As part of our goal to understand the molecular events controlling reproduction we are investigating the following topics on sex hormones. By studying the structure-function and cloning of gonadotropins and their receptors, we hope to produce and test recombinant proteins for immunocontraception and drug targeting. The cloning of different receptor types formed by alternative splicing permits the analysis of signaling events involved in pleiotrophic effects of the hormone. This work is complemented by our molecular genetic approaches which have produced receptor knockout mice (FORKO mice) that could be useful as a model for menopause, aging, obesity and fertility/infertility.

Molecular Reproduction Research
Our focus of research has been the study of the glycoprotein hormones (FSH, LH, TSH) of the pituitary and placenta (hCG) as well as their respective receptors. Although they regulate reproductive events and thyroid function in the body, their effects are indirectly felt on other systems. Being complex glycoproteins these molecules offer a challenging field for investigation and biotechnology. Recent investigations on their molecular biology currently in progress in the laboratory integrate their role in fertility/infertility, cancers of the breast and prostate. Our most recent receptor knockout model in the mouse offers the exciting prospect of being useful in the study of aging, menopause, cardiovascular physiology and cognitive development. These studies are highlighted in the following examples.

Receptor Diversity and Hormone Signal Integration
Gonadotropin receptors are among the most selectively localized interacting systems in the body being expressed preferentially in certain cells of the gonads. However, more recent studies indicating some expression in non-gonadal cells remain to be supported by functional tests. The coordinated expression of gonadotropin receptors in the testis and ovary varies in unison with development and differentiation. Investigating the FSH (Follicle Stimulating Hormone) receptor in the testis and ovary, we have established that a single large gene of 80-100 Kb undergoes extensive alternative splicing to yield receptor proteins with different structural motifs. So far, four receptor types have been cloned in our laboratory and elucidating their signaling function(s) is an important goal of our research. The receptor motifs include a heptahelical G-protein coupled receptor R1 that stimulates adenylate cyclase activation in stably transfected cells. A second receptor R2 of similar size varies from R1 by having a shorter carboxyl terminus and this acts as an inhibitor of adenylate cyclase activation. A third receptor generated by splicing the first eight exons to a DNA segment used in expression of R2 gives rise to a cytokine/growth factor type I receptor that lacks the seven transmembrane region involved in G-protein coupling. This receptor appears to be involved in calcium signaling and cell proliferation. The fourth receptor motif designated R4 is most likely a soluble receptor protein containing part of the large extracellular domain and is coded by the first four exons. As all these receptors have different carboxyl termini, we are currently generating antibodies and receptor probes specific for each type, which can be used to identify stage/cycle specific expression in the ovary and testis. By expressing individual receptors in immortalized gonadal cell lines (that lack receptors) we hope to recreate hormone (FSH) responsive cells to identify various signaling pathways that may account for pleiotropic action(s) of FSH.

A Knockout Model for the Follitropin Receptor and Transgenic Animals
Receptor alterations in man can affect reproductive functions differently depending upon the location of mutations in the genes. Taking a molecular genetic approach to the problem of delineating functional consequences, we have created a mutant animal called the FORKO mouse by eliminating expression of the FSH receptor by gene targeting techniques. This was achieved by deleting the N-terminal region of the mouse FSH receptor coded by the first exon. Receptor (mRNA and protein) expression is absent in the mutants. Mutant males display small testis, partial spermatogenesis failure and reduced fertility. FSH signaling is not essential for initiating spermatogenesis, but is required to sustain viability and motility of sperms. The integrity of the seminiferous tubule and nurturing somatic Sertoli cells is altered to such an extent that there is a general disarray in spermatogenesis. FSH receptor deletion also reduces androgen production indicating the requirement of hormone signaling for inter-cellular communication. The phenotype of mutant females is much more severe. They display thin uteri and small ovaries and are sterile because of a block in follicular development and ovulation. As the hormonal profiles in these genetically altered mice are similar to those that occur in menopausal conditions, we argue that they could be useful in studies related to women's health. We are pursuing studies related to obesity, osteoporosis, cardiovascular problems and cognitive defects. Our future investigations will include generation of transgenic animals expressing known variants of the receptor in these genetically altered mice to establish partial or full restoration of gonadal function. We also expect to localize the fundamental defects in sperms of the mutants that render them immotile and non-functional. Thus, they would become useful in infertility studies and development of male contraceptives.

Contraceptive Vaccines
The possibility of interfering with spermatogenesis by inhibition of FSH secretion/action is an attractive proposition that is undergoing testing in our laboratory. Utilizing our purified recombinant FSH receptor domains we have conducted active immunization of adult male monkeys. Males become infertile without reduction in androgen levels as long as neutralizing receptor antibodies remain in circulation. The method appears to be reversible prompting further studies aimed at developing this as an approach for male contraception. We are presently engaged in analyzing the molecular changes induced in the sperms by flow cytometric techniques.

Functional Role of Glycosylation in Gonadotropic Hormones
The presence of N-linked and/or O-linked glycosylation up to 20-40% carbohydrate suggests that the sugars could serve an important function in signal transduction. By selected chemical methods or site-directed mutagenesis, it has been possible to deplete or remove glycosylation sites in the gonadotropic hormones and assess their functions. These alterations while having no effect on the quaternary structure of the hormones modify their biological profile in a discrete manner. Such deglycosylated (DG) hormones, as they are called, bind to the same receptor(s) as the native hormones, with the same or higher affinity, but are unable to activate the membrane-bound adenylate-cyclase system. Because of these differential characteristics, the DG-hormones can act as specific and competitive antagonists of the action of native hormones, both in in vitro and in vivo model systems. Production of monoclonal antibodies to various gonadotropin antagonists have helped us in understanding hormone-receptor interactions and monitoring hormone (agonist/antagonist) patterns in clinical situations.

Our laboratory is also an important source for highly purified glycoprotein hormones (FSH, LH, TSH and hCG) and their subunits (human and animal origin). The identification and exploitation of potentially new biological activities found in crude pregnancy extracts remain an important goal of our research.