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The objective of this long-term study was to improve the out-of-season breeding rates for ewes by examining effectiveness of dose levels of PG600, a combination of 80-IU/mL pregnant mare serum gonadotropin and 40-IU/mL human chorionic gonadotropin. Each week, during the anestrous season from 1998 through 2016, mature ewes were inserted with 1 of 2 progestogen devices. After 9 to 13 d, at removal of the device, ewes were injected intramuscularly with a specific dose of PG600. A total of 1,402 ewes were treated in this study, with weekly treatment groups ranging from 3 to 12 ewes. Ewes were assigned randomly in equal numbers to 1 of 2 ram pens with one fertile ram in each pen. Rams were fitted with a marking harness. Between 36 and 48 h after removal of the device, rams were switched. Ewes were checked for crayon marks (indicative of estrus) at 36, 48, and 96 h after removal of synchronizing devices. Rams were removed after 1 wk. Pregnancy status was diagnosed at 90 d of gestation using radiography and verified at 120 d or at lambing. To explore the impact of dose level, daylight, and temperature on pregnancy rate, we formulated an ordered multinomial probit model. The model controls statistically for dose level, synchronization device type, device reuse, and natural variation in minimum daily temperature and day of the year (DOY). Predicted pregnancy rate and type of birth at 90 d of gestation were significantly affected by DOY and temperature as well as dose level. Both observed and predicted pregnancy rates were higher for PG600 dose levels between 2.00 and 3.50 mL than for those below 2.00 mL or above 3.99 mL. By giving optimal amounts of PG600 to anestrous ewes, producers will have a greater number of pregnant ewes at lambing during the out-of-season lambing time.

Two experiments were conducted to determine whether mifepristone (RU486) and PGF

In the ruminant ovary, synthesis and secretion of oxytocin begin in the granulosa cells of the preovulatory follicle and are markedly stimulated by the surge of LH and FSH. Luteinization of the granulosa cells results in a further increase in oxytocin gene expression, but translation of mRNA appears to be retarded because the peak concentration of luteal oxytocin occurs later than the maximal accumulation of the message. Several hormones have been shown to stimulate oxytocin secretion from granulosa and luteal cells in vivo or in vitro. However, the role of prostaglandin F2 alpha (PGF2 alpha) in regulating luteal oxytocin secretion has perhaps received more study than other hormones. The mechanism of action of PGF2 alpha has been shown to encompass a phosphoinositide cascade and activation of protein kinase C, events that are associated with luteal secretion of oxytocin. Protein kinase C phosphorylation of the actin-binding protein myristolated alanine-rich C kinase substrate (MARCKS) may be required for exocytosis of oxytocin.

Two experiments were conducted to determine whether mifepristone (RU486) and PGF2α activate the phosphatidylinositol hydrolysis pathway during the midluteal phase of the ovine estrous cycle. In experiment 1, ewes on day 8 of the cycle were given 10 μg RU486 or vehicle into the ovarian artery with removal of the corpus luteum (CL) after 10 min. Blood collected prior to and after treatment was analyzed for progesterone. Aliquots of CL were incubated with 10 μCi of 3H-inositol and in the presence and absence of PGF2α (10 nM) for 15 min. Exposure of CL to RU486 and PGF2α increased phosphatidylinositol hydrolysis (p < 0.05). Serum progesterone was reduced in both control and RU486-treated ewes (p < 0.05) compared to concentrations before treatments. In experiment 2, aliquots of CL collected from ewes on day 8 of the cycle were incubated with 3H-inositol and exposed to RU486 (2 μM) in the presence and absence of PGF2α (1 μM) for 15 min. Treatments stimulated phosphatidylinositol hydrolysis as in Exp 1 (p < 0.05). Progesterone concentrations in incubation medium were increased in response to RU486 and PGF2α (p < 0.05). Collectively, these data suggest that RU486 and PGF2α act to stimulate phosphatidylinositol hydrolysis in the mature ovine CL.

Reduced pregnancy rates often occur in ruminants grazing endophyte-infected (EI) tall fescue. The objectives were to characterize basal and oxytocin-induced PGF2a concentrations in serum and reproductive function in ewes fed tall fescue seed and to determine whether addition of fish meal (FM) to a diet of EI fescue would alter PGF2a production. Ewes were fed a diet with novel or non-toxic endophyte-infected (NE) or EI tall fescue seed containing either corn gluten meal (CG) or FM. Serum concentrations of prolactin, a measure of severity of fescue toxins, were reduced in ewes fed EI compared with NE forage seed (forageday, Pb0.02) and were greater in NEFM than NECG-fed ewes (Pb0.03). Size and number of corpora lutea (CL), determined by trans-rectal ultrasonography, were similar between diets (PN0.10). Serum concentrations of progesterone were reduced in ewes with two CL fed EI compared with NE seed (forageCL numberday, Pb0.001). Oxytocin-induced PGFM concentrations during the luteal phase were determined as a measure of uterine function. On the day of oxytocin administration, peak plasma concentrations of PGFM were reduced in EI compared with NE-fed ewes (foragetime, Pb0.003), but FM did not influence PGFM concentrations. Estrous cycle length was more variable in EI than NE-fed ewes. There appears to be some asynchrony between NE and EI-fed ewes leading to changes in uterine responsiveness to oxytocin. Inclusion of FM did not alter uterine responsiveness to oxytocin. D 2006 Elsevier B.V. All rights reserved.

An experiment was conducted to determine the effect of prostaglandin F2alpha (PGF2alpha) on luteal synthesis of progesterone (P4) and related progestins. Sixteen beef heifers were assigned in equal numbers to four groups in a 2x2 factorial arrangement of treatments. The experiment consisted of two levels of PGF2alpha analog (0 and 500 microg) and two levels of time (4 and 24 h after injection) of corpus luteum collection. All heifers were injected intravenously with saline (2 ml) or PGF2alpha (cloprostenol) on day 8 of the estrous cycle (estrus=day 0). Jugular blood was collected at 0, 1, 2, 3, 4 and 20, 21, 22, 23, and 24 h after injection. Resulting sera were analyzed for P4 by use of radioimmunoassay. Luteal tissue was analyzed by gas chromatography/mass spectrometry for P4, 20beta-hydroxyprogesterone, pregnenolone, and allopregnanolone (3beta-hydroxy-5alpha-pregnan-20-one). Treatment with PGF2alpha reduced serum concentrations of P4 as early as 1 h after injection (P0.005) and steroid levels remained low over 24 h. Similarly, administration of PGF2alpha caused a decline in luteal P4 (P0.005), 20beta-hydroxyprogesterone (P0.10), and pregnenolone (P0.05). In contrast, treatment with PGF(2alpha) caused an increase in luteal allopregnanolone over time (time x treatment interaction; P0.05). These data are interpreted to suggest that PGF2alpha promotes conversion of P4 to the metabolite allopregnanolone.

