A patch-clamp study of Ca²⁺ currents and spectrofluorometric detection of the intracellular Ca²⁺ concentration [Ca²⁺]_i was performed on porcine myometrial cells that had been isolated by collagenase. Isolated myometrial cells were the typical long cylinder shape. The main length and diameter of myometrial cells were 505 ± 20 and 11± 0.5 μm (n = 40), respectively, in the prepartum period and 265 ± 22 and 7 ± 0.4 μm (n = 40), respectively, in the luteal phase. Immunocytochemistry with an antibody against desmin stained 90% of the cells positively, and about 95% of the cells excluded Trypan blue dye. The basal [Ca²⁺]_i of myometrial cells in the luteal phase and the prepartum period was 119 ± 12 (n = 30) and 154 ± 31 nmol l⁻¹ (n = 48), respectively. In prepartum myometrial cells, oxytocin (10⁻⁷ mol l⁻¹) and carbachol (10⁻⁴ mol l⁻¹) increased [Ca²⁺]_i in a biphasic pattern, with a sharp peak followed by a plateau. In cells in the luteal phase, adrenaline (10⁻⁷ mol l⁻¹) plus propranolol (10⁻⁶ mol l⁻¹) produced a biphasic increase in [Ca²⁺]_i. However, in the absence of propranolol, the increase in [Ca²⁺]_i by adrenaline was small. Prostaglandin F_2α (10⁻⁶ mol l⁻¹) induced a monophasic increase in the [Ca²⁺]_i in cells in the luteal phase. By depolarizing the cells from −30 to +50 mV from a holding potential of −50 mV, Ca²⁺ currents were evoked with a threshold at −20 mV, reaching a maximum between 10 and 30 mV. BayK8644 (10⁻⁷ mol l⁻¹), an L-type Ca²⁺ channel agonist, and oxytocin (10⁻⁷ mol l⁻¹) enhanced Ca²⁺ currents by 166 ± 64% and 41 ± 10%, respectively, in prepartum cells. These results suggest that freshly dispersed porcine myometrial cells contain an intact membrane and possess functional voltage-dependent Ca²⁺ channels and receptors for major physiological regulators. Thus, porcine myometrial cells provide a useful model for the study of excitation–contraction coupling and the influence of physiological regulators in the myometrium.

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.

Endothelin, which has potent vasoconstrictor and mitogenic actions, was measured by radioimmunoassay in tissue extracts of sheep endometrium and myometrium and was found to be present in similar amounts in both tissues during the oestrous cycle and in increasing amounts during the first 20 days of pregnancy 250–630 pg g⁻¹ wet weight). Immunoreactive endothelin extracted from endometrium eluted at the same position as standard endothelin-1 on reverse-phase HPLC. Immunohistochemical techniques demonstrated that during the oestrous cycle endothelin immunoreactivity was very low in caruncular and intercaruncular stroma, luminal epithelium, outer and inner glandular epithelium, myometrium and blood vessels until after day 12 (oestrus: day 0). Staining increased in all but the inner glands to day 16 and the most intense staining was found in intercaruncular luminal epithelium and outer glands and in myometrium, although endothelin in tissue extracts did not change over this period. During early pregnancy (days 4–20), staining in intercaruncular areas and in myometrium increased slightly from day 4 to day 12 to a maximum which was maintained from day 15 to day 20. Intensity of staining in caruncles increased only from day 15, particularly in the epithelium. Immunoreactive endothelin was also present in the trophoblast cells of the embryo on day 20 of pregnancy. Strong endothelin immunostaining was observed in uteri from ovariectomized ewes, particularly in epithelial cells and in blood vessels. The intensity of immunostaining in epithelium and stroma was increased slightly by oestradiol and decreased slightly by progesterone treatment, whereas treatment with oestradiol plus progesterone reduced staining intensity of endothelin in all types of tissue. Results of this study therefore demonstrate that immunoreactive endothelin-1 is present in the ovine uterus during the oestrous cycle and in increasing amounts during the first 20 days of pregnancy and is localized in most types of cell. Endothelin-1 is regulated by ovarian steroids and pregnancy-related factors and may play an important role in the regulation of uterine blood flow, in uterine development and the establishment of pregnancy.

