The role of ovarian proteases and their inhibitors in ovulation

To assess the role of inhibitors of proteolytic enzymes, such as plasminogen activator (PA) and collagenase in the ovulatory process, inhibitor activity and mRNA levels were examined in periovulatory rat and human ovaries. In the rat, immature animals received 20 IU of pregnant mare serum gonadotropin (PMSG) followed 52 h later by 10 IU of hCG. Ovaries were removed at intervals from 0 to 20 h after human chorionic gonadotropin (hCG) administration. Inhibitor activity for metalloproteinases, such as collagenase, increased from 60.5 +/- 4.1 inhibitor units/ovary at 0 h (i.e., time of hCG treatment) to a maximum of 218.2 +/- 11.4 units/ovary at 8 h after hCG before decreasing at 12 h (time of ovulation) and 20 h (122.2 +/- 7.9 and 71.6 +/- 8.1 units/ovary, respectively). Human follicular fluid and granulosa cells were obtained from preovulatory follicles of patients in our in vitro fertilization program. Metalloproteinase inhibitor activity was evaluated in follicular fluid as well as the levels of PA and PA inhibitor (PAI) mRNA by Northern analysis. Increasing metalloproteinase inhibitor activity was positively correlated with follicular levels of estradiol (p less than 0.001) and progesterone (p less than 0.02, N = 26). Chromatographic separation of follicular fluid resulted in two peaks of metalloproteinase inhibitor activity. The large molecular weight (MW) inhibitor had an approximate size of 700 kilodaltons (kDa) and may represent alpha 2-macroglobulin, a serum-derived inhibitor. The small MW inhibitor shared many of the characteristics of tissue-derived inhibitors of metalloproteinases. Partial purification of the small MW inhibitor by Concanavalin A-Sepharose and Heparin-Sepharose chromatography demonstrated the inhibitor to be a glycoprotein with an approximate MW = 28-29 K. Northern analysis of human granulosa cell total RNA from preovulatory follicles showed little or no detectable tissue-type PA or urokinase-type PA mRNA. In contrast, two species of PA inhibitor type-1 mRNA were detected in relative abundance. The present findings demonstrate the presence of proteolytic inhibitors in periovulatory ovaries of the rat and human. These ovarian inhibitors may play a role in regulating connective tissue remodeling during follicular rupture.     

Effect of various agents on ovarian plasminogen activator activity during ovulation in pregnant mare's serum gonadotropin-primed immature rats

The plasminogen activator/plasmin synthetic substrate S-2251 was used to measure the effect of indomethacin, cycloheximide, colchicine, dexamethasone, tranexamic acid, and aprotinin on the elevation of ovarian plasminogen activator (PA) that normally occurs during ovulation in the rat. Young Wistar rats were weaned on the morning of Day 21, given 4.0 IU of pregnant mare's serum gonadotropin (PMSG) s.c. at 0800 h on Day 22, and given 10.0 IU of human chorionic gonadotropin (hCG) on Day 24. These animals normally began ovulating between 0000 and 0200 h on Day 25. The induced ovulation rate was 11.5 +/- 2.2 ova/rat, based on the number of ova in the oviducts of control animals at 0900 h on Day 25. In the controls, PA activity in extracts of homogenized ovaries increased 3-fold from 0.125 +/- 0.010 OD units just before the administration of hCG to 0.371 +/- 0.021 at 12 h after hCG, i.e., near the time of ovulation. Indomethacin, in doses of 0.1-1.0 mg/rat, inhibited ovulation but did not inhibit the normal increase in PA activity, whereas indomethacin at the high dose of 10.0 mg/rat inhibited both ovulation and PA activity. Cycloheximide, at a dose of 0.1 mg/rat, was given at 12 h before hCG, immediately after hCG, and at 9 h after hCG. This agent inhibited ovulation most effectively when given at 12 h before hCG, yet it inhibited PA activity most effectively when given immediately after or at 9 h after hCG. Colchicine, at a dose of 0.1 mg/rat, inhibited ovulation, but not PA activity, when it was given 1 h before hCG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Connective tissue breakdown in ovulation

