Effects of N -acetylcysteine on growth, viability and reactive oxygen species levels in small antral follicles cultured in vitro

Abstract

Objective: To investigate the effects of different concentrations of N-acetylcysteine on follicular growth and morphology, as well as on viability, levels of reactive oxygen species (ROS) and meiotic progression of oocytes from in vitro cultured bovine early antral follicles.

Methods: Isolated early antral follicles (about 500 μm) were cultured in TCM-199⁺ alone or supplemented with 1.0, 5.0 or 25.0 mM N-acetylcysteine at 38.5 °C with 5% CO₂ for 8 days. Follicle diameters were evaluated at day 0, 4 and 8 of culture. At the end of culture, the levels of ROS, chromatin configuration and viability (calcein-AM and ethidium homodimer-1 staining) were investigated in the cumulus-oocyte complexes. Comparisons of follicle diameters between treatments were performed. Data on percentages of morphologically normal follicles, growth rates and chromatin configuration in different treatments were compared.

Results: An increase in follicular diameters after culture in all treatments was observed, except for follicles cultured with 25.0 mM N-acetylcysteine. Fluorescence microscopy showed that oocytes cultured in all treatments were stained positively with calcein-AM, and that 5.0 mM N-acetylcysteine reduced fluorescence for ethidium homodimer-1. Intracellular levels of ROS in oocytes from follicles cultured with 1.0 mM N-acetylcysteine showed a significant reduction compared to other treatments. The presence of N-acetylcysteine in culture medium did not influence the rates of oocyte at the germinal vesicle stage.Conclusions:N-acetylcysteine at concentrations of 1.0 and 5.0 mM reduces ROS levels and staining for ethidium homodimer-1 in in vitro cultured follicles, respectively, while 25.0 mM N-acetylcysteine decreases follicular growth and the percentages of continuously growing follicles.

Keywords

Antioxidant / N-acetylcysteine / Antral follicles / Bovine / Reactive oxygen species

Conflict of interest statement

The authors declare that there is no conflict of interest.

Funding

This research was supported by grants from the National Council for Scientific and Technological Development (CNPq, Brazil, grant number 308737/2018-0).

Authors’ contributions

Efigênia B. Cordeiro performed in vitro culture of bovine early antral follicles and statistical analysis of data. Bianca R. Silva isolated the follicles and measured their diameters before and after culture. Laís R. F. M. Paulino isolated the follicles and stained them with fluorescent markers. Pedro A. A. Barroso collected the ovaries in slaughterhouses and prepared in vitro culture media. Laryssa G. Barrozo contributed to the acquisition and analysis of data. Miguel F. de Lima Neto evaluated follicular viability after staining with fluorescent markers. José R. V. Silva designed the work and critically revised the manuscript.

References

[1] Dode MAN, Rodovalho NC, Ueno VG, Alves RGO. Effect of follicle size in nuclear and cytoplasmic maturation of oocytes from zebu cows. Pesq Agropec Bras 2000; 35:207-214.

[2] Pavlok A, Lucas-Hahn A, Niemann H. Fertilization and developmental competence of bovine oocytes derived from different categories of antral follicles. Mol Rep Dev 1992; 31:63-67.

[3] Němcová L, Hulínska P, Ješeta M, Kempisty B, Kaňka J, Machatková M. Expression of selected mitochondrial genes during in vitro maturation of bovine oocytes related to their meiotic competence. Theriogenology 2019; 133:104-112.

[4] Clarke HJ. Regulation of germ cell development by intercellular signaling in the mammalian ovarian follicle. Devel Biol 2018; 7:e294.

[5] da Silva GM, Brito IR, Araújo VR, Silva JRV, Rodrigues APR, Figueiredo JR. In vitro culture of preantral follicles: Main regulatory factors, signaling pathways and advances in murine, bovine, ovine and caprine. Ciênc Anim 2015; 2:13-47.

[6] Paulino LR, de Assis EI, Azevedo VA, Silva BR, da Cunha EV, Silva JR. Why is it so difficult to have competent oocytes from in vitro cultured preantral follicles? Reprod Sci. 2022; 29(12):3321-3334. doi: 10.1007/s43032-021-00840-8.

