Dax1 is required for testis determination.

The orphan nuclear receptor, Dax1, was originally proposed to act as an 'anti-testis' factor. We find, however, that Nr0b1 (also called Dax1 and Ahch, which encodes Dax1) is in fact required for testis differentiation.

Addressing Structural Racism Using a Whole-Scale Planning Process in a Single Academic Center.

Purpose: The murder of George Floyd in 2020 prompted a national demand for cultural transformation to confront the systemic racism prevalent in the country. Academic medical centers were not exempt from this urgent call. This article evaluates the efficacy of a strategic process in fostering cultural transformation within an academic medical system. Methods: A whole-scale strategic planning process was implemented over 13 months, involving multiple working groups representing key stakeholders from each entity across the system, an anonymous survey, a communication plan, and a balanced scorecard to monitor progress. More than 5500 voices, 160 recommendations, 122 data gathering sessions, and town hall meetings contributed to the creation and implementation of vital action items and a strategic framework. The Diversity Engagement Survey (DES) was administered 18 months following the process launch. Results: Of the 45,554 employees, students, faculty, and trainees, 96.5% completed unconscious bias education within the fiscal year and 76% of action items, termed "Just Do Its," were completed. Mission, vision, values, and strategic priorities were crafted to serve as a framework for intermediate and long-term actions. The DES revealed improvement in the "respect" attribute of an inclusive culture, and 64% of respondents confirmed that action for cultural transformation is addressing racism both within and outside of the institution. Conclusion: Implementing a shared purpose, engaging multiple working groups representing key stakeholders, and empowerment of stakeholders to implement changes, in conjunction with the development of a strategic framework addressing structural racism, resulted in the completion of vital action items to initiate cultural change.

ENU mutagenesis in mice identifies candidate genes for hypogonadism.

Genome-wide mutagenesis was performed in mice to identify candidate genes for male infertility, for which the predominant causes remain idiopathic. Mice were mutagenized using N-ethyl-N-nitrosourea (ENU), bred, and screened for phenotypes associated with the male urogenital system. Fifteen heritable lines were isolated and chromosomal loci were assigned using low-density genome-wide SNP arrays. Ten of the 15 lines were pursued further using higher-resolution SNP analysis to narrow the candidate gene regions. Exon sequencing of candidate genes identified mutations in mice with cystic kidneys (Bicc1), cryptorchidism (Rxfp2), restricted germ cell deficiency (Plk4), and severe germ cell deficiency (Prdm9). In two other lines with severe hypogonadism, candidate sequencing failed to identify mutations, suggesting defects in genes with previously undocumented roles in gonadal function. These genomic intervals were sequenced in their entirety and a candidate mutation was identified in SnrpE in one of the two lines. The line harboring the SnrpE variant retains substantial spermatogenesis despite small testis size, an unusual phenotype. In addition to the reproductive defects, heritable phenotypes were observed in mice with ataxia (Myo5a), tremors (Pmp22), growth retardation (unknown gene), and hydrocephalus (unknown gene). These results demonstrate that the ENU screen is an effective tool for identifying potential causes of male infertility.

p21-Activated kinase mediates rapid estradiol-negative feedback actions in the reproductive axis.

Nonclassical estrogen receptor alpha (ERalpha) signaling can mediate E(2) negative feedback actions in the reproductive axis; however, downstream pathways conveying these effects remain unclear. These studies tested the hypothesis that p21-activated kinase 1 (PAK1), a serine/threonine kinase rapidly activated by E(2) in nonneural cells, functions as a downstream node for E(2) signaling pathways in cells of the preoptic area, and it may thereby mediate E(2) negative feedback effects. Treatment of ovariectomized (OVX) rats with estradiol benzoate (EB) caused rapid and transient induction of phosphorylated PAK1 immunoreactivity in the medial preoptic nucleus (MPN) but not the arcuate nucleus. To determine whether rapid induction of PAK phosphorylation by E(2) is mediated by nonclassical [estrogen response element (ERE)-independent] ERalpha signaling, we used female ERalpha null (ERalpha(-/-)) mice possessing an ER knock-in mutation (E207A/G208A; AA), in which the mutant ERalpha is incapable of binding DNA and can signal only through membrane-initiated or ERE-independent genotropic pathways (ERalpha(-/AA) mice). After 1-h EB treatment, the number of pPAK1-immunoreactive cells in the MPN was increased in both wild-type (ERalpha(+/+)) and ERalpha(-/AA) mice but was unchanged in ERalpha(-/-) mice. Serum luteinizing hormone (LH) was likewise suppressed within 1 h after EB treatment in ERalpha(+/+) and ERalpha(-/AA) but not ERalpha(-/ -) mice. In OVX rats, 5-min intracerebroventricular infusion of a PAK inhibitor peptide but not control peptide blocked rapid EB suppression of LH secretion. Taken together, our findings implicate PAK1 activation subsequent to nonclassical ERalpha signaling as an important component of the negative feedback actions of E(2) in the brain.

