Unveiling a novel memory center in human brain: neurochemical identification of the nucleus incertus, a key pontine locus implicated in stress and neuropathology.

BACKGROUND: The nucleus incertus (NI) was originally described by Streeter in 1903, as a midline region in the floor of the fourth ventricle of the human brain with an 'unknown' function. More than a century later, the neuroanatomy of the NI has been described in lower vertebrates, but not in humans. Therefore, we examined the neurochemical anatomy of the human NI using markers, including the neuropeptide, relaxin-3 (RLN3), and began to explore the distribution of the NI-related RLN3 innervation of the hippocampus. METHODS: Histochemical staining of serial, coronal sections of control human postmortem pons was conducted to reveal the presence of the NI by detection of immunoreactivity (IR) for the neuronal markers, microtubule-associated protein-2 (MAP2), glutamic acid dehydrogenase (GAD)-65/67 and corticotrophin-releasing hormone receptor 1 (CRHR1), and RLN3, which is highly expressed in NI neurons in diverse species. RLN3 and vesicular GABA transporter 1 (vGAT1) mRNA were detected by fluorescent in situ hybridization. Pons sections containing the NI from an AD case were immunostained for phosphorylated-tau, to explore potential relevance to neurodegenerative diseases. Lastly, sections of the human hippocampus were stained to detect RLN3-IR and somatostatin (SST)-IR. RESULTS: In the dorsal, anterior-medial region of the human pons, neurons containing RLN3- and MAP2-IR, and RLN3/vGAT1 mRNA-positive neurons were observed in an anatomical pattern consistent with that of the NI in other species. GAD65/67- and CRHR1-immunopositive neurons were also detected within this area. Furthermore, RLN3- and AT8-IR were co-localized within NI neurons of an AD subject. Lastly, RLN3-IR was detected in neurons within the CA1, CA2, CA3 and DG areas of the hippocampus, in the absence of RLN3 mRNA. In the DG, RLN3- and SST-IR were co-localized in a small population of neurons. CONCLUSIONS: Aspects of the anatomy of the human NI are shared across species, including a population of stress-responsive, RLN3-expressing neurons and a RLN3 innervation of the hippocampus. Accumulation of phosphorylated-tau in the NI suggests its possible involvement in AD pathology. Further characterization of the neurochemistry of the human NI will increase our understanding of its functional role in health and disease.

The dual and multifaceted role of relaxin-2 in cancer.

The continuous increase in cancer-associated deaths despite the substantial improvement in diagnosis and treatment has sparked discussions on the need for novel biomarkers and therapeutic strategies for cancer. Although increasing evidence has demonstrated the pivotal role of relaxin-2 in multiple cancers, their role is a double-edged sword with both protumor and antitumor having been reported in various malignant tumors. Considering this dual role, it appears the biological mechanism underpinning the action of relaxin-2 in cancer is not clear and further studies to elucidate their potential as a preventive factor for cancers are of prime importance. Herein, a summarized up-to-date report on the role of relaxin-2 in human cancer including detailed clinical and experimental evidence supporting their tumor-promoting and inhibitory functions in cancer development and progression has been elucidated. Also, signaling pathways and other factors orchestrating the activities of relaxin-2 in the tumor microenvironment has been discussed. Collectively, the evidence from this review has demonstrated the need for further evaluation of the role of relaxin-2 as a diagnostic and or prognostic biomarker for cancer.

Physical Training Reduces Chronic Airway Inflammation and Mediators of Remodeling in Asthma.