A meshwork of collagen over the apical region of the follicle must be breached to permit the ovum to escape. We propose that specific collagenase activity is responsible for collagen breakdown in this region. Immature rats are primed with pregnant mare serum gonadotropin (PMSG), followed at 48 h by hCG. At 8 h after hCG, collagenase activity, measured in extracts of ovarian tissue, is elevated about five-fold. Ovulation follows at 10-12 h. Ovaries from PMSG-primed rats are dissected at 48 h, placed in a perfusion apparatus, and perfused with luteinizing hormone and 3-isobutyl-1-methyl xanthine. The ovulations induced by this treatment can be blocked to the extent of 70% with a synthetic collagenase inhibitor. The activation of procollagenase is believed to involve plasminogen activator and plasmin. In support of this, we find that tranexamic acid at 1 mM inhibits ovulation about 70%. The inhibitor must be added within 3-4 h of LH to be effective. A specific plasmin inhibitor, D-Val-Phe-Lys-chloromethyl ketone, is similarly effective.     

Gelatinase and proteoglycanase activity during the periovulatory period in the rat.

Gelatinase and proteoglycanase are metalloproteinases that govern extracellular matrix remodeling. In the present study, immature rats were primed with eCG (20 IU) and hCG (10 IU). Ovarian gelatinase and proteoglycanase activity were determined at the time of hCG administration (0 h) as well as 4, 8, and 12 h later. Gelatinase and proteoglycanase were extracted by homogenization in Triton and by heating (i.e., heat extraction). An aliquot of the heat extract was reduced and alkylated to destroy metalloproteinase inhibitors. Heat extracts not reduced and alkylated showed low levels of gelatinase and proteoglycanase activity that did not change at the different time points. However, with reduction and alkylation, gelatinolysis increased approximately 4-fold (p less than 0.05) at 4 h, 8 h, and 12 h after hCG priming. Proteoglycanase activity increased approximately 2-fold (p less than 0.05) between 0 and 8 h and declined at 12 h after hCG. The ovarian gelatinolytic activity was due to a metalloproteinase as demonstrated by the inhibition of enzyme activity by phenanthroline and EDTA (97.1 +/- 0.7% and 97.4 +/- 0.6% inhibition respectively). Proteoglycanase activity was not inhibited by phenanthroline (11.5 +/- 3.5%), suggesting that the enzyme activity was not specifically a metal-dependent enzyme. Gelatin gel zymography of the ovarian extracts demonstrated four predominant and distinct gelatin-degrading enzymes of 78, 72, 66, and 62 kDa, similar to the size of gelatinase. The present findings demonstrate a periovulatory increase in ovarian gelatinolytic and proteglycanase activity that may play a pivotal role in connective tissue remodeling associated with ovulation.     

Induction of ovulation in gilts with cloprostenol

Prepuberal gilts were treated with 750 IU pregnant mare serum gonadotropin (PMSG) followed 72 h later by 500 IU human chorionic gonadotropin (hCG) to induce follicular growth and ovulation. In this model, ovulation occurred at 42 +/- 2 h post hCG treatment. When 500 mug of cloprostenol was injected at 34 and of 36 h after hCG injection, 78% of the preovulatory follicles ovulated by 38 h compared with 0% in the control gilts. In addition, plasma progesterone concentrations were significantly higher in the cloprostenol-treated group than in the control group (P<0.01) at 38 h, indicating luteinization along with premature ovulation. These results suggest that prostaglandin F(2)alpha (PGF(2)alpha) or an analog can be used to advance, synchronize or induce ovulation in gilts.     

Increase in ovarian blood volume during ovulation in the gonadotropin-primed immature rat

This study quantifies ovarian blood volume in Wistar rats by measuring the optical density (414 nm) of hemoglobin in ovarian extracts and comparing this measurement to the optical density of known amounts of whole blood. Immature rats were primed with pregnant mare's serum gonadotropin (PMSG), 10 IU s.c., at 23 days of age. On Day 25, the ovulatory process was initiated by human chorionic gonadotropin (hCG), 10 IU s.c., and ova began to appear in the oviducts 10 h later. At 2-h intervals, the ovaries were extirpated and homogenized in 1.0 ml of 0.05 M tris (hydroxymethyl)aminomethane buffer (pH 7.4) for 30 s. Homogenates were centrifuged for 20 min and the supernatant fluids were analyzed with a Gilford RESPONSE UV/VIS spectrophotometer. The hemoglobin in these ovarian extracts had the same peak absorbance of 414 nm characteristic of oxyhemoglobin in whole blood taken by cardiac puncture of the rats. There was a linear relationship between the absorbance and the volume of whole blood in the samples. The volume of blood per ovary from groups of 8 rats was 0.60 +/- 0.07 microL at 0 h after hCG. The volume increased to 1.37 +/- 0.26 microL at 4 h after hCG and reached a peak of 4.55 +/- 0.72 microL at 10 h. Indomethacin treatment (0.3-10.0 mg/rat, s.c.) partially inhibited this 7-fold increase in ovarian blood volume. In conclusion, the increase in ovarian blood volume during ovulation may reflect the vasodilation and hyperemia that are characteristic of inflamed tissues.     

