Control of ovarian follicular growth and maturation by the corpus luteum and the placenta during pregnancy in sheep

Ovarian follicular growth and maturation and its control throughout pregnancy have not been described fully in sheep. Experiment 1 characterized the size and maturation (steroid production in vitro and aromatase activity) of ovarian follicles obtained at days 20, 50, 80 and 110 of pregnancy compared with those obtained at day 12 of the oestrous cycle. There was no difference in the number of small follicles (< 3 mm in diameter) between cyclic and pregnant ewes, regardless of the stage of pregnancy. There was a marked reduction (P < 0.01) in the number of medium follicles (3-5 mm) starting at day 80 of pregnancy. Large follicles (> 5 mm) were not detected at day 110 of pregnancy. In vitro testosterone output by follicles was constant throughout pregnancy. Oestradiol output remained steady until day 80, but decreased markedly at day 110 of pregnancy. This decrease was associated with a reduction in aromatase activity in follicles obtained at this stage. Experiment 2 examined the effect of administration of high concentrations of progesterone between day 100 and day 120 after mating on resumption of follicular growth in ewes that underwent Caesarean section at day 99 of pregnancy. In ewes that underwent Caesarean section, progesterone supplementation was successful in mimicking the profile found in pregnant ewes, but did not prevent re-initiation of follicular growth, as demonstrated by the presence of large follicles (> 5 mm) at day 120 after mating. Experiment 3 examined the effects of PGF(2alpha)-induced regression of the corpus luteum of day 100 of pregnancy on resumption of follicular growth. High concentrations of PGF(2alpha) (0.28 mg kg(-1) body weight) administrated at day 100 of pregnancy were required to initiate regression of the corpus luteum. At day 120 after mating, the mean (+/- SEM) diameter of the largest follicle in PGF(2alpha)-treated ewes (3.40 +/- 0.47 mm) was significantly greater (P < 0.05) than that in control pregnant ewes (2.52 +/- 0.34 mm). Experiment 4 examined the effect of removal of the fetus and of the corpus luteum at day 100 of pregnancy on resumption of ovulation. Removal of the corpus luteum by PGF(2alpha) treatment at the time of removal of the fetus resulted in earlier occurrence of short luteal phases (27.8 versus 40.6 days, PGF(2alpha)-treated versus non-treated) but did not alter the timing of the first normal luteal phases (41 days). In conclusion, the results from these experiments indicate that placental compounds play a major role in inhibiting follicular growth and maturation during late pregnancy in sheep.     

Concentrations of prostaglandins E2, F2 alpha and 6-keto-prostaglandin F1 alpha in the utero-ovarian venous plasma of nonpregnant and early pregnant ewes

The effect of pregnancy on concentrations of prostaglandins E2, F2 alpha and 6-keto-prostaglandin F1 alpha (PGE2, PGF2 alpha and 6-keto-PGF1 alpha) in utero-ovarian venous plasma was examined in ewes on Days 10 through 14 after estrus, an interval which includes the critical period for maternal recognition of pregnancy. The utero-ovarian vein ipsilateral to a corpus luteum was catheterized on Day 9 or 10 in 6 pregnant and 8 nonpregnant ewes. Five blood samples were collected at 30-min intervals for 2 h beginning at 0500 and 1700 h daily. Sampling began at 0500 h on the day after catheterization. The mean and variance within each 2-h collection period were calculated for each ewe. The natural logarithm of the variance in each collection period (ln variance) was used as an estimate of the fluctuations in secretory activity by the endometrial-conceptus complex. Patterns of the mean concentrations of PGE2 were different between pregnant and nonpregnant ewes (P less than 0.01); PGE2 being higher in the pregnant ewes beginning on Day 13. There was a trend for the patterns of ln variance in PGE2 to differ (P less than 0.1) with pregnancy status over the entire period; ln variance was greater in pregnant ewes beginning on Day 13. The patterns of the mean concentrations and ln variances for PGF2 alpha and 6-keto-PGF1 alpha did not differ between pregnant and nonpregnant ewes. There were significant increases in both of these prostaglandins over time, independent of pregnancy status (P less than 0.01). The association of higher concentrations of PGE2 in utero-ovarian venous plasma with early pregnancy is consistent with the hypothesis that PGE2, originating from the uterus and/or conceptus, is one factor involved in maintenance of the corpus luteum of pregnancy.     

Administration of recombinant bovine interferon-alpha I at the time of maternal recognition of pregnancy inhibits prostaglandin F2 alpha secretion and causes luteal maintenance in cyclic ewes.

