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Training Grant in Developmental and Reproductive Biology
Male Reproductive Physiology in the Human
Harvard Reproductive Endocrine Science Center
Genetics of Polycystic Ovarian Syndrome
Aging and the Hypothalamic-Pituitary Reproductive Axis
FSH, Estradiol and Inhibin in Female Reproduction
Modulation of Insulin Action by Testosterone In Men
Establishment of a Human Serum Bank
Physiology and Action of a New Follistatin Related Protein
Establishing the Genetic Etiology for Kallmann Syndrome
Recombinant Human Prolactin for Lactation Insufficiency

 

 

 

 

 

1) Training Grant in Developmental and Reproductive Biology
William F. Crowley (PI)
Project Number: T32 HD07396
Source: NIH
Period: 7/15/91 – 4/30/07

The Reproductive & Developmental Biology Training Program at the Massachusetts General Hospital provides its trainees with an intensive research experience in basic and/or clinical investigation under the attentive mentorship of a skilled faculty and complemented by a strong didactic program underpinning the sciences of reproductive and developmental biology. Trainees are selected from a highly talented applicant pool of M.D.’s, M.D./Ph.D.’s, or Ph.D.’s on the basis of their prior academic and/or research accomplishments, a strong future commitment to an academic career in biomedical investigation, and a personal interview indicating their proper motivation and future potential.

The PI (William Crowley) is a Professor of Medicine at Harvard Medical School and the Director of two NIH-funded Centers. He is an established senior scientist and recognized mentor who shares the governance of this program with an experienced committee of senior faculty members (Drs. Patricia Donahoe, Joel Habener, and Janet Hall) who serve as Co-Directors. The total training faculty consists of 13 active, well-funded scientists whose diverse expertise and investigative interests line a spectrum of reproductive and developmental research from fundamental developmental biology to bench-to-bedside translational clinical investigations.

Trainees are closely supervised by an active and individualized mentorship program overseen by the senior faculty and are encouraged to interact extensively with junior faculty who typically serve as in a co-mentorship role. An extensive program of rigorous didactic sessions interlaces tightly with individually strong, independent laboratory programs that remain at the heart of the trainees’ program and complements their 2-3 years of research experience. Typically, the training period involves several additional years as junior faculty that permits the consolidation of skills required for maximal competitiveness for subsequent independent support, the ultimate goal of their training program.

Click here to view a list Dr. Crowley's current and former trainees.

Research facilities (27,000 ft2) are fully equipped and recently modernized laboratories dedicated to the training faculty. The institutional investigators are highly funded by the NIH, the environment is superb, and reinforcement from the NIH Reproductive Sciences Center program is substantial. The track record of past trainees indicates substantial leadership in reproduction. [Click here to view Dr. Crowley’s list of current and former trainees.] This training grant represents a critical stabilizing element in all of these individual research programs and is thus a crucial link that enables talented trainees to achieve subsequent independent careers in Reproduction and Developmental Biology through stable funding, mentored research projects, and a rich didactic program.

2) Male Reproductive Physiology in the Human
William F. Crowley (PI)
Project Number: 5 R01 HD15788
Source: NIH
Period: 7/1/81-6/30/07

Using the human disease model of men with idiopathic hypogonadotropic hypogonadism (IHH), it is possible to: a) control the timing and pace of their testicular maturation via administration of either exogenous gonadotropins or pulsatile GnRH; b) manipulate their gonadal sex steroid hormonal milieu by administration of steroid hormone inhibitors; and c) vary the dose and frequency of their hypothalamic input of GnRH. This ability to clamp the hypothalamic input to their pituitary via GnRH and contrast the responses of IHH men with those of normal and castrate men (whose hypothalamic-pituitary axis is unconstrained) allows us to dissect the hypothalamic from pituitary sites of action and address several physiologic issues that are not otherwise approachable in the human.

In Specific Aim #1, we explore the relative roles of gonadal sex steroids and Inhibin B (IB) in restraining FSH secretion across the full spectrum of gonadal development, where serum IB levels vary from prepubertal levels to those of normal adult men. Our preliminary results suggest that their relative roles appear to change during gonadal maturation. It also permits us to understand the role of the frequency of GnRH in FSH feedback and thus place it in context with both sex steroids and IB.

