Follicular fluid rheology and the duration of the ovulatory process

The fluid dynamics of ovulation were investigated to understand the mechanical role of follicular fluid in oocyte release. A set of equations describing the flow of fluid from an evacuating follicle was derived from basic principles. These equations demonstrate that, subject to assumptions about the available pressure differential and the source of the expulsive force, the size and shape of the ovulatory orifice have the largest influences on the rate of fluid loss, although the viscosity of the fluid is also an important variable. A thorough rheological examination of pig, bovine and human follicular fluids, performed using a cone-plate viscometer, demonstrated that these fluids have complex, non-Newtonian characteristics. The fluids also undergo time-dependent and spontaneous changes in viscosity at constant shear rates; some fluids were subject to coagulation-like events. Viscosity characteristics were unrelated to broad parameters of follicle development. The models used representative viscosity values to demonstrate that variations in the rate and duration of follicle evacuation, as observed by ultrasonography, could be explained largely by variations in fluid viscosity and the characteristics of the ovulatory orifice.     

Progesterone is a mediator in the ovulatory process of the in vitro-perfused rat ovary

The role of steroids in the ovulatory process of the rat was explored in an in vitro perfusion system. Immature rat ovaries were primed with pregnant mare's serum gonadotropin (20 IU) and perfused in a recirculating perfusion system for up to 20 h. Unstimulated ovaries did not ovulate whereas the addition of luteinizing hormone (LH; 0.1 micrograms/ml) plus 3-isobutyl-1-methylxanthine (IBMX; 0.2 mM) resulted in 13.6 +/- 1.0 ovulations per treated ovary. Addition of an inhibitor of 3 beta-hydroxysteroid dehydrogenase (Compound A; 10 micrograms/ml) significantly (p less than 0.01) decreased the number of ovulations after LH plus IBMX stimulation (1.6 +/- 0.8 ovulations per treated ovary). This inhibition was reversed by the addition of progesterone, with 6.6 +/- 2.1 ovulations at approximately 100 ng/ml progesterone in the perfusion medium and 15.2 +/- 3.4 ovulations at approximately 3000 ng/ml progesterone. The addition of testosterone (10 micrograms/ml) did not reverse the inhibition of ovulations by Compound A. High levels of progesterone in the perfusion medium (greater than 3000 ng/ml) did not significantly (p greater than 0.05) increase the number of ovulations after stimulation with LH plus IBMX (20.2 +/- 4.8 ovulations), and progesterone (greater than 3000 ng/ml) was not by itself able to induce ovulations. Addition of LH plus IBMX resulted in a marked increase in the levels of progesterone, testosterone, and estradiol in the perfusion medium. The production of these steroids was almost completely inhibited by the addition of Compound A, and the levels of testosterone and estradiol were restored by the addition of high concentrations of progesterone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulatory effects of bradykinin on the ovulatory process in the in vitro-perfused rat ovary

The role of bradykinin in the ovulatory process was investigated using an in vitro-perfused rat ovary model. Stimulation with LH (0.1 micrograms/ml) resulted in 2.6 +/- 0.5 (mean +/- SEM) ovulations per ovary, whereas no ovulations occurred in the nonstimulated control group. Bradykinin (5 microM) added to the perfusion system hourly for 10 h induced 2 of 5 ovaries to ovulate, with 2 and 3 ovulations, respectively. When bradykinin (5 microM) was given as a single dose at 5 or 10 h after LH, the ovulation rate was significantly increased to 11.0 +/- 2.8 and 8.6 +/- 2.0 ovulations per ovary, respectively. A competitive bradykinin antagonist, phenylalanine bradykinin, inhibited the bradykinin-induced increase in LH-stimulated ovulations. The addition of LH, but not of bradykinin, increased the levels of prostaglandin endoperoxide synthase in granulosa cells, but the levels of the enzyme in the residual ovarian tissue were negligible. In contrast, prostacyclin synthase was predominantly located in the residual ovarian tissue. This enzyme was not affected by LH or bradykinin. LH increased the tissue levels of prostaglandins, predominantly prostaglandin E2 (PGE2), at 7 h, whereas the stimulatory effect of bradykinin was smaller, with a preferential increase in prostacyclin (prostaglandin I2) levels. This study indicates a modulatory role of bradykinin, possibly involving prostacyclin late in the ovulatory process, in the rat.     