Regulation of Kiss1 and dynorphin gene expression in the murine brain by classical and nonclassical estrogen receptor pathways.

Kisspeptin is a product of the Kiss1 gene and is expressed in the forebrain. Neurons that express Kiss1 play a crucial role in the regulation of pituitary luteinizing hormone secretion and reproduction. These neurons are the direct targets for the action of estradiol-17beta (E(2)), which acts via the estrogen receptor alpha isoform (ER alpha) to regulate Kiss1 expression. In the arcuate nucleus (Arc), where the dynorphin gene (Dyn) is expressed in Kiss1 neurons, E(2) inhibits the expression of Kiss1 mRNA. However, E(2) induces the expression of Kiss1 in the anteroventral periventricular nucleus (AVPV). The mechanism for differential regulation of Kiss1 in the Arc and AVPV by E(2) is unknown. ER alpha signals through multiple pathways, which can be categorized as either classical, involving the estrogen response element (ERE), or nonclassical, involving ERE-independent mechanisms. To elucidate the molecular basis for the action of E(2) on Kiss1 and Dyn expression, we studied the effects of E(2) on Kiss1 and Dyn mRNAs in the brains of mice bearing targeted alterations in the ER alpha signaling pathways. We found that stimulation of Kiss1 expression by E(2) in the AVPV and inhibition of Dyn in the Arc required an ERE-dependent pathway, whereas the inhibition of Kiss1 expression by E(2) in the Arc involved ERE-independent mechanisms. Thus, distinct ER alpha signaling pathways can differentially regulate the expression of identical genes across different brain regions, and E(2) can act within the same neuron through divergent ER alpha signaling pathways to regulate different neurotransmitter genes.

A missense mutation in LRR8 of RXFP2 is associated with cryptorchidism.

Using genome-wide mutagenesis with N-ethyl-N-nitrosourea (ENU), a mouse mutant with cryptorchidism was identified. Genome mapping and exon sequencing identified a novel missense mutation (D294G) in Relaxin/insulin-like family peptide receptor 2 (Rxfp2). The mutation impaired testicular descent and resulted in decreased testis weight in Rxfp2 ( DG/DG ) mice compared to Rxfp2 (+/DG ) and Rxfp2 (+/+) mice. Testicular histology of the Rxfp2 ( DG/DG ) mice revealed spermatogenic defects ranging from germ cell loss to tubules with Sertoli-cell-only features. Genetic complementation analysis using a loss-of-function allele (Rxfp2 (-)) confirmed causality of the D294G mutation. Specifically, mice with one of each mutant allele (Rxfp2 ( DG/-)) exhibited decreased testis weight and failure of the testes to descend compared to their Rxfp2 (+/-) littermates. Total and cell-surface expression of mouse RXFP2 protein and intracellular cAMP accumulation were measured. Total expression of the D294G protein was minimally reduced compared to wild-type, but cell-surface expression was markedly decreased. When analyzed for cAMP accumulation, the EC50 was similar for cells transfected with wild-type and mutant RXFP2 receptor. However, the maximum cAMP response that the mutant receptor reached was greatly reduced compared to the wild-type receptor. In silico modeling of leucine rich repeats (LRRs) 7-9 indicated that aspartic acid 294 is located within the ß-pleated sheet of LRR8. We thus postulate that mutation of D294 results in protein misfolding and aberrant trafficking. The ENU-induced D294G mutation underscores the role of the INSL3/RXFP2-mediated pathway in testicular descent and expands the repertoire of mutations known to affect receptor trafficking and function.

Absence of activating mutations of CXCR4 in pituitary tumours.