The male-oriented ram is a unique and valuable animal model for the study of hormonal, developmental and genetic contributions to sexual partner preference. Unlike most other mammalian models that are in use currently, variations in sexual attraction occur spontaneously in domestic ram populations. It is estimated that as many as 8-10% of rams exhibit a sexual partner preference for other males, classifying them as male-oriented rams. Studies have failed to identify any compelling social factors that can predict or explain the variations in sexual partner preferences of rams. Nor is there consensus on the endocrine and sensory responsiveness of male-oriented rams to other rams. However, a number of studies have reported differences in brain structure and function between male-oriented and female-oriented rams, suggesting that sexual partner preferences are neurologically hard-wired. Recently, we identified a sexually dimorphic nucleus (oSDN) in the sheep preoptic area-anterior hypothalamus. The oSDN is larger in female-oriented rams than in male-oriented rams and similar in size in male-oriented rams and ewes. In addition, mRNA levels for aromatase in the oSDN were higher in males than in females and were higher in female-oriented rams than in male-oriented rams. These results suggest a relationship between steroid hormones, specifically estrogens and oSDN morphology. In this review, we provide an overview of sexual behavior in rams and discuss the multiple factors that may contribute to the development and adult expression of same-sex partner preferences in rams.

Progesterone (P4) has been reported to inhibit oxytocin (OT) binding to its receptor in isolated murine endometrial membranes. The purpose of the present research was to 1) examine the in vivo and in vitro effect of P4 on the binding of OT to its receptor in the ovine endometrium and 2) determine whether the endometrial plasma membranes have high-affinity binding sites for P4. Ovariectomized ewes were pretreated with a sequence of estradiol-17b (2 days) and P4 (5 days) before being treated with estradiol-17b plus either vehicle (corn oil), P4 ,o r P4 1 mifepristone (RU 486) for 3 consecutive days. Treatment of ewes with 10 mg P4/day for 3 days suppressed binding of OT (P , 0.01) compared with that of controls, whereas concomitant treatment with the progestin antagonist RU 486 (10 mg/ day) blocked the effect of P4. Similarly, incubation of endometrial plasma membranes with P4 (5 ng/ml) inhibited binding of OT (P , 0.05), whereas this effect of P4 was blocked by the presence of RU 486 (10 ng/ml). By radioreceptor assay, the endometrial plasma membranes were found to contain a high-affinity binding site for P4 and the progestin agonist promegestone (Kd 1.2 3 10 29 and 1.74 3 10 210 M, respectively). Incubation of endometrial plasma membranes with P4 (5 ng/ml) significantly increased the concentration of progestin binding sites. Binding of labeled promegestone (R 5020) was competitively inhibited by excess unlabeled R 5020, P4, RU 486, and OT but not by estradiol-17b, cortisol, testosterone, and arginine vasopressin. These data suggest a direct suppressive action of P4 on the binding of OT to OT receptors in the ovine endometrial plasma membrane. mechanisms of hormone action, oxytocin, progesterone, progesterone receptor, uterus

The aim of this study was to determine whether endogenous progesterone regulates synthesis and secretion of luteal oxytocin. In Expt 1, mature ewes (n = 5 per group) were assigned randomly to control or mifepristone (RU486) treatment groups. Ewes were injected s.c. twice a day with vehicle or 10 mg RU486 on days 5-7 of the oestrous cycle (oestrus = day 0). On day 8, after an i.v. injection with prostaglandin F(2alpha) (250 microg cloprostenol), venous blood samples were collected at frequent intervals to determine plasma oxytocin concentrations. Plasma oxytocin concentrations of RU486-treated ewes were not significantly different from those of control ewes. In Expt 2, ewes were injected s.c. each day with vehicle or 175 mg RU486 on days 2-5 of the oestrous cycle followed by administration of prostaglandin F(2alpha) on day 6. Four of five RU486-treated ewes showed 'split-oestrus' (oestrous behaviour for 36 h and then again at 84-108 h after the onset of initial oestrus). There was no significant difference in mean plasma oxytocin or progesterone concentrations between treatment groups. The mean masses of mature corpora lutea from control and RU486-treated ewes on day 6 of the oestrous cycle did not differ significantly (394.8 +/- 28.8 versus 319.5 +/- 48.3 mg). RU486-treated ewes contained mature corpora lutea, new corpora lutea (two of four ewes) and preovulatory follicles (>or= 10 mm, two of four ewes). The average interoestrous interval for RU486-treated ewes was 9 days more than that for control animals (26.2 +/- 2.9 versus 17 +/- 0.5 days; P < 0.025).

The aim of the present study was to determine whether the fetal lamb brain has the capacity to aromatize androgens to estrogens during the critical period for sexual differentiation. We also determined whether administration of the aromatase-inhibitor 1,4,6-androstatriene-3,17-dione (ATD) could cross the placenta and inhibit aromatase activity (AA) in fetal brain. Eight pregnant ewes were utilized. On Day 50 of pregnancy, four ewes were given ATD-filled Silastic implants, and the other four ewes received sham surgeries. The fetuses were surgically delivered 2 wk later (Day 64 of gestation). High levels of AA (0.8‐1.4 pmol/ h/mg protein) were present in the hypothalamus and amygdala. Lower levels (0.02‐0.1 pmol/h/mg protein) were measured in brain stem regions, cortex, and olfactory bulbs. The MichaelisMenten dissociation constant (Km) for aromatase in the fetal sheep brain was 3‐4 nM. No significant sex differences in AA were observed in brain. Treatment with ATD produced significant inhibition of AA in most brain areas but did not significantly alter serum profiles of the major sex steroids in maternal and fetal serum. Concentrations of testosterone in serum from the umbilical artery and vein were significantly greater in male than in female fetuses. No other sex differences in serum steroids were observed. These data demonstrate that high levels of AA are found in the fetal sheep hypothalamus and amygdala during the critical period for sexual differentiation. They also demonstrate that AA can be inhibited in the fetal lamb brain by treating the mother with ATD, without harming fetal development. aromatase, behavior, Cyp19, early development, estradiol, neuroendocrinology, sexual differentiation, testosterone

Luminal epithelial cells of porcine endometrium are unresponsive to oxytocin (OT) in vitro although they express the greatest quantity of OT and receptors for OT in vivo. Therefore, the objective of this study was to determine if oxytocin acted in an autocrine manner on luminal epithelial cells to stimulate prostaglandin (PG)F2α secretion. Treatment of endometrial explants or enriched luminal epithelial cells with OT antagonist L-366,948 decreased (P < 0.05) basal secretion of PGF2α. Oxytocin increased (P < 0.01) PGF2α secretion from luminal epithelial cells that were pretreated with 1:5000 or 1:500 OT antiserum for 3 h to immunoneutralize endogenously secreted OT. However, OT only increased (P < 0.05) PGF2α secretion from glandular epithelial cells when pretreated with 1:500 OT antiserum. Pretreatment with OT antiserum did not alter the ability of OT to induce PGF2α secretion from stromal cells. Medium conditioned by culture of luminal epithelial cells stimulated (P < 0.05) phospholipase C activ...

