T Kumar - US grants

DESCRIPTION (provided by applicant): In the male, follicle stimulating hormone (FSH) binds to G-protein coupled transmembrane receptors on the Sertoli cells and controls their development and function. Sertoli cells are the somatic cells of the testis that provide physical scaffolding and various signaling factors to the proliferating and differentiating germ cells. During the mouse testis development, Sertoli cells rapidly divide up to postnatal day 14, thereafter cease to proliferate and terminally differentiate. The number of Sertoli cells determines the germ cell capacity and thus the male reproductive potential. Aberrations in Sertoli cell proliferation and differentiation may cause male infertility. Despite this knowledge, the molecular basis for FSH regulation of Sertoli cell proliferation and differentiation is unknown. A major challenge to study the mechanism of FSH action in the male has been to rapidly isolate pure Sertoli cells (free of contaminating testis cell types) that accurately reflect their in vivo function. The long-term goal of this project is to understand how FSH regulates distinct developmental programs in Sertoli cells by orchestrating various gene/protein networks. In Specific Aim 1, we will isolate pure populations of Sertoli cells by two approaches. In the first approach, a rapid magnetic cell separation method will be used to selectively enrich Sertoli cells based on their unique cell surface expression of FSH-receptors. In the second approach, Sertoli cells will be enriched from two different strains of transgenic mice expressing an enhanced green fluorescent protein (EGFP) marker (driven by either Mullerian inhibiting substance or Pem homoebox promoter) specifically in the Sertoli cell lineage. Additionally, by crossbreeding, the GFP transgenes will be introduced into the FSH-null background. Pure populations of Sertoli cells will be isolated from both these strains of mice (with and without FSH) by flow cytometry. In Specific Aim 2, to begin to identify FSH-responsive genes, we will use subtractive hybridization strategies to enrich Sertoli cell-specific cDNAs or use cDNA microarrays to analyze large-scale gene expression changes in pure populations of Sertoli cells (isolated in Specific Aim 1) during proliferation (day 10) and differentiation (day 42) phases. Collectively, these experiments will enable us to monitor the development of GFP-expressing Sertoli cells in vivo, develop rapid methods to purify Sertoli cells and identify and characterize the FSH-responsive genes in them. Finally, the GFP transgenic mice and the Sertoli cell - specific cDNAs and large-scale gene expression profiles generated in this pilot grant will be valuable reagents and will be made available to a number of investigators engaged in male reproductive endocrinology and developmental biology research.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Male factor infertility is a significant concern throughout the world. The majority of the male infertility cases are idiopathic. In the male, primordial germ cells (PGCs) migrate, proliferate, and colonize the genital ridges to ultimately form testicular cords, where they establish contacts with the Sertoli cells. Key factors that regulate primordial germ cell migration and proliferation are not completely understood. The long-term goal of this project is to delineate the mechanisms of germ cell interactions with Sertoli cells in the testis. A mechanistic understanding of how germ cells develop and function is relevant to clinical conditions of male infertility that manifest as Sertoli cell-only syndrome for which there is currently no treatment. To begin to explore the developmental biology of the male germ cells and to understand the pathobiology of the human Sertoli cell-only syndrome, we have characterized atrichosis, the naturally occurring homozygous recessive mouse mutant. The atrichosis mutant testis histology closely resembles that of Sertoli cell-only syndrome patients and demonstrates tubules lined with only Sertoli cells and contains no germ cells. We will test the central hypothesis that a cell autonomous defect leads to complete absence of germ cells in the atrichosis mutant testis. These studies will identify the gene(s) responsible for the absence of germ cells in the atrichosis mutant mouse and provide a starting point for further loss-of-function and gain-of-function genetic approaches to understand germ cell migration and function. Finally, this work will establish atrichosis mutant as a genetically trackable new mouse model for human male infertility conditions associated with Sertoli cell-only tubules and germ cell aplasia, thus impacting clinical protocols of male fertility restoration.