[7] Telfer EE, Andersen CY. In vitro growth and maturation of primordial follicles and immature oocytes. Fertil Steril 2021; 115:1116-1125.

[8] Vasconcelos EM, Costa FC, Azevedo AVN, Barroso PAA, de Assis EIT, Paulino LRFM, et al. Eugenol influences the expression of messenger RNAs for superoxide dismutase and glutathione peroxidase 1 in bovine secondary follicles cultured in vitro. Zygote 2021; 29:301-306.

[9] Del Collado M, da Silveira JC, Oliveira ML, Alves BM, Simas RC, Godoy AT, et al. In vitro maturation impacts cumulus-oocyte complex metabolism and stress in cattle. Reproduction 2017; 154:881-893.

[10] Soto-Heras S, Paramio MT. Impact of oxidative stress on oocyte competence for in vitro embryo production programs. Res Vet Sci 2020; 132:342-350.

[11] Sá NAR, Bruno JB, Guerreiro DD, Cadenas J, Alves BG, Cibin FWS, et al. Anethole reduces oxidative stress and improves in vitro survival and activation of primordial follicles. Braz J Med Biol Res 2018; 51(8):e7129.

[12] Lins TLBG, Cavalcante AYP, Santos JMS, Menezes VG, Barros VRP, Barberino RS, et al. Rutin can replace the use of three other antioxidants in the culture medium, maintaining the viability of sheep isolated secondary follicles. Theriogenology 2017; 89:263-270.

[13] Mahmoodi M, Mehranjani MS, Shariatzadeh SMA, Eimani H, Shahverdi A. N-acetylcysteine improves function and follicular survival in mice ovarian grafts through inhibition of oxidative stress. Reprod Biomed Online 2015; 30(1):101-110. doi: 10.1016/j.rbmo.2014.09.013.

[14] Fabbri R, Pasquinelli G, Montanaro L, Mozzanega B, Magnani V, Tamburini F, et al. Healthy early preantral follicle can be obtained in a culture of frozen-thawed human ovarian tissue of 32 weeks. Ultrast Pathol 2007; 31:257-262.

[15] Li Q, Zhao Z. Influence of N-acetyl-L-cysteine against bisphenol a on the maturation of mouse oocytes and embryo development: In vitro study. BMC Pharmacol Toxicol 2019; 20:1-9.

[16] Lai FN, Ma JY, Liu JC, Wang JJ, Cheng SF, Sun XF, et al. The influence of N-acetyl-l-cysteine on damage of porcine oocyte exposed to zearalenone in vitro. Tox Appi Pharm 2015; 289:341-348.

[17] Bhardwaj JK, Saraf P. N-acetyl-l-cysteine mediated regulation of DNA fragmentation, an apoptotic event, against methoxychlor toxicity in the granulosa cells of ovarian antral follicles. Mutat Res Genet Toxicol Environ Mutagen 2020; 858-860:503222. doi: 10.1016/j.mrgentox.2020.503222.

[18] Olesen HØ, Pors SE, Jensen LB, Grønning AP, Lemser CE, Nguyen Heimbürger MTH, et al. N-acetylcysteine protects ovarian follicles from ischemia-reperfusion injury in xenotransplanted human ovarian tissue. Hum Rep 2021; 36:429-443.

[19] He Y, Meng K, Wang X, Dong Z, Zhang Y, Quan F. Comparison of bovine small antral follicle development in two-and three-dimensional culture systems. An Acad Bras Cienc 2020; 92(Suppl 2):e20180935. doi: 10.1590/0001-3765202020180935.

[20] Alm H, Kątska-Ksiązkiewicz L, Ryńska B, Tuchscherer A. Survival and meiotic competence of bovine oocytes originating from early antral ovarian follicles. Theriogenology 2006; 65:1422-1434.

[21] Van den Hurk R, Spek ER, Hage WJ, Fair T, Ralph JH, Schotanus K. Ultrastructure and viability of isolated bovine preantral follicles. Hum Reprod Update 1998; 4:833-841.

[22] Campos LB, Silva AMD, Moreira SSJ, Santos CS, Apolinário CAC, Saraiva MVA, et al. Establishment of a protocol for the isolation of ovarian preantral follicles derived from collared peccaries (Pecari tajacu). Zygote 2019; 27(5):315-320.

