Metallothionein-1 messenger RNA transcription in steroid-secreting cells of the rat ovary during the periovulatory period.

An increase in metallothionein 1 (MT-1) mRNA was detected in the ovaries of immature Wistar rats that were primed with s.c. injection of 10 IU eCG followed 48 h later by 10 IU hCG s.c. to initiate the ovulatory process. Ovarian RNA was extracted at 0, 2, 4, 8, 12, 24, 72, 144, and 288 h after the primed animals were injected with hCG. These extracts were used for reverse transcription polymerase chain reaction (RT-PCR) differential display and Northern analyses that yielded complementary gene fragments for MT-1. Expression of MT-1 mRNA increased significantly by 24 h after hCG treatment and reached a peak at 144 h after hCG. In contrast, a disintegrin and metalloproteinase with thrombospondin motifs and a tissue inhibitor of metalloproteinase 1, which were also detected by the RT-PCR differential display procedure, reached a peak at 12 h after hCG and returned to control levels in the ovaries by 72 h after hCG. In situ hybridization indicated that most of the MT-1 mRNA was expressed in the vicinity of the theca interna of preovulatory follicles and in the lutein granulosa of postovulatory follicles. Thus, MT-1 mRNA expression is primarily in the vicinity of steroid-secreting areas of the ovary. The substantial increase in MT-1 mRNA expression might be important in protecting the ovarian tissues from oxidative stress generated by ovarian inflammatory events during the ovulatory process and luteinization.

Gonadotropin-induced expression of pancreatitis-associated protein-III mRNA in the rat ovary at the time of ovulation.

The ovulatory process in mammals involves gross physiological events in the ovary that cause transient deterioration of the ovarian connective tissue and rupture of the apical walls of mature follicles. This gonadotropin-induced process has features similar to an acute inflammatory reaction that affects most of the ovary. The present study reveals that the ovulatory events include induction of mRNA for pancreatitis-associated protein-III (PAP-III). Immature Wistar rats were primed with 10 IU equine chorionic gonadotropin s.c., and 48 h later the 12-h ovulatory process was initiated by 10 IU human chorionic gonadotropin (hCG) s.c. Ovarian RNA was extracted at 0, 2, 4, 8, 12 and 24 h after the animals were injected with hCG. The RNA extracts were used for RT-PCR differential display to detect PAP-III gene expression in the stimulated ovarian tissue. Northern blotting showed that transcription was significantly greater at 4-12 h after the ovaries had been stimulated by hCG. In situ hybridization indicated that PAP-III mRNA expression was limited mainly to the hilar region of the ovarian stroma, with most of the signal emanating from endothelial cells that lined the inner walls of blood vessels, and from small secondary follicles. Treatment of the animals with ovulation-blocking doses of indomethacin (an inhibitor of prostanoid synthesis) or epostane (an inhibitor of progesterone synthesis) revealed that ovarian transcription of PAP-III mRNA was moderately dependent on ovarian progesterone synthesis. In conclusion, the present evidence of an increase in PAP-III gene expression in gonadotropin-stimulated ovaries provides further evidence that the ovulatory process is comparable to an inflammatory reaction.

3alpha-hydroxysteroid dehydrogenase messenger RNA transcription in the immature rat ovary in response to an ovulatory dose of gonadotropin.

The ovulatory process in mammals involves a substantial increase in the metabolism of steroids and eicosanoids in response to a surge in LH or to an injection of hCG into experimental animals. This study provides evidence that the ovulatory stimulus causes induction of the gene for 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD), an enzyme that belongs to several oxidoreductase superfamilies that affect steroid and eicosanoid metabolism. Immature Wistar rats were primed with 10 IU eCG s.c., and 48 h later the 12-h ovulatory process was initiated by 10 IU hCG s.c. Ovarian RNA was extracted at 0, 2, 4, 8, 12, and 24 h after injecting the animals with hCG. The RNA extracts were used for reverse transcription-polymerase chain reaction (PCR) differential display to detect gene expression in the stimulated ovarian tissue. One of the PCR primer sets differentially amplified a cDNA fragment that is 52.3% homologous with a 3alpha-HSD gene in rat liver. Northern analyses revealed that maximum transcription was at 8 h after the animals had been treated with hCG. The Northerns also indicated that the 3alpha-HSD cDNA probe cross-hybridized with as many as six different bands of mRNA on the blots. In situ hybridization localized 3alpha-HSD mRNA in the granulosa and thecal layers of mature follicles and in newly formed corpora lutea at 24 h after the ovulatory stimulus. In conclusion, gene(s) for 3alpha-HSD are transcribed in ovarian follicles in response to an ovulatory dose of gonadotropin. A possible function of the oxidoreductase enzyme that is translated from the 3alpha-HSD mRNA may be to reduce the toxic aldehyde and ketone components of the steroids and eicosanoids that accumulate in the mammalian ovary at the time of ovulation.