DESCRIPTION (provided by applicant): Activins are abundantly expressed in gonads and elicit important autocrine/paracrine actions within the pituitary and hypothalamus. Activins signal through two types of receptors, activin receptor II (ACVR2) and IIB (ACVR2B). ACVR2 is highly conserved evolutionarily and is the predominant form in pituitary gonadotropes. Our genetic studies demonstrated that ACVR2 signaling is important for FSH homeostasis in pituitary, since Acvr2 null mice have suppressed levels of FSH synthesis and secretion. Despite this knowledge, many activin-responsive genes in the reproductive axis remain unknown. Our long-term research goal is to elucidate the in vivo mechanisms of activin signaling pathways that ultimately regulate FSH synthesis and secretion. The central hypothesis is that several critical known/unknown genes downstream of the ACVR2 signaling pathway must be affected in gonadotropes of Acvr2 mutant mice. This hypothesis will be tested in two Specific Aims. In Specific Aim 1, we will use a novel transgenic mouse strain, in which the gonadotropes are labeled with GFP. These mice will be intercrossed to Acvr2 null mice and gonadotropes purified by flow sorting based on GFP fluorescence. This approach will provide pure populations of gonadotropes that have never been exposed to ACVR2 signaling in vivo, at desired time points. In Specific Aim 2, microarray analyses will be performed using gonadotrope RNA samples isolated from control and Acvr2 null mice that express the GFP transgene. These studies should provide new knowledge on activin signaling pathways in gonadotropes and identify novel candidate genes/proteins that are important for FSH synthesis and secretion. Delineation of mechanisms of FSH production is fundamental to physiology of reproduction and offers long-term clinical benefits of diagnosing and treating FSH-dependent fertility/infertility disorders. Finally, because activins regulate multiple processes throughout the reproductive axis and other parts of the body, results obtained from the proposed studies should also provide new mechanistic insights into activin-regulated signaling pathways in other tissues. PROJECT NARRATIVE: These studies should provide new knowledge on how pituitary gland synthesizes and secretes an important hormone called follicle-stimulating hormone (FSH) in response to another locally produced protein called activin. Delineation of mechanisms of FSH production is fundamental to physiology of reproduction and offers clinical benefits of diagnosing and treating FSH-dependent fertility/infertility disorders. These include ovarian hyperstimulation syndrome, amenorrhea and testicular defects.

DESCRIPTION (provided by applicant): Gonadotropes synthesize and secrete heterodimeric glycoproteins luteinizing hormone (LH) and follicle stimulating hormone (FSH) that are critical for reproductive function. The genes encoding the common alpha subunit (Cga) and the hormone-specific beta subunits (Lhb and Fshb) are coordinately regulated by hypothalamic gonadotropin-releasing hormone, steroids and gonadal peptides, inhibin and activin. Although a wealth of knowledge has accumulated on transcriptional control of gonadotropin beta subunits, post- transcriptional mechanisms of these genes are unknown. Recently, micro RNAs (miRNAs) produced by Dicer, an RNA endonuclease III, have emerged as key regulators of gene expression, mRNA stability and protein synthesis. Knowledge on miRNAs that regulate gonadotrope and consequently reproductive function is lacking. Our long-term research goal is to elucidate the in vivo mechanisms that regulate gonadotropin synthesis and secretion. The central hypothesis is that Dicer is a key factor in miRNA biogenesis in gonadotropes and plays essential roles in gonadotropin synthesis and consequently reproductive function. In Specific Aim 1, we will conditionally delete Dicer exclusively in gonadotropes by cre-lox technology and analyze gonadotropin synthesis and secretion. Additionally, we will use novel mouse strains, in which gonadotropes are labeled with GFP on a normal or activin receptor-II (Acvr2) null genetic background. Pure populations of gonadotropes will be isolated from these mice by cell sorting based on GFP fluorescence and miRNAs regulated by Acvr2 identified. In Specific Aim 2, reproductive phenotypes secondary to changes in gonadotropins as a result of Dicer deletion in gonadotropes will be determined. These studies should provide new knowledge on Dicer - dependent miRNAs in gonadotropes and identify novel post-transcriptional mechanisms for gonadotropin secretion. Delineation of mechanisms of gonadotropin secretion is fundamental to physiology of reproduction and offers long-term clinical benefits of diagnosing and treating gonadotropin-dependent fertility/infertility disorders.

