Time Of Luteolysis Research Articles

Profiles of interferon-stimulated genes in multiple tissues and circulating pregnancy-associated glycoproteins and their association with pregnancy loss in dairy cows†.

Pregnancy loss (PL) in lactating dairy cows disrupts reproductive and productive efficiency. We evaluated the expression of interferon-stimulated genes (ISG) in blood leukocytes, vaginal and cervical epithelial cells, luteolysis-related genes, progesterone, and pregnancy-associated glycoprotein (PAG) profiles in lactating dairy cows (n = 86) to gain insight about PL. Expression of ISG on d17, d19, and d21 was greater in cows that maintained the pregnancy (P33) compared to nonpregnant with no PL (NP). Greater ISG differences between groups were observed in the cervix (96.7-fold) than vagina (31.0-fold), and least in blood leukocytes (5.6-fold). Based on individual profiles of ISG and PAG, PL was determined to occur either before (~13%) or after (~25%) d22. For cows with PL before d22, ISG expression was similar on d17 but by d21 was lower and OXTR was greater than P33 cows and similar to NP; timing of luteolysis was similar compared to NP cows suggesting embryonic failure to promote luteal maintenance and to attach to the endometrium (no increase in PAG). For cows with PL after d22, ISG expression was similar to P33 cows on d17, d19, and d21 and luteolysis, when it occurred, was later than NP cows; delayed increase in PAG suggested later or inadequate embryonic attachment. In conclusion, PL before d22 occurred due to embryonic demise/failure to signal for luteal maintenance, as reflected in reduced ISG expression by d21. Alternatively, embryos with PL between d22 and 33 adequately signaled for luteal maintenance (ISG) but had delayed/inadequate embryonic attachment and/or inappropriate luteolysis causing PL.

Effect of cloprostenol on luteolysis in jennies

An intensive farm system has progressively emerged with the rapid development of the donkey industry. One of the primary challenges impeding the development of the donkey industry and the preservation of endangered donkey species is the low reproductive efficiency of jennies (Feng Yang et.al. Equine Veterinary Journal 2021, 53: 1218-1226). In donkeys, luteolysis occurs between days 15 and 17 after ovulation (Carluccio A. et.al. Theriogenology, 2007, 68(6): 914-919). To shorten the estrus cycle, a new estrus cycle may start after luteolysis is induced by the PGF2α analog (Duccio P. et.al. Theriogenology, 2018, 121: 175-180). Treatment of mares and cattle with PGF2α analogs from the fifth day after ovulation induces luteolysis (Bergfelt DR. et.al. Theriogenology, 2006, 65(8): 1605-1619). However, information regarding luteolysis in jennies is limited. In this study, we aimed to investigate the effect of cloprostenol on luteolysis in jennies. The PGF2α analog used in the experiment was cloprostenol (0.2 mg/2 ml, Ningbo Second Hormone Factory, Zhejiang, China). To study the effects of different cloprostenol doses on the estrus cycle, jennies were treated with 0.2 mg (n=30), 0.4 mg (n=30), or 0.6 mg (n=30)cloprostenol (PGF2α), respectively. The time of luteolysis, ovulation interval, follicular size at ovulation, and ovulation rates were compared between the groups. The results showed that luteolysis rates in the jennies within 72 h of treatment were higher than 90% in the three treatment groups. The time required for luteolysis was not statistically different among the three groups. In addition, the estrus interval, ovulation interval, ovulation diameter, and ovulation rate did not differ among the three groups. To examine the timing effect of cloprostenol treatment on the estrus cycle, jennies were treated with 0.4 mg cloprostenol on the third (D 3, n=30), fifth (D 5, n=30), or seventh (D 7, n=30) day following ovulation, respectively. The results showed that the luteal phase of the D 3 treatment group was shorter than that of the D 5 and D 7 treatment groups, and in the D 5 treatment group was shorter than that of the D 7 group (P < 0.05). In can be concluded that treatment with 0.4 mg cloprostenol on the third day following ovulation shortens the length of the luteal phase and estrus cycle considerably.

Ovarian follicle dynamics in ewes treated with intra-vaginal progesterone pessaries. 2. Factors affecting timing of estrus and reproductive outcomes following artificial insemination.

This study extends observations on the effects of intra-vaginal progesterone treatment on the relationships between the time of luteolysis, emergence of the ovulatory follicle, timing of estrus and ewe fertility. Observations were made in progesterone - treated ewes in autumn, the spring equinox and late spring (Experiment 1, Data set 1) and in progesterone - treated ewes and naturally cycling ewes in autumn and the spring equinox (Experiment 1, Data set 2). In Data set 1, the day of emergence of both the first and second ovulatory follicle was positively related to the day luteal regression within each season. In turn, the day of emergence influenced the timing of estrus by means of a season by day of luteal regression interaction (P<0.001) indicating that the relationship was positive in autumn and the spring equinox but negative in late spring. In autumn, older ovulatory follicles were associated with an earlier onset of estrus compared with younger ovulatory follicles. In late spring, this relationship was reversed and was influenced by whether or not ewes were cycling at the time of pessary insertion. In Data set 2, the relationship between the day of follicle emergence and luteal regression was influenced by a treatment by day of regression interaction indicating the relationship was positive in treated ewes and negative in naturally cycling ewes. Timing of estrus was positively related (P<0.001) to both the day of luteal regression and the day of follicle emergence (P<0.05), with both relationships being stronger in naturally cycling ewes than in treated ewes. In Experiment 2, pregnancy rate following artificial insemination in autumn was highest (90.2%) when luteolysis occurred during Days 7-9 of the pessary period compared with Days 1-6 (77.8%, P=0.16), 10 to 12 (68.8%, P<0.05) or Days ≥13 (71.2%, P<0.05). Timing of estrus was not affected. The mean diameter of ovulatory follicles that emerged during Days 7-9 was larger on Day 12 (5.8±0.13mm) compared with other periods (range 4.7±0.05 to 5.6±0.14mm). This study provides two potential strategies to improve the success of AI programs. Firstly, appropriately timed treatment with PGF2α to control the time of emergence of ovulatory follicles and, secondly, earlier treatment with eCG to improve the development of ovulatory follicles that emerge late in the pessary period. Each is likely to be influenced by season and the cyclical status of the ewe.

