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Although rescue of the corpus luteum (CL) is required for pregnancy, luteal function during maternal recognition of pregnancy remains largely unexplored. CL were collected from pregnant cattle on days 14, 17, 20, and 23, to encompass the maternal recognition of pregnancy period. Next-generation sequencing was used to profile mRNA abundance during this time, while tandem mass spectrometry and nanostring technology were used to profile proteins and miRNA, respectively. A total of 1157 mRNA were differentially abundant, while 27 miRNA changed, and 29 proteins tended to change. mRNA that increased were regulators of interferon signaling and DNA repair, while those that decreased were associated with luteolytic processes, such as calcium signaling and matrix metallopeptidase (MMP) signaling, indicating inhibition of these processes. One of these, MMP12, was regulated by prostaglandin F2A in vitro. mRNA that were maximally abundant on day 20 were primarily associated with immune processes. Two of these, C-C motif chemokine ligand 1 and NFKB inhibitor alpha, were regulated by interferon tau in vitro. MiRNA that increased were predicted to inhibit phosphatidylinositol signaling, while those that decreased may be negative regulators of steroidogenesis. One protein that was greater on day 20 than on day 14 was aldehyde dehydrogenase 1 family member A1 (ALDH1A1), which synthesizes retinoic acid. Pharmacological inhibition of this enzyme, or of retinoic acid receptor signaling, led to suppression of progesterone production in vitro. Overall, these data indicate that there are changes in the CL of pregnancy that are important for continued luteal function., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Elevated cytosolic calcium and protein kinase C are well-established mediators of luteolytic actions of prostaglandin F2alpha (PGF2alpha). The objectives of this study were to determine 1) if calcium/calmodulin-dependent kinase kinase 2 (CAMKK2) participates in mediating PGF2alpha actions in developing (Day [d]-4) and mature (d-10) bovine corpus luteum (CL), 2) distal targets of CAMKK2, 3) developmental expression of adenosine monophosphate-activated protein kinase (AMPK), and 4) effects of AMPK activation on progesterone (P4) production. Expression of AMPK increased as the CL matured. Activation of the prostaglandin receptor (FP) induced rapid phosphorylation of AMPK, which was blocked by a CAMKK2 inhibitor. Changes in basal P4 secretion in vitro were determined in response to AMPK activation via metformin (met) or 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) in d-4 and d-10 CL. Production of P4 in d-10 CL decreased with met or AICAR compared to control, similar to activation by PGF2alpha. Therefore, potential distal targets of AMPK in d-10 CL were examined during induced functional regression via exogenous PGF2alpha. Serum and luteal P4 decreased at 2 and 4 h after administration of PGF2alpha. Protein expression of LDLR decreased at 2 and 4 h, while those of ACAT1 and STAR increased 4 h after PGF2alpha. During induced regression, alterations of cholesterol transport proteins contributed to decreased luteal and serum P4. Therefore, developmental differences in signal transduction associated with FP, specifically CAMKK2 and AMPK, partially contribute to differences in the ability of PGF2alpha to induce regression in mature, but not developing, bovine CL. Multiple cholesterol transport proteins, including LDLR, were altered by PGF2alpha and could be potential AMPK targets., (© 2015 by the Society for the Study of Reproduction, Inc.)

Although calcium (Ca(2+)) is accepted as an intracellular mediator of prostaglandin F2 alpha (PGF2alpha) actions on luteal cells, studies defining mechanisms of Ca(2+) homeostasis in bovine corpora lutea (CL) are lacking. The increase in intracellular Ca(2+) concentration ([Ca(2+)]i) induced by PGF2alpha in steroidogenic cells from mature CL is greater than in those isolated from developing CL. Our hypothesis is that differences in signal transduction associated with developing and mature CL contribute to the increased efficacy of PGF2alpha to induce a Ca(2+) signal capable of inducing regression in mature CL. To test this hypothesis, major genes participating in Ca(2+) homeostasis in the bovine CL were identified, and expression of mRNA, protein, or activity, in the case of phospholipase Cbeta (PLCbeta), in developing and mature bovine CL was compared. In addition, we examined the contribution of external and internal Ca(2+) to the PGF2alpha stimulated rise in [Ca(2+)]i in LLCs isolated from developing and mature bovine CL. Three differences were identified in mechanisms of calcium homeostasis between developing and mature CL, which could account for the lesser increase in [Ca(2+)]i in response to PGF2alpha in developing than in mature CL. First, there were lower concentrations of inositol 1,4,5-trisphosphate (IP3) after similar PGF2alpha challenge, indicating reduced phospholipase C beta (PLCbeta) activity, in developing than mature CL. Second, there was an increased expression of sorcin (SRI) in developing than in mature CL. This cytoplasmic Ca(2+) binding protein modulates the endoplasmic reticulum (ER) Ca(2+) release channel, ryanodine receptor (RyR), to be in the closed configuration. Third, there was greater expression of ATP2A2 or SERCA, which causes calcium reuptake into the ER, in developing than in mature CL. Developmental differences in expression detected in whole CL were confirmed by Western blots using protein samples from steroidogenic cells isolated from developing and mature CL. Localization of these genes in steroidogenic luteal cells was confirmed by immunohistochemistry. Therefore, it is concluded that the cellular mechanisms that allow PGF2alpha to induce a calcium signal of greater magnitude in mature than in developing CL involve 1) greater PLCbeta activity with enhanced generation of IP3, 2) an enhanced Ca(2+) release from the ER via unrestrained RYR2 due to a decrease in SRI expression, and 3) a reduction in calcium reuptake to the ER due to lower expression of ATP2A2. Accordingly, the increase in [Ca(2+)]i induced by PGF2alpha in mature large steroidogenic cells had less dependency from extracellular calcium than in those isolated from immature CL.

