Inhibition of human chorionic gonadotropin-induced ovulation and steroidogenesis by short-term hyperprolactinemia in female rabbits

To clarify the possible direct effects of hyperprolactinemia on the ovulatory process, we experimentally established hyperprolactinemia in female rabbits with 4 daily injections of sulpiride (SLP) at different doses and induced ovulation with human chorionic gonadotropin (hCG). Plasma levels of prolactin (PRL) were increased significantly before hCG injection in each SLP-treated group compared with the corresponding values for the controls. The ovulation rates at 14 h after hCG were significantly reduced in the 16 and 24 mg/kg/day SLP-treated groups. An inverse correlation (r = -0.74, P less than 0.001) was found between the ovulation rate and the increasing in plasma PRL measured just prior to hCG injection. The increase in peripheral as well as ovarian venous progesterone and 20 alpha-hydroxypregn-4-en-3-one(20 alpha-OHP) at 4 and 14 h after hCG injection in inhibited ovulation groups was much less than in the control group. However, the estradiol, androstenedione and testosterone concentrations were comparable with the control values. These results indicate that hypersecretion of PRL induced by SLP has a direct effect on ovary by inhibiting follicular rupture induced by hCG and this inhibitory effect was partly due to the suppression of progesterone secretion during the course of ovulation. This may be one of the causes leading to hypogonadism during hyperprolactinemia.     

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.     

Inhibition of ovulation in rabbits by intrafollicular injection of indomethacin and prostaglandin F antiserum

Indomethacin, an inhibitor of prostaglandin biosynthesis, was 100% effective in blocking luteinizing hormone (LH)-induced ovulation when administered via microinjection directly into 22 follicles in twelve rabbits, 5 hours after intravenous injection of the gonadotropin. Ovulation was similarly blocked in 24 of 25 follicles injected with antiserum prepared against prostaglandin F_2α. Antiserum against prostaglandin E₂, at the same dosage (100 μg lyophilized serum per follicle), was considerably less effective, preventing ovulation in only 6 of 14 follicles. Control follicles injected with the phosphate buffer vehicle, or with normal rabbit serum, underwent normal ovulation and luteinization. LH injection caused a striking increase in concentration of F-type prostaglandins in follicles shortly before ovulation, an increase which was prevented by i.v. or intrafollicular injection of ovulation-blocking blocking dosages of indomethacin. These findings provide evidence in support of a role for prostaglandins, acting at the follicular level, in the process of ovulation.     

Cell-associated collagenolytic activity by Candida albicans

Cell associated collagenolytic activity of Candida albicans was quantified by measuring the degradation of synthetic peptide 2-furanacryloyl-Leu-Gly-Pro-Ala (FALGPA), which is a specific substrate for collagenase, by the freeze-thaw procedure method. This collagenolytic activity was enhanced by cells cultured in the presence of bovine serum albumin (BSA) in culture medium. However, this activity was inhibited in the presence of ethylenediaminetetraacetic acid disodium salt (EDTA-2Na), but not by the serine proteinase inhibitor p-amidinophenyl methanesulfonyl fluoride (APMSF), nor the aspartyl proteinase inhibitor pepstatin A. These results suggested the presence of a metalloenzyme on pericellular C. albicans. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cycloheximide inhibition of ovulation, prostaglandin biosynthesis and steroidogenesis in rabbit ovarian follicles

Cycloheximide (5 mg/kg, i.v.) significantly inhibited ovulation in the rabbit when it was administered as early as 20 h before the ovulation process was initiated by hCG, and as late as 1 h after hCG. The ovulation rate was significantly reduced, but follicular biosynthesis of prostaglandins E and F was only partly inhibited. The biosynthesis of progesterone and oestradiol in follicles during the early stages of the ovulation process was also inhibited. Cycloheximide may therefore inhibit ovulation by a mechanism which is different from the action of indomethacin, and this mechanism may involve the suppression of ovarian steroidogenesis.     

Inhibition of rat testicular microsomal steroidogenesis by oxytocin and metyrapone

Capillary gas chromatographic 'steroid profiling' has been utilised to separate and quantify the metabolites (derivatized as methyloximes and/or trimethylsilyl ethers) formed from pregnenolone after incubation with rat testicular microsomes. A wide range of steroid metabolites was found, indicating that both the 5-ene and 4-ene pathways of testosterone biosynthesis were operating, as well as 16 alpha-hydroxylation, 20 beta-reduction and the formation of several C19 steroids (the 16-androstenes). At the concentration used, Metyrapone markedly inhibited 16 alpha- and 17-hydroxylation and side-chain cleavage of 17-hydroxylated C21 steroids. 16-Androstene production was also markedly inhibited and the formation of other metabolites was affected to lesser extents. Oxytocin abolished the formation of all C21 and C19 metabolites of pregnenolone.     