Ovarian follicle dynamics in ewes treated with intra-vaginal progesterone pessaries. 1. Follicle waves and parameters of the estrous cycle

Progesterone treatment for synchrony of estrus is standard in sheep artificial insemination (AI) programs but can be associated with poor outcomes. Potential for improvement exists through a better understanding of the interactions between follicle development, luteal regression, emergence of the ovulatory follicle and timing of estrus. These interactions were examined by comparing progesterone-treated (Day 1 = day of pessary insertion) and naturally cycling ewes (Day 1 = day after estrus) at three times of the year (Autumn, Spring equinox and late Spring). Observations were made from Day 1 until the day of ovulation. Compared with the natural cycle, progesterone treatment (300 mg intra-vaginal pessary for 14 d) reduced the number of follicle waves (2.2 ± 0.18 versus 2.8 ± 0.12; P < 0.05) and increased the length of the ovulatory wave (8.6 ± 0.45 versus 6.6 ± 0.42 d; P < 0.05). The number of follicles per wave, the inter-wave interval and ovulation rate were not affected. However, progesterone treatment induced (P < 0.05) an earlier luteolysis (9.7 ± 0.51 versus 15.4 ± 0.49 d after Day 1), an earlier emergence of the ovulatory follicle (7.5 ± 0.48 versus 11.4 ± 0.46 d after Day 1) and an earlier onset of estrus (26.1 ± 2.95 versus 53.3 ± 2.84 h after Day 14). Time of year also influenced the response to progesterone treatment. In Autumn compared with the Spring equinox and late Spring, there was a reduction (P < 0.05) in follicle wave number (2.4 ± 0.21 versus 2.5 ± 0.29 versus 3.0 ± 0.20 respectively), follicles per wave (2.6 ± 0.27 versus 3.5 ± 0.25 versus 3.2 ± 0.20 respectively), ovulation rate (1.6 ± 0.12 versus 1.9 ± 0.12 versus 2.0 ± 0.10 respectively) and the inter-wave interval was longer (5.3 ± 0.40 versus 4.0 ± 0.32 versus 3.8 ± 0.27 d respectively; P < 0.05). Time of year also influenced (P < 0.05) the time of luteolysis (earliest in late Spring), emergence of the ovulatory follicle (earliest in Autumn) and onset of estrus (earliest in Autumn). It is concluded that (1) the effects of progesterone treatment on follicle waves are relatively minor, (2) the effects of treatment on timing of luteolysis, emergence of the ovulatory follicle and onset of estrus are all significant although the effects on AI outcomes remain to be determined and (3) time of year has a minimal effect on follicle waves but a more significant effect on other parameters of the estrous cycle. A better understanding of these complexities will assist in the development of improved protocols for synchrony of estrus.

Accessory corpus luteum induced by human chorionic gonadotropin on day 7 or days 7 and 13 of the estrous cycle affected follicular and luteal dynamics and luteolysis in lactating Holstein cows