Prostaglandin F2α (PGF2α)-induced secretion of oxytocin by the bovine corpus luteum involves the phosphorylation of a unique protein kinase C (PKC) substrate, myristoylated alanine-rich C kinase substrate (MARCKS) protein. This study was conducted to determine the specific PKC isoform engaged in phosphorylation of MARCKS protein in bovine luteal cells. In experiment 1, dispersed luteal cells recovered from the corpus luteum on d 8 of the estrous cycle were preincubated with [32P] orthophosphate and then exposed to PGF2α alone or in combination with PKC inhibitors. Autoradiography and densitometry of Western blots revealed that MARCKS protein was phosphorylated by a conventional PKC (cPKC) isoform. Experiment 2 was conducted to identify the specific cPKC isoform that phosphorylates MARCKS protein in luteal cells. Corpora lutea were removed from control and PGF2α-treated heifers on d 8 of the cycle, and PKC isoforms associated with membrane and cytosolic fractions were determined. Treatment with PGF2α increased membrane concentrations of PKCα within 5 min after treatment (p

The ruminant corpus luteum, in addition to producing progesterone, synthesizes and secretes oxytocin (OT) during the estrous cycle. Secretion of oxytocin occurs by exocytosis of membrane-encapsulated granules of this hormone. Exocytosis of oxytocin involves transport of granules through a cytoskeletal matrix including an actin cortex closely associated with the plasma membrane (PM). Actin filaments crosslinked by various proteins give rise to the structural integrity of the cortex. Myristoylated alanine-rich C kinase substrate (MARCKS), a protein specifically phosphorylated by protein kinase C (PKC), crosslinks actin filaments and anchors the actin network to the inner leaflet of the PM. There is evidence that the intact actin cortex may serve as a barrier, precluding fusion of transport vesicles with the PM. In some secretory cells, phosphorylation of MARCKS has resulted in its translocation from the PM to the cytoplasm with an associated disassembly of the actin cortex. Prostaglandin F(2alpha) (PGF(2alpha)) stimulation of the bovine corpus luteum during the midluteal phase of the estrous cycle activates PKC, which is associated with an increase in OT secretion in vivo and in vitro. Data are presented demonstrating that stimulation of bovine luteal cells with PGF(2alpha) on Day 8 of the cycle promotes rapid phosphorylation of MARCKS protein and causes its translocation from the PM to the cytoplasm and concomitant, enhanced exocytosis of OT. These data are consistent with the premise that MARCKS plays a role in the exocytotic process.

We tested the hypothesis that progesterone (P4) acts at a local level to inhibit luteal apoptosis. Initial experiments employed aminoglutethimide, a P450 cholesterol side-chain cleavage inhibitor, to inhibit steroid synthesis. Cultured bovine luteal cells were treated with aminoglutethimide (0.15 mM) 6 P4 (500 ng/ ml) for 48 h. Luteal cells were recovered and snap frozen for isolation and analysis of oligonucleosomal DNA fragmentation or fixed for morphological analysis. Medium was collected for analysis of P4 levels by RIA. Aminoglutethimide inhibited P4 synthesis by . 95% and increased the level of apoptosis as evidenced by 32 P-labeled oligonucleosomal DNA fragmentation (. 40%). P4 supplementation inhibited the onset of apoptosis that was induced by aminoglutethimide. These data were further supported by morphological assessment of apoptotic cells utilizing a Hoechst staining technique and together strongly suggest that P4 has anti-apoptotic capacity. Using reverse transcription-polymerase chain reaction, we were able to isolate a 380-base pair cDNA from the bovine corpus luteum (CL) that was 100% homologous to the progesterone receptor (PR) previously found in bovine oviductal tissue. Furthermore, PR transcripts were present in large and small luteal cells. Immunohistochemistry also revealed that PR protein was present in both large and small luteal cells. To determine whether the anti-apoptotic effect of P4 was regulated at the receptor level, luteal cells were cultured in the presence of PR antagonists, RU-486 and onapristone, for 48 h. Both antagonists caused approximately a 40% increase in 32 Plabeled oligonucleosomal DNA fragmentation. Interestingly, there was no difference (P $ 0.05) in P4 levels after treatment with PR antagonists. These observations support the concept that P4 represses the onset of apoptosis in the CL by a PR-dependent mechanism.

The bovine corpus luteum contains a myristoylated alanine-rich C kinase substrate (MARCKS) protein known to crosslink actin filaments in the cytoskeletal cortex associated with the plasma membrane. We conducted experiments to determine whether concentrations of MARCKS mRNA and protein in the bovine corpus luteum varied during the estrous cycle. Using Northern blots probed with a MARCKS cDNA, we found that luteal concentrations of MARCKS mRNA were greatest on d 4, 8, and 12 and markedly reduced on d 16 of the cycle (p0.08). Similarly, Western blot analysis of luteal proteins revealed that concentrations of MARCKS protein were greatest on d 8 and least on d 16 of the cycle (p0.01). Exposure of slices from a d 8 corpus luteum to prostaglandin F2alpha (PGF2alpha) during a 10-min incubation in the presence of [32P]-ortho-phosphate resulted in enhanced phosphorylation of MARCKS in membrane and cytosolic fractions compared to that of controls. We therefore concluded that expression of the luteal MARCKS protein gene may be regulated and that PGF2alpha-induced phosphorylation of this protein is attributable to activation of protein kinase C.

Three experiments were conducted, the objectives of which were to 1) examine the effects of exogenous estradiol (E2) and progesterone (P4) on uterine concentrations of oxytocin receptors (OTR) and OTR mRNA, as well as the effect of exogenous P4 on progesterone receptors (PR) during the late luteal phase of the cycle, and 2) ascertain whether chronic E2 treatment of ewes during the cycle would alter prostaglandin F2alpha (PGF2alpha)-induced secretion of luteal oxytocin (OT). In experiment 1, 15 ewes were assigned to a control (n = 5; 2 ml corn oil [CO] s.c. on Days 4-14 of the estrous cycle) and two treatment groups (n = 5 each) receiving either 250 microg E2 s.c. (Days 4-14) or 10 mg P4 s.c. (CO on Days 4-10 and P4 on Days 11-14). Endometria and corpora lutea were removed on Day 15 of the cycle. Mean luteal weights were greater in treated than in control ewes (p < 0.05). Endometrial concentrations of OTR and OTR mRNA were significantly greater in control than in E2- or P4-treated ewes. In experiment 2, five ewes each were treated s.c. with CO or 10 mg P4 on Days 11-14 of the cycle; endometria were then removed on Day 15 for PR assay. Endometrial concentration of PR did not differ between groups. Experiment 3 consisted of 20 ewes assigned to four groups in a 2 x 2 factorial arrangement. Treatment consisted of two dosages of E2 (0 or 250 microg/day) in 2 ml CO and two dosages of PGF2alpha analogue (0 or 125 microg Estrumate). All ewes were injected s.c. with E2 or CO for 11 days as described for experiment 1. On Day 15, all ewes received an i.v. injection of PGF2alpha or saline (Time 0); then jugular blood was collected at frequent intervals for analysis of serum concentrations of OT. PGF2alpha induced a release of OT in control and E2-treated ewes (p < 0.05) compared to the value in saline-treated ewes. Collectively, these data suggest that in cycling ewes, exposure of the uterus to increased concentrations of E2 or P4 causes down-regulation of OTR as a consequence of suppression of the OTR gene. Chronic E2 treatment of ewes during the cycle does not act directly on the ovary to alter the stores of luteal OT.

