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M. Jedlinska, L. Rozewiecka, and A. J. Ziecik

The influence of exogenous prolactin-induced hyperprolactinaemia and bromocriptine-induced hypoprolactinaemia on LH secretion, endocrine function of testes and the structure of seminiferous tubules was investigated in boars. Treatment with exogenous pig prolactin for 21 days (0.07 mg kg−1 day−1; n = 5) caused a significant increase in mean prolactin concentrations during the experiment (P < 0.05) and during 4 h sampling (P < 0.01). Treatment with bromocriptine (0.2 mg kg−1 per os and 0.05 mg kg−1 i.m.; n = 4) decreased mean prolactin concentrations throughout the experiment as well as during two 4 h sampling periods (P < 0.01). Treatment with exogenous prolactin decreased mean LH concentrations during the treatment (P < 0.01) and during the first 4 h sampling period (P < 0.05), but did not affect the number of pulses, pulse frequency or amplitude. Bromocriptine did not affect LH concentrations. Prolactin increased testosterone concentrations during the treatment (P < 0.01), while boars treated with bromocriptine had lower testosterone concentrations (P < 0.05) than did controls. Both exogenous prolactin and bromocriptine treatments significantly decreased oestradiol concentrations during the course of the experiment. The injections of prolactin and administration of bromocriptine caused a decrease in LH/hCG receptor concentration (P < 0.05) in the cell membrane of testes, without causing a change in binding affinity. The bromocriptine treatment caused premature release of some spermatocytes and the presence of polynucleate cells in the seminiferous tubules. In the seminiferous epithelium of hyperprolactinaemic boars, more mature cells (mainly spermatids) were released into the lumen. In tubules in which many polynucleate cells were found, considerable regression of seminiferous epithelium occurred. It is concluded that both hyper- and hypoprolactinaemia disrupt testicular endocrine function and change the process of spermatogenesis in boars. Hyperprolactinaemia has a greater effect on the testes and may decrease the secretion of LH.

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A. Ziecik, H. J. Shaw, and A. P. F. Flint

Summary. The concentration, affinity constant and occupancy of the LH receptor have been measured in corpora lutea, from 29 pigs at Days 6–16 of the oestrous cycle, and from 25 pigs at Days 12–30 of pregnancy, by using125I-labelled porcine LH tracer. Investigation of the specificity of the receptor showed that cross-reactions of other pituitary hormones were accounted for by their contamination with LH. Luteal concentration of unoccupied receptors doubled between Days 6 and 10 of the cycle, and decreased between Days 12 and 14; it increased 3-fold between Days 20 and 30 of pregnancy, but was lower on Day 12 of pregnancy than at a comparable stage of the cycle. Concentrations of receptors occupied by LH increased early in the oestrous cycle, in parallel with the total receptor concentration; in pregnancy the percentage occupancy dropped dramatically as total receptor concentration increased between Days 20 and 30. Receptor affinity constants increased towards the end of the cycle and decreased between Days 20 and 30 of pregnancy. It is suggested that (1) the lower concentration of receptors in early pregnancy may reflect down regulation by circulating LH, concentrations of which are higher in early pregnancy than during the cycle; (2) the increase in receptor concentration between Days 20 and 30 of pregnancy may be due to a rise in circulating oestrogen levels; and (3) the decrease in occupancy at this time may be caused either by a decrease in affinity constant or by placental production of a chorionic gonadotrophin-like compound.

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D. Pomp, E. J. Eisen, and A. J. Ziecik

Summary. Female mice from lines which had undergone long-term single trait and antagonistic index selection for litter size and body weight were analysed for ovulation rate and LH receptor induction. Compared to randomly selected controls, selection for large litter size increased ovulation rate (60%; P < 0·001) and decreased LH receptor induction per μg ovarian DNA (87%; P < 0·01). Selection for large body weight increased ovulation rate (18%; P < 0·001), but did not lead to a significant correlated response in LH receptor induction. Index selection for large litter size and small body weight increased ovulation rate (14%; P < 0·01) and decreased LH receptor induction (72%; P < 0·01), while index selection for small litter size and large body weight did not significantly alter either ovulation rate or LH receptor induction. LH receptor quantities in testes of males from the 5 lines did not exhibit the among-line profile which was observed in ovaries of females.

