Injection Of PGF2 Alpha Research Articles

Regulation of mRNA encoding low density lipoprotein receptor and high density lipoprotein-binding protein in ovine corpora lutea.

Three experiments were conducted to examine the regulation of steady-state concentrations of mRNA encoding ovine low density lipoprotein receptor (LDL-R) and high density lipoprotein-binding protein (HBP) in corpora lutea. In Experiment 1, corpora lutea were collected from ewes on Days 3, 6, 9, 12 and 15 (Day 0, oestrus) of the oestrous cycle. Enriched preparations of small and large steroidogenic luteal cells were also obtained on Days 6, 9, 12 and 15 of the oestrous cycle. In Experiment 2, 16 ewes were hypophysectomized on Day 5 of the oestrous cycle and received saline, luteinizing hormone (LH), growth hormone (GH) or a combination of LH+GH until collection of luteal tissue on Day 12 of the oestrous cycle. Corpora lutea were also collected from pituitary-intact control ewes on Day 5 and Day 12 of the oestrous cycle. In Experiment 3, 13 ewes on Day 11 or Day 12 of the oestrous cycle were administered prostaglandin F2 alpha (PGF2 alpha) and corpora lutea were collected 4 h, 12 h and 24 h later. Corpora lutea were also collected from 4 non-injected and 4 saline-injected (at 24 h) ewes. Results demonstrated that concentrations of mRNA encoding LDL-R did not differ throughout the oestrous cycle. Luteal tissue collected on Day 3 of the oestrous cycle had higher concentrations of mRNA encoding HBP than luteal tissue collected on any other day of the oestrous cycle. Hypophysectomy increased concentrations of mRNA encoding LDL-R but had no effect on concentrations of mRNA encoding HBP. Twelve hours following PGF2 alpha injection concentrations of mRNA encoding LDL-R were decreased but concentrations of mRNA encoding HBP were increased. Concentrations of both LDL-R and HBP mRNA were decreased 24 h following injection of PGF2 alpha. Thus, long-term positive and acute negative regulation of progesterone secretion from the corpus luteum by luteotrophic and luteolytic hormones was not mediated by changes in steady-state concentrations of mRNA encoding LDL-R or HBP.

Effect of energy balance and somatotropin on insulin-like growth factor I in serum and on weight and progesterone of corpus luteum in heifers.

Holstein heifers (approximately 1 yr old) were fed to be in negative energy balance (n = 14; NEB) or positive energy balance (n = 15; PEB) during four estrous cycles. Each heifer was given PGF2 alpha to control the timing of fourth estrus. From 1 d after PGF2 alpha injection until 10 d after estrus, half of the heifers were injected daily with 14 mg of somatotropin (bST), and the corpus luteum (CL) was collected on d 10. Heifers in NEB had less IGF-I in serum (P < .01), smaller CL (P < .01), and less progesterone in CL (P < .01) than heifers in PEB. Exogenous bST increased (P < .01) IGF-I in serum, but this increase was greater in heifers in PEB than those in NEB (P < .01 for interaction of energy balance and bST). Nonetheless, bST did not alter weight or progesterone content of the CL in either energy balance group. In addition, serum IGF-I during the first 10 d after estrus was not correlated to luteal weight and only weakly correlated (r = .3) with luteal progesterone content. We conclude that exogenous bST given during luteal development does not alter growth or progesterone content of the CL in heifers. Reduced concentration of IGF-I in serum during luteal development does not account for suppressed luteal growth and progesterone synthesis of heifers in NEB.

Protein sources affect follicular dynamics in ewes near the onset of the breeding season.

The influence of source of protein during the luteal phase before a synchronized oestrus on the dynamics of follicular development, observed daily by ultrasonography, was assessed in ewes that were beginning the sexual season. Iso-nitrogenous amounts of soybean meal (SBM) or of a corn-gluten meal-ground-corn grain mixture (CGM-GC), or an iso-energetic amount of ground-corn grain (GC), were fed from four days before to four days after treatment with prostaglandin F2 alpha (PGF2 alpha). Feeding with SBM was associated with a higher frequency of short luteal phases (P < 0.02). Dynamics of follicular population were studied in ewes that ovulated after normal cycles. More follicles > or = 2 mm in diameter were observed on the ovaries of ewes fed SBM four days before PGF2 alpha treatment (P < 0.02), but the highest number was seen in ewes fed CGM-GC at the time of injection of PGF2 alpha (P < 0.08). Ewes fed SBM had larger follicles at last detection and ovulated earlier after PGF2 alpha treatment than their counterparts fed other diets (P < 0.001). Ovulatory follicles developed over a greater range of days in ewes with twin ovulations compared with ewes with single ovulations (P < 0.08). Serum concentrations of insulin were increased after four days of feeding with CGM-GC (P < 0.01), but not with SBM or GC, and reached a peak at the time of oestrus. In summary: (1) the source of dietary protein during the late-luteal phase affected follicular maturation after PGF2 alpha treatment; (2) insulin and glucose may be involved in this response and may play a role in ovarian follicular activity; and (3) twin ovulations appeared to result from both reduced atresia and increased recruitment of follicles.

