Glycoproteins present in human follicular fluid that inhibit the zona-binding capacity of spermatozoa

Previous studies have suggested that human follicular fluid contains factors that reduce the zona-binding capacity of spermatozoa. The present study provides further evidence of the existence of such factors. Using the hemizona binding assay (HZA), we have shown that the inhibitory effect of human follicular fluid on the zona-binding capacity of spermatozoa is concentration-dependent, an inhibitory effect being detected when the concentration of human follicular fluid was > or = 10%. A 1% concentration of human follicular fluid did not possess this inhibitory activity. Heating human follicular fluid at 56 degrees C for 30 min did not affect its inhibitory properties; treatment with proteinase-K abolished such inhibition. Human follicular fluid was fractionated sequentially by concanavalin-A affinity chromatography, Mono Q ion-exchange chromatography and Superose-12 gel filtration. The zona binding inhibitory activity resided in the fraction which bound to the lectin and Mono Q column and contained molecules with native molecular weights of 32 and 192 kDa. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis suggested that the 192 kDa glycoprotein was a tetramer, while the 32 kDa glycoprotein remained as a single molecular species under denaturing conditions. We conclude that two glycoproteins were responsible for the zona binding inhibitory activity of human follicular fluid. The physiological role of these factors remains unclear.     

Internal pH of human spermatozoa: effect of ions, human follicular fluid and progesterone

The internal pH (pHi) of human spermatozoa was measured by the fluorescent indicator 2,7-bicarboxyethyl-5,6-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and the distribution of the radioactive [14C]-methylamine under different external ionic conditions. The effect of the addition of progesterone and human follicular fluid (HFF) on the spermatozoa pHi was also analysed. The pHi values obtained were almost identical with the two probes used. In sodium (NaM) and potassium (KM) media, a linear relationship between the internal and external pH was observed. In NaM, the pHi values were approximately 0.4 pH unit less than the external pH. In KM, the pHi measured was higher than in NaM and only slightly inferior to the external pH (0.1-0.2 pH unit). Addition of 10 microM progesterone, oestradiol 17 beta or 20% HFF to spermatozoa incubated at pH 7.2 in NaM did not induce any rapid variation of the BCECF fluorescence or change in the accumulation of methylamine. A slight change in pH (approximately 0.5 units) occurred with progesterone after 15 min. As a control, addition of 10 mM of NH4Cl induced a rapid alkalinization (0.4 pH unit) of the cell interior while 10 mM lactate produced only a slight acidification (approximately 0.2 pH unit). Under the same conditions (NaM, pH 7.2), the pHi of the spermatozoa prepared by Percoll gradient was found more acidic by 0.2 pH unit than washed unfractionated spermatozoa. Progesterone, oestradiol 17 beta and HFF had no effect on the pHi of these spermatozoa. The results obtained in this study show that it is possible to measure accurately the internal pH of human spermatozoa. Internal pH was found to be dependent upon the pH of the external medium and a quasi-linear relationship exists between the internal and external pH, suggesting no specific pH regulatory mechanisms. Our data suggest instead that the protons, under our experimental conditions, are passively distributed. Progesterone, oestradiol 17 beta and HFF, known to promote both capacitation and the acrosome reaction, do not act through a rapid pHi change.     

High levels of immunoreactive endothelin-1 in human follicular fluids

Follicular fluids were obtained from 180 follicles of 15 women undergoing follicular aspiration for in-vitro fertilization. Follicular development was induced by a combination of buserelin acetate and human menopausal gonadotrophin. Endothelin-1 (ET-1) concentrations in human follicular fluids were determined by specific radioimmunoassay. ET-1-like immunoreactivity (ET-1-LI) ranged from 338 to 928 pg/ml. ET-1-LI concentrations in follicular fluids obtained from immature (< 15 mm) follicles were significantly higher than those from mature (15-25 mm) and post-mature (> or = 25 mm) follicles. No correlation was found between the concentration of ET-1-LI, on the one hand, and that of oestradiol, progesterone, testosterone, prolactin, luteinizing hormone, insulin-like growth factor-I, prostaglandin E2 or platelet activating factor on the other, in follicular fluids. However, a significant positive correlation was observed between ET-1-LI concentration and follicle stimulating hormone and IGF-II concentrations, respectively. These data suggest that the high concentration of ET-1 found in follicular fluids may play some physiological role in follicular development.     

