Ruth Kaplan-Kraicer

The Role of Pigment Epithelium-Derived Factor in the Pathophysiology and Treatment of Ovarian Hyperstimulation Syndrome in Mice

CONTEXT Ovarian hyperstimulation syndrome (OHSS) is a potentially life-threatening complication of assisted reproduction. OHSS is induced by an ovarian release of vasoactive, angiogenic substances that results in vascular hyperpermeability, leakage, and shift of fluids from blood vessels into the extravascular space with consequent ascites and edema that are attributed to vascular endothelial growth factor (VEGF). OBJECTIVE Our objective was to examine a physiological approach for preventing and treating OHSS, based on negating the VEGF network. DESIGN We used a mouse OHSS model and cultured granulosa cells. MAIN OUTCOME Changes in pigment epithelium-derived factor (PEDF) and VEGF were measured by quantitative PCR and Western blot analysis. OHSS was recorded by changes in body weight and in peritoneal vascular leakage, quantified by the modified Miles vascular permeability assay. RESULTS Granulosa cells produced and secreted the anti-angiogenic factor, PEDF, in an inverse fashion to VEGF. The physiological PEDF-VEGF counterbalance was found to be impaired in the mouse OHSS model. Treatment of OHSS-induced mice with low doses of recombinant PEDF (rPEDF) alleviated OHSS signs including edema (P < .001) and vascular leakage (P < .001) and reduced the level of ovarian VEGF mRNA. Low doses of rPEDF also reduced VEGF mRNA levels in granulosa cells in vitro. However, these effects were not seen at higher doses of rPEDF, suggesting a hormetic mechanism of rPEDF action. CONCLUSION These observations provide a new perspective into the pathophysiology of OHSS, namely, high expression level of VEGF together with a nearly undetectable level of PEDF. A replacement therapy with rPEDF is suggested as an innovative physiological treatment for OHSS. Finally, control of the PEDF-VEGF reciprocal relationship could open new therapeutic avenues for other angiogenic-related fertility pathologies.

The relationship between sperm parameters and fertilizing capacity in vitro: a predictive role for swim-up migration.

The relationship between sperm parameters and fertilizing capacity in vitro was examined retrospectively, with the aim of finding predictive criteria for successful in vitro fertilization. Three hundred thirty semen samples were used to inseminate 1462 oocytes. Conventional parameters of sperm concentration and percentage motility in the ejaculate as well as swim-up migration were analyzed in relation to fertilization rate. It was shown that the probability of fertilizing human oocytes in vitro decreases significantly when (a) the sperm concentration is below 20×106 spermatozoa/ml ejaculate (P=0.006), (b) motility is lower than 80% (P=0.002), or (c) less than 4×106 motile spermatozoa/ml are concentrated in the swim-up fraction (P<0.0001). It was also demonstrated that nonfertilizing sperm could not be distinguished from fertilizing sperm by the conventional criteria but rather by the average concentration of motile spermatozoa in the swim-up fraction [12.5±1.5 and 22.3±2.3×106/ml for the 0 and the 100% fertilization groups, respectively (mean±SE;P<0.01)]. Thus, the swim-up migration technique can serve as a predictive test for the in vitro fertilizing capacity of sperm.

Hormonal regulation of pigment epithelium-derived factor (PEDF) in granulosa cells

Angiogenesis is critical for the development of ovarian follicles. Blood vessels are abrogated from the follicle until ovulation, when they invade it to support the developing corpus luteum. Granulosa cells are known to secrete anti-angiogenic factors that shield against premature vascularization; however, their molecular identity is yet to be defined. In this study we address the physiological role of pigment epithelium-derived factor (PEDF), a well-known angiogenic inhibitor, in granulosa cells. We have shown that human and mouse primary granulosa cells express and secrete PEDF, and characterized its hormonal regulation. Stimulation of granulosa cells with increasing doses of estrogen caused a gradual decrease in the PEDF secretion, while stimulation with progesterone caused an abrupt decrease in its secretion. Moreover, We have shown, by time- and dose-response experiments, that the secreted PEDF and vascular endothelial growth factor (VEGF) were inversely regulated by hCG; namely, PEDF level was nearly undetectable under high doses of hCG, while VEGF level was significantly elevated. The anti-angiogenic nature of the PEDF secreted from granulosa cells was examined by migration, proliferation and tube formation assays in cultures of human umbilical vein endothelial cells. Depleting PEDF from primary granulosa cells conditioned media accelerated endothelial cells proliferation, migration and tube formation. Collectively, the dynamic expression of PEDF that inversely portrays VEGF expression may imply its putative role as a physiological negative regulator of follicular angiogenesis.

