier (BTB) via regulatory protein synthesis and the cytoskeleton of Sertoli cells. All of these effects can directly and indirectly maintain and promote spermatogenesis in male testis.

P-468 Organotypic culture of testicular tissue from infant boys with cryptorchidism

Abstract Study question Can organotypic culture support the survival and maturation of germ cells and niche-related cells within testicular tissue from infant boys with cryptorchidism? Summary answer The testicular structure and the number of germ cells were maintained during organotypic culture, whereas Sertoli cells and peritubular myoid cells (PTMCs) matured. What is known already Testicular tissue cryopreservation (TTC) is a strategy to safeguard the fertility of prepubertal boys who face a risk of infertility. Organotypic culture of immature testicular tissue from mice achieved production of spermatozoa. Similarly, the culture of human fetal gonads resulted in the generation of competent spermatids. However , in vitro spermatogenesis by organotypic culture of human prepubertal testicular tissue has not been achieved. It is also unknown whether germ cells as well as its niche-related cells, in testicular tissue from infant boys with cryptorchidism, can maintain and mature under in vitro conditions. Study design, size, duration Testicular tissue was cryopreserved from four infant boys with bilateral cryptorchidism undergoing orchidopexy (age range: 0.5-1.4 years), as part of a fertility preservation program. Culture media with and without retinoic acid were tested. Testicular fragments were harvested at 30 days and 60 days after culture and evaluated by histological assessment of tissue structure, germ cell development, and immunohistochemical staining for germ cell and somatic cell markers. Participants/materials, setting, methods Cryopreserved-thawed testicular tissue was cut into fragments (1-2 mm3) and placed on top of agarose gel stands and cultured at 34oC with 5% CO2 in Minimum Essential Medium-alpha supplemented with knockout serum replacement, human umbilical cord plasma, Activin A, hormones, growth factors, with or without retinoic acid. Immunohistochemical analyses were performed using germ cell markers (MAGE-A, GAGE, and VASA), Sertoli cell maturation markers (AMH, AR, SOX9), PTMC marker (alpha-SMA). Main results and the role of chance Following the 60-day culture, the lumen of the seminiferous tubules had developed. The number of germ cells per tubule remained stable during this period. However, no further germ cell maturation was observed. Germ cells showed different phenotypes of MAGEA, GAGE, and VASA expression with no significant difference in number. The number of SOX9-positive Sertoli cells was significantly increased from 30 days to 60 days of culture (p &amp;lt;0.0001). No difference in AMH expression was observed, while AR expression in Sertoli cells was induced during culture. Alpha-SMA expression was detected in the PTMCs surrounding the seminiferous tubules. The two different culture conditions, with and without retinoic acid in the culture media, did not affect cell survival or maturation. Limitations, reasons for caution The small number of testicular biopsies available is a limitation. Wider implications of the findings Our organotypic culture conditions support the long-term survival of germ cells in testicular tissue from infant boys with cryptorchidism. Thus, further studies are needed to induce the maturation of germ cells under similar experimental conditions. Trial registration number not applicable

FBXO38 Ubiquitin Ligase Controls Sertoli Cell Maturation.

The ubiquitin ligase SCFFBXO38 controls centromeric chromatin by promoting the degradation of the ZXDB protein. To determine the importance of this pathway during development, Fbxo38-deficient mice were generated. The loss of FBXO38 resulted in growth retardation affecting several organs, including the male reproductive system. A detailed analysis of the mutant testes revealed pathological changes in the seminiferous tubules, accompanied by a significant decrease in sperm production and reduced fertility. In adult testes, FBXO38 was specifically expressed in Sertoli cells, a somatic population essential for spermatogenesis initiation and progression. Sertoli cells lacking FBXO38 exhibited stabilized ZXDB protein and upregulated centromeric chromatin. Furthermore, the gene expression profile revealed that the absence of FBXO38 led to a defect in Sertoli cell maturation, specifically characterized by dysregulation in genes controlling retinoic acid metabolism and intercellular communication. Consequently, we documented significant changes in their ability to initiate spermatogonial differentiation. In conclusion, we show that FBXO38 acts as a Sertoli cell maturation factor, affecting the Sertoli cell transcription program, centromere integrity, and, subsequently, the ability to control spermatogenesis.

