Plasma concentrations of 13,14-dihydro-15-keto prostaglandin F2-alpha (PGFM), progesterone and estradiol in pregnant and nonpregnant diestrus cross-bred bitches

The canine corpus luteum (CL) typically sustains elevated plasma progesterone concentrations for 2 months or more, with a peak approximately 15-25 days after ovulation, followed by a slow decline. The processes involved in the slow, protracted regression of the CL over the remaining 1.5-2-month period in nonpregnant bitches and until shortly prepartum in pregnant bitches are not well characterized. The rapid luteolysis that occurs immediately prepartum appears to be a result of a prepartum rise in peripheral PGF. The potential role of PGF in the slow regression process in the several weeks preceding parturition and in nonpregnant bitches after 15-25 days after ovulation is not known. Therefore, plasma concentrations of 13,14-dihydro-15-keto-prostaglandin F2-alpha (PGFM), progesterone (P4) and estradiol (E2) were determined and compared in bitches during nonpregnant diestrus (n = 9) or pregnancy (n = 8). During the gradual decrease in plasma concentrations of progesterone in both groups, the P4 pattern appeared unrelated to changes in either E2 or PGFM concentrations. The PGFM pattern was different between diestrus and pregnant bitches (P > 0.01); there was an apparent progressive but slow increase in PGFM in pregnant bitches from Days 30 to 60, followed by a large increase prior to parturition; concentrations declined immediately postpartum. However, there were no increases in PGFM during the same interval in nonpregnant bitches. Mean estradiol concentrations were sporadically elevated during the last third of pregnancy and less so in nonpregnant diestrus; there was no acute prepartum increase in estradiol associated with the PGFM increase. In summary, although there were no apparent changes in peripheral PGF2alpha concentration involved in regulating the slow protracted phase of luteal regression in nonpregnant bitches, modest increases in PGFM may play a role in ovarian function after mid-gestation in pregnant bitches. Furthermore, the acute prepartum rise in PGFM was not dependent on any concomitant increase in estradiol concentrations.     

Canine corpus luteum regression: apoptosis and caspase-3 activity

The present study evaluated the occurrence of apoptosis and caspase-3 activity in the canine corpus luteum during the period of luteal regression in eight pregnant and nine nonpregnant diestrus bitches. Intact luteal cells were obtained from corpora lutea in both peripartum pregnant bitches and nonpregnant diestrus bitches at approximately 65 d (range 63-68) after estrus, but not at days 75 and 85 in nonpregnant bitches. In all bitches, apoptotic cells were rarely detected and when present, those cells were more easily detected using the hematoxylin and eosin technique than using the critical electrolyte concentration technique. The luteal structures at 75 and 85 d of diestrus had histological characteristics similar to a corpus albicans. Caspase-3 activity was detected in morphologically normal corpora lutea from both pregnant and diestrus bitches around day 65, and also in the later structures considered corpus albicans tissue. These results suggested that apoptosis may not be the major mechanism involved in canine functional luteal regression, and that caspase-3 participated in both functional and morphological luteolysis and in the tissue reorganization involved in corpus albicans formation.     

Comparisons of estradiol,LH and FSH patterns in pregnant and nonpregnant beagle bitches

To characterize plasma estradiol, LH and FSH patterns of secretion during the bitch estrous cycle, blood samples were obtained daily from 15 days before until 135 days after the LH surge in 10 pregnant and 10 nonpregnant beagle bitches. After an initial increase between days 15 and 10 and an expected proestrous peak, estradiol concentrations increased again from days 9-12 (corresponding to cytological metestrus) from basal values observed around day 9 after the LH surge, and remained significantly elevated throughout the luteal phase both in pregnant and nonpregnant animals. Concomitantly with the end of the luteal phase, plasma concentrations of estradiol returned to basal values in both groups. During the mid- to late-luteal phase, mean basal LH secretion was significantly elevated throughout in the pregnant relative to the nonpregnant animals. However, in nonpregnant animals, pulsatility was increased and peaks of higher amplitude were observed. The plasma FSH profiles, determined by a specific homologous RIA, differed significantly between pregnant and nonpregnant bitches during the last two-thirds of the luteal phase with a mean FSH level more elevated during pregnancy. The FSH level then decreased around parturition and low concentrations during lactation period were observed. The FSH concentrations remained steady in nonpregnant luteal phases from early luteal phase through mid-anestrus. The differences in pregnant and nonpregnant LH and FSH concentrations suggest pregnancy differences in regulation of the corpus luteum. Finally, the elevated estradiol concentrations observed during the luteal phase of both pregnant and nonpregnant animals suggest that an ovarian production of estrogens may be involved in overall corpus luteum regulation in dogs as in other species.     