Regulation of division in mammalian oocytes: implications for polar body formation

Meiosis in mammalian oocytes includes two asymmetric meiotic divisions that result in extrusion of the first and second polar bodies (PBI and PBII, respectively). Fyn, an Src family kinase (SFK), colocalizes with filamentous actin (F-actin) at the meiotic cleavage furrow area of mouse oocytes. In this paper, these studies are extended to rat oocytes. Furthermore, inhibition of Fyn decreased the rate of PBs extrusion and led to formation of larger PBs (PBI and PBII). This effect differs from the effect of Fyn inhibition on the first mitotic symmetric cell division where only the rate of cleavage was affected but the two daughter cells were of regular size. Inhibition of Fyn resulted in a significant decrease in cortical F-actin in the oocytes. We suggest a meiotic model for mammalian oocytes in which Fyn is recruited to the meiotic area of cleavage furrow formation and induces polymerization and stabilization of F-actin, possibly by regulating F-actin effectors, such as RhoA, Arp2/3 and formins, thus allowing ingression of the cleavage furrow. In the context of PB formation, we suggest that SFKs are involved in maintaining the precise temporal restrains of the asymmetric divisions and in regulation of PBs size by inducing polymerization and stabilization of F-actin during the formation and ingression of the cleavage furrow.

Molecular cloning of rat sperm galactosyl receptor, a C-type lectin with in vitro egg binding activity.

Rat sperm galactosyl receptor is a member of the C‐type animal lectin family showing preferential binding to N‐acetylgalactosamine compared to galactose. Binding is mediated by a Ca2+‐dependent carbohydrate‐recognition domain (CRD) identical to that of the minor variant of rat hepatic lectin receptor 2/3 (RHL‐2/3). The molecular organization of the genomic DNA, cDNA, and derived amino acid sequence of rat testis galactosyl receptor have been determined and in vitro fertilization studies were conducted to ascertain its role. We have determined that the rat testis galactosyl receptor gene generates two mRNA species: one species, designated liver‐type, is identical to RHL‐2/3; the other, designated testis‐type, contains one unspliced intron (86 nt) which alters the reading frame and changes the amino acid sequence of the carboxyl terminus. As a result, the CRD (glutamine‐proline‐aspartic acid/QPD) and flanked Ca2+‐binding amino acid sequences were not present in the testis‐type protein. Northern and Southern blots demonstrated presence of transcripts with unspliced intron in rat sperm but not liver. Similarly, antibody, raised against a synthetic 12‐amino acid peptide (p12) encoded by the unspliced intron, recognized in immunoblots a 54 kDa receptor protein in protein extracts from testis but not from liver. Immunofluorescence and immunogold electron microscopy studies demonstrated that both protein species localized on the plasma membrane surface of the head and tail of rat sperm. Furthermore, capacitated rat sperm preincubated with polyclonal antisera to RHL‐2/3 or to the CRD of the liver‐type galactosyl receptor showed a statistically significant decrease in the in vitro fertilization rate. We conclude that rat sperm galactosyl receptor may play a role in egg binding and that an undetermined molecular mechanism operates to generate two proteins with identical intracellular amino terminal domain but only one of them displays a CRD and associated Ca2+‐binding sites at the carboxyl terminal extracellular domain. Mol. Reprod. Dev. 56:401–411, 2000.

Myristoylated alanine-rich C kinase substrate, but not Ca2+/calmodulin-dependent protein kinase II, is the mediator in cortical granules exocytosis

Sperm-egg fusion induces cortical granules exocytosis (CGE), a process that ensures the block to polyspermy. CGE can be induced independently by either a rise in intracellular calcium concentration or protein kinase C (PKC) activation. We have previously shown that myristoylated alanine-rich C kinase substrate (MARCKS) cross-links filamentous actin (F-actin) and regulates its reorganization. This activity is reduced either by PKC-induced MARCKS phosphorylation (PKC pathway) or by its direct binding to calmodulin (CaM; CaM pathway), both inducing MARCKS translocation, F-actin reorganization, and CGE. Currently, we examine the involvement of Ca(2)(+)/CaM-dependent protein kinase II (CaMKII) and MARCKS in promoting CGE and show that PKC pathway can compensate for lack of Ca(2)(+)/CaM pathway. Microinjecting eggs with either overexpressed protein or complementary RNA of constitutively active alphaCaMKII triggered resumption of second meiotic division, but induced CGE of an insignificant magnitude compared with CGE induced by wt alphaCaMKII. Microinjecting eggs with mutant-unphosphorylatable MARCKS reduced the intensity of 12-O-tetradecanoylphorbol 13-acetate or ionomycin-induced CGE by 50%, indicating that phosphorylation of MARCKS by novel and/or conventional PKCs (n/cPKCs) is a pivotal event associated with CGE. Moreover, we were able to demonstrate cPKCs involvement in ionomycin-induced MARCKS translocation and CGE. These results led us to propose that MARCKS, rather than CaMKII, as a key mediator of CGE.

