Peritoneal fluid modifies the response of human spermatozoa to follicular fluid

The aim of this study was to elucidate the mechanism involved in the acrosome reaction (AR) induced by follicular fluid (FF) in spermatozoa previously exposed to peritoneal fluid (PF). The influence of progesterone was also investigated. Semen samples were from 18 normozoospermic donors. PF samples were from 13 women with unexplained infertility and from a woman treated with synthetic progestagen. FF samples were collected from six women undergoing IVF/embryo transfer and pooled. Motile spermatozoa were capacitated overnight and a kinetic and inhibition study on the FF-induced AR was performed. Spermatozoa pretreated with PF were challenged with either FF or progesterone. The ability of progesterone- and progestagen-supplemented PF to induce AR was analysed. Enzyme-digested PF was also tested. Pre-incubation with PF for 60 min completely prevented the FF-induced AR; spermatozoa treated with PF were unable to respond to FF or progesterone and this effect was not reversible. Progesterone- and progestagen-supplemented PF stimulated the AR relative to controls. Enzyme-digested PF did not have an inhibitory capacity. These data strongly suggest that there are one or more inhibitory proteins in PF that interact with spermatozoa so as to prevent access of progesterone to its receptor and thus inhibit the occurrence of the AR. The oviduct, or Fallopian tube, provides a place for spermatozoa and egg transport and storage, fertilization and early embryo development. If ovulation has not occurred, spermatozoa may reside in the oviduct for several hours or even a few days, awaiting oocyte arrival. It is assumed that fluids present in the female genital tract may have a role in synchronizing the timing required to guarantee the success of fertilization. We previously observed that the peritoneal fluid that bathes the peritoneal cavity is a suitable medium for sperm survival and we also reported that this fluid could stabilize spermatozoa. In this study we show further evidence that the exposure to peritoneal fluid modifies the response of spermatozoa to oocyte signals.Fil: Caille, Adriana M.. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Berta, Cesar L.. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Cuasnicu, Patricia Sara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Munuce, María J.. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentin

The In Vitro Effect Of Levonorgestrel On The Acrosome Reaction Of Human Spermatozoa From Fertile Men.

The objective of the study was to evaluate the effect of levonorgestrel (LNG) on the occurrence of acrosome reaction (AR) of capacitated spermatozoa from fertile men. A total of 20 semen samples from four fertile men were evaluated. The spermatozoa were separated by swim-up, and subsequently incubated for 20 h under capacitating conditions. Capacitated spermatozoa were exposed to three different concentrations of LNG (200, 400 and 800 ng/mL), follicular fluid (20% v/v), and ethanol or human tubal fluid medium (HTF) as a control. The AR rate and the ratio of live to dead spermatozoa were assessed after 15 and 30 min of incubation at 37 degrees C and 5% CO(2). The different treatments were compared with follicular fluid and HTF medium as positive and negative controls. The main results showed that the AR rate after 15 min of exposure was not affected by LNG and was significantly higher with follicular fluid than with all the other treatments. At 30 min of exposure, the three LNG concentrations induced a greater rate of AR than the HTF and a trend of higher AR rate with greater concentration was observed. Follicular fluid induced a significantly higher rate of AR than the other treatments. In conclusion, the addition of LNG in vitro to capacitated human spermatozoa is associated with a dose-dependent increased rate of AR, but such increase was not as great that induced by follicular fluid.6855-

