The involvement of collagenolysis in ovulation in the rat

Collagenolytic activity in ovarian follicles was previously demonstrated by using synthetic peptides and reconstituted collagen fibers. However, attempts to demonstrate degradation of ovarian collagen and to correlate collagenase activity with ovulation were not successful. By administration of L-(5-3H) proline, we have labeled ovarian and follicular collagen and followed collagenolytic activity by separation of 3H-hydroxyproline (3H-Hyp) from acid hydrolyzates of ovarian tissue by HPLC. The level of ovarian and follicular 3H-Hyp decreased by about 40% on the afternoon of proestrus or after exogenous stimulation of ovulation by human CG (hCG), and this decrease was abolished by blocking the surge of gonadotropins with Nembutal. To verify that the observed reduction in 3H-Hyp was due to the action of a typical collagenase, the collagenous fraction was prepared from ovarian tissue and from preovulatory follicles before and after the ovulatory stimulus. The extracts were treated with trypsin (25 min, 25 C, 0.01 mg/ml) plasmin and p-amino-phenyl-mercuric acetate to fully activate the collagenase extracted along with collagen. Both, enzymatic and chemical activation of collagenase in vitro resulted in degradation of collagen. This degradation could be inhibited by cysteine and EDTA; both are classic inhibitors of mammalian collagenases. The activity of ovarian collagenase increased within 3 h after hCG-stimulation, peaked at 5-fold 6 h after hCG, and declined afterwards. Administration of cysteine (0.001-0.01 mmol) into the bursal cavity of proestrous rats blocked ovulation and breakdown of ovarian collagen in a dose-dependent manner. Cysteine effectively inhibited ovulation even when injected 7 h after the hCG stimulus. Inhibitors of arachidonic acid metabolism prevent ovulation. Indomethacin (inhibitor of cyclooxygenase) and nordihydroguaiaretic acid (inhibitor of lipoxygenase) blocked ovulation and inhibited hCG-induced ovarian collagenolysis. Collectively, these results corroborate the essential role of collagenolysis in follicular rupture in mammals.     

Preovulatory changes in ovarian expression of collagenases and tissue metalloproteinase inhibitor messenger ribonucleic acid: role of eicosanoids.

The preovulatory surge of gonadotropins activates a cascade of proteolytic enzymes resulting in the rupture of the follicular wall and the release of a fertilizable ovum during ovulation. In the rat the process is initiated by a rise in follicular tissue-type plasminogen activator, produced predominantly in granulosa cells. Recent studies revealed a preovulatory increase in ovarian collagenolytic activity in vivo and an increase in activatable collagenase in vitro. In view of the complicated control of mammalian collagenase synthesis and activity by local inhibitors and activators, we examined the expression of ovarian interstitial and type IV collagenases and tissue inhibitor of metalloproteinase (TIMP) mRNA after an ovulatory stimulus. Ovarian mRNA was isolated from immature PMSG-treated rats 3, 6, and 9 h after hCG stimulation. Northern blot analyses revealed a mRNA of 1.7 kilobases (kb) hybridizing with the human interstitial collagenase cDNA probe. The levels of this mRNA showed a 25-fold increase between 3-6 h after hCG stimulation. The human cDNA probe of collagenase IV hybridized with a mRNA of 3.1 kb, which showed only a 4-fold increase 9 h after hCG treatment. The interstitial collagenase mRNA was expressed in both granulosa cells of preovulatory follicles and the residual ovarian tissue, whereas the expression of collagenase IV mRNA was limited to the residual tissue. Inhibitors of eicosanoid synthesis, previously shown to block ovulation and the LH/hCG-induced rise in ovarian collagenolysis, suppressed the gonadotropic stimulation of interstitial collagenase mRNA, but slightly stimulated that of collagenase IV. The mouse cDNA probe of TIMP hybridized with a 0.9-kb mRNA, which was stimulated by hCG to reach a maximum (7- to 8-fold increase) between 6-9 h after stimulation. TIMP was expressed and stimulated in both the granulosa cells and the residual tissue. Inhibitors of eicosanoid synthesis did not affect the gonadotropic stimulation of TIMP mRNA. These data support the suggested role of interstitial collagenase in follicle rupture and the essential role of eicosanoids in the mediation of gonadotropic stimulation of interstitial collagenase production and action. The observed stimulation of TIMP mRNA expression by the gonadotropin and the lack of any effect of eicosanoid synthesis inhibitors on this action of LH/hCG offer an additional mechanism by which these inhibitors may block ovulation. Thus, the suppression of ovulation by inhibitors of eicosanoid synthesis may result from selective inhibition of interstitial collagenase expression and undisturbed gonadotropin-stimulated TIMP expression.     