Tamoxifen and RU39411 synergize with mifepristone to produce preimplantation pregnancy loss by increasing embryo transport (rat)

Tamoxifen is a non-steroidal antiestrogen that possesses antagonistic as well a agonistic properties, while RU39411 is an antiestrogen that is known to possess only antagonistic properties. These steroid antagonists were administered orally to rats, with or without mifepristone, an antiprogestational agent, prior to implantation on Day 2 of pregnancy. The status of pregnancy was assessed on Day 14. Low doses of tamoxifen reduced litter size and weight and inducing embryonic absorption in some animals; a dose of 0.25 mg/Kg prevented pregnancy in all animals. RU39411 also had a dose-dependent effect on pregnancy, but a higher dose (0.5 mg/Kg) was required to achieve the same degree of pregnancy prevention. Addition of mifepristone to both antiestrogens had a synergistic effect on reducing litter size and weight. To determine the mechanism by which antiestrogens terminate pregnancy in rats, oviducts and uteri of treated rats were examined for the presence of embryos on Day 3 and 4 of pregnancy, times when most embryos would be expected to be in the oviducts. Most of the embryos of the treated animals were found in the oviducts on Day 3 of pregnancy. By Day 4, only a few embryos were still present in oviducts. These observations suggest acceleration of embryo transport by Day 4 of pregnancy. There was no accumulation of embryos in the uterus. Since acceleration of embryo transport through the reproductive tract in rat is induced by low doses of estrogens, it is likely that the agonistic action of tamoxifen is responsible for pregnancy prevention in these experiments. The fact that RU39411 also prevents pregnancy by a similar mechanism suggests that this estrogen antagonist also has some estrogen agonist effects on the oviduct. The fact that both antiestrogens also affected embryo weight could suggest the action of the antihormones on other mechanisms controlling embryo development.

Experimental extension of the time interval between oocyte maturation and ovulation: effect on fertilization first cleavage

OBJECTIVE To test the hypothesis that impaired fertility in human patients with high LH concentrations throughout the follicular phase of the menstrual cycle reflects premature maturation of their oocytes. DESIGN Previous information that resumption of meiosis is induced by lower hCG concentrations than that required for stimulation of follicular rupture was confirmed and used for establishment of a rat animal model in which oocyte maturation and ovulation can be separated experimentally. In further experiments hypophysectomized, pregnant mare serum gonadotropin (PMSG)-primed, immature female rats injected with 1.1 IU of hCG, a dose found to induce maturation in 72.9% +/- 6% of the rats with no effect on ovulation, were administered with a second injection of an ovulatory dose (4 IU) of hCG, 24 hours later. The ovulated eggs were subjected to IVF. RESULTS Fertilization and first cleavage in oocytes recovered from our experimental animal model were similar to that observed in control PMSG-primed, either hypophysectomized or intact rats, treated by a single injection of 4 IU of hCG. CONCLUSIONS The extension of the time interval between oocyte maturation and ovulation in the rat does not result in a lower rate of fertilization or a reduced incidence of cleavage. However, an inferior developmental capacity of these embryos cannot be ruled out.

De novo SYNTHESIS OF PROTEIN PHOSPHATASE 1A, MAGNESIUM DEPENDENT, ALPHA ISOFORM (PPM1A) DURING OOCYTE MATURATION

Oocyte maturation in mammals is a multiple-stage process that generates fertilizable oocytes. Ovarian oocytes are arrested at prophase of the first meiotic division characterized by the presence of a germinal vesicle. Towards ovulation, the oocytes resume meiosis and proceed to the second metaphase in a process known as maturation; they undergo nuclear and cytoplasmic changes that are accompanied by translation and degradation of mRNA. Protein phosphatase 1A, magnesium dependent, alpha isoform (PPM1A), which belongs to the metal-dependent serine/threonine protein phosphatase family, is highly conserved during evolution. PPM1A plays a significant role in many cellular functions such as cell cycle progression, apoptosis and cellular differentiation. It works through diverse signaling pathways, including p38 MAP kinase JNK and transforming growth factor beta (TGF-β). Herein we report that PPM1A is expressed in mouse oocytes and that its mRNA level rises during oocyte maturation. Using quantitative real-time polymerase chain reaction (qPCR) and western blot analysis, we found that PPM1A mRNA is synthesized at the beginning of the maturation process and remains elevated in the mature oocytes, promoting the accumulation of PPM1A protein. Since PPM1A function is mainly affected by its level, we propose that it might have an important role in oocyte maturation.

Viscosity and refractive index of follicular fluid in relation to in vitro fertilization

AbstractPurpose: To set the standard values of follicular fluid viscosity and refractive index, and to investigate a possible relationship between these physiological parameters and the outcome of in vitro fertilization treatment. Design and Results: 128 samples of follicular fluid were collected from 40 in vitro fertilization patients. Viscosity determinations (centipoise; mean ± SD) for shear rates of 23, 46, 115, and 230 were 2.04±0.86, 1.84±0.49, 1.48±0.27, and 1.38±0.22, respectively. The average (± SD) refractive index was 1.030±0.002. There was no significant difference between the values of thawed frozen fluids and fresh samples of the same specimens. The data showed no correlation between follicular fluid viscosity or refractive index and the presence of oocytes, their maturation grade or their fertilizing capacity. Conclusions: For the first time, values of the viscosity and refractive index of follicular fluid obtained during in vitro fertilization have been determined. However, these preliminary results did not reveal any relationship between the physiological parameters examined and the outcome of in vitro fertilization treatment.