OBJECTIVE: Mutations of the gsp oncogene are responsible for 30-40% of GH-producing pituitary adenomas and 10% of nonfunctioning pituitary adenomas (NFPAs). However, the pathogenetic mechanism of the remaining pituitary tumours still remains to be identified. Recently, the interaction between the chemokine stromal cell-derived factor 1 and its receptor CXCR4 was found to play an important role in GH production and cell proliferation in various pituitary adenoma cell lines. As CXCR4 is a Gi-coupled chemokine receptor, its constitutive activating mutations may be involved in pituitary tumour formation by cyclic adenosine monophosphate (cAMP)-independent, ERK-related pathways. PATIENTS AND METHODS: We investigated whether somatic activating-mutations of CXCR4 might be a possible tumourigenic mechanism for gsp-negative GH-secreting pituitary adenomas and NFPAs. Direct sequencing of polymerase chain reaction-amplified products for coding exons of CXCR4 were performed using genomic deoxyribonucleic acid samples from 37 GH-producing pituitary tumour tissues that were negative for the gsp mutation and 14 CXCR4 expressing NFPAs. RESULTS: Immunohistochemical analyses and double immunofluorescent staining of sectioned paraffin-embedded pituitary tissues revealed that CXCR4 is highly expressed in GH-producing pituitary adenomas and NFPAs. Direct sequencing showed that two synonymous mutations in exon 2 (87 C > T and 414 C > T) were detected in 4 out of 51 pituitary tumours. CONCLUSION: Our results indicate that an activating mutation of the CXCR4 may not be a common pathogenetic mechanism in GH-producing pituitary tumours and NFPAs.

Aromatase promoter I.f is regulated by estrogen receptor alpha (ESR1) in mouse hypothalamic neuronal cell lines.

Aromatase (CYP19A1) catalyzes the conversion of C(19) steroids to estrogens. Aromatase and its product estradiol (E(2)) are crucial for the sexually dimorphic development of the fetal brain and the regulation of gonadotropin secretion and sexual interest in adults. The regulation of aromatase expression in the brain is not well understood. The aromatase (Cyp19a1) gene is selectively expressed in distinct neurons of the hypothalamus through a distal brain-specific promoter I.f located approximately 36 kb upstream of the coding region. Here, we investigated a short feedback effect of E(2) on aromatase mRNA expression and enzyme activity using estrogen receptor alpha (ESR1; also known as ER alpha)-positive or ESR1-negative mouse embryonic hypothalamic neuronal cell lines that express aromatase via promoter I.f. Estradiol regulated aromatase mRNA expression and enzyme activity in a time- and dose-dependent manner, whereas an E(2) antagonist reversed these effects. The nucleotide -200/-1 region of promoter I.f conferred E(2) responsiveness. Two activator protein 1 (AP-1) elements in this region were essential for induction of promoter activity by E(2). ESR1 and JUN (c-Jun) bound to these AP-1 motifs in intact cells and under cell-free conditions. The addition of an ESR1 mutant that interacts with JUN but not directly with DNA enhanced E(2)-dependent promoter I.f activity. Independently, we demonstrated an interaction between ESR1 and JUN in hypothalamic cells. Knockdown of ESR1 abolished E(2)-induced aromatase mRNA and enzyme activity. Taken together, E(2) regulates Cyp19a1 expression via promoter I.f by enhanced binding of an ESR1/JUN complex to distinct AP-1 motifs in hypothalamic cells. We speculate that this mechanism may, in part, regulate gonadotropin secretion and sexual activity.

Classical estrogen receptor alpha signaling mediates negative and positive feedback on gonadotropin-releasing hormone neuron firing.

