Prostaglandin F2α specific binding in equine corpora lutea

Preliminary studies indicate the presence of PGF_2α specific binding sites in membrane fractions prepared from equine corpora lutea. The equilibrium binding data indicate an apparent dissociation constant of 3.2 × 10^?9M and the concentration of binding sites of ～0.1 pmoles/mg membrane protein. Competition of several natural prostaglandins for equine luteal PGF_2α specific binding sites indicates specificity for the 9α-hydroxyl moiety and the 5,6-cis doublebond. Significant increases in relative binding affinities were demonstrated for PGF_2α analogs with a phenyl ring introduced at carbons 16 or 17. Specific PGF_2α binding was demonstrated in corpora lutea collected at known stages of the estrous cycle. There was no pattern in these values based on the stage of the cycle. While specific ³H-PGE₁ binding could be demonstrated, no high affinity sites could be quantitated. ³H-PGE₁ binding appeared unaffected by changes in temperature or time of incubation, whereas PGF_2α specific binding was significantly modified by both these factors.     

Specific 3H-PGE1 binding sites in rat ovary in estrous cycle and pregnancy.

Stimulation of cAMP synthesis by prostaglandins E series in the rat ovary is consistent with the presence of a prostaglandin receptor in this tissue. Prostaglandin binding sites with specificity for PGE1 in vitro incubation systems have been demonstrated in rat ovary slices and corpora lutea. The binding of 3H-PGE1 was progressively inhibited with increasing amounts of unlabelled PGE1 and PGE2. PGF2alpha inhibitory effect was markedly smaller than that of PGE. 3H-PGE1 binding to the ovary was higher in 3-day-old rats than in 5-day-old and adult animals, when the highest binding was present in estrus. The specific binding of 3H-PGE1 to rat corpora lutea (CL) decreased on days 11 and 13 of pregnancy and then gradually returned to the level found on day 1 during the second half of gestation. This binding of labelled prostaglandin during pregnancy has been studied in relation to the PGE1 stimulation of cAMP synthesis in rat corpora lutea, but no consistent changes were observed in responsiveness.     

Prostaglandin E1 and F2alpha specific binding in bovine corpora lutea: comparison with luteolytic effects.

Preliminary characterization indicated the presence of separate prostaglandin (PG)E1 and (PG)F2alpha binding sites in membrane fractions prepared from bovine corpora lutea. These differ in the rate and temperature dependence of the specific binding. Equilibrium binding data indicate the apparent dissociation constants as 1.32 x 10(-9)M and 1.1 x 10(-8)M for PGE1 and PGF2alpha, respectively. Competition of several natural prostaglandins for the PGE1 and PGF2alpha bovine luteal specific binding sites indicates specificity for the 9-keto or 9alpha-hydroxyl moiety, respectively. Differences in relative ability to inhibit 3H-PG binding were found due to sensitivity to the absence or presence of the 5, 6-cis-double bond as well. Bovine luteal function was affected following treatment of heifers with 25 mg PGF2alpha as measured by reduced estrous cycle length, decreased corpus luteum size and significantly decreased plasma progesterone levels. In contract, treatment with 25 mg PGE1 resulted in cycle lengths comparable to those of non-treated herdmates with no apparent modification in corpus luteum size. However, plasma progesterone levels were increased significantly following PGE1 treatment compared to pretreatment values. In so far as data obtained in vitro on PGF2alpha relative binding affinity to the bovine CL can be compared to data obtained independently in vitro on PGF2alpha induced luteolysis in the bovine, PGF2alpha relative binding to the CL and luteolysis appeared to be associated. By similar reasoning, there was no apparent relationship between PGE1 relative binding affinity in the luteal fractions and luteolysis in estrous cyclic cattle.     