Estrogen synthetase activity in human term placental cells in monolayer culture

Human term placental cells, isolated by trypsin treatment, were grown in culture with medium 199 and 10% fetal bovine serum for up to 1 week. Aromatase specific activity (+/-SE) of freshly isolated cells was low (0.63 +/- 0.04 pmol/min X mg protein; n = 15) compared to that of placental tissue before trypsin treatment (21.30 +/- 0.40 pmol/min X mg protein; n = 6). This activity in attached cells increased 10-fold 24 h after plating (6.32 +/- 0.75 pmol/min X mg protein; n = 19) and continued to increase up to 72-96 h (14.78 +/- 1.09 pmol/min X mg protein; n = 13) before declining to 6.50 +/- 1.40 pmol/min X mg protein (n = 4) after 120 h. The functional activity of the cells was assessed by daily measurements of hCG secretion into the medium. Secretion of hCG was maintained at about 0.3 micrograms/flask up to 48 h in culture, then rose rapidly to about 6.2 micrograms/flask from 96-168 h. The addition of 1 mM (Bu)2cAMP plus 1 mM theophylline to the culture medium 24 h after plating stimulated hCG secretion 7- to 8-fold relative to that by control cells, had no effect on aromatase specific activity 24 h after its addition, but decreased aromatase activity after 48 h. Freshly prepared cells were primarily (approximately 80%) mononucleated. With time in culture, the number and size of the multinucleated cells increased drastically until they accounted for virtually all of the cellular material in culture at 72 h. These morphological and functional changes in hCG secretion and aromatase activity suggest that trypsin-isolated cytotrophoblast cells differentiated in culture to form syncytiotrophoblast cells.     

Inhibition of follicle-stimulating hormone/diethylstilbestrol-stimulated ovarian growth by extracts of pregnancy urine

We devised an in vivo biological assay for ovarian growth inhibiting activity to examine extracts of human pregnancy urine for the presence of ovarian growth inhibiting factor. Diethylstilbestrol (DES) capsules were implanted sc in immature hypophysectomized female rats; FSH was injected sc with or without test substance for 5 days. Rats with unstimulated ovaries were implanted with blank capsules and given the vehicle without FSH. Twenty four hours after the last injection, the ovaries were removed and weighed. The ovarian growth inhibition of the ovarian weight gain achieved in rats treated with DES and FSH. Crude commercial human CG (hCG) preparations, extracted from pregnancy urine, were chromatographed on Sephadex G-100, and the fractions were tested for ovarian growth inhibiting activity. The peak of ovarian growth inhibiting activity was found in fractions eluting from the column in the mol wt range of 12,000-20,000. Ovarian growth inhibiting activity was heat sensitive, not extracted by ether, and precipitated by acetone. Ovarian growth inhibiting activity was stable in acid at pH 2, but was inactivated by digestion with trypsin. The ovarian growth inhibiting activity was purified by chromatography on Concanavalin A-Sepharose and diethylaminoethyl-Sephacel. The active material contained hCG alpha, hCG beta, and beta-carboxyterminal peptide-immunoreactivity and its inhibiting activity could be removed from solution by immunoadsorption with antisera specific for hCG beta. The ovarian growth inhibiting activity was further purified on an anti-hCG alpha-immunoglobulin G affinity column. The activity was eluted from the affinity column at low pH, and eluted material contained all of the immunodeterminants of hCG. Virtually identical dose-response curves of ovarian inhibition were obtained using equivalent doses of beta-carboxyterminal peptide immunoreactivity of purified inhibitor and purified hCG (CR123). The inhibiting activity reached plateau of 80-90% at doses of 50-100 ng hCG/rat. Upon rechromatography on Sephadex G-100, the ovarian growth activity that was pooled from fractions corresponding to the 12,000-20,000 mol wt range was recovered in fractions corresponding to the elution position of hCG. We conclude that the low mol wt inhibiting activity observed in the crude pregnancy extracts is due to hCG and that hCG is a very potent inhibitor of FSH/DES stimulation of ovarian growth.     

Prolactin inhibits plasminogen activator activity in the preovulatory follicles

The present study was designed to determine the effects of PRL on changes in morphology and plasminogen activator (PA) activity in the preovulatory follicles. Rabbit ovaries were perfused with hCG alone or with hCG plus at 10, 10(2), or 10(3) ng/ml. PRL at 10(3) ng/ml directly inhibited the degeneration and decomposition of surface epithelial cells induced by hCG exposure. The subsurface connective tissue was visualized by treatment with sodium dodecyl sulfate, which removed surface epithelial cells from the ovary, thereby exposing collagen fibrils and the basal lamina. Sodium dodecyl sulfate treatment revealed inhibition of connective tissue disruption at the apex of the follicle wall in PRL-treated ovaries. PA activity in mature follicles in perfused rabbit ovaries exposed to hCG increased from 1.40 +/- 0.08 to 28.4 +/- 4.25 IU/g tissue after 4 h of perfusion. The addition of PRL to the perfusate inhibited the hCG-stimulated increase in intrafollicular PA activity in a dose-dependent fashion. Although at 7 h mature follicles treated by hCG alone showed greater intrafollicular PA activity than those treated with hCG plus PRL, this difference was not significant. These results suggest that PRL may act directly by interfering with mechanical events within the ovary that are required for the rupture of mature Graafian follicles, probably via the inhibition of intrafollicular tissue PA activity.     