The antiluteolytic protein, ovine trophoblast protein-1, which is secreted by sheep embryos at about the time of the maternal recognition of pregnancy, exhibits significant structural homology with alpha interferons. Experiments were conducted to examine the effects of intra-uterine and systemic administration of a recombinant bovine interferon-alpha I (rboIFN-alpha I) upon the interoestrus interval, endometrial oxytocin receptor concentrations and secretion of prostaglandin (PG) F2 alpha in cyclic ewes. In Expt 1, each ewe had a cannula placed in the tip of a uterine horn ipsilateral to a corpus luteum, 7 days after an induced oestrus. From day 9 after oestrus until day 19, ewes received either 200 (n = 4), 667 (n = 5) or 2000 (n = 9) micrograms/24 h of rboIFN-alpha I, meclofenamic acid (n = 4) or vehicle (n = 11). Other ewes received 2000 micrograms rboIFN-alpha I/24 h (n = 5) between days 12 and 15 only. All ewes were killed on day 19. Mean luteal phase, as determined by daily plasma progesterone measurements, was significantly longer (P less than 0.01) and mean concentrations of 13,14-dihydro-15-keto PGF 2 alpha (PGFM) in plasma were lower (P less than 0.05) in ewes receiving 667 or 2000 micrograms rboIFN-alpha I between days 9 and 19, or 2000 micrograms between days 12 and 15, than in animals from other treatment or control groups. A similar protocol was used in Expt 2, in which further ewes received either 2000 micrograms rboIFN-alpha I/24 h (n = 5) or vehicle (n = 5) by bolus infusions twice a day into one uterine horn. Mean luteal phase was significantly (P less than 0.05) longer in treated than in control animals, but differences in PGFM concentrations were not significant. In Expt 3, after a synchronized oestrus, ewes received either 2.5 mg rboIFN-alpha I by i.m. injection twice a day between days 12 and 15 (n = 10), 2.5 mg rboIFN-alpha I by i.m. injection twice a day between days 9 and 15 (n = 11), i.m. injection of vehicle alone twice a day (n = 20), or continual intra-uterine infusion of 2 mg rboIFN-alpha I/day between days 12 and 15 (n = 7). The mean luteal phase of ewes receiving rboIFN-alpha I by intrauterine infusion or i.m. injection between days 9 and 15 was significantly longer than for animals from the other two groups (P less than 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of systemic intravenous infusion of PGF-2 alpha and 13,14-dihydro-15-keto-PGF-2 alpha on the release of oxytocin-associated neurophysin from the ovary in the ewe

Systemic intravenous infusion of physiological concentrations of PGF-2 alpha and its major metabolite, 13, 14-dihydro-15-keto-PGF-2 alpha (PGFM) into non-pregnant ewes possessing a corpus luteum induced the release of oxytocin-neurophysin. These results suggest that, during luteolysis, endogenous release of uterine PGF-2 alpha would be able to stimulate the release of ovarian oxytocin and oxytocin-neurophysin from the ovary.     

Ovarian follicular dynamics after cauterization of the dominant follicle in anestrous ewes

An experiment was conducted to ascertain if follicles could reach ovulatory size after the largest follicle (dominant) has been removed at different times during a progestin treatment in anestrous ewes, and secondly to determine if these new follicles could respond to an hCG-induced ovulation and have similar function as corpora lutea. Mature crossbred sheep (n=44) in anestrous were treated with an intravaginal sponge containing 40 mg of FGA (day 0=sponge insertion) for 9 days. Treatments consisted of cauterization of the largest follicle on the experimental day 3 (T1), day 6 (T2) and day 9 (T3); day 12 to ascertain the size of the largest follicle in control ewes. During laparotomies, the diameters of the largest follicle (DF), and those of the second and third largest follicles (SF1 and SF2, respectively) were determined. On day 12, a second laparotomy was performed for those ewes which had their DF cauterized on days 3, 6 and 9, a fourth group was left intact and only laparotomized on day 12. At this time, the size of the new DF, SF1 and SF2 were determined. Immediately after the laparotomy on day 12, all the ewes were treated with 1000 i.u. of hCG to induce ovulation. Blood samples were collected daily from day 0 to 50 and samples were analyzed for progesterone concentrations. The size of the DF at the time of sponge removal was smaller that those observed on day 3 or 6 of sponge suggesting that follicles in ewes treated with this progestin regress and a new wave of follicular development ensues between day 6 and the time of sponge removal. The size of the DF on day 12 was also smaller in ewes that have the largest follicle removed at the time of sponge removal reflecting that these follicles had a shorter period of growth; however, the rate of growth was greater for these follicles than for follicles arising after cauterization on day 3 or 6 after sponge insertion. There were no differences among treatments, in the number of ewes that formed a corpus luteum (CL) in response to hCG. Life span of the corpora lutea did not differ among ewes having their DF removed on day 6 or 9 or those that served as controls, however, ewes that had their DF removed on day 3 developed longer lived CL in a larger proportion of animals. Average progesterone concentration during the life span of the induced corpora lutea was greater in control ewes than in any other experimental group. These observations allow us to conclude that, (a) the follicular dynamics observed in anestrous ewes treated with a progestin intravaginal sponge resembles that observed during the normal estrous cycle in the ewe; (b) the effects of progesterone on life span of the corpus luteum could not be only related to direct effects at the follicle but also involve changes in other components of the uterine-ovarian-hypothalamic axis; (c) the mechanisms controlling luteal life span seem to be different to those mechanisms controlling the function of the induced corpus luteum.     

