Stocktake Sale on now: wide range of books at up to 70% off!
Register      Login
Reproduction, Fertility and Development Reproduction, Fertility and Development Society
Vertebrate reproductive science and technology
RESEARCH ARTICLE (Open Access)

Differential effect of melatonin on ram spermatozoa depending on the allelic variant of the RsaI polymorphism of the MTR1A gene, incubation medium and season

Victoria Peña-Delgado https://orcid.org/0000-0002-4670-3735 A , Agustí Noya https://orcid.org/0000-0002-9552-4709 A , Melissa Carvajal-Serna https://orcid.org/0000-0003-3929-5064 A , José A Abecia https://orcid.org/0000-0003-2827-3054 A , Rosaura Pérez-Pe https://orcid.org/0000-0002-2312-6402 A and Adriana Casao https://orcid.org/0000-0003-1997-4262 A *
+ Author Affiliations
- Author Affiliations

A BIOFITER-IUCA, Universidad de Zaragoza, Facultad de Veterinaria, Miguel Servet 177, 50013 Zaragoza, Spain.

* Correspondence to: adriana@unizar.es

Handling Editor: Ana Villaverde

Reproduction, Fertility and Development 36, RD23233 https://doi.org/10.1071/RD23233
Submitted: 23 December 2023  Accepted: 3 June 2024  Published online: 21 June 2024

© 2024 The Author(s) (or their employer(s)). Published by CSIRO Publishing. This is an open access article distributed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND)

Abstract

Context

The RsaI polymorphism of the melatonin receptor MTNR1A gene affects seasonal reproduction in sheep, but its effect on ram spermatozoa and their response to melatonin is unknown.

Aims

This study aims to evaluate whether RsaI polymorphism of the MTNR1A gene influences the response of ram spermatozoa to in vitro added melatonin.

Methods

Spermatozoa from rams carrying different RsaI allelic variants were incubated with melatonin in a TALP medium or a capacitation-triggering medium during the reproductive and non-reproductive seasons. After incubation, sperm motility, membrane integrity, mitochondria activity, oxidative damage, apoptotic markers and capacitation status were assessed.

Key results

In the reproductive season, the T/T genotype was related to some adverse effects of melatonin when spermatozoa were incubated in TALP medium, whereas the C/C genotype was linked with adverse effects when the hormone was added in a capacitation-triggering medium. The decapacitating effect of melatonin on spermatozoa was also different depending on genotype.

Conclusions

The melatonin effect on spermatozoa from rams carrying different RsaI genotypes differed depending on the season and the medium.

Implications

The knowledge of the RsaI allelic variant of the MTNR1A gene of rams could be helpful when carrying out in vitro reproductive techniques in the ovine species.

Keywords: Capacitation, incubation, melatonin, MTNR1A polymorphism, ram sperm, receptor, RsaI, sheep.

References

Abecia JA, Mura MC, Carvajal-Serna M, Pulinas L, Macías A, Casao A, Pérez-Pe R, Carcangiu V (2020) Polymorphisms of the melatonin receptor 1A (MTNR1A) gene influence the age at first mating in autumn-born ram-lambs and sexual activity of adult rams in spring. Theriogenology 157, 42-47.
| Crossref | Google Scholar | PubMed |

Abecia JA, Heredia A, Pérez-Pe R, Casao A, Carcangiu V, Mura MC, Miranda-De la Lama G (2022) Polymorphisms of the melatonin receptor 1A (MTNR1A) gene affect the sexual performance of Rasa Aragonesa rams without changing their social dominance. Journal of Animal Behaviour and Biometeorology 10, 2231.
| Crossref | Google Scholar |

Abecia JA, Macías A, Laviña A, Martín E, Casao A, Canto F, Carvajal-Serna M, Peña-Delgado V, Pérez-Pe R (2023) Polymorphisms of the melatonin receptor 1A (MTNR1A) gene of Rasa Aragonesa rams affect their fertility after artificial insemination. Animal - Science Proceedings 14, 77-78.
| Crossref | Google Scholar |