Titrating luteinizing hormone surge requirements for ovulatory changes in primate follicles. I. Oocyte maturation and corpus luteum function

The amplitude and duration of the midcycle LH surge required for ovulatory maturation of the follicle and its enclosed oocyte in primates are unknown. To titrate periovulatory LH requirements, female rhesus monkeys received human gonadotropins (FSH with/without LH) for 9 days beginning at menses to promote the development of multiple preovulatory follicles. The next day, animals (n = 4-6/group) received: 1) no ovulatory stimulus; 2) 1000 IU hCG, im; 3) one injection of 100 micrograms GnRH, sc (GnRH-1); 4) three injections of GnRH (GnRH-3) at 3-h intervals (0800, 1100, and 1400 h); or 5) two injections of 50 micrograms GnRH agonist (GnRHa), sc, 8 h apart (0800 and 1700 h) to induce ovulatory maturation. Follicles were aspirated 27 h after the hCG or initial GnRH/GnRHa injection or on days 8 and 10 in animals receiving no ovulatory stimulus. Nuclear maturity of oocytes was evaluated as a marker for reinitiation of meiosis. Estradiol and progesterone levels were determined in daily serum samples by RIA. Levels of LH(-like) bioactivity were measured at selected intervals after hCG injection and within 24 h of GnRH/GnRHa treatment. In all groups, estradiol continuously rose to similar peak levels on day 10. The hCG treatment markedly elevated circulating LH-like bioactivity for up to 3 days. In GnRH-1, bioactive LH increased to 433.1 +/- 170.2 ng/mL (mean +/- SEM; n = 3) within 1-2 h, but then decreased to baseline (4.9 +/- 1.5 ng/mL) within 6 h. GnRH-3 and GnRHa treatment extended the interval of elevated bioactive LH to 8 and 14 h, respectively. There was no difference in the peak levels of LH(-like) bioactivity reached after hCG, GnRH, or GnRHa injection. Functional luteal phases were absent in monkeys receiving no ovulatory stimulus, whereas hCG treatment increased progesterone levels to 101 +/- 9 nmol/L (n = 6) and elicited functional luteal phases of 11.8 +/- 0.4 days. In contrast, only one animal in the GnRH/GnRHa groups (i.e. one GnRH-3 monkey) displayed elevated progesterone levels in the luteal phase. Of the total cohort of oocytes aspirated from follicles, a greater (P less than 0.05) proportion were classified as being in metaphase I or II of meiosis after hCG treatment (86%) compared to no ovulatory stimulus (13%), GnRH-1 (0%), GnRH-3 (43%), and GnRHa (12%). Thus, GnRH elicits a transient LH surge that can be extended by GnRH-3 or GnRHa in stimulated cycles of monkeys.(ABSTRACT TRUNCATED AT 400 WORDS)     

Gonadotropin regulation of tissue-type and urokinase-type plasminogen activators in rat granulosa and theca-interstitial cells during the periovulatory period

Plasminogen activators (PAs) are believed to be involved in ovulation. Because both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) are secreted by cultured rat granulosa cells, we have examined the activities of these proteins in ovarian homogenates as well as granulosa and theca-interstitial (TI) cells during gonadotropin-induced ovulation. Immature rats were injected with 20 IU pregnant mare serum gonadotropin (PMSG) to initiate follicle development, followed by treatment with 10 IU hCG 48 h later to induce ovulation. Ovarian proteins were separated by SDS-PAGE and PA activity determined by fibrin overlay. The activity of tPA, but not uPA, was stimulated following PMSG treatment in ovarian homogenates. Subsequent hCG injection further increased the tPA activity in a time-dependent manner, reaching a maximum (12 h after hCG treatment) immediately prior to ovulation and declined thereafter. Similar preovulatory increases in tPA activity were detected in isolated granulosa cells. Although both tPA and uPA activities were increased in TI cells after PMSG administration, no further increases were detected after hCG treatment. To estimate enzyme secretion, ovarian cells obtained at various preovulatory periods were incubated for 24 h in vitro. The ability of granulosa cells to secrete tPA, but not uPA, increased following in vivo PMSG and hCG treatment in a time-dependent manner, reaching a maximum immediately prior to ovulation. During the preovulatory period, an abrupt increase in tPA secretion by TI cells was also detected. Using immunohistochemical staining for tPA, it was found that ovarian sections from preovulatory rats at 12 h after hCG injection stained positively in granulosa, theca interna, and interstitial gland cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of prostaglandins on collagen synthesis in rabbit ovarian follicles during the ovulatory process

