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Testicular descent is a unique physiological adaptation found in therian mammals allowing optimal spermatogenesis below core body temperature. Recent studies show that INSL3, produced by Leydig cells, and its receptor LGR8 (RXFP2) are essential for mediating the transabdominal phase of testicular descent during early development. However, the origin and genetic basis for this physiological adaptation is not clear. Using syntenic mapping and the functional characterization of contemporary and resurrected relaxin family hormones, we show that derivation of INSL3-mediated testicular descent involved the duplication of an ancestral RLN3-like gene that encodes an indiscriminate ligand for LGR7 (RXFP1) and LGR8. This event was followed by acquisition of the LGR7-selective characteristics by a daughter gene (RLN3) prior to the evolution of the common ancestor of monotremes, marsupials, and placentals. A subsequent mutation of the other daughter gene (INSL3) occurred before the emergence of therian mammals, which then led to the derivation of the reciprocal LGR8-specific characteristics of INSL3. The stepwise evolution of these independent signaling pathways through gene duplication and subsequent divergence is consistent with Darwinian theory of selection and adaptation, and the temporal proximity suggests an association between these genetic events and the concurrent evolution of testicular descent in ancestral therian mammals.
Jae-Il Park, Jenia Semyonov, Chia Lin Chang, Wei Yi, Wesley Warren, and Sheau Yu Teddy Hsu. Genome Res. 2008 June; 18(6): 974–985.
The relaxin peptides are a family of hormones that share a structural fold characterized by two chains, A and B, that are cross-braced by three disulfide bonds. Relaxins signal through two different classes of G-protein-coupled receptors (GPCRs), leucine-rich repeat-containing GPCRs LGR7 and LGR8 together with GPCR135 and GPCR142, now referred to as the relaxin family peptide (RXFP) receptors 1-4, respectively. Although key binding residues have been identified in the B-chain of the relaxin peptides, the role of the A-chain in their activity is currently unknown. A recent study showed that INSL3 can be truncated at the N terminus of its A-chain by up to 9 residues without affecting the binding affinity to its receptor RXFP2 while becoming a high affinity antagonist. This suggests that the N terminus of the INSL3 A-chain contains residues essential for RXFP2 activation. In this study, we have synthesized A-chain truncated human relaxin-2 and -3 (H2 and H3) relaxin peptides, characterized their structure by both CD and NMR spectroscopy, and tested their binding and cAMP activities on RXFP1, RXFP2, and RXFP3. In stark contrast to INSL3, A-chain-truncated H2 relaxin peptides lost RXFP1 and RXFP2 binding affinity and concurrently cAMP-stimulatory activity. H3 relaxin A-chain-truncated peptides displayed similar properties on RXFP1, highlighting a similar binding mechanism for H2 and H3 relaxin. In contrast, A-chain-truncated H3 relaxin peptides showed identical activity on RXFP3, highlighting that the B-chain is the sole determinant of the H3 relaxin-RXFP3 interaction. Our results provide new insights into the action of relaxins and demonstrate that the role of the A-chain for relaxin activity is both peptide- and receptor-dependent.
Hossain MA, Rosengren KJ, Haugaard-Jönsson LM, Zhang S, Layfield S, Ferraro T, Daly NL, Tregear GW, Wade JD, Bathgate RA.
J Biol Chem. 2008 Jun 20;283(25):17287-97. Epub 2008 Apr 22.
