Targeting and repair of adult testicular somatic cells through viral gene therapy

Androgens are essential for the maintenance of male health and wellbeing. A disturbance in androgen signalling has been associated with a number of clinically relevant disorders such as cardiovascular disease, diabetes and metabolic disorders as well as infertility. Primarily produced in the testis in males, the actions of androgens are mediated through binding to androgen receptor (AR), a member of the nuclear receptor superfamily of ligand-activated transcription factors. The somatic cells of the testis are known to have a number of key roles in both testis function and development and the Sertoli, Leydig and Peritubular Myoid cells are known to express AR in adulthood. It is through AR that some testicular functions are mediated; for example, the Sertoli cells support of complete spermatogenesis with Sertoli cell androgen receptor knockout (SCARKO) testis demonstrating a halt of spermatogenesis before meiosis. However, how androgen signalling is impacting testicular function through each of the somatic cell types is not yet fully understood. Currently, treatments for male reproductive disorders such as hypogonadism (low androgens) and infertility are limited to treatment of the symptoms; using androgen replacement therapy and in vitro fertilisation techniques. This has been, up until recently, a result of a lack of understanding of the causes of these conditions and a lack of resources able to treat them, with research suggesting that a genetic component may be responsible in a number of cases. However, due to the limited genetic investigation diagnosis of men with male reproductive disorders, the wider understanding of the genetics underpinning male hypogonadism and infertility is incomplete. Developments in technology for the investigation and editing of the genetic code are triggering a surge in the exploration of genetic disorders and, in parallel, into the fields of gene delivery vectors and editing technologies. These technologies will allow an expansion into the knowledge and understanding of genetic disorders whilst simultaneously affording the opportunity to exploit this understanding for the development of therapeutics. There have been a small handful of previous studies using technologies such as viral vectors to target the testicular somatic cells and deliver exogenous transgenes with the purpose of both gene editing and repair, all with varying degrees of success. Here, techniques to introduce and target the Leydig and Sertoli cells were investigated to determine the most appropriate methodology for gene delivery to and manipulation of the testis. Refinement of injections into the interstitial compartment were carried out before introducing lentiviral vectors and targeting of Leydig cells was validated and optimised. Lentiviral vectors are able to permanently integrate into the host cell. Surprisingly, analysis of testis post lentiviral injection determined that the lentiviral targeted Leydig cells began to undergo apoptosis one week post injection and were subsequently cleared from the testis after ten days. Contrastingly, this was not the case when adenoviral vectors were introduced into the interstitial compartment, with Leydig cells continuing to express the delivered reporter transgene and, importantly, not expressing markers of apoptosis, ten days post injection. This would suggest that using adenoviral vectors to target the Leydig cell population in the adult testis would be more appropriate than using lentiviral vectors. Previous studies have successfully used lentiviral vectors to target the Sertoli cells in the adult testis via the introduction of the particles through the efferent duct. However, this can result in damage to efferent duct, resulting in blockages and subsequently the seminiferous tubules. To circumvent this, introduction of the lentiviral particles through the rete compartment of the testis at a range of lower injection pressures was examined and injecting at a lower pressure through the rete testis was found to reduce the likelihood of introducing negative impacts on testicular histology when targeting the seminiferous tubules. Using these refined methods of introducing lentiviral vectors, targeted Sertoli cells stably expressed the delivered transgene for up to one year post injection. Using viral vector delivered transgenes for both the investigation of testicular genetic disorders and for the development of therapeutics has great potential. To explore this potential, we first generated a mouse model in which AR was ablated from both the Leydig and Sertoli cells using Cre/LoxP technology, termed the SC-LC-ARKO. Alongside providing a potential model to ‘repair’ with viral vectors, the SC-LC-ARKO model also provided an additional model for comparison with other models exhibiting ablation of AR from both single somatic cell types and double somatic cell types. This further enabled a characterisation of the roles of AR in adult testicular function, with results suggesting that loss of AR from more than one cell type results in an additive phenotype when compared to single cell knock outs. Despite providing further insight into the roles of AR in the testis, further analysis of the Cre line used to generate the SC-LC-ARKO model indicated that a small number of Leydig cells were expressing the Cre recombinase, resulting in only a small population of Leydig cells with ablated AR. Considering this, to explore the potential of rescuing Sertoli cell AR using lentiviral vectors, we then utilised an already well characterised Sertoli Cell AR knockout (SCARKO) model. Lentiviral vectors expressing mouse AR and monomeric GFP (moeGFP) downstream of a CMV promoter were generated and injected into the rete testis of WT and SCARKO adult (day 100) males at low pressure. The contralateral testis was injected with a lentiviral vector expressing moeGFP alone (also downstream of a CMV promoter) using the same technique. Analysis of testis sections revealed a reintroduction of AR to Sertoli cells in 100% of SCARKO testis injected with lentivirus expressing mouse AR. As a result of this re-expression of AR in Sertoli cells, 66% of the testis injected with lentivirus expressing mouse AR had evidence of morphologically mature elongated spermatids, indicative of ongoing spermatogenesis. These results suggest that a rescue of the infertility phenotype reported in previous studies of SCARKO testis. Also demonstrated is the reversal of the SCARKO testicular phenotype in tubules targeted by the mAR expressing lentiviral vector. This suggests that absence Sertoli cell AR throughout development does not have a permanent impact on the Sertoli cells capacity to support spermatogenesis in adulthood following rescue of SC AR expression in adulthood. In summary, the results of these studies have provided a refinement in the methodologies for targeting the Sertoli and Leydig cells of the adult testis with viral vectors as well as demonstrating successful rescue of a previously reported mouse model exhibiting infertility through reintroduction of a functional gene. Alongside this, comparisons of AR knockout models have afforded insight into maintenance of testis function through AR

