Limits...
Effect of estrogens on boar sperm capacitation in vitro.

Ded L, Dostalova P, Dorosh A, Dvorakova-Hortova K, Peknicova J - Reprod. Biol. Endocrinol. (2010)

Bottom Line: Individual estrogens have relatively same effect on capacitation progress.According to our results, there is significant difference in the response to tested estrogens at different capacitation time and among individual animals.In animals with a high response to estrogens, there is a concentration-dependent stimulation of capacitation and individual estrogens have relatively the same effect.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Diagnostics for Reproductive Medicine, Institute of Biotechnology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.

ABSTRACT

Background: Mammalian sperm must undergo a series of controlled molecular processes in the female reproductive tract called capacitation before they are capable of penetrating and fertilizing the egg. Capacitation, as a complex biological process, is influenced by many molecular factors, among which steroidal hormone estrogens play their role. Estrogens, present in a high concentration in the female reproductive tract are generally considered as primarily female hormones. However, there is increasing evidence of their important impact on male reproductive parameters. The purpose of this study is to investigate the effect of three natural estrogens such as estrone (E1), 17beta-estradiol (E2) and estriol (E3) as well as the synthetical one, 17 alpha-ethynylestradiol (EE2) on boar sperm capacitation in vitro.

Methods: Boar sperm were capacitated in vitro in presence of estrogens. Capacitation progress in control and experimental samples was analyzed by flow cytometry with the anti-acrosin monoclonal antibody (ACR.2) at selected times of incubation. Sperm samples were analyzed at 120 min of capacitation by CTC (chlortetracycline) assay, immunocytochemistry and flow cytometry with anti-acrosin ACR.2 antibody. Furthermore, sperm samples and capacitating media were analyzed by immunocytochemistry, ELISA with the ACR.2 antibody, and the acrosin activity assay after induced acrosomal reaction (AR).

Results: Estrogens stimulate sperm capacitation of boar sperm collected from different individuals. The stimulatory effect depends on capacitation time and is highly influenced by differences in the response to estrogens such as E2 by individual animals. Individual estrogens have relatively same effect on capacitation progress. In the boar samples with high estrogen responsiveness, estrogens stimulate the capacitation progress in a concentration-dependent manner. Furthermore, estrogens significantly increase the number of acrosome-reacted sperm after zona pellucida- induced acrosomal reaction.

Conclusions: We demonstrate here the stimulatory effect of four different estrogens on boar sperm capacitation in vitro. According to our results, there is significant difference in the response to tested estrogens at different capacitation time and among individual animals. In animals with a high response to estrogens, there is a concentration-dependent stimulation of capacitation and individual estrogens have relatively the same effect. Effects of individual estrogens, differences in the response to them by individual animals, their time and concentration-dependent outcomes further contribute to our knowledge about steroidal action in sperm.

Show MeSH

Related in: MedlinePlus

ACR.2 Acrosomal fluorescent patterns (FITC-conjugated secondary antibody). Representative pictures of three specific ACR.2 acrosomal fluorescent patterns. (A) Uncapacitated, acrosome intact sperm - moderate uniform fluorescence in the acrosomal area; (B) Capacitated, acrosome-intact sperm - intensive fluorescence of the acrosome; (C) Acrosome-reacted sperm - low or no fluorescent signal in the sperm head. Nuclei stained with a Blue DAPI dye.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2908632&req=5

Figure 2: ACR.2 Acrosomal fluorescent patterns (FITC-conjugated secondary antibody). Representative pictures of three specific ACR.2 acrosomal fluorescent patterns. (A) Uncapacitated, acrosome intact sperm - moderate uniform fluorescence in the acrosomal area; (B) Capacitated, acrosome-intact sperm - intensive fluorescence of the acrosome; (C) Acrosome-reacted sperm - low or no fluorescent signal in the sperm head. Nuclei stained with a Blue DAPI dye.

Mentions: ACR.2 immunofluorescent analysis was described previously [22,23]. After the capacitation process, sperm suspensions were centrifuged; the capacitation medium was removed, and kept at -20°C. Sperm were re-suspended in equal volume of phosphate-buffered saline (PBS), smeared onto glass slides, dried and kept at 4°C. During fluorescent specimen preparation, sperm slides were fixed with acetone for 10 min, rinsed with PBS, treated with ACR.2 monoclonal antibody and incubated in a wet chamber for 60 min at 37°C. After thorough washing in PBS, the smears were treated with FITC-conjugated anti-mouse IgG antibody (Sigma, Prague, Czech Republic) and again incubated in a wet chamber for 60 min at 37°C. After washing in PBS and water, smears were mounted by the Vectashield mounting medium with DAPI (Vector Lab., Burlingame, CA). Samples were examined with a Nikon Labothot-2 fluorescent microscope equipped with 40× Nikon Plan 40/0.65 and photographed with a COHU 4910 CCD camera (Inc. Electronics Division, San Diego, USA) using LUCIA imaging software (Laboratory Imaging Ltd., Prague, Czech Republic). Sperm were classified according to their acrosomal staining patterns. (A) Moderate fluorescence in the acrosomal area - uncapacitated, acrosome intact sperm; (B) Intensive fluorescence of the acrosome -- capacitated, acrosome-intact sperm; (C) Low or no fluorescent signal in the sperm head with a remaining positive equatorial segment - acrosome-reacted sperm (Fig. 2). Sperm with nonspecific or intermediate acrosomal status were not selected for subsequent analysis. In each sample, 200 cells were evaluated and the minimal number of evaluated samples was 5.