Several benefits of aerobic training for asthmatic patients have been demonstrated. However, its effects on systemic inflammation and on airway remodeling mediators and lung mechanics are unknown. This prospective study included 21 intermittent and mild asthma patients, and as primary outcomes, the evaluation of pro- and anti-inflammatory and pro- and antifibrotic mediators in exhaled breath condensate (EBC) and blood were performed, beyond the cell counting in blood and in induced sputum. Aerobic training was performed for 3 months, 3 times per week. Aerobic training increased the levels of anti-inflammatory cytokines and of antifibrotic mediators in the breath condensate: IL-1ra (p = 0.0488), IL-10 (p = 0.0048), relaxin-3 (p = 0.0019), and klotho (p < 0.0043), respectively. Similarly, in plasma, increased levels of IL-1ra (p = 0.0147), IL-10 (p < 0.0001), relaxin-3 (p = 0.004), and klotho (p = 0.0023) were found. On contrary, reduced levels of proinflammatory cytokines in the breath condensate, IL-1ß (p = 0.0008), IL-4 (p = 0.0481), IL-5 (p < 0.0001), IL-6 (p = 0.0032), IL-13 (p = 0.0013), and TNF-α (p = 0.0001) and profibrotic markers VEGF (p = 0.0017) and TSLP (p = 0.0056) were found. Similarly, in plasma, aerobic training significantly reduced the levels of proinflammatory cytokines IL-1ß (p = 0.0008), IL-4 (p = 0.0104), IL-5 (p = 0.0001), IL-6 (p = 0.006), IL-13 (p = 0.0341), and TNF-α (p = 0.0003) and of profibrotic markers VEGF (p = 0.0009) and TSLP (p < 0.0076). Fractional exhaled nitric oxide (FeNO) was reduced after the intervention (p = 0.0313). Regarding inflammatory cells in sputum, there was a reduction in total cells (p = 0.008), eosinophils (p = 0.009), and macrophages (p = 0.020), as well as of blood eosinophils (p = 0.0203) and lymphocytes (p = 0.0198). Aerobic training positively modulates chronic airway inflammation and remodeling mediators, beyond to improve systemic inflammation in intermittent and mild asthmatic patients.

Relaxin-2 during pregnancy according to glycemia, continence status, and pelvic floor muscle function.

INTRODUCTION AND HYPOTHESIS: To investigate relaxin-2 concentration comparing gestational diabetes mellitus (GDM) and non-GDM patients during pregnancy according to urinary incontinence (UI) and pelvic function status. METHODS: This is a cross-sectional study evaluating 282 pregnant women from 24 weeks of gestation. The participants were divided into two groups, non-GDM and GDM, according to American Diabetes Association's diabetes mellitus gestational threshold. In addition, according to subanalysis, both groups were subdivided according to the presence of pregnancy-specific urinary incontinence: non-GDM continent, non-GDM incontinent, GDM continent, and GDM incontinent. All participants filled in questionnaires on clinical, obstetric, and urinary continence status (International Consultation on Incontinence Questionnaire-Short Form, ICIQ-SF, and Incontinence Severity Index, ISI), followed by pelvic floor muscle evaluation by the PERFECT scheme in which strength, endurance, and speed of contractions were evaluated. RESULTS: Serum relaxin-2 concentrations were significantly lower in pregnant women with pregnancy-specific urinary incontinence in both non-GDM and GDM patients, but GDM showed the lowest concentration. In addition, the stratification of the groups according to pelvic floor muscle strength showed that pregnant patients with GDM and modified Oxford scale 0-2 had significantly lower levels than those who were non-GDM and GDM with Modified Oxford Scale 3-5. Relaxin-2 level was much lower in GDM incontinent pregnant women with MOS 0-2 compared to the other three groups. CONCLUSIONS: Lower relaxin-2 concentration was associated with the presence of pregnancy-specific urinary incontinence, but the combination of GDM, pregnancy-specific urinary incontinence, and lower levels of pelvic floor strength led to lower levels of relaxin-2 compared to the other three groups.

Preventive and therapeutic effects of relaxin on bisphosphonate related osteonecrosis of the jaw: an experimental study in rats / Efeitos preventivos e terapêuticos da relaxina na osteonecrose da mandíbula relacionada a bisfosfonatos: um estudo experimental em ratos

Objective: Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a challenging complication of chronic bisphosphonate (BP) use. The hormone relaxin is able to induce the multistep differentiation process of human osteoclastogenesis, exhibits antifibrotic and anti-inflammatory actions, and promotes vasodilatation, wound healing, and angiogenesis. The present study aimed to evaluate the effects of relaxin in the prevention and management of BRONJ. Material and Methods: Thirty-six male Sprague Dawley rats were randomly divided into four groups. Rats in group 1 (n = 10) received relaxin and BP simultaneously for 12 weeks. Rats in group 2 (n = 10) received injections of BP for 12 weeks, followed by relaxin for another 12 weeks. Rats in group 3 (n = 10) received only BP injections, and those in group 4 (control, n = 6) received only saline. Necrosis and inflammation in the rats' mandibles were evaluated as indicators of BRONJ. Results: Necrosis and inflammation were not detected in group 1 (BP + relaxin). In group 3 (BP only), incidence rates of necrosis and inflammation were 90% and 60%, respectively. Conclusions: Our findings suggest that relaxin may be potently effective in preventing BRONJ and have some benefit in the treatment of existing BRONJ (AU)

