Kirsten J. McTavish

Subfertile Female Androgen Receptor Knockout Mice Exhibit Defects in Neuroendocrine Signaling, Intraovarian Function, and Uterine Development But Not Uterine Function

Female androgen receptor (AR) knockout mice (AR(-/-)) generated by an in-frame Ar exon 3 deletion are subfertile, but the mechanism is not clearly defined. To distinguish between extra- and intraovarian defects, reciprocal ovarian transplants were undertaken. Ovariectomized AR(-/-) hosts with wild-type (AR(+/+)) ovary transplants displayed abnormal estrus cycles, with longer cycles (50%, P < 0.05), and 66% were infertile (P < 0.05), whereas AR(+/+) hosts with either AR(-/-) or surgical control AR(+/+) ovary transplants displayed normal estrus cycles and fertility. These data imply a neuroendocrine defect, which is further supported by increased FSH (P <0.05) and estradiol (P <0.05), and greater LH suppressibility by estradiol in AR(-/-) females at estrus (P <0.05). Additional intraovarian defects were observed by the finding that both experimental transplant groups exhibited significantly reduced pups per litter (P < 0.05) and corpora lutea numbers (P < 0.05) compared with surgical controls. All groups exhibited normal uterine and lactation functions. AR(-/-) uteri were morphologically different from AR(+/+) with an increase in horn length (P < 0.01) but a reduction in uterine diameter (P < 0.05), total uterine area (P < 0.05), endometrial area (P < 0.05), and myometrial area (P < 0.01) at diestrus, indicating a role for AR in uterine growth and development. Both experimental transplant groups displayed a significant reduction in uterine diameter (P < 0.01) compared with transplanted wild-type controls, indicating a role for both AR-mediated intraovarian and intrauterine influences on uterine physiology. In conclusion, these data provide direct evidence that extraovarian neuroendocrine, but not uterine effects, as well as local intraovarian AR-mediated actions are important in maintaining female fertility, and a disruption of AR signaling leads to altered uterine development.

Follicle-stimulating hormone increases bone mass in female mice

Elevated follicle-stimulating hormone (FSH) activity is proposed to directly cause bone loss independent of estradiol deficiency in aging women. Using transgenic female mice expressing human FSH (TgFSH), we now reveal that TgFSH dose-dependently increased bone mass, markedly elevating tibial and vertebral trabecular bone volume. Furthermore, TgFSH stimulated a striking accrual of bone mass in hypogonadal mice lacking endogenous FSH and luteinizing hormone (LH) function, showing that FSH-induced bone mass occurred independently of background LH or estradiol levels. Higher TgFSH levels increased osteoblast surfaces in trabecular bone and stimulated de novo bone formation, filling marrow spaces with woven rather than lamellar bone, reflective of a strong anabolic stimulus. Trabecular bone volume correlated positively with ovarian-derived serum inhibin A or testosterone levels in TgFSH mice, and ovariectomy abolished TgFSH-induced bone formation, proving that FSH effects on bone require an ovary-dependent pathway. No detectable FSH receptor mRNA in mouse bone or cultured osteoblasts or osteoclasts indicated that FSH did not directly stimulate bone. Therefore, contrary to proposed FSH-induced bone loss, our findings demonstrate that FSH has dose-dependent anabolic effects on bone via an ovary-dependent mechanism, which is independent of LH activity, and does not involve direct FSH actions on bone cells.

Sertoli Cell Androgen Receptor DNA Binding Domain Is Essential for the Completion of Spermatogenesis