Ovulation: a multi-gene, multi-step process.

The luteinizing hormone (LH) surge initiates a cascade of proteolytic events that control ovulation. One of the genes induced by LH is the progesterone receptor (PR). Because mice with a mutant PR gene (PRKO) fail to ovulate and are infertile, we have used them as a model in which to determine PR target genes that might mediate the ovulatory process. The matrix metalloproteinases (MMPs: MMP2, MMP9, and MMP13) appear to be expressed in ovaries of PRKO mice in a manner similar to that in their wild-type littermates. However, the expression of two other types of proteases, cathepsin L (a member of the papain family) and ADAMTS-1 (A Disintegrin And Metalloproteinase with Thrombospondin-like motifs), are selectively induced in granulosa cells of preovulatory follicles by the LH surge. Maximal levels of these proteases are observed at 12-16 h after an LH surge, the time of ovulation. Furthermore, mRNAs encoding cathepsin L and ADAMTS-1 are reduced in the PRKO mice compared to their wild-type littermates. These novel observations indicate that these two proteases regulate some key step(s) controlling ovulation.

Expression of regulator of G-protein signaling protein-2 gene in the rat ovary at the time of ovulation.

The ovulatory process in mammals begins when an endogenous surge in LH circulates to the ovary and couples with receptors in the plasma membranes of granulosa cells in mature ovarian follicles. This study provides evidence that the ovulatory stimulus includes induction of the gene for regulator of G-protein signaling protein-2 (RGS2). Immature Wistar rats were primed with 10 IU eCG s.c., and 48 h later the 12-h ovulatory process was initiated by 10 IU hCG (a homolog of LH) s.c. Ovarian RNA was extracted at 0, 2, 4, 8, 12, and 24 h after injecting the animals with hCG. The RNA extracts were used for reverse transcription-polymerase chain reaction differential display to detect gene expression in the stimulated ovarian tissue. Two of the amplified cDNAs that were upregulated within 2 h after the ovaries had been stimulated by hCG were homologous to segments of the mouse gene for RGS2. In situ hybridization indicated that the RGS2 mRNA was expressed in the granulosa layer of mature follicles. In conclusion, the gene for RGS2, which is known to regulate membrane signaling pathways, is transcribed in ovarian follicles in response to an ovulatory dose of gonadotropin.

Expression of tumor necrosis factor-stimulated gene-6 in the rat ovary in response to an ovulatory dose of gonadotropin.

Current evidence supports the hypothesis that the biochemical events of mammalian ovulation are analogous to an acute inflammatory reaction. This study reveals that tumor necrosis factor-stimulated gene-6 (TSG-6), which encodes a member of the superfamily of hyaluronan-binding proteins that is specifically translated in inflammatory reactions, is expressed in ovarian follicles that have been induced to ovulate. Immature Wistar rats were primed with 10 IU equine CG s.c.; and 48 h later, the 12-h ovulatory process was initiated by 10 IU human CG (hCG), s.c.. Ovarian RNA was extracted at 0, 2, 4, 8, 12, and 24 h after the primed animals were injected with hCG. The RNA extracts were used for RT-PCR differential display of amplified complementary DNAs (cDNAs) that represented gene expression in the stimulated ovarian tissue. Northern analysis of one of the differentially amplified cDNAs confirmed that it was part of a gene that was substantially up-regulated at 4-8 h after the ovaries had been stimulated by hCG. Subcloning and sequence analysis revealed that the cDNA matched the gene for TSG-6. In situ hybridization indicated that the TSG-6 messenger RNA was primarily located in the cumulus mass and the antral granulosa cells of large ovarian follicles. In conclusion, the data show that expression of TSG-6 is an integral part of the cascade of inflammatory-like changes that occur in an ovulatory follicle in response to a trophic hormone that couples with luteinizing hormone/hCG receptors.

Induction of early growth response protein-1 gene expression in the rat ovary in response to an ovulatory dose of human chorionic gonadotropin.