Objetivo: A osteonecrose da mandíbula relacionada ao bisfosfonato (BRONJ) é uma desafiadora complicação do uso crônico de bisfosfonato (BP). O hormônio relaxina é capaz de induzir o processo múltiplo de diferenciação da osteoclastogênese humana, exibe ações anti-fibróticas e anti-inflamatórias e promove vasodilatação, cicatrização de feridas e angiogênese. O presente estudo teve como objetivo avaliar os efeitos da relaxina na prevenção e tratamento do BRONJ. Material e Métodos: Trinta e seis ratos Sprague Dawley machos foram divididos aleatoriamente em quatro grupos. Os ratos do grupo 1 (n = 10) receberam relaxina e BP simultaneamente por 12 semanas. Os ratos do grupo 2 (n = 10) receberam injeções de BP por 12 semanas, seguidos de relaxina por mais 12 semanas. Os ratos do grupo 3 (n = 10) receberam apenas injeções de BP e os do grupo 4 (controle, n = 6) receberam apenas solução salina. Necrose e inflamação nas mandíbulas dos ratos foram avaliadas como indicadores de BRONJ. Resultados: Necrose e inflamação não foram detectadas no grupo 1 (BP + relaxina). No grupo 3 (somente BP), as taxas de incidência de necrose e inflamação foram de 90% e 60%, respectivamente. Conclusões: Nossos resultados sugerem que a relaxina pode ser potentemente eficaz na prevenção do BRONJ e ter algum benefício no tratamento do BRONJ existente.(AU)

Crosstalk between FSH and relaxin at the end of the proliferative stage of rat Sertoli cells.

Follicle-stimulating hormone (FSH) stimulates the proliferation of immature Sertoli cells through the activation of PI3K/AKT/mTORC1 and MEK/ERK1/2 pathways. Mature Sertoli cells stop proliferating and respond to FSH by stimulating cAMP production. To gain insight into possible mechanisms involved in this switch as well as the impact of paracrine factors that stimulate cell proliferation, we analyzed the effects of FSH and relaxin on intracellular signaling pathways involved with proliferation and differentiation in Sertoli cells from 15-day-old rats, which are close to the transition between the two stages. FSH stimulated 3H-thymidine incorporation and cyclin D1 expression, changes associated with proliferation. In contrast, FSH inhibited AKT and ERK1/2 phosphorylation, activated cAMP production and induced changes in several cell cycle genes that were compatible with differentiation. Relaxin also stimulated 3H-thymidine incorporation but increased phosphorylation of ERK1/2 and AKT. When both hormones were added simultaneously, relaxin attenuated FSH-mediated inhibition of ERK1/2 and AKT phosphorylation and FSH-mediated activation of cAMP production. FSH but not relaxin increased CREB phosphorylation, and relaxin but not FSH shifted NF-κB expression from the cytoplasm to the nucleus. Relaxin did not inhibit the effects of FSH on inhibin α and Bcl2 expression. We propose that at this time of Sertoli cell development, FSH starts to direct cells to differentiation through activation of cAMP/CREB and inhibition of ERK1/2 and AKT pathways. Relaxin counteracts FSH signaling through the inhibition of cAMP and activation of ERK1/2, AKT and NF-κB, but does not block the differentiation process triggered by FSH.

Chronic Nicotine Exposure Abolishes Maternal Systemic and Renal Adaptations to Pregnancy in Rats.

Pregnancy is characterized by maternal systemic and intrarenal vasodilation, leading to increases in the renal plasma flow (RPF) and glomerular filtration rate (GFR). These responses are mainly mediated by nitric oxide (NO) and relaxin. The impact of cigarette smoking on the maternal adaptations to pregnancy is unclear. Here we evaluated the effects of chronic exposure to nicotine on systemic and intrarenal parameters in virgin (V) and 14-day pregnant (P) Wistar rats. V and P groups received saline or nicotine (6 mg·kg(-1)·day(-1)) respectively, via osmotic minipumps for 28 days, starting 14 days before pregnancy induction. Nicotine induced a 10% increase in blood pressure in the V group and minimized the characteristic pregnancy-induced hypotension. Renal sympathetic nerve activity (rSNA) and baroreflex sensitivity were impaired by nicotine mainly in the P group, indicating that the effect of nicotine on blood pressure was not mediated by nervous system stimulation. Nicotine had no effect on GFR in the V rats but reduced GFR of the P group by 30%. Renal expression of sodium and water transporters was downregulated by nicotine, resulting in increased fractional sodium excretion mainly in the P group, suggesting that nicotine compromised the sodium and water retention required for normal gestation. There was a reduction in the expression of inducible NO synthase (iNOS) in both the kidney tissue and renal artery, as well as in the expression of the relaxin receptor (LGR7). These results clearly show that nicotine induced deleterious effects in both virgin and pregnant animals, and abolished the maternal capacity to adapt to pregnancy.

