S100 A9 is expressed and secreted by the oviduct epithelium, interacts with gametes and affects parameters of human sperm capacitation in vitro.

Our previous findings demonstrate that some oviductal secretion proteins bind to gametes and affect sperm physiology and gamete interaction. One of these proteins possesses an estimated molecular weight of 14 kDa. The objective of this study was to isolate and identify this 14 kDa protein, to localize it in the human oviduct, to detect gamete binding sites for the protein, and to evaluate its effects on sperm capacitation parameters and gamete interaction. Explants from the human oviductal tissues of premenopausal women were cultured in the presence of [35 S]-Methionine-proteins ([35S]-Met-proteins). De novo synthesized secreted [35 S]-Met-proteins were isolated from the culture media by affinity chromatography using their sperm membrane binding ability and analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using liquid chromatography-tandem mass spectrometry peptide sequencing, human S100 A9 was identified as one of the isolated proteins from the 14 kDa protein band. S100 A9 was detected in oviduct epithelium and oviduct secretion using immunohistochemistry and a Western blot. S100 A9 binding to human oocytes and spermatozoa was assessed by indirect immunofluorescence. The acrosome reaction (AR) affected S100 A9 ability to bind sperm cells. The presence of S100 A9 significantly increased both the induced AR and the sperm protein tyrosine phosphorylation, with respect to controls. However, the protein did not affect sperm-zona pellucida interaction. Results indicate that S100 A9 is present in the human oviduct and that it modulates parameters of sperm capacitation in vitro. Hence, the protein might contribute to the regulation of the reproductive process in the oviductal microenvironment.

Study of the effect of ulipristal acetate on human sperm ability to interact with tubal tissue and cumulus-oocyte-complexes.

OBJECTIVE: Ulispristal acetate (UPA) is a selective progesterone receptor modulator widely used for emergency contraception (EC). The described main mechanism of action is by inhibiting or delaying ovulation; however, the postovulatory effects of the drug are still on debate. Therefore, the aim of this study was to determine whether UPA could interfere with human sperm fertilizing ability. STUDY DESIGN: Human motile spermatozoa were incubated under capacitating conditions with or without UPA, and then used to inseminate human tubal explants, mouse cumulus-oocyte complexes and zona-free hamster eggs. The ability of UPA to interact with human sperm progesterone (P)-binding sites was investigated by incubating the cells with fluorescent-labeled P and analyzing them by fluorescence microscopy. RESULTS: UPA did not affect the ability of human sperm to bind to human tubal tissue explants surface or to penetrate the mouse cumulus mass and the zona-free hamster eggs. In addition, concentrations of UPA much higher than those present in the plasma of EC pill users were required to bind to human sperm P-binding sites. CONCLUSIONS: Our study supports a lack of an agonist or antagonist action of UPA on different functional parameters associated with the fertilizing ability of human sperm. IMPLICATIONS: This study provides new functional evidence supporting that the contraceptive action of UPA is not related to effects on human sperm cells, contributing to a better understanding of the mechanism of action of UPA as EC.

Oviductal secretion and gamete interaction.

Experimental evidence from the last 30 years supports the fact that the oviduct is involved in the modulation of the reproductive process in eutherian mammals. Oviductal secretion contains molecules that contribute to regulation of gamete function, gamete interaction, and the early stages of embryo development. The oviductal environment would act as a sperm reservoir, maintaining sperm viability, and modulating the subpopulation of spermatozoa that initiates the capacitation process. It could also contribute to prevent the premature acrosome reaction and to reduce polyspermy. Many studies have reported the beneficial effects of the oviductal environment on fertilization and on the first stages of embryo development. Some oviductal factors have been identified in different mammalian species. The effects of oviductal secretion on the reproductive process could be thought to result from the dynamic combined action (inhibitory or stimulatory) of multiple factors present in the oviductal lumen at different stages of the ovulatory cycle and in the presence of gametes or embryos. It could be hypothesized that the absence of a given molecule would not affect fertility as its action could be compensated by another factor with similar functions. However, any alteration in this balance could affect certain events of the reproductive process and could perhaps impair fertility. Thus, the complexity of the reproductive process warrants a continuous research effort to unveil the mechanisms and factors behind its regulation in the oviductal microenvironment.

Effects of ulipristal acetate on sperm DNA fragmentation during in vitro incubation.

