Hypocoagulable state of human preovulatory ovarian follicular fluid: role of sulfated proteoglycan and tissue factor pathway inhibitor in the fluid

Ovulation accompanied by tissue damage can cause an increase in the level of tissue factor (TF) in the follicular fluid, triggering the extrinsic coagulation pathway. However, follicular fluid must block fibrin formation and maintain fluidity until the release of the oocyte at ovulation. The combination of sulfated proteoglycan, antithrombin, and TF pathway inhibitor (TFPI) appears to play a critical role in the hypocoagulability of human follicular fluid. When compared with plasma, folicular fluid differs markedly in the levels of a number of important coagulation proteins. Principal among these are 15-fold, 13-fold, and 3.7-fold increases in free TFPI, thrombin-antithrombin complex, and TF, respectively. The excessively prolonged activated partial thromboplastin time (APTT) and prothrombin time (PT) of human ovarian follicular fluid appear to be primarily due to high concentrations of sulfated proteoglycans, which accelerate the inactivation of thrombin and the anti-Xa activity of TFPI. Thus, heparitinase treatment shortened the clotting times of follicular fluid and reduced the inhibition of thrombin by the proteoglycan fraction combined with a fraction containing antithrombin. The remaining prolongation of APTT and PT may be caused by high levels of free TFPI in follicular fluid, which were confirmed by Northern blotting analysis, demonstrating TFPI mRNA expression by granulosa cells.     

Thrombin generation and presence of thrombin receptor in ovarian follicles

Prothrombin, once converted to its enzymatically active form (i.e., thrombin), induces a broad spectrum of cellular responses in both vascular and avascular tissues. Bovine ovarian granulosa cells isolated from healthy follicles of various sizes contain both prothrombin mRNA and immunologically reactive prothrombin that appears to be identical to prothrombin in follicular fluid and plasma. When tissue factor, the primary physiological activator of thrombin generation in plasma, is used to initiate thrombin formation, the profile of prothrombin-to-thrombin conversion is similar in follicular fluid and plasma. The conclusion that biologically functional prothrombin is synthesized by granulosa cells is further supported by evidence that mRNA for gamma-glutamyl carboxylase, an enzyme essential for the vitamin K-dependent posttranslational modification of prothrombin, is expressed in granulosa cells in a manner similar to prothrombin mRNA. Thrombin's biological effects are mediated through selective proteolytic cleavage and activation of specific receptors. Bovine granulosa cells possess thrombin receptor (PAR-1) mRNA, and as seen with prothrombin mRNA and gamma-glutamyl carboxylase mRNA, cells isolated from small follicles possess more PAR-1 mRNA than cells from large follicles. Thrombin receptor expression by cells in close proximity to an active thrombin-generating system suggests that these factors may be important mediators of cellular function in the ovarian follicle.     

Regulation of prostaglandin biosynthesis by human ovarian follicular fluid: a mechanism for ovulation?

We have studied the effect of human ovarian follicular fluid on PG production by bovine seminal vesicles in vitro and found that hFF1 contains a factor of high molecular weight (Mr greater than 30,000) which inhibits PG synthase in a dose-dependent manner. Exposure of this substance to protease activity produced a factor of lower molecular weight (Mr less than 1000) which stimulated PG synthase activity. If this is true of ovarian follicles in vivo, it is possible that increased follicular protease activity stimulates PG synthesis at the time of ovulation.     

Regulation of prostaglandin biosynthesis by human ovarian follicular fluid: A mechanism for ovulation?

We have studied the effect of human ovarian follicular fluid on PG production by bovine seminal vesicles and found that hFF contains a factor of high molecular weight (Mr > 30,000) which inhibits PG synthase in a dose-dependent manner. Exposure of this substance to protease activity produced a factor of lower molecular weight (Mr < 1000) which stimulated PG synthase activity. If this is true of ovarian follicles , it is possible that increased follicular protease activity stimulates PG synthesis at the time of ovulation.     

Lipoprotein-incorporated pregnenolone fatty acid esters act as substrate for ovarian progestin synthesis

Progesterone is of great importance in the normal development of the ovarian follicle and its biosynthesis has been shown to rely on plasma low-density lipoproteins for delivery of cholesterol substrate. However, the pre-ovulatory ovarian follicle has no access to plasma low-density lipoproteins suggesting that other substrates may be involved in pre-ovulatory progestin production. We show in this study using tritiated pregnenolone fatty acid esters incorporated into lipoproteins can give rise to a series of labelled progestins following lipoprotein-mediated internalization. In doing so, progestins are synthesized by shunting the accepted rate limiting step of steroidogenesis, the intracellular P450 side chain cleavage pathway. Furthermore, we demonstrate that follicular fluid high density lipoproteins contain vast endogenous concentrations of this lipoidal pregnenolone metabolite which may indeed contribute to progesterone production in the pre-ovulatory ovarian follicle.     