Prostaglandin F2 alpha (PGF(2alpha)) brings about regression of the bovine corpus luteum (CL). This luteolytic property of PGF(2alpha) is used in beef and dairy cattle to synchronize estrus. A limitation of this protocol is insensitivity of the early CL to luteolytic actions of PGF(2alpha). The mechanisms underlying this differential luteal sensitivity are poorly understood. The developing CL has a maximum number of PGF(2alpha) receptors; therefore, differences in signaling events may be responsible for luteal insensitivity. Hence, differential gene expression at two developmental stages of CL, Day 4 (D-4) and D-10 after estrus, might account for differences in signal transduction pathways associated with luteal sensitivity. This possibility was examined in these studies. Microarray analysis (n = 3 cows per stage) identified 167 genes that were differentially expressed (P < 0.05). These were categorized into genes involved in protein biosynthesis and modification (18.5%), transcription regulation and DNA biosynthesis (18.5%), miscellaneous (17.0%), cell signaling (12.0%), steroidogenesis and metabolism (10.2%), extracellular matrix and cytoskeletal proteins (9.5%), unknown functions (6.0%), protein degradation (5.3%), and antioxidant property (3.0%). Real-time PCR confirmed the differential expression of nine selected genes, including tyrosine 3-monooxygenase/tryptophan 5-monooxygense activation protein zeta polypeptide (YWHAZ) and regulator of G protein signaling 2 24-kDa (RGS2), observed in microarray. Furthermore, the in vivo effect of exogenous PGF(2alpha) (n = 3 cows per stage) on selected genes that were found to be differentially expressed during this developmental transition was examined. PGF(2alpha) increased the expression of a guanine nucleotide-binding protein (G protein) beta polypeptide 1 (GNB1) in D-4 CL and calcium/calmodulin-dependent kinase kinase 2 beta (CAMKK2) in D-10 CL. Therefore, GNB1, CAMKK2, YWHAZ, and RGS2 are candidate genes that may have a significant role in acquisition of luteal sensitivity to PGF(2alpha). Additional evidence supporting the significance of the microarray data was obtained from the observation that the amount of CAMKK2 paralleled the differential mRNA expression observed for this gene when examined by microarray analysis and by real-time RT-PCR. Furthermore, the two types of luteal steroidogenic cells known to be targets for PGF(2alpha) actions were demonstrated to be a cellular source for CAMKK2.

During maternal recognition of pregnancy, the conceptus stimulates endometrial secretion of PGF2alpha and PGE2. However, PGF2alpha is less effective in causing luteal regression in pregnant than in non-pregnant ewes. Experiments were conducted to elucidate mechanisms for reduced luteal sensitivity to PGF2alpha during maternal recognition of pregnancy. Corpora lutea (CL) were collected from pregnant and non-pregnant ewes 0, 4, or 12h following treatment with PGF2alpha on day 12 after estrus. Luteal PTGHS2 mRNA did not differ due to PGF2alpha or pregnancy status. Luteal PTGES mRNA was reduced in both pregnant and non-pregnant ewes after PGF2alpha treatment; while, luteal PTGFS mRNA was reduced 4h after PGF2alpha in pregnant, but not non-pregnant ewes. The result was a greater ratio of PTGES to PTGFS mRNA in pregnant ewes. Luteal mRNA for HPGD did not differ between pregnant and non-pregnant ewes on day 12. Luteal END1 mRNA was reduced in pregnant as compared to non-pregnant ewes prior to PGF2alpha challenge. Luteal END1 mRNA was increased after PGF2alpha in pregnant and non-pregnant ewes; however, ECE1 mRNA was reduced 4h after PGF2alpha in pregnant, but not non-pregnant ewes. The in vitro conversion of PGF2alpha to PGFM was greater in CL of pregnant than non-pregnant ewes at day 14. Luteal conversion of PGF2alpha to PGFM appears to be regulated post-transcriptionally. During maternal recognition of pregnancy, mechanisms of reduced luteal sensitivity to PGF2alpha may include a shift in prostaglandin production to the luteotropin PGE2, a reduction of ECE1 mRNA, and increased catabolism of PGF2alpha.

Expression of PKC alpha, beta I, beta II, epsilon and micro has been demonstrated in the whole bovine CL with PKC epsilon being differentially expressed as a function of development. In experiment 1 we have investigated the amount of mRNA encoding PKC epsilon at different stages of luteal development (days 1, 4, 10 and 17). In experiment 2, the cellular source of luteal PKC isozymes was determined. Enriched steroidogenic (SC) and endothelial (EC) cells from day-10 CL were used to examine this question by Western blot analysis and immuno-histochemistry. In experiment 3, Western blot analysis was used to examine the ability of ET-1 to activate luteal PKC isozymes in day-10 CL. In experiment 4, the role of luteal PKC isozymes in the ET-1 mediated inhibition of P(4) accumulation in steroidogenic cell cultures from day-4 and day-10 CL was examined. Abundance of PKC epsilon mRNA gradually increased from day-1 to -10 with no further increase on day-17. In experiment 2, PKC epsilon was exclusively detected in SC (LLC and SLC). In contrast, PKC alpha, beta I and beta II were detected in both SC and EC, with EC expressing higher amounts of PKC isozymes. In day-10 CL, ET-1 induced cellular redistribution of PKC alpha, beta I, epsilon but not beta II. Inhibitors specific for conventional PKC isozymes as well as PKC epsilon were able to negate the inhibitory effects of ET-1 on P4 accumulation in the day 10 CL. In the day-4 CL, the inhibitory effect of ET-1 might be mediated via conventional PKC. Thus, an exclusive presence of PKC epsilon in luteal steroidogenic cells, its higher expression along with the ability of ET-1 to stimulate its activation in day-10 CL strongly suggests that this PKC isoform may play an important regulatory role in decreasing P(4) during luteal regression. Inhibition of P(4) by ET-1 in the early CL may be mediated via conventional PKC isozymes.