Alquézar-Baeta C, Gimeno-Martos S, Miguel-Jiménez S, Santolaria P, Yániz J, Palacín I, Casao A, Cebrián-Pérez JÁ, Muiño-Blanco T, Pérez-Pé R (2019) OpenCASA: A new open-source and scalable tool for sperm quality analysis. PLOS Computational Biology 15, e1006691.
| Crossref | Google Scholar |

Ashrafi I, Kohram H, Ardabili FF (2013) Antioxidative effects of melatonin on kinetics, microscopic and oxidative parameters of cryopreserved bull spermatozoa. Animal Reproduction Science 139, 25-30.
| Crossref | Google Scholar |

Avdi M, Banos G, Stefos K, Chemineau P (2004) Seasonal variation in testicular volume and sexual behavior of Chios and Serres rams. Theriogenology 62, 275-282.
| Crossref | Google Scholar | PubMed |

Ayoub MA, Couturier C, Lucas-Meunier E, Angers S, Fossier P, Bouvier M, Jockers R (2002) Monitoring of ligand-independent dimerization and ligand-induced conformational changes of melatonin receptors in living cells by bioluminescence resonance energy transfer. Journal of Biological Chemistry 277, 21522-21528.
| Crossref | Google Scholar | PubMed |

Barberino RS, Menezes VG, Ribeiro AEAS, Palheta RC, Jr, Jiang X, Smitz JEJ, Matos MHT (2017) Melatonin protects against cisplatin-induced ovarian damage in mice via the MT1 receptor and antioxidant activity. Biology of Reproduction 96, 1244-1255.
| Crossref | Google Scholar | PubMed |

Bass DA, Parce JW, Dechatelet LR, Szejda P, Seeds MC, Thomas M (1983) Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation. Journal of Immunology 130, 1910-1917.
| Crossref | Google Scholar | PubMed |

Calvo JH, Serrano M, Martinez-Royo A, Lahoz B, Sarto P, Ibañez-Deler A, Folch J, Alabart JL (2018) SNP rs403212791 in exon 2 of the MTNR1A gene is associated with reproductive seasonality in the Rasa aragonesa sheep breed. Theriogenology 113, 63-72.
| Crossref | Google Scholar | PubMed |

Carcangiu V, Mura MC, Vacca GM, Pazzola M, Dettori ML, Luridiana S, Bini PP (2009) Polymorphism of the melatonin receptor MT1 gene and its relationship with seasonal reproductive activity in the Sarda sheep breed. Animal Reproduction Science 116, 65-72.
| Crossref | Google Scholar | PubMed |

Carter WO, Narayanan PK, Robinson JP (1994) Intracellular hydrogen peroxide and superoxide anion detection in endothelial cells. Journal of Leukocyte Biology 55, 253-258.
| Crossref | Google Scholar | PubMed |

Carvajal-Serna M, Cardozo-Cerquera JA, Grajales-Lombana HA, Casao A, Pérez-Pe R (2021) Sperm behavior and response to melatonin under capacitating conditions in three sheep breeds subject to the equatorial photoperiod. Animals 11, 1828.
| Crossref | Google Scholar |

Casao A, Cebrián I, Asumpção ME, Pérez-Pé R, Abecia JA, Forcada F, Cebrián-Pérez JA, Muiño-Blanco T (2010a) Seasonal variations of melatonin in ram seminal plasma are correlated to those of testosterone and antioxidant enzymes. Reproductive Biology and Endocrinology 8, 59.
| Crossref | Google Scholar | PubMed |

Casao A, Mendoza N, Pérez-Pé R, Grasa P, Abecia JA, Forcada F, Cebrián-Pérez JA, Muino-Blanco T (2010b) Melatonin prevents capacitation and apoptotic-like changes of ram spermatozoa and increases fertility rate. Journal of Pineal Research 48, 39-46.
| Crossref | Google Scholar | PubMed |

Casao A, Gallego M, Abecia JA, Forcada F, Pérez-Pé R, Muio-Blanco T, Cebrin-Ṕrez JL (2012) Identification and immunolocalisation of melatonin MT(1) and MT(2) receptors in Rasa Aragonesa ram spermatozoa. Reproduction, Fertility and Development 24, 953-961.
| Crossref | Google Scholar | PubMed |