Our objective was to determine the effect of inducing an accessory corpus luteum (CL) with human chorionic gonadotropin (hCG; 3,300 IU) on d 7 (hCG7) or 2 accessory CL with hCG on d 7 and 13 (hCG7+13) of the estrous cycle in noninseminated lactating Holstein cows. Cows (n = 86) between 39 and 64 DIM were pretreated with an Ovsynch + CIDR protocol, and only synchronized cows were used (n = 64). The day of the last GnRH of Ovsynch was considered d 0 of the estrous cycle. Follicular and luteal dynamics of cows were evaluated daily during an entire estrous cycle by ovarian ultrasonography. Blood samples were collected daily to measure serum concentration of progesterone (P4). Cows were randomly assigned to CON (n = 22, no treatment), hCG7 (n = 20), or hCG7+13 (n = 22) treatments. Two cows from hCG7+13 failed to ovulate after hCG and were removed from the analyses post-hCG treatment. The first day of luteolysis was considered the day that P4 declined to more than 2 SD of the mean for the 4 consecutive P4 concentrations with the greatest mean in late diestrus for each individual cow. The P4 cut-off for complete luteolysis was <1.0 ng/mL. Mean P4 on d 7 (3.23 ± 0.16 ng/mL) did not differ among treatments. Cows treated with hCG had greater total luteal and original CL volume and serum P4 during diestrus than CON. Cows treated with hCG7+13 had greater serum P4 after d 13 of the cycle than hCG7. Cycles were classified as having atypical cycles if the dominant follicle or future dominant follicle at the time of luteolysis did not ovulate (delayed ovulation; CON, n = 2; hCG7, n = 4; hCG7+13, n = 3), had a short cycle (CON, n = 1), delayed (CON, n = 2) or incomplete luteolysis (CON, n = 1; hCG7, n = 4; hCG7+13, n = 5). The remainder of cycles with normal complete luteolysis followed by ovulation were considered to be typical. Based on blood perfusion, the CON cow with incomplete luteolysis had 2 original CL remaining functional after first onset of luteolysis. The rest of the cows with incomplete luteolysis (9/10) had one or more CL regressing and at least one remaining functional after first onset of luteolysis. No specific pattern for CL side (ipsilateral vs. contralateral to a CL with complete regression) was observed for nonregressed CL. Cows with incomplete luteolysis had a second onset of luteolysis to undergo complete functional luteolysis. The proportion of cows with typical cycle was 73% (16/22) for CON, 60% (12/20) for hCG7, and 55% (11/20) for hCG7+13. Cows with typical cycles treated with hCG (hCG7 and hCG7+13) had a later onset of luteolysis, prolonged time to undergo complete luteolysis, and greater proportion of cows with 3 follicular waves than CON, resulting in a longer interovulatory interval for hCG7 and hCG7+13 than CON. In summary, accessory CL induced by hCG during diestrus not only altered follicular and luteal dynamics but also deferred and prolonged the luteolytic process.

235 Uterine Function During Maternal Recognition of Pregnancy Differs Due to Size of the Ovarian Reserve in Beef Heifers

Abstract The relationship of ovarian reserve parameters to fertility in mammalian females continues to be debated. We demonstrated improved uterine function, creating an environment that is more supportive of early embryonic development in beef heifers with increased numbers of follicles. In the present study we hypothesized that timing of luteolysis differs between heifers with increased compared to heifers with diminished numbers of follicles. Angus heifers (n = 20/ group) were classified as low (14.7 ± 1.1) or high (32 ± 1.1) antral follicle number based on ovarian ultrasonography, and artificially inseminated (d0) following a Select Synch protocol. At d16 after insemination, heifers were slaughtered, and reproductive tracts collected. Tracts were flushed with 20 mL of physiological saline to determine pregnancy status by presence of a conceptus and endometrial samples from the horn ipsilateral to the CL were frozen for determination of transcript abundance. Real-time RT-PCR was performed to determine relative transcript abundance of oxytocin receptor (OXTR) and interferon-stimulated gene-15 (ISG15) with glyceraldehyde-3-phospate dehydrogenase as the endogenous reference gene. Transcript abundance was analyzed using the MIXED procedure of SAS with follicle group, pregnancy status and the interaction as fixed effects. Transcript abundance of ISG15 was greater in pregnant endometrium than non-pregnant endometrium (2.75 ± 0.25 vs 0.16 ± 0.22-fold; P &amp;lt; 0.0001) with no influence of follicle group or the interaction. The interaction of follicle group and pregnancy status influenced OXTR transcript abundance (P = 0.06). Luteolytic mechanisms were activated in non-pregnant heifers with diminished numbers of follicles (1.46 ± 0.25-fold) and were not activated in pregnant heifers (0.06 ± 0.22-fold) or heifers with increased numbers of follicles that were not pregnant (0.32 ± 0.30-fold). These data indicate a wider window of recognition of pregnancy in beef heifers with increased numbers of ovarian follicles. USDA is an equal opportunity provider and employer.

Downregulated luteolytic pathways in the transcriptome of early pregnancy bovine corpus luteum are mimicked by interferon-tau in vitro

BackgroundMaintenance of the corpus luteum (CL) beyond the time of luteolysis is essential for establishing pregnancy. Identifying the distinct features of early pregnancy CL remains unresolved, hence we analyzed here the transcriptome of CL on day 18 pregnant (P) and non-pregnant (NP) cows using RNA-Seq. CL of P cows expressed ISGs, verifying exposure to the pregnancy recognition signal, interferon-tau (IFNT), whereas the CL of NP cows had elevated luteal progesterone levels, implying that luteolysis had not yet commenced.ResultsThe DEGs, IPA, and metascape canonical pathways, along with GSEA analysis, differed markedly in the CL of P cows from those of NP cows, at the same day of the cycle. Both metascape and IPA identified similar significantly enriched pathways such as interferon alpha/beta, sonic hedgehog pathway, TNFA, EDN1, TGFB1, and PDGF. However, type-1 interferon and sonic hedgehog pathways were positively enriched whereas most of the enriched pathways were downregulated in the P compared to NP samples. Thirty-four % of these pathways are known to be elevated by PGF2A during luteolysis. Notably, selective DEGs in luteinized granulosa cells were modulated by IFNT in vitro in a similar manner to their regulation in the CL of P cows.ConclusionThis study unraveled the unique transcriptomic signature of the IFNT-exposed, early pregnancy CL, highlighting the abundance of downregulated pathways known to be otherwise induced during luteolysis. These and IFNT-regulated in vitro pregnancy-specific DEGs suggest that IFNT contributes to the characteristics and maintenance of early pregnancy CL.