In Specific Aims #2 & 3, this model allows us to test the ability of FSH to increase Sertoli and germ cell number, testicular size, and sperm counts when administered to IHH men with immature gonads, and to then compare these results with those obtained when FSH stimulation is accompanied by that of LH via pulsatile GnRH therapy. During such studies we also have access to testicular tissue in these men and thus can validate both MIS and IB as markers of Sertoli cell numbers, proliferation, and maturation in a quantitative and statistically valid fashion that has not been previously possible. In collaboration with Dr. Martin Dym, we perform quantitative histomorphometry on these maturing gonads via serial testicular biopsies prior to and following four months of therapy. We then correlate these histologic findings with serial measurements of IB and MIS as well as other clinical and biochemical aspects of gonadal maturation.

Finally, in Specific Aim 4, the availability of serial testicular tissue in these men during Sertoli and early germ cell development allows us to gain insight into the specific genes activated in the human during early testicular development. These studies offer a unique approach to gain new insights into the physiology and developmental biopsy of the male reproductive axis. They also lay the groundwork that provides a context for the exploration of new tools to gain further insights into the complexities of reproductive dysfunction in the male.

3) Harvard Reproductive Endocrine Science Center
William F. Crowley (PI)
Project Number: U54 HD28138
Source: NIH
Period: 4/1/2005-3/31/2010

The Harvard Center was organized 16 years ago by the PI when NICHD first opened the Centers process to competition. From its inception, its scientific goals have strongly reflected its location within Harvard’s academic health center and its strong focus on two evolving themes - human investigation and genetics. FIve years ago our Center dramatically shifted its research focus upon recognizing the opportunity the Human Genome Project would provide for investigation in human reproduction. We also appreciated the unusual opportunity our patients with Idiopathic Hypogonadotropic Hypogonadism (IHH) and Kallmann’s Syndrome (KS) offered. These two disorders have in common GnRH deficiency. Together, this confluence of opportunities presented us with the potential to gain unusual insights into GnRH physiology. Thus, we married these two themes in hopes of providing fundamental insights into the genetic control of GnRH secretion in the human.
Despite GnRH’s acknowledged centrality to reproductive integrity, understanding of the genetic basis of its physiologic control in any species has been elusive. In part, this problem has been because such a high degree of species specificity characterizes its regulatory control. Whereas lower animals rely heavily on pheromones and light dark cycles to synchronize GnRH secretion and reproduction, both are severely attenuated in humans. Thus, across evolution homo sapiens has presumably utilized several different genes or ensembles to modify GnRH secretion as part of its reproductive strategies to adapt and evolve. Specific knowledge of these genes remains one of the great mysteries of biology.
This proposal represents the fruits of this marriage of our unique populations with new genetic approaches. It specifically focuses on 2 novel receptor genes critical to the control of GnRH physiology in the human that our Center’s Investigators and others have identified to cause IHH and KS during this past grant cycle - GPR54, Kiss1, and FGFR1. We have assembled a multidisciplinary team that has demonstrated that: a) mutations in either of these 3 genes cause IHH and KS, conditions which cause infertility in both men and women; b) the breadth of the phenotypic spectrum of these mutations and their full biology within the reproductive system is not fully described; c) their genetics is complex and strongly suggests interactions with other, yet-to-be discovered other genes such as the ligands for these receptors; and d) these genes also play important biological roles in several non-reproductive systems.
Our Center has fulfilled another of NICHD’s programmatic goal in that it has attracted and trained new investigators into this field whose techniques address these problems.

4) The Genetics of Polycystic Ovarian Syndrome
William F. Crowley (PI)
Project Number: U01 HD44417
Period: 9/26/02-3/31/07

The polycystic ovarian syndrome (PCOS) is a phenotypically complex disorder affecting 5% to 10% of women of reproductive age. Its protean manifestations include hyperandrogenemia, insulin resistance with glucose intolerance and an increased incidence of Type II diabetes, and a diffuse metabolic syndrome demonstrating many predisposing risk factors for early cardiovascular disease.

Given our past experience with phenotyping and demonstrating a striking familial tendency of this syndrome, we have initiated a unique collaboration with deCODE Genetics and the Icelandic population to identify the genes associated with this condition. Using Icelandic families, the Icelandic genealogy and the Icelandic health care system, we are exploring the genetic basis for this condition. We then plan to map the genotype/phenotype relationships in Icelandic women and contrast them to US women, to identify the male phenotype within the PCOD families, and to identify the long-term health impact of this condition in women and in men (including the incidence of diabetes, cardiovascular disease, and stroke using the Icelandic healthcare database).

Due to the unique combination of the genotyping facilities and statistical capabilities of deCODE Genetics, access to the Icelandic population and their genealogic history, the Icelandic health care database, and the homogeneous Icelandic population, these studies are much less difficult to undertake. In more heterogeneous populations where such extended family relationships with this disorder are unusual, it would be significantly more expensive and time-consuming to do the research.


