Early Luteolysis Research Articles

Circulating hormone concentrations within a pulse of a metabolite of prostaglandin F2α during preluteolysis and early luteolysis in heifers

Plasma from hourly blood samples from two previous studies in heifers was assayed for hormones that were not considered in the previous reports. The objective was to determine the intrapulse temporal relationships between a prostaglandin F2α metabolite (PGFM) pulse and various hormones during preluteolysis and luteolysis. Hormone concentrations were centralized to the peak of a PGFM pulse (Hour 0) and evaluated from Hours −3 to 3. Experiment 1 ( n = 6) was done during early luteolysis. Progesterone decreased during Hours −3 to 0 and then rebounded, but did not return to prepulse concentrations, and concentration of LH increased between Hours −1 and 2. In Experiment 2 ( n = 7), comparisons were made between the last PGFM pulse of preluteolysis and the first pulse of luteolysis. Intrapulse concentrations of LH increased rapidly between Hours 0 and 1 during preluteolysis and gradually between Hours −2 and 2 during luteolysis. Intrapulse differences in cortisol among hours were not significant for preluteolysis and approached significance ( P < 0.06) during luteolysis, owing primarily to an apparent increase between Hours −2 and 1. Oxytocin concentrations showed only an hour effect ( P < 0.0003) from to an increase between Hours −2 and 0 and a decrease between Hours 0 and 2. Results indicated that oxytocin and PGFM concentrations increased and decreased concomitantly and supported the hypothesis that the reported more prominent rebound in progesterone during the descending portion of a PGFM pulse during preluteolysis than during luteolysis involves a greater transient increase in LH.

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.

212 EXPRESSION OF FIBROBLAST GROWTH FACTOR 18 (FGF18) AND COGNATE RECEPTORS (FGFR3C AND FGFR4) DURING LUTEAL DEVELOPMENT AND INDUCED LUTEOLYSIS IN CATTLE

Fibroblast growth factor receptor 2 (FGFR2) has been shown to induce luteinization in granulosa cells, luteal angiogenesis, and luteal growth. Alternative splicing of 4 genes give rise to 7 subtypes of fibroblast growth factor receptors (FGFR) with varying affinity for different fibroblast growth factors (FGF). Fibroblast growth factor receptor 2 and FGF18 efficiently activate FGFR3C and FGFR4 and may act in cooperation in tissues expressing these receptors. We aimed to determine mRNA expression patterns for FGF18, FGFR3C, and FGFR4 during bovine luteal development and following induced luteolysis. In addition, we assessed FGF18 localization in the bovine CL. Bovine CL were obtained from abattoir ovaries and classed into 4 stages of development: stage 1 =corpus hemorragicum; stage 2 = developing CL; stage 3 = mature or early functional luteolysis CL; and stage 4 = structural luteolysis. To assess FGF18 and FGFR mRNA expression during induced luteolysis, adult cows (Bos taurus Holstein-Friesians) were injected with the PGF2 analogue cloprostenol (500 mg i.m. Intervet, Unterschleissheim, Germany) during the mid-luteal phase of the cycle (Days 8-12). Corpus luteum were collected by transvaginal ovariectomy at 0, 0.5, 2, 4, 12, 24, 48, and 64 hr (n = 5/time point) after PGF2 injection. Tissue samples were submitted to total RNA extraction. Expression of FGF18, FGFR3C, and 4 mRNA during the bovine CL lifespan and induced luteolysis were measured by real-time RT-PCR with oligo-dT in the RT and bovine-specific primers in the PCR. Expression of cyclophilin was used as internal control. The effect of developmental stage and time post-PGF2 on gene expression was tested by ANOVA, followed by Tukey- Kramer HSD test. Immunohistochemical analysis was performed with a commercial human antibody (anti-FGF18; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Fibroblast growth factor 18, FGFR3C, and FGFR4 mRNA was detected in all 4 developmental stages; FGF18 mRNA abundance was higher in stage 3 (2.89 ± 0.05; mean ± SEM) compared with stages 1 (0.3 ± 0.27), 2 (0.56 ± 1.27), and 4 (0.99 ± 0.32). Fibroblast growth factor 18 and FGFR4 mRNA expression did not significantly change during induced luteolysis. Fibroblast growth factor receptor 3C mRNA abundance peaked 4 h after PGF2 injection and significantly decreased at 24 h post-treatment in comparison with peak levels. Immunohistochemical analysis revealed the presence of FGF18 in small and large luteal cells and in blood vessels. In conclusion, the mRNA expression patterns of FGF18 and its receptors suggest their participation in the control of luteal differentiation, particularly during functional luteolysis. The localization of FGF18 protein to blood vessels suggests it may play a role in the control of angiogenesis in the bovine CL. Supported by CAPES/FAPESP.