Experiments were conducted to investigate the mechanisms through which chronic treatment with estradiol-17beta (E2) prolongs the estrous cycle in the ewe. To determine whether treatment with estradiol maintained the corpus luteum (CL), mature ewes were assigned randomly to two groups receiving s.c. injections of either 500 microg E2 in corn oil or vehicle alone for 20 days beginning on Day 4 of the cycle (n = 5 per group). Laparotomy of E2-treated ewes on Day 24 revealed the presence of CL previously marked with India ink on Day 7 of the cycle. Treatment increased the mean interestrous interval for 4 of 5 animals compared with that of controls (p < 0.001). Therefore, to determine whether the increase in the interestrous interval was due to an effect on the function and/or concentration of uterine oxytocin receptors (OTr), ewes (n = 5 per group) were injected with 500 microg E2 or vehicle as described above from Days 4 to 14 of the cycle. On Day 15, a jugular blood sample was collected for progesterone (P4) analysis, after which ewes were given an i.v. injection of oxytocin (OT; 20 IU USP) or saline, and blood samples were collected at frequent intervals for 60 min to determine plasma concentrations of 13,14-dihydro-15-ketoprostaglandin F2alpha (PGFM). Laparotomies were performed after blood collection to remove uterine endometrium for OTr analysis. Mean serum concentrations of P4 were greater in treated than in control ewes on Day 15 (p < 0.05). Treatment of ewes with E2 prolonged luteal function and resulted in reduced uterine concentration of OTr compared with that of controls (p = 0.002). Treatment with E2 reduced OT-induced plasma concentrations of PGFM compared with controls (p < 0.01). Collectively, these data suggest that chronic treatment of ewes with estradiol during the cycle can prolong the interestrous interval by reducing uterine concentration of OTr and hence OT-induced secretion of prostaglandin.

A study was conducted to investigate the effects of exogenous melatonin on serum concentrations of estradiol-17 beta (E2) and progesterone (P4) and uterine prolactin (PRL) receptor concentrations in mated mink. In Experiment 1, two groups of adult, standard dark, female mink were mated to fertile males on March 8 or 9. On March 16, mink in group 1 (N = 8) received an empty Silastic implant inserted sc in the interscapular region and served as controls. Mink in group 2 (N = 8) received an implant containing 10 mg crystalline melatonin. On April 2, all animals were lightly anesthetized and blood samples collected via cardiac puncture were analyzed for serum concentrations of E2 and P4. Animals were subsequently sacrificed and uterine samples collected for analysis of PRL receptor concentrations. In Experiment 2, adult female mink were assigned randomly to three treatment groups and mated to fertile males between March 6 and 9. On March 16, mink in group 1 (N = 6) received empty Silastic implants and served as controls. Mink in group 2 (N = 6) received a Silastic implant containing 10 mg melatonin. Animals in group 3 (N = 6) received an implant containing 10 mg melatonin and in addition each mink was given daily sc injections of P4 (1 mg) from March 21 to April 5. At this time the mink were sacrificed, the number of implantation sites recorded, and uteri collected for quantification of PRL receptors. In Experiment 1, exogenous melatonin reduced serum P4 concentrations to almost nondetectable levels (controls, 8.08 +/- 0.73 vs treated, 0.82 +/- 0. 12 ng/ml; P0.001) and resulted in increased concentrations of E2 (controls, 13.3 +/- 1.9 vs treated, 22.0 +/- 1.9 pg/ml; P0.01). Uterine PRL receptor concentrations decreased (P0.05) from 37.74 +/- 9.37 fmol/mg protein (controls) to 23.74 +/- 9.03 fmol/mg protein in response to melatonin treatment. In those mink treated with melatonin plus P4 (Experiment 2), uterine PRL receptor concentrations were increased to levels not significantly different than those of controls. None of the mink treated with melatonin alone or in combination with P4 exhibited implantation. Uteri of mink treated with melatonin or melatonin plus P4 did not differ in weight but tended to weigh less than uteri of control mink. These data suggest that a high systemic ratio of P4 to E2 is essential for production of the uterine PRL receptor in mink and supports the findings of others that implantation in mink cannot be initiated with P4 alone.

We studied a unique group of rams that would not mate with estrous ewes during extensive testing for sexual behavior. The same rams courted males in preference to females in 30-min sexual preference tests and were classified as male-oriented (n = 6). We compared the following endocrine profiles: systemic steroid concentrations, the capacity of the testes to biosynthesize 1 7a-hydroxyprogesterone and testosterone from 3 H-progesterone in vitro, and the levels of brain aromatase activity (AA) in male-oriented rams vs. rams that were proven breeders and designated as female-oriented (n = 7). After the last behavioral test, sera were collected, and males in each experimental group were killed. Brains and testes were obtained for subsequent determinations of AA and measurements of steroidogenic enzyme activity. All dissections and subsequent assays were performed without knowledge of experimental group assignments. Serum concentration of testosterone (T), dihydrotestosterone (DHT), androstenedione, estrone (E,), and estradiol-1713 (E2) were determined by RIA. AA was quantified by a 3 H20O assay validated for neural tissue of the ram. We studied frontal, parietal and cingulate cortex, cerebellum, hippocampus, olfactory bulb, septum, amygdala, infundibulum-median eminence, and preoptic area (POA). Serum T, E,, and E 2 concentrations of female-oriented subjects were significantly higher (p < 0.05) than those in maleoriented subjects (SEM: 1559 ± 228, 46 ± 2, and 15 ± 3 pg/ ml vs. 874 + 196, 40 + 2, and 8 1 pg/ml serum, respectively). DHT and androstenedione concentrations in the systemic circulation did not differ between groups. Likewise, biosynthesis of labeled T and 17a-hydroxyprogesterone from 3 H-progesterone by testicular homogenates in vitro was significantly higher (p < 0.05) in female-oriented than in male-oriented subjects (28.8 ± 8.1 vs. 12.1 2.3 limol-h -'mg protein-' for T and 416.9 + 100.8 vs. 186.3 ± 30.7 mol-h -mg protein-' for 17othydroxyprogesterone). The highest level of AA was found in the POA, which was significantly greater in female-oriented than in male-oriented rams (472 ± 34 vs. 296 + 24 fmol 3H20-h-'mg protein-', p < 0.05). AA in other brain areas did not differ between experimental groups. Our data suggest that the testes of the male-oriented ram have reduced capacity for T production. In other species, T controls in situ estrogen formation not only by providing substrate for aromatization but also by up-regulating P450.ro mRNA in the POA. Because the POA is part of a neural circuitry that mediates male

