Biomedical subjects

María José Munuce

Publications and source records attributed to María José Munuce.

7 recordsLinked to original sources

PubMed2006

Comparative concentrations of steroid hormones and proteins in human peri-ovulatory peritoneal and follicular fluids.

Despite the fact that both peritoneal (PF) and follicular (FF) fluids have a common ovarian origin, FF is a natural inducer of sperm acrosome reaction (AR) while PF is not. To better understand these effects, concentrations of oestradiol, progesterone and proteins in peri-ovulatory PF and FF were determined and compared. PF was aspirated by laparoscopy at the peri-ovulatory stage from women with unexplained infertility. FF was collected from patients undergoing IVF and pooled. PF and FF were tested for the presence of antisperm antibodies. Oestradiol and progesterone were measured by enzyme immunoassay, and total protein concentration was determined and analysed. The AR was determined in spermatozoa that were exposed to PF alone, progesterone-supplemented PF, progesterone, control medium, or ethanol. No antisperm antibodies were found in any fluid tested. Oestradiol and progesterone and concentrations in PF were significantly lower than in FF. Protein concentration was also significantly lower in PF than in FF, but no differences were observed between the electrophoretic patterns. When capacitated spermatozoa were exposed to progesterone-supplemented PF there was a significant increase in the percentage of AR with respect to those in PF, control medium or ethanol. These results suggest that the lack of AR-stimulating activity of PF was related to its lower progesterone concentration compared with FF.

Acrosome Reaction↗

PubMed2005

Doses of levonorgestrel comparable to that delivered by the levonorgestrel-releasing intrauterine system can modify the in vitro expression of zona binding sites of human spermatozoa.

BACKGROUND: The levonorgestrel-releasing intrauterine system (LNG-IUS) exerts its contraceptive effect by interfering with sperm transport through the female genital tract and with ovulation. However, the possibility cannot be discarded that the device exerts a direct effect on sperm function, thus, helping prevent fertilization. OBJECTIVES: The purpose of this study is to evaluate whether LNG at doses comparable to that measured in the uterus during the use of the LNG-IUS affects the detection of D-mannose binding sites or zona pellucida (ZP) receptors on human spermatozoa. The association with acrosomal status was also investigated. METHODS: Seventeen semen samples from fertile men were used, and spermatozoa were separated using a Percoll gradient and incubated for 22 h at 37 degrees C under 5% CO(2) in air. Capacitated spermatozoa were exposed for 30 min to 1,000 or 10,000 ng/mL of LNG or control medium. D-Mannose binding sites were detected using commercial D-mannosylated bovine serum albumin conjugated with fluorescein isothiocyanate, and the percentage of specific patterns (II and III) was recorded. The acrosome reaction was evaluated using the Pisum sativum technique. RESULTS: Levonorgestrel releasing significantly increased (p < .001) the percentage of spermatozoa with D-mannose receptors localized in pattern III, and this increase was dose dependent and a significant increase (p < .001) in the percentage of acrosome-reacted spermatozoa. Double staining confirmed an association between the location of the zona receptor and acrosomal status. RESULTS: The in vitro exposure of capacitated spermatozoa to the assayed doses of LNG increased the proportion of spermatozoa with fewer chances of interacting with the ZP. Further studies should be carried out to confirm whether this mechanism is part of the contraceptive action of the LNG-IUS.

Acrosome Reaction↗

PubMed2005

In vitro effect of levonorgestrel on sperm fertilizing capacity and mouse embryo development.

The objectives of this study were to assess the expression of alpha-d-mannose binding sites in human spermatozoa, human sperm-oocyte interaction and the development of early stages of mouse embryo in the presence of levonorgestrel (LNG). Semen samples were obtained from 16 normozoospermic men. Spermatozoa were separated by Percoll gradient and incubated overnight for capacitation. The kinetic analysis of the expression of alpha-D-mannose binding sites was determined at 0, 4 and 22 h and in 22 h-capacitated spermatozoa that had been exposed to 1, 10 or 100 ng/mL of LNG or to a control medium for 30 min. Sperm binding sites for alpha-D-mannose were detected using commercial alpha-D-mannosylated bovine serum albumin conjugated with fluorescein isothiocyanate. To evaluate sperm-oocyte interaction, each oocyte was placed in a 100-microL droplet containing one of the three doses of LNG or control medium and inseminated with 1.0 x 10(5) motile spermatozoa/mL, after which the number of bound spermatozoa was evaluated. A total of 157 two-cell embryos recovered from eight mice was pooled and assigned randomly to treatment (1, 10 or 100 ng/mL of LNG) or control groups. There was a significant increase in the expression of specific alpha-d-mannose binding sites (Patterns II and III) during the incubation of spermatozoa under capacitating conditions. In the presence of LNG, results showed that there was no significant difference in the expression of specific alpha-d-mannose binding sites (Patterns II and III) at any LNG concentration tested compared with those spermatozoa in control medium. None of the LNG concentrations were capable of modifying the number of spermatozoa tightly bound to the human zona pellucida. There was no association between the presence or absence of LNG or the different doses of LNG evaluated and mouse embryo development. In conclusion, the hypothesis that in vitro exposure to LNG could interfere with sperm function and could contribute to the mechanism of action of this form of contraception was not confirmed but cannot be ruled out by the results of this study.