DESCRIPTION (provided by applicant): Pituitary adenomas are the most frequent of all pituitary diseases and are observed in both sexes. The prevalence is estimated as ~90 cases per 105 people. The majority arises from the gonadotrope lineage and these tumors do not hypersecrete glycoprotein hormones [i.e., luteinizing hormone (LH) and follicle stimulating hormone (FSH)] and thus are called null cell adenomas. The clinical manifestations include neurological symptoms such as damage to brain tissue and optic chiasm, vision loss, increased intracranial pressure, persistent headache and nausea. Because they have no well-defined diagnostic markers, null cell adenomas often go undetected until they are very advanced and surgery is the only treatment. There are no established model systems or chemoprevention options available for null cell adenomas. We have developed a unique transgenic strain of mice that develops gonadotrope-enriched pituitary adenomas and phenocopies human pituitary null cell adenomas including. Our long-term goal is to understand the mechanisms of origin and progression of null cell adenoma, and develop new strategies of chemoprevention for it. The objective of this proposal is to identify biomarkers and specific targets/pathways responsible for gonadotrope tumor progression such that mechanistic insights into progression and prevention of human null cell adenomas could ultimately be obtained. Our central hypothesis is that both estrogen receptor-1 (ESR1) and Indian hedgehog (IHH) contribute to growth of pituitary gonadotrope adenomas and inhibiting their expression leads to clinical benefit. In Specific Aim 1, we will determine the mechanism by which estrogen signaling influences tumor growth and progression. Effects of estrogen on glycosylation that contribute to hormone secretion failure will be evaluated. Additionally, a novel mouse model in which gonadotrope tumors are fluorescently labeled will be used to identify potential biomarkers. In Specific Aim 2, we will test the efficacyof anti-estrogen therapy to regulate gonadotrope tumor growth. The approach involves the use of mice conditionally overexpressing Esr1 at desired times, and mice lacking either Esr1 or Esr2 on the tumor-prone transgenic background. The in vivo tumor preventive effects of tamoxifen will also be tested. In Specific Aim 3, we will determine the effects of blocking IHH action on gonadotrope tumor development. We will specifically delete Ihh in gonadotrope tumors by using a cre-lox approach. In a second approach, the effects of hedgehog chemoprevention agents will be tested in gonadotrope tumors of transgenic mice. The approach is innovative, because it utilizes a unique transgenic mouse model that develops gonadotrope tumors and uses a combination of in vitro and novel in vivo models. The proposed research is significant because it is expected to vertically advance and expand understanding of how chemopreventive agents targeted to block estrogen and hedgehog signaling regulate gonadotrope/null cell adenomas.

DESCRIPTION (provided by applicant): Pituitary adenomas are the most frequent of all pituitary diseases and are observed in both sexes. The prevalence is estimated as ~90 cases per 105 people. The majority arises from the gonadotrope lineage and these tumors do not hypersecrete glycoprotein hormones [i.e., luteinizing hormone (LH) and follicle stimulating hormone (FSH)] and thus are called null cell adenomas. The clinical manifestations include neurological symptoms such as damage to brain tissue and optic chiasm, vision loss, increased intracranial pressure, persistent headache and nausea. Because they have no well-defined diagnostic markers, null cell adenomas often go undetected until they are very advanced and surgery is the only treatment. There are no established model systems or chemoprevention options available for null cell adenomas. We have developed a unique transgenic strain of mice that develops gonadotrope-enriched pituitary adenomas and phenocopies human pituitary null cell adenomas including. Our long-term goal is to understand the mechanisms of origin and progression of null cell adenoma, and develop new strategies of chemoprevention for it. The objective of this proposal is to identify biomarkers and specific targets/pathways responsible for gonadotrope tumor progression such that mechanistic insights into progression and prevention of human null cell adenomas could ultimately be obtained. Our central hypothesis is that both estrogen receptor-1 (ESR1) and Indian hedgehog (IHH) contribute to growth of pituitary gonadotrope adenomas and inhibiting their expression leads to clinical benefit. In Specific Aim 1, we will determine the mechanism by which estrogen signaling influences tumor growth and progression. Effects of estrogen on glycosylation that contribute to hormone secretion failure will be evaluated. Additionally, a novel mouse model in which gonadotrope tumors are fluorescently labeled will be used to identify potential biomarkers. In Specific Aim 2, we will test the efficacyof anti-estrogen therapy to regulate gonadotrope tumor growth. The approach involves the use of mice conditionally overexpressing Esr1 at desired times, and mice lacking either Esr1 or Esr2 on the tumor-prone transgenic background. The in vivo tumor preventive effects of tamoxifen will also be tested. In Specific Aim 3, we will determine the effects of blocking IHH action on gonadotrope tumor development. We will specifically delete Ihh in gonadotrope tumors by using a cre-lox approach. In a second approach, the effects of hedgehog chemoprevention agents will be tested in gonadotrope tumors of transgenic mice. The approach is innovative, because it utilizes a unique transgenic mouse model that develops gonadotrope tumors and uses a combination of in vitro and novel in vivo models. The proposed research is significant because it is expected to vertically advance and expand understanding of how chemopreventive agents targeted to block estrogen and hedgehog signaling regulate gonadotrope/null cell adenomas.