Prostaglandin F2 alpha inhibition of epinephrine stimulated cyclic AMP and progesterone production by rat corpora lutea of various ages

Epinephrine can mimic the stimulatory effects of LH in vitro on cyclic AMP (cAMP) and progesterone production by isolated rat corpora lutea. The aim of the present study was to test whether the effects of epinephrine in vitro on the rat corpus luteum, as with LH, can be inhibited by prostaglandin F2 alpha (PGF2 alpha). The stimulatory effect of epinephrine on tissue levels of cAMP in 1-day-old corpora lutea was not inhibited by PGF2 alpha. A dose-dependent inhibition by PGF2 alpha (0.5-50 microM) was seen for 3-day-old corpora lutea and this inhibition could not be overcome by higher concentrations of epinephrine (0.165-165 microM). The stimulation by epinephrine on progesterone production was inhibited by PGF2 alpha (5 microM) in 3- and 5-day-old, but not in 1-day-old corpora lutea. Thus, PGF2 alpha can inhibit the stimulatory effect of epinephrine in 3- and 5-day-old corpora lutea, but not in the newly formed corpora lutea (1-day-old) and PGF2 alpha shows in this respect the same age dependent inhibitory pattern as in relation to LH stimulation.     

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.     

Effect of prostaglandin E2 alpha on the hCG-stimulated progesterone production by human corpora lutea

The effect of prostaglandin PGF2 alpha on the hCG stimulated and basal progesterone production by human corpora lutea was examined in vitro. hCG (40 i.u./ml) stimulated progesterone formation in corpora lutea of early (days 16-19 of a normal 28 day cycle), mid (days 20-22) and late (days 23-27) luteal phases. This stimulation was inhibited by PGF2 alpha (10 micrograms/ml) in corpora lutea of mid and late luteal phases. PGF2 alpha alone did not show a consistent effect on basal progesterone production. The inhibition of hCG stimulated progesterone production by PGF2 alpha at times corresponding to luteolysis indicates a role for that prostaglandin in the process of luteolysis in the human corpus luteum.     

Influence of luteinizing hormone and prostaglandin F(2alpha) on progesterone secretion in superfused minced bovine luteal tissue in the early stage of the estrous cycle

Minced luteal tissue of bovine corpora lutea from Day 4, 5, and 6 of the estrous cycle (n = 4 corpora lutea each) was superfused for 9 h, and the progesterone secretion under the influence of 100 ng luteinizing hormone (LH)/ml and/or 1,000 ng prostaglandin F(2alpha) (PGF(2alpha))/ml was determined. In vivo, this period of the estrous cycle is characterized by a transition from PGF(2alpha) refractoriness to PGF(2alpha) sensitivity. The investigations were carried out in order to examine whether this transition is reflected by a change in the hormone secretion pattern in vitro. The basal secretion was higher on Day 6 than on Day 4 and 5 (P < 0.01). PGF(2alpha) slightly increased the progesterone secretion, but there was no statistically significant difference (P > 0.05). LH, however, stimulated the progesterone secretion by about 30% in luteal tissue collected from Day 4 and 5 (P < 0.01). In luteal tissue collected from Day 6, the LH-induced increase in hormone secretion was not statistically significant due to two corpora lutea that showed no response at all to LH. The progesterone secretion of the two other corpora lutea, however, was increased by 30% (P < 0.01). When PGF(2alpha) and LH were simultaneously added, the LH-induced progesterone secretion was not inhibited; PGF(2alpha) even seemed to intensify the action of LH. The difference between the hormone secretion under the influence of LH alone and that under the influence of a combination of LH and PGF(2alpha), however, was not statistically significant. It is concluded that in cattle the end of the refractoriness to PGF(2alpha) in vivo is not reflected by a corresponding change of the hormone secretion pattern in vitro.     

Gonadotropin-releasing hormone 1 directly affects corpora lutea lifespan in Mediterranean buffalo (Bubalus bubalis) during diestrus: presence and in vitro effects on enzymatic and hormonal activities