Chemineau P, Guillaume D, Migaud M, Thiéry JC, Pellicer-Rubio MT, Malpaux B (2008) Seasonality of reproduction in mammals: intimate regulatory mechanisms and practical implications. Reproduction in Domestic Animals 43(Suppl 2), 40-47.
| Crossref | Google Scholar | PubMed |

Colas C, James P, Howes L, Jones R, Cebrian-Perez JA, Muiño-Blanco T (2008) Cyclic-AMP initiates protein tyrosine phosphorylation independent of cholesterol efflux during ram sperm capacitation. Reproduction, Fertility and Development 20, 649-658.
| Crossref | Google Scholar | PubMed |

Dubocovich ML, Markowska M (2005) Functional MT1 and MT2 melatonin receptors in mammals. Endocrine 27, 101-110.
| Crossref | Google Scholar | PubMed |

Espino J, Ortiz Á, Bejarano I, Lozano GM, Monllor F, García JF, Rodríguez AB, Pariente JA (2011) Melatonin protects human spermatozoa from apoptosis via melatonin receptor- and extracellular signal-regulated kinase-mediated pathways. Fertility and Sterility 95, 2290-2296.
| Crossref | Google Scholar | PubMed |

Fernández-Alegre E, Álvarez-Fernández I, Domínguez JC, Casao A, Martínez-Pastor F (2020) Melatonin non-linearly modulates bull spermatozoa motility and physiology in capacitating and non-capacitating conditions. International Journal of Molecular Sciences 21, 2701.
| Crossref | Google Scholar |

Forcada F, Abecia JA, Sierra I (1992) Seasonal changes in oestrus activity and ovulation rate in Rasa Aragonesa ewes maintained at two different body condition levels. Small Ruminant Research 8, 313-324.
| Crossref | Google Scholar |

Fujinoki M (2008) Melatonin-enhanced hyperactivation of hamster sperm. Reproduction 136, 533-541.
| Crossref | Google Scholar | PubMed |

García-López N, Ollero M, Muiño-Blanco T, Cebrián-Pérez JA (1996) A dextran swim-up procedure for separation of highly motile and viable ram spermatozoa from seminal plasma. Theriogenology 46, 141-151.
| Crossref | Google Scholar |

Gillan L, Evans AG, Maxwell WMC (1997) Capacitation status and fertility of fresh and frozen-thawed ram spermatozoa. Reproduction, Fertility and Development 9, 481-488.
| Crossref | Google Scholar | PubMed |

Gimeno-Martos S, Casao A, Yeste M, Cebrián-Pérez JA, Muiño-Blanco T, Pérez-Pé R (2019) Melatonin reduces cAMP-stimulated capacitation of ram spermatozoa. Reproduction, Fertility and Development 31, 420-431.
| Crossref | Google Scholar | PubMed |

Gonzalez-Arto M, Luna C, Pérez-Pé R, Muiño-Blanco T, Cebrián-Pérez JA, Casao A (2016a) New evidence of melatonin receptor contribution to ram sperm functionality. Reproduction, Fertility and Development 28, 924-935.
| Crossref | Google Scholar | PubMed |

González-Arto M, Vicente-Carrillo A, Martínez-Pastor F, Fernández-Alegre E, Roca J, Miró J, Rigau T, Rodríguez-Gil JE, Pérez-Pé R, Muiño-Blanco T, Cebrián-Pérez JA, Casao A (2016b) Melatonin receptors MT1 and MT2 are expressed in spermatozoa from several seasonal and nonseasonal breeder species. Theriogenology 86, 1958-1968.
| Crossref | Google Scholar | PubMed |

Grasa P, Cebrián-Pérez JÁ, Muiño-Blanco T (2006) Signal transduction mechanisms involved in in vitro ram sperm capacitation. Reproduction 132, 721-732.
| Crossref | Google Scholar | PubMed |