A novel macrophage actin-associated protein (MAYP) is tyrosine-phosphorylated following colony stimulating factor-1 stimulation

An approximately 37-kDa cytoplasmic protein is rapidly tyrosine-phosphorylated in the response of mouse BAC1.2F5 macrophages to colony stimulating factor-1 (CSF-1). pp37 was purified from the cytosolic fraction by anti-Tyr(P) affinity chromatography, size exclusion chromatography, and C4 reverse phase high pressure liquid chromatography. The sequences of four peptides derived from the purified protein matched portions of an expressed sequence tag (EST) sequence, and the EST clone was used to obtain cDNA clones encoding the pp37 protein, which shares sequence similarity with the PST PIP (proline, serine, threonine phosphatase interacting protein)/CDC15 family of protein-tyrosine phosphatase substrates. pp37 is predicted to contain a Fes/CIP4 homology (FCH) domain and an actin-binding domain-like sequence. It is expressed selectively in macrophages, macrophage cell lines, and at low levels in macrophage-containing tissues. pp37 is predominantly found in the cytosol, where it is associated with actin. However, approximately 4% resides in the membrane fraction, and the trace amount in the cytoskeletal fraction is increased by CSF-1 stimulation. Termed macrophage actin-associated tyrosine-phosphorylated protein (MAYP), p37 is the major F-actin-associated protein that is tyrosine-phosphorylated in macrophages and is likely to play a role in regulating the CSF-1-induced reorganization of the actin cytoskeleton.     

A bolus of recombinant human follicle stimulating hormone at midcycle induces periovulatory events following multiple follicular development in macaques

The efficacy of follicle stimulating hormone (FSH) as an alternative to luteinizing hormone (LH)/human chorionic gonadotrophin (HCG) for the initiation of periovulatory events in primate follicles is unknown. A single bolus of 2500 IU recombinant (r)-hFSH was compared to 1000 IU r-HCG for its ability to promote oocyte nuclear maturation and fertilization, granulosa cell luteinization and corpus luteum function following r-hFSH (60 IU/day) induction of multiple follicular development in rhesus monkeys. Following the r-hFSH bolus, bioactive luteinizing hormone concentrations were <3 ng/ml. Peak concentrations of serum FSH (1455+/-314 mIU/ml; mean+/-SEM) were attained 2-8 h after r-hFSH, and declined by 96 h. Bioactive HCG concentrations peaked between 2-8 h after r-HCG and remained > or = 100 ng/ml for >48 h, while immunoreactive FSH concentrations were at baseline. The proportion of oocytes resuming meiosis and undergoing in-vitro fertilization (IVF) were comparable for r-hFSH (89%; 47+/-19%) and r-HCG (88%; 50+/-17%). In-vitro progesterone production and expression of progesterone receptors in granulosa cells did not differ between groups. Peak concentrations of serum progesterone in the luteal phase were similar, but were lower 6-9 days post-FSH relative to HCG. Thus, a bolus of r-hFSH was equivalent to r-HCG for the reinitiation of oocyte meiosis, fertilization and granulosa cell luteinization, but a midcycle FSH surge did not sustain normal luteal function in primates.     