Human chorionic gonadotropin and luteinizing hormone-releasing hormone reverse the blockade of ovulation in pregnant mare's serum gonadotropin-primed immature rats by the anti-androgenic drug, hydroxyflutamide

The present study was designed to examine mechanism(s) of the anti-ovulatory action of the anti-androgen, hydroxyflutamide (OH-F). Prepubertal rats were treated with 4 IU pregnant mare's serum gonadotropin (PMSG) (day -2) to induce first estrus and ovulation. They received OH-F in sesame oil or oil alone at 08:00 and 20:00 h on day 0 (the day of proestrus) and ovulations were assessed on the morning of day 1. Eighty-three percent of control animals ovulated with a mean of 7.7 +/- 1.1 corpora lutea per rat. Hydroxyflutamide blocked ovulation in all but 2 of the 12 rats receiving this drug alone. All of OH-F treated rats that received 5 and 25 IU human chorionic gonadotropin (hCG) ovulated with means +/- SEM of 9.1 +/- 0.1 and 7.3 +/- 1.4 corpora lutea per rat, respectively. The dose of 0.2 IU hCG was essentially ineffective, while the effect of 1.0 IU hCG was intermediate. At the dose of 20 ng and above (100 and 500 ng) luteining hormone-releasing hormone (LHRH) completely overcame the ovulation blockade in the OH-F treated animals, while a 4-ng dose was ineffective. At 18:00 h on the day of proestrus, serum LH levels in control animals were 17.56 +/- 2.60 ng/mL, which were 920% above basal levels (1.90 +/- 0.13) indicating a spontaneous LH surge. This surge was suppressed in OH-F treated rats. Injection of LHRH, at the dose of 20 ng and above, reinstated the LH release in OH-F treated animals. Thus, the anti-androgen, OH-F, inhibits ovulation in PMSG-treated immature rats through its interference with the preovulatory LH surge; the inhibition can be reversed by hCG or LHRH. Hydroxyflutamide does not appear to interfere at the level of the pituitary, but may have direct action at the hypothalamic and (or) extrahypothalamic sites involved in the generation of positive feedback signals that control LH release.     

小鼠排卵前后卵巢纤蛋白溶酶原激活因子活性的变化

给幼龄小鼠注射PMSG刺激滤泡生长,随后注射hCG以诱发排卵。在激素处理的不同时间取出卵巢,制备卵巢匀浆液或从卵巢中分离颗粒细胞和卵丘-卵母细胞复合体,并做离体培养。样品中组织型(tPA)和尿激酶型(uPA)纤蛋白溶酶原激活因子经SDS-凝胶电泳分离,用纤蛋白铺盖技术测定。实验结果表明,注射hCG 8h后15％的受试动物排卵,而卵巢匀浆液和颗粒细胞中tPA和uPA活性分别也在hCG注射后4和8h达到高峰。排卵后酶活性下降。卵丘-卵母细胞复合体主要含tPA,注射hCG 12—24h达到高峰。上述资料证明,tPA和uPA都参入小鼠排卵过程。因为排出的卵子中仍含有大量tPA,卵细胞的tPA除参与排卵外,可能对排卵后的一些生理过程也起重要作用。     

Inhibition of ovulation in the perfused rat ovary by the synthetic collagenase inhibitor SC 44463.

