Dietary bisphenol A prevents ovarian degeneration and bone loss in female mice lacking the aromatase gene (Cyp19 ).

We previously generated mice lacking aromatase activity by targeted disruption of Cyp19 (ArKO mice), and reported phenotypes of the female mice, showing hemorrhage formation and follicular depletion in the ovary, diminution in uterine size, and bone loss. In the present study, we examined the influence of dietary bisphenol A (BPA), a monomer used for the production of polycarbonate and known to have estrogenic activity, on these phenotypes of the ArKO mice. When ArKO mice were fed chow diets supplemented with 0.1% or 1% (w/w) BPA for 5 months, they were protected from ovarian degeneration, uterine diminution and bone loss in a dose-dependent manner. Northern blot analyses of ovarian RNA of ArKO mice showed differences in the expression levels of insulin-like growth factor (IGF)-I, IGF-I receptor, growth differentiation factor 9 and bone morphogenetic protein 15 as compared with those in the ovaries of wild-type mice. The differences in the expression levels were restored by dietary BPA. In the ArKO uteri, expression of progesterone receptor and vascular endothelial growth factor mRNAs was diminished, and was restored by BPA to the levels in wild-type mice. In contrast, BPA had little effect on the ovarian, uterine and skeletal structures of wild-type mice. In conclusion, estrogenic effects of BPA on the reproductive tract as well as skeletal tissue were evident in adult female ArKO mice. These results suggest that the ArKO mouse is an animal model suitable for studying effects of estrogenic chemicals as well as estrogen in vivo.     

Localization of androgen receptor and cytochrome P450 aromatase in the follicle and corpus luteum of the porcine ovary

The following study was undertaken to localize androgen receptors (AR) and aromatase cytochrome P450 (P450arom) in porcine ovarian tissue because ovarian androgens may act locally to modulate follicular and luteal function in various species. Androgen receptor was detected immunohistochemically in granulosa and theca cells of preantral as well as in growing antral follicles. The most intensive staining was observed in the antral granulosa layer. Luteinizing granulosa cells of preovulatory follicles, and luteal cells from the early and midluteal phases stained weakly for the androgen receptor. Fully regressed corpora lutea in the early follicular phase of the next cycle did not stain for androgen receptor. In contrast, granulosa cells were very weakly stained for aromatase in early stages of follicular development. The P450arom was maximally expressed with the same intensity in mural and antral layers in large ovulatory follicles. Corpora lutea from the early luteal phase showed positive staining, whereas those from midluteal phase did not stain for aromatase, some cells of regressed corpora lutea unexpectedly exhibited aromatase staining.     

Estrogen actions on follicle formation and early follicle development

Estradiol-17beta (E(2)) affects late follicular development, whereas primordial follicle differentiation and early activation are believed to be independent of E(2). To test this hypothesis we compared numbers of primordial and primary follicles in wild-type and E(2)-deficient, aromatase knockout (ArKO) mice, and the immunohistochemical staining or mRNA expression of Mullerian inhibiting substance (MIS), Wilms tumor 1 (WT-1), and growth differentiation factor (GDF9), which are known to effect early follicular differentiation. Proliferating cell nuclear antigen (PCNA) staining was a marker of proliferative index. The effects of E(2) replacement for 3 wk in 7-wk-old ArKO and wild-type mice on these parameters were also tested. ArKO mice had reduced numbers of primordial and primary follicles compared with wild-type mice (63%, P < 0.001 and 60%, P = 0.062, respectively). This reduction was not corrected by E(2) treatment, suggesting that E(2) affects the initial formation or activation of primordial follicles. There was a significant increase in the diameters of the oocytes in primordial follicles of ArKO mice compared with mice of the wild type. There were no differences in the immunostaining of MIS, WT-1, and PCNA in primordial and primary follicles between wild-type and ArKO mice. The only difference was as a consequence of Sertoli and Leydig cells that develop in ovaries of ArKO mice. GDF9 mRNA expression was markedly increased in ArKO ovaries. E(2) treatment restored the ovarian follicular morphology in ArKO mice, and consequently the immunostaining patterns, but had no effect on early follicle numbers. In conclusion, E(2) has a role in controlling the size of the oocyte and primordial follicle pool in mice.     

