Post Ovulation Research Articles

Regression and resurgence of the CL following PGF 2α treatment 3 days after ovulation in mares

The present study was designed to characterize and compare the physiology and ultrasonographic morphology of the corpus luteum (CL) during regression and resurgence following a single dose of native prostaglandin F 2α (PGF) given 3 days after ovulation, with a more conventional treatment given 10 days after ovulation. On the day of pre-treatment ovulation (Day 0), horse mares were randomly assigned to receive PGF (Lutalyse ®; 10 mg/mare, i.m.) on Day 3 (17 mares) or Day 10 (17 mares). Beginning on either Days 3 or 10, follicle and CL data and blood samples were collected daily until post-treatment ovulation. Functional and structural regression of the CL in response to PGF treatment were similar in both the Day 3 and 10 groups, as indicated by an abrupt decrease in circulating concentrations of progesterone, decrease in luteal gland diameter and increase in luteal tissue echogenicity. As a result, the mean ± S.E.M. interovulatory interval was shorter ( P < 0.0001) in the Day 3 group (13.2 ± 0.9 days) than in the Day 10 group (19.2 ± 0.7 days). Within the Day 3 group, functional resurgence of the CL was detected in 75% of the mares (12 of 16) beginning 3 days after PGF treatment, as indicated by transient major (6 mares) and minor (6 mares) increases ( P < 0.05 and <0.1, respectively) in progesterone. Correspondingly, mean length of the interovulatory interval was longer ( P < 0.03) in mares with major resurgence (15.8 ± 1.6 days) than in mares with minor (11.2 ± 1.2 days) and no resurgences (13.5 ± 0.3 days) in progesterone. Structural resurgence of the CL in the Day 3 group and functional and structural resurgence in the Day 10 group were not detected. In conclusion, PGF treatment 3 days after ovulation resulted in structural and functional regression of the CL and hastened the interval to the next ovulation, despite post-treatment resurgences in progesterone.

In Vivo Infusion of Interleukin-1β and Chorionic Gonadotropin Induces Endometrial Changes that Mimic Early Pregnancy Events in the Baboon

Both human chorionic gonadotropin (hCG) and IL-1beta induce changes in the endometrium that are associated with the establishment of pregnancy. We investigated the synergistic effect of these two embryonic signals on endometrial function using a baboon model of simulated pregnancy. Recombinant hCG (30 IU/d) was infused between d 6 and 10 post ovulation (PO) to mimic blastocyst transit. On the expected day of implantation (d 10 PO), IL-1beta (12 ng/d) or IL-1 receptor antagonist (IL-1Ra; 12 ng/d) was infused for an additional 5 d. Endometria were harvested on d 15 PO. Both hCG and hCG plus IL-1beta induced marked differences in the distribution of alpha-smooth muscle actin, proliferation marker Ki67, decidualization marker IGF-binding protein-1, and cyclooxygenase-1. The most marked effect of IL-1beta was the induction of IGF-binding protein-1 protein in stromal cells close to the apical surface, whereas cyclooxygenase-1 was down-regulated in the glandular epithelium. Protein arrays of uterine flushings showed significant suppression of death receptors, Fas and TNF receptor 1, in the hCG- with or without IL-1beta-treated groups, suggesting an inhibition of apoptosis. Additionally, cytotoxic T lymphocyte antigen-4, matrix metalloproteinase-3, and IL-4 were suppressed in treated animals compared with controls. However, no differences were observed in cytokine profile between hCG-treated and hCG- plus IL-1beta-treated baboons. This study confirms that in preparation for pregnancy, the primate endometrium undergoes both morphological and functional changes, which are modulated by hCG and IL-1beta, that lead to the inhibition of apoptosis and the development of an immunotolerant environment. These changes suggest that infusion of IL-1beta at the time of implantation into the nonpregnant baboon treated with hCG synergizes with hCG and mimics the early endometrial events associated with the presence of an embryo.