A new, powerful, synthetic inhibitor of mammalian tissue collagenases and related metalloproteinases is inhibitory to ovulation in perfused rat ovaries. Ovaries of immature rats, primed with 20 IU of eCG, were dissected and perfused with 0.1 micrograms/ml LH and 0.2 mM 3-isobutyl-1-methylxanthine (IBMX) for 20 h. Addition of SC 44463 (N4-hydroxy-N1-[1S [(4-methoxphenyl)methyl]-2-(methylamino)-2-oxoethyl]- 2R-(2-methylpropyl)butane-diamide) at a concentration of 25 nM inhibited ovulation by 55% (9.6 +/- 1.7 ovulations per ovary, mean +/- SEM, compared to a control value of 21.7 +/- 1.7); and 250 nM inhibited ovulation by 75% (5.3 +/- 1.1 ovulations per ovary). We previously showed that the related compound SC 40827 inhibited ovulation by 70% when used at a concentration of 25 microM (Br?nnstr?m et al., Endocrinology 1988; 122:1715-1721). We now show that SC 44463 is 100, 500, and 75 times more powerful than SC 40827 in blocking ovulation, inhibiting action of ovarian interstitial collagenase, and inhibiting action of the small metalloproteinase of the rat uterus, respectively. SC 44463 also inhibits ovarian type IV collagen-digesting activity 50% at a concentration of 18 nM. Ovulation occurs after 9-12 h of perfusion with LH. Compound SC 44463 (25 nM) showed its full inhibitory capacity when added to the medium as late as 7 h after LH, but there was no significant inhibition when it was added at 9 h. This suggests that the major collagenolytic events occur beyond 7 h after stimulation by LH.     

Interference with the preovulatory luteinizing hormone surge and blockade of ovulation in immature pregnant mare's serum gonadotropin-primed rats with the anti-androgenic drug, hydroxyflutamide

The involvement of androgens in the control of ovulation has been assessed by administration of the androgen antagonist, hydroxyflutamide, to prepubertal rats treated with pregnant mare's serum gonadotropin (PMSG) to induce first estrus and ovulation. Without human chorionic gonadotropin (hCG) injection, only 46% of rats that received six 5-mg, s.c. injections of hydroxyflutamide at 12-h intervals, beginning an hour before s.c. injection of 4 IU PMSG on Day-2 (Day 0 = the day of proestrus), had ovulated a mean of 1.3 +/- 0.4 oocytes per rat when killed on the morning of Day 1, whereas 92% of sesame oil-treated controls had ovulated a mean of 6.9 +/- 0.6 oocytes. After i.p. injection of hCG at 1600 h on Day 0, 92% of hydroxyflutamide-treated rats ovulated a mean of 8.3 +/- 1.2 oocytes compared to 100% of controls, which ovulated 7.3 +/- 0.4 oocytes per rat: these groups were not significantly different from each other, nor from control rats that received no hCG. Thus, exogenous hCG completely overcame the inhibitory effect of hydroxyflutamide on ovulation. Rats treated with PMSG and hydroxyflutamide without hCG were killed either on the morning of Day 0 to determine serum and ovarian steroid levels or on the afternoon of Day 0 to determine serum LH levels. Serum levels of estradiol-17 beta and testosterone in hydroxyflutamide-treated rats were significantly higher (178% and 75%, respectively; p less than 0.01) than levels observed in controls on the morning of Day 0. Ovarian concentrations of the steroids were also elevated in hydroxyflutamide-treated rats (p less than 0.01 for testosterone only).(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of systems for collection of porcine zygotes for DNA microinjection and transfer

Crossbred gilts and sows (n=116) were used for the collection of 1-cell zygotes for DNA microinjection and transfer. Retrospectively, estrus synchronization and superovulation schemes were evaluated to assess practicality for zygote collection. Four synchronization and superovulation procedures were used: 1) sows were observed for natural estrous behavior; 1000 IU human chorionic gonadotrophin (hCG) was administered at the onset of estrus (NAT); 2) cyclic gilts were synchronized with 17.6 mg altrenogest (ALT)/day for 15 to 19 days followed by superovulation with 1500 IU pregnant mares serum gonadotropin (PMSG) and 500 IU hCG (LALT); 3) gilts between 11 and 16 days of the estrous cycle received 17.6 mg ALT for 5 to 9 days and PMSG and hCG were used to induce superovulation (SALT); and 4) precocious ovulation was induced in prepubertal gilts with PMSG and hCG (PRE). A total of 505 DNA microinjected embryos transferred into 17 recipients produced 7 litters and 50 piglets, of which 8 were transgenic. The NAT sows had less (P < 0.05) ovarian activity than gilts synchronized and superovulated by all the other procedures. Synchronization treatments with PMSG did not differ (P > 0.05) in the number of corpora hemorrhagica or unovulated follicles, but SALT and PRE treaments had higher ovulation rates than LALT (24.7 +/- 2.9, 24.3 +/- 1.8 vs 11.6 +/- 2.7 ovulations; X +/- SEM). The SALT and PRE treatments yielded 12.3 +/- 2.6 and 17.7 +/- 1.7 zygotes. Successful transgenesis was accomplished with SALT and PRE procedures for estrus synchronization and superovulation.     