The expression of gonadotropin-releasing hormone (GNRH) receptor (GNRHR) and the direct role of GNRH1 on corpora lutea function were studied in Mediterranean buffalo during diestrus. Immunohistochemistry evidenced at early, mid, and late luteal stages the presence of GNRHR only in large luteal cells and GNRH1 in both small and large luteal cells. Real-time PCR revealed GNRHR and GNRH1 mRNA at the three luteal stages, with lowest values in late corpora lutea. In vitro corpora lutea progesterone production was greater in mid stages and lesser in late luteal phases, whereas prostaglandin F2 alpha (PGF2alpha) increased from early to late stages, and PGE2 was greater in the earlier-luteal phase. Cyclooxygenase 1 (prostaglandin-endoperoxide synthase 1; PTGS1) activity did not change during diestrus, whereas PTGS2 increased from early to late stages, and PGE2-9-ketoreductase (PGE2-9-K) was greater in late corpora lutea. PTGS1 activity was greater than PTGS2 in early corpora lutea and lesser in late luteal phase. In corpora lutea cultured in vitro, the GNRH1 analog (buserelin) reduced progesterone secretion and increased PGF2alpha secretion as well as PTGS2 and PGE2-9-K activities at mid and late stages. PGE2 release and PTGS1 activity were increased by buserelin only in late corpora lutea. These results suggest that GNRH is expressed in all luteal cells of buffalo, whereas GNRHR is only expressed in large luteal phase. Additionally, GNRH directly down-regulates corpora lutea progesterone release, with the concomitant increases of PGF2alpha production and PTGS2 and PGE2-9-K enzymatic activities.     

The influence of embryo presence on prostaglandins synthesis and prostaglandin E2 and F2alpha content in corpora lutea during periimplantation period in the pig

We determined the expression of PGE2 synthase (mPGES-1), PGF synthase (PGFS), carbonyl reductase/prostaglandin 9-ketoreductase (CBR1) genes and the content of PGE2, PGF2alpha in porcine corpora lutea on Days 12-14 of pregnancy and Days 12-14 of the estrous cycle. For this study we used a surgically-generated model in which one of the uterine horns was cut transversely and a part of this horn was detached from the uterine corpus. The expression of mPGES-1, PGFS, and CBR1 genes and mPGES-1/PGFS ratio were significantly higher in corpora lutea of the pregnant gilts compared to the corpora lutea from the parallel ovaries of the cyclic gilts. There was no difference in mPGES-1, PGFS, CBR1 genes expression and mPGES-1/PGFS ratio between corpora lutea ipsi-(CL1) and contralateral (CL2) to the uterine horn with the developing embryos. The highest content of PGE2 was found in CL1 of the pregnant gilts. The PGE2/PGF2alpha ratio was significantly higher in CL1 of the pregnant gilts compared to corpora lutea from parallel ovary of the cyclic gilts. We suggest that the activity of the investigated genes is induced by compounds of embryonic origin which are not distributed only to the ipsilateral ovary but are transported within the mesometrium to both ovaries in a more systemic manner.     

Modulation of 3H-prostaglandin E2 binding sites in the rabbit gastric mucosa

The binding of 3H-prostaglandin E2 (PGE2) to rabbit gastric mucosa was investigated. Binding depended on incubation time, temperature and pH, and was saturable and reversible. Scatchard plot analysis revealed a single class of binding sites with a dissociation constant (Kd) of 5.33 +/- 0.21 nM and a maximum number of binding sites (Bmax) of 138.1 +/- 3.4 fmol/mg protein. PGE1 and 16,16-dimethyl PGE2 potently competed with 3H-PGE2 for the binding sites of gastric mucosa, whereas PGA2, PGF2 alpha, 6-keto PGF1 alpha and thromboxane B2 were less potent. The gastric mucosa prepared from the rabbits given indomethacin (5 mg/kg s.c. three times) showed a lower Kd (2.47 +/- 0.19 nM) for 3H-PGE2 than that from untreated one. Treatment with a PGE1 analog, misoprostol (320 micrograms/kg s.c. three times) lowered the Bmax to 74.1 +/- 2.4 fmol/mg protein without any significant effect on the Kd value. It is concluded that rabbit gastric mucosa has specific binding sites for 3H-PGE2 which may be modulated by the levels of PGs in vivo.     

Inhibition of (3H) prostaglandin F2alpha binding to its receptors by progesterone.