Granulosa cells in a mature ovarian follicle have an abundance of LH/hCG receptors that respond rapidly to an ovulatory surge in gonadotropins. Within minutes, membrane signal transduction sets in motion metabolic changes that lead to follicular rupture. This study provides evidence that the initial ovarian response to such an ovulatory stimulus includes induction of the immediate-early transcription factor gene for early growth response protein-1 (Egr-1). Immature Wistar rats were primed with 10 IU equine CG (eCG), sc, and 48 h later the 12-h ovulatory process was initiated by 10 IU hCG, sc. Ovarian RNA was extracted at 0, 0.5, 1, 2, 4, 8, 12, and 24 h after the primed animals were injected with hCG. The RNA extracts were used for RT-PCR differential display for random detection of gene expression in the stimulated ovarian tissue. Northern analysis of one of the differentially amplified complementary DNAs confirmed that it was part of a gene that was significantly up-regulated within 1 h after the ovaries had been stimulated by hCG. Maximum transcription was at 4 h after hCG, and expression declined to 0 h control levels by 24 h after hCG. Subcloning and sequence analysis revealed that the complementary DNA matched the gene for Egr-1. In situ hybridization indicated that the Egr-1 messenger RNA was in the granulosa layer of mature follicles. Western blotting confirmed the temporal pattern of Egr-1 expression detected by differential display, Northern analysis and in situ hybridization. The Egr-1 protein is approximately 84 kDa. In conclusion, the data show that expression of the zinc finger transcription factor Egr-1 is an early event in the cascade of inflammatory-like changes that occur in an ovulatory follicle in response to a trophic hormone.

Progesterone-regulated genes in the ovulation process: ADAMTS-1 and cathepsin L proteases.

Ovulation is a precisely timed process by which a mature oocyte is released from an ovarian follicle. This process is initiated by the pituitary surge of luteinizing hormone (LH), is temporally associated with transcriptional regulation of numerous genes, and is presumed to involve the synthesis and/or activation of specific proteases that degrade the follicle wall. The progesterone receptor (PR), a nuclear receptor transcription factor, is induced in granulosa cells of preovulatory follicles in response to the LH surge and has been shown to be essential for ovulation, because mice lacking PR fail to ovulate and are infertile. Using these mice as a model in which to elucidate PR-regulated genes in the ovulation process, we show that the matrix metalloproteinases MMP-2 and MMP-9 are not targets of PR during ovulation. In contrast, two other proteases, ADAMTS-1 (A disintegrin and metalloproteinase with thrombospondin-like motifs) and cathepsin L (a lysosomal cysteine protease), are transcriptional targets of PR action. ADAMTS-1 is induced after LH stimulation in granulosa cells of preovulatory follicles and depends on PR. Cathepsin L is induced in granulosa cells of growing follicles by follicle-stimulating hormone, but the highest levels of cathepsin L mRNA occur in preovulatory follicles in response to LH in a PR-dependent manner. The identification of two regulated proteases in the ovary, together with their abnormal expression in anovulatory PR knockout mice, suggests that each plays a critical role in follicular rupture and represents a major advance in our understanding of the proteolytic events that control ovulation.

Ovarian expression of a disintegrin and metalloproteinase with thrombospondin motifs during ovulation in the gonadotropin-primed immature rat.

Mammalian ovulation is a dynamic process that requires degradation of the collagenous connective tissue in the thecal layers of a mature follicle. In this reverse transcription-polymerase chain reaction differential display study, gonadotropin-primed immature rats were used to detect ovarian expression of a relatively new type of disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS-1) that is known to cleave extracellular matrix in acutely inflamed tissues. Immature Wistar rats were primed with 10 IU eCG s. c., and the temporal pattern of expression of the ADAMTS-1 gene was delineated by extracting ovarian RNA at 0, 2, 4, 8, 12, and 24 h after induction of ovulation by injecting the primed animals with 10 IU hCG s.c. The differential display data, Northern analyses, and in situ hybridization micrographs all showed significant up-regulation of ADAMTS-1 gene expression by 8 h after hCG administration. The in situ data indicated that the ADAMTS-1 mRNA was in the granulosa layer of mature follicles. Expression reached a peak at 12 h and remained elevated at 24 h after hCG. ADAMTS-1 gene expression was impaired by the antiprogesterone agent epostane, but this inhibition could be overcome by exogenous progesterone. ADAMTS-1 expression was not affected when ovulation was blocked by treatment of the animals with the anti-eicosanoid agent indomethacin. In conclusion, the temporal pattern of expression of this gene, and its apparent regulation by progesterone, suggests that ADAMTS-1 has a significant role in the inflammatory events of the ovulatory process.