Plasminogen activator activity in cumulus-oocyte complexes of gonadotropin-treated rats during the periovulatory period

Two types of plasminogen activator (tissue-type, tPA; urokinase-type, uPA) have been demonstrated in ovarian granulosa cells, but only tPA activity was found in denuded oocytes. Immature rats were treated subcutaneously with 20 IU pregnant mare's serum gonadotropin (PMSG) to stimulate follicle maturation, followed 2 days later by an injection of 10 IU human chorionic gonadotropin (hCG) to induce ovulation. Cellular plasminogen activator activities were determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis followed by a fibrin-overlay technique. Cumulus-oocyte complexes from rats before and after PMSG treatment contained low amounts of tPA, but not uPA, activity. After hCG treatment, tPA activity showed a time-dependent increase, reaching a maximum at 24 h after injection. At 12 and 24 h after hCG treatment, uPA activity was also detected. The appearance of high molecular weight lysis zones further suggested the formation of plasminogen activator-inhibitor complexes. Morphological analysis indicated that the increases in oocyte tPA activity were correlated with the extent of cumulus cell expansion and dispersion. In denuded oocytes, tPA activity also progressively increased during the periovulatory period to a maximum at 24 h after hCG treatment. In contrast, neither uPA activity nor activator-inhibitor complex was detected. Secretion of the proteases was measured in the conditioned media of cumulus-oocyte complexes cultured for 24 h in vitro. Substantial increases in tPA release were found in complexes obtained at 8 and 12 h after hCG injection, with lower secretion from complexes obtained at 24 h after hCG treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chorionic gonadotropin administration in domestic cats causes an abnormal endocrine environment that disrupts oviductal embryo transport

Fecal steroid analysis was used to investigate relationships between endocrine parameters and embryo characteristics in domestic cats subjected to chorionic gonadotropin stimulation and artificial insemination (AI). In Study 1, normal endocrine patterns were assessed in 12 cycling domestic queens. Fecal estradiol (E) patterns established an anovulatory cycle length of 18.3 +/- 0.4 d with estrus lasting 6.3 +/- 0.3 d. Eight females (67%) exhibited at least one spontaneous ovulation based on sustained increases in fecal progestagens (P). In Study 2, queens were mated during natural estrus (NE, n = 5) or subjected to exogenous i.m. gonadotropin stimulation, 100 IU eCG followed by 75 IU hCG 80 h later, (GS, n = 5). Compared with NE queens, fecal E concentrations were higher (P < 0.05) and remained elevated longer after ovulation induction with hCG. In Study 3, gonadotropin-stimulated queens (n = 7) were artificially inseminated and ovariohysterectomized 160 h after hCG. Ancillary follicles and/or corpora lutea were observed in 5 of 6 (83%) ovulating queens. Both fecal E and number of unovulated follicles observed at ovariohysterectomy were negatively correlated with the percentage of embryos recovered from the uterus (r = -0.91 and r = -0.87, respectively; P < 0.05). In summary, exogenous gonadotropin administration causes an abnormal endocrine environment in domestic cats, likely due to ancillary follicle development. The sustained elevations in estradiol appear to impair oviductal transport of embryos, possibly leading to the reduced fertility typically observed in cats subjected to gonadotropin stimulation and AI.     