Involvement of cytosolic phospholipase A2 in the ovulatory process in gonadotropin-primed immature rats

The preovulatory LH surge induces a remarkable increase in ovarian prostaglandins (PGs) which help to mediate the ovulatory process. We investigated whether cytosolic phospholipase A2 (cPLA2) has a role in this PG production in PMSG/hCG-primed immature rats. The immunoreactive signal for cPLA2 was localized in both thecal and granulosa layers of mature follicles and became evident in response to gonadotropins. The PLA2 activity in the whole ovarian cytosol rose slightly after PMSG stimulation, persisted relatively constant until 24 h after hCG injection and thereafter increased gradually. Intra-ovarian bursal injection of arachidonyl trifluoromethyl ketone, a specific inhibitor for cPLA2 ( 1.0-3.0 mg/ovary), significantly reduced ovarian PGE2 content and the ovulation rate. These results suggest that cPLA2 exists in periovulatory follicles and functions in PG production related to the ovulation process.     

Testosterone immunization blocks the ovulatory process in laying hens without affecting ovarian follicular development

The role of testosterone in the ovulatory process in hens has been largely neglected. The aim of the present study was to evaluate if testosterone plays an important role on the ovulatory process in laying hens. The effect of active and passive immunization against testosterone on ovarian follicular development and oviposition was studied. Egg laying percentage was evaluated in hens actively immunized against testosterone-BSA (T-AI; n = 6) or BSA (BSA-AI; n = 6). Oviposition was reduced as antibody titer increased in T-AI hens (r = -0.67; P < 0.01). Ovarian structures were assessed in three animals from each group. Follicles reached preovulatory size in both groups, nonetheless, in T-AI hens follicles at different stages of regression indicated that ovulation was blocked by treatment. In the remaining animals, preovulatory concentrations of progesterone and testosterone were determined. A preovulatory surge release of progesterone, preceded by a testosterone peak, was observed in the BSA-AI group (P < 0.05). In contrast, progesterone in T-AI animals remained at basal concentrations. Whereas, testosterone concentrations were significantly greater in T-AI as compared with BSA-AI animals (P < 0.05). Finally, to study the effect of passive immunization on oviposition, hens were passively immunized (PI) on four occasions, on alternate days with anti-T serum (T-PI; n = 10) or anti-BSA serum (BSA-PI; n = 8). During the 13-day period that preceded treatment, oviposition averaged 94.1%. Forty-eight hours after the first immunization, no egg was laid by 8 out of the 10 T-PI hens. During the 10 days following the first passive immunization, there was a reduction in the laying percentage that was significantly greater in T-PI hens (reduction of 52% in T-PI versus 29% in P-BSA, P < 0.01). In summary, these studies show that testosterone immunization hampers egg-laying without affecting ovarian follicular development, suggesting that testosterone has an important role in the ovulatory process in laying hens.     

Dynamic aspects of ovarian superoxide dismutase isozymes during the ovulatory process in the rat.

To investigate the role of superoxide dismutase (SOD) in the ovulatory process, SOD isozymes and their mRNAs were determined in the ovary of 22-day-old rats. After treatment with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG), ovarian activity of Mn-SOD decreased markedly while Cu/Zn-SOD remained unchanged. However, the ovarian level of mRNA for Mn-SOD markedly increased after hCG-treatment while that for Cu/Zn-SOD decreased only slightly. Ovulation was inhibited by intravenous injection of a long-acting SOD. These results suggested that superoxide radicals in the ovary might play a critical role in the mechanism for hCG-induced ovulation.     

Correlation between steroids and plasmin in rat ovaries during ovulatory process.

