Direct effects of progesterone and antiprogesterone on human sperm hyperactivated motility and acrosome reaction.

OBJECTIVE: To study the direct effects of progesterone (P) and its antagonist RU486 (mifepristone) on sperm hyperactivation (HA) and acrosome reaction. DESIGN: Prospective evaluation of semen samples incubated in capacitation media with P and/or RU486. SETTING: University-affiliated tertiary care center. PATIENTS: Normal healthy volunteers. INTERVENTIONS: Semen samples were incubated in media with P or RU486 alone or in combination, and aliquots were taken at 10 minutes, 1, 5, and 24 hours for HA analyses by computer-aided sperm analysis system, and at 0, 5, and 24 hours for assessment of acrosome reaction by fluorescein-labeled Pisum sativum (pea) agglutinin. MAIN OUTCOME MEASURES: HA and acrosome reaction. RESULTS: Sperm HA was significantly increased at 10 minutes by P both at 10(-7) M (9.27 +/- 1.59%; mean +/- SEM) and 10(-6) M (9.39 +/- 1.94%) when compared with untreated spermatozoa (5.62 +/- 1.59%). The stimulatory effect of P on sperm HA was transient because this was not observed after 1, 5, and 24 hours of incubation. The antiprogesterone RU486 (10(-6) M) alone had no effect and did not abolish the stimulatory effect of P on HA. The %HA was further enhanced by the addition of RU486 at 10(-6) M to P at 10(-7) M (12.43 +/- 3.31%) or P at 10(-6) M (13.52 +/- 4.10%); however, this effect was not significantly different from P alone. Coincubation of P or RU486 with spermatozoa during capacitation did not stimulate the acrosome reaction in the concentrations tested. CONCLUSION: Progesterone directly stimulates human sperm HA transiently. Progesterone has no significant effect on acrosome reaction in capacitating spermatozoa. The effects of P are rapid and not counteracted by RU486, suggesting that the mechanism of action of P may not be mediated by specific P nuclear receptors.     

The effects of prolactin on human sperm capacitation and acrosome reaction

OBJECTIVE: To study the effects of human prolactin (PRL) on human sperm capacitation and acrosome reaction. DESIGN: Acrosome reactions were induced by the addition of follicular fluid (FF) and progesterone (P). Experiments were performed to determine time and dose-dependent effects of PRL on sperm capacitation, potentiation of the acrosome reaction, decapacitating effects, and potential for PRL to induce an acrosome reaction. RESULTS: An average of 31.5% of spermatozoa underwent acrosome reaction with addition of FF and P. No time- or dose-dependent PRL effects on sperm capacitation or acrosome reaction were found (P greater than 0.05). CONCLUSION: Prolactin does not play an important role in human sperm capacitation or acrosome reaction.     

Induction of the human sperm acrosome reaction by human oocytes

The acrosome reaction of human spermatozoa incubated in the presence or absence of vested human oocytes was investigated. All gametes were obtained from human in vitro fertilization (IVF) cases. Spermatozoa were collected after incubation in insemination medium only and following removal of the oocytes from insemination medium during the IVF procedure. After 16 hours of incubation 18.5% of the spermatozoa in insemination medium alone were acrosome-reacted compared to 31.5% for spermatozoa incubated in medium containing oocytes. The acrosome reaction of spermatozoa incubated with fertilized or unfertilized oocytes was also investigated. The percentage of acrosome reaction did not differ (P greater than 0.05) between the two groups (29.7% in the fertilized cases versus 30.7% in the unfertilized cases). Completion of oocyte nuclear maturation did not affect the proportion of acrosome-reacted spermatozoa observed with unfertilized eggs. A similar (P greater than 0.05) percentage of acrosome reacted spermatozoa were observed regardless of whether the unfertilized oocytes had (29%) or had not (35%) reached metaphase II. These findings indicate the acrosome reaction of human spermatozoa is enhanced in the presence of vested human oocytes. Furthermore, there is no apparent correlation between the percentage of the population of spermatozoa that acrosome react in the medium and the potential of an oocyte for fertilization.     

