Effects of melatonin on specific gene expression in the ovaries of superovulated rats

Sevda Dalkıran; Akın Yakan; Hüseyin Özkan

doi:10.33988/auvfd.1642763

Research Article

Effects of melatonin on specific gene expression in the ovaries of superovulated rats

Year 2025, Accepted Papers, 1 - 11

Sevda Dalkıran , Akın Yakan , Hüseyin Özkan

https://doi.org/10.33988/auvfd.1642763

Abstract

This study investigated the effects of different doses of melatonin administered with superovulation on ovarian health at biochemical and molecular levels. Four groups were created: Control (Con), Superovulation (So), Superovulation+5 mg/kg melatonin (SoM5), and Superovulation+20 mg/kg melatonin (SoM20). It was determined that there was a decrease in ovary tissue MDA levels in groups compared to the control (P<0.01). It was determined that PGR expression increased approximately 7-fold in SoM20 compared to the control (P<0.001). It was found that the expression level of the COX-2 was increased in the experimental groups (So, SoM5, and SoM20) compared to the control (P<0.05). It was determined that the expressions of the EREG in the SoM20 increased approximately 5-fold compared to the control (P<0.001). Positive correlation was determined between serum MDA levels and Bcl-2 and NRF2 genes (P<0.01). A positive correlation was found between COX-2 and PGR and EREG genes (P<0.001), between IL-1β and Bcl-2 and OSGIN1 genes (P<0.05), between Bcl-2 gene and NRF2 and BMP15 genes (P<0.05), between PGR gene and EREG genes (P<0.001), and between NRF2 gene and BMP15 and OSGIN1 genes (P<0.05). Protein–protein interaction network analysis identified COX-2, PGR, and EREG as central nodes within a 28 node, 213 edge network, and MCODE revealed a dense PTGER/PTGES module (score 4.00). Gene enrichment analysis demonstrated significant overrepresentation of reproductive and ovulation related pathways (FDR<0.05). In conclusion, 20 mg/kg melatonin administered during superovulation may support ovarian health and folliculogenesis by increasing the expression of the PGR and EREG genes in the ovary.

