Prostaglandin F2alpha Treatment Research Articles

Efficacy of Second Prostaglandin F2 Alpha Treatment during Ovsynch Protocol on Pregnancy Rate Inseminated with Sex-sorted Semen in Buffaloes

Background: Ovsynch is one of the most adopted estrus synchronization protocols used for synchronization of ovulation and fixed-time artificial insemination however, new corpus luteum (CL) induced by the first GnRH treatment in Ovsynch is inappropriately regressed with a single treatment with prostaglandin F2α. Inadequate CL regression reduces fertility. Considering this rationale, present research was implemented with the effect of second prostaglandin F2 alpha treatment during Ovsynch protocol on pregnancy rate inseminated with sex-sorted semen in buffaloes. Methods: Post-partum true anestrus multiparous buffaloes (n=24) were categorized as a group I (Ovsynch; n=12) and group II (Double PG Ovsynch; n=12). Buffaloes from group I were treated with standard Ovsynch protocol (D=7; G-P-G), while additional second prostaglandin F2 alpha injection was administered on day eighth (D=7; G-P-P-G) in group II (Double PG Ovsynch). All the buffaloes were inseminated 16-20 hrs after the last GnRH injection with sex-sorted semen. Result: The estrus signs exhibited were numerically higher in group II than in group I. The first service pregnancy rate in double PG Ovsynch was higher than the Ovsynch group. The addition of a second prostaglandin F2 alpha injection in the Ovsynch protocol improves the estrus quality and increases pregnancy rate.

Processes involved in prostaglandin F2alpha autoamplification in heifers.

Endometrial and luteal synthesis of prostaglandin F2alpha (PGF2A) occurs before and during luteolysis and is critical for luteal regression. This study demonstrates that PGF2A stimulates further PGF2A synthesis (autoamplification) apparently from the corpus luteum. Understanding the endocrine profile of prostaglandin F2alpha (PGF2A) autoamplification is fundamental to comprehend luteal and endometrial responses to PGF2A. On day 10 of postovulation (preluteolysis), heifers (n = 6/group) were treated intrauterine with saline or PGF2A (0.5 mg; hour 0). A third group received flunixin meglumine + PGF (FM+PGF) to prevent endogenous synthesis of PGF2A. Exogenous PGF2A was metabolized at hour 2 as measured by PGF2A metabolite (PGFM). From hours 5 to 48, concentrations of PGFM were greatest in the PGF group, smallest in the FM+PGF, and intermediate in the control suggesting endogenous synthesis of PGF2A only in PGF group. Progesterone (P4) concentrations decreased transiently between hours 0 and 1 in PGF and FM+PGF groups but rebounded to pretreatment concentrations by hours 6 and 4, respectively. No control or FM+PGF heifers underwent luteolysis during the experimental period. Conversely, in the PGF group, one heifer had complete luteolysis (P4 < 1 ng/mL), two heifers had partial luteolysis followed by P4 and CL resurgence by hour 48, and three heifers did not undergo luteolysis. Endogenous PGF2A appears to be of luteal origin due to the lack of pulsatile pattern of PGFM and lack of endometrial upregulation of oxytocin receptor (typical of endometrial synthesis of PGF2A), whereas luteal downregulation of PGF receptor and HPGD indicates a classic luteal response to PGF2A signaling although other specific mechanisms were not investigated. The hypothesis was supported that a single PGF2A treatment simulating the peak of a natural luteolytic pulse and the uteroovarian transport of PGF2A stimulates measurable endogenous PGF2A production.

Estrus Synchronization of Replacement Gilts Using Estradiol Cipionate and PGF2α and Its Effects on Reproductive Outcomes.

In this study, we evaluated the effectiveness of using estrogen-induced prolonged luteal function followed by prostaglandin F2 alpha (PGF2α) treatment to synchronize estrus in gilts. On day12 of the estrus cycle (D0 = first day of standing estrus), 52 gilts were assigned at random to two experimental groups: non-treated gilts (CON, n = 22), serving as controls, and prolonged luteal function group (CYP, n = 30), receiving a single treatment with 10 mg of estradiol cypionate intramuscularly Starting on day 12, blood samples were collected for estradiol and progesterone assays. Estrus detection started on day 17. Gilts from the CON group were inseminated at the onset of natural estrus. On day 28 CYP gilts were treated with PGF2α to induce luteolysis and inseminated at the onset of estrus. Gilts were slaughtered 5 d after the last insemination. A single treatment with estradiol cypionate prolonged luteal function in 90% of treated gilts. The duration of the estrous cycle was longer (p < 0.0001) for CYP gilts compared to CON gilts. CYP gilts showed synchronized estrus 3.96 ± 0.19 d after induction of luteolysis. The conception rate was similar (p = 0.10) for CON and CYP gilts. No difference was observed in the embryo recovery rate (p = 0.18) and total number of embryos per female (p = 0.06). The percentage of unfertilized oocytes, fragmented embryos and viable embryos was similar among females from CON and CYP groups (p > 0.05). The treatment of gilts with a single application of 10 mg of estradiol cypionate on day 12 of the estrous cycle was effective in prolonging luteal function and treatment with PGF2α resulted in synchronized estrus. Additionally, the synchronization protocol had no deleterious effect on fertility and embryonic development.