Expression of cyclooxygenase 1 and 2 in the canine corpus luteum during diestrus

In the dog, unlike most other domestic animal species, corpus luteum (CL) life span is not affected by hysterectomy. Only in pregnant dogs, during the immediate prepartum decline of progesterone, does PGF2alpha clearly seem to act as an endogenous luteolytic agent. Whether endogenous PGF2alpha plays a role in the slow regression of the corpora lutea of the nonpregnant cycle is not known. To test for possible paracrine/autocrine effects of locally produced PGF2alpha, luteal expression of the key rate-limiting enzymes in prostaglandin biosynthesis, i.e. cyclooxygenase 1 and 2 (Cox1 and Cox2), was examined in dogs during diestrus, including the periods of CL formation, as well as early and late CL regression. Corpora lutea were collected by ovariohysterectomy from nonpregnant bitches 5, 15, 25, 35, 45 and 65 days after ovulation. On the mRNA-level, expression of Cox1 and Cox2 was tested by qualitative and quantitative, Real Time (Taq Man) RT-PCR; on the protein level, expression of Cox2 was studied by immunohistochemistry. The mRNA for Cox1 and Cox2 were detected at all stages of diestrus. Expression of Cox1 was lowest on Day 5 (ovulation = Day 0) and higher and nearly constant thereafter. Expression of Cox2-mRNA was distinctly cycle related and highest on Day 5; it decreased by Day 15 and remained constantly low until Day 65. Immunohistochemistry localized expression of Cox2 in the cytoplasm of luteal cells. Staining was restricted to Days 5 and 15, with stronger signals on Day 5. These data suggested that increased expression of Cox2 is associated with luteal growth and development and not luteal regression. Furthermore, the expression of Cox1 more likely reflected activity of a housekeeping gene.     

Plasma 13,14-dihydro-15-keto-PGF alpha (PGFM) in pregnant and nonpregnant heifers prior to and during surgery and following intrauterine injection of PGF2 alpha

On day 17 postestrus or postmating, heifers were given intrauterine injections of saline (2 pregnant, 2 non-pregnant) or 200 micrograms PGF2 alpha (7 pregnant, 6 nonpregnant) through cannulae installed surgically into the uterine horn ipsilateral to the corpus luteum bearing ovary. Jugular blood samples were collected prior to the laparotomy at which the cannulae were installed during surgery, and for 90 min following the intrauterine injection. Plasma was assayed for progesterone and 13,14-dihydro-15-keto-PGF2 alpha (PGFM). Laparotomies were reopened to confirm proper cannula placement and to determine if blastocysts were present in mated heifers. Concentrations of PGFM were higher in pregnant compared to nonpregnant heifers during the presurgery (68 +/- 26 vs 24 +/- 26 pg/ml; P less than .025) and surgery (186 +/- 47 vs 65 +/- 17 pg/ml; P less than .05) periods. Pregnancy status did not alter the mean concentrations of PGFM (pregnant, 554 +/- 70 pg/ml; nonpregnant, 422 +/- 81 pg/ml) or the half-life of its decline in concentration (18 min) following intrauterine injection of PGF2 alpha. Pregnancy at 17 days in cattle does not appear to influence PGF2 alpha transport from the uterine lumen or its metabolism in the uterus or elsewhere in response to an acute intrauterine injection.     

Apoptosis in canine corpus luteum during spontaneous and prostaglandin-induced luteal regression