297. Reproduction in the arrhythmic Bmal1 knockout mouse

There is strong epidemiological evidence indicating that disruption of the endogenous circadian rhythms can cause a range of health problems ranging from metabolic and cardiovascular disorders to reproductive failure. Circadian rhythmicity is generated by a suite of genes called ‘clock genes’ that are cyclically expressed in the brain and peripheral tissues. The CLOCK and BMAL1 transcription factors regulate the expression of many genes involved in cell growth, angiogenesis and development. The Bmal1 knockout mouse provides an interesting model to analyse the impact of arrhythmicity on reproductive physiology. Female Bmal1–/– mice show a delay in the onset of puberty (WT = 32.7 d, KO = 38.6 d, n = 8–16). Female Bmal1–/– mice reproductive tissues are significantly smaller than in WT mice (Ovaries –40%, Oviduct –25%, Uterus –60%, n = 10). Female Bmal1–/– mice have essentially normal estrus cycles (cycle length WT = 4.2 d, KO = 4.8 d, n = 8) and are able to ovulate and mate but are unable to establish viable pregnancies. They are as responsive to a standard superovulation protocol as their wild type littermates (ovulated oocytes WT = 23.8, KO=22.8, n = 7–10), suggesting the ovaries are developmentally competent. These results suggest disruption of circadian rhythmicity in the mouse affects fertility at multiple sites. Further investigation into the importance of rhythmicity, particularly post ovulation and post fertilisation is required.

Maintenance of ovulation inhibition with the 75- μg desogestrel-only contraceptive pill (Cerazette®) after scheduled 12-h delays in tablet intake

Background In contrast to traditional progestagen-only pills (POPs), the desogestrel-only pill Cerazette® consistently inhibits ovulation. This study was performed to test the hypothesis that desogestrel alone will keep inhibiting ovulation even when pills are taken 12 h late, indicating that delays in tablet intake of up to 12 h do not jeopardize contraceptive efficacy. Methods Women aged between 19 and 40 years with confirmed ovulation were admitted to this open-label pharmacodynamic study. They were treated with Cerazette® for 56 days with three tablets to be taken 12 h late, having been randomized to a regimen with scheduled late tablets on Days 39, 42 and 49 (Group A) or on Days 11, 14 and 21 (Group B). The occurrence of ovulation during treatment was determined by measuring progesterone serum levels every 2 days. Results One of the 103 treated subjects ovulated during treatment. The ovulation incidence thus amounts to 1.0% (two-sided 95% confidence interval 0.02–5.29%). There was no apparent relationship between these ovulations and scheduled late tablets. The minimum time to first posttreatment ovulation was 7 days, whereas it took 17.2 days on average from last tablet intake until ovulation. Conclusions Ovulation inhibition with Cerazette® is maintained after 12-h delays in tablet intake and return of ovulation takes at least 7 days. These properties distinguish Cerazette® from all other POPs.

217LUTEAL REGRESSION AND FOLLICLE DEVELOPMENT FOLLOWING PROSTAGLANDIN-F2± TREATMENT 3 DAYS AFTER OVULATION IN MARES

The corpus luteum (CL) is responsive to exogenous prostaglandin-F2α (PGF) 1 to 2 days after ovulation in the mare (Troedsson et al. 2001 Theriogenology 55, 1891–1899); however, complete and sustained demise of the CL beginning less than 5 days after ovulation in response to PGF treatment has not been documented. The present study was designed to compare the morphological and physiological response of the primary CL to PGF given at early diestrus with a more conventional treatment given at about mid-cycle. In addition, follicle status pre- and post-treatment were examined and compared between the treatment groups. On the day of pretreatment ovulation (Day 0), riding-type horse mares were randomly assigned to receive a single dose of PGF (Lutalyse, Upjohn, Kalamazoo, MI, USA;; 10mg/mare, i.m.) on Day 3 (n=17) or Day 10 (n=17). Beginning on either Days 3 or 10, transrectal ultrasonography was used to determine follicle and CL diameters, determine luteal tissue gray-scale scores (echogenicity), and to detect ovulation. Follicular and luteal measurements and jugular blood samples were collected daily until the post-treatment ovulation. Structural and functional regression of the CL was indicated by: 1) a progressive decrease (day effect;; P&amp;lt;0.0001) in mean diameter of the CL beginning 24h after PGF treatment in the Day 3 and Day 10 groups;; 2) a precipitous decrease (P&amp;lt;0.009) in mean plasma progesterone concentrations within 24h in both groups followed by a more gradual decline to basal concentrations by the second day in the Day 10 group or after the fourth day in the Day 3 group;; and 3) an increase (P&amp;lt;0.02) in mean luteal tissue echogenicity in both groups after the second day following PGF treatment. The mean intervals from PGF treatment to ovulation were not different (P&amp;gt;0.2) between groups (combined, 9.9 days) but the mean (±SEM) interovulatory interval was shorter (P&amp;lt;0.0001) in the Day 3 group (13.2±0.9 days;; range, 7 to 20 days) than in the Day 10 group (19.2±0.7 days;; range, 14 to 26 days). The greater the diameter of the largest follicle at the time of PGF treatment, the shorter the interval to post-treatment ovulation in the Day 3 (r=−0.57, P&amp;lt;0.02) and Day 10 (r=−0.74, P&amp;lt;0.001) groups. Growth rates of the preovulatory follicles were similar (P&amp;gt;0.59) between groups (combined, 3.6mm/day) but the maximum diameter was smaller (P&amp;lt;0.05) in the Day 3 group (40.5±1.2mm) compared to the Day 10 group (43.4±0.8mm). Unexpectedly, more (P&amp;lt;0.03) double ovulations occurred in the Day 3 group (6/17, 35%) than in the Day 10 group (1/17, 6%). In conclusion, an immature CL at early diestrus responded to PGF treatment in a manner comparable to a mature CL at mid-cycle. The Day 3 group ovulated an average of 6 days earlier than the Day 10 group as a result of the difference in timing of the PGF treatment between groups. Thus, these results warrant a reassessment of the prevailing concept that the equine CL is resistant to PGF-induced regression before 5 days after ovulation, especially when considering the potential benefits of a shortened interovulatory interval and an increased double ovulation rate.