Response of the 1-5 day-aged ovine corpus luteum to prostaglandin F2alpha

The hypothesis that, in the ewe, prostaglandin (PG) F2alpha administration on day 3 after ovulation is followed by luteolysis and ovulation was tested using 24 animals. The ewes were treated with a dose of a PGF2alpha analogue (delprostenate, 160 microg) on days 1 (n=8), 3 (n=8) or 5 (n=8) after ovulation, was established by transrectal ultrasonography. Daily scanning and blood sampling were performed to determine ovarian changes and progesterone serum concentrations by radioinmunoassay. The treatment induced a sharp decrease of progesterone concentrations followed by oestrus and ovulation in all ewes treated on days 3 and 5 and in one ewe treated on day 1 (8/8, 8/8, 1/8; P<0.05). Seven ewes treated on day 1 did not respond to PGF2alpha treatment and had an inter-ovulatory cycle of normal length (17.4 +/- 0.5 days). However, the profile of progesterone concentrations during the cycle of these ewes was delayed 1 day (P<0.05) compared with a control cycle. The overall interval between PGF2alpha and oestrus for the 17 responding ewes was 42.4 +/- 2.3 h. In 15 of these ewes the ovulatory follicle was originated from the first follicular wave and the ovulation occurred at 60.8 +/- 1.8 h after PGF2alpha treatment. The other two responding ewes ovulated an ovulatory follicle originated from the second follicular wave between 72 and 96 h after treatment. These results support the hypothesis and suggest that refractoriness to PGF2alpha of the recently formed corpus luteum (CL) may be restricted to the first 1-2 days post-ovulation.     

Effect of the stage of pregnancy and post-partum on the number of gonadotrophin responsive follicles in ewes

The present study was designed to study follicular growth and its interactions with the corpus luteum of pregnancy in sheep during early, middle and late pregnancy and during postpartum anestrus. Ewes with 1 or 2 corpora lutea in one ovary were selected from a larger group of Serres ewes. All pregnant ewes were randomly allocated to two groups, with 10 to 12 ewes per group. Ewes of Group I were treated with 750 IU hCG at Day 25 or 45 or 70 or 100 or 125 of pregnancy. In Group II, ewes were treated with a combination of 1000 IU PMSG + 750 IU hCG either at Day 25 or 45 or 70 or 100 of pregnancy. The results demonstrated the presence of gonadotrophin-responsive follicles during early pregnancy (Days 25 to 45), reduction of their number during mid-pregnancy (Days 70 to 100), and their disappearance during late pregnancy (Day 125). Administration of hCG to Serres ewes at 10 and 20 days postpartum induced ovulation of a high proportion of ewes at 10 days postpartum (62%) with a further increase observed at 20 days postpartum (75%). During pregnancy, as well as during the postpartum period, there was no significant difference in the number of ovulations induced according to the location of the corpus luteum of pregnancy. These data demonstrate that the presence of the corpus luteum of pregnancy does not affect the number of gonadotrophin-responsive follicles until Day 100 of pregnancy. However, during late pregnancy such follicles were no longer present in the ovaries. Gonadotrophin-responsive follicles were again present as soon as Day 10 postpartum.     

Functional and morphological characteristics of the first corpus luteum formed after parturition in ewes

The ability of sheep luteal cells from the first corpus luteum formed after parturition (Group F) to secrete progesterone in the presence or absence of LH was compared with that of luteal cells obtained from normal cyclic ewes (Group C). Luteal concentrations of receptors for LH and prostaglandins (PG) F-2 alpha (PGF-2 alpha) and the cellular composition of corpora lutea from Groups F and C were also compared. Luteal cells from Group F secreted less progesterone in either the presence or absence of LH (P less than 0.01). There was no difference in the number of receptors for LH or PGF-2 alpha per luteal cell between Groups F and C (P greater than 0.1), nor was there a difference in the number of large or small steroidogenic luteal cells (P greater than 0.1). It was concluded that, if short-lived corpora lutea are insensitive to gonadotrophins, this response is not mediated by decreased numbers of receptors for LH. In addition, if the first corpus luteum formed post partum in ewes is more sensitive to the luteolytic effects of PGF-2 alpha, this effect is not mediated by an increased number of receptors for PGF-2 alpha or an increased proportion of PGF-2 alpha-sensitive large luteal cells.     

