The role of testicular growth hormone variants for human reproduction

Regulation of human fertility is a complex process mainly determined by hypothalamic -pituitary - gonadal hormone axes. Additionally, changes in local auto/paracrine growth factor production and action contribute in a modulatory way to gonadal steroidogenesis and gametogenesis. We recently observed that the human gonads of both genders are not only targets for but, surprisingly, eutopically produce the classical somatotrophic factors growth hormone/placental lactogen (GH/PL) and considered these protein hormones as autocrine or paracrine growth or regulatory factors, especially as PL serum levels remained undetectable. In addition to "genuine"; GH and PL this hormone family includes an array of molecular variants which are determined at genomic, posttranscriptional and posttranslational levels, respectively. Unfortunately, only the pituitary- or placental-derived major forms (GH-N, PL-A/B) are available and susceptible to measurement and functional studies. The present project therefore aims at analyzing mechanisms by which eutopically produced gonadal GH/PL genomic and alternative splice variants (hGH-V2, hGH-V, hPL-A2) are involved in the regulation of the reproductive system and how they affect proliferative disorders therein (prostate, testis). In a first approach we intend to develop tools i.e. synthesize recombinant human GH/PL-variants (hGH-V2, hGH-V, hPL-A2) and to establish appropriate selective monoclonal antibodies (mabs) to subsequently monitor and analyze local patterns of eutopic hormone production in human reproductive organs. This should provide the basis for functional studies with recombinant hormones and mabs showing how these variant molecules -in comparison to classical, GH-N, PL-A/B and PRL - interact with their receptors or ligands, subsequently transduce signals, affect steroid hormone biosynthesis and influence proliferative responses in human tumor cell lines (prostate, testis, mammary gland). Thus, ultimately the role of somatotrophic hormone variants for physiology and pathophysiology of human reproduction will be addressed by recombinant proteins and mabs. In preliminary investigations - when cloning the cDNA for GH-V2 - we observed a novel transcript in the human testis encoding for a protein with exciting new features: a putative chimeric cytokine/cystein knot growth factor. This finding was published and well received by others in the field of human male reproduction.

The histogenetic reorganization of the aging human prostate leading to the development of the two most important diseases of this organ, the benign prostatic hyperplasia and prostate cancer, is supported by socalled pleiotropic factors. These are produced locally and are necessary during youth for optimal reproduction but in older age lead to aberrant growth of the prostate. Hormone networks regulate virility, fertility and prostatic growth via the hypothalamic-pituitary-gonadal-prostatic axis. Testicular and prostatic factors act in auto-/para- and lumenocrine ways on cellular differentiation, growth and function and complement the endocrine axes. Disturbances in those networks affect bone metabolism, fertility and sexuality, and influence the development of benign prostatic hyperplasia (BPH) and prostate cancer (PCa). Aims of this study were to identify, isolate, recombinantly synthesize and investigate functions of members of the protein hormone family like growth hormone (GH), prolactin (PRL), placental lactogen (PL) and its variants locally produced in the male reproductive tract. In addition seminal plasma factors, glycoprotein hormones (GPH) and its variants were investigated for their auto-/paracrine effects in the prostate. Previously described splice variants of the GH/PL gene cluster were cloned in complementary 2-way RT-PCR approaches and recombinant proteins produced by the baculovirus system. As shown by confocal microscopy hPL-A2 contains a hydrophobic region responsible for membrane insertion. Thus, surprisingly it might exert its function invers to the classical GH mechanism by serving as a membrane receptor. Seminal plasma (SMP) was analyzed for its content of biological active substances by HPLC. Interestingly, in proliferating prostate epithelial cells a low molecular weight fraction (2-4 kD) of SMP induced p53 and bcl2 dependent apoptosis without activation of caspase 3. Other fractions induced differentiation and hPRL release in prostatic smooth muscle cells (SMC). Moreover, Zn2+-ions which are found in highest concentrations in the prostate and SMP were shown to act in growth-regulatory ways on prostatic cells. Finally, GPH alpha subunit was produced in a differentiation- and age-related manner and inhibited growth of prostate fibroblasts. Our findings on the regulation of growth and differentiation in the human male reproductive tract by protein hormones, glycoprotein hormones and low molecular weight factors were compiled to a hypothesis on the histogenetic reorganization over the adult lifetime and subsequent development of age-related diseases of the aging human prostate.