Ablation of the canonical testosterone production pathway via knockout of the steroidogenic enzyme HSD17B3, reveals a novel mechanism of testicular testosterone production

Male development, fertility, and lifelong health are all androgen-dependent. Approximately 95% of circulating testosterone is synthesized by the testis and the final step in this canonical pathway is controlled by the activity of the hydroxysteroid-dehydrogenase-17-beta-3 (HSD17B3). To determine the role of HSD17B3 in testosterone production and androgenization during male development and function we have characterized a mouse model lacking HSD17B3. The data reveal that developmental masculinization and fertility are normal in mutant males. Ablation of HSD17B3 inhibits hyperstimulation of testosterone production by hCG, although basal testosterone levels are maintained despite the absence of HSD17B3. Reintroduction of HSD17B3 via gene-delivery to Sertoli cells in adulthood partially rescues the adult phenotype, showing that, as in development, different cell-types in the testis are able to work together to produce testosterone. Together, these data show that HS17B3 acts as a rate-limiting-step for the maximum level of testosterone production by the testis but does not control basal testosterone production. Measurement of other enzymes able to convert androstenedione to testosterone identifies HSD17B12 as a candidate enzyme capable of driving basal testosterone production in the testis. Together, these findings expand our understanding of testosterone production in males

Sertoli cell number defines and predicts germ and Leydig cell population sizes in the adult mouse testis

Sertoli cells regulate differentiation and development of the testis and are essential for maintaining adult testis function. To model the effects of dysregulating Sertoli cell number during development or aging, we have used acute diphtheria toxin−mediated cell ablation to reduce Sertoli cell population size. Results show that the size of the Sertoli cell population that forms during development determines the number of germ cells and Leydig cells that will be present in the adult testis. Similarly, the number of germ cells and Leydig cells that can be maintained in the adult depends directly on the size of the adult Sertoli cell population. Finally, we have used linear modeling to generate predictive models of testis cell composition during development and in the adult based on the size of the Sertoli cell population. This study shows that at all ages the size of the Sertoli cell population is predictive of resulting testicular cell composition. A reduction in Sertoli cell number/proliferation at any age will therefore lead to a proportional decrease in germ cell and Leydig cell numbers, with likely consequential effects on fertility and health

Testicular cell selective ablation using diphtheria toxin receptor transgenic mice

Testis development and function is regulated by intricate cell-cell cross talk. Characterization of the mechanisms underpinning this has been derived through a wide variety of approaches including pharmacological manipulation, transgenics, and cell-specific ablation of populations. The removal of all or a proportion of a specific cell type has been achieved through a variety of approaches. In this paper, we detail a combined transgenic and pharmacological approach to ablate the Sertoli or germ cell populations using diphtheria toxin in mice. We describe the key steps in generation, validation, and use of the models and also describe the caveats and cautions necessary. We also provide a detailed description of the methodology applied to characterize testis development and function in models of postnatal Sertoli or germ cell ablation

A Novel Model Using AAV9-Cre to Knockout Adult Leydig Cell Gene Expression Reveals a Physiological Role of Glucocorticoid Receptor Signalling in Leydig Cell Function

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

Specific transcriptomic signatures and dual regulation of steroidogenesis between fetal and adult mouse leydig cells

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