Guthrie HD, Welch GR (2006) Determination of intracellular reactive oxygen species and high mitochondrial membrane potential in Percoll-treated viable boar sperm using fluorescence-activated flow cytometry. Journal of Animal Science 84, 2089-2100.
| Crossref | Google Scholar | PubMed |

Hallap T, Nagy S, Jaakma Ü, Johannisson A, Rodriguez-Martinez H (2005) Mitochondrial activity of frozen-thawed spermatozoa assessed by MitoTracker Deep Red 633. Theriogenology 63, 2311-2322.
| Crossref | Google Scholar | PubMed |

Harrison RA, Vickers SE (1990) Use of fluorescent probes to assess membrane integrity in mammalian spermatozoa. Journal of Reproduction and Fertility 88, 343-352.
| Crossref | Google Scholar | PubMed |

Kumar T, Kumar P, Saini N, Bhalothia SK, Prakash C, Mahla AS, Kumar A (2022) Shielding effect of melatonin improves seminal quality and oxidative stress indices during chilled storage of ram semen. Tropical Animal Health and Production 54, 197.
| Crossref | Google Scholar |

Leahy T, Rickard JP, Aitken RJ, de Graaf SP (2016) Penicillamine prevents ram sperm agglutination in media that support capacitation. Reproduction 151, 167-177.
| Crossref | Google Scholar | PubMed |

Luna C, Mendoza N, Casao A, Pérez-Pé R, Cebrián-Pérez JA, Muiño-Blanco T (2017) c-Jun N-terminal kinase and p38 mitogen-activated protein kinase pathways link capacitation with apoptosis and seminal plasma proteins protect sperm by interfering with both routes. Biology of Reproduction 96, 800-815.
| Crossref | Google Scholar | PubMed |

Luridiana S, Mura MC, Daga C, Cosso G, Bodano S, Farci F, Zidda F, Carcangiu V (2016) Influences of melatonin treatment, melatonin receptor 1A (MTNR1A) and kisspeptin (KiSS-1) gene polymorphisms on first conception in Sarda ewe lambs. Reproduction, Fertility and Development 28, 750-756.
| Crossref | Google Scholar | PubMed |

Martínez-Royo A, Lahoz B, Alabart JL, Folch J, Calvo JH (2012) Characterisation of the Melatonin Receptor 1A (MTNR1A) gene in the Rasa Aragonesa sheep breed: Association with reproductive seasonality. Animal Reproduction Science 133, 169-175.
| Crossref | Google Scholar | PubMed |

Mateescu RG, Lunsford AK, Thonney ML (2009) Association between melatonin receptor 1A gene polymorphism and reproductive performance in Dorset ewes. Journal of Animal Science 87, 2485-2488.
| Crossref | Google Scholar | PubMed |

Messer LA, Wang L, Tuggle CK, Yerle M, Chardon P, Pomp D, Womack JE, Barendse W, Crawford AM, Notter DR, Rothschild MF (1997) Mapping of the melatonin receptor 1a (MTNR1A) gene in pigs, sheep, and cattle. Mammalian Genome 8, 368-370.
| Crossref | Google Scholar | PubMed |

Miguel-Jiménez S, Pina-Beltrán B, Gimeno-Martos S, Carvajal-Serna M, Casao A, Pérez-Pe R (2021) NADPH Oxidase 5 and melatonin: involvement in Ram Sperm Capacitation. Frontiers in Cell and Developmental Biology 9, 655794.
| Crossref | Google Scholar |

Najafi A, Adutwum E, Yari A, Salehi E, Mikaeili S, Dashtestani F, Abolhassani F, Rashki L, Shiasi S, Asadi E (2018) Melatonin affects membrane integrity, intracellular reactive oxygen species, caspase3 activity and AKT phosphorylation in frozen thawed human sperm. Cell and Tissue Research 372, 149-159.
| Crossref | Google Scholar |

Notter DR (2008) Genetic aspects of reproduction in sheep. Reproduction in Domestic Animals 43, 122-128.
| Crossref | Google Scholar | PubMed |

