Involvement of Reactive Oxygen Species in Meiotic Cell Cycle Regulation and Apoptosis in Mammalian Oocytes
Meenakshi Tiwari1, Shilpa Prasad1, Anima Tripathi1, Ashutosh N. Pandey1, Arvind K. Singh2, Tulsidas G. Shrivastav3, and Shail K. Chaube1
1Cell Physiology Laboratory and 2Genetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221005, Uttar Pradesh, India; 3Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, Baba Gang Nath Marg, Munirka, New Delhi 110067, India
Correspondence: firstname.lastname@example.org (S.K.C.)
Tiwari M et al. Reactive Oxygen Species 1(2):110–116, 2016; ©2016 Cell Med Press
(Received: December 18, 2015; Revised: December 21, 2015; Accepted: December 21, 2015)
ABSTRACT | The mammalian ovary is a metabolically active organ and generates a large amount of reactive oxygen species (ROS) during final stages of folliculogenesis. ROS modulate physiological arrest (i.e., diplotene arrest) in follicular oocytes as well as metaphase-II (M-II) arrest in ovulated oocytes in most of the mammalian species. A moderate increase in the level of ROS could be beneficial for meiotic resumption from diplotene and M-II arrest in oocytes. The increased production of ROS, decreased antioxidant system, drug treatment, pathological conditions, stress, and several other factors may lead to accumulation of ROS in the ovary. Increased levels of ROS may generate oxidative stress (OS), which could induce meiotic cell cycle arrest in oocytes. OS triggers granulosa cell apoptosis and thereby reduces the transfer of nutrients and survival factors to the oocytes, leading to apoptosis. In vitro culture conditions, reduced survival factors, and destabilized maturation promoting factor (MPF) may generate ROS and thereby OS in follicular and ovulated oocytes. OS induces apoptosis in diplotene- and M-II-arrested oocytes through mitochondria-mediated pathway. The deterioration in oocyte quality resulting from ROS-mediated apoptosis may negatively impact the outcome of assisted reproductive technology (ART) in several mammalian species, including humans.
KEYWORDS | Apoptosis; Mammalian oocytes; Meiotic cell cycle arrest; Oxidative stress; Reactive oxygen species; Redox signaling
ABBREVIATIONS | ART, assisted reproductive technology; Cdk1, cyclin-dependent kinase 1; M-II, metaphase-II; MPF, maturation promoting factor; OS, oxidative stress; ROS, reactive oxygen species
2. Generation of ROS in the Ovary
3. ROS-Mediated Regulation of Meiotic Cell Cycle at the Diplotene Stage and Apoptosis
4. ROS-Mediated Regulation of Meiotic Cell Cycle at the Metaphase-II Stage and Apoptosis
In mammals, free radicals are produced as by-products of normal cellular metabolism and serve as signal molecules in various physiological and pathological processes [1–4]. Superoxide anion radical (O2˙ˉ), hydrogen peroxide (H2O2), and hydroxyl radical (OH˙) are major reactive oxygen species (ROS) produced in the mammalian ovary [1, 2, 5]. Superoxide is generated when electrons leak from the electron transport chains, whereas dismutation of superoxide results in the formation of H2O2 . Hydroxyl radical is highly reactive, which can modify purine and pyrimidine bases, leading to oxidative DNA damage .
ROS are generated in the body either by non-enzymatic (e.g., Fenton reaction in the presence of transition metal ions) or by the enzymatic (e.g., xanthine dehydrogenase) reactions . Biological reactions, which utilize oxygen molecule as a substrate, also generate a large amount of ROS . The mitochondrial respiratory chain is a main oxygen consuming site in a wide variety of cell types; hence, the majority of ROS are produced from this site under physiological conditions [7–9].
Drug treatment, pathological conditions, and several other factors may generate a high level of ROS in the ovary [10–12]. In addition, depletion of antioxidant system in the ovary may also result in the accumulation of ROS [1, 10–13]. The increased accumulation of ROS could lead to oxidative stress (OS), which directly or indirectly deteriorates oocyte quality by inducing apoptosis of granulosa cells as well as oocytes [10, 14–18].