Hormonal regulation of messenger ribonucleic acid encoding steroidogenic acute regulatory protein in ovine corpora lutea.

Steroidogenic acute regulatory protein (StAR), proposed to be involved in the transport of cholesterol to the inner mitochondrial membrane, has recently been cloned from MA-10 cells. Using reverse transcription-polymerase chain reaction, we generated a complementary DNA encoding 404 base pairs of StAR from ovine luteal tissue to perform studies regarding regulation of the messenger RNA (mRNA) encoding this protein. In Exp 1, ewes were hypophysectomized (HPX) on day 5 of the estrous cycle and administered saline or physiological regimens of LH and/or GH until collection of luteal tissue on day 12 of the estrous cycle (n = 4/group). Luteal concentrations [mean +/- SEM; femtomoles per microgram poly(A)+ RNA] of mRNA encoding StAR were lower (P < 0.05) in the HPX plus saline-treated ewes (26.4 +/- 7.3) than in day 12 pituitary-intact ewes (n = 4; 77.7 +/- 9.3). Replacement of LH (59.1 +/- 13.1), GH (59.1 +/- 12.8), or LH and GH (69.9 +/- 4.5) in HPX ewes increased (P < 0.05) concentrations of mRNA encoding StAR to values not different from those in day 12 controls. In Exp 2, ewes on day 11 or 12 of the estrous cycle were injected with prostaglandin F2 alpha (PGF2 alpha) to induce luteal regression. Corpora lutea were collected 4, 12, or 24 h after injection (n = 4-5/time point) and from untreated control ewes (n = 4) or 24 h after injection of saline (n = 4). Treatment with PGF2 alpha decreased (P < 0.05) concentrations of progesterone in serum 4, 12, and 24 h after injection. Concentrations of StAR mRNA were decreased (P < 0.01) to 47%, 19%, and 8% of control values 4, 12, and 24 h after PGF2 alpha injection, respectively. In Exp 3, ewes received ovarian arterial infusions of saline, PGF2 alpha, or phorbol 12-myristate 13-acetate (PMA), and luteal tissue was collected 0 (no infusion), 4, 12, or 24 h later (n = 3-4/group). Treatment with PGF2 alpha or PMA decreased (P < 0.05) concentrations of progesterone in serum 4, 12, and 24 h postinjection. Steady state concentrations of mRNA encoding StAR (P < 0.05) were 36% and 25% of the control value 12 and 24 h after PGF2 alpha injection. Injection of PMA decreased (P < 0.05) concentrations of StAR mRNA to 75% and 50% of control values at 4 and 12 h, but concentrations of mRNA encoding StAR were not different from control values at 24 h.(ABSTRACT TRUNCATED AT 400 WORDS)

Prostaglandin F2 alpha mediates ovarian sterol carrier protein-2 expression during luteolysis.

