Despite the documented importance of the protein hormone relaxin in reproduction in various mammalian species, the role of relaxin in human reproduction is poorly understood, largely because of the lack of studies in women or in suitable non-human primate models. Here we describe the establishment of a nonhuman primate model of early human pregnancy and its use in defining the actions of relaxin. Results demonstrate that relaxin exerts dramatic uterine effects including pronounced increase in uterine weight and stimulation of endometrial angiogenesis and resident endometrial lymphocyte number. In addition, relaxin decreases endometrial levels of matrix metalloproteinases 1 and 3 and increases levels of their endogenous inhibitor, tissue inhibitor of metalloproteinase 1, resulting in maintenance of endometrial collagen content. Relaxin significantly inhibits endometrial levels of estrogen receptor ␣, but not ␤, and of progesterone receptor isoforms A and B. The findings that relaxin stimulates new blood vessel formation and increases cytokine-containing lymphocyte number while maintaining endometrial connective tissue integrity are consistent with a significant role of relaxin in the establishment and͞or maintenance of early pregnancy.R elaxin is a 6-kDa protein hormone member of the insulinlike growth factor family present in circulation in women during the latter part of the menstrual cycle and throughout pregnancy (1). In many mammalian species, relaxin exerts pronounced effects on the female reproductive tract that are involved in the maintenance of pregnancy and successful parturition (2, 3). Relaxin is important for normal delivery in several mammalian species because of its marked rearrangement of reproductive tract connective tissue (2-4). Despite the documented importance of relaxin in various mammalian species, the role of relaxin in human reproduction is, to date, an important, yet unanswered, question. Elucidation of the role of relaxin in women has been hampered by the inability to perform studies in women and by the lack of studies performed in suitable primate models of human pregnancy. Here we describe the establishment of a non-human primate model of early human pregnancy and its use in defining the actions of relaxin. Results demonstrate that relaxin exerts dramatic uterine effects including pronounced increase in uterine weight, stimulation of endometrial angiogenesis and resident lymphocyte number, maintenance of endometrial connective tissue integrity, and inhibition of endometrial estrogen and progesterone action. These effects are the developmental changes that occur in the human uterus during the late secretory phase of the menstrual cycle and early pregnancy. These findings support the thesis that relaxin acts as an important factor in uterine accommodation to and maintenance of early pregnancy in women.The dramatic species differences in the sources, secretion patterns, and target organs of relaxin have contributed greatly to the lack of understanding of the role of relaxin in human repr...

Extensive evidence demonstrates pronounced effects of relaxin on the differentiation of human endometrial cells in vitro. In vivo data in rhesus monkeys suggest a role for relaxin in the development of endometrial vascular architecture. In women, pregnancy can be established and maintained in the absence of circulating relaxin. Thus, local synthesis by the endometrium is necessary if relaxin plays a physiological role in human endometrial function. Although relaxin protein and the prorelaxin C peptide have been localized to human endometrium, no data for relaxin synthesis have been provided to date. We therefore assessed relaxin mRNA and protein levels in cultured, defined human endometrial cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) techniques were used to demonstrate the presence of relaxin mRNA in human stromal and glandular epithelial cells. Secretion of the protein into the media of cultured cells of both types was also detected. Relaxin stimulated the expression of vascular endothelial growth factor in glandular epithelial and stromal cells that were isolated from tissue that had been taken during the secretory phase of the cycle. Relaxin inhibited the expression of procollagenase from both glandular epithelial cells, with a more marked inhibition demonstrated from cells that were isolated from tissue that had been taken during the secretory phase, and from stromal cells. These data demonstrate that human endometrial cells synthesize relaxin, and they support the concept that relaxin fosters endometrial conditions that are required for implantation in women.

To elucidate the mechanism of relaxin action, we studied the binding characteristics of human relaxin and its effects on intracellular concentrations of cAMP and tyrosine phosphorylation of cellular proteins in a model system of human cervix, human lower uterine segment fibroblasts. Human relaxin labeled with 125I bound specifically to a single class of high-affinity relaxin binding sites, distinct from insulin receptors, with a mean (+/-SEM) dissociation constant (Kd) of 4.36 +/- 1.7 x 10(-9) M and a mean of 3220 +/- 557 binding sites per cell in human lower uterine segment fibroblasts. Relaxin, in quantities that were shown previously to stimulate intracellular levels of cAMP in other cell types, had no effect on intracellular levels of cAMP in human lower uterine segment fibroblasts even in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX). Incubation of the cells with relaxin caused a significant increase in tyrosine phosphorylation of a protein with an apparent Mr of approximately 220 kDa in these cells. In concert with results of recent studies that demonstrated that the Mr of the relaxin receptor is approximately 220 kDa, our data suggest that the phosphorylated protein is likely to be the relaxin receptor.

