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The surgical sterilization of cats and dogs has been used to prevent their unwanted breeding for decades. However, this is an expensive and invasive procedure, and often impractical in wider contexts, for example the control of feral populations. A sterilization agent that could be administered in a single injection, would not only eliminate the risks imposed by surgery but also be a much more cost-effective solution to this worldwide problem. In this study, we sought to develop a targeting peptide that would selectively bind to Leydig cells of the testes. Subsequently, after covalently attaching a cell ablation agent, Auristatin, to this peptide we aimed to apply this conjugated product (LH2Auristatin) to adult male mice in vivo , both alone and together with a previously developed Sertoli cell targeting peptide (FSH2Menadione). The application of LH2Auristatin alone resulted in an increase in sperm DNA damage, reduced mean testes weights and mean seminiferous tubule size, along with extensive germ cell apoptosis and a reduction in litter sizes. Together with FSH2Menadione there was also an increase in embryo resorptions. These promising results were observed in around a third of all treated animals. Given this variability, we discuss how these reagents might be modified in order to increase target cell ablation and improve their efficacy as sterilization agents.

Sertoli cells support the development of sperm and the function of various somatic cells in the interstitium between the tubules. Sertoli cells regulate the function of the testicular vasculature and the development and function of the Leydig cells that produce testosterone for fertility and virility. However, the Sertoli cell-derived factors that regulate these cells are largely unknown. To define potential mechanisms by which Sertoli cells could support testicular somatic cell function, we aimed to identify Sertoli cell-enriched proteins in the testicular interstitial fluid (TIF) between the tubules. We previously resolved the proteome of TIF in mice and humans and have shown it to be a rich source of seminiferous tubule-derived proteins. In the current study, we designed bioinformatic strategies to interrogate relevant proteomic and genomic datasets to identify Sertoli cell-enriched proteins in mouse and human TIF. We analysed proteins in mouse TIF that were significantly reduced after one week of acute Sertoli cell ablation in vivo and validated which of these are likely to arise primarily from Sertoli cells based on relevant mouse testis RNASeq datasets. We used a different, but complementary, approach to identify Sertoli cell-enriched proteins in human TIF, taking advantage of high-quality human testis genomic, proteomic and immunohistochemical datasets. We identified a total of 47 and 40 Sertoli cell-enriched proteins in mouse and human TIF, respectively, including 15 proteins that are conserved in both species. Proteins with potential roles in angiogenesis, the regulation of Leydig cells or steroidogenesis, and immune cell regulation were identified. The data suggests that some of these proteins are secreted, but that Sertoli cells also deposit specific proteins into TIF via the release of extracellular vesicles. In conclusion, we have identified novel Sertoli cell-enriched proteins in TIF that are candidates for regulating somatic cell-cell communication and testis function.

The hypothalamic-pituitary-adrenal (HPA) axis regulates responses to internal and external stressors. Many patients diagnosed with conditions such as depression or anxiety also have hyperactivity of the HPA axis. Hyper-stimulation of the HPA axis results in sustained elevated levels of glucocorticoids which impair neuronal function and can ultimately result in a psychiatric disorder. Studies investigating Glucocorticoid Receptor (GR/NR3C1) in the brain have primarily focused on the forebrain, however in recent years, the hindbrain has become a region of interest for research into the development of anxiety and depression, though the role of GR signalling in the hindbrain remains poorly characterised. To determine the role of glucocorticoid signalling in the hindbrain we have developed a novel mouse model that specifically ablates hindbrain GR to ascertain its role in behaviour, HPA-axis regulation and adrenal structure. Our study highlights that ablation of GR in the hindbrain results in excessive barbering, obsessive compulsive digging and lack of cage exploration. These mice also develop kyphosis, elevated circulating corticosterone and severe adrenal cortex disruption. Together, this data demonstrates a role for hindbrain GR signalling in regulating stress-related behaviour and identifies a novel mouse model to allow further investigation into the pathways impacting stress and anxiety.