Characterization of ovarian carbonyl reductase gene expression during ovulation in the gonadotropin-primed immature Rat.

In this differential-display polymerase chain reaction-based study, four different primer sets generated cDNA fragments of ovarian carbonyl reductase genes that were uniquely expressed during the ovulatory process in eCG-primed immature rats. The temporal pattern of expression of this aldo-keto reductase gene was delineated by extracting ovarian RNA at 0, 2, 4, 8, 12, and 24 h after induction of ovulation via injection of the primed animals with hCG. The results showed that at least four homologous forms of this gene were transcribed during ovulation. Northern blot analyses indicated a 14-fold increase in ovarian mRNA for carbonyl reductase, with expression reaching a peak at 8 h after hCG treatment and then declining to negligible levels during the next 16 h. In situ hybridization revealed that most of the transcription was in the thecal connective tissue of the ovary and was absent from the granulosa layer of ovarian follicles. Treatment of the animals with ovulation-blocking doses of epostane (an inhibitor of progesterone synthesis) or indomethacin (an inhibitor of prostanoid synthesis) did not reduce the expression of ovarian carbonyl reductase. Nevertheless, the temporal pattern of expression of carbonyl reductase after the induction of ovulation suggests that this enzyme activity is at least indirectly associated with the ovulatory process.

Termination of early pregnancy in the rat by a single dose of human menopausal gonadotropin.

Human menopausal gonadotropin (hMG) is commonly used to induce ovarian follicular development and ovulation in infertile women. This report is a preliminary analysis of the ability of hMG to cause folliculogenesis and ovulation in pregnant laboratory animals. Wistar rats were injected subcutaneously with 0.5 mg of hMG on selected days of pregnancy. In addition, 2 days after receiving hMG, one group of animals was injected with 50 IU of human chorionic gonadotropin (hCG) as a substitute for an ovulation-inducing dose of luteinizing hormone. A single dose of hMG caused follicular development and ovulation in the pregnant animals. Furthermore, the results show that such hormone treatment usually terminated the gravid state of the animals. The frequency of pregnancy termination was greater (1) When hMG was administered during the first quarter of the 21-day gestation period; (2) When higher doses of hMG were used; and (3) When the hMG injection was followed 2 days later by hCG. In conclusion, hMG can terminate early pregnancy in a laboratory animal like the rat, and this observation raises the possibility that it may also act as an abortifacient agent in humans. Therefore, a more extensive analysis of the effects of hMG on pregnancy is warranted.

Current status of the hypothesis that mammalian ovulation is comparable to an inflammatory reaction.

This presentation reviews current information on the events that lead to rupture of an ovarian follicle. It contains a summary of the morphological changes that occur at the apex of a follicle wall during ovulation. Existing information shows that the tenacious connective tissue layers of the tunica albuginea and theca externa must be weakened before the follicle wall can dissociate and break open under the force of a modest intrafollicular pressure. These changes are probably dependent on transformation of quiescent thecal fibroblasts into proliferating cells in a manner that is characteristic of tissue responses to inflammatory reactions. The metabolic factors that initiate transformation of the fibroblasts are uncertain, but they are probably generated by gonadotropin-induced changes in the theca interna and granulosa of a follicle as these layers begin to luteinize during the ovulatory process.

Epostane and indomethacin actions on ovarian kallikrein and plasminogen activator activities during ovulation in the gonadotropin-primed immature rat.

Kallikrein and plasminogen activator (PA) are serine proteases that have been implicated in the ovulatory process. Epostane and indomethacin are anti-ovulatory agents that inhibit steroid and eicosanoid synthesis, respectively. This study examines the effects of these two anti-ovulatory agents on ovarian kallikrein and PA activities during ovulation. The proteases were assayed by their actions on chromogenic peptide substrates S-2266 and S-2251, respectively. The ovulatory process was induced in 25-day-old Wistar rats by giving them hCG (10 IU, s.c.) 2 days after the animals had been primed with eCG (10 IU, s.c.). Control animals ovulated approximately 60-70 ova/rat, with the first ova appearing in the oviducts at 10-12 h after hCG administration, and this was the same time ovarian kallikrein and PA activities reached a peak. When doses of epostane ranging from 0.1-5.0 mg/rat or doses of indomethacin ranging from 0.03 to 3.16 mg/rat were administered s.c. at 3 h after hCG, the two drugs inhibited ovulation and ovarian kallikrein and PA activities in a dose-dependent manner. However, the anti-ovulatory action of the two drugs was more closely correlated with suppression of kallikrein activity than with PA activity. Treatment of the animals with exogenous progesterone reversed the inhibitory action of epostane, but not of indomethacin. The results suggest that the increase in ovarian progesterone at the time of ovulation may influence ovarian kallikrein and PA activities.