Administration of gonadotropin-releasing hormone (GnRH) early in the estrous cycle has been shown to cause subsequent altered luteal function. To determine whether membrane-related events may be involved in GnRH-attenuated luteal function, corpora lutea (CL) were removed from beef heifers on day seven of the estrous cycle after iv injection of GnRH or saline on day two of the cycle (n=5/group). Luteal slices were incubated with saline (control), luteinizing hormone (LH), or 8-bromo-cAMP for 2h. In vivo administration of GnRH reduced LH and cAMP-stimulated progesterone production by tissue (p0.01), but basal progesterone production was not affected (p0.05). Luteal adenylyl cyclase activity did not differ between saline and GnRH-treated animals (p0.05). Then to examine if early administration of GnRH alters response of the CL to prostaglandin (PG) F(2α), beefheifers were injected with GnRH as described above (n=4/group), and then injected with PGF(2α) on day eight and the CL removed 60 min later. Blood samples were collected for oxytocin (OT) analysis at frequent intervals after PGF(2α) injection and for progesterone at 0 and 60 min. Induction of the early response gene c-jun or release of OT by PGF(2α) was not altered by GnRH injection (p0.05). Injection of PGF(2α) decreased serum progesterone by 60 min postinjection (p0.05), but concentrations of this steroid were unaffected by GnRH (p0.05). Collectively, these data suggest that GnRH-induced alteration of bovine luteal function may be owing to events distal to cAMP synthesis that do not interfere with PGF(2α)-induced expression of c-jun or OT release, cellular phenomena involved in luteolysis.

Experiments were conducted to determine the in vivo and in vitro effects of metabolites of eicosapentaenoic acid on ovine luteal function. Injection of 750 micrograms methyl eicosapentaenoic acid (EPA) or methyl 12(R),13(S)-dihydroxyeicosapentaenoic acid (12,13-diHEPE) into the ovarian artery of ewes on day 10 of the estrous cycle caused a reduction in serum concentrations of progesterone by 48 h posttreatment compared with levels of this steroid in arachidic acid-treated controls (p0.005). Although mean serum concentrations of progesterone in methyl EPA-treated ewes during the remainder of the cycle did not differ from those in control ewes, levels in methyl 12,13-diHEPE-treated ewes remained significantly suppressed. Duration of the estrous cycle did not differ among treatment groups (p0.05), but more of the methyl 12,13-diHEPE-treated animals (3/5) had exhibited estrus within 3 days after injection than methyl EPA-treated (1/5) or control ewes (0/5). Slices of corpus luteum removed from ewes on day 10 of the estrous cycle were incubated with arachidic acid (controls), 12,13-diHEPE or docosatetraenoic acid (DTA). Regardless of fatty acid treatment, all tissues retained the ability to produce basal levels of progesterone during subsequent incubation. Luteal slices previously exposed to arachidic acid or DTA exhibited an increase in progesterone production in response to subsequent treatment with LH (p0.05). In contrast, luteal slices incubated with 12,13-diHEPE did not respond to LH with a significant increase in production of this steroid above that observed in controls. All tissues displayed a marked increase in progesterone synthesis upon treatment with 8-Br-cAMP relative to incubation of tissue alone (p0.001). Subcellular distribution of [14C]-12,13-diHEPE in luteal cells after incubation revealed that the majority of the fatty acid was associated with the plasma membrane. These data suggest that metabolites of eicosapentaenoic acid with hydroxyl groups on adjacent carbon atoms interfere with luteal function in the ewe, perhaps in part by altering luteal response to LH.

Experiments were conducted to examine the effect of a cyclopropenoid fatty acid (CPFA) on progesterone (P4) production by the ovine corpus luteum (CL) during the estrous cycle. Ewes in Exp 1 and 2 were laparotomized on day 2 of the estrous cycle, and animals with CL in both ovaries were subjected to unilateral ovariectomy. Ewes with CL in one ovary only were not ovariectomized. During surgery, ewes were injected with a mixture of fatty acids (sterculic acid, 39%; palmitic, 29%; linoleic, 12%; malvalic acid, 9%; oleic, 8%; stearic, 3%) containing 500 micrograms sterculic acid (SA; Exp 1), 750 micrograms SA, or 750 micrograms oleic acid (Exp 2) via the artery supplying the ovary bearing the CL. Control ewes were similarly injected with vehicle only (0.1-0.2 ml dimethylsulfoxide; Exp 1 and 2, respectively). Sera from blood samples collected at 15-min intervals for 1 h after injection or once daily on alternate days of the cycle after surgery were analyzed for LH and P4, respectively. In Exp 3, slices of CL removed from five ewes on day 10 of the cycle were incubated for 90 min in medium containing 100 ng/ml SA or vehicle (10 microliters dimethylsulfoxide). Slices were then reincubated for 90 min in medium containing 10 ng/ml oLH or saline (10 microliters). Tissue and medium were analyzed for P4. Injection of 500 micrograms SA suppressed serum levels of P4 (P less than 0.01), but did not alter mean cycle length. Injection of 750 micrograms SA reduced serum concentrations of P4 and shortened estrous cycle duration (P less than 0.005). Oleic acid (750 micrograms) or as much as 1.9 mg of a mixture of fatty acids devoid of CPFA had no effect on cycle length or serum levels of P4, suggesting that altered luteal function was due to the type and not the quantity of fatty acid injected. Treatments had no effect on serum concentrations of LH. Preincubation with SA interfered with the ability of luteal slices to synthesize P4 when subsequently incubated alone or with ovine LH (P less than 0.01). It is concluded that SA acts on the CL to impair steroidogenesis and ultimately cause luteal regression.

This study tested the hypothesis that aromatization of testosterone to estradiol is necessary for sexual differentiation of the sheep brain. Pregnant ewes (n = 10) were treated with the aromatase inhibitor 1,4,6- androstatriene-3,17-dione (ATD) during the period of gestation when the sheep brain is maximally sensitive to the behavior-modifying effects of exogenous testosterone (embryonic d 50-80; 147 d is term). Control (n = 10) ewes received vehicle injections. Fifteen control lambs (7 males and 8 females) and 17 ATD-exposed lambs (7 males and 10 females) were evaluated for sexually dimorphic behavioral and neuroendocrine traits as adults. Prenatal ATD exposure had no significant effect on serum concentrations of androgen at birth, growth rates, expression of juvenile play behaviors, or the onset of puberty in male and female lambs. Rams exposed to ATD prenatally exhibited a modest, but significant, decrease in mounting behavior at 18 mo of age. However, prenatal ATD exposure did not interfere with defeminization of adult sexual partner preferences, receptive behavior, or the LH surge mechanism. In summary, our results indicate that aromatization is necessary for complete behavioral masculinization in sheep. However, before we can conclude that aromatization does not play a role in defeminization of the sheep brain, it will be necessary to evaluate whether intrauterine exposure of male fetuses to higher doses of ATD for a more extended period of time can disrupt normal neuroendocrine and behavioral development.