These results confirm the role of ovulation rate in mediation of the positive genetic correlation between litter size and body weight in mice. Increased ovulation rate in mice selected for large litter size may be due to mechanisms associated with LH receptors as well as factors related to growth. In contrast, increased ovulation rate in mice selected for large body weight may be due exclusively to factors related to growth.

Keywords: ovulation rate; LH receptors; ovary; selection; mice

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A. J. Ziecik, K. L. Esbenshade, and J. H. Britt

Summary. Male (N = 8) and female (N = 8) pigs were assigned to receive saline or a potent GnRH antagonist ([Ac-d 2Nal1,d 4-Cl-Phe2,d-Trp3,d-Arg6, d-Ala10]GnRH*HOAc; 1 mg/kg body weight) at 14 days of age. The GnRH antagonist caused LH to decline (P < 0·01) from 1·7 ng/ml at 0 h to <0·5 ng/ml during 4–32 h in males and females. Concentrations of FSH in gilts declined slowly from 75 ± 8 to 56 ± 5 ng/ml (P < 0·05) at 32 h. In males FSH was low (5·7 ± 0·5 ng/ml) at 0 h and did not change significantly.

To observe the effect of long-term treatment with GnRH antagonist, 10 male and 10 female pigs, 3 days of age, were treated with saline or 1 mg GnRH antagonist per kg body weight every 36 h for 21 days. Concentrations of LH were reduced (P < 0·01) to 0·2–0·4 ng/ml throughout the experimental period in male and female piglets treated with GnRH antagonist. Plasma FSH increased in control females, but remained suppressed (P < 0·001) in females treated with GnRH antagonist. Treatment with the GnRH antagonist suppressed FSH levels in males on Days 8 and 16 (P < 0·05), not on Day 24. Treatment of females with the GnRH antagonist did not influence (P > 0·10) oestradiol-17β concentrations. Administration of GnRH antagonist to males suppressed testosterone and oestradiol-17β values (P < 0·01) and reduced testicular weight (P < 0·01). Concentration of LH/hCG receptors in testes of boars treated with GnRH antagonist was lower (P < 0·10) than in controls, but concentration of FSH receptors was not affected. Basal and potassium-stimulated release of GnRH from the stalk median eminence and medial basal hypothalamus in vitro did not differ between treatment groups. The amount of residual GnRH in hypothalamic tissue was not different in control gilts and in gilts receiving the GnRH antagonist, but it was lower (P < 0·05) in boars treated with GnRH antagonist than in control boars.

Keywords: pig; GnRH antagonist; LH; FSH

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K. Derecka, F. P. Zhang, A. J. Ziecik, and I. Huhtaniemi

In view of the recently documented expression of the LH receptor gene in several non-gonadal reproductive tissues, the aim of this study was to analyse further the ontogeny of expression of this gene in the pig reproductive tract in fetal and neonatal life. RT–PCR was used to investigate the expression of the extracellular and transmembrane receptor domains, and to identify the time of onset of transcription of the full-length LH receptor mRNA and its shorter splice variants. Expression of the LH receptor gene was first detected around day 30 of fetal life in both ovary and testis, coinciding with morphological differentiation. The pattern of expression of LH receptor splice variants did not change during postnatal gonadal maturation. Expression of the LH receptor gene in the pig non-gonadal reproductive tract started during fetal life and continued during sexual maturation. A novel pig LH receptor spliced variant, lacking exon 10, was detected for the first time. The transmembrane receptor domain was expressed in fetal tissues, but not in neonatal control tissues. On the basis of the transmembrane domain of the LH receptor mRNA, it is concluded that the ovary and extragonadal tissues of the pig reproductive tract, like the pig testis, synthesize functional LH receptors during fetal life. The presence of LH receptor mRNA in extragonadal reproductive tissues indicates that LH is involved in the control and regulation of reproductive tract maturation.