Effects of mifepristone on endometrial receptivity

At the development of receptivity the endometrium undergoes specific changes. Several factors have been suggested as markers of endometrial receptivity. A common feature for most of these factors is that they are directly, or indirectly, regulated by progesterone. The effect of various doses and regimens of mifepristone on endometrial development and markers of receptivity has been studied. Timed endometrial biopsies were assessed by immunhistochemistry, reverse transcriptase polymerase chain reaction (RT-PCR) and electron microscopy. In addition the contraceptive efficacy of these regimens was investigated. Administration of 200 mg of mifepristone immediately post ovulation has a pronounced effect on endometrial development and on suggested markers of receptivity. This regimen has been shown to be an effective contraceptive method. When 10 mg is given pre or post ovulation, only minor effects on the endometrium are observed. Our studies show that mifepristone, when administered in low doses that do not affect ovulation, significantly affects some of the studied markers of endometrial receptivity and reduces pregnancy rates; however, these activities are more pronounced with the higher dose, which is more effective. Our findings provide insight into the regulation of progesterone receptors of various suggested markers of endometrial receptivity and the possibility of using mifepristone for endometrial contraception.

Relaxant effects of parathyroid hormone and parathyroid hormone-related peptides on oviduct motility in birds and mammals: possible role of nitric oxide

Parathyroid hormone (PTH) and PTH-related peptides (PTHrP) have previously been shown to modulate the contractile state of numerous types of smooth muscle. The effects of N-terminal PTH and PTHrP on spontaneous in vitro contractility of oviducal smooth muscle using tissues from egg-laying Japanese quail (10–15 h post ovulation), 4 and 9 days pregnant mouse uterus were investigated. Myometrial tissues from both species contracted vigorously for several hours, when incubated in organ baths in De Jalon’s solution gassed with 5%CO 2/95%O 2. Contractions were enhanced in high (1.2–2.5 mM) compared with low (0.1–0.5 mM) calcium (Ca) containing media. Bovine PTH(1–34) (bPTH(1–34)), human PTH(1–34 amide) (hPTHrP(1–34) amide), and hPTHrP(1–40) caused similar concentration-related inhibition of contractions in media containing 1.2 mM Ca over a range of 10 −9 to 10 −7 M, whereas C-terminal hPTHrP(107–139) was devoid of such activity. Responses to bPTH(1–34) in 4 and 9-day pregnant mouse tissues were similar but hPTHrP(1–40) showed substantial loss of activity in 9-day, compared with 4-day pregnant mouse tissues. Repeated exposure of mouse uterine tissue to the peptides resulted in desensitisation of responses. The EC 50 responses of mouse tissues were inhibited by the PTH/PTHrP receptor antagonist, hPTHrP(7–34) amide. Responses to bPTH(1–34) were also inhibited by both non-selective and selective neuronal nitric oxide synthase (NOS) inhibitors N ω-nitro- l-arginine methyl ester (0.01–1 mM) and 7-nitroindazole (0.01–10 μM), respectively. Both NOS inhibitors were more effective in inhibiting bPTH(1–34)-induced relaxation in the absence of l-arginine compared with in the presence of 1 mM l-arginine (a NOS substrate) in the incubation media. It is concluded that relaxant responses to N-terminal PTH and PTHrP peptides are well conserved in oviducal and uterine tissues from avian and mammalian species. The results also suggest that NO may be responsible for mediating relaxant activities of these peptides in pregnant mouse uterine tissue.