48 Timing of luteolysis and conceptus expulsion after induced pregnancy loss at three different timepoints after maternal recognition of pregnancy in cattle

Results from previous research indicate that induced pregnancy loss (IPL) on Day 36 of gestation in cattle resulted in luteolysis by gestation Day ∼45. The aim of this study was to test the hypothesis that IPL at 25, 35, or 55 days of gestation will result in luteolysis and whether the interval between IPL and luteolysis is positively associated with days of gestation at time of treatment. Pregnant non-lactating beef cows were randomly assigned to undergo IPL, consisting of an intrauterine infusion of 120mL of hypertonic saline (7.2%), at Day 25 (IPL25, n=15), Day 35 (IPL35, n=14), or Day 55 (IPL55, n=14). Corpus luteum (CL) volume and embryo/fetal viability were evaluated by transrectal B-mode ultrasonography daily for 19 days following treatment and every other day subsequently until luteolysis was identified or gestation Day 91. Blood flow through the CL was evaluated using colour-Doppler ultrasonography, and luteolysis was defined as the day when &amp;lt;25% of the CL was represented with colour pixels. Conceptus expulsion was defined as the absence of a conceptus and fluid accumulation in the uterus was &amp;lt;25%. Data were analysed using Proc Mixed in SAS (v9.4; SAS Institute Inc.). Cessation of conceptus heartbeat occurred in all animals within 24h of administration of treatment. Day of luteolysis occurred earlier (P&amp;lt;0.01) in IPL25 (5.3±0.4d) than IPL55 (8.9±1.4d) cows, and luteolysis tended (P=0.07) to occur earlier in IPL35 (6.6±0.6d) than IPL55 cows; however, values for this variable in IPL25 and IPL35 cows were similar (P&amp;gt;0.2). The variance in timing of luteolysis was greater for IPL55 than for IPL35 cows, whereas variance for IPL35 was greater than that for IPL25 (P&amp;lt;0.05) cows. Conceptus expulsion was detected earlier (P&amp;lt;0.01) in IPL25 (Day 4.2±0.4) and IPL35 (Day 5.8±0.5) than IPL55 (Day 10.4±1.4) cows; however, IPL25 and IPL35 cows had similar (P&amp;gt;0.1) values for this variable. Values for luteolysis and conceptus expulsion were correlated in IPL35 (r=0.84; P&amp;lt;0.01) and IPL55 (r=0.88; P&amp;lt;0.01) cows, whereas there was no correlation among values for these variables in IPL25 (r=0.31; P=0.3) cows. There were effects of treatment (P&amp;lt;0.01), time (P&amp;lt;0.01), and treatment by time interaction (P&amp;lt;0.01) on CL volume. Volume of the CL was less (P&amp;lt;0.04) in IPL25-treated cows by treatment day 5 compared with that in IPL55-treated cows. Additionally, CL volume was less (P&amp;lt;0.03) in IPL25 by treatment day 6 than in IPL35 cows, and IPL35 cows had a lesser (P&amp;lt;0.01) CL volume starting on treatment day 10 than IPL55 cows. Induction of pregnancy loss at different times of gestation resulted in luteolysis; however, the interval between treatment and luteolysis tended to increase as gestation days increased. Furthermore, the variability in the timing of luteolysis increased as gestation days increased. Results from the present study support the working hypothesis of presence of a continuous mechanism for CL maintenance during pregnancy beyond the classical maternal recognition period in cattle.

Progestogen profiling in plasma during the estrous cycle in cattle using an LC-MS based approach

In many mammalian species, corpus luteum derived progesterone (P4) is the main functional gestagen during the estrous cycle and pregnancy. P4 can be metabolized into various metabolites, of which some are biologically active. While some metabolites target the classical nuclear progesterone receptor (PR), neurosteroids bind the receptors of type A γ-aminobutyric acid (GABAA-r) in the brain. According to the position of reduction within the molecule, metabolites of P4 can be characterized into C20-reduced progestogens (20α-dihydroprogesterone (20α-DHP) and 20β-dihydroprogesterone (20β-DHP)), C3-reduced progestogens (3α-dihydroprogesterone (3α-DHP) and 3β-dihydroprogesterone (3β-DHP)), 5α-reduced progestogens (5α-dihydroprogesterone (5α-DHP), allopregnanolone and isopregnanolone) and 5β-reduced progestogens (5β-dihydroprogesterone (5β-DHP), pregnanolone and epipregnanolone). We questioned whether the reduced progestogens are present in bovine plasma during the estrous cycle and whether their profiles differed from the profile of the common precursor P4 around the time of luteolysis. The analytes were monitored in plasma samples using liquid chromatography mass spectrometry (LC-MS). While progestogens lagged behind the drop of P4 at luteolysis, they followed the profile of P4 during the estrous cycle. The abundance of P4 was predominant followed by allopregnanolone, pregnanolone, epipregnanolone and 20β-DHP. Further studies will need to focus particularly on the period around luteolysis.

Up-regulation of endometrial oxytocin receptor is associated with the timing of luteolysis in heifers with two and three follicular waves†.