Progesterone (0.5 mg/rat) and estradiol-17 beta (10 micrograms/rat) injected(im) to adult female albino rats on the morning of proesterous significantly enhanced the ovarian plasmin activity as measured by the fibrin plate method at the time of ovulation which was confirmed to be at 2.30 a.m. by estimation of ovarian plasmin activity at definite intervals before and after ovulation. The ovarian plasmin activity showed a gradual increase towards ovulation and reached a maximum level at 2.30 a.m. and again decreased after ovulation. However, the nonsteroidal estrogen antagonist, tamoxifen induced inhibitory effects on the ovarian plasmin activity as compared to control and estradiol-17 beta treatment. Thus these studies reveal a positive relationship between steroids and the ovarian plasmin activity during ovulation.     

Increased production of ovarian thromboxane in gonadotropin-treated immature rats: relationship to the ovulatory process

Thromboxane (TX) B2, a stable metabolic product of hydrolysis of TXA2, was measured by radioimmunoassay in tissue extracts of ovaries of immature rats pretreated with pregnant mare's serum gonadotropin and human chorionic gonadotropin. Ovarian concentrations of TXB2 increased before, and remained elevated after, the time of ovulation. In a subsequent study, ovulation was inhibited in a dose-dependent fashion by a reported TXA2 receptor antagonist, AH23848. Nevertheless, inhibition of the preovulatory rise in synthesis of TXB2 by furegrelate (a thromboxane synthetase inhibitor) did not prevent ovulation. Nor was the blockade of ovulation caused by indomethacin (a cyclooxygenase inhibitor) reversed by a TXA2 mimetic (U-46619). It does not appear that a preovulatory increase in ovarian thromboxane is an obligatory component of the ovulatory mechanism of gonadotropin-primed immature rats.     

Increased production of ovarian thromboxane in gonadotropin-treated immature rats: Relationship to the ovulatory process

Thromboxane (TX) B₂, a stable metabolic product of hydrolysis of TXA₂, was measured by radioimmunoassay in tissue extracts of ovaries of immature rats pretreated with pregnant mare's serum gonadotropin and human chorionic gonadotropin. Ovarian concentrations of TXB₂ increased before, and remained elevated after, the time of ovulation. In a subsequent study, ovulation was inhibited in a dose-dependent fashion by a reported TXA₂ receptor antagonist, AH23848. Nevertheless, inhibition of the preovulatory rise in synthesis of TXB₂ by furegrelate (a thromboxane synthetase inhibitor) did not prevent ovulation. Nor was the blockade of ovulation caused by indomethacin (a cyclooxygenase inhibitor) reversed by a TXA₂ mimetic (U-46619). It does not appear that a preovulatory increase in ovarian thromboxane is an obligatory component of the ovulatory mechanism of gonadotropin-primed immature rats.     

Effect of histamine and histamine blockers on the ovulatory process in the vitro perfused rabbit ovary

An increase in the content of histamine in the ovary following luteinizing hormone (LH) release and the inhibition of ovulation in the rabbit by antihistamines suggest that histamine may be involved in the ovulatory process. The effects of various doses of histamine and antihistamines on ovulation were investigated using the in vitro perfused rabbit ovary system. Histamine (100 ng/ml) added to the perfusate at hourly intervals induced ovulation, although at a rate below that observed following human chorionic gonadotropin (hCG) administration. Cimetidine (10 micrograms/ml), an H2 blocker, inhibited histamine-induced ovulation, while the H1 blocker, chlorpheniramine (66.7 micrograms/ml), failed to do so. Neither cimetidine nor chlorpheniramine was able to block ovulation following hCG (50 IU). In all experimental groups in which histamine was used to induce ovulation, both extruded ova and follicular oocytes remained in an immature stage and displayed little evidence of degeneration. In contrast, a high percentage of ova exposed to hCG were mature. Ovarian edema was increased in ovaries in which ovulation occurred, regardless of treatment. A linear correlation was noted between ovulatory efficiency and degree of ovarian edema. Histamine may be an intermediary in the mechanism of follicular rupture, but does not support ovum maturation. However, the inability of H1 and H2 antagonists to block hCG-induced ovulation raises questions regarding the role of histamine in the physiologic process of ovulation.     