Collagen fibers in the ovarian follicle undergo drastic changes at ovulation due to the preovulatory increase of collagenolytic activities. The collagen synthesis in ovaries, however, has not been elucidated yet. To clarify the regulatory role of prostaglandins (PGs) in collagen synthesis of the follicular wall in relation to the ovulatory process, we measured prolyl hydroxylase (PH), as well as lysyl oxidase (LO) activity and the content of hydroxyproline (Hyp) in ovarian follicles of the rabbits treated by hCG, hCG/indomethacin (IM) and hCG/IM/various PGs. The experimental groups consisted of; 1) untreat control group 2) ovulatory group receiving hCG 3) non-ovulatory group given PGs 4) ovulatory group given hCG and PGs 5) group in which hCG-induced ovulation was inhibited by IM (4 mg/kg) 6) group in which IM-inhibited ovulation was recovered by PGF2 alpha (1.5 mg/kg) 7) group in which IM-inhibited ovulation was not restored by PGE1 (0.1 mg/kg) and PGE2 (0.7 mg/kg). The peak activities of PH and LO in ovarian follicles were observed at 12-13 hr after hCG injection, namely, immediately after ovulation. Significant changes of these activities after hCG administration were specific to the ovaries. PH activity in the ovaries was suppressed by the administration of IM, but LO activity was not significantly suppressed. In the hCG/IM/PGF2 alpha-treated ovulatory rabbits (Group 6), PH activity recovered to nearly the level of the hCG-treated rabbits (Group 2). By addition of PGE2, ovulation did not recover but PH activity was restored to about 70% of the hCG-treated rabbits. PGE1 did not have any effect on the reversal of ovulation-blockage or restoration of PH activity. The amount of Hyp after hCG administration tended to decrease from 6 hr to 10 hr but was significantly increased from 10 hr to 13 hr. This increase of Hyp after ovulation significantly correlated with the increase of PH and LO activities. In the hCG/IM/PGF2 alpha-treated rabbits (Group 6), the changes of Hyp were similar to those the hCG-treated rabbits (Group 2). In conclusion, collagen synthetic activity, found to be regulated by PH and LO activities in the ovarian follicles, was activated after follicle rupture, resulting in reconstruction of collagen fibers, and PGs play an important role in the ovulatory process by modifying collagen synthesis.     

Natural and gonadotropin-induced ovarian activity in tigers (Panthera tigris) assessed by fecal steroid analyses

Fecal samples were collected from female tigers (n = 17) to compare endocrine patterns associated with natural ovarian activity with those after chorionic gonadotropin ovulation induction and artificial insemination (AI). Baseline fecal estradiol concentrations were 65.77 +/- 3.61 ng/g with estrual peaks of 167.39 +/- 9.92 ng/g and an anovulatory cycle length of 17.96 +/- 0.70 days. Peak fecal estradiol was higher when females were housed with a male for breeding (262.30 +/- 41.43 vs. 165.30 +/- 3.67 ng/g; P < 0.05). The majority of animals showed some seasonal differences in fecal estradiol however, patterns were inconsistent. Fecal progestagens increased only after breeding confirming tigers are primarily induced ovulators. The non-pregnant luteal phase was 34.50 +/- 1.85 days in duration. In pregnant tigers, fecal progestagens remained elevated for 108 days until parturition and the diagnosis of pregnancy was possible based on the elevated fecal progestagens after 35 days of gestation. Tigers were administered equine chorionic gonadotropin (eCG) to stimulate follicular growth and human chorionic gonadotropin (hCG) to induce ovulation prior to AI [200 IU eCG/100 IU hCG (n = 5); 400 IU eCG/200 IU hCG (n = 2); 500 IU eCG/100 IU hCG (n = 2); 1000 IU eCG/750 IU hCG (n = 11); 1000 IU eCG/1000 IU hCG (n = 4)]. None of the tigers subjected to AI became pregnant (n = 9). Fecal endocrine patterns in gonadotropin-stimulated tigers were considerably different from those observed in naturally bred tigers. In particular, fecal estradiol concentrations were higher than those observed during natural estrus and remained elevated for longer periods of time in tigers administered the higher doses of gonadotropins typically used in conjunction with AI in this species. These abnormal endocrine patterns may help explain the poor success rate of AI in this species.     