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B. Flowers, A. J. Ziecik, and E. V. Caruolo

Summary. We examined the effects of (a) oestrogen and progesterone on concentrations of luteinizing hormone/human chorionic gonadotrophin (LH/hCG) receptors in uterine smooth muscle in vivo and (b) hCG on spontaneous myometrial contractions in vitro. Ovariectomized gilts received 2 ml corn oil (control; n = 5), 2 mg oestradiol benzoate (n = 6) or 20 mg progesterone (n = 5) for 5 days. Gilts were hysterectomized 8 h after the last injection and longitudinal sections of myometrium were incubated in modified Krebs' solution with 0 or 10 i.u. of hCG (n = 10/gilt) for 4 h at 37°C in 95% O2:5% CO2. After incubation, myometrial sections were placed in a tissue chamber perfused with Krebs' solution and mechanical activity was recorded for 30 min. Cell membrane fractions were prepared from myometrial tissue not used for in-vitro studies and analysed for LH/hCG receptors. Treatment with oestradiol benzoate increased (P < 0·01) the number of LH/hCG-binding sites compared with gilts receiving corn oil or progesterone. Incubation of myometrium with hCG reduced (P < 0·01) the frequency and amplitude of spontaneous uterine contractions in gilts treated with oestradiol benzoate. In contrast, hCG had no effect (P > 0·05) on the pattern of myometrial contractions in gilts given corn oil or progesterone. These results indicate that oestradiol promotes the synthesis of LH/hCG receptors in pig myometrium and incubation of oestrogen-primed tissue with hCG has a quiescent effect on myometrial contractility.

Keywords: LH; receptors; myometrium; uterine contractions; chorionic gonadotrophin; pig

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E Przygrodzka, M M Kaczmarek, P Kaczynski, and A J Ziecik

In order to characterize the transition of the corpora lutea (CL) from acquisition of luteolytic sensitivity to rescue of luteal function: i) the expression of 38 factors associated with steroids, prostanoids, and angiogenic systems and ii) concentrations of the main hormones responsible for maintenance of CL function in cyclic and pregnant pigs were examined. Additionally, the effect of prostaglandin (PG) E2 and F2 α on luteal function during the estrous cycle and pregnancy was evaluated in vitro. Significantly up-regulated gene expression was revealed in CL collected on day 14 of the estrous cycle (CYP19A1, ESR2, PTGS2, HIF1A, and EDN1) and on days 12–14 of pregnancy (SCARB1, PGRMC1, STAR, HSD3B1, NR5A1, PTGFR, PTGER4, and VEGFA). Elevated concentrations of estradiol-17β and PGE2 occurred in CL on days 12 and 14 of pregnancy respectively, while an increased intraluteal PGF2 α content was noted on day 14 of the estrous cycle. Both PGs increased the synthesis of progesterone by cultured luteal slices obtained on day 14 of pregnancy, in contrast to the action of PGF2 α on the corresponding day of the estrous cycle. PGE2 stimulated cAMP production via PTGER2 and PTGER4, while PGF2 α elevated the content of CREB in cultured luteal slices from CL of pregnant pigs. In silico analysis showed that infiltration of lymphocytes and apoptosis of microvascular endothelium were activated in CL on day 12 of the estrous cycle vs pregnancy. Summarizing, an abundance of E2 and PGE2 during pregnancy regulates specific pathways responsible for steroidogenesis, the prostanoid signaling system and angiogenesis during rescue from luteolysis in porcine CL.

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T. Krzymowski, J. Kotwica, S. Okrasa, Teresa Doboszyńska, and A. Zieçik

Summary. Prostaglandin F-2α (1·5 mg over 10 h) was infused into the anterior uterine vein of pigs on Days 6, 8, 10, 12, 14 and 15 of the oestrous cycle. At each stage of the cycle PGF-2α suppressed luteal function although the fall in progesterone secretion was much greater and statistically significant when the infusion was performed on Days 12,14 and 15 of the cycle than on Days 6, 8 and 10. The concentration of cAMP was depressed on Days 15 and 17 and fatty degeneration of luteal cells on Days 6–8 or 14 was more pronounced in the ovary ipsilateral to the PGF-2α infusion than in the contralateral ovary. The results are compatible with the local perfusion of PGF-2α from the anterior uterine vein to the ipsilateral ovary, but a systemic effect was also apparent.

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G. Wasowicz, K. Derecka, A. Stepien, L. Pelliniemi, T. Doboszynska, B. Gawronska, and A. J. Ziecik