Despite the importance of relaxin to normal parturition in various species and its potential as an etiological agent in preterm delivery in women, knowledge regarding the mechanisms by which relaxin alters cervical connective tissue is extremely limited. An established in vitro model for human pregnancy cervix, human lower uterine segment fibroblasts, was used to determine the effects of relaxin as well as those of progesterone on the expression of matrix metalloproteinases and tissue inhibitor of metalloproteinase-1. The results demonstrate that relaxin is a positive regulator of matrix metalloproteinase expression, as it stimulates the expression of procollagenase protein and mRNA levels, stimulates prostromelysin-1 protein and mRNA levels, and inhibits tissue inhibitor of metalloproteinase-1 protein expression. Stimulation of procollagenase and prostromelysin-1 expression by relaxin does not involve phorbol-12-myristate-13-acetate- sensitive PKCs. Relaxin-stimulated tyrosine phosphorylation of the putative receptor and inhibition by a receptor tyrosine kinase inhibitor suggest that the relaxin receptor is probably a tyrosine kinase receptor. Inhibition of c-Raf protein expression using an antisense oligonucleotide inhibits relaxin regulation of matrix metalloproteinase and tissue inhibitor of metalloproteinase-1, suggesting that a signaling pathway involving c-Raf kinase mediates relaxin action.

Despite the importance of relaxin to normal parturition in various species and its potential as an etiological agent in preterm delivery in women, knowledge regarding the mechanisms by which relaxin alters cervical connective tissue is extremely limited. An established in vitro model for human pregnancy cervix, human lower uterine segment fibroblasts, was used to determine the effects of relaxin as well as those of progesterone on the expression of matrix metalloproteinases and tissue inhibitor of metalloproteinase-1. The results demonstrate that relaxin is a positive regulator of matrix metalloproteinase expression, as it stimulates the expression of procollagenase protein and mRNA levels, stimulates prostromelysin-1 protein and mRNA levels, and inhibits tissue inhibitor of metalloproteinase-1 protein expression. Stimulation of procollagenase and prostromelysin-1 expression by relaxin does not involve phorbol-12-myristate-13-acetate- sensitive PKCs. Relaxin-stimulated tyrosine phosphorylation of the putative receptor and inhibition by a receptor tyrosine kinase inhibitor suggest that the relaxin receptor is probably a tyrosine kinase receptor. Inhibition of c-Raf protein expression using an antisense oligonucleotide inhibits relaxin regulation of matrix metalloproteinase and tissue inhibitor of metalloproteinase-1, suggesting that a signaling pathway involving c-Raf kinase mediates relaxin action.

To elucidate the potential role of the ras protooncogene proteins in a specific tissue, the present study determined the levels of individual c-ras-encoded p21 proteins in the rat ovary during various stages of physiological function. p21 protein was extracted from ovaries taken from immature normal female rats, mature nonpregnant animals in the metestrus stage of the estrus cycle, rats at various stages of pregnancy, and actively lactating animals. Levels of individual p2ls were evaluated by immunoblot analysis with specific antibodies to the p21 proteins encoded by the Kirsten, Harvey, and neuroblastoma c-ras protooncogenes, c-Ki-ras, c-Ha-ras, and N-ras. Results showed that c-Ki-ras p21 is at its lowest level in the immature ovary and increases with development of the corpora lutea to its highest levels at day 16 of pregnancy, after which levels decline and then rise again during lactation. This pattern, which mimics that of circulating progesterone levels, suggests that ovarian c-Ki-ras p21 levels are regulated and that c-Ki-ras p21 plays a role in the differentiated function of the rat ovary, likely the luteal compartment. In contrast, levels of c-N-ras p21 did not appear to vary with changes in the physiological function of the ovary but appeared to be constitutive. A preferential role for the c-Ki-ras p21 may be due to the documented unique differences in the structure of the carboxyl terminus of this particular c-ras p21.Results of recent studies show the importance of a variety of cellular oncogene products not only in the control of proliferation but also in differentiated function of normal eukaryotic cells (1). Of particular interest is the cellular ras (c-ras) gene family, a group of very highly conserved genes present in yeasts to primates (2, 3). The c-ras genes encode markedly similar 21-kDa proteins (p21) that are localized to the inner face of the plasma membrane, bind guanine nucleotides, and have significant structural homology to other GTP-binding proteins (G proteins), including the adenylate cyclaseassociated G proteins (2,3). Unlike their role in yeasts, however, in mammalian cells c-ras G proteins do not function as part of the cyclase pathway (4). Although various data demonstrate that mammalian c-ras p21 proteins influence the efficacy of membrane receptor signal-transduction pathways (5,6), the specific function of the c-ras p21s in any mammalian cell type has not been defined. In efforts to more clearly define their specific role, examination of total c-ras p21 expression in various normal human and rat tissues has been performed (7-9). Yet these studies have been limited to evaluation of total p21 content rather than determination of the individual p21 proteins and have not defined whether changes in differentiated function of a tissue are associated with variations in p21 protein levels. Since changes in differentiated function of specific compartments in the mammalian ovary are well documented by the reflected patterns in circulating levels of steroid hormones, the ovary i...

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