Multi-omics analysis the effects of Dhx37 deficiency on testis development and nucleolar homeostasis.

A Sertoli Cell-Specific Knockout of Connexin43 Prevents Initiation of Spermatogenesis

Male development depends on the successful development of testes in the embryo, a process beginning with the differentiation of Sertoli cells, directed by the Y-linked gene SRY. The virilisation of the XY embryo is then directed by steroid hormones, which are produced by fetal Leydig cells (FLCs) that reside in the embryonic testis. As only around 20% of XY disorders of sex development (DSDs) are able to be explained at the molecular genetic level, I reasoned that genes involved in the development and function of FLCs may represent an unappreciated source of candidate XY DSD genes. To pinpoint these genes, and to develop a more detailed understanding of the regulatory networks supporting the formation of the somatic cell populations of the developing testes, I developed methods for isolating Sertoli cell, FLC and non-steroidogenic interstitial cell- enriched subpopulations using the Sf1-eGFP transgenic mouse line. RNA-sequencing of the subpopulations at 12.5 dpc, followed by rigorous bioinformatic filtering, identified genes upregulated in Sertoli cells, FLCs and non-steroidogenic interstitial cells. The bioinformatic analysis revealed that expression of components of neuroactive ligand signaling pathways were prevalent in FLCs and Sertoli cells. In addition, I identified 61 genes expressed preferentially in early FLCs with no previous association with FLC specification or differentiation that may be functionally significant. I also sought to identify factors that are possibly involved in signaling between the Sertoli cells, FLCs and non-steroidogenic interstitial cell populations. This study also identified fetal expression of a number of known DSD causing genes in the early somatic cell populations of the gonad, providing further evidence for the fetal origins of gonadal phenotypes in some DSDs. This dataset offers a platform for investigating the biology of FLCs and understanding their role in testicular development. In addition, this dataset provide a foundation for targeted studies aimed at identifying the causes of idiopathic XY DSDs. Conducting a transcriptomic project identified dozens of highly promising gonadogenesis candidate genes and highlighted the challenge of determining gene function when overwhelmed with potential candidate genes. In addition, many candidate genes for human developmental disorders are being identified in rare disease cohorts thanks to whole exome and whole genome sequencing. It is clear that traditional gene targeting methods in mice are too complex and time consuming to clear this backlog, especially when conditional deletion methods are required. To address the need for first pass functional screening methods, I developed a novel technique for assessing gene function, in a knockdown context, by injection of modified antisense morpholino oligonucleotides (MOs) into the heart of mid-gestation mouse embryos. Circulation of the MOs through the embryonic vasculature allowed targeting of multiple organs. Tissues of interest were explanted, cultured and analysed for expression of key markers. As a proof-of-principle, I used MO injection to partially phenocopy known gene knockout phenotypes in the fetal gonads (Stra8, Sox9) and pancreas (Sox9). In addition, I created a novel double knockdown of Gli1 and Gli2, which revealed defects in FLC differentiation. I also investigated the role of Adamts19 and Ctrb1, genes of unknown function in sex determination and gonadal development. This proof-of-principle study demonstrated the utility of this method as a means of first-pass analysis of gene function during organogenesis before undertaking a detailed genetic analysis. In addition, I used MO knockdown to validate a candidate gene for 46,XY DSD. Using whole exome sequencing SART3 was identified as a potential candidate for DSD in 46,XY DSD patients. By performing MO knockdown of SART3 in the fetal mouse gonad I modelled the haploinsufficiency that results from heterozygous deletion of SART3 and provided evidence supporting SART3 as the disease-causing gene in the DSD patients in this study. In addition, this work provided data on the function of SART3 in the developing gonad. Overall, this thesis describes the generation of the most comprehensive somatic cell transcriptome of developing testicular somatic cell populations to date and develops a method for screening the genes of interest that come out of such a study. These resources will be able to be used for the identification and characterisation of genes in gonad development. In addition this work will hopefully provide a platform for identifying new candidates for the fetal origins of DSDs originating from defects in the FLC population.

Disorders of Sex Development.