Production of proteinases in the process of developing cultures of Streptomyces thermovulgaris T-54

The secretion of some proteolytic enzymes by Streptomyces thermovulgaris T 54 was studied using artificial chromogenic substrates of proteinases. Maximum accumulation of the enzymes hydrolysing Z-Glu-pNA and Z-Ala-Ala-Leu-pNA occurred during autolysis of the culture. Two peaks of activity were observed, when DNP-Gly-Gly-Ile-Arg was used as a substrate, one of them being correspondent to prevalence of dense colonies in the culture and the other to the prevalence of friable networks of hyphae.     

Differential effects of RU486 and indomethacin on follicle rupture during the ovulatory process in the rat

Ovulation (i.e., the release of mature oocytes from the ovary) requires spatially targeted follicle rupture at the apex. Both progesterone and prostaglandins play key roles in the ovulatory process. We have studied follicle rupture and ovulation in adult cycling rats treated with a progesterone receptor antagonist (RU486), an inhibitor of prostaglandin synthesis (indomethacin, IM), or both. All rats were treated with LHRH antagonist on the morning (0900 h) of proestrus to inhibit endogenous gonadotropins and with 10 microg of ovine LH (oLH) at 1700 h in proestrus to induce ovulation. Animals were treated from metestrus to proestrus with 2 mg/day of RU486 or vehicle (olive oil) and on the morning of proestrus (1200 h) with 1 mg of IM or vehicle (olive oil). Some rats treated with vehicle or RU486 were killed on the morning of proestrus to assess preovulatory follicle development. The remaining rats were killed on the morning of estrus to study follicle rupture and ovulation. In vehicle-treated rats, oLH induced ovulation in 98% of follicles. In IM-treated rats, spatial targeting of follicle rupture was disrupted. Most oocytes were released to the ovarian interstitium (50%) or to the periovarian space (39%), and a smaller percentage (11%) of oocytes remained trapped inside the luteinized follicle. RU486-treated rats showed, on the morning of estrus, unruptured luteinized follicles. Only occasionally (2.8%), the oocytes were released to the periovarian space. IM treatment induced follicle rupture in RU486-treated rats, and 25% of oocytes were released to the ovarian interstitium. However, the number of oocytes released to the periovarian space (i.e., ovulated) was not increased by IM treatment in rats lacking progesterone actions. Overall, these data indicate that RU486 and IM have opposite effects on follicle rupture and suggest that both progesterone and prostaglandins are necessary for the spatial targeting of follicle rupture at the apex.     

Effects of inhibitors of protein synthesis on the ovulatory process of the perfused rat ovary

The effects of two different protein synthesis inhibitors (cycloheximide and puromycin) on the ovulatory process were examined in vitro using a perfused rat ovary model. Ovaries of PMSG (20 i.u.)-primed rats were perfused for 21 h. Release of cyclic adenosine 3',5'-monophosphate (cAMP) and steroids (progesterone, testosterone, and oestradiol) was measured and the number of ovulations was estimated by counting released oocytes. Unstimulated control ovaries did not ovulate whereas addition of LH (0.1 microgram/ml) plus 3-isobutyl-1-methylxanthine (IBMX; 0.2 mM) resulted in 16.7 +/- 3.5 ovulations per treated ovary. Cycloheximide (5 micrograms/ml) totally inhibited the ovulatory effect of LH + IBMX when present from the beginning of the perfusions and also when added 8 h after LH + IBMX. No inhibition was seen when cycloheximide was added 10 h after LH + IBMX (1-1.5 h before the first ovulation; 15.2 +/- 4.4 ovulations per treated ovary). Puromycin (200 micrograms/ml) completely blocked ovulation when present from the beginning of the perfusions and the inhibition was congruent to 60% (6.5 +/- 2.2 ovulations per treated ovary) when the compound was added 8 h after LH + IBMX. Both inhibitors increased LH + IBMX-stimulated cAMP release substantially, but decreased the release of progesterone, testosterone and oestradiol. These results indicate that de-novo protein synthesis is important late in the ovulatory process for follicular rupture to occur.     