The tamoxifen-inducible Cre system is a popular transgenic method for controlling the induction of recombination by Cre at a specific time and in a specific cell type. However, tamoxifen is not an inert inducer of recombination, but an established endocrine disruptor with mixed agonist/antagonist activity acting via endogenous estrogen receptors. Such potentially confounding effects should be controlled for, but >40% of publications that have used tamoxifen to generate conditional knockouts have not reported even the minimum appropriate controls. To highlight the importance of this issue, the present study investigated the long-term impacts of different doses of a single systemic tamoxifen injection on the testis and the wider endocrine system. We found that a single dose of tamoxifen less than 10% of the mean dose used for recombination induction, caused adverse effects to the testis and to the reproductive endocrine system that persisted long-term. These data raise significant concerns about the widespread use of tamoxifen induction of recombination, and highlight the importance of including appropriate controls in all pathophysiological studies using this means of induction.

The current study aims to identify markers that would reflect the number of Leydig cells present in the testis, to help determine whether labour-intensive methods such as stereology are necessary. We used our well-characterised Sertoli cell ablation model in which we have empirically established the size of the Leydig cell population, to try to identify transcriptional biomarkers indicative of population size. Following characterisation of the Leydig cell population after Sertoli cell ablation in neonatal life or adulthood, we identified Hsd3b1 transcript levels as a potential indicator of Leydig cell number with utility for informing decision-making on whether to engage in time-consuming stereological cell counting analysis.

Leydig cells (LC) are the main testicular androgen-producing cells. In eutherian mammals, two types of LCs emerge successively during testicular development, fetal Leydig cells (FLCs) and adult Leydig cells (ALCs). Both display significant differences in androgen production and regulation. Using bulk RNA sequencing, we compared the transcriptomes of both LC populations to characterize their specific transcriptional and functional features. Despite similar transcriptomic profiles, a quarter of the genes show significant variations in expression between FLCs and ALCs. Non-transcriptional events, such as alternative splicing was also observed, including a high rate of intron retention in FLCs compared to ALCs. The use of single-cell RNA sequencing data also allowed the identification of nine FLC-specific genes and 50 ALC-specific genes. Expression of the corticotropin-releasing hormone 1 ( Crhr1 ) receptor and the ACTH receptor melanocortin type 2 receptor ( Mc2r ) specifically in FLCs suggests a dual regulation of steroidogenesis. The androstenedione synthesis by FLCs is stimulated by luteinizing hormone (LH), corticotrophin-releasing hormone (CRH), and adrenocorticotropic hormone (ACTH) whereas the testosterone synthesis by ALCs is dependent exclusively on LH. Overall, our study provides a useful database to explore LC development and functions.

The organismal roles of the ubiquitously expressed class I PI3K isoform p110β remain largely unknown. Using a new kinase-dead knockin mouse model that mimics constitutive pharmacological inactivation of p110β, we document that full inactivation of p110β leads to embryonic lethality in a substantial fraction of mice. Interestingly, the homozygous p110β kinase-dead mice that survive into adulthood (maximum ~26% on a mixed genetic background) have no apparent phenotypes, other than subfertility in females and complete infertility in males. Systemic inhibition of p110β results in a highly specific blockade in the maturation of spermatogonia to spermatocytes. p110β was previously suggested to signal downstream of the c-kit tyrosine kinase receptor in germ cells to regulate their proliferation and survival. We now report that p110β also plays a germ cell-extrinsic role in the Sertoli cells (SCs) that support the developing sperm, with p110β inactivation dampening expression of the SC-specific Androgen Receptor (AR) target gene Rhox5, a homeobox gene critical for spermatogenesis. All extragonadal androgen-dependent functions remain unaffected by global p110β inactivation. In line with a crucial role for p110β in SCs, selective inactivation of p110β in these cells results in male infertility. Our study is the first documentation of the involvement of a signalling enzyme, PI3K, in the regulation of AR activity during spermatogenesis. This developmental pathway may become active in prostate cancer where p110β and AR have previously been reported to functionally interact.