Endometrial and luteal responses to a prostaglandin F2alpha pulse: a comparison between heifers and mares†.

In heifers and mares, multiple pulses of prostaglandin F2alpha (PGF) are generally associated with complete luteal regression. Although PGF pulses occur before and during luteolysis, little is known about the role of minor PGF pulses during preluteolysis on subsequent luteal and endometrial PGF production that may initiate luteolysis. Heifers (n = 7/group) and mares (n = 6/group) were treated with a single minor dose of PGF (3.0 and 0.5mg, respectively) during mid-luteal phase (12 and 10days postovulation respectively). After treatment, a transient decrease in progesterone (P4) concentrations occurred in heifers between Hours 0 and 2 but at Hour 4 P4 was not different from pretreatment. In mares, P4 was unaltered between Hours 0 and 4. Concentrations of P4 decreased in both species by Hour 24 and complete luteolysis occurred in mares by Hour 48. Luteal and endometrial gene expression were evaluated 4h posttreatment. In heifers, luteal mRNA abundance of PGF receptor and PGF dehydrogenase was decreased, while PTGS2, PGF transporter, and oxytocin receptor were increased. In the heifer endometrium, receptors for oxytocin, P4, and estradiol were upregulated. In mares, luteal expression of PGF receptor was decreased, while PGF transporter and oxytocin receptor were increased. The decrease in P4 between Hours 4 and 24 and changes in gene expression were consistent with upregulation of endogenous synthesis of PGF. The hypotheses were supported that a single minor PGF treatment upregulates endogenous machinery for PGF synthesis in heifers and mares stimulating endogenous PGF synthesis through distinct regulatory mechanisms in heifers and mares.

Effect of the Interval From Prostaglandin F2alpha Treatment to Ovulation on Reproductive Efficiency Rates in a Commercial Equine Embryo Transfer Program

In the present study, 2.228 cycles of 180 Polo Argentino donor mares from an embryo transfer program in Argentina were examined to evaluate the effects of: (1) Interval from Prostaglandin F2alpha analog treatment to ovulation (ITO) on embryo recovery rate (ERR); (2) ITO on number of embryos per flushing (EPF); (3) ITO on multiple ovulation (MO) rate; (4) ITO from donor mare on pregnancy rate (PR) in recipient mares. Mares were inseminated with fresh semen from 31 fertile stallions in the induced estrus. Embryo flushing was performed 7–8 days postovulation. Following embryo flushing, donor mares were treated with prostaglandin F2alpha analog (cloprostenol 250 μg). The ERR increased along with the ITO (P = .01), with the lowest ERR (30.7%) for mares with an ITO of <4 days, and the highest (78.3%) in mares with an ITO of 10 days. The ITO from the donor mare in which the embryo was recovered did not have a significant effect on PR: ITO <6, 6 to 10, and >10 days were 74.6, 81.4, and 77.3%, respectively. The number of EPF and MO rate increased gradually along with the ITO (P < .05). In conclusion, the results of this study indicated that the ITO is positively correlated with the embryo recovery and the multiple ovulation rate.

Profiling of luteal transcriptome during prostaglandin F2-alpha treatment in buffalo cows: analysis of signaling pathways associated with luteolysis.

In several species including the buffalo cow, prostaglandin (PG) F2α is the key molecule responsible for regression of corpus luteum (CL). Experiments were carried out to characterize gene expression changes in the CL tissue at various time points after administration of luteolytic dose of PGF2α in buffalo cows. Circulating progesterone levels decreased within 1 h of PGF2α treatment and evidence of apoptosis was demonstrable at 18 h post treatment. Microarray analysis indicated expression changes in several of immediate early genes and transcription factors within 3 h of treatment. Also, changes in expression of genes associated with cell to cell signaling, cytokine signaling, steroidogenesis, PG synthesis and apoptosis were observed. Analysis of various components of LH/CGR signaling in CL tissues indicated decreased LH/CGR protein expression, pCREB levels and PKA activity post PGF2α treatment. The novel finding of this study is the down regulation of CYP19A1 gene expression accompanied by decrease in expression of E2 receptors and circulating and intra luteal E2 post PGF2α treatment. Mining of microarray data revealed several differentially expressed E2 responsive genes. Since CYP19A1 gene expression is low in the bovine CL, mining of microarray data of PGF2α-treated macaques, the species with high luteal CYP19A1 expression, showed good correlation between differentially expressed E2 responsive genes between both the species. Taken together, the results of this study suggest that PGF2α interferes with luteotrophic signaling, impairs intra-luteal E2 levels and regulates various signaling pathways before the effects on structural luteolysis are manifest.