5) Aging and the Hypothalamic-Pituitary Reproductive Axis
Janet E. Hall (PI)
Project Number: R01 AG13241
Period: 8/1/95-02/28/08
Source: NIH/NICHD

The broad goal of this project is to examine the neuroendocrine mechanisms that contribute to aging of the female reproductive system in the human. The underlying hypothesis is that there are changes in the physiology of hypothalamic gonadotropin-releasing hormone (GnRH) secretion and pituitary responsiveness in women that are specifically related to aging. Our studies concentrate on postmenopausal women in whom the absence of gonadal function makes it possible to investigate the independent effects of aging and gonadal hormones on the brain. Specifically, the age-related changes in pituitary function and the age-related changes in gonadal steroid control of hypothalamic and pituitary function are being explored.

In Aim 1, the pituitary contribution to the decline in gonadotropin secretion with age is being determined in vivo and in vitro, addressing the hypothesis that a decrease in pituitary response to GnRH-which is independent of changes in hypothalamic GnRH input-occurs with aging. Pituitary responsiveness to GnRH is being assessed in the presence of GnRH receptor blockade to control for antecedent GnRH stimulation. This aim also explores whether the decline in gonadotropin secretion that occurs with aging in postmenopausal women is associated with a decrease in gonadotrope number using human autopsy specimens.

Aim 2 investigates whether the negative feedback of estradiol on gonadotrope responsiveness to GnRH is altered with aging in vivo and whether there are changes in estrogen receptor ??(ER?? in vitro. Aim 3 determines the effect of aging on gonadotropin responses to short-term negative and positive feedback effects of gonadal steroids. In these studies, PET scanning is used to differentiate hypothalamic from pituitary sites of action. This aim investigates the hypotheses that negative feedback effects of estradiol are exerted at both the hypothalamic and pituitary levels in postmenopausal women and are maintained with aging, and that estrogen positive feedback is exerted primarily at the pituitary level in the human and declines with age in postmenopausal women.

The information derived from these studies will provide basic insights into the effects of aging on the brain in women and the degree to which aging affects the hypothalamic and pituitary responses to gonadal steroids. These studies may also have important implications for our understanding of the potential neuroendocrine contributions to reproductive aging and menopause.


6) FSH, Estradiol and Inhibin in Female Reproduction
Janet E. Hall (PI)
Project Number: R01 HD42708
Period: 7/10/02-5/31/07
Source: NIH/NICHD

The broad goal of this project is to continue to investigate the neuroendocrine and gonadal regulation of reproduction in women, focusing on the complex interrelationships between FSH, estradiol and the inhibins. There is now evidence that both estradiol and the inhibins contribute to the negative feedback control of FSH in women. However, their relative roles in the precise control of FSH during the follicular and luteal phases of the normal menstrual cycle have not been delineated. There are two unique circumstances in which we have shown that the coordinated secretion of estradiol and the inhibins is altered: in reproductive aging, estradiol levels are increased in the presence of low levels of inhibin, while in African-American women (relative to Caucasian women), normal reproductive cycles are characterized by increased estradiol in the face of identical levels of inhibin. Determining the mechanisms responsible for these changes provides us with important insights into the interactions of inhibin, estradiol, and gonadotropins in normal reproductive physiology.

Aim 1 focuses on the relative contributions of estradiol and the inhibins to the negative feedback regulation of FSH secretion during the follicular and luteal phases of the normal menstrual cycle (using normal and GnRH-deficient women in whom estrogen secretion is altered by aromatase blockade). Aim 2 seeks to elucidate the mechanisms underlying the dichotomy between estradiol and inhibin secretion in reproductive aging by investigating inhibin and estradiol secretion in response to fixed FSH stimulation, as well as by determining the number of granulosa cells, inhibin subunit expression, and aromatase function and expression in antral and preovulatory follicles as a function of reproductive aging.

Aim 3 seeks to determine the feedback and feed-forward interactions between FSH, estradiol and the inhibins that result in an increase in estradiol levels across normal reproductive cycles in African-American women. Feedback is examined using an estrogen infusion protocol, while feed-forward examines the control of estradiol and inhibin secretion in preovulatory follicles. Understanding the feedback and feed-forward dynamics of the hypothalamic-pituitary and ovarian components of the reproductive system is critical to an appreciation of the pathophysiology of reproductive disorders. This information is ultimately required for the design of therapeutic options for patients with reproductive disorders, including infertility.