2. GENERATION OF ROS IN THE OVARY
The ovary is a metabolically active organ and generates excess amounts of ROS during final stages of follicular development and ovulation [1, 2]. The bimodal role of ROS in mammalian ovary has already been documented . Generation of a tonic level of ROS typically acts as a signal mechanism that modulates ovarian physiology , while overproduction of ROS results in OS that may cause detrimental effects on the ovary .
ROS are generated within the follicles during folliculogenesis. This notion is supported by previous studies showing that growing follicles, granulosa cells of graafian follicles, ovulated follicles, and luteal cells are the major sites for the generation of ROS [2, 19, 20]. The OS biomarkers, such as conjugated dienes, lipid hydroperoxide, and thiobarbituric acid-reactive substances, have been reported in human preovulatory follicles . The increased levels of ROS have been reported in the follicular fluid of preovulatory follicles of swines and cows [21, 22]. ROS are also produced within the follicles at the time of follicular rupture just prior to ovulation [2, 23]. Certain amount of ROS within the physiological range is necessary for the normal development of oocytes and it could be used as a marker for healthy developing oocytes [1, 24–26]. Recent studies suggest that a moderate increase of ROS mediates human chorionic gonadotropin (hCG)-induced meiotic resumption in rat oocytes within the follicular microenvironment . The higher level of ROS in human follicular fluid is associated with an increased fertilization rate and implantation [28, 29]. The level of ROS in the follicular fluid may serve as a potential marker for predicting the successful outcomes of in vitro fertilization .
ROS and antioxidants are known to intervene with the reproductive physiology of females . The ovary has its own antioxidant system through which it maintains the redox status during final stages of folliculogenesis [1, 2]. Follicular fluid and cumulus oophorus cells contain superoxide dismutase that protects oocytes from oxidative damage. The activities of superoxide dismutase, catalase, and glutathione peroxidase, as well as the presence of non-enzymatic antioxidants have been reported in the swine follicular fluid . Studies from our laboratory suggest that melatonin increases catalase activity, reduces the ROS level in the ovary, and protects against clomiphene citrate-induced adverse effects at the level of the ovary .
Previous studies from our laboratory also suggest that clomiphene citrate reduces the catalase activity and increases the ROS levels and apoptosis in encircling granulosa cells of preovulatory follicles [32, 33]. The granulosa cell apoptosis disrupts gap junctions and affects the supply of nutrients and maturation-enabling factors required for the achievement of meiotic competency in follicular oocytes [32, 33]. Under this condition, oocytes become more susceptible towards apoptosis which leads to the deterioration of oocyte quality after ovulation. The bioactive ingredients of neem leaf induce ROS-mediated apoptosis in granulosa cells as well as oocytes [17, 18, 34, 35]. The generation of ROS within the follicles and its negative impact on oocyte quality have recently been reviewed [33, 35, 36].
3. ROS-MEDIATED REGULATION OF MEIOTIC CELL CYCLE AT THE DIPLOTENE STAGE AND APOPTOSIS
Diplotene arrest is the longest phase of meiotic cell cycle, which may last for several months to several years depending on the mammalian species [3, 4, 37]. These diplotene-arrested oocytes (also known as dictyate stage oocytes) are morphologically identified by the presence of germinal vesicle (GV) [3, 4, 38, 39]. Meiotic resumption from diplotene arrest could be triggered by ROS within the preovulatory follicles. The beneficial role of ROS comes from the observations that non-enzymatic antioxidants, such as ascorbic acid and 3-tert-butyl-4-hydroxyanisole (BHA), inhibit spontaneous meiotic resumption from diplotene arrest [40, 41]. Our recent studies suggest that a moderate increase in the level of ROS is associated with spontaneous resumption of meiosis from diplotene arrest in rat oocytes cultured in vitro . The increased level of ROS induces Thr14/Tyr15 phosphorylation of cyclin-dependent kinase 1 (Cdk1) and reduces the levels of Thr161 phosphorylated Cdk1 and cyclin B1. Increased phosphorylation of Thr14/Tyr15 and dephosphorylation of Thr161 residues of Cdk1 and reduced cyclin B1 levels destabilize maturation promoting factors (MPF), which eventually results in spontaneous resumption of meiosis in oocytes .