Keywords

Gene expression , MDA , Melatonin , Oxidative stress , Superovulation

Project Number

22.D.018

References

Abdelrahman RS, Abdel-Rahman N (2019): Dimethyl fumarate ameliorates acetaminophen-induced hepatic injury in mice dependent of Nrf-2/HO-1 pathway. Life Sci, 217, 251-260.
Agca C, Yakan A, Agca Y (2013): Estrus synchronization and ovarian hyper-stimulation treatments have negligible effects on cumulus oocyte complex gene expression whereas induction of ovulation causes major expression changes. Mol Reprod Dev, 80, 102-117.
Akison LK, Boden MJ, Kennaway DJ, et al (2014): Progesterone receptor-dependent regulation of genes in the oviducts of female mice. Physiol Genomics, 46, 583-592.
Alshehri FS, Alghamdi BS, Hakami AY, et al (2021): Melatonin attenuates morphine-induced conditioned place preference in Wistar rats. Brain Behav, 11, e2397.
Atasever A, Tekin S, Bolat İ, et al (2025): The effects of melatonin on oxidative stress, inflammation, apoptosis and Nrf2/HO-1 in acrylamide-induced lung injury in rats. Naunyn-Schmiedeberg Arch Pharmacol, 1, 1-18.
Bandariyan E, Mogheiseh A, Ahmadi A (2021): The effect of lutein and Urtica dioica extract on in vitro production of embryo and oxidative status in polycystic ovary syndrome in a model of mice. BMC Complement Med Ther, 21, 1-11.
Benjamini Y, Hochberg Y (1995): Controlling the false discovery rate: a practical and powerful approach to multiple testing. J Roy Stat Soc B, 57, 289-300.
Cabrera RA, Dozier BL, Duffy DM (2006): Prostaglandin-endoperoxide synthase (PTGS1 and PTGS2) expression and prostaglandin production by normal monkey ovarian surface epithelium. Fert Steril, 86, 1088-1096.
Cao Y, Zhao H, Wang Z, et al (2020): Quercetin promotes in vitro maturation of oocytes from humans and aged mice. Cell Death Dis, 11, 965.
Chandras C, Harris TE, Bernal AL, et al (2007): PTGER1 and PTGER2 receptors mediate regulation of progesterone synthesis and type 1 11β-hydroxysteroid dehydrogenase activity by prostaglandin E2 in human granulosa–lutein cells. J Endocrinol, 194, 595-602.
Choi Y, Wilson K, Hannon PR, et al (2017): Coordinated regulation among progesterone, prostaglandins, and EGF-like factors in human ovulatory follicles. J Clin Endocrinol Metab, 102, 1971-1982.
Contreras-Navarro LE, Boeta M, Gonzalez-Lopez R, et al (2020): Melatonin promotes equine cumulus-oocyte complex survival during in vitro maturation. Reprod Biol, 4, 16-30.
Davis BJ, Lennard DE, Lee CA, et al (1999): Anovulation in Cyclooxygenase-2-Deficient Mice Is Restored by Prostaglandin E2 and Interleukin-1β. Endocrinology, 140, 2685-2695.
Espey LL, Richards JS (2002): Temporal and spatial patterns of ovarian gene transcription following an ovulatory dose of gonadotropin in the rat. Biol Reprod, 67, 1662-1670.
Ezzati M, Roshangar L, Rad JS, et al (2018): Evaluating the effect of melatonin on HAS2, and PGR expression, as well as cumulus expansion, and fertility potential in mice. Cell J, 20, 108.
Fan L, Guan F, Ma Y, et al (2022): N-Acetylcysteine improves oocyte quality through modulating the Nrf2 signaling pathway to ameliorate oxidative stress caused by repeated controlled ovarian hyperstimulation. Reprod Fertil Dev, 34, 736-750.
Feng X, Zhang Y, Li N, et al (2023): Melatonin in reproductive medicine: a promising therapeutic target? Curr Med Chem, 30, 3090-3118.
Franceschini A, Szklarczyk D, Frankild S, et al (2012): STRING v9.1: protein-protein interaction networks, with increased coverage and integration. Nucleic Acids Res, 41, D808-D815.
Friberg PA, Larsson DGJ, Billig H (2009): Dominant role of nuclear progesterone receptor in the control of rat periovulatory granulosa cell apoptosis. Biol Reprod, 80, 1160-1167.
Gobbo MG, Costa CFP, Silva DGH, et al (2015): Effect of melatonin intake on oxidative stress biomarkers in male reproductive organs of rats under experimental diabetes. Oxid Med Cell Longev, 2015, 614579.
Hizaki H, Segi E, Sugimoto Y, et al (1999): Abortive expansion of the cumulus and impaired fertility in mice lacking the prostaglandin E receptor subtype EP2. Proc Natl Acad Sci USA, 96, 10501-10506.
Jiang Y, Shi H, Liu Y, et al (2021): Applications of melatonin in female reproduction in the context of oxidative stress. Oxid Med Cell Longev, 2021, 6668365.
Kim EH, Ridlo MR, Lee BC, et al (2020): Melatonin-Nrf2 signaling activates peroxisomal activities in porcine cumulus cell-oocyte complexes. Antioxidants, 9, 1080.
Kim SO, Duffy DM (2016): Mapping PTGERs to the Ovulatory Follicle: Regional Responses to the Ovulatory PGE2 Signal. Biol Reprod, 95, 1-11, 33.
Komuczki D, Stadermann A, Bentele M, et al (2022): High cysteine concentrations in cell culture media lead to oxidative stress and reduced bioprocess performance of recombinant CHO cells. Biotechnol J, 17, 2200029.
Lee M, Ahn J Il, Lee AR, et al (2017): Adverse effect of superovulation treatment on maturation, function and ultrastructural integrity of murine oocytes. Mol Cells, 40, 558-566.