32. The expression of ovulatory mediators by macrophages isolated from the gonadotrophin-stimulated mouse ovary

IL-1beta (IL-1β), TNF alpha (TNFα), nitric oxide (NO) and macrophages have been shown to be important in stimulating the ovulatory event [1-4]. Since macrophages produce these mediators elsewhere, it was our aim to determine if ovarian macrophages are a source of these known ovulatory mediators. Ovaries from gonadotrophin-primed immature mice were removed at various times and enzymatically digested. Macrophages were tagged with the specific antibodies F480 or anti-MHC II (anti- Ia) and isolated using antibody panning. mRNA was isolated from some macrophages and analysed by quantitative RT-PCR; other macrophages were cultured and secreted protein levels measured. mRNA levels, expressed as fold changes from the first isolation time point and normalised to the housekeeper gene HPRT, show that ovarian macrophages exhibit regulated cytokine mRNA profiles in response to hCG. Both IL-1β and TNFα mRNA increased significantly (*) post hCG injection (IL-1β:Ia. 7.3 ± 2*, F480 5.1 ± 0.8*, TNFα:Ia 2.39 ± 0.5, F480 2.47 ± 0.21*). Secreted TNFα protein increased 6 h post hCG and increased further 24 h post ovulation and then returned to pre-hCG levels 48 h after ovulation. Prior to hCG administration IL-1β was not detectable in conditioned media, but could be detected in media from Ia cells following hCG administration. No changes in total NO activity were detected across the stimulated cycle. Therefore, ovarian macrophages exhibit regulated cytokine expression, particularly of TNFα, in response to hCG. Since TNFα is known to modulate follicular rupture, its production by ovarian macrophages may contribute to the ovulatory process.

Birth of piglets after deep intrauterine insemination with flow cytometrically sorted boar spermatozoa

The present study was carried out to determine the pregnancy rates, farrowing rates and litter size in sows with either induced or spontaneous ovulation inseminated with flow cytometric sorted spermatozoa using deep intrauterine insemination technology. Spermatozoa were stained with Hoechst 33342 and sorted by flow cytometry/cell sorting but not separated into separate X and Y populations. In Experiment 1, sows (n=200) were weaned and treated for estrus/ovulation induction with eCG/hCG. Inseminations with either sorted (70 or 140 million) or non-sorted (70 or 140 million) spermatozoa were done using a specially designed flexible catheter. Farrowing rates were 39.1 and 78.7% for 70 million of sorted and non-sorted, respectively, and 46.6 and 85.7% for 140 million of sorted and non-sorted, respectively (P<0.05). The litter size in sows inseminated with sorted spermatozoa showed a tendency to be lower than when non-sorted spermatozoa were inseminated. In Experiment 2, sows (n=140) were inseminated as in Experieriment 1 except that natural estrus was used. The ovaries of these sows were evaluated by transrectal ultrasonography. Farrowing rates were 25 and 77.2% for 70 million of sorted and non-sorted, respectively, and 32 and 80.9% for 140 million of sorted and non-sorted, respectively (P<0.05). These results show that the Deep Intrauterine Insemination technology can be successfully used to produce piglets from sorted spermatozoa when sows are hormonally treated to induce synchronous post weaning oestrus and ovulation.

Characterization and expression of steroidogenic acute regulatory protein and MLN64 cDNAs in trout.

Complementary DNA-encoding proteins with high homology to steroidogenic acute regulatory proteins (StAR) of mammals were cloned from rainbow trout head kidney and a mixture of several brook trout tissues. A cDNA encoding an MLN64 homolog was also cloned from brook trout. The C-terminal domains of rainbow trout StAR and brook trout StAR were very highly conserved compared with StAR of mammals. In rainbow trout, Northern and RT-PCR analyses showed abundant StAR transcripts in head kidney, ovary, and testis, and weaker signals were found in intestine, pyloric caeca, spleen, and kidney. Brief acute stress resulted in elevated plasma cortisol levels and a 2-fold increase in rainbow trout StAR transcripts in head kidneys sampled 3 h after exposure to the stressor. In brook trout, StAR transcripts were detected only in known steroidogenic tissues. Ovarian brook trout StAR mRNA was not seen until the onset of final maturation. Its abundance increased during germinal vesicle breakdown, peaked during and just following ovulation, and decreased by 2 wk post ovulation. Brook trout MLN64 transcripts were found in all tissues tested, and transcript abundance in ovarian samples did not vary during final oocyte maturation and ovulation. Both StAR structure and function appear to be highly conserved throughout the vertebrates.