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.

Driving Human Granulosa-Luteal Cells Recovered From In Vitro Fertilization Cycles Toward the Follicular Phase Phenotype.

Culture systems are available for human granulosa cells (GCs) that perpetuate luteinization. The present study examines the plating density effects and long-term serum-free culture on the in vitro dynamics differentiation of luteinizing human GCs. Cells were cultured in serum-free α-minimum essential medium (α-MEM) or serum-based tissue culture medium (TCM). The time course of GCs morphology and secretion of estradiol (E2), progesterone (P4), and relaxin were analyzed after 48, 96, and 144 hours of culture. Other functional markers as follicle-stimulating hormone/luteinizing hormone receptors and steroidogenic enzymes were investigated at the end of culture. The morphology of an α-MEM cell rather than a TCM cell resembles more closely that seen in vivo. Compared to TCM cultures, α-MEM cells secreted 93.7% and 87.2% more E2 and approximately 7% and 17% of the amount of P4 when cultured at densities of 2 × 10(4) or 4 × 10(4) cells/well, respectively. Relaxin secretion was significantly reduced in α-MEM cultures. α-MEM cells were estrogenic and expressed the CYP19 gene. Levels of CYP17 increased about 8-fold in α-MEM cells above the levels found in TCM cells. Our results reveal new insights into human GCs differentiation in vitro and demonstrate the critical importance of the culture system and cell-plating density on the establishment of estrogenic or progestogenic GC phenotypes.

Evolution of the relaxin/insulin-like gene family in anthropoid primates.

The relaxin/insulin-like gene family includes signaling molecules that perform a variety of physiological roles mostly related to reproduction and neuroendocrine regulation. Several previous studies have focused on the evolutionary history of relaxin genes in anthropoid primates, with particular attention on resolving the duplication history of RLN1 and RLN2 genes, which are found as duplicates only in apes. These studies have revealed that the RLN1 and RLN2 paralogs in apes have a more complex history than their phyletic distribution would suggest. In this regard, alternative scenarios have been proposed to explain the timing of duplication, and the history of gene gain and loss along the organismal tree. In this article, we revisit the question and specifically reconstruct phylogenies based on coding and noncoding sequence in anthropoid primates to readdress the timing of the duplication event giving rise to RLN1 and RLN2 in apes. Results from our phylogenetic analyses based on noncoding sequence revealed that the duplication event that gave rise to the RLN1 and RLN2 occurred in the last common ancestor of catarrhine primates, between ∼ 44.2 and 29.6 Ma, and not in the last common ancestor of apes or anthropoids, as previously suggested. Comparative analyses based on coding and noncoding sequence suggests an event of convergent evolution at the sequence level between co-ortholog genes, the single-copy RLN gene found in New World monkeys and the RLN1 gene of apes, where changes in a fraction of the convergent sites appear to be driven by positive selection.

Cultura de células da granulosa humanas com fenótipo de fase folicular: influência do sistema quimicamente definido na morfologia, ultraestrutura, secreção de esteroides e relaxina / Culture of human granulosa cells with follicular phase phenotype:influence of chemically defined system on morphology, ultrastructure,secretion of steroids and relaxin

Está bem descrito na literatura o padrão de cultivo de células da granulosa (CG) humanas que perpetua a luteinização, simulando a fase lútea do ciclo. Nesse sistema, há redução na secreção de estradiol (E2) e aumento na síntese de progesterona (P4) e relaxina (RLN). Objetivamos padronizar um sistema de cultura livre de soro, com o intuito de reverter o processo de luteinização de CG obtidas em ciclos de fertilização in vitro (FIV), pré-luteinizadas pela gonadotrofina coriônica humana (hCG), para aplicação na maturação in vitro de folículos ovarianos pré-antrais. Foi feito estudo experimental com GC obtidas de 10 mulheres em tratamento de reprodução assistida. As CG foram cultivadas em α-MEM contendo IGF-I, ITS, androstenediona, PVP-40 (meio quimicamente definido) ou TCM-199 contendo FSH/soro. Após 48, 96 e 144 horas, foram avaliados: morfologia das culturas, produção de E2, P4 (Quimioluminescência/Immulite), RLN (Elisa) e ultraestrutura (Microscopia Eletrônica). Os dados foram analisados por Anova e regressão linear com efeitos mistos (SAS versão 9.0). Células cultivadas em α-MEM apresentam alta capacidade estrogênica e padrão de produção hormonal característico da fase folicular, mantendo características morfológicas/ultraestruturais semelhantes a células in vivo. No sistema de cultura padronizado, as CG não completam in vitro o processo de luteinização deflagrado pela hCG, assumindo fenótipo de fase folicular.