In the corpus luteum, prostaglandin F2 alpha (PGF2 alpha) appears to be a physiological agent with both antisteroidogenic and luteolytic actions. It is hypothesized that the antisteroidogenic action of PGF2 alpha acts through altered transport of cholesterol to the mitochondrial cytochrome P450 side-chain cleavage enzyme (P450scc). However, the effect of PGF2 alpha on the expression of the putative cholesterol transport protein, sterol carrier protein-2 (SCP2; 13.2 kilodaltons), has not been examined. In this study, the decline in serum progesterone after PGF2 alpha injection was examined in parallel with altered ovarian SCP2, P450scc, and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) protein and messenger RNA (mRNA) levels. Rats (28 days old) were treated with 8 IU PMSG to induce follicular development and ovulation. Ten days after ovulation, animals were treated with PGF2 alpha (single or multiple injections; 100-250 micrograms each) or left untreated. Ovarian SCP2, P450scc, and 3 beta HSD protein and mRNA levels were examined 0 (time zero), 4, and 8 h post-PGF2 alpha treatment using Western and Northern blot analysis. SCP2 mRNA levels were also examined using a highly sensitive ribonuclease protection assay that detects a 429-base pair SCP2-mRNA specific sequence. The results indicate that serum progesterone was significantly reduced 4 and 8 h after PGF2 alpha injections (P < 0.001; n = 6/time point). The decline in progesterone paralleled a 50-60% reduction in 3 beta HSD protein and mRNA levels by 4 h post-PGF2 alpha. Protein and mRNA levels for 3 beta HSD returned to control values by 8 h post-PGF2 alpha treatment. P450scc expression was also reduced at 4 h (44-54%), but by 8 h, both protein and mRNA levels had increased above the normal control levels (P < 0.02). In contrast, the 0.8-kilobase SCP2-specific mRNA transcript was reduced to 50% and 80% of the pre-PGF2 alpha treatment level at 4 and 8 h, respectively (P < 0.01). SCP2 ribonuclease protection assay analysis also indicated that SCP2 mRNA levels were reduced 65% (P < 0.03) and 85% (P < 0.01) by 4 and 8 h post-PGF2 alpha treatment compared to those in time zero ovarian tissue. Consistent with the loss of SCP2 mRNA expression, Western blot analysis indicated that a 15-kilodalton SCP2-immunoreactive protein (presumably the pro-SCP2 form) was significantly reduced or absent in the PGF2 alpha treated animals (P < 0.04).(ABSTRACT TRUNCATED AT 400 WORDS)

Preliminary findings of altered follicular activity in Holstein cows with coagulation factor XI deficiency.

Factor XI (F XI) deficiency is an autosomal recessive coagulopathy found in Holstein cattle. Affected animals have a 50% greater prevalence of repeat breeding. Therefore, several parameters describing ovarian function were studied. Daily blood sampling revealed that progesterone concentrations were slower to decline from a peak at day 16 (p < 0.01) to values less than 3 nmol/L in F XI-deficient cows (5.14 +/- 0.69 days (mean +/- SD) versus 4.05 +/- 0.63 days in control animals), resulting in an oestrous cycle length of 24.7 +/- 2.1 days compared to 22.9 +/- 3.0 days, respectively. This was not due to an alteration in the availability of prostaglandin F2 alpha (PGF2 alpha) or oxytocin (OT) involved in luteolysis. No significant differences (p > 0.05) were seen between normal (n = 7) and F XI-deficient (n = 7) cows in the peak values or the area under the curve for the pulse in 13,14-dihydro-15-keto PGF2 alpha in response to OT challenge or in the parameters describing the pulse of ovarian OT secretion after PGF2 alpha injection (n = 7 for each) between days 12 and 14. Ovulatory follicular development was assessed by ultrasound monitoring and plasma 17 beta-oestradiol values at 8-h intervals after a luteolytic injection of cloprostenol (n = 6 for each). Follicular diameter was smaller (p < 0.05) and accompanied by lower peak oestradiol values near the time of ovulation in F XI-deficient cows. The results suggest that the oestrous cycle in F XI-deficient cows is characterized by a slower process of luteolysis that may be associated with smaller follicular development.

Differentiation of bovine preovulatory follicles during the follicular phase is associated with increases in messenger ribonucleic acid for cytochrome P450 side-chain cleavage, 3 beta-hydroxysteroid dehydrogenase, and P450 17 alpha-hydroxylase, but not P450 aromatase.

In cattle, a dramatic increase in plasma estradiol occurs during the short 2- to 3-day follicular phase. The objective of this study was to investigate the molecular mechanisms that mediate this critical change, specifically whether increases in the steroidogenic ability of granulosa and thecal cells of the preovulatory follicle are associated with increases in the levels of messenger RNA (mRNA) for steroidogenic enzymes. Luteolysis and a follicular phase were induced cycling Holstein heifers (n=15) by injection of a luteolytic dose of prostaglandin F2 alpha (PGF 2 alpha) on day 6 or 7 of the estrous cycle (day 0 = estrus), and preovulatory follicles were obtained at three stages of differentiation (0, 12, or 24 h post-PGF2 alpha treatment). To assess developmental changes in steroidogenesis in vivo, estradiol and androstenedione were measured in follicular fluid and in culture medium after a 3-h incubation of granulosa and thecal cells in defined medium with or without gonadotropins. To determine whether changes in mRNA for steroidogenic enzymes are associated with changes in follicular steroidogenesis, levels of mRNA for cytochrome P450 side-chain cleavage (P450scc), 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), cytochrome P450 17 alpha-hydroxylase, and cytochrome P450 aromatase (P450arom) were measured in thecal and granulosa cells using ribonuclease protection assays. Concentrations of estradiol in follicular fluid were relatively high at time zero, increased significantly by 12 h, and increased further by 24 h post-PGF2 alpha treatment. However, the aromatizing activity of granulosa cells was high at the time of PGF2 alpha injection and did not increase significantly during the first 24 h after the initiation of luteolysis. The aromatizing activity of granulosa cells was reflected in levels of mRNA for P450arom, which was relatively abundant in granulosa cells obtained before luteolysis and did not increase further during the first 24 h of the follicular phase. Concentrations of androstenedione were virtually undetectable in follicular fluid at time zero and had increased dramatically by 12 and 24 h post-PGF2 alpha treatment. Similarly, thecal cells isolated at 24 h secreted 3-fold more androstenedione than cells isolated at the time of PGF2 alpha injection. Androstenedione production by thecal cells in response to LH was also markedly higher at 12 and 24 h than at the time of PGF2 alpha injection. Likewise; levels of mRNA for P450 17 alpha-hydroxylase increased significantly by 12 h post-PGF2 alpha treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of acute immunoneutralization of inhibin in ewes during the late luteal phase of the oestrous cycle on ovarian hormone secretion and follicular development during the subsequent follicular phase.