Analysis of 20alpha-hydroxysteroid dehydrogenase expression in the corpus luteum of the buffalo cow: effect of prostaglandin F2-alpha treatment on circulating 20alpha-hydroxyprogesterone levels

BackgroundDuring female reproductive cycles, a rapid fall in circulating progesterone (P4) levels is one of the earliest events that occur during induced luteolysis in mammals. In rodents, it is well recognized that during luteolysis, P4 is catabolized to its inactive metabolite, 20alpha-hydroxyprogesterone (20alpha-OHP) by the action of 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) enzyme and involves transcription factor, Nur77. Studies have been carried out to examine expression of 20alpha-HSD and its activity in the corpus luteum (CL) of buffalo cow.MethodsThe expression of 20alpha-HSD across different bovine tissues along with CL was examined by qPCR analysis. Circulating P4 levels were monitored before and during PGF2alpha treatment. Expression of 20alpha-HSD and Nur77 mRNA was determined in CL at different time points post PGF2alpha treatment in buffalo cows. The chromatographic separation of P4 and its metabolite, 20alpha-OHP, in rat and buffalo cow serum samples were performed on reverse phase HPLC system. To further support the findings, 20alpha-HSD enzyme activity was quantitated in cytosolic fraction of CL of both rat and buffalo cow.ResultsCirculating P4 concentration declined rapidly in response to PGF2alpha treatment. HPLC analysis of serum samples did not reveal changes in circulating 20alpha-OHP levels in buffalo cows but serum from pseudo pregnant rats receiving PGF2alpha treatment showed an increased 20alpha-OHP level at 24 h post treatment with accompanying decrease in P4 concentration. qPCR expression of 20alpha-HSD in CL from control and PGF2alpha-treated buffalo cows showed higher expression at 3 and 18 h post treatment, but its specific activity was not altered at different time points post PGF2alpha treatment. The Nur77 expression increased several fold 3 h post PGF2alpha treatment similar to the increased expression observed in the PGF2alpha-treated pseudo pregnant rats which perhaps suggest initiation of activation of apoptotic pathways in response to PGF2alpha treatment.ConclusionsThe results taken together suggest that synthesis of P4 appears to be primarily affected by PGF2alpha treatment in buffalo cows in contrast to increased metabolism of P4 in rodents.

An efficient protocol for inducing pseudopregnancy using estradiol dipropionate and follicular development associated with changes in reproductive hormones after prostaglandin F2alpha treatment in pseudopregnant sows

BackgroundUtilization of estrus synchronization program in livestock industry would provide greater options for reproductive management in herd. To develop a convenient method for estrus synchronization in pigs, we determined the effective protocol using estradiol dipropionate (EDP) for the establishment of pseudopregnancy and investigated follicular development during the estrus synchronization with prostaglandin F2alpha (PGF2alpha) in association with reproductive hormone profiles in pseudopregnant sows.MethodsIn Experiment 1, the effective dose (0, 10, 20, or 30 mg) and timing (5, 8, 11 or 13 days after ovulation) of a single administration of EDP in cyclic pigs for the induction of pseudopregnancy were investigated. In Experiment 2, four pseudopregnant sows were treated with PGF2alpha twice at a 24-h interval between 24 and 28 days after EDP treatment. The changes in plasma concentrations of reproductive hormones were analyzed by time-resolved fluoroimmunoassay. Follicular development and ovulation following PGF2alpha administration were monitored by transrectal ultrasonography.ResultsHigh efficiency (greater than 80%) of pseudopregnancy was achieved with a single treatment with 20 mg of EDP at 8 and 11 days after ovulation (equivalent to 9-13 days after the onset of estrus). Plasma estradiol-17beta concentrations in pseudopregnant sows were significantly higher between 12 h and 7 days than before EDP treatment. Total inhibin concentrations significantly decreased following EDP treatment and remained low for 14 days. The number of small follicles was increased from 6.3 +/- 2.6 at PGF2alpha treatment to 22.8 +/- 4.8 at 3 days later; this was associated with increased plasma concentrations of inhibin. Onset of estrus was detectable in all sows on 5.3 +/- 0.3 days after PGF2alpha treatment and the number of ovulated follicles was 15.5 +/- 1.4 detected at 7.6 +/- 0.2 days after the treatment.ConclusionsThis study has defined the effective dose and timing of EDP treatment for inducing pseudopregnancy in cyclic pigs. Our results also indicated that EDP caused a lowering of inhibin concentrations during pseudopregnancy and small numbers of follicles from 20 to 28 days after EDP. In contrast, EDP-induced pseudopregnancy appears to have no adverse effect on follicular development and subsequent ovulation following PGF2alpha administration.