Inhibition of ovulation in the gonadotropin-primed immature rat by exogenous prostaglandin E2.

In the past two decades there have been innumerable reports that prostaglandins (PGs) are essential for mammalian ovulation. However, we have recently found that a relatively low dose of 0.03 mg indomethacin (INDO) sc to PMSG/hCG-primed immature Wistar rats can significantly reduce ovarian PG levels without inhibiting the control ovulation rate of 60+ ova/rat (1-3). In view of this information, the present study was an effort to duplicate the earlier reports that PGs can reverse the "inhibitory" effect of INDO on ovulation. In control animals, which received PMSG and hCG only, the ovulation rate was 63.8 +/- 4.5 ova/rat. This rate was reduced to 4.1 +/- 1.1 ova/rat when the animals were injected with 1.0 mg INDO at 3 h after hCG. In no instance was this inhibition reversed when the animals were treated with 1.0 mg of PGE2 or PGF2 alpha, or a combination of both prostanoids in either a single dose at 3 h after hCG, or in 4x doses at 2-h intervals beginning at 3 h after hCG. Furthermore, in animals that did not receive INDO, the ovulation rate in PGE2-treated animals was reduced to 20.0 +/- 6.7 ova/rat, and in animals treated with PGE2 and PGF2 alpha (combined) it was reduced to 19.4 +/- 6.5 ova/rat. In summary, not only did the PGs fail to reverse the anti-ovulatory effect of INDO, PGE2 actually suppressed the ovulation rate.

Ovarian hydroxyeicosatetraenoic acids compared with prostanoids and steroids during ovulation in rats.

Hydroxyeicosatetraenoic acid methyl esters (HETEs) are lipoxygenase products of arachidonic acid that are generated along with prostaglandins (PGs) during acute inflammatory reactions. Whereas it is well known that ovarian PG levels increase during the ovulatory process, little is known about ovarian HETEs. This report compares the ovarian changes in 5-, 12-, and 15-HETE with ovarian PGE and PGF, along with progesterone, 17 alpha-hydroxyprogesterone, 4-androstene-3,17-dione, testosterone, and 17 beta-estradiol. Ovulation was induced in immature Wistar rats by sequential treatment with pregnant mare's serum gonadotropin and human chorionic gonadotropin (hCG). Follicles began rupturing 10 h after hCG treatment. The greatest correlation was among 12-HETE, 15-HETE, and progesterone, which increased to peak levels at 10 h after hCG. In contrast, the ovarian levels of 5-HETE, 17 alpha-hydroxyprogesterone, testosterone, and 17 beta-estradiol all declined sharply beginning 4 h after hCG. 2 alpha,4 alpha,7-4,5-Epoxy-17-hydroxy-4,17-dimethyl-3-oxo-androstane-2- carbonitrile (epostane), a potent inhibitor of steroid synthesis and ovulation, sharply reduced the synthesis of all five steroids within 30 min after its injection at 3 h after hCG. Among the five eicosanoids, epostane mainly inhibited 15-HETE. The results suggest that 15-HETE, along with progesterone, may have an important role in ovulation.

Comparison of inhibitory actions of indomethacin and epostane on ovulation in rats.

Indomethacin, an inhibitor of cyclooxygenase that generates prostaglandins (PGs) from arachidonic acid, and 2 alpha,4 alpha,7-4,5-epoxy-17-hydroxy-4,17-dimethyl-3-oxoandrostane- 2-carbonitrile (epostane), an inhibitor of 3 beta-hydroxysteroid dehydrogenase that generates progesterone from pregnenolone, are both potent inhibitors of ovulation. This report compares the dose-dependent effects of these two inhibitors on ovarian levels of 5-, 12-, and 15-hydroxyeicosatetraenoic acid methyl ester (HETEs), prostaglandin E2 (PGE), prostaglandin F2 alpha (PGF), progesterone, 17 alpha-hydroxyprogesterone, 17 beta-estradiol, 4-androstene-3,17-dione, and testosterone during ovulation in 25-day-old immature Wistar rats. The ovulatory process was initiated by 10 IU of human chorionic gonadotropin (hCG). Indomethacin was given at 3 h after hCG in doses ranging from 0.0316 to 10.0 mg/rat. A dose of 0.1 mg/rat was the lowest dose to significantly reduce the ovulation rate from the control level of 70.5 +/- 5.8 ova/rat. This dose also reduced 15-HETE, but not 5-HETE, 12-HETE, or the steroids. PGE and PGF were strongly inhibited by an even lower dose of indomethacin (0.0316 mg/rat), but this dose did not affect the ovulation rate. Epostane was given at 3 h after hCG in doses ranging from 0.1 to 5.0 mg/rat. A dose of 1.0 mg/rat was the lowest dose to significantly inhibit ovulation. This dose also reduced the ovarian levels of 15-HETE and progesterone but not 5-HETE, 12-HETE, PGE, PGF, or the other steroids. The results indicate that the ovulation rate is most closely correlated to ovarian 15-HETE levels.

