Laura Milne

Sertoli Cells Maintain Leydig Cell Number and Peritubular Myoid Cell Activity in the Adult Mouse Testis

The Sertoli cells are critical regulators of testis differentiation and development. In the adult, however, their known function is restricted largely to maintenance of spermatogenesis. To determine whether the Sertoli cells regulate other aspects of adult testis biology we have used a novel transgenic mouse model in which Amh-Cre induces expression of the receptor for Diphtheria toxin (iDTR) specifically within Sertoli cells. This causes controlled, cell-specific and acute ablation of the Sertoli cell population in the adult animal following Diphtheria toxin injection. Results show that Sertoli cell ablation leads to rapid loss of all germ cell populations. In addition, adult Leydig cell numbers decline by 75% with the remaining cells concentrated around the rete and in the sub-capsular region. In the absence of Sertoli cells, peritubular myoid cell activity is reduced but the cells retain an ability to exclude immune cells from the seminiferous tubules. These data demonstrate that, in addition to support of spermatogenesis, Sertoli cells are required in the adult testis both for retention of the normal adult Leydig cell population and for support of normal peritubular myoid cell function. This has implications for our understanding of male reproductive disorders and wider androgen-related conditions affecting male health.

Sertoli cells control peritubular myoid cell fate and support adult Leydig cell development in the prepubertal testis

Sertoli cells (SCs) regulate testicular fate in the differentiating gonad and are the main regulators of spermatogenesis in the adult testis; however, their role during the intervening period of testis development, in particular during adult Leydig cell (ALC) differentiation and function, remains largely unknown. To examine SC function during fetal and prepubertal development we generated two transgenic mouse models that permit controlled, cell-specific ablation of SCs in pre- and postnatal life. Results show that SCs are required: (1) to maintain the differentiated phenotype of peritubular myoid cells (PTMCs) in prepubertal life; (2) to maintain the ALC progenitor population in the postnatal testis; and (3) for development of normal ALC numbers. Furthermore, our data show that fetal LCs function independently from SC, germ cell or PTMC support in the prepubertal testis. Together, these findings reveal that SCs remain essential regulators of testis development long after the period of sex determination. These findings have significant implications for our understanding of male reproductive disorders and wider androgen-related conditions affecting male health.

KATNAL1 regulation of sertoli cell microtubule dynamics is essential for spermiogenesis and male fertility

Spermatogenesis is a complex process reliant upon interactions between germ cells (GC) and supporting somatic cells. Testicular Sertoli cells (SC) support GCs during maturation through physical attachment, the provision of nutrients, and protection from immunological attack. This role is facilitated by an active cytoskeleton of parallel microtubule arrays that permit transport of nutrients to GCs, as well as translocation of spermatids through the seminiferous epithelium during maturation. It is well established that chemical perturbation of SC microtubule remodelling leads to premature GC exfoliation demonstrating that microtubule remodelling is an essential component of male fertility, yet the genes responsible for this process remain unknown. Using a random ENU mutagenesis approach, we have identified a novel mouse line displaying male-specific infertility, due to a point mutation in the highly conserved ATPase domain of the novel KATANIN p60-related microtubule severing protein Katanin p60 subunit A-like1 (KATNAL1). We demonstrate that Katnal1 is expressed in testicular Sertoli cells (SC) from 15.5 days post-coitum (dpc) and that, consistent with chemical disruption models, loss of function of KATNAL1 leads to male-specific infertility through disruption of SC microtubule dynamics and premature exfoliation of spermatids from the seminiferous epithelium. The identification of KATNAL1 as an essential regulator of male fertility provides a significant novel entry point into advancing our understanding of how SC microtubule dynamics promotes male fertility. Such information will have resonance both for future treatment of male fertility and the development of non-hormonal male contraceptives.

Anogenital Distance Plasticity in Adulthood: Implications for Its Use as a Biomarker of Fetal Androgen Action

Androgen action during the fetal masculinization programming window (MPW) determines the maximum potential for growth of androgen-dependent organs (eg, seminal vesicles, prostate, penis, and perineum) and is reflected in anogenital distance (AGD). As such, determining AGD in postnatal life has potential as a lifelong easily accessible biomarker of overall androgen action during the MPW. However, whether the perineum remains androgen responsive in adulthood and thus responds plastically to perturbed androgen drive remains unexplored. To determine this, we treated adult male rats with either the antiandrogen flutamide or the estrogen diethylstilbestrol (DES) for 5 weeks, followed by a 4-week washout period of no treatment. We determined AGD and its correlate anogenital index (AGI) (AGD relative to body weight) at weekly intervals across this period and compared these with normal adult rats (male and female), castrated male rats, and appropriate vehicle controls. These data showed that, in addition to reducing circulating testosterone and seminal vesicle weight, castration significantly reduced AGD (by ∼17%), demonstrating that there is a degree of plasticity in AGD in adulthood. Flutamide treatment increased circulating testosterone yet also reduced seminal vesicle weight due to local antagonism of androgen receptor. Despite this suppression, surprisingly, flutamide treatment had no effect on AGD at any time point. In contrast, although DES treatment suppressed circulating testosterone and reduced seminal vesicle weight, it also induced a significant reduction in AGD (by ∼11%), which returned to normal 1 week after cessation of DES treatment. We conclude that AGD in adult rats exhibits a degree of plasticity, which may be mediated by modulation of local androgen/estrogen action. The implications of these findings regarding the use of AGD as a lifelong clinical biomarker of fetal androgen action are discussed.

