Mark Jimenez

Osteoblast deletion of exon 3 of the androgen receptor gene results in trabecular bone loss in adult male mice.

The mechanism of androgen action on bone was studied in male mice with the AR deleted in mature osteoblasts. These mice had decreased trabecular bone volume associated with a decrease in trabecular number, suggesting that androgens may act directly on osteoblasts to maintain trabecular bone.

Measurement of sex steroids in murine blood and reproductive tissues by liquid chromatography–tandem mass spectrometry

Accurate measurement of sex steroids is essential to evaluate mouse models for human reproductive development and disorders. The recent advent of liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays that match the sensitivity of steroid immunoassay could overcome problems arising from the limited specificity of steroid immunoassay. In this current study we validate a LC-MS/MS assay for the measurement of key sex steroids from murine serum and reproductive tissues. The assay gave excellent dilutional linearity (r(2)> or =0.98) and reproducibility (CV< or =10% of replicate samples) in serum and reproductive tissues with sensitive quantitation limits; testosterone (T; 2pg), dihydrotestosterone (DHT; 10pg), 5alpha-androstane-3alpha,17beta-diol (3alphaDiol; 40pg), 5alpha-androstane-3beta,17beta-diol (3betaDiol; 40pg), estradiol (E2; 0.5pg) and estrone (E1; 0.3pg). Using 0.1mL sample, T was the only consistently detectable steroid (detection limit 20pg/ml) in both male and female mouse serum. In the testis, T and DHT were quantifiable as were both diols at relatively high levels. Prostatic T levels were low and DHT was determined to be the most abundant androgen in this tissue. Uterine and ovarian levels of E2, E1 and T were measurable, with levels varying according to estrous cycle stage. Hence, we demonstrate that this LC-MS/MS method has the sensitivity, specificity and multi-analyte capability to offer accurate steroid profiling in mouse serum and reproductive tissues.

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.

Characterization of Reproductive, Metabolic, and Endocrine Features of Polycystic Ovary Syndrome in Female Hyperandrogenic Mouse Models

Polycystic ovary syndrome (PCOS) affects 5-10% of women of reproductive age, causing a range of reproductive, metabolic and endocrine defects including anovulation, infertility, hyperandrogenism, obesity, hyperinsulinism, and an increased risk of type 2 diabetes and cardiovascular disease. Hyperandrogenism is the most consistent feature of PCOS, but its etiology remains unknown, and ethical and logistic constraints limit definitive experimentation in humans to determine mechanisms involved. In this study, we provide the first comprehensive characterization of reproductive, endocrine, and metabolic PCOS traits in 4 distinct murine models of hyperandrogenism, comprising prenatal dihydrotestosterone (DHT, potent nonaromatizable androgen) treatment during days 16-18 of gestation, or long-term treatment (90 days from 21 days of age) with DHT, dehydroepiandrosterone (DHEA), or letrozole (aromatase inhibitor). Prenatal DHT-treated mature mice exhibited irregular estrous cycles, oligo-ovulation, reduced preantral follicle health, hepatic steatosis, and adipocyte hypertrophy, but lacked overall changes in body-fat composition. Long-term DHT treatment induced polycystic ovaries displaying unhealthy antral follicles (degenerate oocyte and/or > 10% pyknotic granulosa cells), as well as anovulation and acyclicity in mature (16-week-old) females. Long-term DHT also increased body and fat pad weights and induced adipocyte hypertrophy and hypercholesterolemia. Long-term letrozole-treated mice exhibited absent or irregular cycles, oligo-ovulation, polycystic ovaries containing hemorrhagic cysts atypical of PCOS, and displayed no metabolic features of PCOS. Long-term dehydroepiandrosterone treatment produced no PCOS features in mature mice. Our findings reveal that long-term DHT treatment replicated a breadth of ovarian, endocrine, and metabolic features of human PCOS and provides the best mouse model for experimental studies of PCOS pathogenesis.