[23] Chaudhary GR, Yadav PK, Yadav AK, Tiwari M, Gupta A, Sharma A, et al. Necroptosis in stressed ovary. J Biom Sci 2019; 26:1-6.

[24] Sun WS, Jang H, Park MR, Oh KB, Lee H, Hwang S, et al. N-acetyl-L-cysteine improves the developmental competence of bovine oocytes and embryos cultured in vitro by attenuating oxidative damage and apoptosis. Antioxidants 2021; 10:860.

[25] Atkuri KR, Mantovani JJ, Herzenberg LA, Herzenberg LA. N-acetylcysteine-A safe antidote for cysteine/glutathione deficiency. Curr Opin Pharmacol 2007; 7:355-359.

[26] Whitaker BD, Knight JW. Effects of N-acetyl-cysteine and N-acetyl-cysteine-amide supplementation on in vitro matured porcine oocytes. Reprod Domest Anim 2010; 45(5):755-759. doi: 10.1111/j.1439-0531.2009.01344.x.

[27] Matilla E, Martín-Cano FE, González-Fernández L, Sánchez-Margallo FM, Álvarez IS, Macías-García B. N-acetylcysteine addition after vitrification improves oocyte mitochondrial polarization status and the quality of embryos derived from vitrified murine oocytes. BMC Vet Res 2019; 15:1-10.

[28] Naimi M, Shariati M, Naeimi S, Edalatmanesh MA. Effect of N-acetylcystein on ERK gene expression in ovarian tissue of acrylamide-treated adult rats. J Ilmu Ternak dan Veteriner 2020; 25:11-18.

[29] Grinberg L, Fibach E, Amer J, Atlas D. N-acetylcysteine amide, a novel cell-permeating thiol, restores cellular glutathione and protects human red blood cells from oxidative stress. Free Radic Biol Med 2005; 38:136-145.

[30] Aldini G, Altomare A, Baron G, Vistoli G, Carini M, Borsani L, et al. Acetylcysteine as an antioxidant and disulphide breaking agent: The reasons why. Free Radic Res 2018; 52:751-762.

[31] Sanfilippo S, Canis M, Ouchchane L, Botchorishvili R, Artonne C, Janny L, et al. Viability assessment of fresh and frozen/thawed isolated human follicles: reliability of two methods (Trypan blue and Calcein AM/ ethidium homodimer-1). J Assist Reprod Genet 2011; 28:1151-1156.

[32] Neri S. Mariani E, Meneghetti A, Cattini L, Facchini A. Calcein-acetyoxymethyl cytotoxicity assay: Standardization of a method allowing additional analyses on recovered effector cells and supernatants. Clin Diagn Lab Immunol 2001; 8:1131-1135.

[33] Spagnuolo G, D'Antò V, Cosentino C, Schmalz G, Schweikl H, Rengo S, et al. Effect of N-acetyl-L-cysteine on ROS production and cell death caused by HEMA in human primary gingival fibroblasts Biomaterials. 2006; 27:1803-1809.

[34] Zhang X, Wang YN, Zhu JJ, Liu XX, You H, Gong MY, et al. N-acetylcysteine negatively regulates Notch 3 and its malignant signaling. Oneotarget 2016; 7:30855.

[35] Sprong RC, Winkelhuyzen-Janssen AM, Aarsman CJ, van Oirschot JF, van der Bruggen T, van Asbeck BS. Low-dose N-acetylcysteine protects rats against endotoxin-mediated oxidative stress, but high-dose increases mortality. Am J Respir Crit Care Med. 1998; 157: 1283-1293. doi: 10.1164/ajrccm.157.4.9508063.

[36] Moraes NVD, Mello MHD, Souza AMD, Sampaio SV, Queiroz RHC. Potentiation of of methmoglobinizing effect of dapsone by N-acetilcisteinein rats. Ver Bras Cienc Farm 2008; 44:97-104.

[37] Zhang M. Oocyte meiotic arrest. Encycl Reprod 2018; 3:153-158.

[38] Russell DL, Robker RL. Cumulus cells. Encycl Reprod 2018; 2:43-46.