Liu B, Wang JL, Wang XM, et al (2020): Reparative effects of lycium barbarum polysaccharide on mouse ovarian injuries induced by repeated superovulation. Theriogenology, 145, 115-125.
Liu M na, Zhang K, Xu T (2021): The role of BMP15 and GDF9 in the pathogenesis of primary ovarian insufficiency. Hum Fertil, 24, 325-332.
Livak KJ, Schmittgen TD (2001): Analysis of relative gene expression data using real-time quantitative PCR and the 2⁻ΔΔCT method. Methods, 25, 402-408.
Nie X, Dai Y, Zheng Y, et al (2018): Establishment of a mouse model of premature ovarian failure using consecutive superovulation. Cell Physiol Biochem, 51, 2341-2358.
Niringiyumukiza JD, Cai H, Xiang W (2018): Prostaglandin E2 involvement in mammalian female fertility: ovulation, fertilization, embryo development and early implantation. Reprod Biol Endocrinol, 16, 43.
Nuttinck F, Marquant LE, Guienne B, et al (2008): Expression of genes involved in prostaglandin E2 and progesterone production in bovine cumulus-oocyte complexes during in vitro maturation and fertilization. Reproduction, 135, 593-603.
Özkan H, Keçeli HH, Kaya U, et al (2024): Considering potential roles of selected MicroRNAs in evaluating subclinical mastitis and milk quality in California mastitis test (+) and infected bovine milk. Anim Sci J, 95, e13959.
Özkan H, Kaya U, Keçeli HH, et al (2025): Expression patterns of miR-29b and miR-148a in colostrum of Awassi sheep with higher immunoglobulin G levels. Vet Med Sci, 11, e70335.
Pacchiarotti A, Carlomagno G, Antonini G, et al (2016): Effect of myo-inositol and melatonin versus myo-inositol, in a randomized controlled trial, for improving in vitro fertilization of patients with polycystic ovarian syndrome. Gynecol Endocrinol, 32, 69-73.
Park CJ, Lin PC, Zhou S, et al (2020): Progesterone receptor serves the ovary as a trigger of ovulation and a terminator of inflammation. Cell Rep, 31, 1.
Park JY, Su YQ, Ariga M, et al (2004): EGF-like growth factors as mediators of LH action in the ovulatory follicle. Science, 303, 682-684.
Qiao P, Zhang Y, Yang Y, et al (2021): Oral isoniazid causes oxidative stress, oocyte deterioration and infertility in mice. Toxicology, 455, 152749.
Rezaee N, Nematollahi Z, Hoseini E (2016): Effect of sodium metabisulfite on rat ovary and lipid peroxidation. Iran J Toxicol, 10, 23-28.
Rio DC, Ares M, Hannon GJ, et al (2010): Purification of RNA using trizol (TRI reagent). Cold Spring Harb Protoc, 2010, pdb-prot5439.
Sekiguchi T, Mizutani T, Yamada K, et al (2004): Expression of epiregulin and amphiregulin in the rat ovary. J Mol Endocrinol, 33, 281-292.
Shimada M, Hernandez Gonzalez I, Gonzalez Robayna I, et al (2006): Paracrine and autocrine regulation of epidermal growth factor-like factors in cumulus oocyte complexes and granulosa cells: key roles for prostaglandin synthase 2 and progesterone receptor. Mol Endocrinol, 20, 1352-1365.
Sun TC, Liu XC, Yang SH, et al (2020): Melatonin inhibits oxidative stress and apoptosis in cryopreserved ovarian tissues via Nrf2/HO-1 signaling pathway. Front Mol Biosci, 7, 163.
Tamura H, Nakamura Y, Korkmaz A, et al (2009): Melatonin and the ovary: physiological and pathophysiological implications. Fert Steril, 92, 328-343.
Tamura H, Jozaki M, Tanabe M, et al (2020): Importance of melatonin in assisted reproductive technology and ovarian aging. Int J Mol Sci, 21, 1135.
Terrón MP, Delgado Adámez J, Pariente JA, et al (2013): Melatonin reduces body weight gain and increases nocturnal activity in male Wistar rats. Physiol Behav, 118, 8-13.
Trau HA, Brännström M, Curry TEJ, et al (2016): Prostaglandin E2 and vascular endothelial growth factor A mediate angiogenesis of human ovarian follicular endothelial cells. Hum Reprod, 31, 436-444.
Wang Z, Zhang Y, Deng Q, et al (2023): Integrative analysis of androgen receptor interactors aberrations and associated prognostic significance in prostate cancer. Urol J, 20, 318-328.
Xiao P, Nie J, Wang X, et al (2019): Melatonin alleviates the deterioration of oocytes from mice subjected to repeated superovulation. J Cell Physiol, 234, 13413-13422.
Yan X, Lee S, Gugiu BG, et al (2014): Fatty acid epoxyisoprostane E2 stimulates an oxidative stress response in endothelial cells. Biochem Biophys Res Commun, 444, 69-74.
Yang L, Xu H, Chen Y, et al (2021): Melatonin: multi-target mechanism against diminished ovarian reserve based on network pharmacology. Front Endocrinol, 12, 630504.
Yeleswaram K, McLaughlin LG, Knipe JO, et al (1997): Pharmacokinetics and oral bioavailability of exogenous melatonin in preclinical animal models and clinical implications. J Pineal Res, 22, 45-51.
Yoon JD, Jeon Y, Cai L, et al (2015): Effects of coculture with cumulus-derived somatic cells on in vitro maturation of porcine oocytes. Theriogenology, 83, 294-305.
Zhao X, Wang D, Wu Z, et al (2018): Female reproductive performance in the mouse: effect of oral melatonin. Molecules, 23, 1845.