It is well described in the literature the granulosa cells (GC) culture pattern that perpetuates human luteinizing simulating the luteal phase of the cycle. In this system, there is a reduction in the secretion of estradiol (E2) and increased synthesis of progesterone (P4) and relaxin (RLN). We aim to standardize a serum-free culture system, in order to reverse the luteinization process of GC obtained in IVF cycles, pre-luteinized by hCG, for use in in vitro maturation of preantral ovarian follicles. An experimental study was conducted with GC obtained from10 women undergoing treatment for assisted reproduction. The GC were cultured in α-MEM containing IGF-I, STI, androstenedione, PVP-40 (chemically defined medium) or TCM-199 containing FSH/serum. After 48, 96 and 144 h were analyzed: culture morphology, concentrations of E2, P4 (Chemioluminescence/Immulite), and RLN (Elisa), and ultrastructure (ElectronMicroscopy). Data were analyzed by Anova and linear mixed-effects regression (SAS version9.0). Cells cultured in α-MEM present estrogenic capacity and pattern of hormone production characteristic of the follicular phase, maintaining morphological/ultrastructural features similar that in vivo cell. In standard culture system, the CG not completes in vitro luteinization process triggered by hCG, assuming follicular phase phenotype.

INSL4 pseudogenes help define the relaxin family repertoire in the common ancestor of placental mammals.

The relaxin/insulin-like (RLN/INSL) gene family comprises a group of signaling molecules that perform physiological roles related mostly to reproduction and neuroendocrine regulation. They are found on three different locations in the mammalian genome, which have been called relaxin family locus (RFL) A, B, and C. Early in placental mammalian evolution, the ancestral proto-RLN gene at the RFLB locus underwent successive rounds of small-scale duplications resulting in variable number of paralogous genes in different placental lineages. Most placental mammals harbor copies of the RLN2 and INSL6 paralogs in the RFLB. However, the origin of an additional paralog, INSL4 (also known as placentin), has been controversial as its phyletic distribution does not converge with its phylogenetic position. In principle, by searching for INSL4 genes in representative species of all major groups of mammals we can gain insights into when the gene originated and better reconstruct its evolutionary history. Here we identified INSL4 pseudogenes in two laurasiatherian, (alpaca and dolphin) and one xenarthran (armadillo) species. Phylogenetic and synteny analyses confirmed that the identified pseudogenes are orthologs of INSL4. According to these results, the proto-RLN gene in the RFLB underwent two successive tandem duplications which gave rise the INSL6 and INSL4 paralogs in the last common ancestor of placental mammals. The INSL4 gene was subsequently inactivated or lost from the genome in all placentals other than catarrhine primates, where its product became functionally relevant. Our results highlight the contribution of relatively old gene duplicates to the gene complement of extant species.

Intracellular signaling pathways involved in the relaxin-induced proliferation of rat Sertoli cells.

Regulation of Sertoli cell number is a key event to determine normal spermatogenesis. We have previously shown that relaxin and its G-protein coupled receptor RXFP1 are expressed in rat Sertoli cells, and that relaxin stimulates Sertoli cell proliferation. This study examined the mechanisms underlying the mitogenic effect of relaxin in a primary culture of Sertoli cells removed from testes of immature rats. Stimulation with exogenous relaxin increased Sertoli cell number and the expression of the proliferating cell nuclear antigen (PCNA), but did not affect the mRNA level of the differentiation markers cadherins 1 and 2. Relaxin-induced Sertoli cell proliferation was blocked by inhibition of MEK/ERK1/2 or PI3K/AKT pathways, but not by inhibition of PKC or EGFR activity. Relaxin induced a rapid and transient activation of ERK1/2 phosphorylation, which was MEK and SRC-dependent, and involved upstream activation of G(i). AKT activation could be detected 5 min after relaxin stimulation, and was still detected after 24h of stimulation with relaxin. Relaxin-induced AKT phosphorylation was G(i)- but not PKA-dependent, and it was blocked by both PI3K and MEK inhibitors. In conclusion, the mitogenic effect of relaxin in Sertoli cell involves coupling to G(i) and activation of both MEK/ERK1/2 and PI3K/AKT pathways.