Ewes with ovarian autotransplants received either inhibin antiserum (10 ml i.v.; n = 6) or sheep serum (10 ml i.v.; n = 5) on day 10 of the luteal phase with additional daily injections (1 ml i.v.) from 48 h after the initial injection until the end of blood sampling, 9 days later. Luteal regression was induced by injection of prostaglandin F2 alpha (PGF2 alpha) 4.2 days after the initial plasma injection. Jugular and ovarian venous blood samples were taken every 4 h over the experimental period, and more frequent samples (every 10-15 min for 2-3 h) were taken to examine pulsatile secretory responses from the ovary to GnRH-induced (150 ng i.m. 1 and 4 days after initial treatment) or endogenous LH pulses (24 and 48 h after injection of PGF2 alpha). Plasma FSH concentrations, ovarian steroid secretion and ovarian follicular development were measured. Immunization against inhibin resulted in a two- to threefold increase (P < 0.001) in plasma FSH concentrations, which remained higher than controls until injection of PGF2 alpha. Within 24 h of immunization, there was an increase in the number of small ovarian follicles (P < 0.01) and by 4 days after treatment, immunized ewes had four or five (P < 0.01) large ovarian follicles and, despite little change in the basal steroid secretion, a four- to sixfold increase (P < 0.05) in the amplitude of the steroidogenic response to a GnRH-induced LH pulse.(ABSTRACT TRUNCATED AT 250 WORDS)

Estradiol increases amounts of messenger ribonucleic acid for gonadotropin-releasing hormone receptors in sheep.

Two experiments were conducted simultaneously to investigate regulation of amounts of mRNA for GnRH receptors during the periovulatory period in sheep. In the first experiment, amounts of mRNA for GnRH receptors were measured before and after preovulatory surge of LH following regression of the CL by prostaglandin F2 alpha(PGF2 alpha). So that the time of the preovulatory surge of LH could be accurately predicted, ewes received two injections of PGF2 alpha on Day 14 of the estrous cycle. Anterior pituitary glands were collected from 5 control ewes on Day 14 of the estrous cycle (0 h after PGF2 alpha) and at 48, 72, and 96 h after PGF2 alpha (5 ewes per group). The second experiment was conducted to investigate the effects of 17 beta-estradiol on amounts of mRNA for GnRH receptors. On Day 14 of the estrous cycle, 20 ewes were ovariectomized (OVX); 15 of these ewes received estradiol implants when they were OVX (OVXEI). Sixteen hours after OVX, anterior pituitary glands were collected from 5 OVX and 5 OVXEI ewes, and the remaining OVXEI ewes received an i.m. injection of estradiol (25 micrograms in corn oil; OVXEI + E) to induce a preovulatory-like surge of LH. Anterior pituitary glands were collected from OVXEI + E ewes 18 or 54 h after injection of estradiol (n = 5 per group). Half of each anterior pituitary gland was used to measure the number of GnRH receptors. Poly(A)+ RNA was isolated from the remaining half of each anterior pituitary gland, applied to slot blots, and hybridized with a radioactive cDNA probe encoding the ovine GnRH receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostaglandin F2 alpha receptors in the early bovine corpus luteum.