The local relationship between the pregnant uterine horn and the CL during maternal recognition of pregnancy is well-documented. It continues beyond that time; pregnancies were maintained in lutectomized cows when CL were induced on the ovary ipsilateral, but not contralateral, to the uterine horn of pregnancy during Days 28-53. This study evaluated factors affecting maintenance of pregnancy by CL induced after Day 53, in lutectomized cows that had received exogenous progesterone from Day 29 to 15 days after induction of a CL. Twenty-four suckled beef cows were lutectomized on Day 29 of gestation; pregnancy was maintained with progesterone from two controlled internal drug releasing (CIDR) inserts, exchanged every 5 days. Beginning on Day 53, ovaries and viability of pregnancy were evaluated by ultrasonography every 5 days. When a follicle >or=10 mm in diameter was present ipsilateral to the fetus, each cow received 1,000 IU of hCG. Following induction of a CL (20 of 24), progesterone was reduced to a single CIDR for 5 days, then removed. Retention of pregnancy was confirmed by rectal palpation and calving. Cows with induced CL maintained pregnancy to term, including four with the CL contralateral to the fetus. Three cows failed to form normal CL by Day 98 and lost pregnancy after removal of exogenous progesterone. One cow that did not respond to hCG lost pregnancy during exogenous progesterone. In conclusion, CL induced after Day 53 maintained pregnancy to term, even when induced contralateral to the pregnant uterine horn.

A single-cell approach for measuring the concentration of cytoplasmic calcium ions ([Ca(2+)](i)) and a protein kinase C-epsilon (PKCepsilon)-specific inhibitor were used to investigate the developmental role of PKCepsilon in the prostaglandin F(2alpha)(PGF(2alpha))-induced rise in [Ca(2+)](i) and the induced decline in progesterone accumulation in cultures of cells isolated from the bovine corpus luteum. PGF(2alpha) increased [Ca(2+)](i) in Day 4 large luteal cells (LLCs), but the response was significantly lower than in Day 10 LLCs (4.3 +/- 0.6, n = 116 vs. 21.3 +/- 2.3, n = 110). Similarly, the fold increase in the PGF(2alpha)-induced rise in [Ca(2+)](i) in Day 4 small luteal cells (SLCs) was lower than in Day 10 SLCs (1.6 +/- 0.2, n = 198 vs. 2.7 +/- 0.1, n = 95). A PKCepsilon inhibitor reduced the PGF(2alpha)-elicited calcium responses in both Day 10 LLCs and SLCs to 3.5 +/- 0.3 (n = 217) and 1.3 +/- 0.1 (n = 205), respectively. PGF(2alpha) inhibited LH-stimulated progesterone (P(4)) accumulation only in the incubation medium of Day 10 luteal cells. Both conventional and PKCepsilon-specific inhibitors reversed the ability of PGF(2alpha) to decrease LH-stimulated P(4) accumulation, and the PKCepsilon inhibitor was more effective at this than the conventional PKC inhibitor. In conclusion, the evidence indicates that PKCepsilon, an isozyme expressed in corpora lutea with acquired PGF(2alpha) luteolytic capacity, has a regulatory role in the PGF(2alpha)-induced Ca(2+) signaling in luteal steroidogenic cells, and that this in turn may have consequences (at least in part) on the ability of PGF(2alpha) to inhibit LH-stimulated P(4) synthesis at this developmental stage.

We examined the responsiveness of large luteal cells (LLC), small luteal cells (SLC), and endothelial cells of the Day 4 and Day 10 bovine corpus luteum (CL) to prostaglandin (PG) F2alpha and endothelin (ET)-1. Using a single-cell approach, we tested the ability of each agonist to increase the cytoplasmic concentration of calcium ions ([Ca2+]i) as function of luteal development. All tested concentrations of agonists significantly (P = 0.05) increased [Ca2+]i in all cell populations isolated from Day 4 and Day 10 CL. Day 10 steroidogenic cells were more responsive than Day 4 cells to PGF2alpha and ET-1. Response amplitudes and number of responding cells were affected significantly by agonist concentration, luteal development, and cell type. Response amplitudes were greater in LLC than in SLC; responses of maximal amplitude were elicited with lower agonist concentrations in Day 10 cells than in Day 4 cells. Furthermore, on Day 10, as the concentration of PGF2alpha increased, larger percentages of SLC responded. Endothelial cells responded maximally, regardless of agonist concentration and luteal development. In experiment 2, we tested the developmental responsiveness of total dispersed and steroidogenic-enriched cells to the inhibitory actions of PGF2alpha and ET-1 on basal and LH-stimulated progesterone accumulation. The potency of PGF2alpha steroidogenic-enriched cells on Day 4 was lower than on Day 10; in contrast, the potency of ET-1 was not different. Therefore, ET-1 was a tonic inhibitor of progesterone accumulation rather than a mediator of PGF2alpha action. The lower efficacy of PGF2alpha in the early CL more likely is related to signal transduction differences associated with its receptor at these two developmental stages than to the inability of PGF2alpha to up-regulate ET-1.

We have examined the genes of the endothelin system that are targets for regulation by prostaglandin F2alpha (PGF2alpha). The effects of a luteolytic dose of PGF2alpha ) on the mRNA encoding endothelin converting enzyme-1 (ECE-1), pre-pro endothelin-1 (pp ET-1) and the ET receptors ETA, ETB, in bovine corpus luteum (CL) during the early (days 1 and 4), mid (day 10) or late (day 17) luteal phases were examined. The effect of the PGF(2alpha) treatment on ECE-1 protein, Big ET-1 and the biologically active mature ET-1 peptide were also examined. Most importantly, the direct ECE-1 activity was determined. Before day 10 of the cycle, in a PGF2alpha-independent manner, the amounts of mRNA encoding ET-1, ECE-1, ETA, and ETB were increased steadily from day 1. After day 10 of the cycle, expression of mRNA encoding pp ET-1 and ETA acquired responsiveness to exogenous PGF2alpha and both genes were up-regulated by the PGF2alpha treatment. This effect of PGF2alpha was also detected for the proteins corresponding to the mature ET-1. The enzymatic activity of ECE-1 remained unchanged throughout the lifespan of the CL in spite of the detected changes in mRNA and protein. The results suggest that the luteal endothelin system is regulated in a PGF2alpha-independent and -dependent manner. Importantly, an alteration in luteal ET-1 availability is most likely achieved by modulating the expression of mRNA encoding pp ET-1 and not by the amount or activity of ECE-1. This interpretation is supported by the observation that the activity of ECE-1 remained unchanged throughout the ovarian cycle. The combined effects of greater ET-1 availability and gene expression encoding the ETA receptor in the late luteal phase could render the CL, at this developmental stage, more sensitive or responsive to ET-1. If the luteal tissue is responsive to the available ET-1 during the early phase of the ovarian cycle, an additional role for ET-1 should be considered beyond mediating the luteolytic actions of PGF2alpha. Agents blocking the actions of ET-1 might be the best approach to interfere with the luteal ET system and test its physiological role(s) in vivo., (Copyright 2004 Elsevier Inc.)