Markers of Ovarian Antral Follicular Development in Sheep: Differences Between Final (FW) and Penultimate (PW) Wave Pre-Ovulatory Follicles.

Treatment of non-prolific Western white-faced (WWF) ewes with prostaglandin F2alpha (PGF2alpha) and medroxy progesterone acetate (MAP) increased ovulation rate. The increase in ovulation rate was achieved largely by the addition of the ovulation of follicles from the penultimate (PW) follicular wave of the cycle to the expected ovulation of follicles from the final (FW) wave of the cycle. However, the number of lambs born after this treatment was not enhanced, suggesting that the PW follicles ovulating were not functionally viable. We were interested to compare the functional viability of PW and FW follicles. The objective of this study was to evaluate the expression of markers of vascularization and/or angiogenesis: vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), endothelial nitric oxide synthase (eNOS) and Factor VIII; a marker of gap junctional communication, connexin (Cx) 43; and a marker of cellular proliferation, proliferating cell nuclear antigen (PCNA) in preovulatory follicles obtained from the PW and FW in WWF ewes treated as described above. On day 8 of the estrous cycle, 15 ewes were given a single injection of PGF2alpha (15 mg i.m) and an intravaginal MAP sponge, which remained in place for 6 days. Thirty six to forty eight hours after sponge removal, ewes were ovariectomized by mid-ventral laparotomy. Ovaries that contain FW and/PW follicles from 7 ewes were fixed in Carnoy's solution for immunohistochemical detection of selected proteins. Computerized image analysis was used for quantification of protein expression. Granulosa and theca were isolated from FW and PW follicles from the remaining 8 ewes, suspended in PBS and stored at -80°C until RNA extraction. Messenger RNA levels were quantified using real-time PCR. Prostaglandin F2alpha and MAP treatment gave rise to preovulatory follicles emerging both from the FW and PW of the cycle; PW follicles had a longer (P<0.05) lifespan compared to FW follicles (7.1 ± 0.2 vs 3.0 ± 0.2). Protein expression for VEGF, eNOS, Cx43, PCNA and Factor VIII was greater in FW than in PW preovulatory follicles (P<0.05). For granulosa cells, Cx43 mRNA expression was greater (P<0.05) in FW than in PW preovulatory follicles. For theca cells, VEGF mRNA expression was greater (P<0.05) in FW than PW preovulatory follicles. In conclusion, expression of some markers of vascularization/ angiogenesis, gap junctional communication and cell proliferation appeared to be decreased in PW compared to FW preovulatory follicles. This suggests that although the treatment with PGF2alpha and MAP enhanced ovulation rate by extending the lifespan of PW follicles, these follicles were not as functionally viable as FW follicles. Therefore, this treatment provides a useful model to study aged antral follicles. (poster)

Prostaglandin F2-alpha receptor (FPr) expression on porcine corpus luteum microvascular endothelial cells (pCL-MVECs)

BackgroundThe corpus luteum (CL) is a transient endocrine gland and prostaglandin F2-alpha is considered to be the principal luteolysin in pigs. In this species, the in vivo administration of prostaglandin F2-alpha induces apoptosis in large vessels as early as 6 hours after administration. The presence of the prostaglandin F2-alpha receptor (FPr) on the microvascular endothelial cells (pCL-MVECs) of the porcine corpus luteum has not yet been defined. The aim of the study was to assess FPr expression in pCL-MVECs in the early and mid-luteal phases (EL-p, ML-p), and during pregnancy (P-p). Moreover, the effectiveness of prostaglandin F2-alpha treatment in inducing pCL-MVEC apoptosis was tested.MethodsPorcine CLs were collected in the EL and ML phases and during P-p. All CLs from each animal were minced together and the homogenates underwent enzymatic digestion. The pCL-MVECs were then positively selected by an immunomagnetic separation protocol using Dynabeads coated with anti-CD31 monoclonal antibody and seeded in flasks in the presence of EGM 2-MV (Microvascular Endothelial Cell Medium-2). After 4 days of culture, the cells underwent additional immunomagnetic selection and were seeded in flasks until the confluent stage.PCR Real time, western blot and immunodetection assays were utilized to assess the presence of FPr on pCL-MVEC primary cultures. Furthermore, the influence of culture time (freshly isolated, cultured overnight and at confluence) and hormonal treatment (P4 and E2) on FPr expression in pCL-MVECs was also investigated. Apoptosis was detected by TUNEL assay of pCL-MVECs exposed to prostaglandin F2-alpha.ResultsWe obtained primary cultures of pCL-MVECs from all animals. FPr mRNA and protein levels showed the highest value (ANOVA) in CL-MVECs derived from the early-luteal phase. Moreover, freshly isolated MVECs showed a higher FPr mRNA value than those cultured overnight and confluent cells (ANOVA). prostaglandin F2-alpha treatment failed to induce an apoptotic response in all the pCL-MVEC cultures.ConclusionOur data showing the presence of FPr on MVECs and the inability of prostaglandin F2-alpha to evoke an in vitro apoptotic response suggest that other molecules or mechanisms must be considered in order to explain the in vivo direct pro-apoptotic effect of prostaglandin F2-alpha at the endothelial level.