Targeted Loss of Androgen Receptor Signaling in Murine Granulosa Cells of Preantral and Antral Follicles Causes Female Subfertility

ABSTRACT Ovarian granulosa cells display strong androgen receptor (AR) expression, suggesting a functional role for direct AR-mediated actions within developing mammalian follicles. By crossing AR-floxed and anti-Müllerian hormone (AMH)-Cre recombinase mice, we generated granulosa cell-specific androgen receptor knockout mice (GCARKO). Cre expression, assessed by lacZ activity, localized to 70%–100% of granulosa cells in most preantral to antral follicles, allowing for selected evaluation of granulosa cell AR-dependent actions during follicle development. Relative to wild-type (WT) females, GCARKO females were subfertile, producing a 24% reduction in the number of litters (P < 0.05) over 6 mo and an age-dependent decrease in total number of pups born, evident from 6 mo of age (P < 0.05). Follicle dynamics were altered in GCARKO ovaries at 3 mo of age, with a significant reduction in large preantral and small antral follicle numbers compared to WT ovaries (P < 0.05). Global premature follicle depletion was not observed, but increased follicular atresia was evident in GCARKO ovaries at 6 mo of age, with an 81% increase in unhealthy follicles and zona pellucida remnants (P < 0.01). Cumulus cell expansion was decreased (P < 0.01) and oocyte viability was diminished in GCARKO females, with a significant reduction in the percentage of oocytes fertilized after natural mating and, thus, in the rate of progression to the two-cell embryo stage (P < 0.05). In addition, compared with age-matched WT females, 6-mo-old GCARKO females exhibited significantly prolonged estrous cycles (P ≤ 0.05), suggesting altered hypothalamic-pituitary-gonadal feedback signaling. In conclusion, our findings revealed that selective loss of granulosa cell AR actions during preantral and antral stages of development leads to a premature reduction in female fecundity through reduced follicle health and oocyte viability.

Luteinizing Hormone Receptor-Mediated Effects on Initiation of Spermatogenesis in Gonadotropin-Deficient (hpg) Mice Are Replicated by Testosterone

Abstract Testosterone (T) is an absolute requirement for spermatogenesis and is supplied by mature Leydig cells stimulated by LH. We previously showed in gonadotropin-deficient hpg mice that T alone initiates qualitatively complete spermatogenesis bypassing LH-dependent Leydig cell maturation and steroidogenesis. However, because maximal T effects do not restore testis weight or germ cell number to wild-type control levels, additional Leydig cell factors may be involved. We therefore examined 1) whether chronic hCG administration to restore Leydig cell maturation and steroidogenesis can restore quantitatively normal spermatogenesis and testis development and 2) whether nonandrogenic Leydig cell products are required to initiate spermatogenesis. Weanling hpg mice were administered hCG (0.1–100 IU i.p. injection three times weekly) or T (1-cm subdermal Silastic implant) for 6 weeks, after which stereological estimates of germinal cell populations, serum and testicular T content, and testis weight were evaluated. Human CG stimulated Leydig cell maturation and normalized testicular T content compared with T treatment where Leydig cells remained immature and inactive. The maximal hCG-induced increases in testis weight and serum T concentrations were similar to those for T treatment and produced complete spermatogenesis characterized by mature, basally located Sertoli cells (SCs) with tripartite nucleoli, condensed haploid sperm, and lumen development. Compared with T treatment, hCG increased spermatogonial numbers, but both hCG and T had similar effects on numbers of spermatocytes and round and elongated spermatids per testis as well as per SC. Nevertheless, testis weight and germ cell numbers per testis and per SC remained well below phenotypically normal controls, confirming the involvement of non-Leydig cell factors such as FSH for quantitative normalization of spermatogenesis. We conclude that hCG stimulation of Leydig cell maturation and steroidogenesis is not required, and that T alone mostly replicates the effects of hCG, to initiate spermatogenesis. Because T is both necessary and sufficient for initiation of spermatogenesis, it is likely that T is the main Leydig cell secretory product involved and that additional LH-dependent Leydig cell factors are not essential for induction of murine spermatogenesis.

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.