Since the early CL (< or = 4 days after ovulation) does not regress after injection of PGF2 alpha, this study was designed to determine whether number or affinity of PGF2 alpha receptors was lower in the early as compared with the midstage CL. Heifers were randomly assigned to have ovaries removed on Day 2, 4, 6, or 10 (n = 4 heifers per day; Day 0 = day of ovulation). Plasma progesterone concentrations and the weight and size of the CL increased from Day 2 to 6, indicating normal CL development. Plasma membranes from individual CL were evaluated for PGF2 alpha receptor concentration and affinity by Scatchard analysis. CL from each of the 4 days of the estrous cycle were not different with respect to PGF2 alpha receptor concentration (number per microgram of plasma membrane protein) and affinity. To examine tissue specificity, PGF2 alpha binding was evaluated in 12 organs or tissues. High-affinity PGF2 alpha receptors were found in the CL and adrenal medulla but not in granulosa cells or other tissues. In conclusion, a single class of high-affinity PGF2 alpha receptors was present within the bovine CL by 2 days after ovulation; therefore the reported lack of responsiveness to PGF2 alpha in the early CL was not attributable to a deficiency of high-affinity PGF2 alpha receptors.

Changes in the gonadotropin-releasing hormone pulse generator activity during the estrous cycle in the goat.

The hypothalamic GnRH pulse generator activity, which was assessed by the characteristic increases in the multiple unit activity (MUA) specifically associated with the pulsatile LH secretion (MUA volleys), was monitored throughout the estrous cycle of the female goat. Blood samples were collected daily for analysis of progesterone, estradiol and LH levels to monitor ovarian cyclicity. The frequency of the MUA volleys showed a reciprocal relationship with the plasma progesterone profile; being low during the luteal phase, whereas increased during the follicular phase until the onset of the subsequent luteal phase. On the other hand, the duration of the MUA volleys changed in parallel with plasma progesterone levels showing considerable prolongation in the luteal phase. More frequent blood sampling was carried out at 2-hour intervals for timing the LH surge during the follicular phase induced by prostaglandin F2alpha (PGF2alpha) administration. During the PGF2alpha-induced follicular phase, the MUA volley frequency was abruptly increased from 4 h after PGF2alpha injection, and this high frequency was maintained throughout the follicular phase except for the period around the preovulatory LH surge when the MUA volley frequency was lowered temporarily. These results were in accordance with our previous observation that the recurrence of MUA volleys continued with decreased frequency during the estradiol-induced LH surge in ovariectomized goats. The present study revealed that GnRH pulse generator activity changes dramatically during the estrous cycle in the goat, and it is suggested that GnRH pulse generator activity becomes fully potentiated following luteolysis to induce the final follicular development and to stimulate estradiol secretion, which eventually provokes another neural mechanism responsible for the induction of the LH surge.

Studies on the mechanism controlling generation of superoxide radical in luteinized rat ovaries during regression.

The mechanism regulating the luteolytic release of superoxide radical (SOR) was examined in prostaglandin F2 alpha (PGF2 alpha)-treated rats. Tail vein injection of PGF2 alpha caused a rapid increase in SOR in mitochondria and plasma membrane samples prepared from luteinized rat ovaries. The peak in the mitochondria preceded that in the plasma membrane, and both occurred before progesterone concentrations decreased in the blood. The amount of SOR produced was greater when samples from the plasma membrane, mitochondria, and cytosol were combined. In plasma membrane samples, SOR generation was lowered by inhibitors of intracellular signaling pathways, but not by cyanide, which blocks electron transport in respiratory enzymes. In mitochondria samples, however, SOR was blocked by cyanide, but not by inhibitors of intracellular signaling enzymes. The addition of phospholipase-A2, phorbol myristate acetate (protein kinase-C activator), or arachidonic acid stimulated SOR production in plasma membrane samples from ovaries of control rats, and phorbol myristate acetate and arachidonic acid inhibited LH-stimulated progesterone secretion in dispersed rat luteal cells. Also, when mitochondria prepared from ovaries of PGF2 alpha-treated rats were added to dispersed corpus luteum cells, there was an increase in SOR generation and an inhibition of LH-stimulated cAMP formation and progesterone secretion. These results indicate that SOR production in the corpus luteum after PGF2 alpha treatment is generated by several subcellular components. Formation in the plasma membrane may be initiated by SOR generation from the mitochondria and regulated by intracellular signaling pathways. Our results indicate that formation of SOR may lead to the disruption of LH stimulation of progesterone secretion.