Dexamethasone enhances macrophage colony stimulating factor- and granulocyte macrophage colony stimulating factor-stimulated proliferation of bone marrow-derived macrophages

The authors describe a study in which they placed 126 Class V composite resin restorations without mechanical retention, divided into three groups of 42, in 23 patients. They followed the performance of the restorations over a three-year period. For all three groups, restorations were placed using All-Bond 2 dental adhesive and Z100 composite resin; A.R.T. Bond and Brilliant Dentin composite; and Prisma Universal Bond 3 and Variglass VLC polyacid-modified composite resin. The authors evaluated retention as well as color stability, wear resistance, sensitivity, sulcular depth, loss of attachment, bleeding on probing and crevicular fluid flow. Based on their results, the authors propose that restoration of Class V lesions without using mechanical retention could be expected to succeed in seven of 10 restorations over a three-year period using these restorative systems.     

Human follicular fluid proteoglycans in relation to in vitro fertilization

OBJECTIVE: To determine whether the concentrations of proteoglycans and hyaluronan in human follicular fluid (FF) are associated with follicular volume, oocyte fertilization, and ET during IVF. DESIGN: The FF from individual follicles were collected. Enzyme-linked immunosorbent assay methods for quantification of a larger chondroitin sulfate proteoglycan and a smaller composite heparan-chondroitin sulfate proteoglycan were established. Hyaluronan and E2 were measured by RIA techniques. PATIENT(S): Sixteen infertile women participating in the IVF program. MAIN OUTCOME MEASURE(S): Concentrations of the proteoglycans, follicular volume, fertilization, and ET rates. RESULT(S): The follicles contained high concentrations of proteoglycans with an average of 0.8 mg/mL of FF, and approximately 70% consisted of the larger chondroitin sulfate proteoglycan, and 30% of the heparan-chondroitin sulfate proteoglycan. A negative correlation was found between the follicular volume, the chondroitin sulfate proteoglycan (r = -0.43), and hyaluronan (r = -0.56). The percentage of embryos developed in culture was significantly higher in follicles larger than 2 mL. A significant and 35% lower concentration of the chondroitin sulfate proteoglycan was found in larger follicles from which subsequent ET was observed. THe heparan-chondroitin sulfate proteoglycan and hyaluronan were both unrelated to fertilization and ET in vitro. CONCLUSION(S): Lower concentrations of chondroitin sulfate proteoglycan were associated with higher follicular volumes and greater fertilization and ET rates. These associations could merely reflect the maturation of the follicle or a role of the chondroitin sulfate proteoglycan in the fertilization process.     

Treatment of human spermatozoa with follicular fluid can influence lipid content and motility during in vitro capacitation

In order to evaluate the role of human follicular fluid (HFF) on the fertilizing capacity of spermatozoa, we studied the effect of HFF on the lipid composition and on the movement characteristics of human spermatozoa. Spermatozoa (spz) from normospermic patients were prepared with a discontinuous Percoll gradient and incubated in Ménézo B2 medium with or without a supplement of 20% HFF (HFF-Percoll spz and B2-Percoll spz respectively) for 2 and 24 h. After 2 h HFF incubation, percentage progressive motility, straight line velocity (VSL), and amplitude of lateral head displacement (ALH) were improved in HFF-Percoll spz as compared to B2-Percoll spz (P < or = 0.05). After a longer incubation period (24 h), lipid changes appeared in HFF-Percoll spz with lower levels of cholesterol (P = 0.02) and phospholipids (P = 0.05). No modification of the cholesterol/phospholipid ratio after 2 and 24 h of incubation in either B2-Percoll spz or HFF-Percoll spz was observed. Such decreases in lipid content of HFF-Percoll spz may be factors which could be taken into account as constituting part of membrane modifications during the capacitation process.     

Insulin and insulin-like growth factor-1 regulate tau phosphorylation in cultured human neurons