Spontaneous luteal regression and prostaglandin-induced luteolysis in bitches were evaluated by measuring the apoptotic index for DNA fragmentation and the relative level of Bax gene expression in ovaries removed from nine untreated nonpregnant bitches at selected times during diestrus and in nine pregnant bitches after 1 day of administering abortive doses of a PGF-analog gel formulation given intravaginally at selected times during gestation. Nonpregnant diestrus was divided into three periods (early, mid and late) based on vaginal cytology and plasma progesterone concentration. Pregnant bitches were treated with a PGF-analog gel at corresponding stages of pregnancy (early, mid and late) and evaluated by ultrasound. Another eight pregnant bitches were similarly studied and serum progesterone concentrations were determined after 1, 2, 3 or 4 days of PGF-analog gel. Corpora lutea obtained by ovariohysterectomy were analyzed for apoptotic internucleosomal DNA fragmentation relative to that in a control cell line (U937), using an apoptotic DNA ladder kit and gel electrophoresis and for relative expression of the pro-apoptotic Bax gene by RT-PCR and electrophoresis. In nonpregnant bitches, the DNA fragmentation apoptotic index was greater in late than in early diestrus (P < 0.01). The index after 1 day of PGF-analog gel was higher in early pregnant bitches than in early diestrus bitches (P < 0.05); it was highest in midpregnancy (P < 0.05). The degree of apoptosis was related to the number of times PGF-analog gel was administered. Bax mRNA was detected in the corpus luteum (CL) and Bax expression increased from early to middiestrus in nonpregnant subjects (P < 0.05). Potential elevation in Bax due to PGF-analog gel treatment in pregnancy was only significant in relation to normal diestrus during early pregnancy (P < 0.01). In conclusion, we inferred that the effects of endogenous or exogenous prostaglandin on CL life span in bitches involved increases in apoptotic activity and that increased apoptosis was implicated in normal luteal regression in nonpregnant bitches.     

The effects of metergoline combined with PGF2alpha treatment on luteal function and gestation in pregnant bitches

This study was designed to determine the nature of the antiluteotrophic effect of metergoline on pregnant bitches, the occurrence of clinically evident side-effects, and the efficacy of PGF(2alpha) in initiating abortion after a course of metergoline therapy. Starting on Days 18 to 20 after the onset of diestrus, 8 adult pregnant beagle bitches were treated twice daily with 0.4 to 0.5 mg/kg po metergoline for 5 d. After receiving no treatment for the next 5 d, metergoline administration was repeated for a further 3 d, followed by twice-daily intramuscular injections of dinoprost tromethamine at 250 mug/kg. There was an overall trend for the plasma progesterone concentration to decrease during the first and second course of metergoline therapy and to rise during the intervening period of no treatment. None of the bitches aborted during or after the first 5 d of metergoline administration. No side-effects were evident during the metergoline therapy. After pretreatment with metergoline a mean of 4.8 injections of PGF(2alpha) was necessary to ensure complete abortion. All abortions were rapid and uneventful, except for the usual side-effects associated with PGF(2alpha). The plasma progesterone concentration at the onset of PGF(2alpha) treatment was positively correlated to the number of PGF(2alpha) injections needed to complete the abortion process. The plasma progesterone concentration was < 8 nmol/l for several days as a result of metergoline therapy in 6 of the 8 bitches. Only 1 bitch, however, aborted before PGF(2alpha) therapy was initiated. In the other 7 bitches PGF(2alpha) appeared to be necessary for abortion. The results suggest that the effect of metergoline has to be considered incompletely luteolytic at the doses used in this study. Even prolonged suppression of luteal function in early to mid-gestation, however, did not cause abortion without the previously documented luteolytic and/or ecbolic effects of PGF(2alpha). The average interestrus interval of cycles treated with metergoline and PGF(2alpha) was shorter (mean 182.2 d, SD 7.9 d, n = 6) than expected for this group of bitches (mean 211.4 d, SD 31.5 d, n = 7).     

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)     

Plasma homocysteine levels in cycling, pregnant, and spayed bitches

The aim of this study was to evaluate the physiological range of homocysteine (Hcy) exhibited by bitches during the follicular (pro-oestrus), luteal (dioestrus) and anoestrus phases of cycling animals, and in pregnant and spayed bitches. The lowest concentrations of Hcy were observed during dioestrus (3.2+/-1.27mumol/L) and in pregnant bitches (3.9+/-1.72mumol/L), and the highest concentrations during anoestrus (7.8+/-0.6mumol/L) and in spayed bitches (12.1+/-5.16mumol/L).     

In vivo levels of prostaglandin F2 alpha, E2 and prostacyclin in the corpus luteum of pregnant and pseudopregnant rats