Pig seminal proteins PSP-I and PSP-II are major protein components of boars' ejaculate and are present as heterodimers (PSP-dimer) in seminal plasma. These proteins were examined for their ability to modulate pig lymphocyte activity in vitro in mitogen-induced lymphocyte proliferation assays and in one-way mixed lymphocyte reactions. Pig lymphocytes were cultured with phytohaemagglutinin, concanavalin A, or pokeweed mitogen (PWM) in the presence or absence of pig seminal proteins and the amount of cellular [³H]thymidine was used as an indication of proliferation. In the absence of mitogens, none of the three pig seminal proteins affected lymphocyte proliferation suggesting that these proteins are not antigenic or mitogenic. PSP-dimer enhanced lymphocyte proliferation induced by PWM (156–227%, P < 0.05) in a concentration-dependent manner, but had no effect on phytohaemagglutinin- or concanavalin A-induced proliferation. PSP-I enhanced (127–185%, P < 0.05) phytohaemagglutinin-induced proliferation. PSP-II augmented (130–240%, P < 0.05) lymphocyte proliferation induced by concanavalin A and PWM. Lymphocytes from gilts were significantly more responsive to concanavalin A- and PWM-induced lymphocyte proliferation in the presence of PSP-I compared with boars (concanavalin A: gilts 131%, boars 91%; PWM: gilts 188%, boars 134%; P < 0.05). In the mixed lymphocyte reaction, pretreating stimulating cells with increasing concentrations of PSP-I or PSP-II elicited a 400% concentration-dependent increase (P <0.01) in lymphocyte proliferation. The abundance of pig seminal proteins in boar seminal plasma, their ability to enhance lymphocyte proliferation, and their previously reported ability to bind to lymphocytes suggest that these proteins are immunostimulatory and supports the hypothesis that they modulate uterine immune activity to ensure reproductive success.

Summary. Daily afternoon injections of 25 μg melatonin for 12 weeks had no effect on testicular weights of male rats kept in long photoperiod (14L:10D); similarly, exposure of rats to short photoperiod (2L:22D) had no effect on gonadal weight. However, rats maintained in a long or short photoperiod and implanted every 2 weeks with a 15 mm Silastic pellet containing testosterone showed a significant reduction in testicular weight; this effect was more pronounced in rats exposed to a short photoperiod. Melatonin injections in testosterone-treated rats in a long photoperiod exacerbated the inhibitory effects of testosterone alone. Subcutaneous 2-weekly implants of a beeswax pellet containing 1 mg melatonin reversed the effects of the melatonin injections on relative testicular weights but not those due to short photoperiod exposure.

Testosterone implants significantly reduced pituitary LH values in long and short photoperiod-exposed animals, more particularly in those exposed to short photoperiod. Melatonin injections alone or in combination with melatonin pellets did not further exaggerate the depression in pituitary LH due to testosterone alone in long photoperiod-exposed animals; similarly melatonin pellets did not reverse the depression in pituitary LH observed. No significant differences in plasma prolactin concentrations or in thyroxine concentrations or free thyroxine index were observed after any combination of treatments.

We therefore suggest that the effects observed with short photoperiod may be due to melatonin.

