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B. Bagna, C. Schwabe and L. L. Anderson

Summary. Purified pig relaxin (3000 U/mg) was injected i.m. into pregnant Holstein dairy heifers on Day 276 or 277 to determine its effect on parturition and sequential measurements of the pelvic area, cervical dilatation, and peripheral blood-plasma concentrations of progesterone and relaxin. Treatments included phosphate-buffer saline (2ml, Group C, N = 7), relaxin once (1 mg, Group 1R, N = 7), and twice (2mg, 12 h apart; Group 2R, N = 7). Intervals (mean ± s.e.) between the first injection of relaxin or PBS and calving were 64 ± 17, 80 ± 19 and 125 ± 34 h for Groups 2R, 1R and C, respectively. The calving intervals were reduced in Groups 2R (P < 0·01) and 1R (P < 0·05) compared with Group C. The incidence of dystocia was 29% (2 of 7) in Group 2R and 43% (3 of 7) in Group 1R compared with 57% (4 of 7) in Group C (P < 0·01). Body weights and ratios of males to females of the calves were similar (P > 0·05) between groups. Progesterone plasma concentrations decreased (P < 0·01) earlier in Groups IR and 2R compared with Group C, and this acute decrease began within 6 h of treatment. At 24 h after relaxin or PBS injection, progesterone concentrations were 2·7 ± 1·1 ng/ml for Group 2R, 3·5 ± 0·9 ng/ml for Group 1R, and 6·0 ± 0·1 ng/ml for Group C. Relaxin reached peak blood-plasma levels of 19 ± 2·2 ng/ml 1 h after injection of relaxin, but remained unchanged, 0·3 ± 0·01 ng/ml, in Group C. Pelvic area was increased 26%, 22% and 14% and cervical dilatation was increased 109%, 76% and 53% 48 h after injection in Groups 2R, 1R and C, respectively, but these responses were similar among groups at the time of parturition. We conclude that two i.m. injections of relaxin facilitated earlier calving, acutely decreased progesterone secretion, increased cervical dilatation and pelvic area expansion, and decreased the incidence of dystocia in dairy heifers.

Keywords: relaxin; parturition; progesterone; cervix; pelvic area; dystocia; dairy cattle

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O. S. Gazal, Y. Li, C. Schwabe and L. L. Anderson

Pregnant ewes were injected with either the antiprogesterone, RU 486 (4 mg kg−1 body weight, i.m.; n = 5), 3000 iu relaxin (i.m.; n = 9), or diluent (n = 8) at 12:00 h on days 144 and 145, to determine its effect on progesterone and relaxin secretion, and on induction of lambing. RU 486 induced earlier lambing (P < 0.01) compared with diluent treatment, but relaxin treatment did not significantly reduce the interval to parturition. Mean injection–lambing intervals were 31 ± 2, 109 ± 23 and 121 ± 27 h for the RU 486, relaxin and diluent groups, respectively. There was no incidence of difficult birth (dystocia); all lambs were vigorous at birth; and placenta delivery was rapid (within 207 min) with RU 486 and relaxin treatments compared with diluent treated controls. Plasma progesterone concentrations averaged 11 ng ml−1 during the pretreatment period for all animals. RU 486 had a biphasic effect on progesterone concentrations, causing an initial increase (P < 0.05) within 2 h, and then an abrupt drop (P < 0.01) to 6 ng ml−1 by 18:00 h on day 145. Progesterone concentrations remained consistently lower (P < 0.05) in relaxin-treated ewes than in diluent-treated controls from days 144 to 147 and then began a steady decrease to 4 ng ml−1 on the day of parturition (days 149 and 150) in both groups. Immunoreactive relaxin concentrations in control ewes increased (P < 0.05) from 0.6 ng ml−1 to a peak of 3.9 ng ml−1 on day 146, but they were low (0.8 ng ml−1) at the time of parturition (day 150). RU 486 treatment abruptly increased (P < 0.05) circulating relaxin concentration, but the amplitude of this antepartum surge was greatly attenuated compared with that of diluent treated controls. Peak RU 486 concentrations in plasma were 7 and 9 ng ml−1 within 2 h after first and second i.m. injection of the hormone, respectively, and stabilized at 4 ng ml−1 at the time of induced lambing (day 145). The results reveal that an antepartum relaxin surge occurs in sheep 4 days before normal parturition (day 150), but that RU 486 greatly attenuates the relaxin surge and abruptly decreases circulating progesterone concentration with an induced parturition (day 145). The results indicate that RU 486 can precisely control the time of parturition in sheep in late pregnancy without detrimental effects of dystocia, retention of placenta or delayed postpartum fertility.