Experiments were conducted to characterize the nongenomic effects of progesterone (P4) on binding of oxytocin (OT) to its receptor and signal transduction in the ovine endometrium. The dose-response relationship of P4 to OT binding was examined. Membranes from endometrial tissue of ovariectomized hormone-treated ewes were preincubated in the presence of P4 for 1 h followed by OT receptor analysis. P4 interfered with the binding of OT in a dose-dependent manner. Endometrium was then recovered from cyclic ewes and divided into explants. Treatment consisted of two dosages of P4 and two dosages of OT. Explants were analyzed for total inositol monophosphate, bisphosphate (IP(2)), and trisphosphate (IP(3)) content. Preincubation with P4 for 10 min significantly interfered with OT stimulation of IP(2) and IP(3) synthesis. Oxytocin increased monophosphate production, but there was no detectable effect of P4. In the next experiment, endometrial explants were cultured in the absence or the presence of arachidonic acid. Explants were then exposed for 1 h to medium containing vehicle or P4. After incubation, explants were challenged with OT and the media were collected and analyzed for 13,14 dihydro-15-keto prostaglandin F(2alpha) by RIA. Treatment of explants with AA increased PGF(2alpha) content compared with that of controls. Brief exposure to P4 significantly decreased OT-induced PGF(2alpha) secretion from explants previously exposed to medium or AA. Collectively, these data are interpreted to indicate that the observed reduction in OT-induced IP(2) and IP(3) production and OT-induced PGF(2alpha) secretion was due to P4 inhibition of OT binding to its receptor.

Progesterone (P4) has been reported to inhibit oxytocin (OT) binding to its receptor in isolated murine endometrial membranes. The purpose of the present research was to 1). examine the in vivo and in vitro effect of P4 on the binding of OT to its receptor in the ovine endometrium and 2). determine whether the endometrial plasma membranes have high-affinity binding sites for P4. Ovariectomized ewes were pretreated with a sequence of estradiol-17beta (2 days) and P4 (5 days) before being treated with estradiol-17beta plus either vehicle (corn oil), P4, or P4 + mifepristone (RU 486) for 3 consecutive days. Treatment of ewes with 10 mg P4/day for 3 days suppressed binding of OT (P0.01) compared with that of controls, whereas concomitant treatment with the progestin antagonist RU 486 (10 mg/day) blocked the effect of P4. Similarly, incubation of endometrial plasma membranes with P4 (5 ng/ml) inhibited binding of OT (P0.05), whereas this effect of P4 was blocked by the presence of RU 486 (10 ng/ml). By radioreceptor assay, the endometrial plasma membranes were found to contain a high-affinity binding site for P4 and the progestin agonist promegestone (Kd 1.2 x 10-9 and 1.74 x 10-10M, respectively). Incubation of endometrial plasma membranes with P4 (5 ng/ml) significantly increased the concentration of progestin binding sites. Binding of labeled promegestone (R 5020) was competitively inhibited by excess unlabeled R 5020, P4, RU 486, and OT but not by estradiol-17beta, cortisol, testosterone, and arginine vasopressin. These data suggest a direct suppressive action of P4 on the binding of OT to OT receptors in the ovine endometrial plasma membrane.

The aim of the present study was to determine whether the fetal lamb brain has the capacity to aromatize androgens to estrogens during the critical period for sexual differentiation. We also determined whether administration of the aromatase-inhibitor 1,4,6-androstatriene-3,17-dione (ATD) could cross the placenta and inhibit aromatase activity (AA) in fetal brain. Eight pregnant ewes were utilized. On Day 50 of pregnancy, four ewes were given ATD-filled Silastic implants, and the other four ewes received sham surgeries. The fetuses were surgically delivered 2 wk later (Day 64 of gestation). High levels of AA (0.8-1.4 pmol/h/mg protein) were present in the hypothalamus and amygdala. Lower levels (0.02-0.1 pmol/h/mg protein) were measured in brain stem regions, cortex, and olfactory bulbs. The Michaelis-Menten dissociation constant (K(m)) for aromatase in the fetal sheep brain was 3-4 nM. No significant sex differences in AA were observed in brain. Treatment with ATD produced significant inhibition of AA in most brain areas but did not significantly alter serum profiles of the major sex steroids in maternal and fetal serum. Concentrations of testosterone in serum from the umbilical artery and vein were significantly greater in male than in female fetuses. No other sex differences in serum steroids were observed. These data demonstrate that high levels of AA are found in the fetal sheep hypothalamus and amygdala during the critical period for sexual differentiation. They also demonstrate that AA can be inhibited in the fetal lamb brain by treating the mother with ATD, without harming fetal development.

Research was conducted to determine whether seasonal changes in prolactin secretion occur in nonpregnant female African elephants and to examine potential functional interrelationships between secretion of prolactin, cortisol, and progesterone. Weekly blood samples were taken for 18 months from four female African elephants and the sera were analyzed by RIA for progesterone, cortisol, and prolactin concentrations. There was no significant effect of season on serum concentrations of prolactin. Estrous cycles averaged 14 weeks in length and were composed of a 9-week luteal phase and a 5-week follicular phase (based on progesterone concentrations consistently200 and200 pg/ml, respectively). Estrous cycle synchronicity was evident between pairs of elephants. Serum concentrations of prolactin (3.91 +/- 0.69 ng/ml; range: 0.84-15.8 ng/ml) were significantly lower during the luteal, compared with the follicular, phase (P0.0001; t test) and were positively correlated with serum concentrations of cortisol (r = 0.14; P0.05). Mean (+/-SE) serum concentration of cortisol was 5.7 +/- 1.3 ng/ml (range: 1.4-19.3 ng/ml), and concentrations of this adrenal steroid were negatively correlated with progesterone concentrations (r = -0.15; P0.01). Increased serum concentrations of prolactin detected during the follicular phase suggest that this hormone may be regulated by ovarian estrogens and may play a role in modulating ovarian function in the elephant.

Conversion of androgen to estrogen within the brain by cytochrome P450 aromatase is a component of the system controlling the display of normal male reproductive behavior and negative feedback inhibition of LH secretion in rams. In the present study, we used the highly sensitive 3H2O assay to measure aromatase activity in microdissected regions of the basal diencephalon and amygdala of intact and castrated rams. We found that aromatase activity was heterogeneously distributed. The highest activity was found in the medial amygdala, cortical amygdala, and bed nucleus of the stria terminalis. Intermediate levels of aromatase activity were found within the medial preoptic area/anterior hypothalamus, periventricular preoptic area, lateral preoptic area/anterior hypothalamus, ventromedial hypothalamus, and lateral hypothalamus. The lowest activity was present in the septum, infundibulum/median eminence, and dorsal medial hypothalamus. After castration, aromatase was significantly reduced in the medial preoptic area/anterior hypothalamus, periventricular preoptic area, lateral preoptic area/anterior hypothalamus, and infundibular nucleus/median eminence. In contrast, levels in castrate rams were unchanged in several other regions, most notably the bed nucleus of the stria terminalis and medial and cortical amygdala. These results provide a quantitative profile of aromatase activity in discrete regions of the ram hypothalamus and limbic system. They also demonstrate that aromatase activity is reduced in some, but not all brain regions after castration which suggests that different regulatory mechanisms control aromatase within different neuronal populations of the ram.

