Restricted accessResearch articleFirst published online 2025-10
The Effects of Supplemented Coenzyme Q 10 to Extender on the Endoplasmic Reticulum Stress-Related Genes and Sperm Quality Parameters in Cryopreservation of Mouse Spermatozoa
The endoplasmic reticulum (ER) is the organelle responsible for protein folding in the cell. The damage that may occur during the freezing process of the sperm can exceed the protein loading capacity in the ER. Antioxidants, such as coenzyme Q10 (CoQ10), are added to freezing media to protect sperm cells. In this study, the aim was to investigate the expression levels of ER stress-related genes (protein kinase-like ER kinase [PERK], activating transcription factor 4 [ATF4], CCAAT-enhancer-binding-protein homologous protein [CHOP], and nuclear factor erythroid 2-related factor 2 [NRF2]) and quality parameters (viability, motility, acrosome status, and plasma membrane integrity) of mice sperm after freezing with an extender containing CoQ10. Male BALB/c mouse spermatozoa were cryopreserved using a combination of 18% raffinose + 3% skimmed milk and 50 µM CoQ10. The combination of 18% raffinose + 3% skimmed milk without CoQ10 was used as the control group. The results showed that post-thaw sperm motility, viability, plasma membrane integrity, and intact acrosome rates were significantly higher in the CoQ10-supplemented group compared with the control (untreated) group (p < 0.05). The expression of ER stress-related genes was then analyzed to investigate whether CoQ10 attenuates ER stress in frozen–thawed sperm. The results significantly revealed that the addition of 50 µM CoQ10 to the extender increased PERK, ATF4, and CHOP mRNA levels compared with the control group (p < 0.001). Next, NRF2 gene expression was analyzed to investigate whether CoQ10 affects the antioxidant mechanism of post-thaw sperm. It was revealed that the expression of the NRF2 gene significantly increased in the CoQ10 group compared with the control group (p < 0.001). Collectively, these results suggest that the freeze–thaw process induces ER stress in mouse sperm, and the supplementation of CoQ10 to the cryoprotectant agent reduces ER stress-related genes, activates the gene related to the antioxidant defense system, and improves post-thaw sperm quality parameters.
Get full access to this article
View all access options for this article.
References
1.
SchroderM, KaufmanRJ. ER stress and the unfolded protein response. Mutat Res, 2005; 569(1–2):29–63; doi: 10.1016/j.mrfmmm.2004.06.056
2.
SmithMH, PloeghHL, WeissmanJS. Road to ruin: Targeting proteins for degradation in the endoplasmic reticulum. Science, 2011; 334(6059):1086–1090; doi: 10.1126/science.1209235
3.
EllgaardL, HeleniusA. Quality control in the endoplasmic reticulum. Nat Rev Mol Cell Biol, 2003; 4(3):181–191; doi: 10.1038/nrm1052
4.
TajiriS, OyadomariS, YanoS, et al.Ischemia-induced neuronal cell death is mediated by the endoplasmic reticulum stress pathway involving CHOP. Cell Death Differ, 2004; 11(4):403–415; doi: 10.1038/sj.cdd.4401365
RobersonEC, TullyJE, GualaAS, et al.Influenza induces endoplasmic reticulum stress, caspase-12-dependent apoptosis, and c-Jun N-terminal kinase-mediated transforming growth factor-beta release in lung epithelial cells. Am J Respir Cell Mol Biol, 2012; 46(5):573–581; doi: 10.1165/rcmb.2010-0460OC
7.
HummastiS, HotamisligilGS. Endoplasmic reticulum stress and inflammation in obesity and diabetes. Circ Res, 2010; 107(5):579–591; doi: 10.1161/CIRCRESAHA.110.225698
8.
RonD, WalterP. Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol, 2007; 8(7):519–529; doi: 10.1038/nrm2199
9.
YoshidaH. ER stress and diseases. Febs J, 2007; 274(3):630–658; doi: 10.1111/j.1742-4658.2007.05639.x
10.
IwawakiT, AkaiR, YamanakaS, et al.Function of IRE1 alpha in the placenta is essential for placental development and embryonic viability. Proc Natl Acad Sci U S A, 2009; 106(39):16657–16662; doi: 10.1073/pnas.0903775106
11.
YangY, PeiX, JinY, et al.The roles of endoplasmic reticulum stress response in female mammalian reproduction. Cell Tissue Res, 2016; 363(3):589–597; doi: 10.1007/s00441-015-2212-x
12.