Effect of early luteolysis in progesterone-based timed AI protocols in Bos indicus, Bos indicus×Bos taurus, and Bos taurus heifers

The objective of this study was to evaluate the effects of treatment with an intravaginal progesterone-releasing device (CIDR) and estradiol benzoate (EB) on follicular dynamics in Bos indicus (n=23), Bos taurus (n=25), and cross-bred (n=23) heifers. To assess the influence of reduced serum progesterone concentrations during 8 days of treatment with a progesterone-releasing device on follicular dynamics, half of the heifers received PGF at CIDR insertion (Day 0; 3 x 2 factorial design). Mean (+/-S.E.M.) serum progesterone concentrations during CIDR treatment varied (P<0.05) among genetic groups: B. indicus (5.4+/-0.1 ng/mL), B. taurus (3.3+/-0.0 ng/mL), and cross-bred (4.3+/-0.1 ng/mL). Maximum diameter of the dominant follicle (DF) was smaller (P<0.01) in B. indicus heifers (9.5+/-0.5 mm) than in cross-bred (12.3+/-0.4 mm) or B. taurus heifers (11.6+/-0.5 mm). B. indicus experienced lower (P<0.01) ovulation rate (39.1%) than did B. taurus (72.7%) and cross-bred (84.0%). Heifers treated with PGF on Day 0 had lower (P<0.05) serum progesterone concentrations during progesterone treatment. The PGF treatment on Day 0 increased (P<0.01) the diameter of the DF (11.9+/-0.4 mm vs. 10.5+/-0.4 mm). Moreover, greater (P=0.02) ovulation rates (78.8 vs. 54.0%) occurred in heifers treated with PGF on Day 0. In summary, B. indicus heifers had greater serum progesterone concentrations, smaller DF diameter, and a lower ovulation rate compared to B. taurus heifers. Prostaglandin treatment on the day of CIDR insertion reduced serum progesterone during treatment, and resulted in increased maximum DF diameter and ovulation rate.

Luteal phase circulating vascular endothelial growth factor (VEGF), estradiol (E 2) and progesterone (P) levels after GnRH agonist trigger

GnRH agonist trigger is effective in preventing OHSS although the exact mechanism has not been fully elucidated. A single dose of GnRH agonist to induce oocyte maturation leads to early luteolysis and failure of the corpus luteum to secrete steroids and growth factors such as VEGF that may be responsible for OHSS development. The aim of this study was to characterize luteal phase plasma VEGF and serum E2 and P concentrations after GnRH agonist trigger.

P-798: Induction of oocyte maturation with GnRH agonist in high-risk patients undergoing IVF treatment does not adversely affect implantation rate

Previous studies have suggested that GnRH agonist trigger during IVF treatment may adversely affect implantation rates. This may be due to inadequate luteal phase and early pregnancy steroidal concentrations induced by early luteolysis. We evaluated the effect of GnRH agonist trigger on implantation and pregnancy rates in high-risk patients who used a different luteal phase steroidal supplementation protocol than that used in previous studies.