Xenotransplantation in immunodeficient mice to study ovarian follicular development in domestic animals

Nowadays, in vitro study of follicular dynamics of primordial and primary follicular stages is limited because in vitro culture systems for these follicles are lacking, both in domestic animal species and in human. Therefore, additional insights might be generated by grafting ovarian tissue into immunodeficient mice to study activation and maturation of early follicular stages. A considerable amount of data has already been gathered in laboratory animals and through clinical application of human assisted reproduction technologies where live births were reported recently after the use of (cryopreserved) ovarian grafts. However, given that human preantral follicles are difficult to obtain and that there are many similarities between the bovine and human species with regard to ovarian physiology, the bovine model offers exciting additional prospects and is therefore discussed in more detail. This review will focus on recent developments related to preantral follicle and (repeated) ovarian tissue retrieval and xenotransplantation of (bovine) ovarian tissue strips to immunodeficient mice as a model to study preantral follicular dynamics. Different grafting strategies will be discussed as well as the consequences of this procedure on the viability and dynamic behavior of the grafted tissue and follicles.     

A model for the stoichiometric regulation of blood coagulation

We have developed a model of the extrinsic blood coagulation system that includes the stoichiometric anticoagulants. The model accounts for the formation, expression, and propagation of the vitamin K-dependent procoagulant complexes and extends our previous model by including: (a) the tissue factor pathway inhibitor (TFPI)-mediated inactivation of tissue factor (TF).VIIa and its product complexes; (b) the antithrombin-III (AT-III)-mediated inactivation of IIa, mIIa, factor VIIa, factor IXa, and factor Xa; (c) the initial activation of factor V and factor VIII by thrombin generated by factor Xa-membrane; (d) factor VIIIa dissociation/activity loss; (e) the binding competition and kinetic activation steps that exist between TF and factors VII and VIIa; and (f) the activation of factor VII by IIa, factor Xa, and factor IXa. These additions to our earlier model generate a model consisting of 34 differential equations with 42 rate constants that together describe the 27 independent equilibrium expressions, which describe the fates of 34 species. Simulations are initiated by "exposing" picomolar concentrations of TF to an electronic milieu consisting of factors II, IX, X, VII, VIIa, V, and VIIII, and the anticoagulants TFPI and AT-III at concentrations found in normal plasma or associated with coagulation pathology. The reaction followed in terms of thrombin generation, proceeds through phases that can be operationally defined as initiation, propagation, and termination. The generation of thrombin displays a nonlinear dependence upon TF, AT-III, and TFPI and the combination of these latter inhibitors displays kinetic thresholds. At subthreshold TF, thrombin production/expression is suppressed by the combination of TFPI and AT-III; for concentrations above the TF threshold, the bolus of thrombin produced is quantitatively equivalent. A comparison of the model with empirical laboratory data illustrates that most experimentally observable parameters are captured, and the pathology that results in enhanced or deficient thrombin generation is accurately described.     

Lipid transport in the developing bovine follicle: messenger RNA expression increases for selective uptake receptors and decreases for endocytosis receptors

Differences in rates of steroid production and secretion will, eventually, determine the developmental rates of ovarian follicles. The major supply of cholesterol, the precursor for steroid and androgen biosynthesis, to ovarian cells is from circulating lipoproteins via membrane receptors from the low density lipoprotein receptor (LDL) superfamily. This occurs by either endocytosis, which has been described for very low density lipoprotein receptors (VLDLr), for LDL receptors (LDLr), and by the selective uptake pathway described for the scavenger receptor class B type 1 receptor (SRB1) and the recently described ovarian receptor, lipoprotein receptor-related protein 8 (LRP8). In this study, the mRNA expression of these four cholesterol receptors in bovine ovarian cells was determined at different stages of follicular development. In small antral follicles, mRNA expression of the endocytosis receptors was higher than in large antral follicles. Expression of LRP8 mRNA increased linearly with follicular size together with an increase in LDL, VLDL, and cholesterol concentrations in the follicular fluid. SRB1 mRNA expression tended to increase with follicular diameter. Because different mRNA expression patterns were found for the two types of receptor, this may imply different regulation of cholesterol supply at different stages of follicular development. Accumulation of LDL and VLDL particles in the follicular fluid of large antral follicles may enhance cholesterol availability for the intense steroidogenic activity that is essential at these stages.     