The specific binding of [3H] prostaglandin (PG) F2alpha to bovine corpus luteum cell membranes was inhibited by progesterone. Progesterone inhibition of binding was dependent on membrane protein and independent of [3H] PGF2alpha concentrations in the medium. The lower inhibition of binding at high protein concentrations can be overcome by increasing the amounts of progesterone added. Progesterone inhibition of binding appears to be due to a decrease in the receptor number rather than a decrease in the receptor affinities. The kinetic properties (association and dissociation rates) of the remaining receptors were unchanged. The inhibition of [3H] PGF2alpha binding was observed by preincubating the membranes with progesterone or by adding at the beginning but not during incubation. The concentrations of progesterone that inhibited binding by about 50% do occur in bovine corpora lutea of estrous cycle and pregnancy.     

Prostaglandin metabolism in the ovine corpus luteum: catabolism of prostaglandin F(2alpha) (PGF(2alpha)) coincides with resistance of the corpus luteum to PGF(2alpha)

To examine possible mechanisms involved in resistance of the ovine corpus luteum to the luteolytic activity of prostaglandin (PG)F(2alpha), the enzymatic activity of 15-hydroxyprostaglandin dehydrogenase (PGDH) and the quantity of mRNA encoding PGDH and cyclooxygenase (COX-2) were determined in ovine corpora lutea on Days 4 and 13 of the estrous cycle and Day 13 of pregnancy. The corpus luteum is resistant to the action of PGF(2alpha) on Days 4 of the estrous cycle and 13 of pregnancy while on Day 13 of the estrous cycle the corpus luteum is sensitive to the actions PGF(2alpha). Enzymatic activity of PGDH, measured by rate of conversion of PGF(2alpha) to PGFM, was greater in corpora lutea on Day 4 of the estrous cycle (P < 0.05) and Day 13 of pregnancy (P < 0.05) than on Day 13 of the estrous cycle. Levels of mRNA encoding PGDH were also greater in corpora lutea on Day 4 of the estrous cycle (P < 0. 01) and Day 13 of pregnancy (P < 0.01) than on Day 13 of the estrous cycle. Thus, during the early estrous cycle and early pregnancy, the corpus luteum has a greater capacity to catabolize PGF, which may play a role in the resistance of the corpus luteum to the actions of this hormone. Levels of mRNA encoding COX-2 were undetectable in corpora lutea collected on Day 13 of the estrous cycle but were 11 +/- 4 and 44 +/- 28 amol/microgram poly(A)(+) RNA in corpora lutea collected on Day 4 of the estrous cycle and Day 13 of pregnancy, respectively. These data suggest that there is a greater capacity to synthesize PGF(2alpha), early in the estrous cycle and early in pregnancy than on Day 13 of the estrous cycle. In conclusion, enzymatic activity of PGDH may play an important role in the mechanism involved in luteal resistance to the luteolytic effects of PGF(2alpha).     

Receptors for prostaglandins F2 alpha and E2 in ovine corpora lutea during maternal recognition of pregnancy.

Plasma membrane receptors for prostaglandins (PG) F2 alpha and E2 were quantified in ovine corpora lutea obtained from nonpregnant and pregnant ewes on Days 10, 13, and 15 post-estrus, and from additional ewes on Days 25 and 40 of pregnancy. Regardless of reproductive status or day post-estrus, concentrations of luteal receptors for PGF2 alpha were 7- to 10-fold greater than those for PGE2. In pregnant ewes the concentration of receptors for PGF2 alpha was highest on Day 10 (35.4 +/- 2.8 fmol/mg) and lowest on Day 25 (22.3 +/- 2.5 fmol/mg). A difference in the concentration of luteal receptors for PGF2 alpha between pregnant and nonpregnant ewes was apparent only on Day 15 post-estrus, at which time the concentration of receptors for PGF2 alpha was higher in pregnant ewes than in nonpregnant ewes (27.1 +/- 2.7 vs. 17.7 +/- 2.7 fmol/mg). Concentrations of receptors for PGE2 in pregnant ewes were similar (p > 0.05; 2.8 +/- 0.3 to 3.7 +/- 0.2 fmol/mg) between Days 13 and 40 but were higher (p < 0.05) than in corpora lutea obtained from nonpregnant ewes on Days 10 (5.0 +/- 0.4 vs. 4.1 +/- 0.2 fmol/mg) and 15 (3.7 +/- 0.2 vs. 2.0 +/- 0.4 fmol/mg) post-estrus. Although concentrations of receptors for both PGF2 alpha and PGE2 were lowest in corpora lutea obtained from nonpregnant ewes on Day 15, this was not due to luteal regression since the weights and concentrations of progesterone in corpora lutea on Day 15 were not lower than those for corpora lutea obtained on Days 10 and 13.(ABSTRACT TRUNCATED AT 250 WORDS)     