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FR Boockfor, G Fullbright, EE Bullesbach and C Schwabe

Deletion of the relaxin-like factor (RLF) gene in mice causes retention of testicles and infertility. The development of a synthetic RLF has made it possible to investigate the events that connect the genomic event and the basic biological responses that cause gonadal positioning. Anti-RLF antibodies were raised against synthetic RLF, allowing determination of RLF concentrations during the critical period, testing for RLF receptors on the gubernaculum and exploration of the temporal relationship between receptor display and migration of the testes in developing rats. In male rat pups, serum RLF concentrations were high at day 2 before parturition (2.4 ng ml(-1)) and decreased sharply just before parturition. Thereafter, males and females had the same low serum concentrations until RLF concen-trations began to increase in males only, starting at day 10 after parturition and continuing until adult RLF concentrations (0.6 ng ml(-1)) were reached on day 39 after parturition. The testicles are descending into the scrotum during this phase of increasing RLF concentrations and are descended fully by day 19-21 after parturition, before adult hormone concentrations are established. The high prenatal serum RLF concentration coincides with high expression of RLF receptors in the gubernaculum tissue. Competitive binding of RLF per mg of membrane protein prepared from rat gubernacula at various developmental stages showed no increase in receptor density as sexual maturity was reached. Gubernaculum cells in primary culture showed an increased uptake of 5-bromo-2'-deoxyuridine in the presence of RLF compared with controls. These studies demonstrate that the synthetic RLF is biologically active and indicate that the cryptorchid phenotype INSL3(-/-) is a direct consequence of defective gubernaculum growth, caused by the absence of RLF during early phases of development.

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H. H. Juang, A. I. Musah, C. Schwabe, J. J. Ford and L. L. Anderson

Seventy-two Yorkshire boars were used in five experiments to evaluate the temporal changes in relaxin concentrations in peripheral blood plasma during prepubertal development, copulation, after castration and after treatment with hCG. High concentrations of relaxin (484 ± 27 pg ml−1) were detected at 11 weeks of age but there was no positive correlation with testicular development. Relaxin concentrations fluctuated in mature boars but the results do not suggest a diurnal rhythm, although there is the possibility of pulsatile secretion. A decrease (P < 0.05) in circulating relaxin was observed before and immediately after copulation. Castration of boars at 90, 115, 160 and 200 days of age did not significantly decrease relaxin concentrations within 48 h. Administration of hCG significantly depressed relaxin secretion at 90 days of age but not at 160 days of age. These studies suggest a non-gonadal source of boar relaxin that is not correlated with testicular growth or testosterone concentrations, is modulated by copulation and by hCG but only at specific stages of development.

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F P Yuan, X Li, J Lin, C Schwabe, E E Büllesbach, C V Rao and Z M Lei

LH receptor knockout (LhrKO) male mice exhibit a bilateral cryptorchidism resulting from a developmental defect in the gubernaculum during the inguinoscrotal phase of testis descent, which is corrected by testosterone replacement therapy (TRT). In vivo and in vitro experiments were conducted to investigate the roles of the androgen receptor (AR) and RXFP2 signals in regulation of gubernacular development in LhrKO animals. This study demonstrated that AR and RXFP2 proteins were expressed in the gubernaculum during the entire postnatal period. TRT normalized gubernacular RXFP2 protein levels inLhrKO mice. Organ and primary cell cultures of gubernacula showed that 5α-dihydrotestosterone (DHT) upregulated the expression of Rxfp2 which was abolished by the addition of an AR antagonist, flutamide. A single s.c. testosterone injection also led to a significant increase in Rxfp2 mRNA levels in a time-dependent fashion in LhrKO animals. DHT, natural and synthetic insulin-like peptide 3 (INSL3), or relaxin alone did not affect proliferation of gubernacular mesenchymal cells, while co-treatments of DHT with either INSL3 or relaxin resulted in an increase in cell proliferation, and they also enhanced the mesenchymal cell differentiation toward the myogenic pathway, which included a decrease in a mesenchymal cell marker, CD44 and the expression of troponin. These effects were attenuated by the addition of flutamide, siRNA-mediated Rxfp2 knockdown, or by an INSL3 antagonist. Co-administration of an INSL3 antagonist curtailed TRT-induced inguinoscrotal testis descent in LhrKO mice. Our findings indicate that the RXFP2 signaling pathway plays an important role in mediating androgen action to stimulate gubernaculum development during inguinoscrotal testis descent.