We examined the biological importance of Sertoli cell androgen receptor (AR) genomic interaction, using a Cre-loxP approach to selectively disrupt the AR DNA-binding domain (AR-DBD). Sertoli cell (SC)-specific transgenic Abpa or AMH promoters targeted Cre-mediated inframe excision of mouse Ar exon-3, encoding the AR-DBD second zinc-finger (ZF2), generating SC-specific mutant AR(DeltaZF2) lines designated Abp.SCAR(DeltaZF2) and AMH.SCAR(DeltaZF2), respectively. Both SCAR(DeltaZF2) lines produced infertile males exhibiting spermatogenic arrest, despite normal SC numbers and immunolocalized SC nuclear AR. Adult homozygous TgCre((+/+)) SCAR(DeltaZF2) or double-TgCre((+/-)) Abp/AMH.SCAR(DeltaZF2) males displayed equivalent small testes 30% of normal size, representing maximal Cre-loxP-disruption of Sertoli AR function. Hemizygous TgCre((+/-)) vs. homozygous TgCre((+/+)) Abp.SCAR(DeltaZF2) testes were larger (47% normal size) with more postmeiotic development, indicating dose-dependent Cre-mediated disruption of SC-specific AR-DBD activity. SCAR(DeltaZF2) males exhibited adult Leydig cell hypertrophy but normal serum testosterone levels. Sertoli cell-specific Rhox5 and Spinlw1 transcription, regulated by divergent or classical androgen-response elements, respectively, were both decreased in postnatal SCAR(DeltaZF2) vs. control testes, demonstrating SC-specific AR-DBD function as early as postnatal d 5. However, Rhox5 expression declined dose-dependently, whereas Spinlw1 expression increased, in adult TgCre((+/-)) and TgCre((+/+)) SCAR(DeltaZF2) testes, revealing differential temporal control for distinct AR-regulated transcripts. Androgen-repressed Ngfr was not up-regulated in SCAR(DeltaZF2) testes, suggesting maintenance of a nonclassical mechanism independent of AR-DBD. Thus, our unique SCAR(DeltaZF2) paradigm provided dose-dependent Cre-mediated disruption of testicular development and gene expression revealing that the AR-DBD is essential for SC function and postmeiotic spermatogenesis. Nongenomic or AR-DBD-independent pathways appear secondary or play no major independent role in SC function.

Temporal Role of Sertoli Cell Androgen Receptor Expression in Spermatogenic Development

Sertoli cell (SC) androgen receptor (AR) activity is vital for spermatogenesis. We created a unique gain-of-function transgenic (Tg) mouse model to determine the temporal role of SCAR expression in testicular development. The SC-specific rat Abpa promoter directed human Tg AR [Tg SC-specific AR (TgSCAR)] expression, providing strong premature postnatal AR immunolocalized to SC nuclei. Independent Tg lines revealed that TgSCAR dose dependently reduced postnatal and mature testis size (to 60% normal), whereas androgen-dependent mature seminal vesicle weights and serum testosterone levels remained normal. Total SC numbers were reduced in developing and mature TgSCAR testes, despite normal or higher Fshr mRNA and circulating FSH levels. Postnatal TgSCAR testes exhibited elevated levels of AR-regulated Rhox5 and Spinlw1 transcripts, and precocious SC function was demonstrated by early seminiferous tubular lumen formation and up-regulated expression of crucial SC tight-junction (Cldn11 and Tjp1) and phagocytic (Elmo1) transcripts. Early postnatal Amh expression was elevated but declined to normal levels in peripubertal-pubertal TgSCAR vs. control testes, indicating differential age-related regulation featuring AR-independent Amh down-regulation. TgSCAR induced premature postnatal spermatogenic development, shown by increased levels of meiotic (Dmc1 and Spo11) and postmeiotic (Capza3 and Prm1) germ cell transcripts, elevated meiotic-postmeiotic germ:Sertoli cell ratios, and accelerated spermatid development. Meiotic germ:Sertoli cell ratios were further increased in adult TgSCAR mice, indicating predominant SCAR-mediated control of meiotic development. However, postmeiotic germ:Sertoli cell ratios declined below normal. Our unique TgSCAR paradigm reveals that atypical SC-specific temporal AR expression provides a direct molecular mechanism for induction of precocious testicular development, leading to reduced adult testis size and decreased postmeiotic development.

Androgen Initiates Sertoli Cell Tight Junction Formation in the Hypogonadal (hpg) Mouse

ABSTRACT Sertoli cell tight junctions (TJs) form at puberty as a major component of the blood-testis barrier (BTB), which is essential for spermatogenesis. This study characterized the hormonal induction of functional Sertoli cell TJ formation in vivo using the gonadotropin-deficient hypogonadal (hpg) mouse that displays prepubertal spermatogenic arrest. Androgen actions were determined in hpg mice treated for 2 or 10 days with dihydrotestosterone (DHT). Follicle-stimulating hormone (FSH) actions were studied in hpg mice expressing transgenic human FSH (hpg+tgFSH) with or without DHT treatment. TJ formation was examined by mRNA expression and immunolocalization of TJ proteins claudin-3 and claudin-11, and barrier functionality was examined by biotin tracer permeability. Immunolocalization of claudin-3 and claudin-11 was extensive at wild-type (wt) Sertoli cell TJs, which functionally excluded permeability tracer. In contrast, seminiferous tubules of hpg testes lacked claudin-3, but claudin-11 protein was present in adluminal regions of Sertoli cells. Biotin tracer permeated throughout these tubules, demonstrating dysfunctional TJs. In hpg+tgFSH testes, claudin-3 was generally absent, but claudin-11 had redistributed basally toward the TJs, where function was variable. In hpg testes, DHT treatment stimulated the redistribution of claudin-11 protein toward the basal region of Sertoli cells by Day 2, increased Cldn3 and Cldn11 mRNA expression, then induced the formation of functional TJs containing both proteins by Day 10. In hpg+tgFSH testes, TJ protein redistribution was accelerated and functional TJs formed by Day 2 of DHT treatment. We conclude that androgen stimulates initial Sertoli cell TJ formation and function in mice, whereas FSH activity is insufficient alone, but augments androgen-induced TJ function.