Dynamic Expression of Caspase-2, -3, -8, and -9 Proteins and Enzyme Activity, But Not Messenger Ribonucleic Acid, in the Monkey Corpus Luteum during the Menstrual Cycle

Studies were designed to determine whether: 1) changes in caspase expression or activity occur in the macaque corpus luteum (CL) during its lifespan in the menstrual cycle, and 2) LH acting directly or via ovarian steroids regulates luteal caspases. Caspase-2, -3, -8, and -9 mRNAs were detectable by semiquantitative RT- or real time-PCR in CL, but levels did not differ between the early, mid, mid-late, late, and very-late luteal phases. Immunostaining for caspase-2 and -3 proteins was observed in luteal cells and appeared to peak by mid to mid-late stage. Enzyme activity for caspase-2, -3, -8, and -9 increased (P < 0.05) by mid-late stage, and then declined by the very-late stage. Treatment with GnRH antagonist + LH at the mid-late stage increased caspase-2, -8, and -9, but not -3, activity, compared with controls. Coadministration of a steroid synthesis inhibitor (trilostane) with GnRH antagonist + LH reduced (P < 0.05) caspase-2, -8, and -9 activity. Progestin (R5020) replacement during trilostane treatment did not restore caspase activity. Thus, initiator and effector caspases are present during CL development and regression in the menstrual cycle. The increased caspase activity at mid-late stage suggests that apoptosis is involved in early luteolysis in primates. Gonadotropin, perhaps via local steroids, modulates initiator caspases in the primate CL.

The effects of lipopolysaccharide and interleukins-1alpha, -2 and -6 on oxytocin receptor expression and prostaglandin production in bovine endometrium.

Up-regulation of endometrial oxytocin receptor (OTR) expression followed by an increase in pulsatile endometrial prostaglandin (PG) F(2alpha) secretion causes luteolysis in cattle. Inhibition of luteolysis is essential for the maternal recognition of pregnancy but also occurs in association with endometritis. The factors regulating OTR expression at this time are unclear. The OTR gene promoter region contains binding elements for acute phase proteins but their function has not been established. This study investigated the effects of various cytokines on OTR expression and on PGF(2alpha) and PGE(2) production in explant cultures of bovine endometrium. Endometrium was collected in the late luteal phase (mean day of cycle 15.4+/-0.50) or early luteolysis (mean day of cycle 16.4+/-0.24) as determined by the initial concentration of endometrial OTR. Explants were treated for 48 h with: (i) lipopolysaccharide (LPS) and/or dexamethasone (DEX), (ii) ovine interferon-tau (oIFN-tau), or (iii) human recombinant interleukin (IL)-1alpha, -2 or -6. OTR mRNA was then measured in the explants by in situ hybridisation and the medium was collected for measurement of PGF(2alpha) and PGE(2) by RIA. LPS treatment stimulated production of PGF(2alpha), whereas DEX either alone or in combination with LPS was inhibitory to both PGF(2alpha) and PGE(2). Neither of these treatments altered OTR mRNA expression. oIFN-tau reduced OTR mRNA expression but stimulated production of both PGF(2alpha) and PGE(2). In endometrial samples collected in the late luteal phase, IL-1alpha, -2 and -6 all inhibited OTR mRNA expression, but IL-1alpha and -2 both stimulated PGF(2alpha) production. In contrast, when endometrium was collected in early luteolysis, none of the interleukins altered OTR expression or caused a significant stimulation of PGF(2alpha) production but IL-2 increased PGE(2). Neither IL-1alpha nor -2 altered OTR promoter activity in Chinese hamster ovary cells transfected with a bovine OTR promoter/chloramphenicol acetyl transferase reporter gene construct. In conclusion, the action of interleukins on both OTR mRNA expression and endometrial prostaglandin production alters around luteolysis. Pro-inflammatory interleukins suppress OTR expression in the late luteal phase, while LPS stimulates PGF(2alpha) without altering OTR mRNA expression. IL-I and -2 and LPS are therefore unlikely to initiate luteolysis but may cause raised production of PGF(2alpha) during uterine infection.