Effect of genetically defined oocyte depletion on production of androgens and oestrogens by ovaries of suckling mice

Steroid production and histological features of ovaries were compared either among normal +/+ mice of 3-12 days of age or among 12-day old mutant mice with various degrees of oocyte depletion. Whole ovaries were cultured in the medium containing [3H]progesterone and hCG or 4-androstene-3,17-dione and FSH; amounts of [3H]androgens or oestrogens released from the ovaries were assayed. FSH-responsive aromatase activity was detectable in ovaries of +/+ mice on day 3 after birth (2.6 +/- 0.4 pmol/2 ovaries/48 h), but the activity producing androgens from progesterone, under stimulation of hCG, was not detectable even on day 6 after birth (less than 0.1 pmol/2 ovaries/48 h). The androgen-producing activity appeared on day 9 after birth (1.16 +/- 0.25 pmol/2 ovaries/48 h), when follicles with more than two layers of granulosa cells developed. The ovaries of 12-day old Sl/Slt mice contained a considerable number of follicles with a single layer of granulosa cells, but did not contain any follicles with more than two layers of granulosa cells. The ovaries of Sl/Slt mice possessed aromatase activity (3.3 +/- 0.4 pmol/2 ovaries/48 h) but, not androgen-producing activity (less than 0.1 pmol/2 ovaries/48 h). The present results suggest that development of follicles with more than two layers of granulosa cells may induce the activity producing androgens from progesterone under stimulation of LH in suckling mouse ovaries, though the FSH-responsive aromatase activity is present even in follicles with a single layer of granulosa cells.     

Morphological and biochemical correlates of equine ovarian follicles as a function of their state of viability or atresia.

The histological features and hormonal content of follicular fluid of antral follicles during oestrus were correlated. As a result it was possible to characterize several categories of viable and atretic follicles. A seemingly important stage in maturation appeared to be at 3 cm in diameter since follicular oestrogens and androgens underwent a 3-fold increase in concentration at that size. Evidence was obtained to suggest that oestrogens are anti-atretogenic. However, a drop in oestrogens was not the cause of atresia since degeneration commenced when levels were high. Contrary to the concept that androgens are atretogenic in some species, it was also evident that elevated androgens did not precipitate spontaneous atresia. Theca epithelioid cells not only underwent histological luteinization in viable follicles as they matured toward ovulation but occasionally in atretic follicles as well. Elevated prostaglandin F levels were associated with follicles in the transitory states of either luteinization or atresia. Granulosa cells of viable follicles only were capable of specifically binding hCG. It was not determined whether loss of binding capacity or atresia occurred first. Follicular atresia in the mare appears to be a gradual process of which the initiating cause remains unknown.     

A role for the androgen receptor in follicular atresia of estrogen receptor beta knockout mouse ovary.

Estrogen receptor beta (ERbeta) is highly expressed, but ERalpha is not detectable in granulosa cells in the mouse ovary. In ERbeta knockout (BERKO) mice, there is abnormal follicular development and very reduced fertility. At 3 wk of age, no significant morphologic differences were discernable between wild type (WT) and BERKO mouse ovaries, but by 5 mo of age, atretic follicles were abundant in BERKO mice and there were very few healthy late antral follicles or corpora lutea. At 2 yr of age, unlike the ovaries of their WT littermates, BERKO mouse ovaries were devoid of healthy follicles but had numerous large, foamy lipid-filled stromal cells. The late antral and atretic follicles in BERKO mice were characterized by a high level of expression of the androgen receptor (AR) and IGF-1 receptor. These proteins were abundantly expressed in granulosa cells of preantral and early antral follicles in both genotypes, but their expression was extinguished in late antral follicles of WT mice. Healthy late antral follicles and corpora lutea were restored in BERKO ovaries after 15 days of treatment of mice with the antiandrogen flutamide. The results suggest that in the absence of ERbeta there was a loss of regulation of AR. Because androgens enhance recruitment of primordial follicles into the growth pool and cause atresia of late antral follicles, the inappropriately high level of AR probably is related to the follicular atresia and to the early exhaustion of follicles in BERKO mice.     