Dynamics of human sperm acrosome reaction: relation with in vitro fertilization

OBJECTIVE: Acrosomal status has been studied on human sperm prepared for in vitro fertilization (IVF) and related to the rate of fertilization. DESIGN AND PATIENTS: A group of 41 men with normal classical semen parameters, included in the IVF program of University of Nice for feminine tubal obstruction (n = 37) or unexplained infertility (n = 4), were evaluated in a prospective study and compared with a control group of 10 fertile donors. MAIN OUTCOME MEASURES: Evaluation of acrosome status (spontaneous and A23187-induced acrosome loss) after 6 hours incubation in Ménézo's B2 medium was made by flow cytometry on suspended cells with a new immunofluorescence test recently reported by the authors based on a monoclonal antibody GB24. RESULTS: Spontaneous acrosome loss remained low even after 6 hours capacitation (mean + 1 SD, 6.5% + 4.9%). Response to A23187 increased with the duration of preincubation with a marked response after 6 hours (29.5% + 8.9%). Low spontaneous acrosome loss (less than mean + 1 SD) and high response to A23187 (greater than mean - 1 SD) were observed in 25 out of 26 cases of group A with a high fertilization rate (greater than 50% fertilized oocytes). A high level of spontaneous acrosome loss and/or a lack of response to A23187 was observed in 2 of 7 cases of group B (fertilization rate less than 50%) and 6 of 8 cases of group C (unexplained unsuccessful fertilization). CONCLUSION: Impaired acrosomal status can be associated with unexplained unsuccessful fertilization.     

Evaluation of acrosome reaction and viability of human sperm with two fluorescent dyes

Objective: To evaluate the usefulness of the Hoechst 33258/FITC-Pisum sativum agglutinin (FITC-PSA) staining for simultaneous assessment of viability and acrosome reaction rate (%AR) of human sperm. Material and Methods: Fresh sperm was collected 13 fertile donors provided fresh semen. We used motile sperm selected by the ”swim-up” procedure using modified HTF. Hoechst 33258 was added and co-incubated with sperm for 10 min. Samples were washed free of unbound stain and the sperm were mounted as smears on glass slides. After drying, sperms were incubated with FITC-PSA for 30 min. Sperm were examined by fluorescence microscopy. Also, FITC-Concanavalin A (FITC-ConA) staining and vital staining with yellowish eosin were performed simultaneously. The correlation of viability and %AR were analyzed. Results: Four different staining patterns were observed and clearly distinguished as follows: a) Viable acrosome-reacted sperm, b) Viable acrosome-intact sperm, c) dead acrosome-reacted sperm, d) dead acrosome-intact sperm. There was significant correlation between the results obtained by Hoechst 33258 and vital staining methods in viability of human sperm (r=0.927, P<0.001). There was significant correlation between the two methods (FITC-PSA and FITC-ConA) in %AR of human sperm (r=0.92, P<0.001). Conclusion: Viability and acrosome status of a human sperm can be easily assessed simultaneously by Hoechst 33258/FITC-PSA staining method. This combination method is considered useful to evaluate sperm function. Received: 8 May 2000 / Accepted: 31 July 2000     

Correlation between sperm penetration assay and acrosome reaction in sperm treated with human follicular fluid.

In this study, we determined whether there was a correlation between sperm penetration assay (SPA) and acrosome reaction (AR) in sperm after being treated with human follicular fluid (hFF). Sperm specimens were obtained from 13 men participating in our in vitro fertilization program. After washing and preincubation, aliquots of sperm samples were treated with either hFF or Ham's F-10 medium (control). Approximately 1.0 x 10(6) motile sperm from the control and hFF-treated samples were coincubated individually with zona-free hamster eggs for SPA. Sperm remaining in the suspension after coincubation with eggs were taken to determine the incidence of AR by a triple-stain technique. The data revealed that an increased SPA score in hFF-treated samples was related to a higher incidence of acrosome-reacted sperm.     

The influence of various molecules on the occurrence of the human sperm acrosome reaction