7) Modulation of Insulin Action by Testosterone In Men
Frances J Hayes (PI)
Project Number: 1RO1 DK071168-01A2
Source: NIH
Period: 7/1/2006-6/31/11

Insulin resistance plays a key role in the pathogenesis of type 2 diabetes, a disease now reaching epidemic proportions and anticipated to affect 220 million people worldwide by 2010. Thus, elucidating the factors that alter insulin sensitivity has important public health implications. While epidemiologic studies consistently demonstrate an inverse relationship between insulin resistance and testosterone (T) levels in men, data on causality are lacking. In addition, it is unclear whether T might modulate insulin action by a direct effect mediated through the androgen receptor or an indirect effect mediated by aromatization to estradiol (E2). Thus, the overall goal of this proposal is to define the role of gonadal sex steroids in modulating insulin action in men. Specific Aim (SA) #1 will examine the impact of acute (24 h) and short-term (4 weeks) suppression of sex steroids on insulin sensitivity in normal men (Protocol 1). A gonadotropin-releasing hormone antagonist will be used to induce hypogonadism. Changes in insulin sensitivity will be assessed by a hyperinsulinemic-euglycemic clamp. The mechanisms underlying changes in insulin sensitivity will be explored by analyzing changes in body composition, and detailed studies of fat metabolism (rates of lipolysis & lipid oxidation) and skeletal muscle (muscle fiber type, intramyocellular lipid content, & gene expression profiles). Protocol 2 will examine the impact of 3 months of T therapy on all components of the metabolic syndrome (SA #2), the mechanism underlying any changes in insulin sensitivity using the methods outlined for SA #1, and the role of E2 in mediating the effect of T on insulin sensitivity (SA# 3). The present study, by combining carefully designed physiologic experiments with state of the art genetic and imaging studies, affords the opportunity to determine, if and how, T modulates insulin sensitivity in men. It is currently estimated that 20% of men over 60 yr have low T levels. Therefore, if low T levels are shown to play a role in the pathogenesis of insulin resistance, T may well represent an important therapeutic modality for both preventing and treating the metabolic syndrome and type 2 diabetes in men.


8) Title of Project: Establishment of a Human Serum Bank
Frances Hayes (PI)
Source: Abbott Pharmaceuticals
Period: 12/01/01-

Determination of the serum levels of reproductive hormones is a key component of the clinical evaluation and diagnosis of disorders of the reproductive system. Availability of gender and age-specific reference ranges is critical to their interpretation. The diversity of clinical applications for measurement of sex steroids requires assays that are highly sensitive, precise and accurate over wide analytical ranges. Current automated systems have not been well characterized with respect to clinical diagnosis. In order to establish normal ranges with a direct correlation to clinical diagnosis and to create an independent standard for comparing assay systems, it is imperative that reference ranges be carefully established for each system based on precise phenotyping of patients.

Accordingly, the aim of this study is to establish reference ranges for reproductive hormones: LH, FSH, testosterone, estradiol, progesterone, sex hormone binding globulin, inhibin A, and inhibin B based on carefully phenotyped normal volunteers and specific patient groups comprising women with polycystic ovary syndrome and women undergoing in vitro fertilization for infertility. This type of validation is beyond the financial capabilities or scope of practice of the routine clinical laboratory and can best be accomplished by collaboration between an academic research laboratory, a clinical investigator, and an industrial sponsor, as outlined in this protocol.


9) Physiology and Action of a new Follistatin Related Protein
Alan Schneyer (PI)
Project Number: 5 R01 HD039777
Source: NIH/NIDDK
Period: 8/01/01-7/31/06 (no cost extension)

Follistatin-related protein (FSRP) is a recently discovered glycoprotein whose gene structure and activin binding properties are highly homologous to the activin binding protein follistatin (FS). FSRP was originally cloned as an overexpressed protein in a B-cell leukemia and is expressed in numerous cancer cell lines, suggesting that it may be involved with, or be a useful marker for a wide array of tumors. Unlike FS, however, FSRP is most highly expressed in the placenta and testis (FS is highest in the ovary and kidney), indicating that FSRP has unique functions in these tissues. Immunocytochemical studies have demonstrated that FSRP is highly concentrated in the nucleus of all cell lines and primary cells tested, but is secreted only by cells with the highest FSRP expression levels.
These observations suggest that regulation of FSRP intracellular trafficking is complex and unique, and further, that FSRP may have nuclear activities distinct from strictly non-nuclear FS. Overexpression of FSRP in transgenic mice disrupts follicular development, resulting in female infertility.

Thus, the broad goal of this project is to determine the biological functions of FSRP in normal and pathophysiological circumstances, as well as to elucidate the biochemical features of this protein which govern its unique biology and distribution. Transgenic and knockout mice, along with several in vitro bioassays, are utilized to identify the normal and pathophysiological actions of FSRP (Specific Aim 1).