Characterization and Expression of Steroidogenic Acute Regulatory Protein and MLN64 cDNAs in Trout

Complementary DNA-encoding proteins with high homology to steroidogenic acute regulatory proteins (StAR) of mammals were cloned from rainbow trout head kidney and a mixture of several brook trout tissues. A cDNA encoding an MLN64 homolog was also cloned from brook trout. The C-terminal domains of rainbow trout StAR and brook trout StAR were very highly conserved compared with StAR of mammals. In rainbow trout, Northern and RT-PCR analyses showed abundant StAR transcripts in head kidney, ovary, and testis, and weaker signals were found in intestine, pyloric caeca, spleen, and kidney. Brief acute stress resulted in elevated plasma cortisol levels and a 2-fold increase in rainbow trout StAR transcripts in head kidneys sampled 3 h after exposure to the stressor. In brook trout, StAR transcripts were detected only in known steroidogenic tissues. Ovarian brook trout StAR mRNA was not seen until the onset of final maturation. Its abundance increased during germinal vesicle breakdown, peaked during and just following ovulation, and decreased by 2 wk post ovulation. Brook trout MLN64 transcripts were found in all tissues tested, and transcript abundance in ovarian samples did not vary during final oocyte maturation and ovulation. Both StAR structure and function appear to be highly conserved throughout the vertebrates.

Caspase-3 is a pivotal mediator of apoptosis during regression of the ovarian corpus luteum.

Because caspase-3 is considered a primary executioner of apoptosis and has been implicated as a mediator of luteal regression, we hypothesized that corpora lutea (CL) derived from caspase-3 null mice would exhibit a delayed onset of apoptosis during luteal regression, when compared with CL derived from wild-type (WT) mice. To test this hypothesis, ovulation was synchronized in immature (postpartum d 24-27) WT and caspase-3-deficient female littermates by exogenous gonadotropins. Individual CL were isolated by manual dissection, 30 h after ovulation, and placed in organ culture dishes in the absence of serum and growth factors. At the time of isolation (0 h) and after 24, 48, and 72 h in culture, the CL were removed and assessed for the presence of processed (active) caspase-3 enzyme and for apoptosis by multiple criteria. There was no evidence of active caspase-3 enzyme or apoptosis in either WT or caspase-3-deficient CL before culture. However, CL derived from the WT mice exhibited a time-dependent increase in the level of active caspase-3 and apoptosis during culture. By comparison, CL derived from caspase-3-deficient mice, cultured in parallel, failed to exhibit any detectable active caspase-3 and showed attenuated rates of apoptosis. To extend these findings derived from ex vivo culture experiments, ovaries were collected from WT and caspase-3 null female littermates at 2, 4, or 6 d post ovulation, and the occurrence of apoptosis within the CL was analyzed. Whereas ovaries of WT mice had only residual luteal tissue at d 6 post ovulation, ovaries collected from caspase-3-deficient mice retained many CL, at d 6 post ovulation, that were similar in size to those observed in the early luteal phase of WT mice. Importantly, there was no dramatic increase in apoptosis in CL of caspase-3-deficient mice at any time point examined post ovulation, indicating that the involution process had indeed been delayed. In contrast, the levels of progesterone declined regardless of genotype. These data provide the first direct evidence that caspase-3 is functionally required for apoptosis to proceed normally during luteal regression. However, caspase-3 is not a direct mediator of the decrease in steroidogenesis associated with luteolysis.

Chromosomal abnormalities and developmental kinetics in in vivo-developed cattle embryos at days 2 to 5 after ovulation.