Effect of estrogens on boar sperm capacitation in vitro.

Ded L, Dostalova P, Dorosh A, Dvorakova-Hortova K, Peknicova J - Reprod. Biol. Endocrinol. (2010)

ACR.2 Acrosomal fluorescent patterns (FITC-conjugated secondary antibody). Representative pictures of three specific ACR.2 acrosomal fluorescent patterns. (A) Uncapacitated, acrosome intact sperm - moderate uniform fluorescence in the acrosomal area; (B) Capacitated, acrosome-intact sperm - intensive fluorescence of the acrosome; (C) Acrosome-reacted sperm - low or no fluorescent signal in the sperm head. Nuclei stained with a Blue DAPI dye.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2908632&req=5

Figure 2: ACR.2 Acrosomal fluorescent patterns (FITC-conjugated secondary antibody). Representative pictures of three specific ACR.2 acrosomal fluorescent patterns. (A) Uncapacitated, acrosome intact sperm - moderate uniform fluorescence in the acrosomal area; (B) Capacitated, acrosome-intact sperm - intensive fluorescence of the acrosome; (C) Acrosome-reacted sperm - low or no fluorescent signal in the sperm head. Nuclei stained with a Blue DAPI dye.
Mentions: ACR.2 immunofluorescent analysis was described previously [22,23]. After the capacitation process, sperm suspensions were centrifuged; the capacitation medium was removed, and kept at -20°C. Sperm were re-suspended in equal volume of phosphate-buffered saline (PBS), smeared onto glass slides, dried and kept at 4°C. During fluorescent specimen preparation, sperm slides were fixed with acetone for 10 min, rinsed with PBS, treated with ACR.2 monoclonal antibody and incubated in a wet chamber for 60 min at 37°C. After thorough washing in PBS, the smears were treated with FITC-conjugated anti-mouse IgG antibody (Sigma, Prague, Czech Republic) and again incubated in a wet chamber for 60 min at 37°C. After washing in PBS and water, smears were mounted by the Vectashield mounting medium with DAPI (Vector Lab., Burlingame, CA). Samples were examined with a Nikon Labothot-2 fluorescent microscope equipped with 40× Nikon Plan 40/0.65 and photographed with a COHU 4910 CCD camera (Inc. Electronics Division, San Diego, USA) using LUCIA imaging software (Laboratory Imaging Ltd., Prague, Czech Republic). Sperm were classified according to their acrosomal staining patterns. (A) Moderate fluorescence in the acrosomal area - uncapacitated, acrosome intact sperm; (B) Intensive fluorescence of the acrosome -- capacitated, acrosome-intact sperm; (C) Low or no fluorescent signal in the sperm head with a remaining positive equatorial segment - acrosome-reacted sperm (Fig. 2). Sperm with nonspecific or intermediate acrosomal status were not selected for subsequent analysis. In each sample, 200 cells were evaluated and the minimal number of evaluated samples was 5.

Bottom Line: Individual estrogens have relatively same effect on capacitation progress.According to our results, there is significant difference in the response to tested estrogens at different capacitation time and among individual animals.In animals with a high response to estrogens, there is a concentration-dependent stimulation of capacitation and individual estrogens have relatively the same effect.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Diagnostics for Reproductive Medicine, Institute of Biotechnology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.

ABSTRACT

Background: Mammalian sperm must undergo a series of controlled molecular processes in the female reproductive tract called capacitation before they are capable of penetrating and fertilizing the egg. Capacitation, as a complex biological process, is influenced by many molecular factors, among which steroidal hormone estrogens play their role. Estrogens, present in a high concentration in the female reproductive tract are generally considered as primarily female hormones. However, there is increasing evidence of their important impact on male reproductive parameters. The purpose of this study is to investigate the effect of three natural estrogens such as estrone (E1), 17beta-estradiol (E2) and estriol (E3) as well as the synthetical one, 17 alpha-ethynylestradiol (EE2) on boar sperm capacitation in vitro.

Methods: Boar sperm were capacitated in vitro in presence of estrogens. Capacitation progress in control and experimental samples was analyzed by flow cytometry with the anti-acrosin monoclonal antibody (ACR.2) at selected times of incubation. Sperm samples were analyzed at 120 min of capacitation by CTC (chlortetracycline) assay, immunocytochemistry and flow cytometry with anti-acrosin ACR.2 antibody. Furthermore, sperm samples and capacitating media were analyzed by immunocytochemistry, ELISA with the ACR.2 antibody, and the acrosin activity assay after induced acrosomal reaction (AR).

Results: Estrogens stimulate sperm capacitation of boar sperm collected from different individuals. The stimulatory effect depends on capacitation time and is highly influenced by differences in the response to estrogens such as E2 by individual animals. Individual estrogens have relatively same effect on capacitation progress. In the boar samples with high estrogen responsiveness, estrogens stimulate the capacitation progress in a concentration-dependent manner. Furthermore, estrogens significantly increase the number of acrosome-reacted sperm after zona pellucida- induced acrosomal reaction.

Conclusions: We demonstrate here the stimulatory effect of four different estrogens on boar sperm capacitation in vitro. According to our results, there is significant difference in the response to tested estrogens at different capacitation time and among individual animals. In animals with a high response to estrogens, there is a concentration-dependent stimulation of capacitation and individual estrogens have relatively the same effect. Effects of individual estrogens, differences in the response to them by individual animals, their time and concentration-dependent outcomes further contribute to our knowledge about steroidal action in sperm.

Show MeSH
Related in: MedlinePlus