Temporal Associations among Pulses of 13,14-Dihydro-15-keto-PGF2alpha, Luteal Blood Flow, and Luteolysis in Cattle1

Luteal blood flow was studied in heifers by transrectal color-Doppler ultrasound. Data were normalized to the decrease in plasma progesterone to <1 ng/ml (Day 0 or Hour 0). Blood flow in the corpus luteum (CL) was estimated by the percentage of CL area with color flow signals. Systemic prostaglandin F2alpha (PGF) treatment (25 mg; n=4) resulted in a transient increase in CL blood flow during the initial portion of the induced decrease in progesterone. Intrauterine treatment (1 or 2 mg) was done to preclude hypothetical secondary effects of systemic treatment. Heifers were grouped into responders (luteolysis; n=3) and nonresponders (n=5). Blood flow increased transiently in both groups; induction of increased blood flow did not assure the occurrence of luteolysis. A transient increase in CL blood flow was not detected in association with spontaneous luteolysis when examinations were done every 12 h (n=6) or 24 h (n=10). The role of PGF pulses was studied by examinations every hour during a 12-h window each day during expected spontaneous luteolysis. At least one pulse of 13,14-dihydro-15-keto-PGF2alpha (PGFM) was identified in each of six heifers during the luteolytic period (Hours -48 to -1). Blood flow increased (P<0.02) during the 3-h ascending portion of the PGFM pulse, remained elevated for 2 h after the PGFM peak, and then decreased (P<0.03) to baseline. Results supported the hypothesis that CL blood flow increased and decreased with individual PGFM pulses during spontaneous luteolysis.

Improving the Reproductive Efficiency by Zoo-Technical Methods at a Dairy Farm

This trial was conducted on a Hungarian dairy farm between July 2001 and December 2004. The objective of this work was to improve the reproductive efficiency with relatively high milk production. At the beginning of this trial blood and fodder samples were taken for checking the metabolic status of the animals in order to determine their health condition. The nutritive value of the daily ration for all groups met with the requirements of the Hungarian National Standard and almost all serum metabolic parameters differed between the milking and pregnant animals. Early pregnancy detection (by ultrasound) and ovulation synchronizing methods were introduced to optimize reproductive performance. The oestrus cycle was also checked by ultrasound and open cows were treated by the appropriate method in order to inseminate them as soon as possible. Efficiency of artificial insemination (AI) followed by a single prostaglandin F2alpha (PGF2alpha) and Ovsynch treatment was similar (30.8% and 29%) and less effective than AI after natural heat detection (37.1%). Provsynch (Pre-synch followed by Ovsynch) was the most effective ovulation synchronization method (conception rate=42.6%; p<0.01). Although milk production increased between 2002 and 2004 by approximately 600 kg per cow, the calving period decreased by 20 days and the number of AIs per pregnancy was also improved (0.8 AI per conception). These findings are really beneficial economically because the decrease in the calving interval returns profit for the dairy farms; one open day costs euro2.5/cow. Economical analysis showed a high profit ratio from the reproductive 'investment' on this farm. Every invested euro yielded approximately euro10.

Acquisition of luteolytic capacity: changes in prostaglandin F2alpha regulation of steroid hormone receptors and estradiol biosynthesis in pig corpora lutea.