Keywords: melatonin, testosterone pellets, photoperiod, LH, prolactin, T₄, T₃

Immunoreactive β-endorphin (ir-BEND) and GnRH (ir-GnRH) have been identified in the pig uterus. The study reported here examined (i) possible biochemical differences between ir-BEND and ir-GnRH present in pig uterine fluids and standard synthetic peptides, and (ii) the secretory profiles of uterine ir-BEND and ir-GnRH during the oestrous cycle and early pregnancy for two breeds of pig, Large White and the highly prolific Chinese Meishan. Reverse phase (RP)-HPLC analysis of concentrated uterine fluids indicated that the majority of ir-BEND eluted with a hydrophobicity similar to that of synthetic BEND 1–31 (BEND) and α-N-acetylated BEND 1–31 (Nac-BEND), with a ratio of BEND to Nac-BEND of approximately 1.8:1. The RP-HPLC profiles of ir-GnRH demonstrated a major peak coeluting with synthetic GnRH, along with a minor peak eluting at the void volume. Total content of ir-BEND (pg per uterus) was affected by the interaction of breed with day (P < 0.001), but was independent of reproductive status. In Large White gilts, uterine fluid content of ir-BEND was higher (P < 0.05) on days 10 and 11 than on days 8, 12 and 14; however, in Meishan gilts, ir-BEND decreased from day 8 to days 10 and 11, and remained low on days 12 and 14. Compared to Meishan gilts, Large White gilts had higher ir-BEND concentrations on day 10 (P < 0.01), day 11 (P < 0.0001), day 12 (P < 0.01) and day 14 (P < 0.02). Similar patterns were detected when ir-BEND in uterine fluid was expressed as pg mg⁻¹ protein. Differences in total ir-GnRH content of uterine fluid were influenced by the interaction of breed, status and day (P < 0.02). For Large White gilts, total ir-GnRH content was similar on days 8, 10, 11 and 12 between cyclic and pregnant gilts, but increased markedly (P < 0.0001) on day 14 of pregnancy. In pregnant Meishan gilts, total ir-GnRH content was increased on days 8, 12 and 14, compared with cyclic Meishan gilts on the same days. However, concentrations of ir-GnRH in uterine fluid (pg mg⁻¹ protein) depended only upon the interaction of status and day (P < 0.03). These results suggest that ir-BEND and ir-GnRH in uterine fluids possess biochemical similarities to their respective synthetic peptides. In addition, the uterine secretory profile of ir-BEND is different in Large White and Meishan pigs but the significance of this difference is unclear. The marked increase in ir-GnRH in uterine fluids between days 12 and 14 of early pregnancy suggests that uterine ir-GnRH may play a role in early pregnancy.

The corpus luteum undergoes tremendous growth, development and regression each oestrous or menstrual cycle. These changes are reflected by equally impressive growth and regression of the luteal vasculature. We have previously shown that angiogenic factors from corpora lutea are primarily heparin binding and that one of these factors is similar to vascular endothelial growth factor (VEGF). In an effort to identify this factor, and to define its role in luteal vascular development, the cDNA for the coding region of ovine VEGF was sequenced and a sensitive RNase protection assay was developed to quantitate mRNA encoding VEGF in luteal tissues from ewes in the early (days 2–4), mid- (day 8) and late (days 14–15) stages of the oestrous cycle. In addition, an N-terminal peptide was synthesized from the translated ovine cDNA sequence for VEGF and an antiserum was raised against this peptide for use in western immunoblotting procedures. Nested reverse transcriptase (RT)-PCR of RNA from ovine corpora lutea resulted in three products that correspond in size to the alternatively spliced variants of VEGF VEGF₁₂₀, VEGF₁₆₄, and VEGF₁₈₈) predicted from other species. The RNase protection assay revealed that the proportion of mRNA encoding VEGF was 2- to 3-fold greater on days 2–4 than on day 8 or days 14–15. Densitometric analysis of gels from the RNase protection assay showed that VEGF₁₂₀ represented approximately one third of the total mRNA encoding VEGF in the corpus luteum and that this proportion did not vary with stage of the oestrous cycle. SDS-PAGE and western immunoblot analysis of a homogenate from corpora lutea showed a single 18 kDa protein. These data demonstrate that VEGF is expressed in luteal tissue throughout the ovine oestrous cycle and that expression of mRNA encoding VEGF is upregulated during the period of rapid luteal development, when luteal vascular growth is at its maximum.