While DIS3 functions in male germ cells are emerging, its ribonuclease activity governing Sertoli cell RNA metabolism, essential for testicular development and spermatogenesis, remains undefined. This study identifies a critical role for exosome-associated DIS3 ribonuclease in regulating Sertoli cell maintenance and maturation during spermatogenesis. Dis3 deficiency in Sertoli cells causes severe testicular atrophy, marked by rapid depletion of both Sertoli and germ cells. This phenotype results from impaired Sertoli cell proliferation, elevated apoptosis, disrupted maturation, and compromised blood-testis barrier integrity, culminating in defective spermatogenesis and infertility. scRNA-seq analysis reveals altered cell-cell communication, alongside heightened p53 and Wnt signaling activity in Dis3 cKO Sertoli cells. p53 inhibitor treatment mitigates overt apoptosis in Dis3 cKO Sertoli cells. Similarly, inhibition of the Wnt/β-Catenin pathway increases the abundance of both Sertoli and germ cells while improving Sertoli cell maturation. Together, these findings establish Sertoli cell-specific DIS3 as essential for testicular development and spermatogenesis in mice, and implicate the p53 and Wnt/β-Catenin pathways as potential mechanistic contributors.

Decision letter: The single-cell chromatin accessibility landscape in mouse perinatal testis development

Male sex determination hinges on the development of testes in the embryo, beginning with the differentiation of Sertoli cells under the influence of the Y-linked gene SRY. Sertoli cells then orchestrate fetal testis formation including the specification of fetal Leydig cells (FLCs) that produce steroid hormones to direct virilization of the XY embryo. As the majority of XY disorders of sex development (DSDs) remain unexplained at the molecular genetic level, we reasoned that genes involved in FLC development might represent an unappreciated source of candidate XY DSD genes. To identify these genes, and to gain a more detailed understanding of the regulatory networks underpinning the specification and differentiation of the FLC population, we developed methods for isolating fetal Sertoli, Leydig, and interstitial cell-enriched subpopulations using an Sf1-eGFP transgenic mouse line. RNA sequencing followed by rigorous bioinformatic filtering identified 84 genes upregulated in FLCs, 704 genes upregulated in nonsteroidogenic interstitial cells, and 1217 genes upregulated in the Sertoli cells at 12.5 days postcoitum. The analysis revealed a trend for expression of components of neuroactive ligand interactions in FLCs and Sertoli cells and identified factors potentially involved in signaling between the Sertoli cells, FLCs, and interstitial cells. We identified 61 genes that were not known previously to be involved in specification or differentiation of FLCs. This dataset provides a platform for exploring the biology of FLCs and understanding the role of these cells in testicular development. In addition, it provides a basis for targeted studies designed to identify causes of idiopathic XY DSD.

Loss of Atg5 in Sertoli cells enhances the susceptibility of cadmium-impaired testicular spermatogenesis in mice.

Predominant Sertoli cell deficiency in a 46,XY disorders of sex development patient with a new NR5A1/SF-1 mutation transmitted by his unaffected father

Disorders of sex development (DSD) are defined as congenital conditions in which development of chromosomal, gonadal or anatomical sex is atypical. In 46, XY DSD, the genotype is XY, but the external genitalia is incompletely virilised, ambiguous, or completely female. The objectives of this prospective study are to evaluate the testicular Sertoli and Leydig cell functions, to establish the genetic basis and to determine the relative prevalence of etiologies in Chinese patients with 46,XY DSD in Hong Kong. All patients with 46,XY DSD (either new or known) presented to five paediatric departments in Hong Kong from July 2009 till June 2011 were recruited. They were assessed by paediatric endocrinologists. Comprehensive evaluation of testicular and adrenal functions was performed using serum hormonal assays and urine steroid profiling. Based on the hormonal results, mutational analyses of the candidate genes by polymerase chain reaction and direct DNA sequencing were conducted to delineate the genetic basis of the etiologies. Sixty-five patients (54 male and 11 female) with 46,XY DSD were recruited. Their age ranged from birth to 27 years. Sixty-one (94%) patients presented with ambiguous external genitalia, two presented with delayed puberty and one each with primary amenorrhoea and inguinal hernia. Definitive diagnoses were made in 25 (38%) patients. Eleven (17%) patients had 5-alpha reductase 2 deficiency. Androgen insensitivity was confirmed by genetic analysis in eight (12%) patients. There was one patient with each of the following etiologies: Swyer syndrome, SF-1 mutation, Frasier syndrome, cholesterol side-chain cleavage deficiency, persistent Mullerian duct syndrome and mixed gonadal dysgenesis. Genetic basis of the etiologies was delineated in 23 (35%) patients. A total of 10 novel mutations were identified. The longest follow up period was 27 years, none of the patients requested change of gender sex so far. In conclusion, 46,XY DSD is a heterogeneous group with diverse etiologies. Although 5-alpha reductase 2 deficiency is believed to be rare, it is not uncommon in Hong Kong.