Western blotting was used to identify the array of protein kinase C (PKC) isozymes expressed in the early (Day 4) and midcycle (Day 10) bovine corpus luteum (CL). PCKalpha, betaI, betaII, epsilon, and micro isozymes were detected in total protein samples prepared from both Day-4 and Day-10 corpora lutea. In contrast, specific antibodies for PKCgamma, eta, lambda, and theta isozymes failed to detect protein bands in the luteal samples. PKCbetaII and epsilon isozymes were expressed differentially at these two developmental stages of the bovine CL. In the Day-4 luteal samples, PKCepsilon was barely detectable; in contrast, in the Day-10 samples, the actin-corrected ratio for PKCepsilon was 1.16 +/- 0.13. This ratio was higher than the detected ratio for PKCbetaI and micro at this developmental phase of the CL (P < 0.01), but it was comparable with the ratio detected for the PCKalpha and betaII. The amount of PKCbetaII was, although not as dramatic, also greater in the Day-10 CL (actin-corrected ratio was 0.85 +/- 0.2) than in the Day-4 CL (0.35 +/- 0.09 [P < 0.01]). The actin-corrected ratios for all other PKC isozymes, alpha (Day 4 = 0.93 +/- 0.16, Day 10 = 0.97 +/- 0.09), betaI (Day 4 = 0.54 +/- 0.073, Day 10 = 0.48 +/- 0.74), and micro (Day 4 = 0.21 +/- 0.042, Day 10 = 0.21 +/- 0.38) were not different at these 2 days of the cycle. An experiment was designed to test whether activation of specific isozymes differed between CL that do or do not regress in response to PGF(2alpha). Bovine CL from Day 4 and Day 10 of the estrous cycle were collected and 1 mm CL fragments were treated in vitro for 0, 2.5, 5, 10 or 20 min with PGF(2alpha) (0.1, 1.0, and 10 nM) or minimal essential medium-Hepes vehicle. Translocation of PKC from cytoplasm to membrane fraction was used as indication of PKC activation by PGF(2alpha). Evidence for PKC activation was observed in both Day-4 and Day-10 luteal samples treated with 10 nM PGF(2alpha). Therefore, if PKC, an intracellular mediator associated with the luteal PGF(2alpha) receptor, contributes to the lesser sensitivity of the Day-4 CL, it is likely due to the differential expression of the epsilon and betaII isozymes of PKC at this stage and not due to an inability of the PGF(2alpha) receptor to activate the isozymes expressed in the early CL.

Experiments were performed to test the null hypotheses that embryonic survival is not affected by the presence of regressing corpora lutea in progestogen-supplemented ewes, and that the embryotoxic effects of regressing corpora lutea do not act locally on embryos in the uterine horn adjacent to the regressing corpora lutea. In Expt 1, laparotomies were performed on day 4 after mating, and progestogen supplementation was initiated and continued until pregnancy was diagnosed by ultrasonography on day 25. On day 4 after mating, ewes were lutectomized (n = 17) or sham lutectomized (n = 15), and injected (i.m.) with 5 mg PGF2alpha at 8 h intervals for 2 or 3 days. Controls (n = 14) were sham lutectomized and injected with saline as described above. Pregnancy rates did not differ in ewes treated with PGF2alpha for 2 rather than 3 days. Pregnancy rates were lower in ewes treated with PGF 2a compared with controls (P < 0.01). In ewes treated with PGF2alpha, lutectomy resulted in an increase in pregnancy rates (59%) compared with ewes subjected to sham lutectomy (33%; P < 0.05). In Expt 2, progestogen supplementation was initiated in the morning of day 4 after mating and continued until pregnancy diagnosis on day 25. In the afternoon of day 4, one ovary selected at random was lutectomized in ewes (n = 34) with at least one corpus luteum on each ovary, and the uterine horns were isolated by ligation to impede intraluminal transfer of luteal or uterine products that might initiate embryonic death. On days 5-8 after mating, equal numbers of ewes were injected i.m. with either saline or 5 mg PGF2alpha at 8 h intervals. Pregnancy rates did not differ between isolated uterine horns contralateral and ipsilateral to the regressing corpus luteum; however, pregnancy rates were lower in PGF2alpha-treated ewes than in saline-treated ewes (34 and 77%; P < 0.05). In conclusion, regressing corpora lutea exert an embryotoxic effect; however, there is no evidence that this effect occurs through systemic pathways.

Recent evidence in the cow suggests that endothelin-1 (ET-1) plays a role during prostaglandin (PG) F(2alpha)-induced luteal regression. We have examined the effects of treatment with PGF(2alpha) during the early and midluteal phases on three components of the endothelin system: endothelin-converting enzyme-1 (ECE-1), ET type A receptor (ET(A)), and ET-1 in the bovine corpus luteum (CL). Cyclic beef cows were injected (0 h) on Day 4 or 10 with either saline or the PGF(2alpha) analogue Lutalyse (15 mg). The CL were collected at 2 (n = 11), 10 (n = 23), 24 (n = 15), or 48 h (n = 12) after treatment. The cows in which CL were removed after 10 h comprised of two experimental groups. The first group (n = 11) received one injection; the second group (n = 12) received two injections, one at 0 h and one at 8 h. The cows in which CL were collected after 24 and 48 h received one injection every 8 h. Semiquantitative reverse transcriptase-polymerase chain reaction was used to evaluate the mRNA encoding ECE-1, ET(A), and ET-1. The ECE-1 and ET(A) proteins were evaluated by semiquantitative Western blot analysis. The ET-1 was the most likely component of the endothelin system target for PGF(2alpha) regulation during the midluteal phase. The ET(A) and ECE-1 genes were constitutively expressed in the Day 4 and Day 10 CL. A practical application of this observation is that it may be possible to target the ET-1 gene as a way to manipulate the luteolytic action of PGF(2alpha).