Regulation of STUB1 expression and its biological significance in mouse Sertoli cells

STIP1 Homology and U-Box Containing Protein 1 (STUB1), a ubiquitin E3 ligase initially involved in immune responses, has recently emerged as a pleiotropic regulator of different biological systems, including skeletal and male reproduction systems. On the latter, a homozygous mutation in the STUB1 gene has been identified in patients with hypogonadism. However, the pattern of expression and biological actions of STUB1 in testis remains so far unexplored. Herein, we report analyses on the testicular expression of STUB1 in human testes with impaired spermatogenesis and paracrine regulation of STUB1 expression in mouse testis development and the direct effects of ablation STUB1 on Sertoli cell (SC) functions. STUB1 was expressed abundantly in pachytene spermatocytes and SCs, and weakly in spermatogonia and differentiating spermatids in normal human testis. In contrast, Sertoli-specific expression of STUB1 was significantly decreased in the human testes with impaired spermatogenesis. Throughout postnatal development of mouse testis, however, STUB1 was expressed exclusively in the nuclei of the functionally mature SCs. The adjacent germ cell (GC)-derived IL-1α overtly regulated STUB1 expression through promoting the ETS domain transcription factor Elk-1 (ELK1)-mediated transactivation. Importantly, ablation of endogenous STUB1 caused lipid accumulation and senescence in GC co-incubated SCs. Together with previous reports on the stimulatory effects of IL-1α on cell senescence, our findings suggest that STUB1 may serve as an important negative feedback signaling to modulate the magnitude of GCs-derived IL-1α, which is normally maintained at low levels within testis.

Corifollitropin Alfa Combined With Human Chorionic Gonadotropin in Adolescent Boys With Hypogonadotropic Hypogonadism.

Adolescent males with hypogonadotropic hypogonadism (HH) have traditionally been treated with exogenous testosterone (T) or human chorionic gonadotropin (hCG) to produce virilization; however, those modalities do not result in growth of the testes and may promote premature maturation and terminal differentiation of Sertoli cells prior to their proliferation, which may impact future fertility. Another option is to use gonadotropins in those individuals to induce testicular growth, proliferation and maturation of Sertoli cells, and production of endogenous T with consequent virilization. We examined the efficacy and safety of corifollitropin alfa (CFA) combined with hCG for the induction of testicular growth and pubertal development in adolescent boys with HH. This was a 64-week, multicenter, open-label, single-group study of CFA in adolescent boys, aged 14 to younger than 18 years, with HH. Seventeen participants initiated a 12-week priming period with CFA (100 μg if weight ≤ 60 kg, or 150 μg if weight > 60 kg) given subcutaneously once every 2 weeks, after which they entered a 52-week combined treatment period with CFA, once every 2 weeks, and subcutaneous hCG, twice-weekly (hCG dose adjusted between 500 IU and 5000 IU to keep total T and estradiol levels within protocol-specified ranges). The primary efficacy end point was change from baseline in testicular volume (TV), measured as the sum of volumes of left and right testes by ultrasound. After 64 weeks of therapy with CFA/CFA combined with hCG, geometric mean fold increase from baseline in TV was 9.43 (95% CI, 7.44-11.97) (arithmetic mean of change from baseline at week 64, 13.0 mL). Hormonal, Tanner stage, and growth velocity changes were consistent with initiation and progression of puberty. Treatment was generally well tolerated. No participant developed anti-CFA antibodies. Treatment of adolescent boys with HH with CFA alone for 12 weeks followed by CFA combined with hCG for 52 weeks induced testicular growth accompanied by pubertal progression, increased T, and a pubertal growth spurt (EudraCT: 2015-001878-18).

Changes in the testicular histomorphometry and their association with genes expression pattern of testes from birth to puberty in Beetal goat kids

In the present study, twenty-eight (n = 28) longitudinally rear male goat kids were slaughtered at monthly intervals (n = 4/group) till the 6th month of age; to examine the testicular development and establishment of spermatogenesis through histo-architecture along with mRNA expression of related genes. The results showed that seminiferous tubular diameter was linearly increased (P < 0.001) at birth (40 µm) to 6th months (200 µm). The epithelial height was 18.41 ± 0.65 µm at birth and 164 ± 2.82 µm at 6th months. Whereas, at 3rd months age greatest increase was observed (74.08 ± 1.66 µm), possibly due to a rapid increase in the numbers of spermatocytes and spermatids (4.0 ± 0.3 and 0.9 ± 0.04, respectively). At the same time point (4–6 months of age), a dramatic increase (P < 0.001) was observed in the mRNA level of genes expressed in haploid germ cells including Protamine (PRM1), Outer dense fiber protein (ODF2) and Stimulated by Retinoic Acid (STRA8). In addition, the maturation of Sertoli cells (SCs) was appeared to be completed at the start of 1st month (5.6 ± 0.5), which was further validated by sudden repression (P < 0.001) of SC-specific SOX9 and FSHR mRNA levels during this period. The spermatids that appeared at the end of the 3rd month reflect the start of spermatogenesis. However, a significant (P < 0.001) number of elongated spermatids and mature spermatozoa was observed at the end of 6 months, which indicated pubertal onset (9.8 ± 0.7 and 9.9 ± 0.9, respectively). The mRNA expression of other genes altered during this developmental phase was Heat-shock Protein 8 (HSPA8) and Aromatase (CYP19A1), both was important in sperm maturation and testosterone level. In conclusion, marked changes in cellular and molecular events were observed from the 3rd month; when active spermatogenesis initiated, and mature spermatozoa appeared in the 6th month at pubertal onset in Beetal bucks.