Hyperphosphorylated tau is the major component of paired helical filaments in neurofibrillary lesions associated with Alzheimer's disease. Hyperphosphorylation reduces the affinity of tau for microtubules and is thought to be a critical event in the pathogenesis of this disease. Recently, glycogen-synthase kinase-3 has been shown to phosphorylate tau in vitro and in non-neuronal cells transfected with tau. The activity of glycogen-synthase kinase-3 can be down-regulated in response to insulin or insulin-like growth factor-1 through the activation of the phosphatidylinositol 3-kinase pathway. We therefore hypothesize that insulin or insulin-like growth factor-1 may affect tau phosphorylation through the inhibition of glycogen-synthase kinase-3 in neurons. Using cultured human neuronal NT2N cells, we demonstrate that glycogen-synthase kinase-3 phosphorylates tau and reduces its affinity for microtubules and that insulin and insulin-like growth factor-1 stimulation reduces tau phosphorylation and promotes tau binding to microtubules. We further demonstrate that these effects of insulin and insulin-like growth factor-1 are mediated through the inhibition of glycogen-synthase kinase-3 via the phosphatidylinositol 3-kinase/protein kinase B signaling pathway.     

Concentration of tissue inhibitor of metalloproteinases (TIMP)-1 in ovine follicular fluid and serum

Tissue inhibitor of metalloproteinases (TIMP)-1 mRNA and protein localize within granulosal cells of post-gonadotropin-surge follicles and luteal tissue in ewes. Our objectives were to test the hypotheses that 1) follicular fluid concentration of TIMP-1 increases following a gonadotropin surge induced by LHRH agonist (Exp. 1) and 2) luteal status affects peripheral serum concentration of TIMP-1 (Exp. 2 and 3). In Exp. 1, the concentration of TIMP-1 within antral fluid from post-surge follicles (28.7 +/- 6.65 microg/mL) was greater (P < .02) than from pre-surge follicles (2.37 +/- 2.47 microg/mL). In Exp. 2, serum concentration of TIMP-1 did not differ among d 0 to 6 (1.27 +/- .55 microg/mL) of the estrous cycle or among periods of luteal maintenance (1.29 +/- .06 microg/mL), spontaneous luteal regression (1.19 +/- .09 microg/mL), or luteal development (1.22 +/- .08 microg/mL). However, serum concentration of TIMP-1 was greater ( P < .001) during the period of luteal maintenance (1.14 +/- .04 microg/mL) than during PGF2alpha-induced luteolysis (d 26; .85 +/- .06 microg/mL) and induced luteal absence (d 27 to 33; .95 +/- .05 microg/mL). In Exp. 3, ewes (n = 14) were bled daily from d 1 to 19 (d 0 = estrus) and at 12-min intervals for 6 h on d 3, 10, and 17. Although concentration of TIMP-1 varied considerably within and among ewes, mean concentration of TIMP-1 per ewe per day increased ( P < .05) from d 3 to 17. These data indicate that follicular fluid concentration of TIMP-1 increases following a gonadotropin surge, but the contribution of ovarian derived TIMP-1 to peripheral serum concentration is negligible.     

Presence of dioxins in human follicular fluid: their possible stage-specific action on the development of preimplantation mouse embryos

Examination of human follicular fluid revealed the presence of polychlorinated dibenzodioxins (PCDDs) and dibenzofurans (PCDFs) at concentrations of approximately 1 pg/ml (0.01 pg TEQ/ml). To study their possible action, two-cell mouse embryos were cultured in the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at concentrations between 0.5 and 100 pM and evaluated at 24-h intervals for their development to the eight-cell and blastocyst stages. The percentage of eight-cell embryos exposed to TCDD at 1, 2, and 5 pM concentrations was significantly lower than that of controls. However, blastocyst formation of the surviving eight-cell embryos was accelerated, with the number of cells in the blastocysts increased in a dose-dependent manner. Findings suggest that PCDDs and PCDFs may be present in human reproductive fluid and may exert some stage-specific effects on early embryonic development.     