EhmckeJ, SchlattS. Animal models for fertility preservation in the male. Reproduction, 2008; 136(6):717–723; doi: 10.1530/REP-08-0093
13.
SetyawanEM, KimMJ, OhHJ, et al.Maintaining canine sperm function and osmolyte content with multistep freezing protocol and different cryoprotective agents. Cryobiology, 2015; 71(2):344–349; doi: 10.1016/j.cryobiol.2015.08.010
14.
AgarwalA, VirkG, OngC, et al.Effect of oxidative stress on male reproduction. World J Mens Health, 2014; 32(1):1–17; doi: 10.5534/wjmh.2014.32.1.1
15.
SalehRA, AgarwalA. Oxidative stress and male infertility: From research bench to clinical practice. J Androl, 2002; 23(6):737–752.
16.
TrzcinskaM, BrylaM. Apoptotic-like changes of boar spermatozoa in freezing media supplemented with different antioxidants. Pol J Vet Sci, 2015; 18(3):473–480; doi: 10.1515/pjvs-2015-0062
17.
KimSH, YuDH, KimYJ. Effects of cryopreservation on phosphatidylserine translocation, intracellular hydrogen peroxide, and DNA integrity in canine sperm. Theriogenology, 2010; 73(3):282–292; doi: 10.1016/j.theriogenology.2009.09.011
18.
FangY, BlairH, ZhongR, et al.Optimizing the freezing rate for ovine semen cryopreservation: Phospholipid profiles and functions of the plasma membrane and quality and fertilization of spermatozoa. Small Ruminant Research, 2016; 139:46–51.
19.
CaoSS, KaufmanRJ. Endoplasmic reticulum stress and oxidative stress in cell fate decision and human disease. Antioxid Redox Signal, 2014; 21(3):396–413; doi: 10.1089/ars.2014.5851
20.
SaidTM, GaglaniA, AgarwalA. Implication of apoptosis in sperm cryoinjury. Reprod Biomed Online, 2010; 21(4):456–462; doi: 10.1016/j.rbmo.2010.05.011
21.
SiW, BensonJD, MenH, et al.Osmotic tolerance limits and effects of cryoprotectants on the motility, plasma membrane integrity and acrosomal integrity of rat sperm. Cryobiology, 2006; 53(3):336–348; doi: 10.1016/j.cryobiol.2006.09.001
22.
AmidiF, PazhohanA, Shabani NashtaeiM, et al.The role of antioxidants in sperm freezing: A review. Cell Tissue Bank, 2016; 17(4):745–756; doi: 10.1007/s10561-016-9566-5
23.
SilvaSV, SoaresAT, BatistaAM, et al.In vitro and in vivo evaluation of ram sperm frozen in tris egg-yolk and supplemented with superoxide dismutase and reduced glutathione. Reprod Domest Anim, 2011; 46(5):874–881; doi: 10.1111/j.1439-0531.2011.01758.x
24.
MasoudiR, SharafiM, Zare ShahnehA, et al.Supplementation of extender with coenzyme Q10 improves the function and fertility potential of rooster spermatozoa after cryopreservation. Anim Reprod Sci, 2018; 198:193–201; doi: 10.1016/j.anireprosci.2018.09.019
25.
LenazG, FatoR, Di BernardoS, et al.Localization and mobility of coenzyme Q in lipid bilayers and membranes. Biofactors, 1999; 9(2–4):87–93; doi: 10.1002/biof.5520090202
26.
CraneFL. Biochemical functions of coenzyme Q10. J Am Coll Nutr, 2001; 20(6):591–598; doi: 10.1080/07315724.2001.10719063
27.
HyunDH, EmersonSS, JoDG, et al.Calorie restriction up-regulates the plasma membrane redox system in brain cells and suppresses oxidative stress during aging. Proc Natl Acad Sci U S A, 2006; 103(52):19908–19912; doi: 10.1073/pnas.0608008103
MorrisG, AndersonG, BerkM, et al.Coenzyme Q10 depletion in medical and neuropsychiatric disorders: Potential repercussions and therapeutic implications. Mol Neurobiol, 2013; 48(3):883–903; doi: 10.1007/s12035-013-8477-8
30.