OBJECTIVE: Ulipristal acetate (UPA) acts as an emergency contraceptive by inhibiting ovulation. This study explores possible additional effects on the fragmentation of sperm DNA during in vitro incubation. METHODS: Motile spermatozoa from healthy donors were selected by swim-up and incubated under capacitating conditions in control medium or with UPA (1, 10, 100, 1,000 or 10,000 ng/ml). In some experiments, 200 µM of H2O2 were added to induce oxidative stress. The sperm chromatin dispersion test was performed to analyse DNA integrity (400 cells; 1000×). Lipid peroxidation (thiobarbituric acid assay), induced-acrosome reaction (AR) and sperm vitality (Eosin Y) were also evaluated in spermatozoa exposed to UPA and/or H2O2. RESULTS: During sperm incubation, the percentage of fragmented DNA increased significantly, from 15.0 ± 1.3 to 41.0 ± 4.5% (p < 0.001). In the presence of UPA, DNA fragmentation decreased significantly (p < 0.05), in a dose-dependent manner. At 100 and 1000 ng/ml, UPA also counteracted the effect of H2O2 and prevented DNA fragmentation. No effect on sperm vitality, lipid peroxidation or induced-AR was found with any treatment. CONCLUSIONS: During in vitro sperm capacitation DNA fragmentation increased but the latter was counteracted in the presence of UPA, which possibly acted as a scavenger of reactive oxygen species produced by spermatozoa.

Effect of exposure to ulipristal acetate on sperm function.

OBJECTIVE: A pill containing ulipristal acetate (UPA) is used for emergency contraception (EC). Considering that, following its intake, spermatozoa may be exposed to UPA in the female genital tract we intended to evaluate sperm functions after incubation with this compound. METHODS: Motile spermatozoa were selected by swim-up and were incubated under capacitating conditions with UPA (at concentrations of 1, 10, 100, 1,000, and 10,000 ng/ml) or control medium. The main outcome measures were sperm vitality, sperm protein tyrosine phosphorylation (TyrP), spontaneous acrosomal reaction (AR), and human follicular fluid (hFF)-induced AR. RESULTS: Sperm vitality and TyrP pattern were similar between spermatozoa exposed to UPA or control. In addition, spontaneous AR ranged from 14.0 ±1.5% to 18.0 ±1.9% after exposure to UPA or control medium without significant differences, and UPA did not prevent hFF-induced AR. CONCLUSIONS: Incubation of sperm with UPA at concentrations around the expected plasma levels after ingestion of this EC pill (˜100-200 ng/ml) did not modify the signal transduction of TyrP involved in sperm capacitation. Moreover, UPA showed no agonist effect on progesterone receptors because it did not induce AR. Considering that progesterone in hFF is essential for AR induction, and UPA did not prevent the hFF-induced AR, an antagonist action of UPA on the AR is unlikely.

Detection of mammaglogin A in blood from breast cancer patients, before and after treatment, using a one-tube nested PCR protocol. Association with the absence of tumor estrogen receptors.

OBJECTIVE: A one-tube nested RT-PCR protocol was set up and used to detect mammaglobin A (MGA) expression in blood samples from breast cancer patients. The correlation of MGA detection with prognostic factors was analyzed. DESIGN AND METHODS: Total RNA from nucleated blood cells was extracted from 65 breast cancer patients (before surgery and after the treatments) and 18 healthy subjects and used to detect MGA expression by a modified nested RT-PCR. RESULTS: MGA expression was detected in 38.4% of patients before surgery, and in 50% and 36.8% of post-treatment samples from patients that expressed MGA or were MGA negative before surgery, respectively. MGA detection was associated with the absence of tumor estrogen receptors (p=0.004). CONCLUSIONS: MGA detection by the modified nested RT-PCR is a specific marker for circulating tumor cells in patients with breast carcinoma and a negative prognostic factor for the disease.

Human tubal secretion can modify the affinity of human spermatozoa for the zona pellucida.