Analysis of the generation and inhibition of activated coagulation factor X in pure systems and in human plasma

The overall generation and inhibition of human factor Xa have been studied in pure systems and plasma to determine the kinetic characteristics of inhibition during factor Xa generation. Generation curves were measured amidolytically in a pure system containing factor X and antithrombin, which was activated with the factor X-activating enzyme of Russell's viper venom (RVV-X). The measured change in factor Xa level with time was fitted to a 3-parameter 2-exponential model to determine apparent first-order rates of inhibition. With antithrombin at 4.5 microM, the inhibition rate constant thus obtained was very close to the known rate of inhibition of exogenous enzyme. Factor Xa generation curves were also analyzed in plasma; however, to reduce interference in the assay of thrombin, congenitally prothrombin-deficient plasma was used containing 0.5 microM D-Phe-Pro-Arg-chloromethylketone. In plasma, factor Xa generated in the presence of phospholipid and Ca2+ ions by RVV-X, factor IXa, or tissue factor was inhibited more slowly than exogenous enzyme. The reduction was particularly severe with tissue factor activation, where the rate was 0.04-0.06 min-1. This protection by tissue factor was also observed in pure systems and apparently required factor VII.     

Mouse ovarian granulosa cells produce urokinase-type plasminogen activator, whereas the corresponding rat cells produce tissue-type plasminogen activator

It is well established that rat ovarian granulosa cells produce tissue plasminogen activator (tPA). The synthesis and secretion of the enzyme are induced by gonadotropins, and correlate well with the time of follicular rupture in vivo. We have found that in contrast, mouse granulosa cells produce a different form of plasminogen activator, the urokinase-type (uPA). As with tPA synthesis in the rat, uPA production by mouse granulosa cells is induced by gonadotropins, dibutyryl cAMP, and prostaglandin E2. However, dexamethasone, a drug which has no effect on tPA synthesis in rat cells inhibits uPA synthesis in the mouse. Results of these determinations made in cell culture were corroborated by examining follicular fluid, which is secreted in vivo predominantly by granulosa cells, from stimulated rat and mouse ovarian follicles. Rat follicular fluid contained only tPA, and mouse follicular fluid only uPA, indicating that in vivo, granulosa cells from the two species are secreting different enzymes. The difference in the type of plasminogen activator produced by the rat and mouse granulosa cells was confirmed at the messenger RNA level. After hormone stimulation, only tPA mRNA was present in rat cells, whereas only uPA mRNA was found in mouse cells. Furthermore, the regulation of uPA levels in mouse cells occurs via transient modulation of steady-state levels of mRNA, a pattern similar to that seen with tPA in rat cells.     

Surface-mediated control of blood coagulation: the role of binding site densities and platelet deposition

A mathematical model of the extrinsic or tissue factor (TF) pathway of blood coagulation is formulated and results from a computational study of its behavior are presented. The model takes into account plasma-phase and surface-bound enzymes and zymogens, coagulation inhibitors, and activated and unactivated platelets. It includes both plasma-phase and membrane-phase reactions, and accounts for chemical and cellular transport by flow and diffusion, albeit in a simplified manner by assuming the existence of a thin, well-mixed fluid layer, near the surface, whose thickness depends on flow. There are three main conclusions from these studies. (i) The model system responds in a threshold manner to changes in the availability of particular surface binding sites; an increase in TF binding sites, as would occur with vascular injury, changes the system's production of thrombin dramatically. (ii) The model suggests that platelets adhering to and covering the subendothelium, rather than chemical inhibitors, may play the dominant role in blocking the activity of the TF:VIIa enzyme complex. This, in turn, suggests that a role of the IXa-tenase pathway for activating factor X to Xa is to continue factor Xa production after platelets have covered the TF:VIIa complexes on the subendothelium. (iii) The model gives a kinetic explanation of the reduced thrombin production in hemophilias A and B.     