Intracellular trafficking, regulation of biosynthesis, and nuclear functions of FSRP are being examined in HeLa and human granulosa cells using pulse chase labeling, immunoprecipitation, and affinity chromatography (Specific Aim 2). The binding affinity and ligand specificity, as well as functional domains of FSRP responsible for these activities and FSRP’s nuclear localization are being examined using site directed mutagenesis and domain swapping with FS (Specific Aim 3). The results of this research will define the role(s) of FSRP in normal physiology, determine the mechanism whereby FSRP overexpression disrupts folliculogenesis, and define the novel regulatory mechanisms that result in nuclear localization and activity of a protein that is also secreted.

10) Establishing the Genetic Etiology for Kallmann Syndrome
Stephanie Seminara, M.D. (PI)
Project Number: 5 R01 HD43341
Source: NIH/NICHD
Period: 04/01/03-08/31/09

In all mammalian species, gonadotropin-releasing hormone (GnRH) is the first hormone in a complex reproductive cascade. GnRH is released by the hypothalamus and stimulates the secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH) from the pituitary; these gonadotropins then stimulate the gonads to produce sex steroids and follicles/sperm. The actions of GnRH are complex--it is secreted in a pulsatile, as opposed to constitutive, fashion, and a variable frequencies throughout the reproductive cycle. Understanding what signals modulate the developmental fate and secretory actions of GnRH neurons remains a major question for reproductive biologists.

This grant addresses this issue using a human disease model in which GnRH secretion is defective or absent. Patients with this condition, idiopathic hypogonadotropic hypogonadism (IHH), fail to undergo puberty and are infertile if untreated. Although congenital IHH is a rare disease and family sizes are typically small, a large inbred family of French Canadian descent has been identified with IHH and anosmia. A genome-wide scan has been performed and a chromosomal locus for the genetic defect has been identified. In this proposal, the candidate region will be further refined, a complete transcript map for the region will be developed, and RT-PCR will be used to screen the candidate gene for the precise genetic mutation. The mutation spectrum will then be juxtaposed against the baseline clinical/biochemical features of the patients, their neuroendocrine phenotypes, as well as their responses to physiologic replacement with exogenous pulsatile GnRH to develop robust genotype/phenotype correlations. The spatiotemporal pattern of expression of the gene will be studied and in vitro model systems developed to study the phyysiology of the newly-identified gene. It is hoped that this information will ultimately be used to understand numerous human diseases defined by abnormalities in GnRH secretion, including constitutional delay of puberty, hypothalamic amenorrhea, and central precocious puberty.

11) Recombinant Human Prolactin for Lactation Insufficiency
Corrine Welt (PI)
Source: March of Dimes
Period:
6/1/04-5/31/07

Breast milk is the nutrition of choice for infants based on the medical and psychological advantages for both the mother and infant. Breast milk is particularly important for premature and low birth weight infants because it is more easily digested, appears to decrease the rates of infection and necrotizing enterocolitis and may enhance neurocognitive development. Mothers of premature or low birth weight infants in the neonatal intensive care unit often need to maintain their milk supply using a breast pump because infants are too ill or weak to suckle. Even when mothers follow strict pumping guidelines, however, breast milk production tends to decrease after 4-6 weeks, resulting in lactation insufficiency. Although dopamine antagonists are available to increase prolactin and, therefore, milk production, the medications are not FDA approved for this purpose and have side effects for both the mother and baby.

The broad goal of this proposal is to examine the efficacy and safety of recombinant human prolactin to treat lactation insufficiency in mothers of premature infants who are pumping to provide breast milk. The hypotheses include: 1) Recombinant human prolactin administration will increase breast milk quantity in women with lactation insufficiency; 2) There is a direct relationship between prolactin level and milk quantity; and 3) Enhancing breast milk production with recombinant human prolactin is safe for infants and mothers. To test these hypotheses, recombinant human prolactin or placebo will be administered subcutaneously, in a randomized, placebo-controlled manner, to mothers who are pumping breast milk for their premature infants and develop lactation insufficiency. Breast milk volume will be monitored at baseline and during prolactin or placebo injections and correlated with serum prolactin levels. Side effects in mothers and infants will be documented. An increase in milk volume during recombinant human prolactin, but not placebo, administration will provide insight into the importance of prolactin in the maintenance of milk volume during lactation. The data will also be necessary for development of recombinant human prolactin for treatment of lactation insufficiency, thus directly impacting the nutrition and outcome of premature infants.


 

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