The role of steroids in follicular growth

The steroidogenic pathway within the ovary gives rise to progestins, androgens and oestrogens, all of which act via specific nuclear receptors to regulate reproductive function and maintain fertility. The role of progestins in follicular growth and development is limited, its action confined largely to ovulation, although direct effects on granulosa cell function have been reported. Consistent with these findings, progesterone receptor knockout mice are infertile because they cannot ovulate. Androgens have been shown to promote early follicular growth, but also to impede follicular development by stimulating atresia and apoptosis. The inability of androgens to transduce a signal in mice lacking androgen receptors culminates in reduced fertility. Oestrogens are known to exert effects on granulosa cell growth and differentiation in association with gonadotrophins. Studies with oestrogen receptor knockouts and oestrogen depleted mice have shown us that oestrogen is essential for folliculogenesis beyond the antral stage and is necessary to maintain the female phenotype of ovarian somatic cells. In summary, the action of steroids within the ovary is based on the developmental status of the follicle. In the absence of any single sex steroid, ovarian function and subsequently fertility, are compromised.     

Decreased oocyte DAZL expression in mice results in increased litter size by modulating follicle-stimulating hormone-induced follicular growth

While the germ cell-specific RNA binding protein, DAZL, is essential for oocytes to survive meiotic arrest, DAZL heterozygous (het) mice have an increased ovulation rate that is associated with elevated inhibin B and decreased plasma follicle-stimulating hormone (FSH). The relationship between decreased oocyte DAZL expression and enhanced follicular development in het mice was investigated using in vitro follicle cultures and in vivo modulation of endogenous FSH, by treating mice with inhibin and exogenous FSH. In vitro, follicles from het mice are more sensitive to FSH than those of wild-type (wt) mice and can grow in FSH concentrations that are deleterious to wild-type follicles. In vivo, despite no differences between genotypes in follicle population profiles, analysis of granulosa cell areas in antral follicles identified a significantly greater number of antral follicles with increased granulosa cell area in het ovaries. Modulation of FSH in vivo, using decreasing doses of FSH or ovine follicular fluid as a source of inhibin, confirmed the increased responsiveness of het antral follicles to FSH. Significantly more follicles expressing aromatase protein confirmed the earlier maturation of granulosa cells in het mice. In conclusion, it is suggested that DAZL expression represses specific unknown genes that regulate the response of granulosa cells to FSH. If this repression is reduced, as in DAZL het mice, then follicles can grow to the late follicular stage despite declining levels of circulating FSH, thus leading to more follicles ovulating and increased litter size.     

Alternations in hepatic expression of fatty-acid metabolizing enzymes in ArKO mice and their reversal by the treatment with 17beta-estradiol or a peroxisome proliferator

We generated aromatase gene knockout mice (ArKO mice) by targeting disruption of Cyp19, which encodes an enzyme responsible for conversion of androgens to estrogens. We found that ArKO males developed hepatic steatosis spontaneously with aging, indicating that the function of Cyp19 is required to maintain constitutive lipid metabolism in male mice. Plasma lipoprotein analysis using a gel permeation chromatography revealed that high density lipoprotein (HDL)-cholesterol levels were slightly higher in ArKO males than in wild-type males, whereas no other obvious alternations in the profiles were detected. Nevertheless, analysis of lipoprotein compositions by SDS-polyacrylamide gel electrophoresis demonstrated apparent reduction in the amounts of apolipoprotein E, functioning in receptor-mediated clearance of lipoproteins in the liver, in the IDL/LDL fraction of ArKO males as compared with that of wild-type males. Biochemical analysis on the ArKO livers revealed suppression of mRNA expression and activity of enzymes involved in fatty acid beta-oxidation. The impairment was reversed to the wild-type levels by treatment with 17beta-estradiol or bezafibrate, the latter is a synthetic peroxisome proliferator. These findings indicated a pivotal role of estrogen in supporting constitutive hepatic expression of genes involved in fatty acid beta-oxidation and in maintaining lipid homeostasis.     