Research was conducted to determine the nature of the isoforms of protein kinase C (PKC) present in the bovine (Bos taurus) corpus luteum (CL) and their subcellular distribution. Western blot analysis was performed using isozyme-specific polyclonal antibodies and revealed that the bovine corpus luteum contains the alpha and epsilon isozymes of PKC, but not the beta, gamma, delta or zeta isozymes. Subcellular fractionation demonstrated that the alpha and epsilon isozymes of PKC were present in both the cytosolic and plasma membrane fractions. Densitometric measurements of Western blots of the subcellular luteal PKC fractions indicated that PKC alpha was found predominantly in the cytosolic fraction, whereas the majority of PKC epsilon was associated with the plasma membrane.

Two experiments were conducted to evaluate the effects of bromocriptine, melatonin (MLT), and 17 beta-estradiol (E2) on uterine physiology in mink (Mustela vison). In Expt. 1, summer-anestrous mink were injected sc daily with 2 mg bromocriptine or vehicle (n = 20 each) for 14 days. On Day 14, both groups were divided into two subgroups and injected sc with either 100 micrograms E2 or vehicle. Mink were bled immediately prior to euthanasia (24 hr after E2) and the sera analyzed for prolactin (PRL), E2 and progesterone (P4). At necropsy, aliquots of uterine tissue (n = 5) were used to measure in vitro oxidation of [14C]glucose, incorporation of [3H]thymidine into DNA and [14C]leucine into protein, and nuclear concentrations of estrogen receptor (ER) and P4 receptor (PR). In Expt. 2, anestrous mink were assigned to one of two treatment groups or a control group (n = 5 each). In mid-summer, groups 1 and 2 were implanted with 10 mg Silastic MLT implants. Seventeen weeks later, mink in group 1 received 100 micrograms E2 (sc) while group 2 and nonimplanted controls (group 3) were injected with vehicle. Mink were sacrificed 24 hr after injection and levels of PRL, E2, P4, ER, and PR determined. Bromocriptine suppressed serum concentrations of PRL (P < 0.001), increased serum levels of E2 (P < 0.05) and levels of PR (P < 0.01), but had no effect on levels of P4, uterine weight, glucose oxidation, DNA and protein synthesis, or concentrations of ER.(ABSTRACT TRUNCATED AT 250 WORDS)

The first two papers in this issue of Biology of Reproduction are minireviews derived from talks delivered at the 36th Annual Meeting of the Society for the Study of Reproduction, July 19–July 22, 2003, in Cincinnati, Ohio. The minisymposium, titled Steroid Regulation of Uterine Function in Domestic Animals, was organized by Dr. Fredrick Stormshak and included presentations by Dr. Thomas E. Spencer, Dr. Alan K. Goff, and Dr. Michael J. Fields. The papers included here were submitted to the Editorial office of Biology of Reproduction and were subjected to the same standards of peer review as all manuscripts published in the journal.

A review of the current state of knowledge of oxytocin production by the preovulatory follicle and corpus luteum is presented. Corpora lutea of a number of mammalian species have been found to synthesize oxytocin. However, the synthesis and secretion of this nanopeptide by the corpus luteum of the ruminant has been most extensively studied because of the potential role of this peptide in facilitating luteal regression. While much information exists relative to various biochemical and endocrine factors that impact on oxytocin gene expression, this aspect about luteal synthesis of this peptide hormone remains enigmatic. Prostaglandin F-2α (PGF-2α) has been shown to be a primary endogenous hormone responsible for triggering luteal secretion of oxytocin. Details are provided regarding the PGF-2α-induced intracellular signal transduction pathway that ultimately results in exocytosis of luteal oxytocin. Evidence is also presented for potential autocrine/paracrine actions of oxytocin in regulating progesterone production by luteal and granulosa cells. Concluding remarks highlight aspects about luteal oxytocin production that require further research.

Experiments were conducted to examine the effects of exogenous GnRH and LH on serum concentrations of progesterone (P4) in the ewe. Ewes in Exp. 1 and 2 were laparotomized on d 2 of an estrous cycle and ewes with corpora lutea (CL) in both ovaries were unilaterally ovariectomized. Ewes with CL in one ovary only were not ovariectomized. While they were anesthetized, ewes (n = 5) were injected with 25 micrograms GnRH (Exp. 1) or 50 ng GnRH (Exp. 2) into the artery supplying the ovary bearing the CL. Control ewes (n = 5 in each experiment) were injected similarly with saline. In Exp. 3, six ewes were injected i.v. (jugular) on d 2 with 100 micrograms oLH (t = 0) and 50 micrograms oLH at 15, 30 and 45 min; six control ewes were injected similarly with saline. Jugular blood was collected from all ewes at frequent intervals after treatment for LH analysis and on alternate days of the cycle through d 10 or 11 for P4 analysis. Treatment with 25 micrograms GnRH increased serum concentrations of LH at 15, 30, 45 and 60 min postinjection (P less than .001) and reduced serum concentrations of P4 on d 7 through 11 (treatment x day interaction; P less than .05). Injection with 50 ng GnRH caused a slight increase in serum concentrations of LH at 15 min but had no effect on serum concentrations of P4.(ABSTRACT TRUNCATED AT 250 WORDS)

An experiment was conducted to evaluate the effects of diet on fur colour and quality of bromocriptineand melatonin-induced winter pelage in mink. At weaning, 90 standard dark female kit mink were assigned randomly to one of two dietary regimens. Diet 1 (no. = 30) was a basal ranch mink diet, and diet 2 (no. = 60) was the basal diet, supplemented with liver and eggs. On 26 June 1986, mink given each diet were assigned to treatment (no. = 15) and control groups (no. = 15). Animals receiving diet 1 were treated with 120 mg melatonin implants (silastic) while those given diet 2 were treated with 120 mg melatonin implants, 60 mg bromocriptine pellets, or 60 mg bromocriptine pellets plus 18·6 mg melatonin implants (silastic). Control groups on each diet received no implants. Subsequently, fur growth was measured at biweekly intervals. Mink treated with 120 mg melatonin, 60 mg bromocriptine, and 60 mg bromocriptine plus 18·6 mg melatonin all moulted 1 month earlier than controls and exhibited significantly greater fur growth during the months of August and September (P < 0·05). Mink implanted with 120 mg melatonin, and bromocriptine plus melatonin were considered to be in fully prime pelage by mid October and were pelted by October 23. Mink receiving bromocriptine alone were not considered to be in prime pelage in October and were pelted in December with controls. Neither diet supplementation with liver and eggs, nor treatment to induce early fur growth had an effect on fur colour or quality. It was also concluded that treatment of mink with bromocriptine may not completely mimic the effect of exogenous melatonin on winter fur growth.