Plasmin cleaves tumor necrosis factor alpha exodomain from sheep follicular endothelium: implication in the ovulatory process

Ovulation in the sheep is predicated on plasmin up-regulation at the ovarian surface-follicular interface, release of tumor necrosis factor (TNF) alpha from contiguous endothelium, and apoptotic cell death. The objectives of this investigation were to determine whether plasmin elicits TNFalpha secretion from thecal endothelium of ovine follicles, to characterize the site(s) of enzymatic attack, and to assess the physiological consequence of soluble TNFalpha action. Endothelial cells of thecal tissues isolated from antral follicles of eCG-primed anestrous ewes shed (histochemical depletion) TNFalpha into incubation medium (ovarian cell DNA fragmentation bioassay, Western blot detection) upon exposure to plasmin. Immunopurification and N-terminal sequence analysis indicated that TNFalpha was excised from its transmembrane precursor at the Arg79-Ser80 and Lys88-Pro89 linkages. Microinjection of TNFalpha into the apical wall of explanted follicles induced cellular apoptosis and stigma development. We suggest that plasmin-mediated cleavage of TNFalpha exodomain from its membrane anchor along thecal endothelium is a determinant of tissue dissolution within the formative ovulatory rupture site of ewes.     

Isolation and characterization of extra- and intra-cellular metal proteinases produced in the spawn-running process ofHypsizygus marmoreus

Isolation and characterization of extra-(PE-1) and intra-cellular (PE-2) metal proteinases produced during the spawn-running process ofHypsizygus marmoreus were carried out. These enzymes were the most active toward Hammarsten casein at pH 7.0 (PE-1) and pH 6.5–7.5 (PE-2). The molecular weight and pl value of PE-1 were 29,500, 8.8 and those of PE-2 were 21,500, 8.4. Km values against the synthetic peptide substrate Z-Gly-l-Leu-NH₂ were 0.9×10⁻³M (PE-1) and 1.2×10⁻³M (PE-2). PE-1 was strongly inhibited by phosphoramidon, whereas PE-2 was weakly inhibited. These enzymes are considered to play an important role in providing nitrogenous substrates during fruit-body formation.     

Bone morphogenetic protein-1/Tolloid-like proteinases process dentin matrix protein-1

Bone morphogenetic protein-1 (BMP-1)/Tolloid-like metalloproteinases play key roles in formation of mammalian extracellular matrix (ECM), through the biosynthetic conversion of precursor proteins into their mature functional forms. These proteinases probably play a further role in formation of bone through activation of transforming growth factor beta-like BMPs. Dentin matrix protein-1 (DMP1), deposited into the ECM during assembly and involved in initiating mineralization of bones and teeth, is thought to undergo proteolysis in vivo to generate functional cleavage fragments found in extracts of mineralized tissues. Here, we have generated recombinant DMP1 and demonstrate that it is cleaved, to varying extents, by all four mammalian BMP-1/Tolloid-like proteinases, to generate fragments similar in size to those previously isolated from bone. Consistent with possible roles for the BMP-1/Tolloid-like proteinases in the physiological processing of DMP1, NH2-terminal sequences of products generated by BMP-1 cleavage of DMP1 match those predicted from processing at the predicted DMP1 site that shows greatest cross-species conservation of sequences. Moreover, fibroblasts derived from mouse embryos homozygous null for genes encoding three of the four mammalian BMP-1/Tolloid-like proteinases appear to be deficient in processing of DMP1. Thus, a further role for BMP-1-Tolloid-like proteinases in formation of mineralized tissues is indicated, via proteolytic processing of DMP1.