Kinase Activity of Fission Yeast Mph1 Is Required for Mad2 and Mad3 to Stably Bind the Anaphase Promoting Complex

Summary Defects in chromosome segregation result in aneuploidy, which can lead to disease or cell death [1, 2]. The spindle checkpoint delays anaphase onset until all chromosomes are attached to spindle microtubules in a bipolar fashion [3, 4]. Mad2 is a key checkpoint component that undergoes conformational activation, catalyzed by a Mad1-Mad2 template enriched at unattached kinetochores [5]. Mad2 and Mad3 (BubR1) then bind and inhibit Cdc20 to form the mitotic checkpoint complex (MCC), which binds and inhibits the anaphase promoting complex (APC/C). Checkpoint kinases (Aurora, Bub1, and Mps1) are critical for checkpoint signaling, yet they have poorly defined roles and few substrates have been identified [6–8]. Here we demonstrate that a kinase-dead allele of the fission yeast MPS1 homolog (Mph1) is checkpoint defective and that levels of APC/C-associated Mad2 and Mad3 are dramatically reduced in this mutant. Thus, MCC binding to fission yeast APC/C is dependent on Mph1 kinase activity. We map and mutate several phosphorylation sites in Mad2, producing mutants that display reduced Cdc20-APC/C binding and an inability to maintain checkpoint arrest. We conclude that Mph1 kinase regulates the association of Mad2 with its binding partners and thereby mitotic arrest.

Pituitary Androgen Receptor Signalling Regulates Prolactin but Not Gonadotrophins in the Male Mouse

Production of the androgen testosterone is controlled by a negative feedback loop within the hypothalamic-pituitary-gonadal (HPG) axis. Stimulation of testicular Leydig cells by pituitary luteinising hormone (LH) is under the control of hypothalamic gonadotrophin releasing hormone (GnRH), while suppression of LH secretion by the pituitary is controlled by circulating testosterone. Exactly how androgens exert their feedback control of gonadotrophin secretion (and whether this is at the level of the pituitary), as well as the role of AR in other pituitary cell types remains unclear. To investigate these questions, we exploited a transgenic mouse line (Foxg1Cre/+; ARfl/y) which lacks androgen receptor in the pituitary gland. Both circulating testosterone and gonadotrophins are unchanged in adulthood, demonstrating that AR signalling is dispensable in the male mouse pituitary for testosterone-dependent regulation of LH secretion. In contrast, Foxg1Cre/+; ARfl/y males have a significant increase in circulating prolactin, suggesting that, rather than controlling gonadotrophins, AR-signalling in the pituitary acts to suppress aberrant prolactin production in males.

Sertoli Cells Modulate Testicular Vascular Network Development, Structure, and Function to Influence Circulating Testosterone Concentrations in Adult Male Mice

The testicular vasculature forms a complex network, providing oxygenation, micronutrients, and waste clearance from the testis. The vasculature is also instrumental to testis function because it is both the route by which gonadotropins are delivered to the testis and by which T is transported away to target organs. Whether Sertoli cells play a role in regulating the testicular vasculature in postnatal life has never been unequivocally demonstrated. In this study we used models of acute Sertoli cell ablation and acute germ cell ablation to address whether Sertoli cells actively influence vascular structure and function in the adult testis. Our findings suggest that Sertoli cells play a key role in supporting the structure of the testicular vasculature. Ablating Sertoli cells (and germ cells) or germ cells alone results in a similar reduction in testis size, yet only the specific loss of Sertoli cells leads to a reduction in total intratesticular vascular volume, the number of vascular branches, and the numbers of small microvessels; loss of germ cells alone has no effect on the testicular vasculature. These perturbations to the testicular vasculature leads to a reduction in fluid exchange between the vasculature and testicular interstitium, which reduces gonadotropin-stimulated circulating T concentrations, indicative of reduced Leydig cell stimulation and/or reduced secretion of T into the vasculature. These findings describe a new paradigm by which the transport of hormones and other factors into and out of the testis may be influenced by Sertoli cells and highlights these cells as potential targets for enhancing this endocrine relationship.

Sertoli cell androgen receptor signalling in adulthood is essential for post-meiotic germ cell development.