The acrosome reaction is essential for fertilization, but the mechanism of the acrosome reaction of human spermatozoa is not clear at the present time. We studied the mechanism to analyze the cause of unexplained infertility, the appropriate timing of insemination, and the environment of spermatozoa prior to fertilization. For this study, we examined the effects of Ca++, Mg++, Kallikrein, Phospholipase A2, p-bromophenacyl bromide (Phospholipase A2 specific inhibitor), Lysophosphatidyl choline, Arachidonic acid, and Glyceryl monooleate using in vitro penetration assay employing zona- free hamster eggs. Results obtained were as follows. When human spermatozoa were incubated in mBWW with Ca++ or (and) Mg++ free medium, the acrosome reaction was inhibited. When human spermatozoa were incubated in mBWW with Kallikrein (1.0-4.0 KU ml), the acrosome reaction was promoted. When Phospholipase A2 was used at concentrations of 0.2 and 2.0 unit/ml, penetration rates showed the same tendency as in the control. But when p-bromophenacyl bromide was tested at concentrations of 1 X 10(-5) - 1 X 10(-3)M, penetration rates were inhibited when compared with the control. When human spermatozoa were incubated in medium containing Lysophosphatidyl choline (50 micrograms/ml), Arachidonic acid (5-50 micrograms/ml), and Glyceryl monoleate (300-400 micrograms/ml), the acrosome reaction was accelerated.     

Progesterone requires heat shock protein 90 (HSP90) in human sperm to regulate motility and acrosome reaction

Purpose

The aims of this paper were to study whether heat shock protein 90 (HSP90) is a regulator of sperm functions and to determine its association with oligoasthenozoospermia.

Methods

The levels of HSP90 in sperm lysates were measured by ELISA. Localization of HSP90 and its isoforms was evaluated by immunofluorescence. Sperm motility and kinetics were assessed by computer-assisted sperm analysis. Acrosome reaction was determined by lectin staining.

Results

The levels of HSP90 were lower in oligoasthenozoospermic men and correlated positively with the number of motile spermatozoa. In capacitated human spermatozoa, HSP90α was mostly found in residual nuclear envelope, and the HSP90β isoform was higher in the flagella. Inhibition of HSP90 by geldanamycin or 17-AAG did not affect basal motility, but suppressed progesterone-mediated forward progressive motility, hyperactivation and acrosome reaction. Progesterone treatment dephosphorylated both HSP90α and HSP90β at Ser/Thr-Pro residues, but not Tyr residues.

Conclusion

HSP90 levels are downregulated in oligoasthenozoospermia, and its functional inhibition attenuates progesterone-mediated sperm motility and acrosome reaction.

     

Comparative flow cytometric analysis of the human sperm acrosome reaction using CD46 antibody and lectins

Purpose: Our purpose was to determine the most suitable marker for the human sperm acrosome reaction, based on detection of CD46 antibody binding compared with lectin binding. Methods: Flow cytometric analysis of CD46 antibody versus lectins (PNA, PSA, and Con A) was used to quantify the acrosome reaction of human sperm. Results: Neither PSA nor Con A was able to detect significant changes in the spontaneous and ionophore-induced acrosome reactions compared to CD46 antibody. However, PNA was found to exhibit a binding pattern similar to that observed with CD46 and could be used to quantify measurable changes in acrosomal response to ionophore, albeit of a lower magnitude than the responses detected by CD46. Conclusions: We conclude that PNA binds to the inner acrosomal membrane of acrosome-reacted sperm and is suitable for use as a marker of the acrosome reaction by flow cytometry. Data are presented which clarify the assessment of the acrosome reaction when CD46 and lectins are used.     

Inhibition of the human sperm acrosome reaction by a high molecular weight factor from human seminal plasma

Human seminal plasma possesses a factor (acrosome reaction-inhibitory factor) that is precipitated by high speed centrifugation and that inhibits the ionophore- and dbc AMP-induced acrosome reaction of capacitated human spermatozoa but only if it is added toward the end of the capacitation period. Acrosome reaction-inhibitory factor can be partially purified by cation exchange chromatography and appears to differ from another factor that can be obtained by ultracentrifugation of human seminal plasma and that prevents the fertilization of mouse gametes.     

Effect of fatty acids on boar sperm motility viability and acrosome reaction

Aim  The present study was undertaken to determine which fatty acids improve motility viability and increase acrosome reaction (AR) in boar spermatozoa. Methods  Boar spermatozoa were washed, swum-up and incubated at 37°C for 4 h in TALP medium supplemented with myristic, palmitic, stearic, lignoceric, oleic, linoleic, arachidonic, docosahexaenoic and palmitoleic acid. Sperm motility, viability and AR were evaluated during 4 h of incubation. Results  Results show that oleic and linoleic acid significantly improved (P < 0.05) the motility and viability of boar spermatozoa. The AR was significantly improved (P < 0.05) by oleic and arachidonic acid in almost all incubation periods. When combinations of oleic, linoleic and arachidonic acid were studied for motility, viability and AR, it was found that oleic plus linoleic acid significantly increased (P < 0.05) motility, whereas arachidonic plus oleic acid significantly increased (P < 0.05) AR. Conclusion  Unsaturated fatty acids, especially arachidonic acid, can improve boar sperm motility and AR. A combination of arachidonic and oleic acid is important for inducing boar sperm AR.     