Experiments were conducted in beef cows without a primary CL, in which pregnancy had been maintained with exogenous progestogen. In preliminary trials, replacement CL induced ipsilateral to the embryo and after, rather than before, d 36 of pregnancy, maintained more pregnancies after withdrawal of exogenous progestogen (13/13 vs 2/6; P < 0.05). In Exp. 1, in cows with replacement CL induced by treatment with hCG on d 28 of pregnancy, treatment with flunixin meglumine on d 31 through 37 did not increase maintenance of pregnancy. Experiment 2 was conducted to evaluate directly the effects of concentrations of PGF2alpha and estradiol-17beta during d 31 through 35 of pregnancy on maintenance of pregnancy by replacement CL induced between d 28 and 31. In cows that maintained pregnancy while progestogen was provided, maintenance of pregnancy after withdrawal of exogenous progestogen tended to be greater with high (5/5) than with low (2/6; P < 0.10) concentrations of PGF2alpha and greater with low (6/7) than with high (2/6; P = 0.10) concentrations of estradiol-17beta. Secretion of progesterone by replacement CL was greater (P < 0.05) in cows with high than in those with low concentrations of PGF2, during d 31 through 35. Prostaglandin F2alpha may facilitate attachment of the bovine embryo (d 30 to 40) in a manner similar to that reported for implantation in other species. Cows that did not form CL in response to hCG on d 28 to 31 responded well when retreated after d 36. Again, maintenance of pregnancy was greater when replacement CL were induced after (9/9) rather than before d 36 (8/16; P < 0.05).

Three experiments were conducted to examine gene expression during induced luteal regression in the cow; the initial purpose was the identification of potential embryotoxins. In experiment 1, changes in gene expression in the corpus luteum (CL) were identified by differential display reverse transcription-polymerase chain reaction (DD-PCR) during the first 72 h of luteal regression in cows treated with prostaglandin F(2alpha) (PGF(2alpha)) on Days 4-7 after estrus. Expression of insulin-like growth factor-binding protein-1 (IGFBP-1) was up-regulated, with greatest expression at 24 h (P < 0.05) after treatment with PGF(2alpha) began. In experiment. 2, IGFBP-1 and its mRNA were quantified in CL collected 24 or 48 h after treatment with PGF(2alpha) on Day 4 or 10 after estrus. Because local mechanisms for exchange of hormones between the ovary and uterus are known in ruminants, uterine flushings were assayed for IGFBP-1 to seek evidence of local transfer of luteal IGFBP-1 to the uterus. IGFBP-1 mRNA was increased (P < 0.05) in CL 24 h after treatment when PGF(2alpha) that began on Day 10, and by 48 h after treatment that began on Day 4. Concentrations of IGFBP-1 increased (P < 0.05) in a pattern similar to mRNA, by 24 h on Day 10, and by 48 h on Day 4. Concentrations of IGFBP-1 in uterine flushings did not change on either day. Concentrations of progesterone decreased (P < 0.05) by 8 h after treatment with PGF(2alpha) that began on Day 10, but not until 24 h after treatment that began on Day 4. In experiment 3, cows received either saline or PGF(2alpha) and CL were collected 2 or 10 h after a single treatment, or 2 h after a second treatment that was given 8 h after the first. Expression of IGFBP-1 was increased by 2 h after treatment with PGF(2alpha) on both Days 4 and 10 after estrus. In conclusion, secretion of IGFBP-1 is increased during luteolysis, and may inhibit the steroidogenic effects of insulin-like growth factor-I (IGF-I), but no evidence was found to implicate IGFBP-1 in the embryotoxic effect of regressing CL.

Early luteal regression in cattle has an embryotoxic effect that is not overcome by replacement with progesterone, but is prevented by removal of the regressing CL. Two experiments were designed to test the null hypothesis that the luteal component of the embryotoxic effect is delivered by a systemic pathway. Beef heifers and cows (n = 39) received two good quality embryos, one placed into each uterine horn on Day 6 or 7 of the estrous cycle. Treated animals (n = 20) received 15 mg of PGF2alpha three times per day from Day 7 (n = 11; Experiment 1) or 5 (n = 9; Experiment 2) through 8; controls (n = 19) received saline. Progestogen replacement therapy (12 mg flurogestone acetate daily, s.c.) was provided from Day 6 (Experiment 1) or 4 (Experiment 2) until ultrasonographic diagnosis of embryo survival on Day 35 after estrus. The effects of treatment, location of the embryo and location by treatment interaction on embryo survival were tested by Chi square. In Experiment 1, there was no significant difference in embryo survival rate between PGF2alpha-treated and control recipients. In Experiment 2, only 6 of 18 embryos survived to Day 35 when transferred to animals treated with PGF2alpha compared to 12 of 18 in control animals (P< 0.05). The survival of embryos did not differ with location (adjacent or opposite to the regressing CL) or location by treatment interaction. Thus no evidence was obtained to support a local effect of the regressing CL. The embryo mortality associated with luteolytic doses of PGF2alpha in cows receiving replacement therapy with progestogen probably involves compounds that either act systemically or are transported via the uterine lumen to the uterine horn contralateral to the regressing CL.