We read with interest the paper of Pizzo et al. [1], confirming that adolescence is a key period for the diagnosis of 46,XY disorders of sex development (DSD) [2]. However, in our opinion, some points should be better addressed. Among these, we have the following. The authors stated that the reported girl showed “normal intellectual function,” but this information had little relevance because mental function is not impaired in females with 46,XY DSD, reaching adolescence without any clinical suspicions. Indeed, this statement might be stigmatizing for these persons. The statement that this adolescent was affected by hypergonadotropic hypogonadism (HH) is correct, according to the high LH and FSH values shown in Table 1 [1]. Very low levels of both 17β-estradiol and testosterone were also shown [1]. This endocrine pattern is not typical of late adolescent and young adult females with complete androgen insensitivity syndrome (CAIS) and intact testes. On the contrary, these persons show high/normal levels of both 17β-estradiol (for a person with a 46,XY karyotype) and testosterone [3–5]. In addition, adolescent/young adult women with CAIS did not show HH: LH is in high normal range or slightly elevated (due to the androgen resistance at central level), but FSH is in normal range (due to unaffected inhibin secretion from Sertoli cells) [3–5]. In addition, SHBG was reported within adult male range by Pizzo et al. [1], suggesting a normal sensitivity to the low levels of androgens in this girl [6]. Indeed, SHBG is within normal female range in women with CAIS, due to peripheral androgen resistance [5]. All these findings show poor agreement with the affirmed diagnosis of CAIS [1]. The clinical phenotype of the reported girl did not match with the phenotype of adolescents with CAIS; in fact, the latter shows normal breast development related to the relatively high normal estrogen levels and unopposed androgen action [3, 4, 7] and does not show hypotrophic breasts [1]. It is quite surprising that hormonal replacement therapy was started before any diagnosis was established. Histological findings do not completely agree with the diagnosis of CAIS, in particular the absence of Leydig cells, which are abundant in adolescent females with CAIS and sometimes formed aggregates up to 2 mm in diameter [8, 9]. 46,XY karyotype, female phenotype, and absence of mullerian derivatives may be present in several 46,XY DSD; they should be excluded before diagnosis of CAIS by optimal clinical, endocrine, and genetic investigations [10]. For example, the testosterone/Δ4-androstenedione ratio in the adolescent reported by Pizzo et al. [1] is very low (0.13; normal values >0.8 [11]). Thus, diagnoses of 17β-hydroxysteroid-dehydrogenase deficiency type 3 or 46,XY gonadal dysgenesis [11, 12] must be considered in the diagnostic process. Risk of gonadal cancer largely varies among 46,XY DSD. For example, in CAIS is very low at least during the first two decades of life [13–15]. Thus, delayed gonadal removal can be recommended to permit both full sexual development [15] and better bone health [16]. If diagnosis of CAIS is certain, surgery can be postponed—at least until the legal age at which the propositus can participate in decision making [1, 15, 17–19]. We are concerned and in complete disagreement with the decision to perform gonadectomy without full disclosure and assent of the adolescent [17–19]. In conclusion, some findings are poorly consistent with a diagnosis of CAIS, which should be confirmed by molecular analysis of androgen receptor gene [2, 7]. In our experience, more than 25% of the females referred to our departments with clinical/endocrine diagnosis of CAIS did not have this diagnosis confirmed by genetic analysis [20]. Clinical approach should be changed according to the new guidelines for management of persons with 46,XY DSD and directly involving the girl in the decision process [2, 18, 21]. We also stress that multidisciplinary team evaluation in tertiary centers with documented experience in this field must be guaranteed to each person with 46,XY DSD for optimal holistic management [21], especially before performing irreversible surgical procedures.

Testicular dysgenesis syndrome: mechanistic insights and potential new downstream effects

Sertoli cells are main players in the male gonads development and their study may shed light on 46,XY disorders of sex development (DSD). Mature primary Sertoli cells are incapable of proliferating in prolonged in vitro cultures and the available Sertoli cell models have several limitations since they derive from mouse or human cancer tissues. We differentiated human fibroblasts (HFs)-derived induced pluripotent stem cells into Sertoli-like cells (SLC) and, in order to characterize this new Sertoli cell model, we performed gene expression analyses by NextGeneration Sequencing techniques. This approach revealed that our putative SLC have reduced expression of pluripotency markers and expressed Sertoli cell markers such as SRY-Related HMG-Box 9 (SOX9), vimentin (VIM), and claudin-11 (CLDN-11). More in detail, the transcriptional profile analysis suggested that these cells are in an early stage of Sertoli cells maturation. Harnessing the power of induced pluripotent stem cells, we were able to generate SLC that show genetic and functional similarities to human Sertoli cells (HSerCs). SLC could become an excellent source of patient-specific Sertoli cells that could be of paramount benefit for both basic research and personalized medicine in sex development and reproductive medicine.