Jugular levels of 13,14-dihydro-15-keto-prostaglandin F and progesterone around luteolysis and early pregnancy in the ewe

Six non-pregnant ewes at day 12 of the estrous cycle each had a day-12 embryo transferred into the uterine horn ipsilateral to the corpus luteum, and 4 non-pregnant ewes at day 13 each had a day-13 embryo similarly transferred. Four control ewes, 2 at day 12 and 2 at day 13 received sheep serum into the uterine horn ipsilateral to the corpus luteum. Jugular blood samples were taken at 2-hourly intervals for 3 days post-surgery, then twice-daily for a further 4 days, and the plasma radioimmunoassayed for progesterone and 13,14-dihydro-15-keto-prostaglandin F. All control ewes exhibited estrus within the expected time range and pulsatile peaks of 13,14-dihydro-15-keto-prostaglandin F occurred coincident with declining progesterone levels. With one exception, the recipient ewes had prolonged cycles and those ewes found pregnant at necropsy, 30 days after transfer, showed no progesterone decline and no pulsatile peaks of prostaglandin during days 12 to 16 after estrus. These observations suggest that the presence of the embryo at a critical stage after mating suppresses the release of uterine prostaglandin F_2α.     

Evidence that progesterone may influence subsequent luteal function in the ewe by modulating preovulatory follicle development

Ovulation was induced in seasonally anoestrous ewes by repeated 2-h injections of 250 ng Gn-RH, after 12 days (Group 1, N = 7; Group 2, N = 8), 2 days (Group 3, N = 8) or no (Group 4, N = 7) progesterone pretreatment. A preovulatory LH peak occurred spontaneously at a mean (+/- s.e.m.) time of 43.1 +/- 2.0 h, 38.5 +/- 3.1 h and 26.8 +/- 1.7 h after the start of Gn-RH treatment in Groups 1, 3 and 4 respectively, and was artificially induced in ewes in Group 2, after 24 h of treatment, by a single i.v. injection of 150 micrograms Gn-RH. Normal luteal function occurred in all progesterone-pretreated ewes, but in only 1/7 animals not treated with progesterone. These results demonstrate that, although normal luteal function in progesterone-primed ewes induced to ovulate with repeated injections of low doses of Gn-RH is associated with a delayed preovulatory LH peak, it is not this extended period of follicle development which is responsible for functional competence of the resultant corpus luteum. Since as little as 2 days of exposure to elevated plasma progesterone concentrations is effective, it is suggested that progesterone may act directly on the preovulatory follice.     

Reduction by human chorionic gonadotropin of the luteolytic effect of prostaglandin F2 alpha in ewes

The ability of human chorionic gonadotropin (HCG) to reduce the luteolytic effect of prostaglandin (PGF2 alpha) was demonstrated in cycling ewes. As expected, treatment with 10 mg of PGF2 alpha alone on Day 10 of the estrous cycle exerted a potent negative effect on the function and structure of corpus luteum (CL) as indicated by reduced plasma progesterone, CL progesterone, and CL weight. However, the identical PGF2 alpha treatment failed to significantly reduce either luteal function or luteal weight when administered to ewes that were also treated with HCG on Days 9 and 10 of the estrous cycle. Treatment with HCG alone had a positive effect on CL as indicated by increased plasma progesterone, CL progesterone, and CL weight. Treatment with HCG did not render the CL totally insensitive to the negative effects of PGF2 alpha because plasma progesterone was reduced when the dose of PGF2 alpha was doubled. Whether CL regressed or continued to function after treatment with both HCG and PGF2 alpha appeared to depend upon a balance between the positive and negative effects of the two hormones.     

What regulates placental steroidogenesis in 90-day pregnant ewes?