Estradiol Induction of Spermatogenesis Is Mediated via an Estrogen Receptor-α Mechanism Involving Neuroendocrine Activation of Follicle-Stimulating Hormone Secretion

Both testosterone and its nonaromatizable metabolite dihydrotestosterone (DHT) induce spermatogenesis in gonadotropin-deficient hpg mice. Surprisingly, because aromatization is not required, estradiol (E2) also induces spermatogenesis and increases circulating FSH in hpg mice, but the mechanism remains unclear. We studied E2-induced spermatogenesis in hpg mice on an estrogen receptor (ER)-alpha (hpg/alphaERKO) or ERbeta (hpg/betaERKO) knockout or wild-type ER (hpg/WT) background treated with subdermal E2 or DHT implants for 6 wk. In hpg/WT and hpg/betaERKO, but not hpg/alphaERKO mice, E2 increased testis and epididymal weight, whereas DHT-induced increases were unaffected by ERalpha or ERbeta inactivation. E2 but not DHT treatment increased serum FSH (but not LH) in hpg/WT and hpg/betaERKO but not hpg/alphaERKO hpg mice. DHT or E2 alone increased (premeiotic) spermatogonia and (meiotic) spermatocytes without significant change in Sertoli cell numbers. DHT alone increased postmeiotic spermatids, regardless of ER presence, compared with variable ERalpha-dependent E2 postmeiotic responses. An ERalpha-mediated effect was confirmed by treating hpg mice for 6 wk by subdermal selective ER-alpha (16alpha-LE(2)) or ERbeta (8beta-VE(2)) agonist implants. ERalpha (but not ERbeta) agonist increased testis and epididymal weight, Sertoli cell, spermatogonia, meiotic, and postmeiotic germ cell numbers. Only ERalpha agonist markedly increased serum FSH, whereas either agonist induced small rises in serum LH. Administration of ERalpha agonist or E2 in the presence of functional ERalpha induced prominent gene expression of specific Sertoli (Eppin, Rhox5) and Leydig cell (Cyp11a1, Hsd3b1) markers. We conclude that E2-induced spermatogenesis in hpg mice involves an ERalpha-dependent neuroendocrine mechanism increasing blood FSH and Sertoli cell function.

Androgen receptor gene expression in leucocytes is hormonally regulated: implications for gender differences in disease pathogenesis

Objective  There is evidence that male sex hormones influence the rate of progression of inflammatory and cardiovascular diseases. We have previously shown that human leucocytes and arterial cells isolated from male donors express more androgen receptor (AR) than those from female cells, with potentially pro‐atherogenic effects. We now investigate whether the gender difference in AR expression is due to genetic or hormonal regulation.

Validation of an Ultrasensitive and Specific Immunofluorometric Assay for Mouse Follicle-Stimulating Hormone

Abstract Sensitive and specific measurement of FSH is critical to research in reproductive biology, and the increasing availability of transgenic mouse models has created a need for a robust, sensitive, and specific mouse (m) FSH assay. The present study evaluated a time-resolved immunofluorometric assay (IFMA) for mFSH using monoclonal antibody to human (h) FSHβ as a capture antibody and a biotinylated polyclonal antibody to rat α subunit as a detection probe, with signaling amplified by europium-labeled streptavidin. The mFSH IFMA lowered the detection limit 34-fold (5 vs. 170 pg/sample) compared with standard mFSH RIA. The mFSH IFMA demonstrated parallelism of response to dilutions of castrated mouse serum and rat FSH but no cross-reactivity with hFSH and mLH or hLH, whereas the RIA demonstrated nonparallel cross-reactivity with hFSH. The IFMA has a wide analytical range, with a good precision profile for within- and between-assay reproducibility. Because the IFMA is a sandwich-type assay with strict dimer-specificity by design, the lower readings and recovery obtained were compared with the RIA when both assays used a pituitary-purified mFSH assay standard that contained isolated or fragmented subunits as well as intact dimeric FSH. When used with mouse serum sample, the mFSH IFMA demonstrated the expected increases following orchidectomy as well as markedly enhanced sensitivity to very low levels of endogenous mFSH in gonadotropin-deficient mice. Furthermore, the IFMA measured mFSH with fidelity in both intact and orchidectomized male mice without any interference from transgenic hFSH. The greatly enhanced sensitivity, specificity, and technical convenience of this mFSH IFMA will allow wider application of FSH measurements to very small blood samples in immature and mature mice as well as transgenic models.