Gene turnover and differential retention in the relaxin/insulin-like gene family in primates.

The relaxin/insulin-like gene family is related to the insulin gene family, and includes two separate types of peptides: relaxins (RLNs) and insulin-like peptides (INSLs) that perform a variety of physiological roles including testicular descent, growth and differentiation of the mammary glands, trophoblast development, and cell differentiation. In vertebrates, these genes are found on three separate genomic loci, and in mammals, variation in the number and nature of genes in this family is mostly restricted to the Relaxin Family Locus B. For example, this locus contains a single copy of RLN in platypus and opossum, whereas it contains copies of the INSL6, INSL4, RLN2 and RLN1 genes in human and chimp. The main objective of this research is to characterize changes in the size and membership composition of the RLN/INSL gene family in primates, reconstruct the history of the RLN/INSL genes of primates, and test competing evolutionary scenarios regarding the origin of INSL4 and of the duplicated copies of the RLN gene of apes. Our results show that the relaxin/INSL-like gene family of primates has had a more dynamic evolutionary history than previously thought, including several examples of gene duplications and losses which are consistent with the predictions of the birth-and-death model of gene family evolution. In particular, we found that the differential retention of relatively old paralogs played a key role in shaping the gene complement of this family in primates. Two examples of this phenomenon are the origin of the INSL4 gene of catarrhines (the group that includes Old World monkeys and apes), and of the duplicate RLN1 and RLN2 paralogs of apes. In the case of INSL4, comparative genomics and phylogenetic analyses indicate that the origin of this gene, which was thought to represent a catarrhine-specific evolutionary innovation, is as old as the split between carnivores and primates, which took place approximately 97 million years ago. In addition, in the case of the RLN1 and RLN2 genes of apes our phylogenetic trees and topology tests indicate that the duplication that gave rise to these two genes maps to the last common ancestor of anthropoid primates. All these genomic changes in gene complement, which are particularly prevalent among anthropoid primates, might be linked to the many physiological and anatomical changes found in this group. Given the various roles of members of the RLN/INSL-like gene family in reproductive biology, it might be that changes in this gene family are associated to changes in reproductive traits.

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 in the male reproductive system

xin (RLN) belong s to a family of hormones structurally related to insulin and presents a broad spectrum of actions. H umans have three forms of RLN , encoded by three different genes ( RLN1 , RLN2 and RLN3 ) , but nonprimate vertebrate s have only two forms of relaxin (RLN1 and RLN3) . RLN1 of these animals is encoded by Rln1 , orthologous to the h uman RLN 2 gene , and both genes , Rln1 and human RLN2 , encode the major form of relaxin found in the male reproductive system . In the reproductive tract of human male s , RLN is mainly produced by the prostate and secreted into the seminal fluid, where it seems to play a role in sperm function. R LN may also play a role in prostate cancer progression. A lack of RLN in animal models impairs male fertili ty , and RLN knockout mice display decreased sperm maturation . T he precise role of RLN in the male reproductive system , however , is still far from clear. RLN actio n is due to its interaction with the G - protein coupled receptor RXFP1. Studies from our labora tory have shown that RLN and RXFP1 are e x pressed in rat Sertoli cells, and e x ogenous RLN stimulates Sertoli cell proliferation. RLN receptors can also be detected in rat germ cells at different stages of development, suggesting that RLN may play a direct r ole in spermatogenesis. The distribution of RLN/RXFP1 , however , appears to be species - dependent, because i n the boar testis RLN production seems restricted to the Leydig cells, whereas RXFP1 is found in Leydig, Sertoli and germ cells. The co - expression of RLN and RXFP1 in several regions of the male reproductive system suggest s that the peptide may act in an autocrine/paracrine fashion.(AU)