Decrease in ovarian platelet-activating factor during ovulation in the gonadotropin-primed immature rat

Platelet-activating factor (PAF) is a biologically active phospholipid that is released locally during acute inflammatory reactions and tissue injury. Since there is evidence that the biochemical events of mammalian ovulation resemble an inflammatory reaction, the objective of this study was to determine whether ovarian levels of PAF change during ovulation. At 2-h intervals during the ovulatory process in gonadotropin-primed 25-day-old Wistar rats, the ovaries were extirpated, homogenized, and extracted for lipids. The extracts were subjected to thin-layer chromatography (TLC), and the portion of the silica gel that comigrated with PAF was re-extracted and assayed for PAF activity. The PAF was measured (in fmole equivalents of synthetic PAF) by a bioassay based on the capacity of aliquots of the extracts to release [3H]-serotonin from platelets isolated from whole blood of rabbits and prelabeled with [3H]-serotonin. The ovarian level of PAF decreased (p less than 0.01) by 36% from 6.67 +/- 0.77 to 4.27 +/- 0.45 fmoles/mg ovary by 2 h after treatment with human chorionic gonadotropin (hCG), and it declined another 14% by 4 h after hCG. The ovarian PAF remained at this reduced level for up to 24 h after hCG. The administration of indomethacin (5 mg/rat, s.c.) or epostane (5 mg/rat, s.c.) at 1 h after hCG prevented ovulation, but neither drug affected the decline in ovarian PAF. Preliminary tests showed that the lipid extracts from the ovaries also contained PAF inhibitor(s) that comigrated with PAF on the TLC plates. Similar to PAF, the lipid-soluble inhibitor(s) decreased (p less than 0.05) in the ovaries within 4 h after hCG treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Types I and IV collagenolytic and plasminogen activator activities in preovulatory ovarian follicles

During ovulation, enzymatic degradation of the extracellular matrix occurs within and around the graafian follicles. In this study, the activities of several different proteolytic enzymes were measured in the culture media of follicles taken from pregnant mare serum gonadotropin (PMSG)-primed immature rats. At 52 h after PMSG, the follicles were cultured for 2 to 15 h in media with or without human chorionic gonadotropin (hCG). Type I collagenase activity in hCG-stimulated follicles gradually increased within 6 h to 3.3-fold above that of the controls. Relatively pure populations of granulosa cells produced type I collagenase to a similar extent. Likewise, type IV collagenase increased 3.8-fold by 6 h after exposure of the follicles to hCG. In contrast, plasminogen activator activity increased by 3.9-fold at 2 h after hCG, but was negligible at 4, 6, and 15 h after incubation. These results suggest that plasminogen activator may activate both type I and type IV collagenase in hCG-stimulated ovulatory follicles.     

Collagen, collagenase and collagenolytic activity in rat Graafian follicles during follicular growth and ovulation

Follicles were dissected from the ovaries of immature rats at intervals after subcutaneous injection of 20 IU of pregnant mare's serum gonadotropin. A surge of luteinizing hormone was observed at 54 h and ovulation occurred at 64-66 h. The follicular volume between 36 and 48 h, then doubled again shortly before ovulation. The collagen content of the follicles increased 3-fold from 35 to 56 h, but decreased significantly (25%) from 61 to 66 h. Follicle homogenates, activated with trypsin or aminophenylmercuric acetate, digested Type I collagen at 28 degrees C to produce typical of a true collagenase. Collagenolytic activity assayed against endogenous collagen at 37 degrees C did not change significantly between 38 and 66 h.     

Synchronization of ovulation in cyclic gilts with porcine luteinizing hormone (pLH) and its effects on reproductive function

The overall objective was to evaluate the use of porcine luteinizing hormone (pLH) for synchronization of ovulation in cyclic gilts and its effect on reproductive function. In an initial study, four littermate pairs of cyclic gilts were given altrenogest (15 mg/d for 14 d). Gilts received 500 microg cloprostenol (Day 15), 600 IU equine chorionic gonadotropin (eCG) (Day 16) and either 5mg pLH or saline (Control) 80 h after eCG. Blood samples were collected every 4h, from 8h before pLH/saline treatment to the end of estrus. Following estrus detection, transcutaneous real-time ultrasonography and AI, all gilts were slaughtered 6d after the estimated time of ovulation. Peak plasma pLH concentrations (during the LH surge), as well as the amplitude of the LH surge, were greater in pLH-treated gilts than in the control (P=0.01). However, there were no significant differences between treatments in the timing and duration of estrus, or the timing of ovulation within the estrous period. In a second study, 45 cyclic gilts received altrenogest for 14-18d, 600 IU eCG (24h after last altrenogest), and 5mg pLH, 750 IU human chorionic gonadotropin (hCG), or saline, 80 h after eCG. For gilts given pLH or hCG, the diameter of the largest follicle before the onset of ovulation (mean+/-S.E.M.; 8.1+/-0.2 and 8.1+/-0.2mm, respectively) was smaller than in control gilts (8.6+/-0.2mm, P=0.05). The pLH and hCG groups ovulated sooner after treatment compared to the saline-treated group (43.2+/-2.5, 47.6+/-2.5 and 59.5+/-2.5h, respectively; P<0.01), with the most synchronous ovulation (P<0.01) in pLH-treated gilts. Embryo quality (total cell counts and embryo diameter) was not significantly different among groups. In conclusion, pLH reliably synchronized ovulation in cyclic gilts without significantly affecting embryo quality.     