The frequency of chromosome abnormalities was investigated in cattle embryos (n = 256) derived from superovulated heifers (n = 35) on Days 2, 3, 4, and 5 postovulation (PO). Interphase nuclei (n = 4358) were analyzed for chromosome abnormalities using fluorescent in situ hybridization with chromosome 6- and chromosome 7-specific probes and the developmental rate was described by scoring cell numbers. We found that 93%, 85%, 84%, and 69% of the embryos from Days 2, 3, 4, and 5 PO, respectively, displayed a normal diploid chromosome number in all cells. Of the embryos containing abnormal cells, mixoploidy was significantly more frequent than polyploidy. The percentage of mixoploidy at Days 2, 3, 4, and 5 PO was 5%, 13%, 16%, and 31%, respectively, whereas the percentages of polyploidy were 2%, 2%, 0%, and 0%, respectively. The mean number of cells per embryo was 4.7, 8, 11.5, and 48.3, respectively, at Days 2, 3, 4, and 5 PO. Thus, in vivo-developed embryos were significantly more advanced than the in vitro-produced (IVP) embryos except for Day 2. In conclusion, a significantly lower frequency of chromosomally abnormal embryos, in particular displaying polyploidy early after fertilization, was seen in in vivo versus IVP embryos, and these chromosomal abnormalities may be inherent to the process of IVP in cattle.

Meloxicam inhibits rabbit ovulation

The nonsteroidal antiinflammatory, selective cyclo-oxygenase-2 (COX-2) inhibitor, meloxicam, was tested to assess its effect on rabbit ovulation. Meloxicam in different doses was administered intraperitoneally (ip) to adult female Californian rabbits at 2, 5, 8, and 24 h postcoitus with sperm-positive rabbits. Rabbits were killed on Day 10 of gestation. Meloxicam produced significant inhibition of ovulation in rabbits. This inhibition of ovulation by meloxicam was dose- and time-dependent. Ovulation in rabbits was completely inhibited by a single ip administration of meloxicam (20 mg/kg) when the drug was administered at 2 and 5 h postcoitus, whereas neither ovulation nor implantation were inhibited (pregnancy rate 75%) by the same dose administered 24 h postcoitus (approximately 14 h post ovulation). Further, ovulation was completely inhibited by 10 mg/kg of meloxicam when the drug was administered at 5 or 8 h postcoitus, but there was less inhibition of ovulation when 10 mg/kg of the drug was administered at 2 or 24 h postcoitus (pregnancy rate 25 and 80%, respectively). Corpora lutea, maternal plasma progesterone, ovary fresh weight, and maternal body weight gain were affected by meloxicam treatment. Histopathological findings observed in the ovaries of treated rabbits included microscopic dilatation of graffian follicles, particularly mature follicles. Some of the follicles were cystically dilated in addition to severe hemorrhage within the follicles which lost ova. These results show that ovulation can be inhibited in rabbits by meloxicam. Further studies are needed to assess the value of selective COX-2 inhibitors as potential nonhormonal contraceptive agents.

Termination of mid-gestation pregnancy in bitches with aglepristone, a progesterone receptor antagonist

Six pregnancies were terminated in mid-gestation with aglépristone, a progesterone receptor antagonist, in 5 beagle bitches in order to determine the effects of aglépristone on plasma concentrations of prolactin and progesterone, the duration of the luteal phase, and the interestrous interval. In addition, the effects of aglépristone on the condition of the uterus and fetuses were examined by ultrasonography. After confirmation of pregnancy by ultrasonography, the dogs received 10 mg, sc aglépristone per kg body weight on 2 consecutive days at about 30 d post ovulation. Before, during and after treatment with aglépristone, plasma samples were collected for determination of the concentrations of prolactin and progesterone. The condition of the uterus and fetuses was assessed by ultrasonography the day before and at least 3 times a week for at least 2 wk after aglépristone administration. Termination of pregnancy occurred within 4 to 7 d after the start of aglépristone treatment, which was well tolerated, with no side-effects except slight vaginal discharge. The results of ultrasonographic examination indicated that aglépristone leads to abortion but not to fetal resorption. Elevated plasma concentrations of prolactin were observed during aglépristone treatment, while plasma progesterone levels remained unchanged. Pregnancy termination with aglépristone resulted in premature cessation of luteal function. In addition, the interestrous interval was shortened. The latter effects may be the consequence of actions of the progesterone receptor antagonist at the hypothalamus-pituitary level. In conclusion, aglépristone proved to be a safe and effective abortifacient in mid-gestation in the bitch. The results of the present study also indicated that aglépristone directly or indirectly influences pituitary function.

Identification of genes expressed in the epithelium of porcine oviduct containing early embryos at various stages of development.