Mechanisms involved in the contraceptive effects of ulipristal acetate

The use of emergency contraception (EC) methods is increasing worldwide as it constitutes an effective way to prevent unplanned pregnancy after unprotected sexual intercourse. During the last decade, ulipristal acetate (UPA), a selective progesterone receptor modulator, has emerged as the most effective EC pill, and it is now recommended as first-line hormonal treatment for EC in several countries. Its principal mechanism of action involves inhibition or delay of follicular rupture, but only when administered during the follicular phase before the luteinizing hormone (LH) peak. However, considering the high efficacy of UPA, it is possible that it also exerts contraceptive effects besides ovulation. In the present review, we summarize and discuss the existing evidence obtained on the effect of UPA on sperm function and post-ovulatory events as potential additional mechanisms to prevent pregnancy. The bulk of evidence collected so far indicates that UPA would not affect gamete function; however, it could impair embryo?uterine interaction. Thus, besides the described effects on ovarian function, UPA contraceptive effectiveness might also be attributed to post-ovulatory effects, depending on the moment of the female cycle in which the drug is administered.Fil: Munuce, María José. Universidad Nacional de Rosario; ArgentinaFil: Gómez Elías, Matías Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Caille, Adriana María. Universidad Nacional de Rosario; ArgentinaFil: Bahamondes, Luis. Universidade Estadual de Campinas; BrasilFil: Cuasnicu, Patricia Sara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Cohen, Débora J.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

Fibroblast Growth Factor Receptors (FGFRs) in Human Sperm: Expression, Functionality and Involvement in Motility Regulation.

Fibroblast growth factors receptors (FGFRs) have been widely characterized in somatic cells, but there is scarce evidence of their expression and function in mammalian gametes. The objective of the present study was to evaluate the expression of FGFRs in human male germ cells, to determine sperm FGFR activation by the FGF2 ligand and their participation in the regulation of sperm motility. The expression of FGFR1, 2, 3 and 4 mRNAs and proteins in human testis and localization of these receptors in germ cells of the seminiferous epithelium was demonstrated. In ejaculated sperm, FGFRs were localized to the acrosomal region and flagellum. Sperm exposure to FGF2 caused an increase in flagellar FGFR phosphorylation and activation of extracellular signal-regulated kinase (ERK) and protein kinase B (PKB or Akt) signaling pathways. Incubation with FGF2 led to a significant increase in the percentage of total and progressive sperm motility, as well as in sperm kinematics. All responses were prevented by sperm preincubation with BGJ398, a specific inhibitor of FGFR tyrosine kinase activity. In addition to confirming the expression of FGFRs in germ cells of the human testis, our study describes for the first time the presence, localization and functionality of human sperm FGFRs, and provides evidence of the beneficial effect of FGF2 upon sperm motility

Expression of FGFRs in human testis and sperm.

<p>(<b>A</b>) Messenger RNA expression of testicular and sperm FGFRs assessed by RT-PCR. Messenger RNA extracted from MCF7 cells served as positive controls; negative controls without reverse transcriptase (RT Control) and without template (PCR Control) are shown. The amplicon sizes are indicated on the right. (<b>B</b>) Detection of testis and sperm FGFR protein forms using Western immunoblotting. Protein extracts from human testis and sperm were subjected to SDS-PAGE and Western immunoblotting using anti FGFR antibodies or rabbit IgG as control. The estimated molecular weights of the protein bands are indicated on the right. The experiments were performed at least 3 times obtaining similar results. Typical results are shown.</p

Activation of sperm FGFRs in response to FGF2.

<p>Localization of sperm pFGFRs by immunocytochemistry. Sperm were incubated for 4 h and exposed to FGF2 (0, 1, 10 and 100 ng/ml) for the last 15 min. In some aliquots, sperm were incubated for 15 min with BGJ398 (0.1 μM) before the addition of FGF2. Sperm were processed for immunocytochemistry, stained with anti pFGFR and FITC-conjugated secondary antibody; nuclei were stained with propidium iodide. (<b>A</b>) pFGFR immunolocalization in sperm incubated in the absence of FGF2 (Control), with 100 ng/ml FGF2, and with BGJ398 + 100 ng/ml FGF2. Bar: 10 μm. (<b>B</b>) Percentage of sperm cells stained with anti pFGFR antibody after exposure to different concentrations of FGF2 in the absence (<b>left</b>) or presence of BGJ398 (<b>right</b>). Results are expressed as mean ± SEM, n = 4. * <i>P</i> < 0.05; ** <i>P</i> < 0.01 compared with Control.</p

Activation of FGFR-related intracellular pathways in sperm exposed to FGF2.