By day-90, the placenta secretes half of the circulating progesterone and 85% of the circulating estradiol-17beta [Weems YS, Vincent D, Tanaka Y, et al. Effects of prostaglandin F(2alpha) on sources of progesterone and pregnancy in intact, ovariectomized, and hysterectomized 90-100 day pregnant ewes. Prostaglandins 1992;43:203-22; Weems YS, Vincent DL, Nusser K, et al. Effects of prostaglandin F(2alpha) (PGF(2alpha)) on secretion of estradiol-17beta and cortisol in 90-100 day hysterectomized, intact, or ovariectomized pregnant ewes. Prostaglandins 1994;48:139-57]. Ovariectomy (OVX) or prostaglandin (PG) F(2alpha) (PGF(2alpha)) does not abort intact or OVX 90-day pregnant ewes and PGF(2alpha) regresses the corpus luteum, but does not affect placental progesterone secretion in vivo [Weems YS, Vincent D, Tanaka Y, et al. Effects of prostaglandin F(2alpha) on sources of progesterone and pregnancy in intact, ovariectomized, and hysterectomized 90-100 day pregnant ewes. Prostaglandins 1992;43:203-22]. Luteal progesterone secretion in vitro at day-90 of pregnancy in ewes is regulated by PGE(1)and/or PGE(2), not by ovine luteinizing hormone (LH; 3). Concentrations of PGE in uterine or ovarian venous plasma averaged 6 ng/ml at 90-100 days of pregnancy in ewes [Weems YS, Vincent DL, Tanaka Y, Nusser K, Ledgerwood KS, Weems CW. Effect of prostaglandin F(2alpha) on uterine or ovarian secretion of prostaglandins E and F(2alpha) (PGE; PGF(2alpha)) in vivo in 90-100 day hysterectomized, intact or ovariectomized pregnant ewes. Prostaglandins. 1993;46:277-96]. Ovine placental PGE secretion is regulated by LH up to day-50 and by pregnancy specific protein B (PSPB) after day-50 of pregnancy [Weems YS, Kim L, Humphreys V, Tsuda V, Weems CW. Effect of luteinizing hormone (LH), pregnancy specific protein B (PSPB), or arachidonic acid (AA) on ovine endometrium of the estrous cycle or placental secretion of prostaglandins E(2) (PGE(2)) and F(2alpha) (PGF(2alpha)), and progesterone in vitro. Prostaglandins Other Lipid Mediators 2003;71:55-73]. Indomethacin (INDO), a prostaglandin synthesis inhibitor [Lands WEM. The biosynthesis and metabolism of prostaglandins. Annu Rev Physiol 1979;41:633-46], lowers jugular venous progesterone [Bridges PJ, Weems YS, Kim L, et al. Effect of prostaglandin F(2alpha) (PGF(2alpha)), indomethacin, tamoxifen or estradiol-17beta on pregnancy, progesterone and pregnancy specific protein B (PSPB) secretion in 88-90 day pregnant ewes. Prostaglandins Other Lipid Mediators 1999;58:113-24] and inferior vena cava PGE of pregnant ewes with ovaries by half at day-90 [Bridges PJ, Weems YS, Kim L, LeaMaster BR, Vincent DL, Weems CW. Effect of prostaglandin F(2alpha) (PGF(2alpha)), indomethacin, tamoxifen or estradiol-17beta on prostaglandin E (PGE), PGF(2alpha) and estradiol-17beta secretion in 88-90 day pregnant sheep. Prostaglandins Other Lipid Mediators 1999;58:167-78]. In addition, treatment of 90 day ovine diced placental slices with androstenedione in vitro increased placental estradiol-17beta, but treatment with PGF(2alpha)in vitro did not decrease placental progesterone secretion, which indicates that ovine placenta progesterone secretion is resistant to the luteolytic action of PGF(2alpha) [Weems YS, Bridges PJ, LeaMaster BR, Sasser RG, Vincent DL, Weems CW. Secretion of progesterone, estradiol-17beta, prostaglandins (PG) E (PGE), F(2alpha) (PGF(2alpha)), and pregnancy specific protein B (PSPB) by day 90 intact or ovariectomized pregnant ewes. Prostaglandins Other Lipid Mediators 1999;58:139-48]. This also explains why ovine uterine secretion of decreased around day-50 [Weems YS, Kim L, Humphreys V, Tsuda V, Weems CW. Effect of luteinizing hormone (LH), pregnancy specific protein B (PSPB), or arachidonic acid (AA) on ovine endometrium of the estrous cycle or placental secretion of prostaglandins E(2) (PGE(2)) and F(2alpha) (PGF(2alpha)), and progesterone in vitro. Prostaglandins Other Lipid Mediators 2003;71:55-73], when