Changes in Levels of Messenger Ribonucleic Acid for Cytochrome P450 Side-Chain Cleavage and 3β-Hydroxysteroid Dehydrogenase during Prostaglandin F2α-Induced Luteolysis in Cattle1

Regression of the CL causes a dramatic decrease in plasma progesterone levels. To test the hypothesis that the decrease in progesterone involves the down-regulation of mRNA encoding the steroidogenic enzymes, cytochrome P450 side-chain cleavage (P450scc) and/or 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), levels of plasma progesterone and luteal mRNA for P450scc and 3 beta-HSD were measured and correlated during induced luteolysis. Holstein heifers (n = 25) were injected with 25 mg prostaglandin F2 alpha (PGF2 alpha) on Day 6 or 7 of the estrous cycle (Day 0 = estrus) to induce luteal regression. To determine acute changes in plasma progesterone during luteolysis, jugular blood samples were obtained from 5 heifers hourly for 12 h, beginning immediately before injection of PGF2 alpha, and assayed for progesterone by RIA. A significant decrease in plasma progesterone levels was observed as early as 1 h after the PGF2 alpha injection (3.62 vs. 2.72 ng/ml, p < 0.05). Progesterone levels continued to decline with time through 12 h after administration of PGF2 alpha. The other 20 animals were ovariectomized at 0 (n = 6), 2 (n = 4), 12 (n = 4), or 24 (n = 6) h after PGF2 alpha. Levels of P450scc and 3 beta-HSD mRNA were determined in extracts of total luteal RNA by ribonuclease (RNase) protection assay. By 2 h after PGF2 alpha, 3 beta-HSD mRNA levels had decreased by about 40% as compared with levels at time 0 (p < 0.05), and a further significant decrease occurred between 2 and 12 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of protein kinase-C, calpains, and calpastatin in prostaglandin F2 alpha-induced oxytocin secretion from the bovine corpus luteum.

An experiment was conducted to determine whether prostaglandin F2 alpha (PGF2 alpha)-induced secretion of oxytocin (OT) by the bovine corpus luteum (CL) was associated with changes in the activities of protein kinase-C (PKC), calpains, and calpastatin. On day 8 of the estrous cycle (estrus = day 0), beef heifers were restrained and given a 500-micrograms iv injection of cloprostenol, a PGF2 alpha analog. Corpora lutea were surgically removed from beef heifers 0, 2, 7.5, or 30 min (n = 4 animals/time period) after cloprostenol injection. Blood samples were collected before injection and at frequent intervals after injection. Distribution of PKC activity in cytosol and membrane fractions and activities of microcalpain, millicalpain, and calpastatin were determined for all CL. OT was measured in plasma and tissue by RIA. Relative to mean plasma levels of OT at time zero (85 +/- 7 pg/ml), peak plasma levels occurred between 1.5-10 min (270 +/- 36 pg/ml) for all animals. The mean luteal concentration of OT was greater at 0, 2, and 7.5 min (145 +/- 27, 232 +/- 82 and 269 +/- 115 ng/g, respectively) than at 30 min (93 +/- 33), but differences in tissue OT over time were not significant (P > 0.05). PKC activities (percentage over nonactivated control values) in the membrane or cytosolic fractions did not differ significantly among the times of CL removal; however, membrane PKC activity was positively correlated with the plasma OT level at the time of CL removal (r = 0.82; P < 0.0025). Luteal millicalpain activity was approximately twice that of microcalpain at each time point (P < 0.001), although the activities of the individual calpains over time after PGF2 alpha injection did not change. Calpastatin activity was significantly higher at 30 min (515 +/- 28 U/g tissue) than at 0, 2, or 7.5 min (373 +/- 26, 423 +/- 26, and 426 +/- 24 U/g tissue, respectively). PKC activity in the membrane appears to be positively correlated with OT secretion from the bovine CL, and increased calpastatin activity after PGF2 alpha injection may inhibit calpains present in the CL, thereby maintaining an active pool of PKC.

Involvement of protein kinase-C, calpains, and calpastatin in prostaglandin F2 alpha-induced oxytocin secretion from the bovine corpus luteum