Effects of day of estrous cycle and induced luteal regression on follicular dynamics in ewes were examined. In Exp. 1, ewes received PGF2 alpha (i.m.) on d 5, 8, or 11 after estrus (d 0) or saline on d 14 (n = 27, 27, 31, and 29, respectively). Ewes were laparotomized before treatment and again 48 h after induced estrus to record numbers, sizes, and locations of follicles > or = 2 mm and corpora lutea (CL). The two largest follicles (designated F1 and F2) and any of equal size (F1.5 and F2.5) were marked with India ink in stroma around the follicle. Ewes treated on d 8 ovulated fewer F1 and F1.5 (39%) than ewes treated on d 5 (69%; P < .05); d 11 (51%) and 14 (56%) were intermediate. In Exp. 2, ewes received PGF2 alpha on d 5 or 8 (n = 15/d). Ovaries were examined with transrectal ultrasonography daily from 4 d before PGF2 alpha until ovulation, and on d 5 and 8 after ovulation. Diameters, locations, and ranks (at time of PGF2 alpha) of follicles > or = 2 mm and CL were recorded. Ewes treated on d 8 ovulated fewer F1 and F1.5 (35%) than ewes treated on d 5 (86%; P < .05). Ovulatory follicles at time of PGF2 alpha were larger on d 5 (4.7 +/- .2 mm) than on d 8 (4.0 +/- .2 mm; P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)

This study was conducted to determine the effects of the corpus luteum (CL) of pregnancy and(or) the previously gravid uterine horn (g-UH) on the incidence and site of ovulation and the duration of luteal function in postpartum (PP) ewes. Ewes were laparotomized at d 60 to 80 of gestation and assigned to one of four groups: 1) single g-UH, CL left intact (SI, n = 35); 2) single g-UH, CL removed (SR, n = 30); 3) twin g-UH, CL on both ovaries left intact (TI, n = 21); and 4) twin g-UH, CL removed from one ovary (TR, n = 32). On d 20 PP, the number of follicles on each ovary was determined at laparoscopy, and ewes received (i.m.) 750 IU of hCG. Ovarian structures were determined at laparoscopy on d 24 PP. Blood samples were collected daily from d 20 to 40 PP and assayed for progesterone. more follicles (P < .05) were observed at laparoscopy on d 20 PP in SI/TI than in SR/TR groups (8.4 vs 6.0, respectively; SE = .4). Overall, 52% of the ewes had visible CL 4 d after hCG, and 73% of the ewes had elevated levels of progesterone by 7 d after hCG. The number of g-UH (S vs T) or status of the CL (I vs R) did not affect these responses. For SR ewes with hCG-induced CL on a single ovary, the incidence of CL formation on the ovary adjacent to g-UH was less (P < .05) than that for SI ewes (0 vs 57, respectively). Removal of the CL from S ewes, but not from T ewes, reduced follicular development and ovulation in response to hCG. Perhaps there is a positive systemic effect of the CL of pregnancy on postpartum ovarian function in ewes.

In postpartum cows expected to have corpora lutea (CL) of normal (norgestomet-treated) compared to short (control) life spans, function of the largest follicle increases after an increase in concentrations of prostaglandin F2 alpha (PGF). To determine whether PGF alters follicular growth and subsequent life span of the CL, 43 crossbred beef cows (19 to 22 d postpartum) were assigned to one of four treatments: 1) control (C; n = 10), 2) control+PGF (CPGF; n = 10), 3) norgestomet (N; n = 13), 4) norgestomet+flunixin meglumine (NFM; n = 10). Flunixin meglumine inhibits prostaglandin endoperoxide synthase. On day 0, N and NFM cows received a 6 mg implant of norgestomet. From days 3 through 8, CPGF and NFM cows were injected every 8 hr with 10 mg PGF im or 1 g FM iv, respectively. Implants were removed on day 9. On day 11, each cow received 1000 IU of hCG im to induce formation of CL. Follicular growth was monitored by daily ultrasonography from days 6 through 11. In a majority of the cases (25/32), the largest follicle present on day 6 was still the largest on day 11; frequency of persistence did not differ with treatment. Rate of growth of the largest follicle was greater in CPGF than in N cows (.6 +/- .1 vs .3 +/- .1 mm/d, respectively; P less than .05) but did not differ between C and NFM cows (.4 +/- .1 and .5 +/- .1 mm/d, respectively). Concentrations of estradiol in NFM cows were higher (P less than .05) on day 3 and declined to concentrations similar to those of the other treatments on day 9.(ABSTRACT TRUNCATED AT 250 WORDS)

Two experiments were designed to examine whether hormonal profiles were related to luteal life span in pluriparous postpartum anestrous beef cows. Cows (Exp. 1, n = 34; Exp. 2, n = 23) received norgestomet (N) for 9 d or served as controls (C). Each cow received 1,000 IU human chorionic gonadotropin (hCG) 48 h after removal of N (d 0). Blood samples collected every 15 min for 8 h on d -5, 3 and 5 (Exp. 1) or on d -10 and -1 (Exp. 2) were assayed for luteinizing hormone (LH) and follicle stimulating hormone (FSH). Cortisol was determined in hourly samples collected on d -5 and in samples collected every 2 min during suckling on the same day (Exp. 1). Concentrations of 15-keto-13,14-dihydro-PGF2 alpha (PGFM) were determined in samples collected at 15-min intervals for 2 h on d -5, 3, 5 and 10 (Exp. 1). Estradiol-17 beta was measured in samples collected on d -5 (Exp. 1) or on d -10 and -1 (Exp. 2). Life span of induced corpora lutea was longer (P less than .05) in N than C cows. Percentages of N cows in which corpora lutea, formed in response to hCG, exhibited a normal life span were 83% on farm 1 and 25% on farm 2 (Exp. 1), and 90% (Exp. 2), compared with 0% in C cows. Concentrations of FSH were not affected by N but were lower (P less than .05) on d -5 in cows on farm 2 (.6 +/- .1 ng/ml) than in cows on farm 1 (.8 +/- .1 ng/ml). On d -5, a treatment X farm interaction (P less than .05) for mean LH was observed and frequency of pulses of LH was higher (P less than .01) in N than C cows (2.7 +/- .4 vs. .8 +/- .8 pulses/8 h). Neither cortisol nor PGFM was affected by N. Estradiol was increased in d -1 (6.1 +/- .5 vs 2.6 +/- .8 pg/ml; P less than .01) by N. It is suggested that pre-treatment with N enhanced life span of induced corpora lutea, in part, by influencing secretion of LH and development of follicles, but a threshold concentration of FSH was required for N to exert this effect.