Recombinant human OVGP1 increases intracellular calcium and further potentiates the effects of progesterone on human sperm

PurposeTo investigate the effects of recombinant human oviduct–specific glycoprotein (rHuOVGP1) alone and in combination with progesterone (P4) on intracellular Ca²⁺ concentration [Ca²⁺]_i and to investigate if rHuOVGP1 in combination with P4 can further enhance tyrosine phosphorylation (pY) of sperm proteins during human sperm capacitation.MethodsFluorometric flow cytometry was performed to examine the effects of rHuOVGP1 on [Ca²⁺]_i in human sperm during capacitation. Confocal microscopy was used in conjunction with live cell imaging to analyze the influence of rHuOVGP1 and P4 on [Ca²⁺]_i in the sperm tail and to examine the involvement of CatSper channels in their effect on [Ca²⁺]_i. Western blot analysis was performed to assess the protein levels of p105, a major tyrosine-phosphorylated sperm protein.ResultsrHuOVGP1 increases [Ca²⁺]_i in human sperm at the beginning of capacitation and further increases and sustains the level of [Ca²⁺]_i in the sperm tail following the addition of P4. Inhibition of CatSper channels impedes the effects of rHuOVGP1 on [Ca²⁺]_i in the sperm tail. P4 alone can increase pY of a major human sperm protein, p105, yet yields a further increase when used in combination with rHuOVGP1.ConclusionThe present study revealed that rHuOVGP1 may work with P4 to upregulate [Ca²⁺]_i at the beginning of capacitation in part through CatSper channels which, in turn, leads to the downstream event of pY of sperm proteins and enhancement of sperm capacitation.Supplementary informationThe online version contains supplementary material available at 10.1007/s10815-022-02591-0.     

Modern human sperm freezing: Effect on DNA,chromatin and acrosome integrity

Objective

Presence of vitrification method in sperm freezing and the introduction of solid surface vitrification beside rapid freezing in vapour, opens an easy and safe way to help infertility centres. While the effects of cryopreservation on motility, morphology and viability of sperm are documented, the question of the probable alteration of sperm DNA, chromatin and acrosome integrity after freezing and thawing procedures in different methods is still controversial.

Effect of peritoneal fluid from endometriosis patients on sperm motion characteristics and acrosome reaction

OBJECTIVE: To determine whether peritoneal fluid from women with endometriosis contributes to infertility by impairing sperm motion and functional characteristics. METHODS: Women with endometriosis (n = 20) underwent laparoscopy for infertility or pelvic pain. Patients undergoing tubal ligation served as controls (n = 14). Peritoneal fluid was aspirated from women with endometriosis, or from women undergoing laparoscopic tubal ligation. Sperm motility, motion characteristics and acrosome reaction were assessed following incubation with peritoneal fluid. RESULTS: Sperm motility, motion characteristics, and acrosome reaction did not differ significantly between the two groups after 3, 5, or 24 hours of incubation with peritoneal fluid. CONCLUSIONS: Sperm motion or functional characteristics showed no significant impairment when sperm from normal donors were incubated with peritoneal fluid from patients with endometriosis. It is unlikely that peritoneal fluid in these patients contributes to infertility.     

Flow cytometric quantitation of the expression of membrane cofactor protein as a marker for the human sperm acrosome reaction.

A mAb (TRA-2-10, IgG1) to an embryonal carcinoma cell line (2102ep) that recognizes an antigen termed membrane cofactor protein (CD 46, TLX antigen) binds to human sperm after chemical induction of acrosomal loss by Ca2+ ionophore. An indirect immunofluorescence assay was developed in which sperm membrane cofactor protein was detected by flow cytometry on acrosome-reacted living human sperm. The expression of the membrane cofactor protein antigen on acrosome-reacted sperm may represent a marker that can be used in a rapid, quantitative, and reproducible flow cytometric assay for the evaluation of human sperm acrosomal loss.