During the female reproductive cycle, the neuroendocrine action of estradiol switches from negative feedback to positive feedback to initiate the preovulatory GnRH and subsequent LH surges. Estrogen receptor-alpha (ERalpha) is required for both estradiol negative and positive feedback regulation of LH. ERalpha may signal through estrogen response elements (EREs) in DNA and/or via ERE-independent pathways. Previously, a knock-in mutant allele (ERalpha-/AA) that selectively restores ERE-independent signaling onto the ERalpha-/- background was shown to confer partial negative but not positive estradiol feedback on serum LH. The current study investigated the roles of the ERE-dependent and ERE-independent ERalpha pathways for estradiol feedback at the level of GnRH neuron firing activity. The above ERalpha genetic models were crossed with GnRH-green fluorescent protein mice to enable identification of GnRH neurons in brain slices. Targeted extracellular recordings were used to monitor GnRH neuron firing activity using an ovariectomized, estradiol-treated mouse model that exhibits diurnal switches between negative and positive feedback. In wild-type mice, GnRH neuron firing decreased in response to estradiol during negative feedback and increased during positive feedback. In contrast, both positive and negative responses to estradiol were absent in GnRH neurons from ERalpha-/- and ERalpha-/AA mice. ERE-dependent signaling is thus required to increase GnRH neuron firing to generate a GnRH/LH surge. Furthermore, ERE-dependent and -independent ERalpha signaling pathways both appear necessary to mediate estradiol negative feedback on serum LH levels, suggesting central and pituitary estradiol feedback may use different combinations of ERalpha signaling pathways.

Estrogen actions in the male reproductive system involve estrogen response element-independent pathways.

The estrogen receptor-alpha (ERalpha) acts through multiple pathways, including estrogen response element (ERE)-dependent (classical) and ERE-independent (nonclassical) mechanisms. We previously created a mouse model harboring a two-amino-acid mutation of the DNA-binding domain (E207A, G208A) that precludes direct binding of ERalpha to an ERE. After crossing heterozygous mutant mice with an ERalpha knockout (ERKO) line, it was possible to assess the degree of physiological rescue by the isolated ERalpha nonclassical allele (-/AA; AA) when compared with ERKO mice (-/-) and to wild type (+/+; WT). In male ERKO mice up to 8 months of age, testosterone levels were high, although LH levels were similar to WT. Testosterone was normal in the AA mice, indicating that the AA allele rescues the enhanced testosterone biosynthesis in ERKO mice. Male ERKO mice exhibited distention of the seminiferous tubules as early as 2-3 months of age as a consequence of decreased water resorption in the efferent ducts. By 3-4 months of age, ERKO mice had impaired spermatogenesis in approximately 40% of their tubules, and sperm counts and motility declined in association with the histological changes. In the AA mice, histological defects were greatly reduced or absent, and sperm counts and motility were rescued. Levels of aquaporins 1 and 9, which contribute to water uptake in the efferent ducts, were reduced in ERKO mice and partially or fully rescued in AA mice, whereas another water transporter, sodium-hydrogen exchanger-3, was decreased in both ERKO and AA mice. We conclude that non-ERE-dependent estrogen pathways are sufficient to rescue the defective spermatogenesis observed in ERKO mice and play a prominent role in ERalpha action in the testis, including pathways that regulate water resorption and androgen biosynthesis.

HoxD10 gene delivery using adenovirus/adeno-associate hybrid virus inhibits the proliferation and tumorigenicity of GH4 pituitary lactotrope tumor cells.

Prolactinoma is one of the most common types of pituitary adenoma. It has been reported that a variety of growth factors and cytokines regulating cell growth and angiogenesis play an important role in the growth of prolactinoma. HoxD10 has been shown to impair endothelial cell migration, block angiogenesis, and maintain a differentiated phenotype of cells. We investigated whether HoxD10 gene delivery could inhibit the growth of prolactinoma. Rat GH4 lactotrope tumor cells were infected with adenovirus/adeno-associated virus (Ad/AAV) hybrid vectors carrying the mouse HoxD10 gene (Hyb-HoxD10) or the beta-galactosidase gene (Hyb-Gal). Hyb-HoxD10 expression inhibited GH4 cell proliferation in vitro. The expression of FGF-2 and cyclin D2 was inhibited in GH4 cells infected with Hyb-HoxD10. GH4 cells transduced with Hyb-HoxD10 did not form tumors in nude mice. These results indicate that the delivery of HoxD10 could potentially inhibit the growth of PRL-secreting tumors. This approach may be a useful tool for targeted therapy of prolactinoma and other neoplasms.

New insights into the classical and non-classical actions of estrogen: evidence from estrogen receptor knock-out and knock-in mice.