As a first step toward elucidation of the action of factors secreted by the epithelium of oviduct, differential display reverse transcription-polymerase chain reaction (DDRT-PCR) was used in this study to identify transcripts of such oviductal factors in gilts carrying various stages of early embryo development post hormone-induced ovulation. A total of 13 differentially expressed transcripts were identified between 50 and 120 hr post-hCG injection (between 1- and 8-cell embryonic stages). Twelve of these transcripts were found to be initially expressed at 96 hr post-hCG injection (at 4-cell embryonic stage) and beyond. Three of such genes were shown by sequence analysis to be the porcine transforming growth factor-alpha, the porcine transforming growth factor-beta-binding protein II and a porcine astral natriuretic factor receptor-like transcript. Only one differentially expressed gene was detected between 50-60 and 85 hr post-hCG injection, and this gene turned out to be the porcine follicle-stimulating hormone receptor. The remaining eight transcripts detected by DDRT-PCR were novel. Moreover, most of these newly expressed genes were found to be turned on at a time coincidental with that of the 4-cell block of porcine embryos cultured in vitro. Our results demonstrate that DDRT-PCR is a feasible approach for rapid identification of genes that are differentially expressed in oviductal epithelium. Some of the genes thus identified may be important for unhindered development of embryos in the oviduct.

Ovarian dynamics, serum estradiol and progesterone concentrations during the interovulatory interval in goats

Ovarian changes determined by daily transrectal ultrasonic scanning, and its correlation with serum progesterone (P4) and estradiol (E2) concentrations were studied in seven cyclic Saanen goats. Estrous cycles were synchronized with 2 injections of a PGF 2α analogue 9 d apart. All follicles ≥2 mm in diameter and CL were measured each day. One goat showed a longer interestrous interval, associated with development of a cystic-luteinized structure. The mean interovulatory interval for the other 6 goats was 20.8±0.4 d. The incidence of goats with 4, 3, and 2 follicular waves was 3, 1 and 2 respectively; follicular waves emerged on Days 0.5±0.6, 7.2±0 7, 10.7±0.5 and 13.7±0.8 for Wave 1, 2, 3 and the Ovulatory wave, respectively. The largest follicle of Wave 2 was smaller (4.9±0.1 mm) than the largest follicles of Wave 3 (6.2±0.1 mm; P≤0.01) and of the Ovulatory wave (7.0±0.5 mm; P≤0 01), and tended to be smaller than the largest follicle of Wave 1 (6.3±0.6 mm; P≤0 09). Interval between emergence of Wave 1 and Wave 2 was longer than interval between emergence of Wave 2 and Wave 3 (7.3±0.9 d vs 4.0±0.4 d; P≤0.01), and between Wave 3 and the Ovulatory wave (3.8±1.1 d; P≤0.05). Two days before ovulation, the diameter of the ovulatory follicle was larger (P≤0.01) than the first subordinate follicle Serum E2 concentrations increased from the day of ovulation (2 7±0 3 pg/mL) to Day 2 (7.6±0.9 pg/mL; P≤0.01), associated with the early-mid growing phase of the largest follicle of Wave 1, and then decreased to basal levels on Day 5 (P≤0.01) and peaked again (16 5±2 4 pg/mL) 2 d before ovulation. The CL were detected ultrasonically on Day 3 post ovulation and attained a mean maximum diameter of 13.5±0.8 mm between Days 8 and 14 The following characteristics were observed: 1) ovarian follicular development in goats is wave-like; 2) increased P4 concentrations may be promoting follicular wave turnover; 3) it is suggested that the presence of follicular dominance and the production of E2 are different among waves. While in Wave 1 and in the Ovulatory wave, follicular dominance is present and production of E2 is consistent, no changes in serum E2 concentrations were found in other stages of the interovulatory interval. In the intervening waves, no indicators of follicular dominance could be firmly documented

Activity of Ovarian Nitric Oxide Synthase (NOs) during Ovulatory Process in the Rat: Relationship with Prostaglandins (PGs) Production

Nitric oxide (NO) is synthesized by the rat ovary and a role in the follicular development, the ovulation, and the luteal formation has been postulated. The aims this study were to determine the activity of nitric oxide synthase (NOs) enzyme during the ovulatory process and to demonstrate the existence of a relationship between the ovarian NO production and the synthesis of prostaglandins (PGs) involved in the follicular rupture. Prepuberal rats treated with PMSG/hCG to induce ovulation were used. The NOs activity, measured by [14C]citrulline formation, showed an increase after PMSG administration and reached a maximum at 10 h after hCG injection. NOs activity remained high up to 24 h post ovulation. At 10 h after the hCG injection, the activity of Ca2+-dependent NOs (constitutive NOs) was similar to that seen at 0 h, and the activity of Ca2+-independent NOs (inducible NOs) increased from 14.4 to 51% of total activity. The in vitro ovarian production of PGE and PGF2α was inhibited by L-NAME and stimulated by 3-morpho-linosydnonimine (SIN-1), a NO donor. The in vivo production of ovarian prostaglandins was also inhibited by the intrabursal administration of two NOs inhibitors, NG-nitro-l-arginine methyl ester (L-NAME) and NG-monomethyl-l-arginine (L-NMMA). Our results suggest that the inducible NOs (iNOs) is the main isoform involved in the ovulatory process and that the NO produced stimulates the synthesis of both PGE and PGF2α from the cyclooxygenase pathway, to enhance the process of follicle rupture.