Cell-secreted vesicles in equine ovarian follicular fluid contain miRNAs and proteins: a possible new form of cell communication within the ovarian follicle

Proper cell communication within the ovarian follicle is critical for the growth and maturation of a healthy oocyte that can be fertilized and develop into an embryo. Cell communication within the follicle involves many signaling molecules and is affected by maternal age. Recent studies indicate that cell communication can be mediated through secretion and uptake of small membrane-enclosed vesicles. The goals of this study were to 1) identify cell-secreted vesicles (microvesicles and exosomes) containing miRNAs and proteins within ovarian follicular fluid and 2) determine if miRNA level differs in exosomes isolated from follicular fluid in young compared to old mares. We demonstrate the presence of vesicles resembling microvesicles and exosomes in ovarian follicular fluid using transmission electron microscopy and CD63-positive and RNA containing vesicles using flow cytometry. Moreover, proteomics analysis reveals that follicular fluid-isolated exosomes contain both known exosomal proteins and proteins not previously reported in isolated exosomes. MicroRNAs were detected in microvesicle and exosomes preparations isolated from follicular fluid by real-time PCR analysis. Uptake of fluorescent-labeled microvesicles by granulosa cells was examined using in vitro and in vivo approaches. MicroRNA expression profiling reveals that miRNAs in microvesicle and exosome preparations isolated from follicular fluid also are present within surrounding granulosa and cumulus cells. These studies revealed that cell communication within the mammalian ovarian follicle may involve transfer of bioactive material by microvesicles and exosomes. Finally, miRNAs present in exosomes from ovarian follicular fluid varied with the age of the mare, and a number of different miRNAs were detected in young vs. old mare follicular fluid.     

Amounts of selected coagulation factors in pre- and post-mortem follicular fluid are similar and do not correlate with molecular mass

This study was designed to evaluate the amounts of coagulation factors and to determine whether the protein profile in pre-ovulatory ovarian follicular fluid aspirated from ovaries collected from mares at slaughter are representative of that in follicular fluid collected from live animals. The proteins evaluated included, (i) albumin, ceruloplasmin and fibronectin, (ii) the procoagulant plasma proteins, Factor V (FV), Factor VII (FVII), Factor X (FX) and prothrombin, and (iii) the anticoagulant plasma proteins, antithrombin and alpha2-macroglobulin. The amounts of the individual proteins were similar in both types of follicular fluid. There was no correlation between the activity of FV, FVII, FX or prothrombin in follicular fluid and their molecular size although a correlation was found for the other proteins. These results suggest that the procoagulant proteins in follicular fluid are not likely derived from plasma. The total protein content of follicular fluid samples collected from both sources was similar and the results determined with the Biuret, Lowry and Biorad methods were also not significantly different (P>0.05).     

Follicular fluid adrenomedullin concentrations in spontaneous and stimulated cycles: relationship to ovarian function and endothelin-1 and nitric oxide

The objective of this study was to determine concentration of adrenomedullin (AM) in follicular fluid and whether a correlation exists between AM and nitric oxide (NO) or endothelin-1 (ET-1) levels in follicular fluid, serum 17beta-estradiol or other parameters of ovarian function in spontaneous and gonadotrophin stimulated ovarian cycles. Follicular fluid samples were obtained at oocyte retrieval from 50 women who underwent an in vitro fertilization (IVF) program: 40 undergoing ovarian hyperstimulation with recombinant FSH and 10 had spontaneous ovarian cycles. AM, ET-1, and NO were detected in all of the follicular fluid samples and their concentrations were similar in spontaneous and stimulated cycles. In patients undergoing ovarian stimulation, follicular fluid AM levels correlated with serum 17beta-estradiol concentration. No correlation was found between follicular AM concentration and parameters of ovarian function. Similarly, no relationship was observed between ET-1, NO, and AM follicular fluid concentrations in either spontaneous or stimulated cycles.This study suggests a possible regulatory effect of the sexual hormones on AM production by the ovary during the ovulatory process. The site of AM secretion and its function (if any), however, remain to be established.     

Physiological and pathophysiological roles of lysophosphatidic acids produced by secretory lysophospholipase D in body fluids

Recently, a family of phospholipid mediators has received much attention because of its variety of biological activities. Lysophosphatidic acid (LPA) is a central member of the phospholipid autacoid family that exerts diverse effects through binding to and activation of several specific receptors coupled to G-proteins. In accordance with its function as a receptor agonist, there are pathways for extracellular generation of LPA in vivo. One pathway involves a novel lysophospholipase D activity that was originally found in rat plasma. LPA is also produced in significant amounts after incubation of various plasma-derived body fluids such as human follicular fluid at 25-37 degrees C. In animal models, LPA was shown to stimulate oocyte maturation, embryonic development and transport in the oviduct. An increase in serum lysophospholipase D activity was observed during pregnancy in human. These results suggest that LPA generated by lysophospholipase D is likely to play an important role in reproductive biology. LPA produced by lysophospholipase D activity in body fluids has also been observed under pathophysiological conditions: serum and ascitic fluid from ovarian cancer patients and serum from hypercholesterolemic rabbits. Hence, excess generation of LPA by lysophospholipase D activity in body fluids has been suggested to be relevant to the pathogenesis of cancer and atherosclerosis.     