Three experiments were conducted to examine the relationship between systemic concentrations of luteinizing hormone (LH) and estradiol-17 beta (E2) after withdrawal of progesterone in cycling ewes. In Exp. 1, ewes were assigned randomly to one of three treatments: laparotomy (C), removal of the luteal ovary (ULO), or ULO plus anesthesia with sodium pentobarbital for 6 h beginning 4 h after surgery. Anesthesia was used in an attempt to block the expected increase in tonic secretion of LH. Patterns of LH and E2 in these three groups did not differ during the 24-h experimental period. In Exp. 2, a longer period of anesthesia was utilized. Forty-eight ewes were assigned at random to one of four treatments: C, ULO, lutectomy or an intrafollicular injection of prostaglandin F2 alpha (PGF2 alpha). One-half of the ewes in each group were anesthetized with sodium pentobarbital from initiation of treatment (0 h) until 10 h after surgery. Sodium pentobarbital did not suppress the increases in LH and E2 after progesterone withdrawal. The regression of concentrations of E2 on concentration of LH was not significant. In Exp. 3, ewes were infused with either saline or dopamine after receiving an im injection of PGF2 alpha. Tonic secretion of LH increased after 4 h in ewes infused with saline, but not in ewes infused with dopamine. Despite the suppression of LH, concentrations of E2 increased in dopamine-treated ewes as in control ewes. Therefore, the initial increase in E2 after a decline of progesterone in cycling ewes is independent of increases in LH.

A study was done to test whether ovulatory follicles destined to form subfunctional corpora lutea differed from normal ovulatory follicles in steroidogenic function. Twenty-five ewes were treated with prostaglandin F2 alpha on d 11 of the estrous cycle, then unilaterally ovariectomized before (n = 13) or after (n = 12) the surge of luteinizing hormone (LH) at the induced estrus to collect "control" follicles, which would have produced normal corpora lutea. In 15 ewes, the second ovary was removed 63 to 84 h later to collect "treated" follicles before (n = 7) or after (n = 8) the second expected surge of LH. Five ewes (control) were allowed to ovulate from the remaining ovary at first estrus and another five (treated) at the second estrus (3 to 4 d later). Treated ewes had lower serum progesterone than control ewes during the ensuing cycle (P less than .05). Treated follicles contained less estradiol in the theca (4.4 +/- .6 vs 10.0 +/- 2.5 ng; P less than .05), less androstenedione (.1 +/- .1 vs 1.0 +/- .2 ng) and estradiol (.5 +/- .1 vs 2.9 +/- 2.2 ng) in the granulosa (P less than .05) and less progesterone in the follicular fluid (.8 +/- .4 vs 3.3 +/- .8 ng; P less than .05) than control follicles, when removed before the surge of LH. Follicles removed after the surge of LH did not differ. In conclusion, ovulatory follicles with low steroidogenic function became corpora lutea that secreted lower-than-normal quantities of progesterone.

Prostaglandin F2 alpha (PGF2 alpha) was injected on d 5, 8 or 11 postestrus in ewes to determine how stage of the estrous cycle would affect PGF2 alpha-induced changes in concentrations of ovarian and pituitary hormones and intervals to the onset of estrus and the preovulatory surge of luteinizing hormone (LH). Initial concentrations of progesterone and average values during the 12 h after PGF2 alpha were related positively to the day of cycle on which PGF2 alpha was administered. Patterns of decline in progesterone after injection of PGF2 alpha were similar among the 3 d. Concentrations of LH in plasma increased in a similar manner from 0 to 12 h in all ewes. After 12 h LH continued to increase, plateaued or declined in ewes treated on d 5, 8 or 11, respectively. Initial concentrations of follicle stimulating hormone (FSH) in plasma were related positively to day of treatment. After treatment with PGF2 alpha, FSH increased within 2 h on d 5 but declined by that time on d 8 or 11. Concentrations of estradiol following treatment did not vary with day. The onset of estrus and the preovulatory surge of LH occurred at 36 and 35, 40 and 45, and 48 and greater than 48 h in ewes treated on d 5, 8 or 11, respectively. It is concluded that: 1) the initial increase in LH is dependent on a decrease in plasma progesterone and 2) differences in patterns of secretion of gonadotropins before the preovulatory surge of LH might be caused by differences in progesterone or progesterone:-estradiol ratio when luteal regression is induced on different days of the estrous cycle.

Two studies were conducted to investigate the effect of a progesterone-releasing intrauterine contraceptive device (IUD) on uterine secretion of prostaglandin (PG) F2alpha associated with IUD-induced luteolysis in sheep. In bilaterally ovulating ewes receiving an IUD on day 3 (estrus = day 0) intrauterine release of progesterone (90 microgram per day) by the device did not prevent premature luteolysis or the increased concentration of PGF2alpha in uterine venous plasma on day 5 caused by conventional IUD's. In the second study a progesterone-releasing IUD (645 microgram per day) inserted adjacent to the intact ovary at unilateral ovariectomy on day 14 inhibited early luteolysis in the subsequent estrous cycle and reduced the uterine secretion of PGF2alpha on day 5, without affecting concentrations of progesterone in the peripheral circulation at the intervening estrus. It is suggested that locally delivered intrauterine progesterone can inhibit IUD-induced luteolysis in the ewe by suppressing the uterine secretion of PGF2alpha.

The objective of this study was to determine if corpora lutea anticipated to have short lifespans were more responsive to the luteolytic action of prostaglandin F2 alpha (PGF2 alpha) than corpora lutea anticipated to have normal lifespans. Sixteen cows were allotted randomly to a hysterectomized-control (HC) or hysterectomized-progestogen (norgestomet) implant (HN) group. To verify that progestogen treatment of postpartum cows prior to induction of ovulation with gonadotropin-releasing hormone (GnRH) results in an increased number of cows exhibiting normal-length luteal phases, 21 additional cows were allotted randomly to a uterine intact-control (IC) or a uterine intact-progestogen implant (IN) group. Cows allotted to the HN and IN groups received norgestomet ear implants for 9 d beginning 17 to 21 d postcalving. All cows were injected (i.m.) with 100 micrograms GnRH 28 to 32 d postcalving (48 h after implant removal in the HN and IN groups) to induce ovulation. Two or 3 d after GnRH injection (d 0), cows in the HC (n = 8) and HN (n = 8) groups were hysterectomized to remove the major endogenous source of PGF2 alpha, and on d 7 cows were injected (i.m.) with 10 mg PGF2 alpha to assess luteal sensitivity. The proportion of corpora lutea having normal lifespans was greater (P less than .1) for the IN than for the IC group. In HC and HN groups, concentration of progesterone (P) increased similarly from d 0 to 6. Injection of PGF2 alpha in HC and HN groups on d 7 decreased (P less than .01) concentration of P approximately 50% by 6 h after injection (similar for both groups). Complete luteolysis was induced by PGF2 alpha in none of eight and two of eight cows in the HC and HN groups, respectively. In remaining cows (HC and HN groups) concentration of P increased (P less than .01; similar for HC and HN groups) beginning 24 h after PGF2 alpha and remained elevated through d 30 to 34 (end of experimental-period). In summary, corpora lutea anticipated to be short-lived were not more responsive to PGF2 alpha than corpora lutea anticipated to have normal lifespans.

