High incidence of imperforate vagina in ADGRA3-deficient mice.

BACKGROUND: Ten percent of the female population suffers from congenital abnormalities of the vagina, uterus, or oviducts, with severe consequences for reproductive and psychological health. Yet, the underlying causes of most of these malformations remain largely unknown. ADGRA3 (GPR125) is involved in WNT signaling and planar cell polarity, mechanisms vital to female reproductive tract development. Although ADGRA3 is a well-established spermatogonial stem cell marker, its role within the female urogenital system remains unclear. RESULTS: In this study, we found Adgra3 to be expressed throughout the murine female urogenital system, with higher expression pre-puberty than after sexual maturation. We generated a global Adgra3-/- mouse line and observed imperforate vagina in 44% of Adgra3-/- females, resulting in distension of the reproductive tract and infertility. Ovarian morphology, plasma estradiol, ovarian Cyp19a1, and vaginal estrogen receptor α (Esr1) expression were unaffected. However, compared to controls, a significantly lower bone mineral density was found in Adgra3-/- mice. Whereas vaginal opening in mice is an estrogen-dependent process, 17ß-estradiol treatment failed to induce vaginal canalization in Adgra3-/- mice. Furthermore, a marked reduction in vaginal and ovarian progesterone receptor expression was observed concomitant with an upregulation of apoptotic regulators Bcl2, Bid, and Bmf in adult Adgra3-/- females with a closed vagina. CONCLUSIONS: Our collective results shed new insights into the complex mechanisms by which the adhesion receptor ADGRA3 regulates distal vaginal tissue remodeling during vaginal canalization via altered sex hormone responsiveness and balance in apoptotic regulators. This highlights the potential of ADGRA3 as a target in diagnostic screening and/or therapy for obstructive vaginal malformations in humans.

Loss of Adgra3 causes obstructive azoospermia with high penetrance in male mice.

The adhesion receptor ADGRA3 (GPR125) is a known spermatogonial stem cell marker, but its impact on male reproduction and fertility has not been examined. Using a mouse model lacking Adgra3 (Adgra3-/- ), we show that 55% of the male mice are infertile from puberty despite having normal spermatogenesis and epididymal sperm count. Instead, male mice lacking Adgra3 exhibited decreased estrogen receptor alpha expression and transient dilation of the epididymis. Combined with an increased estradiol production, this indicates a post-pubertal hormonal imbalance and fluid retention. Dye injection revealed a blockage between the ejaculatory duct and the urethra, which is rare in mice suffering from infertility, thereby mimicking the etiologies of obstructive azoospermia found in human male infertility. To summarize, male reproductive tract development is dependent on ADGRA3 function that in concert with estrogen signaling may influence fluid handling during sperm maturation and storage.

Embryonic mammary gland development.

Embryonic mammary gland development involves the formation of mammary placodes, invagination of flask-shaped mammary buds and development of miniature bi-layered ductal trees. Currently there is a good understanding of the factors that contribute to ectodermal cell movements to create these appendages and of pathways that lead to mammary specification and commitment. Gene expression profiles of early bipotent mammary stem cells populations as well as cell surface proteins and transcription factors that promote the emergence of unipotent progenitors have been identified. Analyses of these populations has illuminated not only embryonic mammary development, but highlighted parallel processes in breast cancer. Here we provide an overview of the highly conserved pathways that shape the embryonic mammary gland. Understanding the dynamic signaling events that occur during normal mammary development holds considerable promise to advance attempts to eliminate cancer by restoring differentiative signals.

Ltbp1L is focally induced in embryonic mammary mesenchyme, demarcates the ductal luminal lineage and is upregulated during involution.

INTRODUCTION: Latent TGFß binding proteins (LTBPs) govern TGFß presentation and activation and are important for elastogenesis. Although TGFß is well-known as a tumor suppressor and metastasis promoter, and LTBP1 is elevated in two distinct breast cancer metastasis signatures, LTBPs have not been studied in the normal mammary gland. METHODS: To address this we have examined Ltbp1 promoter activity throughout mammary development using an Ltbp1L-LacZ reporter as well as expression of both Ltbp1L and 1S mRNA and protein by qRT-PCR, immunofluorescence and flow cytometry. RESULTS: Our data show that Ltbp1L is transcribed coincident with lumen formation, providing a rare marker distinguishing ductal from alveolar luminal lineages. Ltbp1L and Ltbp1S are silent during lactation but robustly induced during involution, peaking at the stage when the remodeling process becomes irreversible. Ltbp1L is also induced within the embryonic mammary mesenchyme and maintained within nipple smooth muscle cells and myofibroblasts. Ltbp1 protein exclusively ensheaths ducts and side branches. CONCLUSIONS: These data show Ltbp1 is transcriptionally regulated in a dynamic manner that is likely to impose significant spatial restriction on TGFß bioavailability during mammary development. We hypothesize that Ltbp1 functions in a mechanosensory capacity to establish and maintain ductal luminal cell fate, support and detect ductal distension, trigger irreversible involution, and facilitate nipple sphincter function.

Optopharmacological tools for precise spatiotemporal control of oxytocin signaling in the central nervous system and periphery.