Control of oocyte growth and maturation by follicular cells and molecules present in follicular fluid. A review

The aim of this review is to summarize the interactions between the oocyte and its surrounding granulosa cells which are involved in the control of oocyte growth or apoptosis as well as those playing a key role in the ability of the oocyte to undergo nuclear (resumption as meiosis to reach the MII stage) or cytoplasmic maturation (ability to fertilize and develop to the blastocyst stage). The respective roles of the oocyte and of the granulosa cells in controlling the initiation of growth are poorly understood. During the preantral follicular stage when most oocyte growth is achieved, a local regulation appears to be in operation involving growth factors such as fibroblast growth factor (FGF) or epidermal growth factor/transforming growth factor alpha (EGF/TGF alpha), together with two proteins (c-kit present on the oocyte's membrane and its ligand KL produced by granulosa cells). In-situ techniques used to detect apoptosis demonstrate apoptotic oocytes in the reserves of primordial follicles but seldom within preantral follicles (because it is too fast?). Proteins involved in cell death (bax) or cell survival (bcl2) are present in oocytes as well as compounds (TNF alpha, Fas) involved in the initiation of apoptosis. However, the molecular and cellular mechanisms triggering oocyte apoptosis are not fully clarified. Three approaches have been used to identify compounds which are relevant to the oocyte's nuclear or cytoplasmic maturation. a) Correlation between amounts of specific compounds in follicular fluid or within follicle cells and the oocyte's ability to mature. b) Analysis of the consequences of pharmacological disruption of mechanisms such as steroidogenesis on oocyte maturation. c) Analysis of the consequences of addition of graded amounts of specific compounds on oocyte maturation in defined media. Factors playing a key role in stimulating nuclear maturation appear to be epidermal growth factor (EGF) and the inhibin (cattle)/activin (rodents) family, while testosterone has an inhibitory effect. Cytoplasmic maturation of the oocyte appears to be stimulated by oestradiol, EGF and inhibin.     

Isolation and characterization of maturation inhibiting compound in bovine follicular fluid

The protein fraction which is responsible for the inhibition of maturation of bovine oocytes in vitro was isolated from cow follicular fluid by means of column chromatography on a Sephadex G-200 and a Sepharose 4B, both in 0.1 M ammonium acetate, pH 6.7. The molecular weight of the maturation inhibiting protein fraction is approximately 60 kDa. At a concentration of 2.0 mg/mL in cultivation medium, 100% of the oocytes were arrested at the germinal vesicle stage. At a concentration of 0.25 mg/mL, the protein fraction still had some meiosis inhibiting effects, but 56% of the oocytes were capable of maturing to the metaphase of the second meiosis (MII). Without compact cumulus the inhibiting fraction had no meiosis retarding effect on the oocytes. Cow follicular fluid also exhibited this inhibitory effect on oocyte maturation in vitro. However, the follicular fluid from follicles of 2.5-5.0 mm diameter showed higher meiosis inhibiting effects than the follicular fluid from follicles of 5-10 mm diameter.     

Soluble intercellular adhesion molecule-1 in ovarian follicles: production by granulosa luteal cells and levels in follicular fluid

OBJECTIVE: To determine the concentration of the soluble form of intercellular adhesion molecule (ICAM)-1 in granulosa luteal cell-conditioned media and in follicular fluid (FF). DESIGN: Granulosa cells and FF samples were obtained at the time of oocyte retrieval for IVF. In 10 women, a total of 33 fluids were obtained from individual follicles, whereas in 70 women, the follicular aspirates were pooled. SETTING: Clinica "L. Mangiagalli" and Reproductive Center, San Raffaele Hospital, Milan, Italy. PATIENT(S): Eighty women referred for IVF for tubal factor or male factor infertility. INTERVENTION(S): Women underwent ovarian hyperstimulation. MAIN OUTCOME MEASURE(S): Soluble ICAM-1 was measured by an ELISA, and its levels were correlated with follicular size, the number of retrieved oocytes, and the number of follicles with a diameter of >15 mm. RESULT(S): The concentration of soluble ICAM-1 in granulosa luteal cell-conditioned media was 17.8 +/- 1.8 ng/5 x 10(5) cells. Interleukin-1beta can stimulate soluble ICAM-1 release in a dose-dependent manner. A significant positive correlation was demonstrated between levels of soluble ICAM-1 in pooled FF and the number of retrieved oocytes or the number of follicles with a diameter of >15 mm. CONCLUSION(S): Soluble ICAM-1 can be released by granulosa luteal cells and can be detected in FF after ovarian hyperstimulation. Levels of soluble ICAM-1 in FF correlate directly with some indices of ovarian function.     