Androgen synthesis during follicular development: evidence that rat granulosa cell 17-ketosteroid reductase is independent of hormonal regulation

Although androgens have been implicated in follicular atresia, ovarian follicular androgen synthesis is required for preovulatory follicular growth. To localize the site(s) of androgen biosynthesis and to obtain a better understanding of the regulation of the androgenic pathway(s) in rat ovarian follicles we examined the relative abilities of developing follicles to accumulate specific androgens [testosterone (T) and dihydrotestosterone (DHT)] using both radioimmunoassay (RIA) and 3H-substrate metabolism techniques. Small antral and preovulatory follicles were obtained from control or human chorionic gonadotropin (hCG)-primed immature rats, respectively (Richards and Bogovich, 1982). Small antral follicles, theca and granulosa cells produced little immunoassayable androgen (T + DHT) when incubated with or without 8-bromo-cAMP. In contrast, preovulatory follicles and theca produced more androgen than small antral tissues and in a manner acutely stimulable by cAMP. Granulosa cells produced little androgen under these conditions. Inclusion of [3H] androstenedione in the incubates yielded increased accumulation of [3H] T and [3H] DHT for all small antral and preovulatory tissues. Indeed, granulosa cells from both small antral and preovulatory follicles possessed a remarkable ability to accumulate [3H] T. This ability was not altered by hypophysectomy or subsequent treatment with estradiol and/or follicle-stimulating hormone (FSH). These results suggest that 17-ketosteroid reductase may be a constitutive enzyme in granulosa cells.     

Aromatase-deficient (ArKO) mice accumulate excess adipose tissue

Aromatase is the enzyme which catalyses the conversion of C₁₉ steroids into C₁₈ estrogens. We have generated a mouse model wherein the Cyp19 gene, which encodes aromatase, has been disrupted, and hence, the aromatase knockout (ArKO) mouse cannot synthesise endogenous estrogens. We examined the consequences of estrogen deficiency on accumulation of adipose depots in male and female ArKO mice, observing that these animals progressively accrue significantly more intra-abdominal adipose tissue than their wildtype (WT) litter mates, reflected in increased adipocyte volume and number. This increased adiposity was not due to hyperphagia or reduced resting energy expenditure, but was associated with reduced spontaneous physical activity levels, reduced glucose oxidation, and a decrease in lean body mass. Elevated circulating levels of leptin and cholesterol were present in 1-year-old ArKO mice compared to WT controls, as were elevated insulin levels, although blood glucose was unchanged. Associated with these changes, the livers of ArKO animals were characterised by a striking accumulation of lipid droplets. Our findings demonstrate an important role for estrogen in the maintenance of lipid homeostasis in both males and females.     

Autoimmune targeted disruption of the pituitary-ovarian axis causes premature ovarian failure

Premature ovarian failure (POF) is characterized by amenorrhea and high serum levels of follicle-stimulating hormone (FSH). POF causes female infertility and represents a substantial women's health risk affecting 1% of women by age 40. Although ovarian autoimmunity has been associated with POF, the identity of ovarian Ags recognized is unknown. In this study, we show that autoimmune-targeted disruption of the pituitary-ovarian axis leads to POF. Immunization of SWXJ female mice with the p215-234 peptide derived from mouse inhibin-alpha activates CD4(+) T cells and induces experimental autoimmune oophoritis with a unique biphasic phenotype characterized by an early stage of enhanced fertility followed by a delayed stage of POF. Affected mice show high serum levels of inhibin-alpha-neutralizing Abs that prevent inhibin-mediated down-regulation of activin-induced pituitary FSH release. The loss of activin/FSH down-regulation leads to prolonged metestrus-diestrus, superovulation, increased numbers of mature follicles, increased offspring, accelerated depletion of primordial follicles, and ultimately premature infertility. Thus, inhibin-alpha-targeted experimental autoimmune oophoritis is initiated by CD4(+) Th1 T cells that stimulate B cells to produce inhibin-alpha-neutralizing Abs directly capable of mediating POF and transferring disease into naive recipients. Our inhibin-alpha autoimmune model of POF shows how premature infertility may develop in the context of elevated FSH levels thereby closely mimicking the hallmark features of human POF.     

Alternations in hepatic expression of fatty-acid metabolizing enzymes in ArKO mice and their reversal by the treatment with 17β-estradiol or a peroxisome proliferator