Embryotoxicity of a Regressing Corpus Luteum in Beef Cows Supplemented with Progestogen1

Postpartum cows with short-lived corpora lutea produce embryos that arrive at the uterus, but pregnancy rates are low even with exogenous progestogen. Four experiments were conducted to determine whether prostaglandin (PG) F2 alpha, known to cause early luteolysis, could have a direct effect on embryonic loss. Exogenous progestogen was injected s.c. twice daily in each experiment, starting 3 or 4 days after estrus (day of estrus = Day 0). Nonlactating, cycling beef cows were mated and injected i.m. every 8 h on Days 4 through 7 (experiment 1) or 5 through 8 (experiment 3) with either 15 mg PGF2 alpha or 3 ml saline. In experiment 1, cows in a third group received 1 g flunixin meglumine i.m. every 8 h. Ten of 18 PGF2 alpha-treated cows in experiment 3 were luteectomized on Day 5. Pregnancy rates were higher (p < 0.05) in cows given saline or flunixin meglumine (5 of 7) than in cows given PGF2 alpha (1 of 5) in experiment 1, and in cows given saline (6 of 9) or given PGF2 alpha, and luteectomized (8 of 10) than in cows given PGF2 alpha (2 of 8) in experiment 3. Postpartum beef cows, mated at weaning-induced first estrus, received i.m. injections every 8 h on Days 4 through 9 of 3 ml saline or 1 g flunixin meglumine (experiment 2); 14 flunixin meglumine-treated cows were luteectomized on Day 7. Pregnancy rates were higher in cows given flunixin meglumine and luteectomized (7 of 14) than in cows given saline (4 of 15) or flunixin meglumine alone (3 of 15; p < 0.05). In experiment 4, postpartum cows were luteectomized or sham-operated on Day 5. Pregnancy rates (2 of 13 and 2 of 14, respectively) did not differ. Thus, both reduction of endogenous PGF2 alpha and luteectomy were required for embryo survival in postpartum cows with short-lived corpora lutea, whereas luteectomy alone prevented effects of exogenous PGF2 alpha in cycling cows.

Effect of treatment with estradiol valerate on endocrine changes and ovarian follicle populations in dairy cows

A linear-array ultrasound instrument was used to monitor the dynamics of follicular cyst formation following estradiol valerate (EV) administration in postpartum dairy cattle. Twelve cyclic cows were given two intramuscular (i.m.) injections of prostaglandin and F 2α (PGF 2α) 12 d apart to synchronize estrus. On Day 16 (Day 0 = day of estrus) six cows received 10 mg of EV in 1 ml sesame oil; the remaining six cows were treated with 1 ml sesame oil. The ovaries of all cows were scanned rectally each morning from Day 9 until 14 or 30 d post treatment. Plasma concentrations of luteinizing hormone (LH) and progesterone (P 4) were also determined as objective indices of treatment effects. Day 0 to 16 ultrasound pictures of the ovaries of both control and treated cows were characterized by the presence of a corpus luteum (CL; 19 to 38 mm), several small follicles (<5 mm) and a medium-sized follicle (6 to 28 mm). Following treatment in control cows, the CL regressed gradually, and a preovulatory follicle was identifiable by Day 17 to 18, it increased in size and reached a maximum of 28 to 30 mm by Day 20 after ovulation and was identifiable throughout the rest of the cycle. Administration of 0 mg of EV resulted in a rapid reduction in the size of the CL. Growth of a large follicle was observed in all treated animals around Days 16 to 20, but having reached a maximum diameter of 12 to 24 mm it regressed without resulting in ovulation. Subsequent ultrasound pictures of EV-treated cows were characterized by the absence of a new CL and the presence of medium-sized persistent follicles. Estradiol valerate treatment induced early luteolysis (43 ± 05 h post EV vs 101 ± 22 h) and an LH surge (41 ± 11 h vs 125 ± 17 h).

Are pony and larger mares similar as recipients for non‐surgical transfer of Day 7 embryos?

SummaryNon‐surgical transfer of equine embryos to nine recipient pony mares failed to result in any pregnancies whereas transfer of nine embryos to larger Selle‐Français recipient mares resulted in four pregnancies. Plasma progesterone profiles in the recipient and sham transfer control mares showed the occurrence of early luteolysis (before Day 12 after ovulation) in three of the nine recipient pony mares whereas luteolysis occurred at the normal time (Day 14 to 15) in the control mares and the non‐pregnant Selle‐Français and pony recipient mares. Daily plasma PGFM measurements showed the occurrence of PGF release on the day before luteolysis in all groups, except the recipient pony mares which underwent early luteolysis. In these animals, luteolysis occurred four days after the initial PGF release. PGFM profiles in closely timed serial blood samples taken on the day of transfer showed that cervical transfer induced PGF release from 1 min to 6 h after transfer in both breeds. This release was not directly responsible for non‐pregnancy in the pony recipient mares; in all three pony recipient mares which underwent early luteolysis, this immediate post‐transfer PGF release was followed by an additional release on the day after transfer. This suggests that the uteri of pony mares respond differently to those of larger mares with regard to mechanical stimulation of the cervix and/or bacterial or other types of endometritis. Clearly, pony and Selle‐Français mares are not similar as recipients for non‐surgical transfer of Day 7 equine embryos.