Chronic exposure of the developing corpus luteum in monkeys to chorionic gonadotropin: persistent progesterone production despite desensitization of adenylate cyclase

The transient steroidogenic response of the macaque corpus luteum to chronic human CG (hCG) treatment beginning on days 9-10 of the luteal phase (i.e. stimulated early pregnancy) is associated with decreased numbers and affinity of available receptors for gonadotropin and homologous desensitization of adenylate cyclase. This study determined if similar changes in the receptor-adenylate cyclase system accompany the persistent steroidogenic response which occurs when hCG treatment begins earlier in the luteal phase. Female rhesus monkeys received increasing doses of hCG (15 up to 5760 LU) twice daily beginning 5-6 days after the midcycle LH surge. The levels of circulating progesterone increased (P less than 0.05) within 24 h of initial hCG exposure and did not decrease throughout the 10-day regimen. The corpus luteum was removed after 0 (n = 8), 6 (n = 4), or 10 (n = 4) days of hCG treatment. Whereas the numbers of available [125I]hCG binding sites in luteal particulates remained unchanged by 10 days of hCG exposure, the dissociation constant (Kd) for gonadotropin binding was greater than at day 0 (6.17 +/- 1.41 vs. 0.91 +/- 0.06 X 10(-10) M, P less than 0.05). Since the number of binding sites occupied by injected hCG increased with treatment (7.81 +/- 1.55 fmol/mg wet wt at day 10), the total number (available + occupied) of gonadotropin receptors was 3-fold greater (P less than 0.05) at day 10 than at day 0. Adenylate cyclase activity in luteal homogenates, assessed by conversion of [alpha-32P]ATP to [32P]cAMP, was stimulated on day 0 by hCG (2.7 +/- 0.7 X control, at 250 nM hCG), prostaglandin E2 (2.5 + 0.5 X control, at 0.5 mM), and prostaglandin I2 (2.3 +/- 0.5 X control at 0.5 mM) as well as forskolin (100 microM) and 5'-guanylyl-imidodiphosphate (50 microM). In contrast, cAMP production by day 6 of treatment was insensitive to hCG, but remained responsive to prostaglandin E2, prostaglandin I2, and nonhormonal activators. We conclude that CG treatment in the early luteal phase did not prevent the development of gonadotropin receptors to levels typically observed in the functional corpus luteum of the menstrual cycle. Also, many changes in the gonadotropin receptor-adenylate cyclase system in macaque luteal tissue were similar after CG treatment beginning on days 5-6 or days 9-10 of the luteal phase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Developmental changes in luteinizing hormone/human chorionic gonadotropin steroidogenic responsiveness in marmoset granulosa cells: effects of follicle-stimulating hormone and androgens

Factors regulating LH/hCG responsiveness in primate granulosa cells were examined in the marmoset monkey (Callithrix jacchus). Granulosa cells were isolated and pooled from small antral (0.5-1.0 mm) and large preovulatory (greater than or equal to 2 mm) follicles from mid- to late follicular phase ovaries of cyclic marmosets. The cells from small and large follicles were cultured in serum-free medium for 48 h in the absence or presence of increasing concentrations of hCG (0.1-100 ng/ml) with or without 0.1 microM androgen [testosterone or 5 alpha-dihydrotestosterone (DHT]). Granulosa cells from small follicles were also cultured in the absence or presence of a constant concentration of human FSH (30 ng/ml) with or without androgen for 48 h before exposure to hCG for an additional 48 h. Steroidogenic responsiveness was assessed by measuring progesterone accumulation in culture medium and aromatase activity in washed monolayers. Granulosa cells from large follicles showed dose-dependent increases in both progesterone accumulation and aromatase activity in response to treatment with hCG. In contrast, granulosa cells from small follicles were unresponsive to hCG. However, pretreatment of granulosa cells from small follicles for 48 h with FSH stimulated hCG responsiveness. The effects of both testosterone and DHT on hCG-stimulated aromatase activity and progesterone accumulation by granulosa cells from large preovulatory follicles were inhibitory. Testosterone and DHT also suppressed basal (no hCG) progesterone accumulation in these cells, but had no effect on basal aromatase activity. The effects of androgens on FSH-induced hCG responsiveness in immature granulosa cells were variable. The results show a development-related increase in marmoset granulosa cell responsiveness to LH/hCG and provide evidence that FSH and androgens interact to regulate the onset and expression of this critical event during preovulatory follicular development in the primate ovary.     

Variable ovarian response to gonadotropin-releasing hormone antagonist-induced gonadotropin deprivation during different phases of the menstrual cycle