Estrogen receptor alpha (ERalpha) mediates estrogen (E2) actions in the brain and is critical for normal reproductive function and behavior. In the classical pathway, ERalpha binds to estrogen response elements (EREs) to regulate gene transcription. ERalpha can also participate in several non-classical pathways, including ERE-independent gene transcription via protein-protein interactions with transcription factors and rapid, non-genotropic pathways. To distinguish between ERE-dependent and ERE-independent mechanisms of E2 action in vivo, we have created ERalpha null mice that possess an ER knock-in mutation (E207A/G208A; "AA"), in which the mutant ERalpha cannot bind to DNA but retains activity in ERE-independent pathways (ERalpha(-/AA) mice). Understanding the molecular mechanisms of ERalpha action will be helpful in developing pharmacological therapies that differentiate between ERE-dependent and ERE-independent processes. This review focuses on how the ERalpha(-/AA) model has contributed to our knowledge of ERalpha signaling mechanisms in estrogen regulation of the reproductive axis and sexual behavior.

Recommendations to Sustain the Academic Mission Ecosystem at U.S. Medical Schools.

Academic medical center (AMC) faculty, administrators, and leaders have the critical tasks of teaching and training the next generation of health care providers and biomedical researchers, as well as generating new knowledge that improves the health of all. In the United States, medical schools and their affiliated hospitals train remarkably high-quality physicians and scientists, and the research conducted at these institutions results in advances in health. To that end, AMCs have become essential engines for driving better health in the United States and the rest of the world; they also have become essential engines driving the economies of their respective communities and regions. The education and research missions, however, require subsidization because tuition and extramural grant funding do not cover the costs of these endeavors. This subsidization largely has come from revenues generated by AMCs' clinical endeavors. The viability of this cross-subsidization, however, is increasingly threatened in the current clinical environment. The authors of this Perspective discuss these issues in depth and provide some concrete recommendations to address these challenges. They hope to stimulate discussion and, ultimately, ensure the financial viability of U.S. AMCs-a national resource of utmost importance. Recommendations to sustain research include creating strategic biomedical research plans, developing a defined and sustained model to support National Institutes of Health funding that keeps pace with inflation, and evolving funding mechanisms. Recommendations to sustain medical education include limiting student debt, creating more cost-effective curricula, and ensuring that clinical training opportunities that meet national standards are available to students.

What a medical school chair wants from the dean.

Economic pressure has led the evolution of the role of the medical school dean from a clinician educator to a health care system executive. In addition, other dynamic requirements also have likely led to changes in their leadership characteristics. The most important relationship a dean has is with the chairs, yet in the context of the dean's changing role, little attention has been paid to this relationship. To frame this discussion, we asked medical school chairs what characteristics of a dean's leadership were most beneficial. We distributed a 26-question survey to 885 clinical and basic science chairs at 41 medical schools. These chairs were confidentially surveyed on their views of six leadership areas: evaluation, barriers to productivity, communication, accountability, crisis management, and organizational values. Of the 491 chairs who responded (response rate =55%), 88% thought that their dean was effective at leading the organization, and 89% enjoyed working with their dean. Chairs indicated that the most important area of expertise of a dean is to define a strategic vision, and the most important value for a dean is integrity between words and deeds. Explaining the reasons behind decisions, providing good feedback, admitting errors, open discussion of complex or awkward topics, and skill in improving relations with the teaching hospital were judged as desirable attributes of a dean. Interestingly, only 23% of chairs want to be a dean in the future. Financial acumen was the least important skill a chair thought a dean should hold, which is in contrast to the skill set for which many deans are hired and evaluated. After reviewing the literature and analyzing these responses, we assert that medical school chairs want their dean to maintain more traditional leadership than that needed by a health care system executive, such as articulating a vision for the future and keeping their promises. Thus, there appears to be a mismatch between what medical school chairs perceive they need from their dean and how the success of a dean is evaluated.

Distinct roles for steroidogenic factor 1 and desert hedgehog pathways in fetal and adult Leydig cell development.