Expression of Cyclooxygenase-1 and -2 in the Baboon Endometrium during the Menstrual Cycle and Pregnancy

Cyclooxygenase (COX) is the rate-limiting enzyme in the biosynthesis of PGs. PGs together with ovarian steroids play important regulatory roles in the establishment and maintenance of pregnancy in a number of different species. In the primate, little is known about the role of PGs in these processes. In this study, the uterine expression of COX-1 and COX-2 throughout the menstrual cycle [late follicular, day 5 postovulation (PO), day 10 PO, and day 14 PO] and pregnancy (days 12–18, day 39, day 51, and near term) was analyzed using semiquantitative RT-PCR, in situ hybridization, and immunocytochemistry. During the menstrual cycle, the highest expression of COX-1 occurred in luteal phase endometrium and was localized to the surface and glandular epithelium. The stromal cells did not express detectable levels of COX-1 at any time. COX-2 messenger RNA (mRNA) expression, as measured by RT-PCR, was evident at all stages of the menstrual cycle, and in situ hybridization showed specific localization for this mRNA in the epithelial cells during the cycle. Treatment of animals with the antiprogestin (ZK 137.316) for 9 days (beginning on the day of the LH surge) inhibited COX-1 expression in the epithelium when the tissue was analyzed on day 10 PO, whereas COX-2 expression disappeared in the epithelium and increased in the stroma. With the onset of pregnancy, COX-1 expression in epithelial cells decreased dramatically. In contrast, COX-2 continued to be detected on the surface epithelium and was also strongly expressed specifically in the stromal cells at the site of implantation. Immunocytochemical staining for COX-2 showed the same pattern of expression for the protein as the message. Finally, near-term decidua expressed very little COX-1 or COX-2 mRNA. These studies suggest that in the baboon endometrium, COX-1 expression is regulated primarily by progesterone, whereas regulation of COX-2 expression may involve additional mediators of embryonic origin at the site of implantation.

Expression of cyclooxygenase-1 and -2 in the baboon endometrium during the menstrual cycle and pregnancy.

Cyclooxygenase (COX) is the rate-limiting enzyme in the biosynthesis of PGs. PGs together with ovarian steroids play important regulatory roles in the establishment and maintenance of pregnancy in a number of different species. In the primate, little is known about the role of PGs in these processes. In this study, the uterine expression of COX-1 and COX-2 throughout the menstrual cycle [late follicular, day 5 postovulation (PO), day 10 PO, and day 14 PO] and pregnancy (days 12-18, day 39, day 51, and near term) was analyzed using semiquantitative RT-PCR, in situ hybridization, and immunocytochemistry. During the menstrual cycle, the highest expression of COX-1 occurred in luteal phase endometrium and was localized to the surface and glandular epithelium. The stromal cells did not express detectable levels of COX-1 at any time. COX-2 messenger RNA (mRNA) expression, as measured by RT-PCR, was evident at all stages of the menstrual cycle, and in situ hybridization showed specific localization for this mRNA in the epithelial cells during the cycle. Treatment of animals with the antiprogestin (ZK 137.316) for 9 days (beginning on the day of the LH surge) inhibited COX-1 expression in the epithelium when the tissue was analyzed on day 10 PO, whereas COX-2 expression disappeared in the epithelium and increased in the stroma. With the onset of pregnancy, COX-1 expression in epithelial cells decreased dramatically. In contrast, COX-2 continued to be detected on the surface epithelium and was also strongly expressed specifically in the stromal cells at the site of implantation. Immunocytochemical staining for COX-2 showed the same pattern of expression for the protein as the message. Finally, near-term decidua expressed very little COX-1 or COX-2 mRNA. These studies suggest that in the baboon endometrium, COX-1 expression is regulated primarily by progesterone, whereas regulation of COX-2 expression may involve additional mediators of embryonic origin at the site of implantation.