The gonadotropin dependence of ovarian follicular maturation and corpus luteum function can now be examined in women using antagonistic analogs of GnRH. We studied the responses of three groups of women throughout a control cycle and during the administration of a potent GnRH antagonist, detirelix ([N-Ac-D-Nal(2)1,D-pCl-Phe2,D-Trp3,D-hArg(Et2)6,D-Ala10++ +] GnRH, Syntex Research). Detirelix (10 mg, sc) was administered for 3 consecutive days during the midfollicular phase (n = 4), preovulatory phase (n = 4), and early luteal phase (n = 4). The pituitary response to detirelix was similar throughout the three phases of the menstrual cycle. Immunoreactive LH concentrations decreased to 35% (mean +/- SEM) of pretreatment values within 8 h after the initial injection and remained suppressed for 72 h after discontinuance of treatment. Immunoreactive FSH concentrations decreased to 73 +/- 3% of pretreatment levels within 8 h and returned to baseline within 24 h after the third injection. In contrast, the ovarian response to detirelix varied markedly during different phases of the cycle. Midfollicular phase treatment was associated with a decline in estradiol (E2) levels from pretreatment values of 246 +/- 48 to 81 +/- 15 pmol/L within 24 h of the last injection. Vaginal bleeding ensued in three of four women. Follicular recruitment was then reinitiated, and an ovulatory LH surge occurred 18.2 +/- 2.9 days after the last injection. Similarly, treatment during the early luteal phase produced a decline in E2 concentrations from 286 +/- 29 to 70 +/- 7 pmol/L and a decline in progesterone concentrations from 20 +/- 1.6 to 1.9 +/- 0.3 nmol/L within 24 h after the last injection. Luteolysis was associated with menstrual bleeding in all four women. The subsequent ovulatory LH surge occurred 16.5 +/- 1.0 days after discontinuance of treatment. In contrast, treatment during the preovulatory phase resulted in a decline in E2 concentrations from 844 +/- 66 to 429 +/- 132 pmol/L during the first 48 h of treatment. Gonadotropin and E2 concentrations subsequently recovered from suppression, growth of the dominant follicle resumed, and a LH surge occurred 5.8 +/- 1.4 days after the last injection. These data indicate that the GnRH antagonist detirelix produces rapid and consistent suppression of pituitary gonadotropin secretion. The magnitude of suppression and preferential suppression of LH vs. FSH are similar throughout the cycle. In contrast, the ovarian response to gonadotropin deprivation varies during the menstrual cycle.(ABSTRACT TRUNCATED AT 400 WORDS)     

Endocrine profiles after triggering of final oocyte maturation with GnRH agonist after cotreatment with the GnRH antagonist ganirelix during ovarian hyperstimulation for in vitro fertilization

In a randomized multicenter study, the efficacies of two different GnRH agonists were compared with that of hCG for triggering final stages of oocyte maturation after ovarian hyperstimulation for in vitro fertilization. Ovarian stimulation was conducted by recombinant FSH (Puregon), and the GnRH antagonist ganirelix (Orgalutran) was coadministered for the prevention of a premature LH rise. Luteal support was provided by daily progestin administration. Frequent blood sampling was performed at midcycle in the first 47 eligible subjects included in the current study, who were randomized for a single dose of 0.2 mg triptorelin (n = 17), 0.5 mg leuprorelin (n = 15), or 10,000 IU hCG (n = 15). Serum concentrations of LH, FSH, E2, and progesterone (P) were assessed at variable intervals. LH peaked at 4 h after both triptorelin and leuprorelin administration, with median LH levels of 130 and 107 IU/liter (P < 0.001), respectively. LH levels returned to baseline after 24 h. Subjects receiving hCG showed peak levels of 240 IU/liter hCG 24 h after administration. A rise in FSH to 19 IU/liter (P < 0.001) was noted in both GnRH agonist groups 8 h after injection. Within 24 h the areas under the curve for LH and FSH were significantly higher (P < 0.001) in both GnRH agonist groups compared with that for hCG. E2 and P levels were similar for all groups up to the day of oocyte retrieval. Luteal phase areas under the curve for P and E2 were significantly elevated (P < 0.001) in the hCG group. The mean (+/-SD) numbers of oocytes retrieved were 9.8 +/- 5.4, 8.7 +/- 4.5, and 8.3 +/- 3.3; the percentages of metaphase II oocytes were 72%, 85%, and 86%; and fertilization rates were 61%, 62%, and 56% in the triptorelin, leuprorelin, and hCG group, respectively (P = NS for all three comparisons). These findings support the effective induction of final oocyte maturation in both GnRH agonist groups. In summary, after treatment with the GnRH antagonist ganirelix for the prevention of premature LH surges, triggering of final stages of oocyte maturation can be induced effectively by a single bolus injection of GnRH agonist, as demonstrated by the induced endogenous LH and FSH surge and the quality and fertilization rate of recovered oocytes. Moreover, corpus luteum formation is induced by GnRH agonists with luteal phase steroid levels closer to the physiological range compared with hCG. This more physiological approach for inducing oocyte maturation may represent a successful and safer alternative for in vitro fertilization patients undergoing ovarian hyperstimulation.     

Human chorionic gonadotropin stimulation of immunoreactive prostaglandin synthase in the rat ovary

Follicular prostaglandins (PG) increase markedly in the hours after the preovulatory gonadotropin rise in the rat. The present investigation was performed to determine if the increased prostaglandins result from elevation of the amount of the principal enzyme in the conversion of arachidonic acid to prostaglandins i.e. PG synthase. PG synthase was purified from sheep seminal vesicles and rat ovaries for the preparation of monoclonal antibodies. A monoclonal antibody was utilized in a competitive, microtiter plate-based enzyme immunoassay to quantitate PG synthase protein. Follicular development was stimulated in 26-day-old rats by injection of 20 IU of PMSG, and 51 h later 20 IU of hCG was injected. Ovaries were removed from rats before and 8 h after the hCG injection for quantitation of PG synthase by enzyme immunoassay. PG synthase immunologie activity was increased three-fold by hCG stimulation. These findings support the hypothesis that the preovulatory gonadotropin rise causes increased PG synthase protein in the rat ovary.     