Testicular Leydig cells produce testosterone and provide the hormonal environment required for male virilization and spermatogenesis. In utero, fetal Leydig cells (FLCs) are necessary for the development of the Wolffian duct and male external genitalia. Steroidogenic factor 1 (Sf1) is a transcriptional regulator of hormone biosynthesis genes, thus serving a central role in the Leydig cell. Desert hedgehog (Dhh), a Sertoli cell product, specifies the FLC lineage in the primordial gonad through a paracrine signaling mechanism. Postnatally, FLCs are replaced in the testis by morphologically distinct adult Leydig cells (ALCs). To study a putative interaction between Sf1 and Dhh, we crossed Sf1 heterozygous mutant mice with Dhh homozygous null mice to test the function of these two genes in vivo. All of the compound Sf1(+/-); Dhh(-/-) mutants failed to masculinize and were externally female. However, embryonic gonads contained anastomotic testis cords with Sertoli cells and germ cells, indicating that sex reversal was not attributable to a fate switch of the early gonad. Instead, external feminization was attributable to the absence of differentiated FLCs in XY compound mutant mice. ALCs also failed to develop, suggesting either a dependence of ALCs on the prenatal establishment of Leydig cell precursors or that Sf1 and Dhh are both required for ALC maturation. In summary, this study provides genetic evidence that combinatorial expression of the paracrine factor Dhh and nuclear transcription factor Sf1 is required for Leydig cell development.

Estrogen response element-independent estrogen receptor (ER)-alpha signaling does not rescue sexual behavior but restores normal testosterone secretion in male ERalpha knockout mice.

Estrogen receptor (ER)-alpha mediates estradiol (E(2)) actions in the male gonads and brain and is critical for normal male reproductive function. In the classical pathway, ERalpha binds to estrogen response elements (EREs) to regulate gene transcription. ERalpha can also regulate gene transcription independently of EREs via protein-protein interactions with transcription factors and additionally signal via rapid, nongenomic pathways originating at the cell membrane. This study assessed the degree to which ERE-independent ERalpha signaling can rescue the disrupted masculine sexual behaviors and elevated serum testosterone (T) levels that have been shown to result from ERalpha gene deletion. We utilized male ERalpha null mice that possess a ER knock-in mutation (E207A/G208A; AA), in which the mutant ERalpha is incapable of binding to DNA and can signal only through ERE-independent pathways (ERalpha(-/AA) mice). We found that sexual behavior, including mounting, is virtually absent in ERalpha(-/-) and ERalpha(-/AA) males, suggesting that ERE-independent signaling is insufficient to maintain any degree of normal sexual behavior in the absence of ERE binding. By contrast, ERE-independent signaling in the ERalpha(-/AA) mouse is sufficient to restore serum T levels to values observed in wild-type males. These data indicate that binding of ERs to EREs mediates most if not all of E(2)'s effects on male sexual behavior, whereas ERE-independent ERalpha signaling may mediate E(2)'s inhibitory effects on T production.

Effects of loss of classical estrogen response element signaling on bone in male mice.

The role of estrogen signaling in the male skeleton via estrogen receptor (ER)-alpha is now well established. ERalpha can elicit responses through either classical estrogen response elements (ERE) pathways or nonclassical, non-ERE pathways. In the present study, we examined the effects of either the attenuation or loss of classical ERalpha signaling on the murine male skeleton. To accomplish this, we crossed male mice heterozygous for a knock-in mutation [nonclassical ERalpha knock-in (NERKI)], which abolishes the ERE-mediated pathway with female heterozygous ERalpha knockout mice (ERalpha+/-) and studied the F1 generation ERalpha+/+, ERalpha+/-, ERalpha+/NERKI, and ERalpha-/NERKI male progeny longitudinally using bone density and histomorphometry. The only ERalpha allele present in ERalpha-/NERKI mice is incapable of classical ERE-mediated signaling, whereas the heterozygous ERalpha+/NERKI mice have both one intact ERalpha and one NERKI allele. As compared with ERalpha+/+ littermates (n=10/genotype), male ERalpha+/NERKI and ERalpha-/NERKI mice displayed axial and appendicular skeletal osteopenia at 6, 12, 20, and 25 wk of age, as demonstrated by significant reductions in total bone mineral density (BMD) at representative sites (areal BMD by dual-energy x-ray absorptiometry at the lumbar vertebrae and femur and volumetric BMD by peripheral quantitative computed tomography at the tibia; P<0.05-0.001 vs. ERalpha+/+). The observed osteopenia in these mice was evident in both trabecular and cortical bone compartments. However, these decreases were more severe in mice lacking classical ERalpha signaling (ERalpha-/NERKI mice), compared with mice in which one wild-type ERalpha allele was present (ERalpha+/NERKI mice). Collectively, these data demonstrate that classical ERalpha signaling is crucial for the development of the murine male skeleton.