The long-term goal of this project is to study mechanisms of pituitary control of ovarian and bone function in aging women. Normal ovarian function is dependent on follicle-stimulating hormone (FSH), a pituitary derived heterodimeric glycoprotein consisting of a ?-and a ?-subunit. Both the subunits are glycosylated with two N- linked sugar chains in each subunit. This fully glycosylated form is designated FSH24. Glycosylation plays a major role in secretion, serum half-life and biological actions of FSH. It is known that glycosylation of pituitary gonadotropins is also estrous/menstrual cycle- and age-specific. Biochemical and physiological studies in several species have identified unique hypo-glycosylated variants consisting of sugar chains only in the ? but either one or none on the ? subunit. These variants are known as hypo-glycosylated FSH glycoforms and designated as FSH21, FSH18 or FSH15. Most importantly, the ratio of hypo- to fully-glycosylated FSH forms is found age-dependent, with high levels of fully-glycosylated variant predominantly present in peri/post- menopausal women and may contribute to the aging-associated bone loss. However, the distinct in vivo biological functions of these FSH glycoform variants are unknown in normal and aging ovarian and bone physiology. The central hypothesis is that glycosylation on FSH is an age-related switch that changes target tissue specificity from ovary to bone. This hypothesis will be tested using genetically engineered mouse models in two specific Aims. In Aim 1, we will test ovarian development and function progressively with aging using Fshb null mice expressing individual glycosylated forms of FSH. This genetic strategy will allow us to test systematically the in vivo biological actions of each glycosylated FSH variant in ovarian physiology in the absence of endogenous mouse FSH. In Aim 2, first, we will use the FSH glycoform-expressing mice and test bone development as a function of aging. To unequivocally test the direct actions of FSH on bone, in one approach, we will engineer mice in which Fshr will be selectively deleted in osteoclasts by a Cre-lox genetic approach. In a second approach, we will develop a genetically engineered mouse line that permits creating temporal loss of FSH at desired times. Functional analyses with these genetically altered mouse models will identify distinct biological actions of FSH variants in vivo during ovarian aging and by extrapolation, in human ovarian senescence. These novel mouse models will also allow us to directly test whether aging-associated bone loss is dependent on FSH ligand or FSH receptor-mediated signaling in osteoclasts in the bone. Our studies may unravel a novel phenomenon of age-dependent N-glycosylation switch on a pituitary glycoprotein hormone that results in alterations in target tissue specificity (ovary versus bone) and may potentially lead to new therapeutic options for intervention of bone loss in aging women.

PROJECT SUMMARY The long-term goal of this project is to study age-dependent mechanisms of follicle-stimulating hormone (FSH) actions in the ovary. FSH is a pituitary glycoprotein consisting of an ?-and a ?-subunit. Both the subunits are glycosylated with two N-linked sugar chains on each subunit. Glycosylation of FSH is estrous/menstrual cycle- and age-specific. Macro-heterogeneity results in FSH variants consisting of 2 sugar chains on the ? subunit but either one or none on the ?. These variants are known as hypo-glycosylated FSH glycoforms, FSH21, and FSH18, in contrast to the fully glycosylated FSH24. Interestingly, the abundance of hypo- and fully glycosylated FSH is age-dependent, with high levels of FSH21/18 glycoforms predominant in young women and FSH24 predominant in peri/post-menopausal women. This shift suggests a role of FSH24 in ovarian aging. In vitro studies and pharmacological rescue of Fshb null mice with recombinant hypo- and fully glycosylated FSH glycoforms indicate differences in regulation of known FSH-responsive ovarian genes and proteins downstream of FSH receptor (FSHR) signaling. However, the mechanisms by which these FSH glycoforms regulate ovarian signaling pathways in vivo are unknown. The central hypothesis is that age-specific FSH glycoforms act via FSHRs but regulate distinct downstream signaling cascades to elicit different gene/protein expression signatures in the ovary. This hypothesis will be tested using genetically engineered novel mouse models. In Aim 1, we will perform a trans-omics analysis by overlaying the gene, protein and phosphoprotein expression signatures in ovaries of Fshb null mice expressing individual FSH glycoforms to identify signaling networks regulated by each FSH glycoform. In Aim 2, we will test the hypothesis that the FSH glycoform specificity in FSHR-mediated signaling is achieved by recruitment of distinct protein complexes to activate different downstream gene/protein networks. Fshb null mice expressing individual FSH glycoform and His - tagged FSHRs in granulosa cells will be used. Pull-down experiments with an His tag-specific antibody followed by mass spectrometry analysis of ovarian proteins will allow us to identify the FSH glycoform-specific FSH receptor and receptor co-factor protein complexes in each case. In Aim 3, we will evaluate the direct effects of recombinant FSH glycoforms in secondary follicles obtained from reproductively young and old mice. Gene and protein expression profiling will be performed and how FSH glycoforms impact follicle growth and gamete quality in vitro will be determined. Successful completion of the proposed studies will advance our understanding of the mechanisms by which FSH glycoforms regulate selective recruitment of distinct FSHR - co-factor partner complexes to achieve FSHR-mediated signal transduction pathways in vivo in ovaries and provide a direct read out of FSH glycoform actions during in vitro folliculogenesis and oogenesis. Our mechanistic studies serve as the foundation for novel therapeutic opportunities to preserve ovarian function and will enable improved design of ovarian induction protocols, in alignment with the NICHD mission.