<p>Sperm were incubated for a total 4-h period and exposed to FGF2 (0, 1, 10 and 100 ng/ml) for the last 15 min. In some aliquots, sperm were incubated for 15 min with BGJ398 (0.1 μM) before the addition of FGF2. (<b>A</b>) Immunolocalization of pERK and pAkt in sperm incubated in the absence of FGF2 (Control), with 100 ng/ml FGF2, and with BGJ398 + 100 ng/ml FGF2. Sperm were processed for immunocytochemistry, stained with anti pERK or pAkt and FITC-conjugated secondary antibodies; nuclei were stained with propidium iodide. Bar: 10 μm. (<b>B</b>) Percentage of sperm cells stained with anti pERK and anti pAkt after exposure to FGF2 in the absence (<b>left</b>) or presence of BGJ398 (<b>right</b>). Results are expressed as mean ± SEM, n = 4. * <i>P</i> < 0.05; ** <i>P</i> < 0.01 compared with Control. (<b>C</b>) Phosphorylation of ERK and Akt assessed by Western immunoblotting. Protein extracts from human sperm were subjected to SDS-PAGE and Western immunoblotting using anti pERK, ERK, pAkt and Akt antibodies. The estimated molecular weights of the protein bands are indicated on the right. (<b>D</b>) Densitometric analysis of Western immunoblotting results for pERK normalized to ERK and pAkt normalized to Akt. Results are expressed as mean ± SEM, n = 5 for ERK and n = 5 for Akt. * <i>P</i> < 0.05 and ** <i>P</i> < 0.01 compared with Control.</p

Localization of FGFRs in human sperm.

<p>Sperm cells were stained with anti FGFR1, FGFR2, FGFR3 and FGFR4 or rabbit IgG and a secondary antibody labeled with Cy3. The corresponding fields stained with FITC-PSA to assess acrosomal status are shown. Bar: 10 μm. On the right, a representative image of individual sperm is depicted; (<b>A</b>) sperm stained with anti FGFR antibody and Cy3-conjugated secondary antibody, (<b>B</b>) FITC-PSA, (<b>C</b>) merge.</p

Localization of FGFRs in human seminiferous epithelium.

<p>Immunohistochemical analysis of FGFRs in human testis using anti FGFR antibodies; rabbit IgG was included as control. The specimens were counterstained with hematoxylin. S: Sertoli cell, Sg: spermatogonia, Sc: spermatocyte, St: spermatid. Arrows indicate immunoreactivity for FGFRs in the flagellum of elongating/elongated spermatids and the arrow head indicates FGFR4 immunoreactivity in spermatid acrosome. Bar: 20 μm.</p

Effect of sperm incubation with FGF2 on sperm motility.

<p>Sperm were incubated with 0, 10 and 100 ng/ml FGF2 in the absence or in the presence of 0.1 μM BGJ398 and subjected to computer-assisted sperm analysis. (<b>A</b>) Percentages of progressive (Grade a + b) and total motility (Grade a + b + c) for aliquots incubated in the absence (<b>left</b>) or in the presence of BGJ398 (<b>right</b>). Results are expressed as mean ± SEM, n = 5. ** <i>P</i> < 0.01 compared with Control. (<b>B</b>) Individual recordings of the effect of sperm incubation with FGF2 (0, 10 and 100 ng/ml) on the percentage of total sperm motility in samples with low and high sperm motility. Each sample is identified with a different symbol (n = 12). (<b>C</b>) The percentages of sperm with Grade a, b, c and d motility in each condition (as defined in Materials and Methods) is depicted. * <i>P</i> < 0.05; ** <i>P</i> < 0.01 compared with the same Grade in Control (n = 12).</p