Effect of time and dose of indomethacin on follicular prostaglandins and ovulation in the rabbit

This study determined specifically when ovarian prostaglandins (PGs) increase during ovulation and how effectively different doses of indomethacin inhibit PGs and ovulation. Rabbit ovarian follicles were removed at hourly intervals after stimulating the animals with hCG (50 IU/kg). The follicles were homogenized in 0.1 M acetate buffer (pH 4.5), and the PGE and PGF in the extracts were measured by RIA. Ovulation rates were determined by calculating the percentage of mature follicles that ruptured after stimulation by hCG. Before hCG, the normal levels of PGE and PGF were 111.1 +/- 14.7 and 51.0 +/- 6.6 pg/mg follicle, respectively. By 2 h after hCG treatment, PGE and PGF both increased to 162.6 +/- 17.0 and 80.6 +/- 13.3 pg/mg follicle, respectively. Approximately 5 h later, there was a second, sharper increase in both PGs which peaked at 652.6 +/- 63.5 and 345.4 +/- 32.3 pg/mg follicle, respectively, 10 h after hCG treatment, i.e. at the expected time of ovulation. We found that regardless of whether indomethacin was given early or late during the ovulation process, this agent significantly reduced follicular PG production within 5 min after its administration. For example, only 5 min after 10 mg/kg indomethacin were administered 8 h after hCG, PGE and PGF dropped from 317.7 +/- 50.0 and 125.0 +/- 10.5 pg/mg follicle to 93.3 +/- 17.4 and 49.3 +/- 10.5 pg/mg follicle, respectively. Unexpectedly, when graded doses of indomethacin were administered either 1 or 8 h after hCG, there was not a statistically significant correlation between follicular PG levels and ovulation rate. For example, when doses of 1.25, 2.5, 5.0, and 10.0 mg/kg indomethacin were given 8 h after hCG, the PGE and PGF levels at the expected time of ovulation (i.e. 2 h later) were always equal to or less than the PG levels in follicles that had not been stimulated by hCG, yet ovulation proceeded at rates of 46.7 +/- 12.3%, 30 +/- 7.7%, 14.3 +/- 9.3%, and 0%, respectively. Therefore, the results raise questions about the specific role of PGs in the ovulation process.     

Are ovarian steroids required for ovum maturation and fertilization? Effects of cyanoketone on the in vitro perfused rabbit ovary

An isolated perfused rabbit ovary preparation was used to determine the effects of cyanoketone, a potent inhibitor of 3 beta-hydroxysteroid dehydrogenase, on ovulation, ovum maturation and fertilizability, and steroid production. In the first experiment, cyanoketone (10(-4) M) was added to the perfusate of one ovary. The contralateral control ovary was perfused with medium alone. Thirty minutes after the onset of perfusion, hCG (50 IU) was added to the perfusate of both ovaries. The ovulatory efficiency of ovaries treated with cyanoketone plus hCG (82.3 +/- 4.6%) was similar to that of ovaries treated with hCG alone (84.8 +/- 4.4%). No difference was observed in the degree of ovum maturity or degeneration between control and cyanoketone-treated ovaries. Progesterone and estradiol production were significantly reduced by cyanoketone treatment; concentrations in the perfusate of ovaries treated with cyanoketone were 9.7% and 8.0% of the control values, respectively, 2 h after exposure to hCG. The concentration of 17-hydroxypregnenolone was not affected by cyanoketone treatment. Exposure to cyanoketone resulted in a significant (P less than 0.005) reduction in the fertilizability of ova ovulated and fertilized in vitro. In the second experiment, the percentage of ova that showed evidence of normal fertilization was significantly (P less than 0.025) increased in ovaries perfused with cyanoketone plus estradiol (64.5%) compared to that in ovaries perfused with cyanoketone alone (32.4%). In the third experiment, the addition of progesterone to the perfusate did not affect fertilizability of ovulated ova in ovaries perfused with cyanoketone plus estradiol. These results suggest that the presence of estradiol in the ovarian steroid environment may be essential for fertilizability of ova, but not for the processes of ovulation or meiotic maturation.     