Serum samples for immunoreactive INSL3 concentration analysis were assayed in duplicate by using a commercial RIA (Phoenix Pharmaceutical, Belmont, CA). The assay is based on a polyclonal rabbit antiserum raised against full-length human INSL3 and on 125I-labeled INSL3 as the tracer. The assay performance was checked before analysis by verifying specificity, sensitivity, precision, and accuracy. To detect the specificity, a cross-reactivity test was conducted via a binding assay with decreasing concentrations of human insulin, INSL4, INSL5, INSL6, and INSL7. The sensitivity was determined as the lower detection limit (28). The precision was checked by using replicates of a serum pool control to measure intraassay and interassay variability. Finally, to detect the accuracy of the method, linearity of dilution was determined by serially diluting serum pool control, and recovery was evaluated by measuring pooled serum samples spiked with increasing standard human INSL3 concentrations before analysis in the RIA. Cross-reactivity with human insulin, INSL4, INSL5, INSL6, and INSL7, used to evaluate the specificity of the INSL3 assay, was 0%. The sensitivity determined as the lower detection limit was 1.8 pg/ml. The intraassay and interassay coefficients of variation were 3.1 and 6.8%, respectively. Suitability of the assay to measure INSL3 accurately was demonstrated by results of linearity and recovery (slope of 1; mean of recovery of 104%), which demonstrated the absence of bias.
Alessandra Gambineri, Laura Patton, Rosaria De Iasio, Federica Palladoro, Uberto Pagotto and Renato Pasquali .The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 6 2066-2073
The administration of testosterone plus a progestogen functions as a male contraceptive by inhibiting the release of pituitary gonadotropins. After 3 to 4 months of treatment, most men are azoospermic or severely oligospermic ( 1 million sperm/mL). However, 10% to 20% of men have persistent sperm production despite profound gonadotropin suppression. Since insulin-like factor 3 (INSL3) has been shown to prevent germ cell apoptosis in mice, we hypothesized that INSL3 might be higher in men with persistent spermatogenesis during treatment with male hormonal contraceptives. In a retrospective analysis, we measured serum INSL3 in 107 men from 3 recent male hormonal contraceptive studies and determined the relationship between suppression of spermatogenesis and serum INSL3. At the end of treatment 63 men (59%) were azoospermic and 44 men (41%) had detectable sperm in their ejaculates. Baseline INSL3 did not predict azoospermia; however, end of treatment serum INSL3 was significantly higher in nonazoospermic men compared with those with azoospermia (median [interquartile range]: 95 [73–127] pg/mL vs 80 [67–101] pg/mL; P = .03). Furthermore, serum INSL3 was positively correlated with sperm concentration (r = .25; P = .009) at the end of treatment and was significantly associated with nonazoospermia by multivariate logistic regression (P = .03). After 6 months of treatment with a hormonal male contraceptive regimen, higher serum INSL3 concentrations were associated with persistent sperm production. INSL3 may play a role in preventing complete suppression of spermatogenesis in some men on hormonal contraceptive regimens. This finding suggests that INSL3 may be a potential target for male contraceptive development.
Serum insulin-like factor 3 at the end of treatment by study and combined in subjects with azoospermia (A shaded) compared with those with nonazoospermia (A top) and in those with severe oligospermia (SO shaded) compared with those with nonsevere oligospermia (B bottom) by study and combined. Boxes represent median and interquartile ranges, with outliers depicted as circles. P for the comparisons between groups is included above the line above the box and whiskers plot.
Serum INSL3 was measured by a radioimmunoassay (RIA) (Phoenix Pharmaceuticals Inc, Belmont, Calif). The normal range was 291 to 1132 pg/mL. The lower limit of detection was 16 pg/mL; the intraassay coefficients of variation were 7.6%, 5.2%, and 5.7% and the interassay coefficients of variation were 30%, 13%, and 8.1% for low, mid, and high pools, respectively.
JOHN K. AMORY et al. Journal of Andrology, Vol. 28, No. 4, July/August 2007
Ablation of LGR7-activation activity of AncRFLC and restoration of lost LGR7-activation activity in human INSL3 by point mutation. (A–C) LGR7 (RXFP1)- and LGR8 (RXFP2)-activation activity of AncRFLC RB12H, zRFLC1 RB12H, and zRFLC2 RB12H peptides (A), opossum INSL3 peptide (oINSL3) (B), and INSL3 HB12R and INSL3 HB12A peptides (C). Mean ± SEM, N = 4.
Jae-Il Park, Sheau Yu Teddy Hsu, et al. Genome Res. 2008 June; 18(6): 974–985.
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