Ulla Simanainen

Molecular insights into androgen actions in male and female reproductive function from androgen receptor knockout models

BACKGROUND Androgens and the androgen receptor (AR) have well known roles in male reproduction, and recent genetic mouse models inactivating the Ar gene have conclusively defined a role for androgens in female reproduction. In males, AR gene inactivation severely disrupts spermatogenesis by interrupting completion of meiosis, thereby eliminating production of mature sperm leading to male sterility. These effects have overshadowed the study of additional post-meiotic androgen effects required for the production of fully functional spermatozoa, as well as the production of females with complete androgen insensitivity which cannot be produced by natural breeding. However, these limitations have been overcome by the creation of global and cell-specific AR knockout (ARKO) mouse models using Cre-LoxP genetic engineering. METHODS Pubmed searches were carried out using the following search terms: androgen receptor, knockout mouse and fertility. Articles published before the end of November 2009 were included. RESULTS These experimental models have identified cell-specific AR-mediated androgen actions in testis and androgen actions in sex accessory glands independent of testicular effects which are crucial for sperm maturation, motion and fertilizing ability. The ability to produce homozygous ARKO females has revealed that AR-mediated androgen actions are important for normal female fertility. AR function is required for full functionality in follicle health, development and ovulation through both intra-ovarian and neuroendocrine mechanisms. CONCLUSIONS ARKO mouse models provide valuable tools to unravel novel roles of AR-mediated actions in male and female reproductive function, and new insights into the role of androgens in human reproductive function.

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.

Macronutrient balance, reproductive function, and lifespan in aging mice

Significance A fundamental tenet of life-history theory is that reproduction and longevity trade off against one another. Experiments on invertebrates show that, rather than competing for limiting resources, reproduction and lifespan are optimized on different dietary macronutrient compositions. In mice, studies have yet to establish the relationship between macronutrient balance, reproduction, and lifespan. We evaluated the effects of macronutrients and energy on lifespan and reproductive function. Indicators of reproductive function (uterine mass, ovarian follicle number, testes mass, epididymal sperm counts) were optimized by high protein (P), low carbohydrate (C) diets whereas lifespan was greatest on low P:C diets. Corpora lutea and estrous cycling were higher in females on lower P:C diets. Macronutrient balance has profound and opposing effects on reproduction and longevity. In invertebrates, reproductive output and lifespan are profoundly impacted by dietary macronutrient balance, with these traits achieving their maxima on different diet compositions, giving the appearance of a resource-based tradeoff between reproduction and longevity. For the first time in a mammal, to our knowledge, we evaluate the effects of dietary protein (P), carbohydrate (C), fat (F), and energy (E) on lifespan and reproductive function in aging male and female mice. We show that, as in invertebrates, the balance of macronutrients has marked and largely opposing effects on reproductive and longevity outcomes. Mice were provided ad libitum access to one of 25 diets differing in P, C, F, and E content, with reproductive outcomes assessed at 15 months. An optimal balance of macronutrients exists for reproductive function, which, for most measures, differs from the diets that optimize lifespan, and this response differs with sex. Maximal longevity was achieved on diets containing a P:C ratio of 1:13 in males and 1:11 for females. Diets that optimized testes mass and epididymal sperm counts (indicators of gamete production) contained a higher P:C ratio (1:1) than those that maximized lifespan. In females, uterine mass (an indicator of estrogenic activity) was also greatest on high P:C diets (1:1) whereas ovarian follicle number was greatest on P:C 3:1 associated with high-F intakes. By contrast, estrous cycling was more likely in mice on lower P:C (1:8), and the number of corpora lutea, indicative of recent ovulations, was greatest on P:C similar to those supporting greatest longevity (1:11).

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.

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.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure on bone material properties

Dioxins are known to decrease bone strength, architecture and density. However, their detailed effects on bone material properties are unknown. Here we used nanoindentation methods to characterize the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on nanomechanical behaviour of bone matrix. Pregnant rats were treated with a single intragastric dose of TCDD (1 microg/kg) or vehicle on gestational day 11. Tibias of female offspring were sampled on postnatal day (PND) 35 or 70, scanned at mid-diaphysis with pQCT, and evaluated by three-point bending and nanoindentation. TCDD treatment decreased bone mineralization (p<0.05), tibial length (p<0.01), cross-sectional geometry (p<0.05) and bending strength (p<0.05). Controls showed normal maturation pattern between PND 35 and 70 with decreased plasticity by 5.3% and increased dynamic hardness, storage and complex moduli by 26%, 13% and 12% respectively (p<0.05), while similar maturation was not observed in TCDD-exposed pups. In conclusion, for the first time, we demonstrate retardation of bone matrix maturation process in TCDD-exposed animals. In addition, the study confirms that developmental TCDD exposure has adverse effects on bone size, strength and mineralization. The current results in conjunction with macromechanical behaviour suggest that reduced bone strength caused by TCDD is more associated with the mineralization and altered geometry of bones than with changes at the bone matrix level.

Dose-response analysis of short-term effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in three differentially susceptible rat lines.