The non-luteolytic effect of prostaglandin F2α at the beginning of the bovine oestrous cycle: The role of oestrogen?

After the preovulatory gonadotrophin surge, antral follicles ovulate or become atretic; whatever their evolution, they stop secreting oestradiol. Since it was demonstrated that oestrogens were necessary for luteolysis to occur after PGF(2)alpha treatment, their absence could explain the non-luteolytic effect of PGF(2)alpha injected early in the cycle. Thus, cyclic cows received a PGF(2)alpha analogue and oestradiol valerate together on day 3. This treatment did not affect the life span of the corpus luteum. The absence of oestrogens in the blood does not explain the failure of PGF(2)alpha to cause luteolysis in young corpora lutea.     

Release and specific binding of prostaglandins in bovine pineal gland

Incubated bovine pineal glands released prostaglandin E-and prostglandin F-like material (304 +/- 20 and 582 +/- 56 pg/mg dry tissue wt/h, respectively) and the release was increased 2.2 2.9-fold by adding 10(-4)-10(-6)M of norepinephrine to the medium. Binding assays revealed the existence of high affinity binding of 3H-prostaglandin E2 (3H-PGE2) and 3H-prostaglandin F1 alpha (3H-PGF2 alpha) in low speed supernatants of pineal homogenates. Binding was increased by increasing Ca++ concentration in medium up to 2 mM, was heat labile and was depressed following incubation with trypsin. In subcellular fractionation studies maximal 3H-PG binding was found in the 27000 x g pellet. Scatchard analysis of 3H-PGE2 binding revealed the presence of a single population of binding sites with a Kd= 1.2 nM and a binding site concentration of 1-2 pmoles/g protein. A single population of binding sites for 3H-PGF2 alpha was also detected with a Kd= 1.7 nM and a similar binding site concentration. Non-radioactive PGE1 and PGE2 were almost equally effective to compete for 3H-PGE1 binding sites (ED50= 5 and 2 nM, respectively). Unlabeled PGF1 was relatively ineffective to compete for 3H-PGE2 binding (ED50 greater than 1000 nM) but displaced effectively 3H-PGF2 alpha binding (ED20=1.2 nM).     

Specific PGF-2 alpha binding by the corpus luteum of the pregnant and non-pregnant mare.

The binding of prostaglandin (PG) F-2 alpha to corpora lutea (CL) from pregnant and non-pregnant Pony mares was examined. Studies of the rates of association and dissociation indicated that [3H]PGF was bound specifically and reversibly to a luteal cell membrane preparation (MP) that was isolated by high speed (100,000 g) ultracentrifugation. Various PGs and PG metabolites displaced [3H]PGF from the receptors in the following decreasing order: PGF-2 alpha greater than 13, 14-dihydro-PGF-2 alpha = 13,14-dihydro-15-keto PGF-2 alpha greater than PGD-2 greater than PGF-1 alpha = PGE-2 greater than PGE-2 beta greater than PGE-1. These data implicate the 9 alpha-OH and 5,6 cis double bond as major contributors to PGF receptor recognition. The membrane preparation appeared to contain at least two receptor populations, a high affinity, low capacity and a low affinity, high capacity receptor. The binding of PGF (pg/mg MP protein +/- s.e.m. (n)) to CL of the non-pregnant mare increased from 4.09 +/- 11.6 (4), on Day 4 after ovulation, to reach maximal levels by Day 12, 15.01 +/- 2.5 (4), and declined thereafter. In pregnancy the binding of PGF continued to increase until Day 18, reaching 27.47 +/- 1.7 (3), before it declined on Day 20. The reduction in binding by Day 16 in the non-pregnant mare may reflect the process of luteolysis, while high PGF binding capacity of CL between Days 16 and 18 of pregnancy indicated that luteal maintenance during pregnancy is not associated with a reduction of PGF binding capabilities.     