We generated aromatase gene knockout mice (ArKO mice) by targeting disruption of Cyp19, which encodes an enzyme responsible for conversion of androgens to estrogens. We found that ArKO males developed hepatic steatosis spontaneously with aging, indicating that the function of Cyp19 is required to maintain constitutive lipid metabolism in male mice. Plasma lipoprotein analysis using a gel permeation chromatography revealed that high density lipoprotein (HDL)-cholesterol levels were slightly higher in ArKO males than in wild-type males, whereas no other obvious alternations in the profiles were detected. Nevertheless, analysis of lipoprotein compositions by SDS-polyacrylamide gel electrophoresis demonstrated apparent reduction in the amounts of apolipoprotein E, functioning in receptor-mediated clearance of lipoproteins in the liver, in the IDL/LDL fraction of ArKO males as compared with that of wild-type males. Biochemical analysis on the ArKO livers revealed suppression of mRNA expression and activity of enzymes involved in fatty acid β-oxidation. The impairment was reversed to the wild-type levels by treatment with 17β-estradiol or bezafibrate, the latter is a synthetic peroxisome proliferator. These findings indicated a pivotal role of estrogen in supporting constitutive hepatic expression of genes involved in fatty acid β-oxidation and in maintaining lipid homeostasis.     

Estrogen and adiposity--utilizing models of aromatase deficiency to explore the relationship

Estrogen has an important role to play in energy homeostasis in both men and mice. Lack of estrogen results in the development of a metabolic syndrome in humans and rodents, including excess adiposity, hepatic steatosis (in male but not female aromatase knockout (ArKO) mice) and insulin resistance. Estrogen replacement results in a prompt reversal of the energy imbalance symptoms associated with estrogen deficiency. A corollary to the perturbed energy balance observed in the ArKO mouse is the death by apoptosis of dopaminergic neurons in the hypothalamic arcuate nucleus of male ArKO mice, an area of the brain pivotal to the regulation of energy uptake, storage, and mobilisation. An extension of our work exploring the relationship between estrogen and adiposity has been to examine the role played by androgens in energy balance. We have demonstrated that an increased androgen to estrogen ratio can promote visceral fat accumulation in the rodent by inhibiting AMPK activation and stimulating lipogenesis. Therefore, understanding the regulation of energy homeostasis is becoming an increasingly fascinating challenge, as the number of contributors, their communications, and the complexity of their interactions, involved in the preservation of this equilibrium continues to increase. Models of aromatase deficiency, both naturally occurring and engineered, will continue to provide valuable insights into energy homeostasis.     

Selective expression of KrasG12D in granulosa cells of the mouse ovary causes defects in follicle development and ovulation

Activation of the RAS family of small G-proteins is essential for follicle stimulating hormone-induced signaling events and the regulation of target genes in cultured granulosa cells. To analyze the functions of RAS protein in granulosa cells during ovarian follicular development in vivo, we generated conditional knock-in mouse models in which the granulosa cells express a constitutively active KrasG12D. The KrasG12D mutant mice were subfertile and exhibited signs of premature ovarian failure. The mutant ovaries contained numerous abnormal follicle-like structures that were devoid of mitotic and apoptotic cells and cells expressing granulosa cell-specific marker genes. Follicles that proceeded to the antral stage failed to ovulate and expressed reduced levels of ovulation-related genes. The human chorionic gonadotropin-stimulated phosphorylation of ERK1/2 was markedly reduced in mutant cells. Reduced ERK1/2 phosphorylation was due, in part, to increased expression of MKP3, an ERK1/2-specific phosphatase. By contrast, elevated levels of phospho-AKT were evident in granulosa cells of immature KrasG12D mice, even in the absence of hormone treatments, and were associated with the progressive decline of FOXO1 in the abnormal follicle-like structures. Thus, inappropriate activation of KRAS in granulosa cells blocks the granulosa cell differentiation pathway, leading to the persistence of abnormal non-mitotic, non-apoptotic cells rather than tumorigenic cells. Moreover, those follicles that reach the antral stage exhibit impaired responses to hormones, leading to ovulation failure. Transient but not sustained activation of RAS in granulosa cells is therefore crucial for directing normal follicle development and initiating the ovulation process.     

Ovarian follicle apoptosis in bovine growth hormone transgenic mice

Growth hormone directly or via insulin like-growth factor-I has been shown to inhibit preovulatory follicle apoptosis, which is the underlying mechanism of follicular atresia. We studied the levels of apoptosis in the ovaries of transgenic mice expressing bovine growth hormone. Female bovine growth hormone transgenic mice (n = 10) and nontransgenic litter mates (n = 8) were killed at early proestrus. Ovaries were collected, sectioned, and processed using a nonradioactive in situ method for apoptosis detection. Follicles were classified and counted on the basis of size and level of apoptosis. Our results demonstrate that the percentage of ovarian follicles containing apoptotic cells was lower in transgenic versus normal mice (30% vs. 46%; P < 0.05). The percentage of follicles undergoing heavy apoptosis was lower (P < 0.05) in transgenic versus control animals in preovulatory and early antral follicles, but it was not different in preantral follicles. The percentage of healthy preovulatory follicles was also higher in transgenic versus normal mice (7.4% vs. 4.3%; P < 0.05). These results indicate that growth hormone overexpression in transgenic mice significantly decreases follicle apoptosis, and thus atresia in the mouse ovary, therefore leading to increased propensity for ovulation in these animals.     