Relaxin in the male reproductive system

xin (RLN) belong s to a family of hormones structurally related to insulin and presents a broad spectrum of actions. H umans have three forms of RLN , encoded by three different genes ( RLN1 , RLN2 and RLN3 ) , but nonprimate vertebrate s have only two forms of relaxin (RLN1 and RLN3) . RLN1 of these animals is encoded by Rln1 , orthologous to the h uman RLN 2 gene , and both genes , Rln1 and human RLN2 , encode the major form of relaxin found in the male reproductive system . In the reproductive tract of human male s , RLN is mainly produced by the prostate and secreted into the seminal fluid, where it seems to play a role in sperm function. R LN may also play a role in prostate cancer progression. A lack of RLN in animal models impairs male fertili ty , and RLN knockout mice display decreased sperm maturation . T he precise role of RLN in the male reproductive system , however , is still far from clear. RLN actio n is due to its interaction with the G - protein coupled receptor RXFP1. Studies from our labora tory have shown that RLN and RXFP1 are e x pressed in rat Sertoli cells, and e x ogenous RLN stimulates Sertoli cell proliferation. RLN receptors can also be detected in rat germ cells at different stages of development, suggesting that RLN may play a direct r ole in spermatogenesis. The distribution of RLN/RXFP1 , however , appears to be species - dependent, because i n the boar testis RLN production seems restricted to the Leydig cells, whereas RXFP1 is found in Leydig, Sertoli and germ cells. The co - expression of RLN and RXFP1 in several regions of the male reproductive system suggest s that the peptide may act in an autocrine/paracrine fashion.

Métodos farmacológicos de indução do trabalho de parto: qual o melhor? / Pharmacological methods for induction of labor: what's the best?

A indução do parto consiste em estimular artificialmente as contrações uterinas coordenadas e efetivas antes de seu início espontâneo, levando ao desencadeamento do trabalho de parto em mulheres que ultrapassaram a 22ª semana de gravidez. A antecipação do parto pode ser necessária em diversas situações obstétricas, como gestação prolongada, diabetes, ruptura prematura das membranas e pré-eclâmpsia. Estima-se que mais de 15% de todas as gestantes apresentem alguma indicação de indução do parto. Vários métodos de indução do parto são propostos, tanto naturais como artificiais e, dentre estes, os métodos farmacológicos merecem especial destaque. Realizou-se uma revisão da literatura baseada nos melhores níveis de evidências e considerando os graus de recomendação. De acordo com a literatura, a utilização de estrogênio, propranolol, relaxina, mefepristone e hialuronidade não deve ser estimulada por não existirem evidências suficientes para a sua recomendação. O seu uso, portanto, deve ser limitado a protocolos de pesquisas. Ocitocina é um método de indução efetivo que pode ser usado em pacientes com ruptura das membranas amnióticas. Prostaglandinas (PG) e misoprostol (um éster sintético da PGE1) são efetivos para a indução do parto independentemente da integridade das membranas. Prostaglandinas devem ser administradas preferencialmente por via vaginal. Habitualmente, o misoprostol é preferido devido a questões práticas, como o baixo custo e a facilidade de administração e estocagem. Doses baixas de misoprostol devem ser utilizadas e a atualmente recomendada é de 25 g a cada 4 ou 6 horas. Tanto a via oral como a via vaginal podem ser utilizadas.

Induction of labor consists of stimulation of effective and coordinated uterine contractions before their spontaneous onset for the purpose of bring on labor in women who have surpassed the 22nd week of pregnancy. In several obstetrical situations, such as prolonged pregnancy, diabetes, premature rupture of membranes and preeclampsia, anticipation of labor and delivery may be necessary. It is estimated that more than 15% of all pregnant women eventually present any indication for induction of labor. Several natural and artificial methods for induction are proposed. Among them, pharmacological methods are the most relevant. A literature review was carried out based on the highest levels of evidence and on the grade of recommendations. According to the literature, the use of estrogens, relaxin, mifepristone and hyaluronidade should not be stimulated because there are not enough evidences for their recommendation, so their utilization should be limited to research protocols. Oxytocin is an effective method for induction of labor that may be used in patients with ruptured membranes. On the other hand, prostaglandins and misoprostol (a prostaglandin E1 analog) are effective for induction of labor independently on the membrane integrity. Vaginal administration should be preferred for prostaglandins. Misoprostol is habitually preferred due to practical questions, such as low cost and facility for storage and administration. Low doses of misoprostol should be used, and the currently recommended dose is 25 g every four or six hours. Both vaginal and oral routes of administration can be used.

Locally produced relaxin may affect testis and vas deferens function in rats.