Effects of epostane on ovarian levels of progesterone, 17 beta-estradiol, prostaglandin E2, and prostaglandin F2 alpha during ovulation in the gonadotropin-primed immature rat.

The antiovulatory action of epostane, an inhibitor of 3 beta-hydroxysteroid dehydrogenase activity and progesterone synthesis, was studied in the immature rat. The ovulatory process was induced in 25-day-old rats by injecting them with hCG (10 IU, sc) 2 days after the animals had been primed with PMSG (10 IU). Epostane was administered at different times between 20 h before and 11 h after hCG. Maximum inhibition of ovulation occurred when the drug was given at 3 h after hCG. Epostane inhibited ovulation in a dose-dependent manner when administered in doses ranging from 1.0-50 mg/rat, while exogenous doses of progesterone restored the ovulation rate. A dose of 3.1 mg epostane/rat 3 h after hCG reduced ovarian progesterone levels within 15 min, but the production of this steroid rebounded within 2 h and approached normal levels by 12 h after hCG, i.e. when the follicles began to rupture in control animals. 17 beta-Estradiol synthesis was inhibited just as rapidly, but it remained suppressed for up to 12 h after hCG administration. The ovarian levels of prostaglandins E2 and F2 alpha decreased approximately 30% within 2 h after the administration of epostane, but such a moderate reduction in the synthesis of ovarian prostanoids is usually not sufficient to block ovulation. The results show that epostane has a rapid, but transient, effect on ovarian progesterone synthesis. The temporary decline in the local progesterone level is apparently sufficient to interfere with the normal sequence of metabolic events that lead to the rupture of follicles.

Characteristics and control of the normal menstrual cycle.

The most important activity during the follicular phase of the cycle is the secretion of gonadotropins, which control folliculogenesis and influence uterine proliferation. The dominant events of the periovulatory phase are the LH surge and ovulation. The significant change during the luteal phase is the production of a nutritive mucus by the endometrial glands in preparation for an embryonic blastocyst. The cardinal passage of the menstrual phase is the menstrual flow itself. These different events (and the metabolic processes that regulate them) have wide-ranging effects on the integrity of the body. As Havelock Ellis, the eminent English psychologist, stated at the turn of this century, "the omnipresent process of sex, as it is woven into the whole texture of our body, is the pattern of all the process of our life" (source unknown). The sweeping influences of the menstrual cycle illustrate the extent to which the process of reproduction is indeed woven into the whole of the human body.

Increase in ovarian kallikrein activity during ovulation in the gonadotrophin-primed immature rat.

The ovulatory process was initiated in 25-day-old rats by injecting them with hCG (10 i.u., s.c.) 2 days after the animals had been primed with PMSG (10 i.u., s.c.). At 2-h intervals after hCG, the ovaries were extracted and assayed for glandular kallikrein activity by using a chromogenic substrate (H-D-Val-Leu-Arg-p-nitroanilide) which exhibits optical density (at 405 nm) upon hydrolysis. In 0-h control ovaries the activity was 12.5 x 10(-3) kallikrein units (KU)/mg protein and it increased to a peak of 56.6 x 10(-3) KU/mg at 12 h after hCG, when the follicles first began to rupture. The kallikrein activity was distinguishable from ovarian plasminogen activator activity on the basis of pH optima and response to trypsin inhibitor (SBTI). The activity was inhibited by a s.c. dose of indomethacin of 0.3 mg/rat, or higher, and this dosage inhibited ovulation. The results suggest that kallikrein activity contributes to the degradation of Graafian follicles during ovulation in mammals.