The pig corpora lutea (CL) acquires luteolytic capacity at about Day 13 of the estrous cycle, after which luteolysis occurs in response to prostaglandin F2alpha (PGF2alpha) treatment. We postulated that differences in transcription factors such as the steroid hormone receptors may be responsible for the differences in PGF2alpha-induced gene expression after acquisition of luteolytic capacity. In these studies, we evaluated the effect of PGF2alpha on luteal expression of receptors for progesterone (nuclear and membrane progesterone receptor [PR]), estradiol (ERalpha and ERbeta), glucocorticoid, androgens, and two enzymes in estradiol synthesis (P450-17alpha and aromatase). Two experiments were conducted to examine the early (0.5 h, experiment I) and late (10 h, experiment II) effects of PGF(2alpha) on the expression of these receptors in CL with (Day 17) or without (Day 9) luteolytic capacity. PGF2alpha decreased ERalpha mRNA (35%) and increased ERbeta mRNA (558%) and protein (376%) only in Day 17 CL and not Day 9. The estradiol biosynthetic pathway was upregulated by PGF2alpha in Day 17 CL but not Day 9 CL, with a dramatic increase in aromatase mRNA and luteal estradiol content. Nuclear PR was not affected by PGF2alpha, but was greater (176%) in Day 9 CL, while a putative membrane PR was greater (156%) in Day 17 than Day 9 CL. There were no detectable changes in glucocorticoid or androgen receptor mRNA. Thus, luteolytic capacity is associated with upregulation of estradiol biosynthesis, which in conjunction with increased ERbeta expression and altered PR expression may promote luteolysis in the pig CL.

Accumulation of caspase-3 messenger ribonucleic acid and induction of caspase activity in the ovine corpus luteum following prostaglandin F2alpha treatment in vivo.

Caspase-3, a vertebrate homologue of the protein encoded by the Caenorhabditis elegans cell death gene, ced-3, induces apoptosis when overexpressed in eukaryotic cells. Since apoptosis occurs during corpus luteum (CL) regression in many species, including the ewe, these studies were conducted to 1) isolate a cDNA encoding ovine caspase-3, 2) measure steady state amounts of caspase-3 mRNA in the CL during luteolysis induced by prostaglandin F2alpha (PGF2alpha) and during the time of maternal recognition of pregnancy, and 3) measure changes in caspase activity during PGF2alpha-initiated luteal regression. Oligonucleotide primers corresponding to a human caspase-3 cDNA sequence were combined with total RNA from ovine CL in a reverse transcription-polymerase chain reaction-based procedure to amplify a 640-base pair partial cDNA with a nucleotide sequence 86% and 81% identical to the human and rat caspase-3 cDNAs, respectively. CL were collected from ewes at 0, 12, or 24 h after treatment with PGF2alpha on Day 10 of the estrous cycle and from nonpregnant and pregnant ewes on Day 12 or Day 14 of the cycle. Northern blot analysis of total cellular RNA from ovine CL and a radiolabeled ovine caspase-3 cRNA probe indicated the presence of a single mRNA transcript of approximately 2.5 kilobases. Levels of caspase-3 mRNA were approximately 3-fold higher (p < 0.05) in CL at 12 h and 24 h after PGF2alpha in comparison to those levels measured in matched CL from untreated ewes. There were no differences (p > 0.05) in amounts of caspase-3 mRNA in CL on Day 12 or Day 14 of the estrous cycle compared to Day 12 or Day 14 of pregnancy, respectively. Caspase activity in CL (measured by the ability of CL lysates to cleave an artificial caspase substrate) was also significantly (p < 0.05) increased in CL collected after treatment with PGF2alpha compared to CL collected from nontreated ewes. We conclude that physiological cell death during PGF2alpha-induced luteal regression in the ewe is mediated, at least in part, via increased expression and activity of the caspase family of pro-apoptotic proteases.

Prostaglandin f2alpha treatment in vivo, but not in vitro, stimulates protein kinase C-activated superoxide production by nonsteroidogenic cells of the rat corpus luteum.

Luteal regression is associated with the generation of reactive oxygen species (ROS). To determine the nature of the ROS generator, cells isolated from luteinized rat ovaries were examined for ROS production using luminol-amplified chemiluminescence (LCL). Cells cultured for 2-48 h exhibited minimal LCL, but there was a significant (30- to 50-fold), rapid (maximum at 3-5 min), and dose-dependent increase in LCL in response to phorbol ester (phorbol 12-myristate 13-acetate; TPA; ED50 = 0.03 microM) and diacylglycerol (1,2-dioctanoyl-glycerol; ED50 = 30 microM). The TPA-induced response was cell number dependent and was virtually abolished by superoxide dismutase, freezing, or heating (95 degrees C for 5 min). Zymosan, known to induce a phagocytic response in leukocytes, stimulated a superoxide (O2-.) response with a slow onset (maximum at 40 to 60 min) and a maximum about one third of that observed for TPA. The response to TPA and zymosan was inhibited by the NADPH/NADH-oxidase inhibitor, diphenylene iodonium (ID50 = 5 microM for TPA), but not by the mitochondrial inhibitors, potassium cyanide, rotenone, or sodium azide. Fractionation of cells by centrifugal elutriation showed that TPA-stimulated O2-. production coeluted with the nonsteroidogenic cells and that little, if any, O2-. generation coeluted with the steroidogenic cells. Cells isolated 1, 2, and 4 h after in vivo treatment with a luteolytic dose of prostaglandin F2alpha (PGF2alpha) showed a significant increase in TPA-stimulated O2-. production at 2 h, whereas luteal cells or corpora lutea incubated directly with 1 microM PGF2alpha did not show any increase in response. Corpora lutea isolated from naturally regressed ovaries (18 days after ovulation) showed a significantly elevated level of TPA-stimulated O2-. production. In conclusion, there is a superoxide generator in luteinized ovaries that is activated through a protein kinase C pathway, localized in nonsteroidogenic cells, transiently increased during PGF2alpha-induced luteolysis in vivo, and elevated during natural luteal regression.