Entamoeba histolytica: collagenolytic activity and virulence

Several axenic strains of pathogenic and nonpathogenic Entamoeba histolytica were tested for their capacity to digest native radioactive type I collagen gels and to produce liver abscesses when injected into the liver of newborn hamsters. The results demonstrate that the pathogenic strains of amebas (HM1:IMSS, HM3:IMSS, HM38:IMSS and HK9) have a collagenolytic activity that closely correlates with their in vivo capacity to produce liver lesions. The nonpathogenic isolate (Laredo) did not show collagenolytic activity and failed to produce lesions in the liver of newborn hamsters. The results also demonstrate that type I collagen obtained from rodents and cats is degraded less by amebic collagenase than is bovine collagen, which is similar to human collagen. These findings suggest that species susceptibility to invasive infection may depend, among other factors, on the characteristics of the extracellular components of host tissues.     

Role of green tea polyphenols in the inhibition of collagenolytic activity by collagenase

Inhibitory effect of green tea polyphenols viz., catechin and epigallocatechin gallate (EGCG) on the action of collagenase against collagen has been probed in this study. Catechin and EGCG treated collagen exhibited 56 and 95% resistance, respectively, against collagenolytic hydrolysis by collagenase. Whereas direct interaction of catechin and EGCG with collagenase exhibited 70 and 88% inhibition, respectively, to collagenolytic activity of collagenase against collagen and the inhibition was found to be concentration dependent. The kinetics of inhibition of collagenase by catechin and EGCG has been deduced from the extent of hydrolysis of (2-furanacryloyl-L-leucyl-glycyl-L-prolyl-L-alanine), FALGPA. Both catechin and EGCG exhibited competitive mode of inhibition against collagenase. The change in the secondary structure of collagenase on treatment with catechin and EGCG has been monitored using circular dichroism spectropolarimeter. CD spectral studies showed significant changes in the secondary structure of collagenase on treatment with higher concentration of catechin and EGCG. Higher inhibition of EGCG compared to catechin has been attributed to the ability of EGCG to exhibit better hydrogen bonding and hydrophobic interaction with collagenase.     

Triple-helical peptide analysis of collagenolytic protease activity

Matrix metalloproteinase (MMP) family members are involved in the physiological remodeling of tissues and embryonic development as well as pathological destruction of extracellular matrix components. To study the mechanisms of MMP action on collagenous substrates, non-fluorogenic and fluorogenic triple-helical peptide models of MMP-1 cleavage sites in interstitial collagens have been constructed. Triple-helical peptides were assembled by either (a) covalent branching or (b) self-association driven by hydrophobic interactions. Fluorogenic triple-helical peptide (fTHP) substrates contained the fluorophore/quencher pair of (7-methoxycoumarin-4-yl)acetyl (Mca) and N-2,4-dinitrophenyl (Dnp) in the P5 and P5' positions, respectively. Investigation of MMP family hydrolysis of THPs showed kcat/Km values in the order of MMP-13 > MMP-1 approximately MMP-1(delta243-450) approximately MMP-2 > MMP-3. Studies on the effect of temperature on fTHP and an analogous fluorogenic single-stranded peptide (fSSP) hydrolysis by MMP-1 showed that the activation energies between these two substrates differed by 3.4-fold, similar to the difference in activation energies for MMP-1 hydrolysis of type I collagen and gelatin. The general proteases trypsin and thermolysin were also studied for triple-helical peptidase activity. Both of these enzymes exhibited similar activation energies to MMP-1 for hydrolysis of fTHP versus fSSP. These results suggest that 'triple-helical peptidase' activity can be distinguished from 'collagenolytic' activity, and that mechanistically distinct enzymes convergently evolved to develop collagenolytic activity.     

Dissociation of copulation from ovulation in pregnant rabbits

Experiments were designed to determine why copulation in the pregnant rabbit does not terminate pregnancy while treatment with ovulatory doses of luteinizing hormone (LH) human chorionic gonadotropin (hCG) or luteinizing hormone-releasing hormone (LHRH) is known to do so. Pregnant rabbits (Day 8) were mated or were injected with hCG (25 IU/doe) or LHRH (1, 10 micrograms/kg). Serial blood samples were collected over the next 72 h and analyzed for content of LH, follicle-stimulating hormone (FSH) and progesterone. At sacrifice, uteri and ovaries from these animals were examined for viability of the embryos and for signs of recent ovulation. Injection of hCG or LHRH into pregnant animals led to ovulation and to patterns of LH, FSH and progesterone secretion like those which precede ovulation in estrous rabbits. However, mating the pregnant does did not lead to ovulation or to any changes in the circulating hormones. To investigate whether the elevated levels of progesterone during pregnancy were responsible for the dissociation of coitus from ovulation, nonpregnant rabbits were injected with progesterone (2 mg/kg) and then mated or injected with hCG or LHRH. In virtually every respect, the numbers of ovulations and the patterns of hormone secretion in the progesterone-treated, nonpregnant rabbits mimicked those observed in the 8-day pregnant animals; injection of hCG or LHRH caused ovulation and hormonal surges while hCG caused ovulation only. Mating did not lead to ovulation or any change in blood levels of LH, FSH or progesterone. Taken together, the results show that the elevated circulating levels of progesterone, characteristic of pregnancy, are probably responsible for the dissociation of copulation from gonadotropin release in pregnant rabbits.