Relaxin affects cell organization and early and late stages of spermatogenesis in a coculture of rat testicular cells.

Relaxin and its receptor RXFP1 are co-expressed in Sertoli cells, and relaxin can stimulate proliferation of Sertoli cells. In this study, we investigated a role of relaxin in spermatogenesis, using a short-term culture of testicular cells of the rat that allowed differentiation of spermatogonia to spermatids. Sertoli, germ, and peritubular myoid cells were the predominant cell types in the culture. Sertoli and germ cells expressed RXFP1. Cultures were incubated without (control) or with 0.5% fetal bovine serum (FBS) or 100 ng/mL H2 relaxin (RLN) for 2 days. Cell organization, number, and differentiation were analyzed after 2 (D2), 5 (D5) or 8 (D8) days of culturing. Although the proportion of germ cells decayed from D2 to D5, the relative contribution of HC, 1C, 2C, and 4C germ cell populations remained constant in the control group during the whole culture. RLN did not affect the proportion of germ cell populations compared with control, but increased gene and/or protein expression of the undifferentiated and differentiated spermatogonia markers PLZF and c-KIT, and of the post-meiotic marker Odf2 in D5. RLN favored organization of cells in tubule-like structures, the arrangement of myoid cells around the tubules, arrangement of c-KIT-positive spermatogonia at the basal region of the tubules, and expression of the cell junction protein ß-catenin close to the plasma membrane region. Knockdown of relaxin with small interfering RNA (siRNA) reduced expression of ß-catenin at the cell junctions, and shifted its expression to the nucleus. We propose that relaxin may affect spermatogenesis by modulating spermatogonial self renewal and favoring cell contact.

Gene duplication and positive selection explains unusual physiological roles of the relaxin gene in the European rabbit.

The relaxin gene family is a group of genes involved in different physiological roles, most of them related to reproduction. In vertebrates the genes in this family are located in three separate chromosomal locations, and have been called relaxin family locus (RFL) A, B, and C. Among mammals the RFLA and RFLC are the most conserved as no gene copy-number variation has been observed thus far. The RFLB locus is also conserved on most mammals other than primates, where there are several gene gains and losses. Interestingly, the relaxin gene found on the RFLB locus in the European rabbit has acquired a novel role. In addition to the classical reproductive roles, this gene is expressed in tracheobronchial epithelial cells and its expression has been linked to squamous differentiation. We reconstructed the evolutionary history of the European rabbit RFLB locus using the tools of comparative genomics and molecular evolution. We found that the European rabbit possess a RFLB locus which is unique among mammals in that there are five tandemly arranged relaxin gene copies, which contrast with the single relaxin copy gene found in most mammals. In addition we also found that the ancestral pre-duplication gene was subject to the action of positive selection, and several amino acid sites were identified under the action of natural selection including the sites B12 and B13 which are part of the receptor recognition and binding site.

Relaxin prevents the development of severe acute pancreatitis.

AIM: To investigate the severity of acute pancreatitis (AP) is associated to the intensity of leukocyte activation, inflammatory up-regulation and microcirculatory disruption associated to ischemia-reperfusion injury. Microvascular integrity and inhibition of pro-inflammatory mediators are key-factors in the evolution of AP. Relaxin is an insulin-like hormone that has been attributed vasorelaxant properties via the nitric oxide pathway while behaving as a glucocorticoid receptor agonist. METHODS: AP was induced by the bilio-pancreatic duct-outlet-exclusion closed-duodenal-loops model. Treatment with relaxin was done at different time-points. Nitric oxide synthase inhibition by L-NAME and glucocorticoid receptor (GR) blockage by mifepristone was considered. AP severity was assessed by biochemical and histopathological analyses. RESULTS: Treatment with relaxin reduced serum amylase, lipase, C-reactive protein, IL-6, IL-10, hsp72, LDH and 8-isoprostane as well as pancreatic and lung myeloperoxidase. Acinar and fat necrosis, hemorrhage and neutrophil infiltrate were also decreased. ATP depletion and ADP/ATP ratio were reduced while caspases 2-3-8 and 9 activities were increased. L-NAME and mifepristone decreased the efficiency of relaxin. CONCLUSION: Relaxin resulted beneficial in the treatment of AP combining the properties of a GR agonist while preserving the microcirculation and favoring apoptosis over necrosis.

Relaxin has a minor role in rat mammary gland growth and differentiation during pregnancy.

Growth and differentiation of mammary gland is associated with numerous hormones and a variety of cell-cell, cell-matrix interactions. This study addressed the role of relaxin (Rlx) on these processes. Morphologic and biochemical changes that occur throughout the second half of pregnancy are reported. Temporal patterns and spatial distributions of markers useful to evaluate proliferation, secretion, and collagen remodeling were established. To evaluate the role of Rlx, an ablation/replacement animal model was used. Considering Rlx secretion pattern, two periods were selected: d 11 through d 13, and d 20 through d 23. In the stroma, the extracellular compartment showed changes associated with the lack of Rlx. Collagen remodeling within the lobuloalveolar structure, measured by a significant increase in collagen birefringence, decreased at d 12, d 21, and d 22. Parenchymal structures were less sensitive to the absence of Rlx than stroma. Epithelial cell proliferation was lower in Rlx-deficient rats only at d 12, and alpha-lactalbumin expression decreased at d 21 and d 22. Both lobuloalveolar diameter and percentage of area occupied by these structures showed no changes. In the absence of Rlx, some of the studied markers showed statistically significant differences in scattered days; these do not make clear trends. No differences were found on d 23 on any of the studied parameters suggesting that compensatory mechanisms might be activated to overcome the effects of the absence of Rlx. Unlike the critical role of Rlx either in uterine cervix dilation or in nipple development during rat pregnancy, Rlx had a minor role in growth and differentiation of rat mammary gland.

Role of relaxin and estrogen in the control of eosinophilic invasion and collagen remodeling in rat cervical tissue at term.

Ripening and dilation of the rat cervix at term involves a widespread reduction in the density and organization of collagen fibers following a polymorphonuclear eosinophilic invasion. These are hormonally regulated events; however, the correlation between hormonal milieu and these morphological changes is not well understood. To investigate the role of preparturient relaxin and estradiol-17ss (E2) in cervical collagen remodeling and eosinophilic infiltration, pregnant rats were either sham-ovariectomized (group C) or ovariectomized in the morning of Day 22. Ovariectomized rats were treated with E2 (group OE), relaxin (group OR), E2 plus relaxin (group OER), or hormone vehicles (group O). There were 4 or 5 animals per group. Cervices were taken from animals killed approximately 1 h before expected parturition. Five-micrometer serial sections of paraffin-embedded cervices were stained with either Sirius Red in alkaline solution to measure eosinophil infiltration or in Picrosirius to measure collagen birefringence. Ovariectomized rats treated with E2 (group OE or OER) showed high levels of eosinophilic infiltration that did not differ from those in group C intact controls. However, the values of eosinophilic infiltration in ovariectomized rats treated with relaxin or hormone vehicles (groups OR and O) were low and far below (p < 0.01) those of groups OE and C. In ovariectomized rats treated with E2 alone (group OE), the widespread reduction in collagen organization that occurred in group C controls was not observed. It was only in ovariectomized rats treated with relaxin or E2 + relaxin (groups OR and OER) that the values of birefringence were low, and they were as low as in control group C. In conclusion, this study indicates that eosinophilic infiltration and collagen remodeling in the rat cervix at term are under the control of different hormones: E2 stimulates eosinophilic invasion, and relaxin promotes a widespread reorganization of collagen fibers.