Resumen
The aim of this study was to identify the presence of NADPH oxidase 5 (NOX5) in ram spermatozoa and
to investigate if melatonin could modulate this enzyme during in vitro capacitation. Semen from nine
Rasa Aragonesa rams was collected and pooled. Seminal plasma free spermatozoa were selected by a
swim-up procedure (control sample). Spermatozoa were then incubated in TALP medium without (TALP
sample) or with cAMP-elevating agents (cocktail sample, Ck) for 3 h at 39 °C and 5% CO2. 1 µM
melatonin was added to TALP and cocktail samples (TALP-Mel and Ck-Mel) (n=6). Capacitation status
was evaluated by chlortetracycline (CTC) staining. Identification and distribution of NOX5 in ram
spermatozoa was investigated by western-blot and indirect immunofluorescence (IIF) with the anti- rabbit
NOX5 C-terminal antibody (ab191010, Abcam, Cambridge, UK). At least 200 spermatozoa were scored
per sample in CTC and IIF assays. Differences between experimental groups in CTC staining and NOX5
immunolabeling were compared by means of chi-square test using GraphPad InStat software (Version
3.01). As expected, the inclusion of cAMP-elevating agents in the cocktail sample increased the
capacitated-sperm pattern by CTC compared with TALP sample after in vitro capacitation (p< 0.001),
whereas the presence of melatonin at 1 µM in both samples increased the non-capacitated-pattern relative
to samples without hormone (p<0.001). Regarding the presence of NOX5 in ram spermatozoa, Western
blot analyses revealed a band of 86 kDa compatible with that reported to NOX5 in human (Musset et al.,
The journal of biological chemistry, 287: 9376-9383,2012) and equine (Sabeur and Ball, Reproduction
134:263-270, 2007) spermatozoa. IIF revealed six differences immunotypes depending on the presence of
NOX5 in the ram sperm: I: apical region II: acrosome , III: post-acrosome, IV: apical and post-acrosomal,
V: acrosome and post-acrosome (all subtypes with midpiece labelling) and VI: labelling in the midpiece
of the spermatozoa. In swim-up selected (control) ram spermatozoa, the predominant NOX5
immunotypes were I and II. After incubation in capacitating conditions, these inmunotypes decreased in
TALP samples and increased those III and V (p< 0.001) when compared to control. In cocktail samples,
there was also an increase in the rate of spermatozoa with labelling only in the midpiece of the flagellum
(type VI, p<0.001). However, the presence of melatonin in TALP medium (TALP-Mel) increased II
subtype and in cocktail sample (Ck-Mel) increased V immnutype (p< 0.001),spermatozoa presented a
NOX5 distribution very similar to that observed control and TALP samples respectively. In conclusion,
these preliminary results reveal for the first time that NOX5 is present in ram spermatozoa, and that
melatonin can prevent the NOX5 distribution changes associated with sperm capacitation.
Acknowledgements: AGL2017-83799-R, DGA-A07_17R , BES-2015-07203, red PIVEV AGL2016-
81890-REDT.
to investigate if melatonin could modulate this enzyme during in vitro capacitation. Semen from nine
Rasa Aragonesa rams was collected and pooled. Seminal plasma free spermatozoa were selected by a
swim-up procedure (control sample). Spermatozoa were then incubated in TALP medium without (TALP
sample) or with cAMP-elevating agents (cocktail sample, Ck) for 3 h at 39 °C and 5% CO2. 1 µM
melatonin was added to TALP and cocktail samples (TALP-Mel and Ck-Mel) (n=6). Capacitation status
was evaluated by chlortetracycline (CTC) staining. Identification and distribution of NOX5 in ram
spermatozoa was investigated by western-blot and indirect immunofluorescence (IIF) with the anti- rabbit
NOX5 C-terminal antibody (ab191010, Abcam, Cambridge, UK). At least 200 spermatozoa were scored
per sample in CTC and IIF assays. Differences between experimental groups in CTC staining and NOX5
immunolabeling were compared by means of chi-square test using GraphPad InStat software (Version
3.01). As expected, the inclusion of cAMP-elevating agents in the cocktail sample increased the
capacitated-sperm pattern by CTC compared with TALP sample after in vitro capacitation (p< 0.001),
whereas the presence of melatonin at 1 µM in both samples increased the non-capacitated-pattern relative
to samples without hormone (p<0.001). Regarding the presence of NOX5 in ram spermatozoa, Western
blot analyses revealed a band of 86 kDa compatible with that reported to NOX5 in human (Musset et al.,
The journal of biological chemistry, 287: 9376-9383,2012) and equine (Sabeur and Ball, Reproduction
134:263-270, 2007) spermatozoa. IIF revealed six differences immunotypes depending on the presence of
NOX5 in the ram sperm: I: apical region II: acrosome , III: post-acrosome, IV: apical and post-acrosomal,
V: acrosome and post-acrosome (all subtypes with midpiece labelling) and VI: labelling in the midpiece
of the spermatozoa. In swim-up selected (control) ram spermatozoa, the predominant NOX5
immunotypes were I and II. After incubation in capacitating conditions, these inmunotypes decreased in
TALP samples and increased those III and V (p< 0.001) when compared to control. In cocktail samples,
there was also an increase in the rate of spermatozoa with labelling only in the midpiece of the flagellum
(type VI, p<0.001). However, the presence of melatonin in TALP medium (TALP-Mel) increased II
subtype and in cocktail sample (Ck-Mel) increased V immnutype (p< 0.001),spermatozoa presented a
NOX5 distribution very similar to that observed control and TALP samples respectively. In conclusion,
these preliminary results reveal for the first time that NOX5 is present in ram spermatozoa, and that
melatonin can prevent the NOX5 distribution changes associated with sperm capacitation.
Acknowledgements: AGL2017-83799-R, DGA-A07_17R , BES-2015-07203, red PIVEV AGL2016-
81890-REDT.
| Idioma original | Inglés |
|---|---|
| Estado | Publicada - 1 sep. 2019 |
| Evento | 35th Annual Meeting of the European Embryo Transfer Association - Murcia, Espana Duración: 13 sep. 2019 → 14 sep. 2019 |
Conferencia
| Conferencia | 35th Annual Meeting of the European Embryo Transfer Association |
|---|---|
| Título abreviado | 35th AETE |
| País/Territorio | Espana |
| Ciudad | Murcia |
| Período | 13/09/19 → 14/09/19 |
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