Initiation of luteolysis in ruminants is variable due to ill-defined mechanisms. Cycles of two follicular waves are shorter and have earlier luteolysis than three-wave cycles. This study validated a cytobrush technique for evaluating dynamics of endometrial gene expression and associated changes in mRNA with timing of luteolysis, based on circulating progesterone and ultrasound-determined changes in blood flow and volume of corpus luteum (CL). On day 8 (ovulation=day 0), Holstein heifers were randomized into two groups: cytobrush group (n=9) had an endometrial sample collected every 48h from day 8 until end of luteolysis (CL blood flow ≤ 20%) and control group was sampled only once either before (day 12; n=4) or at the end of luteolysis (n=5). Concentrations of progesterone, CL blood flow, CL volume, and the frequency of two and three-wave cycles were similar between groups. Endometrial mRNA for progesterone receptors and estradiol receptors 1 and 2 was greater on day 8 and decreased thereafter similarly in two and three-wave cycles. Oxytocin receptor mRNA increased earlier in two vs three-wave cycles (day 14 vs 18), and the increase was associated with the onset of luteolysis. In conclusion, the cytobrush technique allowed in vivo collection of multiple endometrial samples during the estrous cycle. Endometrial mRNA expression of steroid receptors did not explain the variability in timing of onset of luteolysis in heifers while the later onset of luteolysis in three-wave cycles was associated with later up-regulation of oxytocin receptor mRNA.

Novel concepts on the role of prostaglandins on luteal maintenance and maternal recognition and establishment of pregnancy in ruminants

In ruminants, the corpus luteum (CL) of early pregnancy is resistant to luteolysis. Prostaglandin (PG)E2 is considered a luteoprotective mediator. Early studies indicate that during maternal recognition of pregnancy (MRP) in ruminants, a factor(s) from the conceptus or gravid uterus reaches the ovary locally through the utero-ovarian plexus (UOP) and protects the CL from luteolysis. The local nature of the embryonic antiluteolytic or luteoprotective effect precludes any direct effect of a protein transported or acting between the gravid uterus and CL in ruminants. During MRP, interferon tau (IFNT) secreted by the trophoblast of the conceptus inhibits endometrial pulsatile release of PGF2α and increases endometrial PGE2. Our recent studies indicate that (1) luteal PG biosynthesis is selectively directed toward PGF2α at the time of luteolysis and toward PGE2 at the time of establishment of pregnancy (ESP); (2) the ability of the CL of early pregnancy to resist luteolysis is likely due to increased intraluteal biosynthesis and signaling of PGE2; and (3) endometrial PGE2 is transported from the uterus to the CL through the UOP vascular route during ESP in sheep. Intrauterine co-administration of IFNT and prostaglandin E2 synthase 1 (PGES-1) inhibitor reestablishes endometrial PGF2α pulses and regresses the CL. In contrast, intrauterine co-administration of IFNT and PGES-1 inhibitor along with intraovarian administration of PGE2 rescues the CL. Together, the accumulating information provides compelling evidence that PGE2 produced by the CL in response to endometrial PGE2 induced by pregnancy may counteract the luteolytic effect of PGF2α as an additional luteoprotective mechanism during MRP or ESP in ruminants. Targeting PGE2 biosynthesis and signaling selectively in the endometrium or CL may provide luteoprotective therapy to improve reproductive efficiency in ruminants.

Efficacy of induction of luteolysis in superovulated cows is dependent on time of prostaglandin F2alpha analog treatment: effects on plasma progesterone and luteinizing hormone profiles

The objectives were to (1) evaluate the effectiveness of induction of luteolysis in superovulated (SOV) cows at two distinct time points after embryo flushing; and (2) compare the pattern of LH release after treatment with PGF in cows with single vs. multiple ovulations. In the first experiment, Holstein cows were SOV with 400 IU of FSH following standard procedures. Uterine flushing for embryo recovery was performed 7 days after artificial insemination (Day 0), and cows were randomly allocated into two groups to receive PGF (0.5-mg sodium cloprostenol, intramascular) either immediately after flushing (Day 7 group, N = 19) or 4 days later (Day 11 group, N = 20). Time of luteolysis was determined on the basis of plasma progesterone (P4) concentrations. There was no difference (P > 0.05) in plasma P4 before treatment between Day 7 and Day 11 groups. A decline in plasma P4 was observed 48 hours after PGF treatment in both the groups (P < 0.0001). In Day 11 cows, P4 continued to decrease thereafter, whereas Day 7 animals had no further reduction in plasma P4. Luteolysis (P4 < 1 ng/mL) occurred in all Day 11 cows. In the Day 7 group, however, luteolysis failure was observed for 11 of 19 cows (57.9%). In cows without luteolysis, plasma P4 increased after the initial PGF–induced decline. The second experiment compared luteolysis in (SOV, N = 6) vs. non-SOV (control, N = 8) cows. Both groups received a single PGF treatment on Day 11 after estrus, and luteolysis was monitored daily by ovarian ultrasonography and plasma P4 measurements. In addition, plasma LH was measured in blood samples taken every 20 minutes for 1 hour during five consecutive days after treatment. A similar percentage of reduction in P4 was observed in both groups 24 hours after treatment; however, SOV cows only reached plasma P4 values similar (P > 0.05) to controls 96 hours after treatment. There was no difference in initial LH values between SOV and controls (P > 0.05). The slower decrease in plasma P4 in the SOV group prevented an increase in LH for up to 96 hours after luteolysis induction, whereas LH values increased (P < 0.05) in controls 24 hours after treatment. In conclusion, (1) luteolysis may fail or be incomplete when PGF treatment is given on the day of uterine flushing (Day 7) in SOV cows; (2) induction of luteolysis 4 days later (Day 11) is effective, but the initial high-plasma P4 concentrations result in a slower slope of P4 decline to basal levels, and consequently, delayed increase in LH pulses.