Prostaglandin F2 alpha (PGF2 alpha) is a well-known luteolytic factor in the rat corpus luteum. To investigate a possible luteal origin of PGF2 alpha, measurements of this prostaglandin were performed in different luteal tissues in vivo. Prostaglandin E2 (PGE2) and the stable metabolite of prostacyclin, 6-keto-PGF1 alpha, were assayed simultaneously. Corpora lutea of different ages from 57 pregnant and pseudopregnant rats (mated with sterile males) were rapidly excised, dissected in 0 degree C indomethacin solution, homogenized, and extracted for prostaglandins with solid-phase extraction cartridges. Prostaglandins were determined by radioimmunoassay. Plasma levels of progesterone and 20 alpha-dihydroprogesterone were also monitored. In the adult pseudopregnant rat model, luteolysis occurs at Day 13 +/- 1, and maximal levels of all three prostaglandins were detected on Day 13 of pseudopregnancy: 0.40 +/- 0.02, 2.6 +/- 0.29, and 1.76 +/- 0.24 pmol/mg protein (mean +/- SEM, n=7) for PGF2 alpha, PGE2, and 6-keto-PGF1 alpha respectively. In pregnant rats, on the corresponding day, levels were considerably lower: 0.15 +/- 0.02, 0.90 +/- 0.13, and 0.50 +/- 0.06 pmol/mg protein (mean +/- SEM, n=9, p less than 0.0001), respectively. Luteal levels in pregnant rats showed a continuous decline on Days 13 and 19 for all prostaglandins measured, whereas in pseudopregnant rats an increment of PGF2 alpha was noted between Days 7 and 13 and remained high on Day 19. PGE2 closely followed levels of PGF2 alpha, but at a 5- to 10-fold higher level. The coefficient of correlation between PGF2 alpha and PGE2 in the luteal compartment of both models was 0.87 (p less than 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Luteolytic effects of prostaglandin F2alpha on day 8 to 19 corpora lutea in the bitch

Luteolysis was induced in 5 experimental Beagle (8 cycles) and 7 client-owned bitches treated with 150 to 200 microg/kg, sc of prostaglandin F2alpha administered twice daily for 4 d, starting on Days 8 to 19 after the onset of cytological diestrus. Five experimental Beagle bitches had been mated during the estrus preceding treatment, and copulation had been confirmed in 2/7 client-owned bitches presented for termination of unwanted pregnancy. Serum progesterone concentration (mean +/- SD) declined from 26.1 +/- 66 ng/ml before treatment to 0.3 +/- 0.4 ng/ml on the fourth day of treatment One of the 7 client-owned bitches maintained her pregnancy even though serum progesterone concentrations were less than 0.5 ng/ml on the third and fourth day of treatment. Mean (+/- SEM) inter-estrous intervals before and following prostaglandin-induced luteolysis were 207.3 +/- 12.4 (n = 11 cycles in 6 bitches) and 95.5 +/- 20.0 d (n = 6 cycles in the same 6 bitches; P < 0.0001), respectively These results suggest that effective prostaglandin-induced luteolysis can be achieved with administration of 180 microg/kg during the third week of diestrus in pregnant and nonpregnant bitches.     

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.     

Synthesis of uterine endometrial proteins during early diestrus in the cyclic and pregnant dog, and after estrogen and progesterone treatment.

The objectives of this study were to identify and characterize dog uterine endometrial proteins synthesized de novo in explant culture during early luteal phase, to examine distribution of these proteins prior to the embryo's entering the uterus and during its free-floating period prior to implantation, and to examine regulation of endometrial proteins by estrogen and progesterone (P4) treatments. Uterine endometrium was collected from cyclic and pregnant bitches on diestrus Days 3, 7, and 10 as determined by loss of cornification of vaginal epithelium, and from ovariectomized dogs after treatment with corn oil, estrogen, P4, or estrogen followed by 1 or 2 wk of P4. Tissue was incubated in an explant culture system in the presence of [3H]leucine or [35S]methionine. The rate of incorporation of [3H]leucine into nondialyzable macromolecules indicated no significant change in rates of incorporation by status (pregnant vs. nonpregnant), day, or steroid treatment. Uterine endometrial-conditioned culture medium, analyzed by two-dimensional SDS-PAGE and fluorography, revealed a complex array of at least ten proteins or protein complexes in cyclic and pregnant bitches. No difference in protein pattern was detected by status; however, differences in distribution were apparent by day of cycle or early pregnancy. Two major proteins, cP5 (M(r) 54,686) and cP6 (M(r) 23,010) appeared to be differentially expressed. Expression of cP5, maximal on diestrus Day 3, decreased as the cycle or pregnancy progressed to diestrus Day 10. In contrast, expression of cP6, a minor protein on diestrus Day 3, appeared to be up-regulated for each status to Day 10, with increased intensity and multiple isoelectric and molecular-weight variants. In ovariectomized steroid-treated dogs, two-dimensional SDS-PAGE showed that pattern and distribution of specific proteins were affected by treatment. Acidic protein cP1 (M(r) 87,600), synthesized after corn oil and P4 treatment, was suppressed with estradiol (E2). Proteins cP2 (M(r) 40,000 and M(r) 42,000), present with all treatments, were intensified with P4. A high-M(r) basic protein complex (cP3) and acidic protein cP4 were expressed with E2 and maintained with P4 treatment. Proteins cP5 and cP6, while not induced by E2 or P4 alone, required E2 priming for P4 induction. Protein cP5 was down-regulated while cP6 was up-regulated with P4 for 2 wk. Proteins induced by estrogen followed by 1 or 2 wk of P4 treatments were similar to those released by endometrial explants collected from pregnant and cyclic bitches on Days 3, 7, and 10 of spontaneous diestrus.(ABSTRACT TRUNCATED AT 400 WORDS)     