OBJECTIVE: To study the effect of the human tubal tissue conditioned medium (CM) on sperm parameters related to sperm-zona pellucida interaction. DESIGN: Controlled experimental laboratory study. SETTING: Research laboratory. SUBJECT(S): Semen samples from donors with normozoospermia. Human tubal tissue obtained from women undergoing hysterectomies. Human follicular fluids (hFF) and oocytes collected from patients undergoing IVF-ET. INTERVENTION(S): Incubation of spermatozoa with CM proteins obtained from human tubal tissue culture; sperm binding to the zona pellucida assessment. MAIN OUTCOME MEASURE(S): Explants' viability was assessed by tissue DNA analysis. Sperm ability to interact with zona was tested with use of the whole oocyte test. Expression of d-mannose binding sites was assessed with use of a fluorescent probe on mannose coupled to bovine serum albumin. Human FF-induced acrosome reaction was assessed by the Pisum sativum technique. RESULT(S): Although treatment with 0.8 microg/microL of CM allowed sperm binding to the zona and the expression of d-mannose binding sites comparable with sperm in control medium, with 3.2 microg/mL of CM resulted in a significant decrease of both parameters. No effect of CM on spontaneous or hFF-induced acrosome reaction or in sperm viability was observed. CONCLUSION(S): The results indicate that the incubation of spermatozoa in the presence of CM reduces sperm affinity for the zona pellucida. This effect can be partly explained by the decreased expression of d-mannose binding sites on the sperm surface.

Effect of human oviductal in vitro secretion on human sperm DNA integrity.

PURPOSE: In the female genital tract spermatozoa interact with the oviductal secretion. The aim of the study was to evaluate the effect of conditioned media (CM) from cultures of human oviductal tissue, on sperm DNA integrity. The effect of H(2)O(2) on sperm DNA integrity, before and after incubation under capacitating conditions, was also investigated. METHODS: Motile sperm obtained from normozoospermic semen samples were incubated (4 h or 22 h) in the presence or absence of CM and further exposed to H(2)O(2). DNA damage was detected by the comet assay. RESULTS: The CM significantly reduced the DNA damage associated with sperm incubation, and also decreased the effect of H(2)O(2) after 4 h incubation, compared to controls. The H(2)O(2) caused a dose-dependent deleterious effect on sperm DNA integrity both before and following 22 h of capacitation. CONCLUSION: The oviductal tissue CM increased the stabilization of the sperm DNA structure under culture conditions.

Búsqueda de células tumorales en sangre periférica de pacientes con carcinoma mamario : determinación de mamaglobina A (MGA): Influencia del tratamiento y valor pronóstico

Los objetivos de este estudio son investigar la expresión del mRNA del gen de la MGA en células de sangre periférica en pacientes diagnosticadas con cáncer de mama, previo al tratamiento y posteriormente al mismo, en busca de micrometástasis y correlacionar la detección del marcador con la presencia o ausencia de metástasis, con otros factores pronósticos y con la recurrencia de la enfermedad. Además, para evaluar la extensión del daño sobre el DNA antes y después del tratamiento con agentes genotóxicos externos en la terapia para el cáncer de mama se utilizará el ensayo del cometa sobre linfocitos de sangre periférica de las pacientes.

Búsqueda de células tumorales en sangre periférica de pacientes con carcinoma mamario : determinación de mamaglobina A (MGA): Influencia del tratamiento y valor pronóstico

Los objetivos de este estudio son investigar la expresión del mRNA del gen de la MGA en células de sangre periférica en pacientes diagnosticadas con cáncer de mama, previo al tratamiento y posteriormente al mismo, en busca de micrometástasis y correlacionar la detección del marcador con la presencia o ausencia de metástasis, con otros factores pronósticos y con la recurrencia de la enfermedad. Además, para evaluar la extensión del daño sobre el DNA antes y después del tratamiento con agentes genotóxicos externos en la terapia para el cáncer de mama se utilizará el ensayo del cometa sobre linfocitos de sangre periférica de las pacientes.

Búsqueda de células tumorales en sangre periférica de pacientes con carcinoma mamario : determinación de mamaglobina A (MGA): Influencia del tratamiento y valor pronóstico

Los objetivos de este estudio son investigar la expresión del mRNA del gen de la MGA en células de sangre periférica en pacientes diagnosticadas con cáncer de mama, previo al tratamiento y posteriormente al mismo, en busca de micrometástasis y correlacionar la detección del marcador con la presencia o ausencia de metástasis, con otros factores pronósticos y con la recurrencia de la enfermedad. Además, para evaluar la extensión del daño sobre el DNA antes y después del tratamiento con agentes genotóxicos externos en la terapia para el cáncer de mama se utilizará el ensayo del cometa sobre linfocitos de sangre periférica de las pacientes.