ManciniA, BalerciaG. Coenzyme Q(10) in male infertility: Physiopathology and therapy. Biofactors, 2011; 37(5):374–380; doi: 10.1002/biof.164
31.
SafarinejadMR. Efficacy of coenzyme Q10 on semen parameters, sperm function and reproductive hormones in infertile men. J Urol, 2009; 182(1):237–248; doi: 10.1016/j.juro.2009.02.121
32.
BalerciaG, BuldreghiniE, VigniniA, et al.Coenzyme Q10 treatment in infertile men with idiopathic asthenozoospermia: A placebo-controlled, double-blind randomized trial. Fertil Steril, 2009; 91(5):1785–1792; doi: 10.1016/j.fertnstert.2008.02.119
RamadanLA, Abd-AllahAR, AlyHA, et al.Testicular toxicity effects of magnetic field exposure and prophylactic role of coenzyme Q10 and L-carnitine in mice. Pharmacol Res, 2002; 46(4):363–370; doi: 10.1016/s1043661802001718
35.
AslFR, KhosraviM, HajikhaniR, et al.Complementary effects of coenzyme Q10 and Lepidium sativum supplementation on the reproductive function of mice: An experimental study. Int J Reprod Biomed, 2021; 19(7):607–618; doi: 10.18502/ijrm.v19i7.9471
36.
Lozano-HernandezR, VelascoJ, PachecoL, et al.Protective effect of coenzyme Q10 in mouse testes ınfected with staphylococcus epidermidis. Austin Andrology, 2018; 3(3):1026.
LewinA, LavonH. The effect of coenzyme Q10 on sperm motility and function. Mol Aspects Med, 1997;(18 Suppl):S213–S9; doi: 10.1016/s0098-2997(97)00036-8
39.
TakeoT, NakagataN. In vitro fertilization in Mice. Cold Spring Harb Protoc, 2018; 2018(6); doi: 10.1101/pdb.prot094524
40.
QuinnP, KerinJF, WarnesGM. Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubal fluid. Fertil Steril, 1985; 44(4):493–498; doi: 10.1016/s0015-0282(16)48918-1
41.
MaklerA. A new chamber for rapid sperm count and motility estimation. Fertil Steril, 1978; 30(3):313–318; doi: 10.1016/s0015-0282(16)43518-1
42.
OrganisationWH. WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus İnteraction. Cambridge University Press. 1999.
43.
BahamondesL, FazanoF, De LucioMA, et al.Evaluation of human sperm membrane integrity using the water test and the hypoosmotic test. Andrologia, 2001; 33(2):75–77; doi: 10.1046/j.1439-0272.2001.00374.x
44.
MihaiAD, SchroderM. Glucose starvation and hypoxia, but not the saturated fatty acid palmitic acid or cholesterol, activate the unfolded protein response in 3T3-F442A and 3T3-L1 adipocytes. Adipocyte, 2015; 4(3):188–202; doi: 10.4161/21623945.2014.989728
45.
ZhangG, LingX, LiuK, et al.The p-eIF2alpha/ATF4 pathway links endoplasmic reticulum stress to autophagy following the production of reactive oxygen species in mouse spermatocyte-derived cells exposed to dibutyl phthalate. Free Radic Res, 2016; 50(7):698–707; doi: 10.3109/10715762.2016.1169403
46.
ZhangS, JiangC, LiuH, et al.Fluoride-elicited developmental testicular toxicity in rats: Roles of endoplasmic reticulum stress and inflammatory response. Toxicol Appl Pharmacol, 2013; 271(2):206–215; doi: 10.1016/j.taap.2013.04.033
47.
YangSH, HeJB, YuLH, et al.Protective role of curcumin in cadmium-induced testicular injury in mice by attenuating oxidative stress via Nrf2/ARE pathway. Environ Sci Pollut Res Int, 2019; 26(33):34575–34583; doi: 10.1007/s11356-019-06587-9
48.
JiYL, WangH, ZhaoXF, et al.Crosstalk between endoplasmic reticulum stress and mitochondrial pathway mediates cadmium-induced germ cell apoptosis in testes. Toxicol Sci, 2011; 124(2):446–459; doi: 10.1093/toxsci/kfr232
49.
GongZK, WangSJ, HuangYQ, et al.Identification and validation of suitable reference genes for RT-qPCR analysis in mouse testis development. Mol Genet Genomics, 2014; 289(6):1157–1169; doi: 10.1007/s00438-014-0877-6
50.