An experiment was conducted to determine whether prostaglandin F2 alpha (PGF2 alpha)-induced secretion of oxytocin (OT) by the bovine corpus luteum (CL) was associated with changes in the activities of protein kinase-C (PKC), calpains, and calpastatin. On day 8 of the estrous cycle (estrus = day 0), beef heifers were restrained and given a 500-micrograms iv injection of cloprostenol, a PGF2 alpha analog. Corpora lutea were surgically removed from beef heifers 0, 2, 7.5, or 30 min (n = 4 animals/time period) after cloprostenol injection. Blood samples were collected before injection and at frequent intervals after injection. Distribution of PKC activity in cytosol and membrane fractions and activities of microcalpain, millicalpain, and calpastatin were determined for all CL. OT was measured in plasma and tissue by RIA. Relative to mean plasma levels of OT at time zero (85 +/- 7 pg/ml), peak plasma levels occurred between 1.5-10 min (270 +/- 36 pg/ml) for all animals. The mean luteal concentration of OT was greater at 0, 2, and 7.5 min (145 +/- 27, 232 +/- 82 and 269 +/- 115 ng/g, respectively) than at 30 min (93 +/- 33), but differences in tissue OT over time were not significant (P > 0.05). PKC activities (percentage over nonactivated control values) in the membrane or cytosolic fractions did not differ significantly among the times of CL removal; however, membrane PKC activity was positively correlated with the plasma OT level at the time of CL removal (r = 0.82; P < 0.0025). Luteal millicalpain activity was approximately twice that of microcalpain at each time point (P < 0.001), although the activities of the individual calpains over time after PGF2 alpha injection did not change. Calpastatin activity was significantly higher at 30 min (515 +/- 28 U/g tissue) than at 0, 2, or 7.5 min (373 +/- 26, 423 +/- 26, and 426 +/- 24 U/g tissue, respectively). PKC activity in the membrane appears to be positively correlated with OT secretion from the bovine CL, and increased calpastatin activity after PGF2 alpha injection may inhibit calpains present in the CL, thereby maintaining an active pool of PKC.

Norgestomet and gonadotropin-releasing hormone enhance corpus luteum function and fertility of postpartum suckled beef cows.

In Exp. 1,101 suckled beef cows were administered two injections of prostaglandin F2 alpha (PGF2 alpha) 11 d apart. Cows in Group 1 received no additional treatment. Cows in Groups 2 and 3 received 250 micrograms of GnRH plus 47-h calf removal. Cows in Group 3 also received a norgestomet ear implant that was inserted 4 d after the first PGF2 alpha injection and left in situ for 8 d. The second PGF2 alpha injection was administered approximately 28 h before the time of norgestomet implant removal. The GnRH was administered approximately 30 h after the time of norgestomet implant removal (or 58 h after the second PGF2 alpha injection). Calf removal was the period from the time of implant removal to AI. All cows were artificially inseminated once 75 h after the second PGF2 alpha injection (47 h after implant removal). Reproductive status before the time of GnRH treatment (presynchronization) was determined by multiple sampling (2, 13, and 23 d before GnRH treatment) for blood progesterone concentrations (62% anestrous and 38% cyclic). The GnRH treatment increased (P < .01) the ovulation response and norgestomet reduced (P < .01) the incidence of short luteal phases in the presynchronization anestrous cows. Norgestomet treatment increased (P < .05) the timed breeding pregnancy rates for both presynchronization anestrous and cyclic cows. In Exp. 2, 174 suckled beef cows were administered Syncro-Mate B (which includes norgestomet). Cows in Group 1 received no additional treatment, whereas cows in Group 2 received 250 micrograms of GnRH (30 h after norgestomet implant removal).(ABSTRACT TRUNCATED AT 250 WORDS)

Repeated Administration of Prostaglandin F2α during the Early Luteal Phase Causes Premature Luteolysis in the Pig1

Previous investigators considered pig corpora lutea refractory to the luteolytic effects of prostaglandin (PG) F2 alpha before Day 12 of the estrous cycle. This study was designed to determine whether multiple injections of PGF2 alpha would result in a sustained reduction of serum progesterone and luteolysis, leading to significant shortening of the estrous cycle and interestrous interval. On Days 5-10 of an estrous cycle, gilts (n = 4) received injections of 12.5 mg PGF2 alpha (dinoprost tromethamine) i.m. every 12 h, or vehicle (PBS; n = 4) according to the same schedule. Mean interestrous interval in PGF2 alpha-treated gilts was reduced (p < 0.001) to 13.3 +/- 0.5 days compared with 19.8 +/- 0.6 days for control gilts. Serum progesterone declined below 1 ng/ml by Day 10.5 in PGF2 alpha-treated gilts compared to Day 17.5 in control animals. Serum concentrations of estradiol-17 beta (E2) reached maximal levels in PGF2 alpha-treated gilts earlier (Day 12.5) in the cycle than in control gilts (Day 19.5). Peak E2 and LH concentrations coincided with the periestrous period, suggesting that PGF2 alpha-induced estrus is accompanied by normal follicular development and ovulation. These results demonstrate that the pig is susceptible to the luteolytic effects of PGF2 alpha before Day 12 if repeated injections are given from Day 5 through Day 10.