Small-Molecule-Driven Direct Reprogramming of Fibroblasts into Functional Sertoli-Like Cells as a Model for Male Reproductive Toxicology.

Sertoli cells (SCs) are vital to providing morphological and nutritional support for spermatogenesis. Defects in SCs often lead to infertility. SCs transplantation is a promising potential strategy to compensate for SC dysfunction. However, isolation of SCs from testes is impractical due to obvious and ethical limitations. Here, a molecular cocktail is identified comprising of pan-BET family inhibitor (I-BET151), retinoic acid, and riluzole that enables the efficient conversion of fibroblasts into functional Sertoli-like cells (CiSCs). The gene expression profiles of CiSCs resemble those of mature SCs and exhibit functional properties such as the formation of testicular seminiferous tubules, engulfment of apoptotic sperms, supporting the survival of germ cells, and suppressing proliferation of primary lymphocytes in vitro. Moreover, CiSCs are sensitive to toxic substances, making them an alternative model to study the deleterious effects of toxicants on SCs. The study provides an efficient approach to reprogram fibroblasts into functional SCs by using pure chemical compounds.

What Does Androgen Receptor Signaling Pathway in Sertoli Cells During Normal Spermatogenesis Tell Us?

Androgen receptor signaling pathway is necessary to complete spermatogenesis in testes. Difference between androgen binding location in Sertoli cell classifies androgen receptor signaling pathway into classical signaling pathway and non-classical signaling pathway. As the only somatic cell type in seminiferous tubule, Sertoli cells are under androgen receptor signaling pathway regulation via androgen receptor located in cytoplasm and plasma membrane. Androgen receptor signaling pathway is able to regulate biological processes in Sertoli cells as well as germ cells surrounded between Sertoli cells. Our review will summarize the major discoveries of androgen receptor signaling pathway in Sertoli cells and the paracrine action on germ cells. Androgen receptor signaling pathway regulates Sertoli cell proliferation and maturation, as well as maintain the integrity of blood-testis barrier formed between Sertoli cells. Also, Spermatogonia stem cells achieve a balance between self-renewal and differentiation under androgen receptor signaling regulation. Meiotic and post-meiotic processes including Sertoli cell - Spermatid attachment and Spermatid development are guaranteed by androgen receptor signaling until the final sperm release. This review also includes one disease related to androgen receptor signaling dysfunction named as androgen insensitivity syndrome. As a step further ahead, this review may be conducive to develop therapies which can cure impaired androgen receptor signaling in Sertoli cells.

Genomic Identification, Evolution, and Expression Analysis of Bromodomain Genes Family in Buffalo.

Bromodomain (BRD) is an evolutionarily conserved protein–protein interaction module that is critical in gene regulation, cellular homeostasis, and epigenetics. This study aimed to conduct an identification, evolution, and expression analysis of the BRD gene family in the swamp buffalo (Bubalus bubalis). A total of 101 BRD protein sequences deduced from 22 BRD genes were found in the buffalo genome. The BRD proteins were classified into six groups based on phylogenetic relationships, conserved motifs, and conserved domains. The BRD genes were irregularly distributed in 13 chromosomes. Collinearity analysis revealed 20 BRD gene pairs that had remarkable homologous relationships between the buffalo and cattle, although no tandem or segmental duplication event was found in the buffalo BRD genes. Comparative transcriptomics using a 10x sequencing platform analysis showed that 22 BRD genes were identified in the Sertoli cells (SCs) at different developmental stages of buffalo. Further, the mRNA expression levels of bromodomain and the extraterminal (BET) family in SCs at the pubertal stage were higher than that at the prepubertal stage of buffalo. However, the SMARCA2, PHIP, BRD9, and TAF1 genes exhibited the opposite trend. The maturation process of SCs may be regulated by the BRD family members expressed differentially in SCs at different developmental stages of buffalo. In summary, our findings provide an understanding of the evolutionary, structural, and functional properties of the buffalo BRD family members, and further characterize the function of the BRD family in the maturation of SCs. It also provides a theoretical basis for further understanding in the future of the mechanism of SCs regulating spermatogenesis.