Pig umbilical cord, like that of humans, contains two arteries and a vein surrounded by Wharton's jelly with amnion covering the exterior surface. The aim of the present study was to investigate whether LH–hCG receptors are present in the pig umbilical cord, using light microscope immunohistochemistry, semiquantitative autoradiography, western blotting and reverse transcription–polymerase chain reaction. Umbilical cords were collected on days 48, 71 and 103 of fetal life (n = 6). Monoclonal and polyclonal anti-LH receptor antibodies were used to study receptor distribution. Immunoreactivity was observed in the umbilical blood vessels, the epithelium of umbilical amnion and cells in the Wharton's jelly. No differences in LH–hCG receptor distribution related to the sex of the fetus, period of fetal life or section of the umbilical cord were observed. Strong immunostaining was observed in umbilical vein and in umbilical arteries. However, in the arteries, the tunica media expressed weaker receptor immunostaining than did the tunica intima and tunica adventitia. No immunoactivity was detected in non-target tissue (skeletal muscle) but LH receptors were immunostained in the pig ovary. Topical autoradiography showed that vein and arteries in the umbilical cord bind125I-labelled hCG, which was highly diminished after co-incubation with an excess of unlabelled hCG. The binding of125I-labelled hCG to the Wharton's jelly and epithelial amnion was less intense than it was to vessels. Gonadotrophin binding sites were not present in the skeletal muscle. The pig umbilical arteries, vein and Wharton's jelly contained a 75 kDa immunoreactive LH–hCG receptor protein similar to that found in corpora lutea. Southern blot analysis of reverse transcription–polymerase chain reaction products, performed to enhance the sensitivity and specificity of LH receptor transcripts determination in umbilical cord tissues, revealed that the expected fragments of 740 and 470 bp were present in the arteries, vein, Wharton's jelly and corpora lutea (positive control). An additional product of 670 bp was found in the corpora lutea and arteries of umbilical cord, but not in the vein and Wharton's jelly. This is probably the first reported evidence of the presence of LH–hCG receptors in the umbilical cord of a non-human female mammal.

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B. Gawronska, T. Paukku, I. Huhtaniemi, G. Wasowicz, and A. J. Ziecik

The current studies investigated the concentration and distribution of LH receptors in the oviduct of ovariectomized gilts at various times after administration of oestradiol benzoate (10 μg kg−1 body weight) to determine whether LH participates in the regulation of oviductal contractions. Polyclonal antibodies to the LH receptor were used in immunocytochemical and western blot analyses of oviductal tissues. The mechanical activity of the isthmus and ampullar segments of oviduct, collected from 16 cyclic gilts, was recorded for 30 min after LH or hCG treatment. In the oviduct, there was little competition for receptor occupancy between hCG and pig FSH, bovine thyroid-stimulating hormone (TSH), pig growth hormone (GH) and pig prolactin (1.2, 0.1, 0.01 and < 0.001%, respectively) but pig LH could completely inhibit the binding of [125I]hCG. Oestradiol benzoate increased (P < 0.01) the number of LH binding sites in oviduct 24, 48 and 72 h (0.60 ± 0.08, 1.62 ± 0.15, 2.48 ± 0.35 fmol mg−1 protein; n = 4 per treatment, respectively) after injection compared with the control gilts treated with corn oil (0.20 ± 0.04 fmol mg−1 protein; n = 4). The affinity of oviductal LH/hCG binding sites (K a) varied from 4.0 to 8.5 × 1010l mol−1 and was similar to that of luteal cell binding sites (6.1 × 1010l mol−1). Oestradiol benzoate also resulted in more intense LH receptor immunostaining of the tubal mucosal epithelium, smooth muscle cells and blood vessels as compared with controls. Western blotting has revealed that the pig oviduct, similar to the corpus luteum, contains 75, 48 and 45 kDa immunoreactive LH receptor proteins. Treatment with LH in vitro (100 ng ml−1) affected the contractility of oviduct. During the peri-ovulatory stage of the oestrous cycle, the amplitude, frequency and area under curve(s) of the isthmus decreased (P < 0.05), as did the frequency and area under curve (P < 0.05 and P < 0.01, respectively) of the ampulla (n = 4). The frequency and area under curve of the oviductal contractions were also significantly reduced during the early follicular phase of the oestrous cycle (P < 0.05). There was no effect of LH (or hCG) on the frequency and area under curve of the oviductal contractions during luteal stages of the oestrous cycle (n = 8). These data indicate that (1) the pig oviduct possesses immunoreactive and functional LH receptor, (2) oestradiol promotes the synthesis of LH receptor in the epithelium and smooth muscles, and (3) LH causes the relaxation of oviduct, especially during the peri-ovulatory stage of the oestrous cycle. In summary, the results of the present study indicate that LH can control oviductal contractions directly and may be partially responsible for the relaxation of isthmus during fertilization in pigs.