placental estradiol-17beta secretion is increasing [Weems C, Weems Y, Vincent D. Maternal recognition of pregnancy and maintenance of gestation in sheep. In: Reproduction and animal breeding: advances and strategies. Enne G, Greppi G, Lauria A, editors, Elsevier Pub., Amsterdam 1995. p. 277-93]. Treatment of 90 day pregnant ewes with estradiol-17beta+ PGF(2alpha), but not either treatment alone, caused a linear increase in both estradiol-17beta and PGF(2alpha) and ewes were aborting [Bridges PJ, Weems YS, Kim L, Sasser RG, LeaMaster BR, Vincent DL, Weems CW. Effect of prostaglandin F(2alpha) (PGF(2alpha)), indomethacin, tamoxifen or estradiol-17beta on pregnancy, progesterone and pregnancy specific protein B (PSPB) secretion in 88-90 day pregnant ewes. Prostaglandins Other Lipid Mediators 1999;58:113-24; Bridges PJ, Weems YS, Kim L, LeaMaster BR, Vincent DL, Weems CW. Effect of prostaglandin F(2alpha) (PGF(2alpha)), indomethacin, tamoxifen or estradiol-17beta on prostaglandin E (PGE), PGF(2alpha) and estradiol-17beta secretion in 88-90 day pregnant sheep. Prostaglandins Other Lipid Mediators 1999;58:167-78]. Pregnant ewes OVX on day 83 of pregnancy and placental slices cultured in vitro secretes 2-3-fold more estradiol-17beta, PSPB, PGE, and progesterone than placental slices from 90 day intact pregnant ewes, but placental PGF(2alpha) secretion by placental slices from intact or OVX ewes did not change [Denamur R, Kann G, Short R V. How does the corpus luteum of the sheep know that there is an embryo in the uterus? In: Pierrepont G, editor. Endocrinology of pregnancy and parturition, vol. 2. Cardiff, Wales, UK: Alpha Omega Pub Co.; 1973. p. 4-38]. The objective of these experiments was to determine what regulates ovine placental progesterone and estradiol-17beta secretion at day-90 of pregnancy, since the hypophysis [Casida LE, Warwick J. The necessity of the corpus luteum for maintenance of pregnancy in the ewe. J Anim Sci 1945;4:34-9] or ovaries [Weems CW, Weems YS, Randel RD. Prostaglandins and reproduction in female farm animals. Vet J 2006;171:206-28] are not necessary after day-55 to maintain pregnancy. In Experiment 1, diced placental slices from day-90 intact or OVX pregnant ewes that were ovariectomized or laparotomized and ovaries were not removed on day 83 were collected on day-90 and incubated in vitro in M-199 with Vehicle, ovine luteinizing hormone (oLH), ovine follicle stimulating hormone (oFSH), ovine placental lactogen (oPL), PGE(l), PGE(2), PGD(2), PGI(2), insulin-like growth factor (IGF) 1 or 2 (IGF(l); IGF(2)), leukotriene C(4) (LTC(4)), platelet activating factor (PAF) 16 or 18 (PAF-16; PAF-18) at doses of 0, 1, 10, or 100ng/ml for 4h. In Experiment 2, placental slices from day-90 intact and OVX (intact or OVX laporotomized 7 days earlier) pregnant ewes were incubated in vitro with vehicle, INDO, Meclofenamate (MECLO), PGE(l), PGE(2), INDO+PGE(1), MECLO+PGE(l), INDO+PGE(2), or MECLO+PGE(2) for 4h. Media were analyzed for progesterone, estradiol-17beta, PGE, or PGF(2alpha) by RIA. Hormone data in media were analyzed in Experiment 1 by a 2x3x13 and in Experiment 2 by a 2x9 Factorial Design for ANOVA. In Experiment 1, placental progesterone, PGE, or estradiol-17beta secretion were increased (P< or =0.05) two-fold by OVX. Progesterone was not increased (P> or =0.05) by any treatment other than OVX and only FSH increased (P< or =0.05) estradiol-17beta secretion by placental slices in both OVX and intact ewes 90-day pregnant ewes. In Experiment 2, INDO or MECLO decreased (P< or =0.05) placental progesterone secretion by 88% but did not decrease (P> or =0.05) placental estradiol-17beta secretion from intact or OVX ewes. PGE(l) or PGE(2) increased (P< or =0.05) progesterone secretion only in ewes treated with INDO or MECLO. It is concluded that FSH probably regulates day-90 ovine placental estradiol-17beta secretion, while PGE(l) or PGE(2) regulates day-90 placental progesterone secretion.     