Oxytocin is a neuropeptide critical for maternal physiology and social behavior, and is thought to be dysregulated in several neuropsychiatric disorders. Despite the biological and neurocognitive importance of oxytocin signaling, methods are lacking to activate oxytocin receptors with high spatiotemporal precision in the brain and peripheral mammalian tissues. Here we developed and validated caged analogs of oxytocin which are functionally inert until cage release is triggered by ultraviolet light. We examined how focal versus global oxytocin application affected oxytocin-driven Ca2+ wave propagation in mouse mammary tissue. We also validated the application of caged oxytocin in the hippocampus and auditory cortex with electrophysiological recordings in vitro, and demonstrated that oxytocin uncaging can accelerate the onset of mouse maternal behavior in vivo. Together, these results demonstrate that optopharmacological control of caged peptides is a robust tool with spatiotemporal precision for modulating neuropeptide signaling throughout the brain and body.

Cadherins and catenins in breast cancer.

Recent studies show that cadherins and catenins are hormonally regulated and carry out physiological roles during mammary development but have pathological effects when deregulated. E-cadherin expression is irreversibly lost in invasive lobular breast cancer (ILC). Animal models of ILC provide mechanistic insight, confirming that E-cadherin serves as both a tumor suppressor and an invasion suppressor in ILC. Ductal breast cancer involves complex, reversible, epigenetic modulation of multiple cadherins. Transcriptional regulators of E-cadherin have been identified that induce epithelial-to-mesenchymal transitions. Catenins are lost or mislocalized in tumors lacking cadherins. However, beta-catenin signaling is upregulated by numerous pathways in >50% of breast tumors and animal models suggest its oncogenic function in breast relates to its role in mammary progenitor cell expansion.

Choreographing metastasis to the tune of LTBP.

Latent Transforming Growth Factor beta (TGFß) Binding Proteins (LTBPs) are chaperones and determinants of TGFß isoform-specific secretion. They belong to the LTBP/Fibrillin family and form integral components of the fibronectin and microfibrillar extracellular matrix (ECM). LTBPs serve as master regulators of TGFß bioavailability, functioning to incorporate and spatially pattern latent TGFß at regular intervals within the ECM, and actively participate in integrin-mediated stretch activation of TGFß in vivo. In so doing they create a highly patterned sensory system where local changes in ECM tension can be detected and transduced into focal signals. The physiological role of LTBPs in the mammary gland remains largely unstudied, however both loss and gain of LTBP expression is found in breast cancers and breast cancer cell lines. Importantly, elevated LTBP1 levels appear in two gene signatures predictive of enhanced metastatic behavior. LTBP may promote metastasis by providing the bridge between structural and signaling components of the epithelial to mesenchymal transition (EMT).

Gpr125 is a unifying hallmark of multiple mammary progenitors coupled to tumor latency.

Gpr125 is an orphan G-protein coupled receptor, with homology to cell adhesion and axonal guidance factors, that is implicated in planar polarity and control of cell movements. By lineage tracing we demonstrate that Gpr125 is a highly specific marker of bipotent mammary stem cells in the embryo and of multiple long-lived unipotent basal mammary progenitors in perinatal and postnatal glands. Nipple-proximal Gpr125+ cells express a transcriptomic profile indicative of chemo-repulsion and cell movement, whereas Gpr125+ cells concentrated at invasive ductal tips display a hybrid epithelial-mesenchymal phenotype and are equipped to bind chemokine and growth factors and secrete a promigratory matrix. Gpr125 progenitors acquire bipotency in the context of transplantation and cancer and are greatly expanded and massed at the pushing margins of short latency MMTV-Wnt1 tumors. High Gpr125 expression identifies patients with particularly poor outcome within the basal breast cancer subtype highlighting its potential utility as a factor to stratify risk.

Distinct function of androgen receptor coactivator ARA70α and ARA70ß in mammary gland development, and in breast cancer.

Steroid receptor coactivators are important in regulating the function of the receptors in endocrine organ development and in cancers, including breast. Androgen receptor (AR) coactivator ARA70, was first identified as a gene fused to the ret oncogene and later characterized as an AR coactivator. We previously reported that the full length ARA70α functions as a tumor suppressor gene and that ARA70ß functions as an oncogene in prostate cancer. Here we show that both ARA70α and ARA70ß function as AR and estrogen receptor (ER) coactivators in breast cancer cells. However, ARA70α and ARA70ß serve different functions in mammary gland development and breast cancer tumorigenesis. We observed hypoplastic development of mammary glands in MMTV driven ARA70α transgenic mice and overgrowth of mammary glands in ARA70ß transgenic mice at virgin and pregnant stages. We determined that ARA70α inhibited cell proliferation, and that ARA70ß promotes proliferation in MCF7 breast cancer cells. These effects were observed in hormone-free media, or in media with androgen or estrogen, though to varying degrees. Additionally, we observed that ARA70ß strongly enhanced the invasive ability of MCF7 breast cancer cells in in vitro Matrigel assays. Significantly, decreased ARA70α expression is associated with increased tendency of breast cancer metastasis. In summary, ARA70α and ARA70ß have distinct effects in mammary gland development and in the progression of breast cancer.