The corpora lutea proangiogenic state of VEGF system components is turned to antiangiogenic at the later phase of the oestrous cycle in cows

Blood vessel expansion and reduction in the corpus luteum (CL) is regulated by the vascular endothelial growth factor (VEGF) system and linked to the maintenance of the CL. The VEGF system has both angiogenic and antiangiogenic ligands and receptors. Our objective was to evaluate the relationship between the mRNA expression of angiogenic and antiangiogenic members of the VEGF system in the CL, throughout the luteal phase of the oestrous cycle in cows. The CL of 18 cows were collected by transvaginal surgery on days 4, 6, 9, 12, 15 and 18 of the oestrous cycle and the mRNA expression of VEGF system components was evaluated by quantitative real-time PCR. The mRNA expression of VEGF ligands and receptors increased (P<0.05) from the early- and mid-luteal phase (days 4 to 12) reaching its maximum expression on day 15 of the cycle. We found no expression of VEGF164b throughout the cycle. Expression of sVEGFR1 did not change during the oestrous cycle and exceeded that of the VEGFR1 by 100 times. Nonetheless, as VEGFR1 increased, the relationship between the soluble and membrane receptor decreased (P<0.01). In contrast, the expression of VEGFR2 was higher than that of its soluble isoform for all days studied, however, the ratio between the membrane-bound and its soluble counterpart decreased continuously throughout the cycle (P<0.01). Our results show that the expression levels for VEGF ligands, receptors and their antagonistic counterparts are adjusted during CL development and regression, to upregulate angiogenesis early in the oestrous cycle and restrict it at the time of luteolysis.

The Buserelin Enigma; How Does Treatment with this GnRH Analogue Decrease Embryo Mortality?

The use of the GnRH analogue buserelin administered in mid dioestrus has been shown to increase early pregnancy rate, reduce embryo loss and increase litter size in several species. The mechanism by which this works is unclear and may differ between species. In cattle it is believed that buserelin, by reducing oestrogen concentrations, causes the development of endometrial oxytocin receptors to be delayed. Luteal oxytocin binds with these receptors to promote the formation and secretion of prostaglandin. Any delay in the release of PGF2α would allow an underdeveloped embryo extra time to produce and release the maternal recognition of pregnancy signal, which might otherwise be lost following luteolysis. This may also be the mechanism in sheep and pigs although an increase in progesterone concentrations could also be important. However in the mare, following buserelin administration at 9 or 10 days after the detection of ovulation, pregnancy rates are already increased by 12-13 days compared with untreated controls, i.e. before the time of luteolysis, without any change in progesterone concentrations. In addition to an increased pregnancy rate, the twin embryo rate is also increased and subsequent embryo loss rate reduced. The possible mechanisms by which this may result in the mare are discussed.

Intrauterine Inhibition of Prostaglandin Transporter Protein Blocks Release of Luteolytic PGF2alpha Pulses Without Suppressing Endometrial Expression of Estradiol or Oxytocin Receptor in Ruminants1

In ruminants, prostaglandin F2 alpha (PGF2(alpha)) is synthesized and released in a pulsatile pattern from the endometria luminal epithelial (LE) cells during the process of luteolysis. Prostaglandin transporter (PGT) is a 12-transmembrane solute carrier organic anion transporter protein that facilitates transport of PGF2(alpha). The present study determined the effects of inhibition of PGT protein on pulsatile release of luteolytic PGF2(alpha) and the underlined cell-signaling mechanisms. The results indicate that intrauterine inhibition of the PGT protein inhibits the pulsatile release of PGF2(alpha) from the endometrium and maintains a functional corpus luteum. Surprisingly, inhibition of PGT-mediated luteolytic pulses is not associated with spatial regulation of estrogen and oxytocin receptors in the LE of the endometrium and is also not accompanied by decreased biosynthesis of PGF2(alpha) or increased catabolism of PGF2(alpha) by the endometrium. Importantly, PGT inhibitor increases expression of pERK1/2 proteins in the LE of the endometrium. Knock down of ERK1/2 genes in LE cells reverses the inhibitory effects of PGT inhibitor on release of PGF2(alpha). In conclusion, intrauterine inhibition of PGT inhibits the pulsatile release of PGF2(alpha) from the endometrium without modulating spatial expressions of estrogen and oxytocin receptor proteins and metabolism of PGF2(alpha) at the time of luteolysis. Activation of ERK1/2 pathways and interactions between ERK1/2 and PGT protein appear to be important cell-signaling mechanisms that control PGT-mediated efflux transport function. PGT emerges as an important final component in the luteolytic machinery that controls the release of luteolytic pulses of PGF2(alpha) from the endometrium in sheep.