KouadjoKE, NishidaY, Cadrin-GirardJF, et al.Housekeeping and tissue-specific genes in mouse tissues. BMC Genomics, 2007; 8:127; doi: 10.1186/1471-2164-8-127
51.
LivakKJ, SchmittgenTD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 2001; 25(4):402–408; doi: 10.1006/meth.2001.1262
52.
BaileyJL, BilodeauJF, CormierN. Semen cryopreservation in domestic animals: A damaging and capacitating phenomenon. J Androl, 2000; 21(1):1–7.
53.
DesrosiersP, LegareC, LeclercP, et al.Membranous and structural damage that occur during cryopreservation of human sperm may be time-related events. Fertil Steril, 2006; 85(6):1744–1752; doi: 10.1016/j.fertnstert.2005.11.046
54.
ObletteA, RivesN, DumontL, et al.Assessment of sperm nuclear quality after in vitro maturation of fresh or frozen/thawed mouse pre-pubertal testes. Mol Hum Reprod, 2017; 23(10):674–684; doi: 10.1093/molehr/gax048
55.
SaeedAM, El-NagarHA, WafaWM, et al.Effect of coenzyme Q10 as an antioxidant added to semen extender during cryopreservation of buffalo and cattle semen. J Animal Poultry Production, 2016; 7(11):403–408.
56.
TasDO, OzkavukcuS, InancI, et al.The effects of coenzyme Q10 and curcumin supplementation in freezing medium for human sperm cryopreservation. Eur J Obstet Gynecol Reprod Biol, 2023; 287:36–45; doi: 10.1016/j.ejogrb.2023.05.040
57.
NogueiraBG, PereiraRR, BitencourtJL, et al.Coenzyme Q10 and melatonin protect cryopreserved equine sperm against lipoperoxidation. Anim Reprod Sci, 2022; 243:107027; doi: 10.1016/j.anireprosci.2022.107027
58.
TaleviR, BarbatoV, FiorentinoI, et al.Protective effects of in vitro treatment with zinc, d-aspartate and coenzyme q10 on human sperm motility, lipid peroxidation and DNA fragmentation. Reprod Biol Endocrinol, 2013; 11:81; doi: 10.1186/1477-7827-11-81
59.
Abd-ElhafizH, IssaN, SweedE. Protective effects of coenzyme Q10 against cisplatin-induced toxicity on testis in experimental rats. Inter J Cancer and Biomedical Res, 2021; 5(4)(:109–120.
60.
StanescuM, BirtoiuAI. Freezing of dog’s sperm: A review. Romanian Biotechnological Letters, 2012; 17(5):7709–7716.
61.
ParkHJ, KooYK, ParkMJ, et al.Restoration of spermatogenesis using a new combined herbal formula of epimedium koreanum nakai and angelica gigas nakai in an luteinizing hormone-releasing hormone agonist-ınduced rat model of male ınfertility. World J Mens Health, 2017; 35(3):170–177; doi: 10.5534/wjmh.17031
62.
SikkaSC. Role of oxidative stress and antioxidants in andrology and assisted reproductive technology. J Androl, 2004; 25(1):5–18; doi: 10.1002/j.1939-4640.2004.tb02751.x
63.
GualtieriR, BarbatoV, FiorentinoI, et al.Treatment with zinc, d-aspartate, and coenzyme Q10 protects bull sperm against damage and improves their ability to support embryo development. Theriogenology, 2014; 82(4):592–598; doi: 10.1016/j.theriogenology.2014.05.028
64.
YousefianI, EmamverdiM, Karamzadeh-DehaghaniA, et al.Attenuation of cryopreservation-induced oxidative stress by antioxidant: Impact of Coenzyme Q(10) on the quality of post-thawed buck spermatozoa. Cryobiology, 2018; 81:88–93; doi: 10.1016/j.cryobiol.2018.02.005
65.
YousefianI, Zare-ShahnehA, ZhandiM. The effect of coenzyme Q10 and α-Tocopherol in skim milk–based extender for preservation of caspian stallion semen in cool condition. J Equine Veterinary Sci, 2014; 34(8):949–954.
66.
AppiahMO, Asante-BaduB, ZhaoJ, et al.Possible protective mechanisms of coenzyme Q10 action on spermatozoa during cryopreservation or cooled-stored condition. Cryo Letters, 2020; 41(5):246–256.