Effect of dose and time of injection of prostaglandin F(2alpha) in cycling ewes

A study was done to evaluate the efficacy of graded doses of prostaglandin F(2alpha) (PGF(2alpha)) to induce regression of the corpus luteum and hence estrus, in cycling ewes when given on various days of the estrous cycle. One hundred cycling cross-bred ewes were observed twice daily (08:00 and 20:00 h) for marking by raddled vasectomized rams. After estrus was confirmed in marked ewes by assay of plasma progesterone concentration, the ewes were treated in pairs with 0, 5, 10, 15 or 20 mg PGF(2alpha) on day 2, 3, 4, 7, 8, 9, 12, 13, 14 or 15 of an estrous cycle and then exposed to a raddled ram of known libido and fertility. Plasma progesterone levels were determined on the day of, and on the day following PGF(2alpha)-treatment to monitor luteal function. Ewes marked between one and five days after treatment and having a decrease in plasma progesterone were considered to have responded to the treatment. The percentages of ewes responding were 10, 35, 60, 70 and 95 to doses of 0, 5, 10, 15 and 20 mg PGF(2alpha) respectively. Differences due to dose were significant (P < 0.01) with the two higher doses being more effective. There were differences due to the day of injection, with treatments on days 2 and 3 being less effective.     

Secretion and metabolism of prostaglandin F2 alpha by endometrial tissue obtained at different days of the oestrous cycle in cattle

Two mature heifers were slaughtered on days 3, 6-7, 10-11, 16, 18-19 or on day 21 of the oesterus cycle. Endometrium was incubated in quadruplicates with medium-199 at 37 C and a water saturated gas phase of 95% O2 + 5% CO2. Half ml medium samples were taken after 6, 12 and 24 h of incubation for determination of PGF2 alpha and PGFM. PGF2 alpha was secreted by endometrium at each stage of the oestrous cycle. Maximal secretion was measured around oestrus (p less than 0.01) compared with days 6-16 of the cycle. Concentration of PGFM in medium had a similar trend. Highest ability of endometrium for PGF2 alpha metabolism (indicated by the ratio PGF2 alpha:PGFM) was on days 6-16 of the cycle. Data suggest that PGF2 alpha metabolism by the endometrium may depend on ovarian steroids and that this metabolism may also protect the corpus luteum from the luteolytic action of PGF2 alpha besides reduced production of this prostaglandin during the luteal phase.     

Regulation of prostaglandin synthesis by progesterone in the bovine corpus luteum

The objective of the present study was to investigate the influence of progesterone on prostaglandin synthesis by the corpus luteum (CL). Corpora lutea were obtained from dairy cows on days 4, 6, 10, and 18 of the estrous cycle, dissociated, and placed in serum-free culture. The addition of luteinizing hormone (LH) resulted in a slight, but non-significant (p greater than 0.05), increase in levels of 6-keto-PGF1 alpha, and had no effect on PGF2 alpha. Progesterone treatment caused a significant, dose-dependent decrease in both PGF2 alpha and 6-keto-PGF1 alpha in 6-day and 10-day corpora lutea, but not in 4-day or 18-day corpora lutea. In the 6- and 10-day corpora lutea, progesterone treatment resulted in a greater inhibition of PGF2 alpha than 6-keto-PGF1 alpha production. Therefore, progesterone treatment brought about an increase in the 6-keto-PGF1 alpha to PGF2 alpha ratio in these cells (12.9 vs. 21.3). It is concluded from these studies that progesterone can modulate luteal prostacyclin and PGF2 alpha synthesis, suggesting an interaction of progesterone and prostaglandin production within the corpus luteum.     

Is nitric oxide luteolytic or antiluteolytic?

Nitric oxide (NO) has been reported to be luteolytic based on treatment of cows in vivo with an inhibitor of nitric oxide synthase (NOS-produces NO), which delayed the decline in progesterone by two to three days [Jaroszewki J, Hansel, W. Intraluteal administration of a nitric oxide synthase blocker stimulates progesterone, oxytocin secretion and prolongs the life span of the bovine corpus luteum. Proc Soc Exptl Biol Med 2000;224:50-5; Skarzynski D, Jaroszewki J, Bah, M, et al. Administration of nitric oxide synthase inhibitor counteracts prostaglandin F(2alpha)-induced luteolysis in cattle. Biol Reprod 2003;68:1674-81]. The objective of this experiment was to determine the effect of a long acting NO donor or a NOS inhibitor infused chronically into the interstitial tissue of the ovarian vascular pedicle adjacent to the ovary with a corpus luteum on secretion of progesterone during the ovine estrous cycle. Ewes were treated either with Vehicle (N=5); Diethylenetriamine (DETA-control for DETA-NONOate; N=5); (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolate (DETA-NONOate-long acting NO donor; N=6); or l-nitro-arginine methyl ester (l-NAME-NOS inhibitor; N=6) every 6 h from 24:00 h (0 h) on day 8 through 18:00 h on day 18 of the estrous cycle. Jugular venous blood was collected every 6h for analysis for progesterone and corpora lutea were collected at 18:00 h on day 18 and weighed. Weights of corpora lutea were heavier (P< or =0.05) in DETA-NONOate-treated ewes when compared to Vehicle, DETA, or l-NAME-treated ewes, which did not differ amongst each other (P> or =0.05). Profiles of progesterone in jugular venous blood on days 8-18 differed (P< or =0.05) in DETA-NONOate-treated ewes when compared to Vehicle, DETA, or l-NAME-treated ewes did not differ (P> or =0.05) amongst each other. It is concluded that NO is not luteolytic during the ovine estrous cycle, but may instead be antiluteolytic and prevent luteolysis.     