Immunohistochemical localization of PGF2 alpha receptor in the rat ovary.

As a step towards understanding the role of prostaglandin F2 alpha (PGF2 alpha) in ovarian function, a rabbit antiserum against purified PGF2 alpha receptor (PGF2 alpha-R) was produced. This report details the use of this antiserum in immunohistochemical staining of ovaries of non-pregnant and pregnant rats to ascertain which cell types, in vivo, possess PGF2 alpha-R. In non-pregnant rats, three ovarian cell subpopulations contain immunoreactive PGF2 alpha-R. These include: a subpopulation of the cells found in corpora lutea, a subpopulation of the thecal cells surrounding secondary and mature (Graafian) follicles, and a subpopulation of primary and secondary interstitial cells. The ovarian tissues and cell types in which immunoreactive PGF2 alpha-R cannot be demonstrated include: the serosa overlying the ovary and its vessels, the coelomic epithelium and its underlying cortical stroma, medullary stroma and vessels, granulosa cells of primary, secondary and mature follicles, the oocyte, and the blood vessels and stroma within corpora lutea. PGF2 alpha-R immunohistochemical staining of corpora lutea from non-pregnant animals was examined both prior to the start of luteolysis and during luteolysis. During luteolysis, cells undergoing apoptosis stained for the presence of PGF2 alpha-R. PGF2 alpha-R immunohistochemical staining was also examined in corpora lutea during pregnancy and until 4 days postpartum. The major findings here were the apparent large increase in staining intensity of granulosa-lutein cells during pregnancy, and the loss of PGF2 alpha-R immunopositivity of the granulosa-lutein cells during the postpartum period. In summary, three ovarian cell subpopulations, all of which can secrete steroids, possess immunoreactive PGF2 alpha-R.     

Cyclic AMP formation of isolated human corpora lutea in response to HCG - interference by PGF2 alpha.

Human corpora lutea of defined ages were excised at operation, cut into pieces and incubated in the presence of HCG, PGF2 alpha and PGE2 alone or in combination. Following incubation cAMP formation in tissue and medium was determined. HCG-stimulated tissue cAMP content was most pronounced at a corpus luteum age of 7-10 days after ovulation. This stimulation was antagonized by PGF2 alpha in corpora lutea older than 6 days. PGE2 stimulated cAMP formation per se and this effect was more pronounced when HCG and PGE2 were combined. A possible role for PGF2 alpha as a luteolytic substance in the human is suggested.     

Changes in corpus luteum content of prostaglandin F2 alpha and E in the adult pseudopregnant rat

Conflicting reports exist regarding the source of luteolytic PGF2 alpha in the rat ovary. To assess the quantities of different PGs, measurements of PGF2 alpha, PGE and PGB were performed by radioimmunoassay in the adult pseudopregnant rat ovary throughout the luteal lifespan. Ovaries of 84 rats were separated by dissection into two compartments, corpora lutea of pseudopregnancy and remainder of ovary. Tissue samples were homogenized and prostaglandins extracted and determined by radioimmunoassay. During the mid-luteal and late-luteal phases, levels of PGs were significantly higher in the corpora lutea of pseudopregnancy than in the remainder of ovary. An increase of PGF2 alpha-content in the corpus luteum was registered with peak-levels of 53.9 +/- 8.5 (mean +/- SEM, N = 18) ng/g tissue wet weight at day 13 of pseudopregnancy. PGE-levels reached peak-values at day 11 of pseudopregnancy (271.6 +/- 28.4 ng/g w w, mean +/- SEM, N = 12). PGB-levels were below detection limits in all compartments for all ages studied. The present study demonstrates increased availability of PGF2 alpha in the corpus luteum during the luteolytic period, and points toward either increased luteal synthesis or luteal binding of PGF2 alpha during the luteolytic period.