67.
LanconiR, CeleghiniECC, GiuliV, et al.Coenzyme Q-10 improves preservation of mitochondrial functionality and actin structure of cryopreserved stallion sperm. Anim Reprod, 2021; 18(1):e20200218; doi: 10.1590/1984-3143-AR2020-0218
68.
NogueiraBG, SampaioBF, SouzaMI, et al.Coenzyme Q10 and alpha-Tocopherol prevent the lipid peroxidation of cooled equine semen. Reprod Domest Anim, 2015; 50(6):1003–1010; doi: 10.1111/rda.12627
69.
NathAK, BasuS, DattaU. Coenzyme Q10 and soyphosphatidylcholine in EK extender on preservation of Rhode Island Red poultry semen. J Adv Vet Anim Res, 2015; 2(2):134–140.
70.
IsachenkoE, IsachenkoV, WeissJM, et al.Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose. Reproduction, 2008; 136(2):167–173; doi: 10.1530/REP-07-0463
71.
HashemEZ, HaddadR, EslamiM. Evaluation of ram semen enrichment with oleic acid on different spermatozoa parameters during low temperature liquid storage. Small Ruminant Research, 2017; 150:30–39.
72.
OmurAD, CoyanK. Protective effects of the antioxidants curcumin, ellagic acid and methionine on motility, mitochondrial transmembrane potential, plasma membrane and acrosome integrity in freeze-thawed Merino ram sperm. Vet Med, 2016; 61(1):10–16.
73.
LibbyRT, GouldDB. Endoplasmic reticulum stress as a primary pathogenic mechanism leading to age-related macular degeneration. Adv Exp Med Biol, 2010; 664:403–409; doi: 10.1007/978-1-4419-1399-9_46
74.
LinS, ZhangJ, ChenH, et al.Involvement of endoplasmic reticulum stress in capsaicin-induced apoptosis of human pancreatic cancer cells. Evid Based Complement Alternat Med, 2013; 2013:629750; doi: 10.1155/2013/629750
75.
OslowskiCM, UranoF. Measuring ER stress and the unfolded protein response using mammalian tissue culture system. Methods Enzymol, 2011; 490:71–92; doi: 10.1016/B978-0-12-385114-7.00004-0
76.
Hebert-SchusterM, RottaBE, KirkpatrickB, et al.The ınterplay between glucose-regulated protein 78 (GRP78) and steroids in the reproductive system. Int J Mol Sci, 2018; 19(7); doi: 10.3390/ijms19071842
77.
KarnaKK, ShinYS, ChoiBR, et al.The role of endoplasmic reticulum stress response in male reproductive physiology and pathology: A review. World J Mens Health, 2020; 38(4):484–494; doi: 10.5534/wjmh.190038
78.
SelvarajuS, ParthipanS, SomashekarL, et al.Occurrence and functional significance of the transcriptome in bovine (Bos taurus) spermatozoa. Sci Rep, 2017; 7:42392; doi: 10.1038/srep42392
79.
ChenX, WangY, ZhuH, et al.Comparative transcript profiling of gene expression of fresh and frozen-thawed bull sperm. Theriogenology, 2015; 83(4):504–511; doi: 10.1016/j.theriogenology.2014.10.015
80.
WangM, KaufmanRJ. Protein misfolding in the endoplasmic reticulum as a conduit to human disease. Nature, 2016; 529(7586):326–335.
81.
KabelAM, ElkhoelyAA. Ameliorative effect of coenzyme Q10 and/or candesartan on carboplatin-ınduced nephrotoxicity: Roles of apoptosis, transforming growth factor-beta1, nuclear factor kappa-B and the Nrf2/HO-1 pathway. Asian Pac J Cancer Prev, 2017; 18(6):1629–1636; doi: 10.22034/APJCP.2017.18.6.1629
82.
El-AgamyDS, El-HarbiKM, KhoshhalS, et al.Pristimerin protects against doxorubicin-induced cardiotoxicity and fibrosis through modulation of Nrf2 and MAPK/NF-kB signaling pathways. Cancer Manag Res, 2019; 11:47–61; doi: 10.2147/CMAR.S186696
83.
WangSW, XuY, WengYY, et al.Astilbin ameliorates cisplatin-induced nephrotoxicity through reducing oxidative stress and inflammation. Food Chem Toxicol, 2018; 114:227–236.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