Androgens are key drivers of spermatogenesis, and germ cells in mice lacking androgen receptor (AR), specifically from Sertoli cells, arrest in meiosis (reviewed in Smith and Walker, 2014). When Sertoli‐cell AR is ablated during fetal life (De Gendt and Verhoeven, 2012), however, it is impossible to determine whether the meiotic‐arrest phenotype observed in adults results from perturbed Sertoli cell development or perturbed function in adulthood. We used a lentiviral approach to determine if Sertoli‐cell AR is essential for supporting spermatogenesis specifically in adult testes—an organ where tamoxifen‐inducible knockout may present off‐target effects. Specifically, we introduced Cre recombinase into the Sertoli cells of adult male ARflox mice (De Gendt et al., 2004) to generate adult Sertoli‐Cell AR Knockout (aSCARKO) mice. Lentiviral particles contained both CMV‐Cre recombinase and tRFP635 (red fluorescent protein) transgenes separated by an IRES, or CMV‐tRFP635 alone. Shuttle vectors were packaged using a third‐generation lentiviral vector pseudotyped for VSV‐G, produced at a viral titer of >1 × 109. Virus was introduced into the seminiferous tubules of adult male ARflox/Y via injection into the efferent ducts, using 10 μl of Cre virus, tRFP635 control virus, or optiMEM (vehicle); an additional sham operated, but not injected, control was also evaluted. To control for systemic effects, combinations of Cre/control Cre/optiMEM, Cre/sham, control/optiMEM, control/sham, were generated in testes from individual mice (one treatment per testis; n = 10 per group). Tissues were collected 40 days after surgery (one complete cycle of spermatogenesis). Body and seminal vesicle weight (a biomarker of circulating androgen concentrations) did not differ between any treatment group (data not shown), but the weight of testes injected with Cre recombinase virus was significantly reduced (sham, 92.52 ± 4.29; OptiMEM, 100.12 ± 4.96; tRFP635 control, 70.22 ± 17.31; Cre, 35.48 ± 3.45 mg) to a final weight consistent with developmental‐SCARKO mice (De Gendt et al., 2004). tRFP635 was specifically detected in the cytoplasm of Sertoli cells (Fig. ​(Fig.1A,B),1A,B), but not in other testicular cell types. AR expression was observed in all somatic cells in both sham‐operated and control tRFP635 lentivirus‐injected testes. In contrast, injection of testes with Cre recombinase virus resulted in Sertoli cell‐specific localisation of tRFP with a loss of AR expression only in Sertoli cells; AR was retained in other testicular somatic cell types. Thus, AR had been selectively ablated in adult Sertoli cells whilst leaving the remainder of the testis untouched. Figure 1 Immunolocalisation of tRFP635 and AR, and testicular and epididymal morphology in virus‐injected adult testes. A–B: Sertoli cells retain AR expression (white arrows) following injection of control virus (A), whereas injection of Cre‐encoding ... Forty days post‐injection, seminiferous tubules from control testes retained normal spermatogenesis, with no obvious defects (Fig. ​(Fig.1A,C).1A,C). Testes injected with Cre recombinase virus, however, displayed evidence of germ‐cell arrest during meiosis (Fig. ​(Fig.1B,D),1B,D), similar to that observed in SCARKO mice (De Gendt et al., 2004). Furthermore, epididymides continuous with the Cre‐encoding virus‐injected testes contained no mature spermatozoa (Fig. ​(Fig.1F).1F). Therefore, loss of Sertoli‐cell AR in adulthood recapitulates the spermatogenic‐block phenotype observed in developmentally induced SCARKO models, unequivocally demonstrating that Sertoli cell AR is essential for continuous spermatogenesis in adults.

Leukemia Inhibitory Factor-Receptor is Dispensable for Prenatal Testis Development but is Required in Sertoli cells for Normal Spermatogenesis in Mice

Leukemia inhibitory factor (LIF), a pleiotropic cytokine belonging to the interleukin-6 family, is most often noted for its role in maintaining the balance between stem cell proliferation and differentiation. In rodents, LIF is expressed in both the fetal and adult testis; with the peritubular myoid (PTM) cells thought to be the main site of production. Given their anatomical location, LIF produced by PTM cells may act both on intratubular and interstitial cells to influence spermatogenesis and steroidogenesis respectively. Indeed, the leukemia inhibitory factor receptor (LIFR) is expressed in germ cells, Sertoli cells, Leydig cells, PTM cells and testicular macrophages, suggesting that LIF signalling via LIFR may be a key paracrine regulator of testicular function. However, a precise role(s) for testicular LIFR-signalling in vivo has not been established. To this end, we generated and characterised the testicular phenotype of mice lacking LIFR either in germ cells, Sertoli cells or both, to identify a role for LIFR-signalling in testicular development/function. Our analyses reveal that LIFR is dispensable in germ cells for normal spermatogenesis. However, Sertoli cell LIFR ablation results in a degenerative phenotype, characterised by abnormal germ cell loss, sperm stasis, seminiferous tubule distention and subsequent atrophy of the seminiferous tubules.