Dioxins are persistent organic pollutants interfering with endocrine systems and causing reproductive and developmental disorders. The objective of our project was to determine the impact of an in utero exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on reproductive function of male and female offspring in the rat with a special emphasis on the immature period. We used a low dose of TCDD (unique exposure by oral gavage of 200 ng/kg at 15.5 days of gestation) in order to mirror a response to an environmental dose of TCDD not altering fertility of the progeny. We choose a global gene expression approach using Affymetrix microarray analysis, and testes of 5 days and ovaries of 14 days of age. Less than 1% of the expressed genes in gonads were altered following embryonic TCDD exposure; specifically, 113 genes in ovaries and 56 in testes with 7 genes common to both sex gonads. It included the repressor of the aryl hydrocarbon receptor (Ahrr), the chemokines Ccl5 and Cxcl4 previously shown to be regulated by dioxin in testis, Pgds2/Hpgds and 3 others uncharacterized. To validate and extend the microarray data we realized real-time PCR on gonads at various developmental periods of interest (from 3 to 25 days for ovaries, from 5 to the adult age for testes). Overall, our results evidenced that both sex gonads responded differently to TCDD exposure. For example, we observed induction of the canonic battery of TCDD-induced genes coding enzymes of the detoxifying machinery in ovaries aged of 3-14 days of age (except Cyp1a1 induced at 3-10 days) but not in testes of 5 days (except Ahrr). We also illustrated that inflammatory pathway is one pathway activated by TCDD in gonads. Finally, we identified several new genes targeted by TCDD including Fgf13 in testis and one gene, Ptgds2/Hpgds regulated in the two sex gonads.

Androgens such as testosterone and dihydrotestosterone (DHT) are essential for male sexual development, masculinisation, and fertility. Testosterone is produced via the canonical androgen production pathway and is essential for normal masculinisation and testis function. Disruption to androgen production can result in disorders of sexual development (DSD). In the canonical pathway, 17β-hydroxysteroid dehydrogenase type 3 (HSD17B3) is viewed as a critical enzyme in the production of testosterone, performing the final conversion required. HSD17B3 deficiency in humans is associated with DSD due to low testosterone concentration during development. Individuals with HSD17B3 mutations have poorly masculinised external genitalia that can appear as ambiguous or female, whilst having internal Wolffian structures and testes. Recent studies in mice deficient in HSD17B3 have made the surprising finding that testosterone production is maintained, male mice are masculinised and remain fertile, suggesting differences between mice and human testosterone production exist. We discuss the phenotypic differences observed and the possible other pathways and enzymes that could be contributing to testosterone production and male development. The identification of alternative testosterone synthesising enzymes could inform the development of novel therapies to endogenously regulate testosterone production in individuals with testosterone deficiency.

Glucocorticoids are steroids involved in key physiological processes such as development, metabolism, inflammatory and stress responses and are mostly used exogenously as medications to treat various inflammation-based conditions. They act via the glucocorticoid receptor (GR) expressed in most cells. Exogenous glucocorticoids can negatively impact the function of the Leydig cells in the testis, leading to decreased androgen production. However, endogenous glucocorticoids are produced by the adrenal and within the testis, but whether their action on GR in Leydig cells regulates steroidogenesis is unknown. This study aimed to define the role of endogenous GR signalling in adult Leydig cells. We developed and compared two models; an inducible Cre transgene driven by expression of the Cyp17a1 steroidogenic gene (Cyp17-iCre) that depletes GR during development and a viral vector-driven Cre (AAV9-Cre) to deplete GR in adulthood. The delivery of AAV9-Cre ablated GR in adult mouse Leydig cells depleted Leydig cell GR more efficiently than the Cyp17-iCre model. Importantly, adult depletion of GR in Leydig cells caused reduced expression of luteinising hormone receptor (Lhcgr) and of steroidogenic enzymes required for normal androgen production. These findings reveal that Leydig cell GR signalling plays a physiological role in the testis and highlight that a normal balance of glucocorticoid activity in the testis is important for steroidogenesis.