Selenium Levels and Glutathione Peroxidase Activity in Blood,Plasma and Reproductive Organs in Dairy Cows

Selenium levels and glutathione peroxidase activity were determined in blood and reproductive organs in 12 Norwegian dairy cows at different stages of the oestrus cycle. Blood samples were collected before slaughter, and samples from genital organs were obtained as soon as possible after slaughter. Blood and plasma selenium levels were significantly correlated with selenium levels in follicular fluid, and in ovarian and uterine tissues, but not with the levels in corpora lutea. The activity of blood glutathione peroxidase was significantly correlated with that in ovarian and uterine tissue, but not with activity in corpora lutea and follicular fluid. No effect of stage of oestrus cycle on selenium content or glutathione peroxidase in reproductive tissue was observed.     

Ovarian epidermal growth factor-like activity. Concentrations in porcine follicular fluid during follicular enlargement

Numerous data indicate that epidermal growth factor has important effects on cultured granulosa cells. However, most of the few attempts to detect epidermal growth factor in ovarian tissue have been unrevealing. In this study, ovarian epidermal growth factor-like activity was easily detected by a radioreceptor assay based on the A431 cell line but not by an immunoassay for mouse epidermal growth factor. The concentration of this activity in follicular fluid from small porcine ovarian follicles was higher than that in fluid from medium or large follicles or serum (p less than 0.01), but lower than that in salivary gland extracts. Receptor-active epidermal growth factor-like peptides could function as local ovarian regulators.     

Activation of human blood coagulation factor XI independent of factor XII. Factor XI is activated by thrombin and factor XIa in the presence of negatively charged surfaces

Human blood coagulation factor XI was activated by either autoactivation or thrombin. These reactions occurred only in the presence of negatively charged materials, such as dextran sulfate (approximately Mr 500,000), sulfatide, and heparin. During the activation, factor XI was cleaved at a single Arg-Ile bond by thrombin or factor XIa to produce an amino-terminal 50-kDa heavy chain and a carboxyl-terminal 35-kDa light chain. This activation pattern is identical to that produced by factor XIIa. The addition of a small amount of thrombin and sulfatide to factor XII-deficient plasma produced shorter clotting times than when these agents were added to factor XI/factor XII combined-deficient plasma. These results suggest that the activation of factor XI by thrombin and possibly the autoactivation of factor XI proceed in plasma to lead fibrin clot formation. These reactions may have a role on an appropriate negatively charged surface in normal hemostasis.     

Regulation of anti-Müllerian hormone production in the cow: a multiscale study at endocrine, ovarian, follicular, and granulosa cell levels

Anti-Müllerian hormone (AMH) is an endocrine marker that can help predict superovulatory responses to treatments administered to cows for embryo production. However, the optimal time of the estrous cycle at which a blood test should be performed for a highly reliable prognosis has not yet been established. Moreover, little is known about the regulation of AMH production. To answer these questions, a study was designed to investigate the regulation of AMH production in cows selected for their high or low ovulatory responses to superovulation. At the granulosa cell level, AMH production was inhibited by follicle-stimulating hormone but enhanced by bone morphogenetic proteins. At the follicular level, the expression of AMH within the follicle was dependent on the stage of follicular development. At the ovarian level, the size of the pool of small antral growing follicles determined ovarian AMH production. At the endocrine level, AMH followed a specific dynamic profile during the estrous cycle, which occurred independently of the follicular waves of terminal follicular development. Cows selected for their high or low responses to superovulation did not differ in the regulation of AMH production, but cows with higher responses had higher plasma AMH concentrations throughout the cycle. The optimal period of the estrous cycle at which to measure AMH concentrations with the aim of selecting the best cows for embryo production was found to be at estrus and after Day 12 of the cycle. Based on this multiscale study, we propose a model that integrates the different regulatory levels of AMH production.