The progesterone receptor antagonist, onapristone has differential effects on the timing and control of the luteolytic mechanism depending on timing of administration in sheep

Cyclic ewes were treated with control vehicle or progesterone receptor antagonist (onapristone; 100mg i.m. twice daily) during either early (day 3–5) or late (day 12–14) luteal phase and plasma samples collected for hormone analysis and to determine endogenous and oxytocin induced PGF2α release. On day 14 and 17, ewes were euthanised and reproductive tracts collected for ovarian morphology and endometrium for oxytoxin and steroid hormone receptor analysis. Early treatment increased LH, but not progesterone or oestradiol, while late treatment elevated all three hormones. Early treatment delayed the up-regulation of endometrial oxytocin receptors and responsiveness to oxytocin challenge, delaying luteolysis. Late treatment advanced development of oxytocin receptors and responsiveness to oxytocin though not timing of luteolysis. Patterns of hormone receptor mRNA were differentially disrupted by treatments. Results provide mechanistic insight into hormonal control of the oestrous cycle and identify the ability of the luteolytic mechanism to dissociate from functional luteolysis.

Expression and localization of inhibitor of differentiation (ID) proteins during tissue and vascular remodelling in the human corpus luteum

Members of the transforming growth factor-β (TGF-β) superfamily are likely to have major roles in the regulation of tissue and vascular remodelling in the corpus luteum (CL). There are four inhibitor-of-differentiation (ID1-4) genes that are regulated by members of the TGF-β superfamily and are involved in the transcriptional regulation of cell growth and differentiation. We studied their expression, localization and regulation in dated human corpora lutea from across the luteal phase (n = 22) and after human chorionic gonadotrophin (hCG) administration in vivo (n = 5), and in luteinized granulosa cells (LGCs), using immunohistochemistry and quantitative RT-PCR. ID1-4 can be localized to multiple cell types in the CL across the luteal phase. Endothelial cell ID3 (P < 0.05) and ID4 (P < 0.05) immunostaining intensities peak at the time of angiogenesis but overall ID1 (P < 0.05) and ID3 (P < 0.05) expression peaks at the time of luteolysis, and luteal ID3 expression is inhibited by hCG in vivo (P < 0.01). In LGC cultures in vitro, hCG had no effect on ID1, down-regulated ID3 (P < 0.001), and up-regulated ID2 (P < 0.001) and ID4 (P < 0.01). Bone morphogenic proteins (BMPs) had no effect on ID4 expression but up-regulated ID1 (P < 0.01 to P < 0.005). BMP up-regulation of ID2 (P < 0.05) was additive to the hCG up-regulation of ID2 expression (P < 0.001), while BMP cancelled out the down regulative effect of hCG on ID3 regulation. As well as documenting regulation patterns specific for ID1, ID2, ID3 and ID4, we have shown that IDs are located and differentially regulated in the human CL, suggesting a role in the transcriptional regulation of luteal cells during tissue and vascular remodelling.

Intraluteal Prostaglandin Biosynthesis and Signaling Are Selectively Directed Towards PGF2alpha During Luteolysis but Towards PGE2 During the Establishment of Pregnancy in Sheep1

In ruminants, endometrial prostalgandin (PG) F(2alpha) causes functional luteolysis, whereas luteal synthesis of PGF(2alpha) is required for structural luteolysis. PGE(2) is considered to be a luteoprotective mediator. Molecular aspects of luteal PGF(2alpha) and PGE(2) biosynthesis and signaling during the estrous cycle and establishment of pregnancy are largely unknown. The objectives of the present study were 1) to determine the regulation of proteins involved in PGF(2alpha) and PGE(2) biosynthesis, catabolism, transport and signaling in the corpus luteum (CL); 2) to investigate the transport of interferon tau (IFNT), PGF(2alpha), and PGE(2) from the uterus to the ovary through the vascular utero-ovarian plexus (UOP); and 3) to compare the intraluteal production of PGF(2alpha) and PGE(2) on Days 12, 14, and 16 of the estrous cycle and pregnancy in sheep. Our results indicate that luteal PG biosynthesis is selectively directed towards PGF(2alpha) at the time of luteolysis and towards PGE(2) during the establishment of pregnancy. Moreover, the ability of the CL of early pregnancy to resist luteolysis is due to increased intraluteal biosynthesis of PGE(2) and PGE(2) receptor (PTGER) 2 (also known as EP2)- and PTGER4 (also known as EP4)-mediated signaling. We also found that IFNT protein is not transported through the UOP from the uterus to the ovary; in contrast, a large proportion of endometrial PGE(2) is transported from the uterus to the ovary through the UOP. These results indicate that endometrial PGE(2) stimulated by pregnancy is transported locally to the ovary, which increases luteal PGE(2) biosynthesis and hence activates luteal PTGER2 and PTGER4 signaling, thus protecting the CL during the establishment of pregnancy in sheep.

Selective Increased Luteal Prostaglandin E2 Compared to F2 Alpha Biosynthesis and Signaling Are Critical for Luteal Protection and Resistance During Establishment of Pregnancy in Sheep.