Effect of interval from melengestrol acetate to prostaglandin F2 alpha on timed and synchronized pregnancy rates of beef heifers and cows.

The objective of this experiment was to determine the optimal interval from the last day of melengestrol acetate (MGA) feeding to prostaglandin F2 alpha (PGF2 alpha) treatment on pregnancy rates of beef heifers and cows. All females (149 heifers and 399 postpartum cows) were fed MGA (.5 mg) daily for 14 d and then administered PGF2 alpha (25 mg Lutalyse) 13, 15, and 17 d (Groups 1, 2, and 3, respectively) after the last day of MGA feeding. Females not in estrus the first 52 h after PGF2 alpha treatment were artificially inseminated 72 h after PGF2 alpha treatment. Females in estrus 0 to 52 h and 78 h to 6 d after PGF2 alpha treatment were inseminated at estrus. Blood sera (collected immediately before and 3 d after PGF2 alpha treatment) were assayed for progesterone concentrations. Pregnancy was determined 44 to 47 d after the 72-h AI by rectal examination. The intervals from MGA feeding to PGF2 alpha that had the highest 72-h AI pregnancy rates were 17 d for heifers (43%) and 15 d for cows (43%). Heifers with a 17-d interval had a higher (P < .05) 72-h AI pregnancy rate than heifers with 13-d and 15-d intervals, and cows with a 15-d interval had a higher (P < .05) 72-h AI pregnancy rate than cows with a 17-d interval. The 4-d synchronized pregnancy rates (the 72-h AI and the succeeding 3 d inseminations) for both heifers (44%) and cows (53%) were not different (P > .05) among groups. Fewer (P < .05) cows with a 17-d interval from MGA to PGF2 alpha had corpora lutea regression (by 72 h) after PGF2 alpha treatment than cows with 13-d and 15-d intervals. We interpret the results to indicate that the interval from MGA to PGF2 alpha treatment may influence 72-h AI pregnancy rates, that optimal intervals are 17 d for heifers and 15 d for cows, and that pregnancy rates are improved by insemination for 3 d after the 72-h AI.

Modulation of Cholesteryl Ester Hydrolase Messenger Ribonucleic Acid Levels, Protein Levels, and Activity in the Rat Corpus Luteum1

Previous studies have shown that the induction of functional luteolysis (loss of progesterone production) with either prostaglandin F2 alpha (PGF2 alpha) treatment or hypophysectomy (APX) diminished neutral cholesteryl ester hydrolase (CEH) activity in the corpus luteum (CL) and that prolactin (PRL) replacement of APX animals prevented luteolysis and maintained CEH activity at control levels. More recent studies have shown that CEH is the same protein as hormone-sensitive lipase (HSL) and that CEH/HSL activity may be regulated by phosphorylation. However, the possibility that CEH/HSL activity may be under transcriptional and/or translation control has not been excluded. Therefore, in the present study we examined whether PGF2 alpha treatment, APX, or inhibition of PRL secretion by bromocryptine (BrC) treatment modulated CEH/HSL mRNA and/or protein levels in a coordinate fashion with CEH activity. Furthermore, we examined whether CEH/HSL mRNA and/or protein levels changed after luteinization of the ovary and after natural functional regression. PGF2 alpha treatment and APX significantly reduced CEH activity; and PGF2 alpha treatment, APX, and BrC treatment significantly reduced CEH/HSL protein and mRNA levels. PRL replacement after APX substantially blocked the reductions in CEH activity, CEH/HSL protein, and CEH/HSL mRNA levels. PRL replacement during BrC treatment significantly inhibited the reductions in CEH/HSL protein and mRNA levels. CEH/HSL mRNA levels increased twofold after luteinization. Whereas CEH/HSL mRNA levels remained elevated after natural luteal regression, CEH/HSL protein significantly decreased. In summary, the luteolytic actions of PGF2 alpha, APX, and BrC resulted in coordinate reductions in luteal CEH activity, protein levels, and mRNA levels; PRL replacement significantly reversed the luteolytic effects of APX and BrC; natural luteal regression resulted in a reduction in CEH/HSL protein without a concomitant reduction in CEH/HSL mRNA. These results suggest that ovarian CEH activity is controlled at the level of both transcription and translation, and that PRL is important for continued CEH/HSL mRNA transcription in the CL.