Handling during pregnancy in the blue fox (Alopex lagopus): the influence on the fetal gonadal function

In previous studies it has been shown that handling produced stress in pregnant blue fox vixens increasing plasma level and adrenal in vitro production of cortisol. Moreover, this treatment increased plasma cortisol levels in the fetuses. The present study was designed to examine effects of a 1-min daily handling stress applied to pregnant blue fox vixens on fetal gonadal steroidogenesis. Plasma concentrations of oestradiol and testosterone, gonadal content and gonadal in vitro production of these steroids, and response to exogenous hCG as well as gonadal weights, and anogenital distances were measured in control (C, n=69) and stressed (S, n=54) fetuses on 47-48 days of pregnancy. Maternal stress induced a suppression of gonadal steroidogenesis in the fetuses. The decreased testosterone content in the testes and oestradiol content in the ovaries were demonstrated in stressed fetuses compared with control (testosterone: 4.91+/-0.46 vs. 7.35+/-0.87 ng/both testes, P<0.05; oestradiol: 29.1+/-3.4 vs. 46.5+/-4.9 ng/both ovaries, P<0.05). The ovarian oestradiol in vitro production in female fetuses from stressed mothers was decreased in comparison with control (3.69+/-0.39 vs. 7.52+/-1.51 pg/ovary/h, P<0.05). The same difference was observed between stressed and control male fetuses in the testosterone testicular response to hCG (5.34+/-0.64 vs. 8.73+/-0.40 ng/testis/h, P<0.05). The ovarian weight from stressed fetuses was lower in comparison with control (12.9+/-0.7 vs. 16.8+/-0.6 mg, P<0.05). The anogenital distance in female fetuses from stressed vixens was also reduced (0.6+/-0.03 vs. 0.8+/-0.02 cm, P<0.01). These results indicate that prenatal stress resulted in a significant reduction of hormonal and morphometric measures of the reproductive system in blue fox fetuses with more drastic effects in female fetuses.     

Concentration of cyclic AMP in rabbit ovaries in the preovulatory period after administration of luteinizing hormone or human chorionic gonadotrophin

Ovarian adenosine 3'-5'-monophosphate (cAMP) concentrations were determined in untreated female rabbits and in those sacrificed at various times (0.5-6.0 h) after administration of luteinizing hormone (LH, 50 micrograms) or human chorionic gonadotrophin (hCG, 100 IU). Both LH and hCG caused a rapid increase in ovarian cAMP concentrations within 0.5 h. After LH stimulation the elevated levels declined rapidly and by 2 h they were almost back to control levels. In the hCG-treated animals the decline in ovarian cAMP from the initial peak concentrations was much more gradual and the levels were significantly higher than control even at 6 h. The different response to the two gonadotrophins is discussed in relation to the physiologic importance earlier attributed to the prolonged elevation of cAMP in rabbit ovaries after stimulation with hCG.     

Short loop feedback control of the estrogen-induced luteinizing hormone surge in pigs

This study examines whether hCG will block the estradiol-induced LH surge in ovariectomized gilts. Twenty post-puberal cross-bred gilts were ovariectomized at 6-7 months of age. Approximately 2 months later, the experiment was conducted, and all gilts were given estradiol benzoate (EB; 10 micrograms/kg, im) at 0 h. Controls (n = 6) received im saline 24 and 48 h after EB. Two groups of gilts received 2000 IU hCG im, at 24 h (hCG24; n = 5) or 48 h (hCG48; n = 5) after EB. The fourth group (n = 4) received hCG at 48 h and was then given iv a LHRH agonist (des-Gly10, [D-Ala6]LHRH ethylamide) in 100-ng boluses hourly from 54-96 h after EB. Blood samples for determination of LH and FSH were collected every 6 h from 0-96 h. In controls, EB alone suppressed LH from 3.9 +/- 1.9 ng/ml at 0 h to 1.0 +/- 0.2 during 6-48 h (negative feedback), but LH then increased to 4.5 +/- 0.5 between 54 and 96 h (positive feedback), with the peak of the surge (6.7 +/- 1.6) occurring at 72 h. Treatment with hCG did not alter LH during the negative feedback phase (1.1 +/- 0.1 and 1.0 +/- 0.1 for hCG24 and hCG48, respectively). However, there was no LH surge in gilts given hCG at 24 or 48 h (2.4 +/- 0.2 and 2.2 +/- 0.1 form 54-96 h; P less than 0.05). Hourly injections of the LHRH agonist evoked a surge in LH (8.3 +/- 1.3) and maintained elevated LH (4.5 +/- 0.6) between 54 and 96 h, similar (P greater than 0.05) to values for controls. Generally, FSH in gilts given hCG followed the same pattern as LH secretion during the negative feedback stage; however, due to randomization, means for the period from 0-48 h for gilts treated with hCG 24 or 48 h after EB were lower (P less than 0.05) than for controls or gilts given LHRH agonist (62.2 +/- 2.8 and 63.0 +/- 2.7 vs. 79.3 +/- 3.2 and 93.3 +/- 4.2 ng/ml, respectively). During the positive feedback phase (54-96 h), FSH was lower in gilts given hCG (hCG24, 63.4 +/- 2.3; hCG48, 67.3 +/- 2.0) than in controls (86.0 +/- 4.0), but in gilts given LHRH agonist, FSH was higher (1001.1 +/- 7.7) than in controls.(ABSTRACT TRUNCATED AT 400 WORDS)