Ollero M, Muiño-Blanco T, López-Pérez MJ, Cebrián-Pérez JA (1996) Viability of ram spermatozoa in relation to the abstinence period and successive ejaculations. International Journal of Andrology 19, 287-292.
| Crossref | Google Scholar | PubMed |

Ooi EH, Smith DJ, Gadêlha H, Gaffney EA, Kirkman-Brown J (2014) The mechanics of hyperactivation in adhered human sperm. Royal Society Open Science 1, 140230.
| Crossref | Google Scholar |

Parrish JJ, Susko-Parrish J, Winer MA, First NL (1988) Capacitation of bovine sperm by heparin. Biology of Reproduction 38, 1171-1180.
| Crossref | Google Scholar | PubMed |

Pelletier J, Bodin L, Hanocq E, Malpaux B, Teyssier J, Thimonier J, Chemineau P (2000) Association between expression of reproductive seasonality and alleles of the gene for Mel(1a) receptor in the ewe. Biology of Reproduction 62, 1096-1101.
| Crossref | Google Scholar | PubMed |

Pulinas L, Starič J, Cosso G, Curone G, Mura MC, Carcangiu V, Luridiana S (2022) MTNR1A gene polymorphisms and reproductive recovery after seasonal anoestrus in different Mediterranean sheep breeds. Animal Reproduction Science 236, 106905.
| Crossref | Google Scholar |

Reiter RJ, Tan DX, Osuna C, Gitto E (2000) Actions of melatonin in the reduction of oxidative stress. A review. Journal of Biomedical Science 7, 444-458.
| Crossref | Google Scholar |

Rosa HJD, Bryant MJ (2003) Seasonality of reproduction in sheep. Small Ruminant Research 48, 155-171.
| Crossref | Google Scholar |

Starič J, Farci F, Luridiana S, Mura MC, Pulinas L, Cosso G, Carcangiu V (2020) Reproductive performance in three Slovenian sheep breeds with different alleles for the MTNR1A gene. Animal Reproduction Science 216, 106352.
| Crossref | Google Scholar |

Succu S, Berlinguer F, Pasciu V, Satta V, Leoni GG, Naitana S (2011) Melatonin protects ram spermatozoa from cryopreservation injuries in a dose-dependent manner. Journal of Pineal Research 50, 310-318.
| Crossref | Google Scholar | PubMed |

Trecherel E, Batailler M, Chesneau D, Delagrange P, Malpaux B, Chemineau P, Migaud M (2010) Functional characterisation of polymorphic variants for ovine MT1 melatonin receptors: Possible implication for seasonal reproduction in sheep. Animal Reproduction Science 122, 328-334.
| Crossref | Google Scholar | PubMed |

van Vuuren RJ, Pitout MJ, van Aswegen CH, Theron JJ (1992) Putative melatonin receptor in human spermatozoa. Clin Biochem 25, 125-127.
| Crossref | Google Scholar | PubMed |

Vermes I, Haanen C, Steffens-Nakken H, Reutellingsperger C (1995) A novel assay for apoptosis flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. Journal of Immunological Methods 184, 39-51.
| Crossref | Google Scholar | PubMed |

Ward CR, Storey BT (1984) Determination of the time course of capacitation in mouse spermatozoa using a chlortetracycline fluorescence assay. Developmental Biology 104, 287-296.
| Crossref | Google Scholar | PubMed |

Weaver DR, Liu C, Reppert SM (1996) Nature’s knockout: the Mel1b receptor is not necessary for reproductive and circadian responses to melatonin in Siberian hamsters. Molecular Endocrinology 10, 1478-1487.
| Crossref | Google Scholar | PubMed |

Zhao D, Yu Y, Shen Y, Liu Q, Zhao Z, Sharma R, Reiter RJ (2019) Melatonin synthesis and function: evolutionary history in animals and plants. Frontiers in Endocrinology 10, 249.
| Crossref | Google Scholar | PubMed |

Zhu Z, Li R, Lv Y, Zeng W (2019) Melatonin protects rabbit spermatozoa from cryo-damage via decreasing oxidative stress. Cryobiology 88, 1-8.
| Crossref | Google Scholar |