Basigin plays important roles in both male and female reproduction because basigin (Bsg) null male and female mice are infertile. The aim of the present study was to determine whether basigin expression in reproductive organs requires estrogen receptor-α (ESR1, ERα) or -β (ESR2, ERβ). Expression of basigin protein in the testis, ovary, and male and female reproductive tracts was studied in adult wild-type (WT), Esr1-null (αERKO), and Esr2-null (βERKO) mice by immunohistochemistry and immunoblotting. Basigin mRNA levels in ovary and uterus were examined by quantitative RT-PCR. In females, basigin protein expression was observed mainly in granulosa and interstitial cells of the ovary and epithelial cells of the proximal oviduct in all genotypes. Basigin protein was also expressed in the uterine epithelium at proestrus and estrus in WT and βERKO mice but not in αERKO mice. However, a higher level of basigin mRNA was observed in uteri of αERKO mice compared with WT and βERKO mice. In males, basigin was expressed in Leydig cells and all germ cells except spermatogonia in all genotypes. Basigin was present in epithelial cells lining the efferent ductules in WT and βERKO mice, but expression was greatly reduced in αERKO mice. In epididymal ducts, basigin expression was observed in epithelial cells in the caput and cauda in all genotypes. These data suggest that expression of basigin protein requires ESR1, but not ESR2, in the uterus and efferent ductules, but is independent of estrogen receptor in the ovary, oviduct, testis, and epididymis.

Fibroblast growth factor 2 (FGF2), a member of FGF family, binds with FGF receptors (FGFR) to initiate biological functions in various somatic cells. However, little is known regarding the role of FGF2/FGFR on oocyte meiosis. In this study, we investigated expression patterns and functions of FGF2/FGFR during in vitro maturation (IVM) of mouse cumulus-oocyte complexes (COCs). Among four FGFRs, Ffgr1 was the most abundant in COCs. The transcripts for Fgf2 and Ffgr1 in COCs increased during IVM. Ffgr1 was present in oocytes and cumulus cells, while Fgf2 was present in only cumulus cells. Treatment of COCs with the selective FGFR inhibitor SU5402 blocked oocyte meiotic progression and downregulated expression of Bmp15 and Gdf9. In contrast, supplement of FGF2 promoted oocyte meiotic progression and upregulated Bmp15 and Gdf9 expression. Inhibition of FGFR with SU5402 reduced cumulus expansion and expressions of Ptx3, Has2 and Tnfaip6. Treatment with FGF2 increased Ptx3 and Has2 expression. Inhibition of FGFR had no effect on meiotic progression of denuded oocytes (DOs). However, co-culture of DOs with COCs or supplementation with FGF2 promoted meiotic progression of DOs. Inhibition of FGF2/FGFR signaling also downregulated Ffgr1 expression, while supplemental FGF2 upregulated Fgfr1 expression. Furthermore, inhibition of FGFR in COCs interrupted the c-Mos/MAPK pathway and maturation-promoting factor (MPF), as indicated by downregulation of oocyte c-mos and Ccnb1 transcripts, respectively. Overall, this study suggests that FGF2 produced by cumulus cells, activates a FGF2/FGFR autocrine/paracrine loop within COCs to regulate cumulus expansion and oocyte meiosis. These findings reveal a novel role for FGF2/FGFR signaling during in vitro maturation of COCs.

Leptin produced by the placental syncytiotrophoblasts participates in a number of processes in pregnancy including implantation, proliferation of the cytotrophoblasts, and nutrient transfer across the placenta. Despite the functional significance of leptin in pregnancy, the regulation of leptin synthesis is poorly understood in human placental syncytiotrophoblasts. In this study, we investigated the role of endogenous human chorionic gonadotropin (hCG) in the regulation of leptin production as well as the underlying mechanism involving the cross talk between cAMP and p38 mitogen-activated protein kinase (MAPK) pathways. We found that neutralization of endogenous hCG with its antibody dose dependently decreased leptin mRNA level and secretion, whereas exogenous hCG increased leptin mRNA level and secretion. Activation of the cAMP pathway with dibutyryl cAMP (db cAMP) or forskolin recapitulated the stimulatory effect of hCG on leptin expression. Inhibition of protein kinase A with H89 not only reduced the basal leptin expression but also attenuated the induced leptin expression by hCG. Treatment of the syncytiotrophoblasts with db cAMP and hCG phosphorylated p38 MAPK. Inhibition of p38 MAPK with SB203580 not only reduced the basal leptin production but also attenuated the leptin-induced production by both hCG and db cAMP. These data suggest that endogenous hCG plays a significant role in maintaining leptin production in human placental syncytiotrophoblasts, and this effect involves a cross talk between cAMP and p38 MAPK pathways.