Two experiments were conducted in peripuberal ewe lambs to determine (a) the influence of the first luteal structure [most frequently a transient (i.e., 1 to 4 d) structure] on subsequent ovarian activity and (b) a role for the uterus in its demise. In Exp. 1, 21 lambs were assigned randomly on the day of the first rise in progesterone in the plasma to (1) sham-operation, (2) removal of the nonluteal ovary, (3) removal of the luteal ovary and (4) removal of the luteal ovary plus progesterone replacement (5 mg given three times 12 h apart, initiated at surgery). No effect of treatment on subsequent ovarian activity was observed. In Exp. 2, four of 14 lambs were assigned randomly to be hysterectomized before their first rise in progesterone. The remaining 10 lambs, (five each) were sham-operated or hysterectomized on the day of the first rise in progesterone. All hysterectomized lambs (N = 9) exhibited a rise in progesterone and maintained elevated concentrations of progesterone, whereas sham-operated lambs initiated estrous cycles. Oviducts and uteri collected from lambs hysterectomized on the day of the first rise in progesterone in Exp. 2 were flushed for presence of oocytes and none were found. Similarly, no retained oocytes were found in histological preparations of first luteal structures obtained from eight lambs ovariectomized in Exp. 1. Concentrations of luteinizing hormone (LH) and prolactin were determined in daily samples collected from 19 lambs in Exp. 1. Luteinizing hormone increased and became more variable as lambs matured, whereas prolactin decreased with no detectable change in variability. It is concluded that the transient luteal structure is not required for sexual maturation and that its lifespan is uterine dependent.

The objectives of this study were to determine 1) the ability of charcoal-extracted bovine follicular fluid (bFF) to suppress endogenous follicle stimulating hormone (FSH) at various stages of the estrous cycle and 2) the effects of suppression of FSH on luteal function and lengths of the current and subsequent estrous cycles. Twenty-six mature ewes were assigned randomly to receive 5 ml of either bFF or saline, subcutaneously, at 8-h intervals on d 1 through 5 (bFF n = 6; saline n = 3), d 6 through 10 (bFF n = 6; saline n = 3) or d 11 through 15 (bFF n = 6; saline n = 2) of the estrous cycle (d 0 = estrus). Blood was collected daily beginning at estrus and continued until the third estrus (two estrous cycles) or 40 d; more frequent samples were collected 2 h prior to initiation of treatment (0600), hourly for the first 8 h of treatment, then every 4 h until 0800 on the first day after treatment, and finally at 1600 and 2400 on that day. Plasma concentrations of FSH were lower (P less than .001) in bFF-treated than in saline-treated ewes. Treatment with bFF reduced (P less than .05) plasma concentrations of progesterone during the current but not during the subsequent estrous cycle. Treatment with bFF did not affect plasma concentrations of estradiol-17 beta. Administration of bFF on d 11 through 15 of the estrous cycle lengthened the interval from the decline in progesterone to estrus and the inter-estrous interval by approximately 3 and 4 d, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Seventy-three multiparous beef cows (21 to 35 d postpartum) were used in an experiment with a 2 X 4 factorial design to test the effects of pretreatment with norgestomet and preovulatory gonadotropic stimulation on the life span of corpora lutea induced by human chorionic gonadotropin (hCG). Cows were implanted subcutaneously for 9 d with norgestomet (3 mg; 35 cows) or sham-implanted (38 cows). HCG (1,000 IU) was administered in to all cows 36 h (time = 0) after implant removal. Each cow received one of the following treatments: (1) pregnant mare's serum gonadotropin (PMSG; 750 IU) in a single subcutaneous injection 48 h prior to hCG (-48 h); (2) follicle stimulation hormone (FHS-P) in saline (10 mg) in a series of four subcutaneous injections (2.5 mg/injection) at -48, -36, -24 and -12 h; (3) FSH-P in 5% beeswax-sesame oil (10 mg) administered as in 2; or (4) saline, administered as in 2. Samples of serum taken at the time of implantation, at the time of implant removal and daily on d 5 to 20 after administration of hCG were assayed for progesterone. Samples taken at 12-h intervals from -48 to +12 h were assayed for estradiol-17 beta. Cows were observed for estrus at 6-h intervals from 24 to 60 h after implant removal and twice daily until d 22. Seventy-one percent of the cows (25/35) were in standing estrus from 29 to 60 h after removal of a norgestomet implant, compared to 16% of the sham-implanted cows (six of 38). As determined by progesterone in serum and by palpation of the ovaries, 88% of the cows (64/73) formed corpora lutea in response to hCG, with a tendency (P less than or equal to .10) for a positive effect of pretreatment with norgestomet (94 vs 82%). The life span of corpora lutea was longer (P less than or equal to .01) in cows treated with norgestomet (19.6 d) than in sham-implanted cows (13.4 d). Level of function of the induced corpora lutea as indicated by the pattern of daily concentrations of progesterone in serum was increased by pretreatment with either norgestomet (P less than .01) or gonadotropin (P less than .05), but there was no interaction of these treatments. The mean level of estrogen before ovulation was increased (P less than .01) by pretreatment with norgestomet or any regimen of gonadotropin, and, again, there was no significant interaction. Pretreatment with progestogen was associated with a higher pattern of concentration of estrogen over time (P less than .01).