Animals↗

PubMed2005

The in vitro effect of emergency contraception doses of levonorgestrel on the acrosome reaction of human spermatozoa.

INTRODUCTION: The aim of this study was to evaluate the effect of three concentrations of levonorgestrel (LNG) comparable to the levels found in serum following ingestion of LNG as emergency contraception (EC) on the acrosome reaction (AR) of capacitated and noncapacitated spermatozoa of fertile men. MATERIALS AND METHODS: A total of 24 semen samples from three fertile men were evaluated. The spermatozoa were selected by Percoll gradient. Twelve samples were subsequently incubated with human tubal fluid medium supplemented with bovine serum albumin (HTF/BSA) for 20 h under capacitating conditions. The capacitated spermatozoa and the spermatozoa from the remaining 12 samples were exposed to LNG at 1, 10 and 100 ng/mL, to follicular fluid (FF) (20 %v/v) and to HTF medium. The ratio of live to dead spermatozoa was assessed after 1, 2 and 3 h of incubation at 37 degrees C and 5% CO2. After 30 min of exposure to the different LNG concentrations, aliquots were divided into two parts. In the first part, spermatozoa were immediately stained with Hoescht 33258 and fluorescein isothiocyanate-pisum sativum agglutinin (FITC-PSA) in order to assess AR rate and to repeat evaluation of the live-to-dead ratio. After 3 h of incubation, the remaining part of the aliquots were submitted to the same procedures. Each concentration of LNG was then compared with FF and HTF medium as positive and negative controls, respectively. RESULTS: The results showed that in vitro exposure to the three different LNG concentrations did not induce AR. CONCLUSION: This study failed to show any in vitro effect on AR of LNG concentrations similar to those found in serum following intake of LNG as EC. If this effect exists or if there is any other that influences sperm fertilizing capacity, in vitro experiments are probably not an appropriate way of testing it.

Acrosome Reaction↗

PubMed2005

Effects of human oviductal in vitro secretion on spermatozoa and search of sperm-oviductal proteins interactions.

It has been suggested that oviductal proteins could be involved in modulating sperm function and fertilizing ability through as yet not well-known mechanisms. The objective of the study was to investigate the pattern of proteins secreted by human oviductal tissue cultures and the effects of their conditioned media (CM) on sperm function under capacitating conditions and in phosphate buffered saline (PBS). In addition, interactions between spermatozoa and oviductal proteins were examined. The oviductal tissue was obtained from pre-menopausal patients scheduled for hysterectomies because of uterine fibromyoma. Normozoospermic semen samples were obtained from healthy donors. Cultures of human fallopian tissue were carried out and CM were collected for analysis of the de novo production of [35S]-methionine-labelled proteins by SDS-PAGE. Motile spermatozoa were incubated under capacitating conditions and in PBS, with or without CM, and sperm fertilizing ability was assessed by ionophore-induced acrosome reaction (AR) and the acrosome reaction to ionophore challenge (ARIC) score. The ionophore-induced AR was evaluated by the Pisum sativum technique. Sixteen de novo produced proteins were detected in CM. One of these proteins (molecular weight 79 kDa) was detected in extracts from spermatozoa pre-incubated with CM. Sperm survival and motility were maintained in the presence of CM, although results showed a significant decrease in ARIC score (p < 0.05), with respect to controls. The presence of CM significantly decreased sperm fertilizing ability, without affecting sperm survival. These results suggest that the oviductal secretion could contribute to preserve sperm viability and motility, and to prevent a premature response of spermatozoa to AR inducers.

Cell Survival↗

PubMed2003

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.

Acrosome↗

PubMed2003

Modulation of human sperm function by peritoneal fluid.

OBJECTIVE: To examine the effect of peritoneal fluid on various parameters of sperm function in vitro. DESIGN: Prospective study. SETTING: Basic research laboratory. PATIENT(S): Semen samples were obtained from normozoospermic volunteers (n = 43). Peritoneal fluids were aspirated laparoscopically from women with unexplained infertility (n = 14). Follicular fluid and oocytes were collected from patients undergoing IVF-ET. INTERVENTION(S): Sperm incubated under capacitating conditions were exposed to peritoneal fluid, and functional variables were evaluated in vitro. MAIN OUTCOME MEASURE(S): Sperm viability and motility, follicular fluid and calcium ionophore-induced acrosome reactions, protein tyrosine phosphorylation, expression of D-mannose binding sites, and ability of sperm to interact with zona pellucida. RESULT(S): Exposure of sperm to peritoneal fluid for up to 6 hours did not affect sperm viability or motility. Unlike follicular fluid, peritoneal fluid did not induce the acrosome reaction. Moreover, incubation of sperm with > or =20% v/v peritoneal fluid for 1 hour prevented the follicular fluid and the ionophore-induced acrosome reaction. Although treatment with peritoneal fluid allowed protein tyrosine phosphorylation during capacitation, it resulted in a significant decrease in the expression of D-mannose binding sites and sperm-zona pellucida binding. CONCLUSION(S): Peritoneal fluid maintains sperm survival and decreases sperm ability to respond to inducers of the acrosome reaction and bind to the zona pellucida in vitro, indicating that this fluid might modulate sperm function in vivo.

Acrosome Reaction↗