Influence of oxytocin infusion during oestrus and the early luteal phase on progesterone secretion and the establishment of pregnancy in ewes

In Experiment 1, an osmotic minipump containing oxytocin was implanted s.c. in ewes for 12 days beginning on Day 10 of the oestrous cycle, producing approximately 100 pg oxytocin/ml in the plasma. Two days after the start of infusion, all ewes were injected with 100 micrograms cloprostenol and placed with a fertile ram. At slaughter 22 days later, 9 (75%) of the 12 control (saline-infused) ewes were pregnant compared with 1 (11%) of the 9 ewes infused with oxytocin. In the control group, midcycle plasma concentrations of oxytocin were significantly higher in nonpregnant than in pregnant ewes. In Experiment 2, an infertile ram was used throughout to avoid any possible effects of pregnancy and oxytocin infusions were given at different stages of the oestrous cycle. Otherwise the protocol was similar to that in Exp. 1. Oxytocin infusion during luteolysis and the early follicular phase had no effect on the subsequent progesterone secretion pattern, but infusions beginning the day before cloprostenol-induced luteolysis and lasting for 7 or 12 days and infusions beginning on the day of oestrus for 4 days all delayed the subsequent rise in plasma progesterone by approximately 3-4 days. In these animals, the cycle tended to be longer. It was concluded that an appropriate oxytocin secretion pattern may be necessary for the establishment of pregnancy in ewes and that a high circulating oxytocin concentration during the early luteal phase delays the development of the young corpus luteum.     

Prostaglandin F-2 alpha is transferred from the uterus to the ovary in the sheep by lymphatic and blood vascular pathways

[3H]Prostaglandin F-2 alpha (PGF-2 alpha) was infused into a uterine lymphatic vessel or a uterine vein for up to 1 h, or injected into the uterine lumen of anaesthetized non-pregnant sheep 7-15 days after oestrus. After an intraluminal injection, labelled PGF-2 alpha was recovered in uterine lymph and peak radioactivity was reached 50 min after injection. [3H]PGF-2 alpha infused at a constant rate into a uterine lymphatic vessel resulted in a maximum concentration of radioactivity in plasma which was 5.6- and 1.7-fold higher in the adjacent utero-ovarian and ovarian vein, respectively, than in carotid arterial plasma. Estimation of the amount of infusate transferred from a lymphatic into ovarian venous blood gave a value (0.4%) similar to that for transfer from a uterine vein (0.3%). Evidence for local transfer was substantiated by the presence of significantly higher concentrations of 3H-labelled compounds in the ovary and corpus luteum adjacent to the site of intra-lymphatic infusion compared with those in the opposite organs. The concentrations in the adjacent ovary and corpus luteum were significantly greater when an intra-lymphatic rather than intra-uterine vein infusion was adopted. The results show that [3H]PGF-2 alpha is transferred locally from uterine lymphatic vessels into the adjacent ovary, corpus luteum and ovarian vein.     

Effect of hysterectomy on the short life-cycle corpus luteum produced after GnRH-induced ovulation in the anoestrous ewe

Anoestrous Romney Marsh ewes were treated with small-dose (250 ng) multiple injections of GnRH. Ewes in Groups 1 and 3 were hysterectomized 2 weeks before treatment, while those in Groups 2 and 4 were intact controls. Groups 1 and 2 were primed with progesterone (+P) and treated with 2 h injections of GnRH (250 ng) for 36 h, while Groups 3 and 4 were not pretreated (-P) but were given 2 h injections of GnRH (250 ng) for 18 h. Both treatment regimens were terminated with a bolus injection of GnRH (125 micrograms), given to synchronize the timing of the LH surge and subsequent luteal progesterone production. The plasma progesterone profiles of 5/5 animals in Group 2 (+P controls) and 2/5 animals in Group 4 (-P controls) were indicative of normal luteal function, while the remaining 3/5 animals in Group 4 produced plasma progesterone profiles typical of abnormal luteal function. However, in all the hysterectomized animals (Groups 1 and 3) peripheral plasma progesterone concentrations rose to reach a mean peak value of 1.3 ng/ml plasma on Day 8 which was maintained in all animals irrespective of progesterone pretreatment. The absence of a fall in progesterone concentrations precluded the identification of any animal in Group 4 showing abnormal luteal function. It was also noted that, after hysterectomy, although the corpus luteum was maintained, it was with reduced secretory capacity. The prevention of the expected proportion (70%) of -P animals from displaying a decline in plasma progesterone concentration after hysterectomy provides firm evidence that the uterus is involved in the premature regression of the short-cycle corpus luteum.