Serum androstenedione and testosterone concentrations during pregnancy and nonpregnant cycles in dogs

Concentrations of testosterone and of androstenedione were determined by radioimmunoassay in serum samples collected every 2-5 days throughout the periovulatory and luteal phases of the ovarian cycles of pregnant and nonpregnant beagle bitches. Testosterone levels were consistently lower than those of androstenedione, reached peaks of 29 +/- 4 ng/dl near the time of the preovulatory luteinizing hormone peak, and were reduced to near the limits of detection (less than or equal to 5-10 ng/dl) throughout the luteal phase. Androstenedione levels reached preovulatory peaks of 73 +/- 13 ng/dl, were 54 +/- 7 ng/ml during early estrus, increased (P less than 0.05) to early luteal phase peaks of 76 +/- 8 ng/dl between Days 6 and 18, and then declined to 41 +/- 5 ng/dl by Day 35-40 in both pregnant (n = 8) and nonpregnant (n = 4) bitches. Subsequent protracted increases in androstenedione occurred in 4 of 8 pregnancies but in none of the nonpregnant bitches. From Days 42 to 64 the differences in mean levels between pregnant (45 +/- 2 ng/ml) and nonpregnant (32 +/- 3 ng/ml) bitches was not significant (P greater than 0.05). At parturition androstenedione levels fell (P less than 0.05) abruptly from 39 +/- 7 to 13 +/- 3 ng/dl. These results suggest that, in the bitch, androstenedione is the major circulating androgen during the follicular and luteal phases and that patterns of androstenedione levels during the luteal phase parallel those reported for progesterone in pregnant and nonpregnant bitches, including maintenance of elevated levels throughout gestation and an abrupt decline at parturition.     

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.     

Oxytocin (OT) action in uterine tissues of cyclic and early pregnant gilts: OT receptors concentration, prostaglandin F(2)alpha secretion, and phosphoinositide hydrolysis

Oxytocin (OT) is involved in the regulation of luteolysis in pigs. However, it is still not clear if OT is responsible for initiation of luteal regression in this species. The objectives of the study were: (1) to compare OT receptors (OTr) concentrations in endometrium and myometrium of cyclic and early pregnant pigs, (2) to examine the effect of OT on plasma PGF(2)alpha secretion during the progressive luteal regression, (3) to ascertain the effect of OT on inositol phosphates (IPs) accumulation in endometrial and myometrial cells of cyclic and early pregnant pigs. Concentrations of OTr on the endometrium and myometrium of cyclic (n = 33) (days 2-4; 11-13; 14-16; 18-20; day 21) and early pregnant (n = 4) (days 14-16) gilts were determined and they ranged from 7 +/- 3 (days 11-13) to 377 +/- 113 fmol/mg protein (day 21) in the endometrium and from 33 +/- 11 (days 2-4) to 167 +/- 28 fmol/mg protein (days 18-20) in the myometrium. In both tissues, concentrations of OTr were low during the luteal phase and increased (P < 0.01) during the follicular phase. In contrast to myometrial OTr, endometrial OTr during pregnancy were undetectable. In next experiment, mature gilts (n = 12) were injected with OT (20IU; i.v.) for three consecutive days starting on days 14 and 15 of the oestrous cycle and plasma PGF(2)alpha metabolite-13,14-dihydro-16-keto PGF(2)alpha (PGFM) concentration was determined. On days 15-16 and 16-17, OT increased plasma PGFM level. This effect was not observed on days 14-15 of the estrous cycle. A negative correlation was noticed between plasma concentrations of PGFM and progesterone (r = -0.3; P < 0.05). In last experiment, OT (100 nM) augmented (P < 0.01) an accumulation of inositol phosphates (IPs) in isolated myometrial cells on days 14-16 (n = 4) and 18-20 (n = 3) of the estrous cycle and on days 14-16 (n = 4) of pregnancy. Oxytocin-stimulated accumulation of IPs was not observed in endometrial cells. In summary: (1) concentrations of OTr on both the endometrium and myometrium were the highest during perioestrus-period in pigs, (2) myometrium of early pregnant sows possessed functional OTr, (3) oxytocin increased plasma PGFM concentration after initiation of luteolysis; and (4) OT-stimulated accumulation of IPs in myometrial, but not in endometrial cells. In conclusion, OT appears to not be involved in the initiation of luteal regression in sows and functional OTr are still present in the myometrium during early pregnancy (days 14-16).     