Luteal maintenance in cattle after conceptus death during the first trimester of gestation

The objectives of this experiment were to evaluate effect of age of the conceptus at death on subsequent interval to estrus and to determine whether prostaglandin F2 alpha (PGF) treatment after induced conceptus death would reduce interval to estrus. Sixty-four beef cows were diagnosed pregnant by ultrasonography between 24 and 50 d of gestation. Nine pregnancy groups were identified; each encompassed a 3-d interval from d 24 through 50 of gestation. Cows were allocated at random within pregnancy group to be treated with colchicine to induce conceptus death. Forty-eight hours after colchicine treatment, conceptus death was verified by ultrasonography and each animal received either saline or PGF. There was no evidence of an effect of age of conceptus at death on the subsequent interval to estrus after either saline or PGF. Interval to estrus after saline was 13.5 +/- .8 d, which was greater (P less than .01) than the 6.2 +/- .8 d after PGF. After saline, the estimate for the linear effect of time on serum progesterone (P4) was -1.32 +/- .36 ng/d and was less (P less than .05) than -2.83 +/- .36 ng/d for cows given PGF. Cows were exposed to fertile bulls at the first postabortion estrus. Pregnancy rates were 37.5% for cows given saline vs 31.3% for those given PGF. These results indicate that interval to estrus after conceptus death between 24 and 50 d of gestation is not affected by the age of the conceptus and can be reduced by PGF treatment.

Behandlung von Eileiterschwangerschaften mit Prostaglandinen

In a study, the clinical use of prostaglandin F2 alpha in local and systemic application in women with ectopic pregnancies were studied (1, 9). Two different treatment schedules were defined and applied. In group A, patients with a diagnosed ectopic and beta-HCG level lower than 850 mIU/ml were treated with prostaglandin F2 alpha i.m. injected only. In group B, prostaglandin F2 alpha were injected in the chorionic cavity of the ectopic by laparoscopy after localisation with a thin needle. In spite of prostaglandin F2 alpha treatment, 6 of 30 patients (20.0%) had to be operated by microsurgery because of increasing serum beta-HCG levels. A control of tubal patency 6 month later showed one closed tube only (4.5%). Up to now, 8 spontaneous intrauterine pregnancies occurred in our study groups after successful prostaglandin F2 alpha treatment; one pregnancy was seen in a women with a single fallopian tube. The conserving treatment of one ectopic pregnancy using prostaglandin F2 alpha yields positive results, if serum beta-HCG levels are below 2000 mIU/ml.

Effect of PG F2 alpha treatment on conception rates of dairy cows treated with a GnRH agonist 12 to 14 days after Artificial Insemination

The effect of a GnRH agonist (10 ug Buserelin) on conception rate was determined when injected into dairy cows 12 to 14 days after Artificial Insemination (AI). The following factors were taken into account: previous treatment prior to AI with Prostaglandin F2 alpha, clinical history recorded prior to AI since last calving, parity and milk yield. A number of 118 cows, from one large dairy herd, were involved in this study. A total of 210 AI's were performed, followed by 140 GnRH treatments and 70 saline injections. Reproductive events were then recorded. The rank of AI was equally distributed among the two groups. 153 AI's were preceded by a Prostaglandin F2 alpha (Dinoprost - 25 mg) treatment among which 103 were subsequently GnRH-treated and 50 were controls. Plasma progesterone concentrations were determined daily for 34 days after AI in 13 treated and 13 control cows. An Early Pregnoncy Diagnosis (EPD) from milk progesterone concentration was performed on Day 21 after AI in all cases. Post-AI GnRH agonist treatment resulted in a significant enhancement of the conception rate: 60% vs 44%; p < 0.01, respectively for treated and control animals. PG F2 alpha treatment prior to GnRH injection had a major influence on the conception rate (62% vs 40%; p < 0.01). No effect was seen (54 % vs 55 %) in non-PG F2 alpha treated females which were subsequently injected respectively with GnRH or saline after AI. Previous clinical reproductive disorders, parity and milk yield had no significant effect. In non-pregnant treated animals, GnRH agonist treatment resulted in an increased rate of heat detection on days 20 – 25 after AI (91 % vs 74 %) and a higher fertility rate on the following AI was seen (59 % vs 44 %; p < 0.05). In conclusion, GnRH agonist treatment 12 to 14 days after AI only enhanced the conception rate of females which had previously been treated with PG F2 alpha. In non-pregnant cows, this treatment had also a benficial effect on heat detection and improved the conception rate at the subsequent AI.