Line A, B, and C rats were selectively bred from TCDD-resistant Han/Wistar (Kuopio; H/W) and TCDD-sensitive Long-Evans (Turku/AB; L-E) rats. Line A rats are the most resistant to TCDD acute lethality followed by line B and line C rats. The resistance in line A rats is associated with a mutated H/W-type aryl hydrocarbon receptor (Ahr) allele (Ahr(hw)) and in line B rats the resistance is associated with an allele of an unknown gene B (B(hw)), while line C rats are almost as sensitive to TCDD as L-E rats. The dose-responses of characteristic short-term effects (day 8 postexposure) of TCDD were used to evaluate the efficacy (magnitude of effect) and potency relationships between these lines. Line A rats showed similar efficacies as line C (line A:line C efficacy ratio more than 0.7) for thymus weight, EROD activity, and incisor tooth defects. In contrast, efficacies in line A were decreased (efficacy ratios 0.19-0.37) for body weight change, serum bilirubin, and FFA levels, and serum ASAT activity. For most endpoints the efficacies in line B rats seem to be lower than in line C rats. The potencies were close to each other in line A and B rats, but somewhat lower than in line C rats. The results support our previous concept of two different AHR-mediated signaling pathways leading to dioxin type I and type II endpoints. Rats with the Ahr(hw/hw) genotype show a markedly decreased efficacy for type II endpoints, but B(hw) allele had only a minor effect on efficacies for most endpoints. Both H/W-type resistance alleles also decreased the potency of TCDD. However, the potency differences in short-term toxicity seem not to explain, at least alone, the differences seen in acute lethality among the rat lines.

Severe subfertility in mice with androgen receptor inactivation in sex accessory organs but not in testis.

Androgen action on sex accessory organs influences rodent fertility, but the mechanisms remain unclear and investigation is difficult without the ability to restrict androgen action in specific tissues. We used Cre-LoxP technology to generate male mice with prostate epithelial-specific androgen receptor deficiency (denoted PEARKO). In addition to prostate, these males have reduced androgen action due to tissue-selective androgen receptor inactivation in seminal vesicle, epididymis, and vas deferens, whereas the testis is unaffected. We find that fertility of PEARKO males was severely reduced, compared with littermates with prominent defects in copulatory plug formation, which were smaller, softer, and more friable than controls. Despite normal testis sperm production, sperm numbers were reduced in caput but increased in cauda epididymis, suggesting alterations in sperm epididymal transit kinetics associated with increased rate of spontaneous acrosome reaction and abnormal flagellar morphology in PEARKO cauda epididymal sperm. Whereas the quantitative in vitro fertilizing ability of PEARKO epididymal sperm was normal, fewer fertilized oocytes were flushed from the oviducts of females after natural mating with PEARKO males. These data show that sperm formed in mice with impaired androgen action restricted to accessory glands and epididymis are quantitatively normal in number and in vitro fertilizing function but that severe in vivo subfertility reflects other functions related to sperm transport and survival in female reproductive tract that determine fertility in vivo.

Androgen Receptor Inactivation Resulted in Acceleration in Pubertal Mammary Gland Growth, Upregulation of ERα Expression, and Wnt/β-Catenin Signaling in Female Mice

The androgen receptor (AR) is widely expressed in mammary cells of female mammals including humans and mice, indicating a possible role for AR-mediated androgen actions in breast development, function, and pathology, although the specific mechanisms remain unclear. To elucidate the mechanisms of androgen action in mammary gland physiology and development, we used AR-knockout (AR(Δex3)KO) female mice with a universally expressed, transcriptionally inactive AR protein harboring an in-frame deletion of its second zinc finger. Although in sexually mature wild-type (WT) and AR(ex3Δ)KO females, the mammary epithelial growth was fully extended to the edge of the fat pad, during puberty, AR(ex3Δ)KO females exhibit significantly accelerated mammary ductal growth and an increased number of terminal end buds compared with WT females. Accelerated AR(ex3Δ)KO female mammary growth was associated with significantly increased mammary epithelial ERα expression and activated Wnt/β-catenin signaling as shown by increased Wnt4 expression and accumulation of nuclear β-catenin. These findings are consistent with increased mammary estrogen exposure although ovarian estradiol content was unchanged compared with WT females. Furthermore, treatment with the potent pure androgen DHT markedly reduced ductal extension and terminal end bud numbers in WT but not in AR(Δex3)KO females, further supporting the concept that AR-mediated, androgen-induced suppression of murine mammary growth is a physiological characteristic of puberty. In summary, our findings reveal an inhibitory role of AR-mediated androgen actions in pubertal mammary gland development by reducing epithelial cell proliferation and could be mediated by regulation of Wnt/β-catenin signaling.

Development and Characterization of Uterine Glandular Epithelium Specific Androgen Receptor Knockout Mouse Model

ABSTRACT While estrogen action is the major driver of uterine development, androgens acting via the androgen receptor (AR) may also promote uterine growth as suggested by uterine phenotypes in global AR knockout (ARKO) female mice. Because AR is expressed in uterine endometrial glands, we generated (Cre/loxP) uterine gland epithelium-specific ARKO (ugeARKO) to determine the role of endometrial gland-specific androgen actions. However, AR in uterine gland epithelium may not be required for normal uterine development and function because ugeARKO females had normal uterine development and fertility. To determine if exogenous androgens acting via AR can fully support uterine growth in the absence of estrogens, the ARKO and ugeARKO females were ovariectomized and treated with supraphysiological doses of testosterone or dihydrotestosterone (nonaromatizable androgen). Both dihydrotestosterone and testosterone supported full uterine regrowth in wild-type females while ARKO females had no regrowth (comparable to ovariectomized only). These findings suggest that androgens acting via AR can promote full uterine regrowth in the absence of estrogens. The ugeARKO had 50% regrowth when compared to intact uterine glands, and histomorphologically, both the endometrial and myometrial areas were significantly (P < 0.05) reduced, suggesting glandular epithelial AR located in the endometrium may indirectly modify myometrial development. Additionally, to confirm Cre function in endometrial glands, we generated uge-specific PTEN knockout mouse model. The ugePTEN knockout females developed severe endometrial hyperplasia and therefore present a novel model for future research.