Estradiol-17β has a Biphasic Effect on Oxytocin Secretion by Bovine Granulosa Cells1

The peptide hormone oxytocin (OT) and its mRNA are synthesized by bovine granulosa cells. Bovine granulosa cells isolated before the preovulatory LH/FSH surge secrete low amounts of OT and respond to gonadotropins with an increase in OT secretion. After the LH/FSH surge, basal OT secretion increases 20-fold. At the same time, dramatic changes in steroidogenesis occur in the preovulatory follicle. Estradiol-17 beta production is high before and declines after the LH surge. The objective of this study was to determine whether steroid hormones, particularly estradiol-17 beta, influence OT secretion by bovine granulosa cells. Heifers (n = 5) were injected with prostaglandin F2 alpha (PGF2 alpha) on Day 7 of the estrous cycle to induce luteolysis. Preovulatory follicles were obtained during the ensuing follicular phase, 24 h after PGF2 alpha injection (about 24-36 h before the expected time of the preovulatory LH/FSH surge). Granulosa cells were isolated and cultured for 5 days in defined medium containing graded doses (0, 0.001, 0.01, 0.1, 1, 10 micrograms/ml) of estradiol-17 beta, testosterone, or progesterone. Media were collected daily and assayed for OT by radioimmunoassay. Addition of 0.001 or 0.01 micrograms estradiol-17 beta/ml stimulated OT secretion by granulosa cells by about 75% during the last 2 days of culture (p < 0.01). In contrast, estradiol-17 beta at high doses (1 and 10 micrograms/ml) inhibited OT secretion by 45-85% (p < 0.01). Therefore, estradiol-17 beta had a biphasic effect on OT secretion by granulosa cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of Messenger Ribonucleic Acid Encoding 3β-Hydroxysteroid Dehydrogenase/Δ5-Δ4 Isomerase in the Ovine Corpus Luteum1

Three experiments were conducted to determine how steady-state levels of mRNA encoding 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) in the ovine corpus luteum vary 1) between the two steroidogenic luteal cell types, 2) during the estrous cycle, and 3) during prostaglandin F2 alpha (PGF2 alpha)-induced luteolysis. In the first experiment, RNA (10 micrograms) was isolated from purified preparations (n = 4) of large or small steroidogenic luteal cells. Large luteal cells contained 42% more (p < 0.05) message for 3 beta-HSD per microgram RNA than did small luteal cells, while the amount of mRNA for tubulin did not differ between the two types of luteal cells. To determine whether luteal levels of mRNA for 3 beta-HSD differ during the estrous cycle, corpora lutea were collected from cycling ewes (n = 3/day) on Days 3, 6, 9, 12, and 15 postestrus. Levels of mRNA for 3 beta-HSD were similar on Days 3, 6, 9, and 12 but were lower (p < 0.05) on Day 15 postestrus, while levels of mRNA for tubulin were unchanged. In the final experiment, ewes were treated on Day 10 postestrus with two injections of PGF2 alpha (5 mg each) or saline (control) at a 4-h interval. Corpora lutea were collected from ewes (n = 4/treatment) 1 h or 8 h after the second injection of PGF2 alpha or 8 h after the second saline injection.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of suppressing plasma FSH on ovarian follicular dominance in cattle.

The importance of FSH during development of the dominant follicle of the first wave of follicular growth in cattle was assessed by injecting heifers twice daily with 20 ml saline (n = 6) or 20 ml charcoal-extracted bovine follicular fluid (bFF; n = 10), containing inhibin, on days 6 and 7 of the oestrous cycle. All animals received a luteolytic dose of PGF2 alpha on day 8. The interval to oestrus after PGF2 alpha in controls was 2.6 +/- 0.2 days. Five of ten bFF-treated heifers exhibited oestrus at the same time as controls (2.7 +/- 0.1 days), whereas time to oestrus in the remaining five bFF-treated heifers was significantly longer (6.8 +/- 0.6 days; P < 0.05). Treatment with bFF suppressed plasma FSH during the 48 h injection period (P < 0.05). Concentrations of FSH were not different in bFF-treated animals that did not display delayed oestrus compared with those in animals that exhibited delayed oestrus. In controls, the dominant follicle of the first wave continued growing during treatment and ovulated after injection of PGF2 alpha. In all ten bFF-treated animals, growth of the dominant follicle of the first wave was arrested during the treatment period. In bFF-treated animals that did not exhibit delayed oestrus, the dominant follicle resumed growth after luteolysis and ovulated. In bFF-treated animals that displayed delayed oestrus, the dominant follicle regressed after luteolysis and the ovulatory follicle was selected from a newly recruited (second) wave of follicular growth.(ABSTRACT TRUNCATED AT 250 WORDS)