In ruminants, pulsatile secretion of prostaglandin F2 alpha (PGF2a) by the endometrium is locally transported to corpus luteum (CL) and stimulates intraluteal PGF2a biosynthesis and induces luteal regression/luteolysis. Interestinlgy, luteolytic PGF2a fails to accomplish regression of CL of early pregnancy. This increased resistance of the CL to PGF2a may suggest existence of luteal protective mechanisms. Early physiological studies proposed prostaglandin (E2) PGE2 as luteal protective mediator because PGE2 counteracted the effects of PGF2a and protected the CL from regression. However, the molecular and cellular aspects of resistance of the CL to luteolytic PGF2a are largely unknown. The objective of this study is to determine biosynthesis, transport, and signaling of PGF2a and PGE2 in CL during the estrous cycle and early pregnancy in sheep. Suffolk cross-bred ewes were ovariohysterectomized on days 12, 14 and 16 of the estrous cycle (ECY) or pregnancy (PNY), the CLs were collected, and temporal expression of PGF2a and PGE2 biosynthetic, transport and signaling proteins were determiend by western blot. Blood samples were collected from the ovarian artery and ovarian vein and transport of PGF2a, PGFM, PGE2, and PGEM were determiend by ELISA. Results indicated that: (i) COX-2 protein was abundantly expressed on days 12-16 of ECY and PNY and not affected by the ECY or PNY status, and COX-1 protein was expressed at very low level; (ii) PGES-1 protein was abundantly expressed on days 12-14 of ECY and PNY, and then decreased (4 fold) on day 16 of the ECY compared to that of PNY, expression of PGES-2 protein was not modulated by ECY and PNY, and expression of PGES-3 protein was decreased (5 fold) on days 12-16 of ECY compared to that of PNY; (iii) expression of PGFS-AKR1C1 protein was not modulated by ECY and PNY, and PGFS-AKR1C3 protein was expressed at steady sate level on day 10-16 of ECY but decreased (several fold) on days 12-16 of PNY; (iv) expression of FP, EP1 and EP3 proteins were not modulated by ECY and PNY, but interestingly, expression of EP2 and EP4 proteins were decreased (4 fold) on day 16 of ECY compared to that of PNY; (v) PGT protein was abundantly expressed on days 12-16 of ECY and PNY and its expression was decreased on day 16 of ECY compared to that of PNY; and (vi) PGDH protein expression was increased on days 14-16 of PNY compared to that of ECY. (vi) Ovarian venous plasma concentrations of PGF2a, PGFM, PGE2 and PGEM were 1500-2000 pg/ml, 400-500 pg/ml, 1000-1250 pg/ml, and 2-5 pg/ml on days 14-16 of the estrous cycle, respectively, and 400-500 pg/ml, 2000-2500 pg/ml, 3000-3500 pg/ml, and 3-5 pg/ml on days 14-16 of pregnancy, respectively. These results together suggest that the luteal PG biosynthetic and signaling machinery is selectively directed towards PGF2a at the time of luteolysis, by contrast; towards PGE2 at the time of establishment of pregnancy. In addition, CL of pregnancy has innate capacity to catabolize PGF2 to inactive PGFM but not PGE2 to PGEM and favors intraluteal PGE2 production. Thus, increased intraluteal PGE2 production and action are the critical mechanisms that resist/protect CL of early pregnancy to luteolytic PGF2a. Selective inhibition of PGES-1 or EP2 and EP4 along with PGF2a could be the potential future therapeutic strategy to improve reproductive efficiency in ruminants. Supported by USDA award 2008-35203-19101 to JAA. (poster)

Evaluation of genes involved in prostaglandin action in equine endometrium during estrous cycle and early pregnancy

The aim was to evaluate expression of genes involved in the biosynthesis of prostaglandins (PTG), Prostaglandin H Synthase-1 (PTGS1) and PTGS2, PGF synthase (PTGFS), and PGE synthase (PTGES), PGF receptor (PTGFR), PGE receptors (PTGER2 and PTGER4), prostaglandin transporter (SLCO2A1) and hydroxyprostaglandin dehydrogenase-15 (HPGD). Endometrial biopsies were obtained from mares on day of ovulation (d0, n = 4), late diestrus (LD, n = 4), early luteolysis (EL, n = 4) and after luteolysis (AL, n = 4) during the cycle. Stages of the cycle were confirmed by plasma progesterone concentrations measured daily and ultrasound examinations. Biopsies were also taken on days 14 (P14; n = 4), 15 (P15, n = 4), 18 (P18, n = 4) and 22 (P22; n = 4) of pregnancy. Relative mRNA expressions were quantified using real-time RT-PCR. A mixed model was fitted on the normalized data and least significant difference test ( α = 0.05) was employed. Expression of PTGS1 mRNA was low throughout the estrous cycle and early days of pregnancy, but upregulated on P18 and P22. PTGS2 expression was increased on EL, but it was suppressed by pregnancy on P15, P18, and P22. PTGFS expression was upregulated in both cyclic and pregnant mares compared to d0 and its level was the highest on LD. PTGFR expression was transiently increased on LD and EL and was suppressed during early pregnancy. Both PTGES and PTGER2 expressions were increased on LD, EL, and early pregnancy, but were decreased after the luteolysis in cyclic mares as they remained high on P18 and P22. PTGER4 expression did not change throughout the cycle and early pregnancy. Levels of HPGD and SLCO2A1 were significantly increased only on P22. In conclusion, PTGS2 expression increases around the time of luteolysis and concurrent upregulation of PTGFS and PTGES indicates that equine endometrium has increased capability of PTG production around the time of luteolysis. However, pregnancy reduces PTGS2 expression, but maintains the high levels of PTGES during early pregnancy along with PTGER2 while PTGFR expression was suppressed. These findings suggest that possible luteotrophic action of PGE 2 is required in early equine pregnancy. PTGS1 is only upregulated later in the early pregnancy suggesting that it is not involved in luteolysis, but could be the main PTGS enzyme at this time during early pregnancy. An increase in HPGD and SLCO2A1 levels on P22 indicates a tight regulation of PTG action by pregnancy.