Sertoli cells are essential for testis development and normal spermatogenesis by providing support and nutrients. Pre-messenger RNA (pre-mRNA) processing is the basic mechanism required for gene expression, and members of the serine/arginine-rich protein (SR) family are key components of the machines that perform these basic processing events. Serine/arginine-rich splicing factor 2 (SRSF2) is an important member of the SR family; however, the physiological functions of SRSF2 in Sertoli cells are still unclear. Here, we found that SRSF2 was localized in the nuclei of Sertoli and germ cells in male mice at all stages by breeding Amh-Cre mice obtained with Srsf2-specific knockout in Sertoli cells to define the function of SRSF2 in Sertoli cells. The experimental results showed that specific deletion of SRSF2 impaired fetal Sertoli cell proliferation and induced abnormal apoptosis and severe DNA damage in seminiferous tubules, resulting in severe testicular dysplasia, seminiferous tubule atrophy, and almost no normal seminiferous tubules at postnatal day 14. Eventually, these changes resulted in failure to produce normal sperm and infertility. Further RNA-seq results showed that many key genes related to proliferation and apoptosis were downregulated; Racgap1 mRNA undergoes exon skipping. Thus, SRSF2-dependent Sertoli cells are essential for testicular development and male reproduction.

TAR DNA binding protein of 43 kD (TDP-43) is an evolutionarily conserved, ubiquitously expressed transcription factor and RNA-binding protein with major human health relevance. TDP-43 is present in Sertoli and germ cells of the testis and is aberrantly expressed in the sperm of infertile men. Sertoli cells play a key role in spermatogenesis by offering physical and nutritional support to male germ cells. The current study investigated the requirement of TDP-43 in Sertoli cells. Conditional knockout (cKO) of TDP-43 in mouse Sertoli cells caused failure of spermatogenesis and male subfertility. The cKO mice showed decreased testis weight, and low sperm count. Testis showed loss of germ cell layers, presence of vacuoles, and sloughing of round spermatids, suggesting loss of contact with Sertoli cells. Using a biotin tracer, we found that the blood-testis barrier (BTB) was disrupted as early as postnatal day 24 and worsened in adult cKO mice. We noted aberrant expression of the junction proteins connexin-43 (gap junction) and N-cadherin (ectoplasmic specialization). Oil Red O staining showed a decrease in lipid droplets (phagocytic function) in tubule cross-sections, Sertoli cells cytoplasm, and in the lumen of seminiferous tubules of cKO mice. Finally, qRT-PCR showed upregulation of genes involved in the formation and/or maintenance of Sertoli cell junctions as well as in the phagocytic pathway. Sertoli cells require TDP-43 for germ cell attachment, formation and maintenance of BTB, and phagocytic function, thus indicating an essential role for TDP-43 in the maintenance of spermatogenesis.

We have developed an in vivo model to examine testicular cord formation by isolated Sertoli and myoid cells when implanted under the kidney capsule of severe combined immunodeficient (SCID) mice. Neonatal porcine Sertoli (92.5% +/- 3.5%) and myoid (2.2% +/- 0.7%) cellular aggregates were transplanted underneath the kidney capsule of SCID mice. Grafts were removed between 0 and 60 days posttransplantation and examined histologically for the progressive development of structures resembling testicular cords. Aggregates began to reorganize by day 3, and cord structures were present at day 7 posttransplantation. These structures became larger and more defined as the time progressed after implantation. To localize Sertoli and peritubular myoid cells, grafts were immunostained for the Sertoli cell proteins, vimentin, DNA transcription factor GATA-4, and Müllerian inhibiting substance (MIS), as well as for a myoid cell protein, smooth muscle alpha-actin. In the "seminiferous" epithelial layer, the Sertoli cells were arranged with their nuclei along the basal edge adjacent to the peritubular myoid cells that were surrounding the tubules. Moreover, the expression of MIS mimicked that during porcine testicular development, suggesting the Sertoli cells were developing normally. In addition, proliferating cell nuclear antigen (PCNA) was detected in the Sertoli cells at all time points, indicating the proliferation of Sertoli cells in the grafts, which is consistent with Sertoli cell proliferation prior to puberty in the native porcine testis. These results suggest that the specific factors required for cord formation and prepubertal development are inherent in the transplanted cells. Moreover, we have developed a novel in vivo transplantation model to study seminiferous cord formation and prepubertal development.