The role of arginine vasotocin and prostaglandin F 2α on oviposition and luteolysis in the common snapping turtle Chelydra serpentina

The administration of arginine vasotocin (AVT) to gravid snapping turtles with steroidogenically active corpora lutea and high plasma progesterone concentration (1480 ± 155 pg/ml) did not trigger oviposition, whereas 12 days after ovulation when luteolysis occurred and plasma progesterone concentration was low (570 ± 78 pg/ml), treatment with AVT caused oviposition. Controls with high plasma progesterone concentration (1605 ± 185 pg/ml) oviposited 15–23 days after ovulation when plasma progesterone concentration dropped to 201 ± 35 pg/ml. Deluteinization-induced oviposition was initiated 15 hr after surgery and was completed by 30 hr. Oviposition of complete clutches occurred and was correlated with a significant drop in plasma progesterone. Sham-operated turtles did not exhibit oviposition and no significant change in progesterone concentration was observed. A single injection of prostaglandin F 2α (PGF) in recently ovulated turtles induced early luteolysis and a significant decrease in plasma progesterone concentration after 24–30 hr. A single administration of PGF caused the disappearance of steroidogenic features such as the smooth endoplasmic reticulum and mitochondria with tubular cristae 48 hr later. Also PGF triggered the invasion of a hyaline-like material from the luteal theca into the luteal cell mass which eventually induced luteolysis. The role of AVT, PGF, and progesterone in relation to egg retention and oviposition is discussed.

Effects of alloxan-induced diabetes on corpus luteum function in the pseudopregnant rat.

The effects of alloxan-induced diabetes mellitus on rat ovarian structure and function were examined throughout pseudopregnancy (PSP). Animals received either saline (C) or alloxan (40 mg/kg) treatment on the day of proestrus (PA) preceding PSP or on day 1 (D-1A) of PSP (day 0 = ovulation). Serum samples were analyzed by radioimmunoassay for progesterone (P) and 17-beta-estradiol (E) levels and compared with the corresponding changes in ovarian and uterine weights in C, PA, and D-1A rats. In addition, the effects of daily treatment with 6 IU ovine insulin (AI) on serum P levels were assessed in D-1A-treated rats and compared with controls. Alloxan treatment effectively elevated blood glucose levels (P less than or equal to 0.01) in PA and D-1A groups as compared with controls or AI rats. Alloxan treatment reduced both ovarian and uterine weights of PA and D-1A groups as compared with C and AI rats. Serum P levels were significantly reduced in PA (P less than or equal to 0.01) and D-1A (P less than or equal to 0.05-0.01) rats as compared with control rats throughout PSP. Daily insulin treatment reversed the suppressive effects of D-1A treatment on serum P levels, but did not restore luteal function to control levels. Neither C nor D-1A groups exhibited any marked differences in serum E levels throughout PSP. The results of these studies indicate that the administration of alloxan before the onset of PSP effectively inhibits luteal function, whereas D-1A treatment induces early luteolysis as compared with controls.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of a progesterone-releasing intrauterine contraceptive device on uterine secretion of prostaglandin F2α and life-span of corpora lutea in the ewe

Two studies were conducted to investigate the effect of a progesterone-releasing intrauterine contraceptive device (IUD) on uterine secretion of prostaglandin (PG) F2α 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 μg per day) by the device did not prevent premature luteolysis or the increased concentration of PGF2α in uterine venous plasma on day 5 caused by conventional IUD's. In the second study a progesterone-releasing IUD (645 μg 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 PGF2α 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 PGF2α.