Induction of ovulation by clomiphene citrate in the Indian vespertilionid bat, Scotophilus heathi

The ovulation induction property of clomiphene citrate (CC) and human chorionic gonadotropin (hCG) was studied in Scotophilius heathi, an Indian tropical vespertilionid bat, during the period of delayed ovulation between December to early January. The results of the study showed that 10 microg of CC alone was ineffective to induce ovulation, whereas 100 microg CC and 10 IU hCG alone induced ovulation. A significant (P < 0.01) increase in the ovulation rate was observed when 10 microg CC followed by 10 IU hCG, compared to 10 IU hCG and 100 microg CC alone groups. Finally, CC at a 100 microg dose, followed by 10 IU hCG, produced superovulation (14.00 +/- 0. 70), which is significantly different in comparison to all other groups. This is the first report of ovulation induced by CC in the Indian tropical bat as well as in any animal model that exhibits temporary anovulation similar to polycystic ovary syndrome (PCOD) during the normal physiology of reproduction.     

Expression of matrix metalloproteinases and their tissue inhibitor messenger ribonucleic acids in macaque periovulatory granulosa cells: time course and steroid regulation.

Progesterone appears essential for ovulation and luteinization of the primate follicle, but specific gene targets of progesterone action remain elusive. Limited evidence supports a role for progesterone in the induction of collagenolytic activity in the periovulatory follicle of primate and nonprimate species. This study was designed to elucidate the pattern of expression and progesterone regulation of mRNAs for the matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in macaque granulosa cells during controlled ovarian stimulation cycles before (0 h) and after (up to 36 h) administration of an ovulatory hCG bolus. Levels of mRNAs for interstitial collagenase, gelatinase A, matrilysin, TIMP-1 and TIMP-2 increased (p < 0.05) within 12 h of hCG, while gelatinase B mRNA increased later, by 36 h after hCG. Administration of a 3beta-hydroxysteroid dehydrogenase inhibitor (Trilostane [TRL]) during hCG treatment decreased (p < 0.05) mRNA levels for interstitial collagenase, gelatinase B, matrilysin, TIMP-1, and TIMP-2. Progestin (R5020) replacement during hCG+TRL treatment returned interstitial collagenase and TIMP-1 mRNAs to control levels. These data suggest that one action of progesterone, and possibly other steroids, in the cascade of events leading to ovulation and luteinization of the primate follicle is to regulate the expression of specific ovarian proteases and protease inhibitors.     

Effect of hCG on the 13,14-dihydro-prostaglandin F2-alpha forming capacity of the ovary after ovulation in PMSG-primed immature female rats

We have examined the change in the ovarian 13,14-dihydro-prostaglandin F2 alpha (13,14H2-PGF2 alpha) forming capacity after the first ovulation induced by injection of pregnant mare serum gonadotropin (PMSG 5 IU, sc) at 26 days of age. After ovulation, the 13,14H2-PGF2 alpha forming capacity in the whole ovary (WO) and in non-luteal ovarian tissues (WO-CL) gradually decreased, whereas a rapid decrease of the synthesizing capacity was observed in corpus luteum (CL). The capacity in WO 4 days after ovulation (33 days of age) was markedly stimulated by human chorionic gonadotropin (hCG 10 IU, ip) administration, whereas CL at 33 days of age did not respond to the stimulatory effect of hCG. A single injection of hCG on day 7 after hypophysectomy resulted 12 hrs later in a significant increase in the forming capacity of 13,14H2-PGF2 alpha in WO-CL. These results indicate that the 13,14H2-PGF2 alpha forming capacity in CL rapidly decreases after the first ovulation and the WO-CL, but not CL, retain the ability to form 13,14H2-PGF2 alpha in response to exogenous gonadotropin for a long time.