Bone marrow transplantation restores follicular maturation and steroid hormones production in a mouse model for primary ovarian failure

Recent studies suggest that bone marrow stem cells (BMSCs) are promising grafts to treat a variety of diseases, including reproductive dysfunction. Primary ovarian failure is characterized by amenorrhea and infertility in a normal karyotype female, with an elevated serum level of follicle-stimulating hormone (FSH) and a decrease level of estrogen caused by a mutation in FSH receptor (FSHR) gene. Currently, there is no effective treatment for this condition. The phenotype of FSHR (-/-) mouse, FORKO (follitropin receptor knockout), is a suitable model to study ovarian failure in humans. Female FORKO mice have elevated FSH, decreased estrogen levels, are sterile because of the absence of folliculogenesis, and display thin uteri and small nonfunctional ovaries. In this study, we determined the effects of BMSC transplantation on reproductive physiology in this animal model. Twenty four hours post BMSC transplantation, treated animals showed detectable estroidogeneic changes in daily vaginal smear. Significant increase in total body weight and reproductive organs was observed in treated animals. Hemotoxylin and eosin (H&E) evaluation of the ovaries demonstrated significant increase in both the maturation and the total number of the follicles in treated animals. The FSH dropped to 40-50% and estrogen increased 4-5.5 times in the serum of treated animals compared to controls. The FSHR mRNA was detected in the ovaries of treated animals. Our results show that intravenously injected BMSCs were able to reach the ovaries of FORKO mice, differentiate and express FHSR gene, make FSHR responsive to FSH, resume estrogen hormone production, and restore folliculogenesis.     

Sex- and age-related response to aromatase deficiency in bone

Deficiency of sex steroids causes osteoporosis, but the relationship between estrogen and androgen is not clear because androgen is converted into estrogen by aromatase. In this study, we characterized bone metabolism in the aromatase-deficient (ArKO) mouse. At 9 weeks old, a marked loss of cancellous bone due to increased bone resorption was observed not only in female ArKO mice but also in males. The degree of bone loss in ArKO males was similar to that in females, and treatment with 17beta-estradiol completely restored the bone mass in both sexes. At 32 weeks old, female ArKO mice showed severe loss of cancellous and cortical bone. Male ArKO mice of this age also showed reduced bone mass, but the degree of bone loss in females was more marked than that in males. Here, we report sex- and age-related responses to aromatase deficiency in bone.     

Bone phenotype of the aromatase deficient mouse

Estrogens are important for normal bone growth and metabolism. The mechanisms are incompletely understood. Thus, we have undertaken characterization of the skeletal phenotype of aromatase (ArKO) deficient mice. No abnormalities have been noted in skeletal patterning in newborns. Adult ArKO mice show decreased femur length and decreased peak Bone Mineral Density (BMD) with accelerated bone loss by 7 months of age in females. Magnetic resonance microscopy (MR) and microCT (μCT) imaging disclosed decreased cancellous connectivity and reduced cancellous bone volume in ArKO females. Bone formation rate (BFR) is increased in ArKO females and decreased in ArKO males. Estradiol therapy reverses these changes. This anabolic effect of estradiol in the male skeleton is supported by 18-F⁻ Positron Emission Tomography (PET) imaging, which clearly demonstrates decreased spinal uptake, but marked increase after estradiol therapy. Serum IGF-1 levels are high in young female ArKO mice but low in young ArKO males. The reduced BMD in ArKO females, despite the presence of elevated serum IGF 1, suggests that other mechanism(s) are operative. There is increased B-cell lymphopoiesis in adult female ArKO bone marrow cells. These results show that ArKO mice show the effects of estrogen deficiency on bone growth, mass, metabolism, microarchitecture and the hematopoietic microenvironment.