We have previously shown that the rat testis and vas deferens contain high levels of the relaxin receptor, RXFP1. The present study was undertaken to determine the expression of relaxin in these tissues, and the effect of exogenous relaxin on Sertoli cell proliferation and on the mRNA levels of some proteins that may contribute to epithelial secretion and tissue reorganization in the vas deferens. Relaxin mRNA levels in testis and vas deferens were much lower than in the prostate. Sertoli cells seem to be an important source of relaxin mRNA in testis. Relaxin immunoreactivity was detected in the seminiferous epithelium but not in the interstitial compartment. The relaxin precursor was expressed in the vas deferens, and relaxin immunoreactivity was detected in apical cells of the vas deferens. Castration, but not treatment with the anti-estrogen ICI 182,780, dramatically reduced relaxin mRNA levels in the prostate and vas deferens, and this effect was prevented by testosterone. Rxfp1 mRNA levels in the vas deferens and prostate were not affected by castration or treatment with ICI 182,780. Exogenous relaxin increased the incorporation of (3)H-thymidine in cultured Sertoli cells, and treatment of the vas deferens with 100 ng/ml relaxin increased the mRNA levels for the cystic fibrosis chloride channel (cystic fibrosis transmembrane regulator) about three times, and doubled mRNA levels for the inducible form of nitric oxide synthase and metalloproteinase 7. These results suggest that locally produced relaxin acts as an autocrine or paracrine agent in the testis and vas deferens to affect spermatogenesis and seminal fluid composition.

Relaxina en suero de mujeres embarazadas, a nivel del mar y en la altura / Relaxin in serum of pregnant women, at sea level as high altitude

Se estudió los niveles en suero de relaxina en el 3°, 4° y 5° mes de gestación, así como los niveles en suero de mujeres posparto y en recién nacidos dentro de las primeras 24 horas, tanto a nivel del mar como de altura. El estudio se realizó en 24 mujeres embarazadas (18 a 32 años), en el tercer, cuarto o quinto mes de gestación natural de Cerro de Pasco, (4200 msnm) y 20 mujeres embarazadas de la misma edad y estado gestacional nativas del nivel del mar (Lima, 150 m). Asimismo, se estudiaron los niveles en suero de relaxina en 09 mujeres y sus 09 recién nacidos dentro de las siguientes 24 horas después del parto para el grupo de altura, y 20 mujeres con sus 20 recién nacidos dentro de las siguientes 24 horas, a nivel del mar. La relaxina fue medida por RIA (Radioinmunoanálisis), con el uso de kits de los Laboratorios Immunodiagnostik (Alemania) marcada con 1-125 y con el uso del equipo Contador de centelleo gamma del laboratorio del Instituto de Investigaciones Clínicas de la Universidad Nacional Mayor de San Marcos. Los resultados nos indican que no hay diferencia significativa entre los niveles hormonales de relaxina en el 3°, 4° Y 5° mes de gestación en las mujeres del nivel del mar y altura; existiendo una gradual disminución de los niveles en suero del 3° al 5° mes tanto a nivel del mar como en la altura. En las mujeres post parto, los niveles de relaxina en suero fueron significativamente menores en las mujeres de altura con respecto a las del nivel del mar. El radio relaxina madre/relaxina hijo es igual en la altura como en el nivel del mar.

We studied serum levels of relaxin in the 3th, 4th and 5th month of pregnancy, women postpartum and newborns within the first 24 hours both at sea level as high altitude. The study was performed in 24 pregnant women (18 to 32 years) in the third, fourth or fifth month of gestation in (Cerro de Pasco, 4300 m) and 20 pregnant women of the same gestational age at sea level (Lima, 150 m). Also, we studied the serum levels of relaxin in 09 women and 09 newborns within 24 hours postpartum at the high altítude group and 20 women and 20 newborns within 24 hours at sea level. Relaxin was measured by RIA (Radioimmunoassay), using kits Immunodiagnostic Laboratories (Germany) labeled with 1-125 and using the gamma scintillation counter computer in lab of the Institute of Clinical Research at the National University of San Marcos. The results indicate no significant difference between the hormone levels of relaxin in the 3 th, 4 th and 5 th month of pregnancy in women at high altitude and sea level, showing a gradual decrease in serum levels of 3 th to 5 th month both at sea level and at altitude. In postpartum women, levels of relaxin in serum were significantly lower in women at high altitude with regard to sea level. The radio mother relaxin / child relaxin is the same at high altitude as the sea level.