Reproductive failure in mice expressing transgenic follicle-stimulating hormone is not caused by loss of oocyte quality†

Abstract Human female reproductive aging features declining ovarian follicle reserve and oocyte quality, and rising levels of circulating follicle-stimulating hormone (FSH).We determined the effects of elevated FSH on oocyte–embryo development in mature mice exhibiting premature infertility caused by progressively rising transgenic human FSH (TgFSH) levels. Oocyte–embryo developmental competence and quality were examined using oocyte maturation and aneuploidy rates, biomarkers of oocyte quality, and reciprocal embryo transfers assessed for implantation and pregnancy. In vitro maturation suggested that TgFSH exposure only hindered oocyte developmental competence in old females, as significantly more oocytes from ≥12-month-old TgFSH females remained at germinal vesicle stage compared with age-matched control oocytes. Aneuploidy rates were equivalent in oocytes from aging TgFSH compared with wildtype females. Cumulus cell expression levels of candidate biomarker Inhba, Egfr, and Rgs2 transcripts were elevated in associated aneuploid vs euploid oocytes from both TgFSH and wildtype females. In vivo, embryos transferred from subfertile 6-month-old TgFSH females to wildtype recipients yielded normal implantation rates and more pups born compared with controls. Transfer of wildtype embryos rescued the fertility of 6-month-old TgFSH-recipient females, although pup birth weight was reduced in TgFSH vs wildtype recipients. Our current findings show that elevated FSH had minimal disruption of either embryo developmental capacity or uterine function when examined in isolation, and the subfertility of TgFSH female mice was not caused by altered oocyte aneuploidy or quality. Summary Sentence Elevated FSH causes minimal disruption of either embryo developmental capacity or uterine function when examined in isolation, and the subfertility of TgFSH female mice is not caused by altered oocyte aneuploidy or quality.

265. Increased FSH activity increases primordial follicle reserve and enhances preovulatory follicle survival in transgenic FSH female mice

The mammalian female reproductive lifespan is determined by the depletion rate of the finite ovarian follicle reserve established before or shortly after birth. Follicle formation, initiation and early growth are thought to be independent of follicle-stimulating hormone (FSH), whereas antral follicle development requires FSH stimulation. Rising serum FSH is one of the earliest signs of reproductive ageing in women, coinciding with declining fecundity and an accelerated decline in remaining follicle reserves, but whether or not increased FSH plays a direct or feed-forward role in accelerating reproductive ageing remains undetermined. We previously described transgenic (Tg) mice with rising serum human FSH that produced larger litter sizes <20 weeks of age, then rapidly declining litter size from 20–40 weeks old (wo) culminating in premature infertility1. Despite declining fertility, ageing TgFSH females maintained ovulation rates ~3-fold higher than wt females. Follicle quantitation revealed that ovarian antral follicle numbers at diestrus were equivalent in 26 wo TgFSH and wt females. The elevated ovulation rates in TgFSH females may reflect increased preovulatory follicle survival during proestrus, as ~70% of large antral follicles go on to ovulate in TgFSH females, compared with only 30% in wt females. In contrast to the view that higher FSH may increase follicle development and consequently accelerate follicle depletion, examination of follicle reserve revealed that subfertile or infertile 26–52 wo TgFSH females exhibited increased total ovarian primordial follicle numbers (60%, P < 0.05) with no significant change in primary follicle numbers compared with age-matched wt females. Therefore, increased FSH activity appeared to act as a survival factor for primordial follicles. Our current analysis of increased FSH actions in female mice suggests that FSH may enhance the survival of both early (primordial) and late (preovulatory) follicle populations. (1) McTavish KJ et al. Endocrinology. 2007 Sep;148(9):4432–9.