Binding of estradiol to a specific endometrial cytoplasmic receptor and translocation of the estrogen-receptor complex to the nucleus was studied in ewes on days 0 (estrus), 3, 6 and 10 of the estrous cycle. In addition, total endometrial cytoplasmic and nuclear receptors were determined by use of the estradiol exchange assay. The equilibrium dissociation constant for the estradiol-receptor complex and the maximum concentration of binding sites were determined to be 8.30 nM and 13,700 molecules per cell, respectively. As determined by exchange assay, total estradiol bound to the cytoplasmic receptor was greater (P < 0.01) on days 0 and 3 (6.84 and 9.56 fmol/μg DNA, respectively) than on days 6 and 10 (1.59 and 2.80 fmol/μug DNA, respectively). Total nuclear-bound estradiol averaged 1.21 ± 0.18 (SE) fmol/μg DNA on days 0, 3 and 6; however, it declined to 0.64 fmol/μg DNA on day 10. For each stage of the cycle studied, incubation of endometrium with 2 × 10-8M [3H]estradiol resulted in translocation of 77 / ...

Experiments were conducted to determine if prolactin receptors were present in the mink ovary, and to examine the relationship between receptor numbers and serum levels of prolactin (PRL) during embryonic diapause and blastocyst reactivation. For analysis of the physicochemical properties of prolactin receptors, ovaries were obtained from anestrous mink. All binding determinations were made using 125I-ovine prolactin (125I-oPRL), and 20 micrograms of tissue protein from the 100,000 X g particulate fraction. To quantify prolactin receptors during gestation, 20 primiparous mink were mated twice on consecutive days between 4 and 10 March and assigned randomly to one of two groups. Mink in Group 1 (N=8) were killed on 13 March when blastocysts were completing their migration into the uterus and entering a state of diapause. Animals in Group 2 (N=10) were killed on 26 March during the period of blastocyst reactivation, just prior to implantation. To determine serum levels of prolactin during gestation, an additional 20 primiparous mink were similarly mated and bled every 4 days from 15 March to 8 April, and then every 7 days until 23 April. Prolactin concentrations were determined by a heterologous double antibody radioimmunoassay using porcine PRL for both tracer and standards. Optimum conditions for binding 125I-oPRL to ovarian membranes were attained at 25 degrees C after 12 h. Scatchard analysis revealed a single class of high affinity binding sites with a Kd of 6.14 X 10(-11) M. The total concentration of receptors during anestrus was 85 fmol/mg protein, which increased significantly during embryonic diapause to 484 fmol/mg protein, then declined to 16 fmol/mg during blastocyst reactivation.

The present study was conducted to determine if specific binding sites for prolactin (PRL) are present in the uterus of the mink. Uteri of anestrous mink were homogenized and subjected to differential centrifugation into three particulate fractions, 1500, 15 000 and 50 000 X g. Binding of [125I]oPRL to membranes in an aliquot (200-400 micrograms protein) of the 50 000 X g particulate fraction was quantified. Time and temperature for optimal binding were 18 h at 25 degrees C. Scatchard plot analysis revealed a single set of binding sites for PRL with a Kd of 8.25 X 10(-11) +/- 0.68 M. The maximum amount of [125I]oPRL bound was 28 fmoles/mg protein. Prolactin binding sites were detected in both the uterus and kidney of mink, but not in skeletal muscle, spleen, diaphragm or lung. These data indicate that uterine cell membranes of the mink contain sites that bind prolactin with high affinity.

An experiment was conducted to evaluate the effect of exogenous gonadotropin releasing hormone (GnRH) on ovulation and embryonic survival in pubertal gilts. Gilts were assigned in replicates to a control (n = 10) and treatment (n = 10) group. Treatment consisted of an iv injection of 200 micrograms of GnRH immediately after initial mating on the first day of detected estrus. Control gilts were similarly injected with physiological saline. Blood samples were collected from the anterior vena cava immediately prior to injection, thereafter at 15-min intervals for 90 min, and subsequently, before slaughter on d 30 of gestation. Serum samples were analyzed for luteinizing hormone (LH) and progesterone by radioimmunoassay. Treatment with GnRH increased the quantity of LH released (P less than .05), with highest serum concentrations (ng/ml, means +/- SE) of gonadotropin in treated gilts (17.3 +/- 3.5) occurring at 75 min post-injection. In control gilts, serum concentrations of LH were not affected by injection of saline. Mean number of ovulations in treated gilts was also greater (P less than .05) than that of control animals (14.5 +/- .7 vs 12.1 +/- .6). However, treatment with GnRH did not enhance the number of attached conceptuses (normal and degenerating) present (treated, 10.9 +/- .9 vs control, 10.5 +/- .7) nor the percentage of viable fetuses (treated, 74.7 +/- 6.9 vs control, 83.5 +/- 5.0%) on d 30 of gestation. Although GnRH increased ovulation rate, mean weight of corpora lutea of treated and control gilts did not differ (402.8 +/- 16.3 vs 389.5 +/- 11.3 mg, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

A study was conducted to determine the effect of the milk-ejection reflex on exogenous gonadotropin releasing hormone (GnRH)-induced release of luteinizing hormone (LH) after shortterm calf removal. Twenty-four postpartum multiparous beef cows were assigned randomly to groups arranged in a 23 factorial arrangement. Factors consisted of two levels of suckling [suckled (S) or nonsuckled (NS)], treatment with GnRH [saline(C) or 200 pg GnRH] and days postpartum (d 1 and 14). Dams were isolated from their calves for 4 h on d 1 and 14 postpartum. Atthe end of 4 h dams were reunited with their calves in S+C and S+GnRH groups, while dams of calves in NS+C and NS+GnRH groups remained separated an additional 2 h. Cows were injected iv with saline or GnRH following the 4-h isolation period, 5 min after calves had begun suckling or nuzzling the udder. Sera from jugular blood samples collected 15 min prior to the end of the 4-h isolation period, immediately prior to injection (0 h) and at 15 min intervals thereafter for 120 min were analyzed for LH. Serum concentrations of LH in control cows did not differ due to suckling or stage of the postpartum period and averaged 2.3 s .1 ng/ml. Pituitary response to GnRH was determined by computing the rate of LH release. Rate of LH release (rig LH.ml -I -rain -1 ) in response to GnRH on d 14 was greater (P

The role of ovarian steroids and prostaglandin F2α (PGF2α) in regulating constriction of ovine uterine arteries was studied. Five unilaterally ovulating ewes were sacrificed on each of days 0 (estrus), 3, 6 and 10 of the estrous cycle. A segment of uterine artery supplying each horn was removed, cannulated and subjected to 10-min perfusions of saline (control vehicle), PGF2α, norepinephrine (NE) and neurotransmitter antagonists. At the end of each perfusion, vasoconstriction was provoked by stimulation of periarterial sympathetic nerves. Ovaries having the greatest follicular development (follicular ovaries) contained a corpus luteum in 16 of 20 ewes. Regardless of stage studied, uterine arteries adjacent to follicular ovaries (ipsilateral arteries) responded to nerve stimulation (NS) with increased constriction compared to the low consistent responses of contralateral arteries. Responses of ipsilateral arteries to NS following perfusions of saline, PGF2α or NE increased from day 0 to 10 of the cycle. Per...