Leptin concentrations in the follicular phase of spontaneous cycles and cycles superovulated with follicle stimulating hormone

It has been reported that oestradiol may play a role in the production of leptin from adipocytes. To investigate this relationship further, nine normally ovulating women were studied during two menstrual cycles, i.e. an untreated spontaneous cycle and a cycle treated with follicle stimulating hormone (FSH) from cycle day 2 until the day of human chorionic gonadotrophin (HCG) injection. Serum leptin values on cycle day 2 did not differ significantly between the spontaneous and the FSH cycles. In the spontaneous cycles, leptin values declined gradually and significantly up to day 7 and then increased progressively up to the day of luteinizing hormone (LH) surge onset, at which point they achieved the highest values. In the FSH cycles, serum leptin values increased gradually and significantly up to day 6, remaining stable thereafter, and were in the midfollicular phase significantly higher than in the spontaneous cycles. Significant positive correlations were found between mean values of leptin and mean values of oestradiol during the second half of the follicular phase in the spontaneous cycles and during the first half in the FSH cycles. A significant negative correlation was found between these two parameters in the spontaneous cycles during the first half of the follicular phase. Serum leptin levels were significantly higher in the midluteal than in the follicular phase in both cycles. These results demonstrate for the first time significant changes in leptin values during the follicular phase of the human menstrual cycle and a significant increase during superovulation induction with FSH. It is suggested that oestradiol may be involved in the regulation of leptin production in women.     

Increase in transforming growth factor beta1 in ovarian follicular fluid following ovarian stimulation and in-vitro fertilization correlates to pregnancy

Talin, an actin-binding protein from smooth muscle, is shown to bind to myosin in such a way that it stimulates the ATPase activity of myosin irrespective of the phosphorylation state of myosin. The binding site is shown to be localized at the N-terminal, 47 KDa fragment. The position of the actin-binding site at the C terminal suggests that talin may work as a crosslinker between myosin and actin.     

Engineering the C-region of human insulin-like growth factor-1: implications for receptor binding

Recombinant wild-type human IGF-1 and a C-region mutant in which residues 28-37 have been replaced by a 4-glycine bridge (4-Gly IGF-1) were secreted and purified from yeast. An IGF-1 analogue in which residues 29-41 of the C-region have been deleted (mini IGF-1) was created by site-directed mutagenesis and also expressed. All three proteins adopted the insulin-fold as determined by circular dichroism. The significantly raised expression levels of mini IGF-1 allowed the recording of two-dimensional NMR spectra. The affinity of 4-Gly IGF-1 for the IGF-1 receptor was approximately 100-fold lower than that of wild-type IGF-1 and the affinity for the insulin receptor was approximately 10-fold lower. Mini IGF-1 showed no affinity for either receptor. Not only does the C-region of IGF-1 contribute directly to the free energy of binding to the IGF-1 receptor, but also the absence of flexibility in this region eliminates binding altogether. As postulated for the binding of insulin to its own receptor, it is proposed that binding of IGF-1 to the IGF-1 receptor also involves a conformational change in which the C-terminal B-region residues detach from the body of the molecule to expose the underlying A-region residues.     

Mitogenic activity of peptides related to the sequence of human fibroblast growth factor-1

Linear synthetic peptides related to the human Fibroblast Growth Factor-1 (hFGF-1) segment [112-147] were tested for their capacity of mimicking FGF mitogenic activity, binding to heparin-Sepharose columns, stimulating DNA synthesis and competing with hFGF-1 for the cellular receptors. The results obtained indicated that the activity of these compounds is dependent on the presence of the sequence WFVGLK in their structures. The affinity for the cellular receptors increased when this sequence was elongated in order to incorporate amino acid residues that are important for FGF-heparin binding.