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.     

Preliminary report on the effect of prostaglandin F(2)alpha on the duration of the oestrus interval in beagle bitches

In an attempt to shorten the oestrus interval of bitches, ten nonpregnant beagles were treated with prostaglandin F (PGF(2)alpha) within eight weeks of oestrus. The dose varied from 60 to 500 mug/kg/day administered over three to six days. Fifteen untreated bitches served as controls. The average oestrus interval of treated bitches was four months, while that of the controls was 6.55 months.     

Prostaglandin biosynthesis and its regulation in the bovine endometrium: A comparison between nonpregnant and pregnant status

Prostaglandin F(2alpha) (PGF(2alpha)), an arachidonic acid metabolism product of the prostaglandin synthetase pathway, is synthesized and released from the endometrium during luteolysis in nonpregnant animals. When proper conception occurs, the synthesis and release pattern is changed to maintain the corpus luteum (CL) function. The biosynthesis of prostaglandins in the bovine endometrium was highest in the microsomes but of low order. In nonpregnancy, the formation of prostaglandins from labelled precursor acid was higher than in pregnancy. Besides the prostaglandin synthetase, an inhibiting activity on the conversion of arachidonic acid to prostaglandins was found in both the nonpregnant and pregnant endometrium. During luteolysis (Day 17), a low inhibiting capacity was seen in comparison with other days of the estrous cycle (Days 1, 4 and 14). The inhibitory capacity was very high on Days 16 to 20, 25, and 31 of pregnancy. In the nonpregnant endometrium at Day 17, a very low inhibitor potency, calculated as IC(50) values, was found both in the cytoplasma and in the microsomes, whereas during early pregnancy (Days 17, 18, and 20) both cytoplasma and microsomes possessed very high inhibitor potency. This finding indicates that the bovine endometrium contains both prostaglandin synthetase and an unknown potent inhibitor of prostaglandin biosynthesis that regulates prostaglandin biosynthesis both during the estrous cycle and early pregnancy.     

Expression of 3beta-hydroxysteroid dehydrogenase, cytochrome p450 17alpha-hydroxylase/17,20-lyase and cytochrome p450 aromatase enzymes in corpora lutea of diestrous and early pregnant mares

In the pregnant mare, luteal estrogen production increases at the onset of equine chorionic gonadotropin (eCG) secretion by endometrial cups. In previous studies, we have demonstrated that eCG stimulates luteal androgen and estrogen production in pregnant mares. To further elucidate the regulation of steroidogenesis within the equine corpus luteum (CL) of pregnancy, we examined the expression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), cytochrome P450 17alpha-hydroxylase/17,20 lyase (P450(17alpha)) and cytochrome P450 aromatase (P450(arom)) in luteal tissue samples collected during diestrus (Days 7 to 10) and pregnancy before (Days 29 to 35) and after (Days 42 to 45) the onset of eCG secretion. Immunoblot analyses revealed a single protein per enzyme with molecular weights of 48 kDa (3beta-HSD), 58 kDa (P450(17alpha)) and 56 kDa (P450(arom)). Steady-state levels of 3beta-HSD were lower in luteal tissue of diestrus than pregnancy, but expression did not change during pregnancy. Steady-state expression of P450(17alpha) in CL of diestrus was not significantly different from that of pregnancy. During pregnancy, P450(17alpha) expression was significantly higher after the onset of eCG secretion. Steady-state expression of P450(arom) in CL of diestrus was not significantly different from that of pregnancy. During pregnancy, luteal expression of P450(arom) was significantly lower after the onset of eCG secretion. These data support the hypotheses that eCG has a differential effect on the expression of luteal steroidogenic enzymes, that the eCG-induced increase in luteal estrogen production is the result of an increase in available aromatizable androgen due to an increase in P450(17alpha) expression and activity, and that increased luteal estrogen production is not due to an increase in aromatase expression.