ROS act as a double-edged sword and serve as key signal molecules in meiotic cell cycle progression and also have a role in the pathological processes, such as cell cycle arrest and apoptosis [1, 33, 35]. Our previous studies suggest that less than 60 ng/oocyte is a physiological level of ROS present in diplotene-arrested oocytes [10, 11]. Further, generation of a tonic level of ROS or a small accumulation of ROS (i.e., a relatively higher physiological range) may trigger meiotic resumption in follicular oocytes [10, 11]. Our quantitative analysis suggests that a level of ≥60 ng/oocyte (i.e., 60–80 ng/oocyte) may be considered as a moderate increase of ROS, which is beneficial for meiotic resumption from diplotene arrest [10, 11]. On the other hand, due to drug treatment or pathological conditions, or stress and several other factors, ROS levels may increase beyond the physiological range (>80 ng/oocyte), which could induce cell cycle arrest and thereby apoptosis in follicular oocytes [10–12] (Figure 1). Based on these studies, we propose that the increased levels of ROS and the resulting OS could be one of the reasons for the deterioration of oocyte quality and depletion of germ cells from the ovary just prior to ovulation under various pathophysiological conditions.
4. ROS-MEDIATED REGULATION OF MEIOTIC CELL CYCLE AT THE METAPHASE-II STAGE AND APOPTOSIS
In freshly ovulated oocytes, metaphase-II (M-II) arrest is a physiological event in mammals [3, 38]. However, spontaneous exit from M-II arrest has been reported in several mammalian species [42–45]. Recent studies from our laboratory suggest that a moderate increase of ROS is associated with spontaneous exit from M-II arrest . A transient increase of ROS or a small rise in the level of ROS modulates specific phosphorylation status of Cdk1 [16, 46]. ROS increase Thr14/Tyr15 phosphorylation and decrease Thr161 phosphorylated Cdk1 levels [16, 46]. The modulation in specific phosphorylation status of Cdk1 may result in dissociation and degradation of cyclin B1, leading to MPF destabilization. The destabilized MPF in turn triggers meiotic exit from M-II arrest [16, 46].
Handling of ovulated oocytes under in vitro culture conditions during various ART programs may increase the ROS level to the extent which could lead to OS . Such a high level of ROS could induce meiotic cell cycle arrest at M-II stage and apoptosis under in vitro culture conditions. This possibility is further supported by our studies demonstrating that verapamil (a non-specific L-type calcium channel blocker that causes increased formation of ROS in cells) induces meiotic cell cycle arrest and apoptosis in rat oocytes [48, 49]. Likewise, the calcium ionophore A23187 induces generation of ROS, which also results in meiotic cell cycle arrest and apoptosis in rat oocytes [14, 49, 50]. Further, roscovitine, a specific Cdk1 inhibitor, induces generation of ROS, maintenance of M-II arrest, and apoptosis in rat oocytes cultured in vitro . These studies suggest that increased levels of ROS due to drug treatment could be involved in cell cycle arrest and apoptosis in oocytes. In addition, exogenous supplementation of H2O2 in the culture medium induces meiotic cell cycle arrest and apoptosis in rat oocytes cultured in vitro  (Figure 1).
ROS induce meiotic cell cycle arrest and apoptosis in oocytes through mitochondria-mediated pathway [33–36]. This notion is further supported by the observations that the increased levels of ROS modulate the Bax/Bcl-2 ratio in mitochondrial membrane and thereby the membrane potential [33, 35, 36]. Changes in the mitochondrial membrane potential trigger the release of cytochrome c into the cytoplasm of oocytes [33, 35, 36]. Cytochrome c binds to apoptotic protease activating factor 1, resulting in the activation of upstream and downstream caspases [10, 36, 47–50]. The activated caspase-3 cleaves key structural and regulatory proteins, leading to several biochemical and morphological changes associated with oocyte apoptosis [36, 47–50]. Thus, high levels of ROS-mediated cell cycle arrest and apoptosis could be interlinked.
FIGURE 1. Reactive oxygen species in meiotic cell cycle regulation and apoptosis in oocytes. This scheme illustrates the possible factors that induce generation of reactive oxygen species (ROS) in the ovary and the impact of sustained high levels of ROS and the resulting oxidative stress on meiotic cell cycle arrest and apoptosis in follicular as well as ovulated oocytes.
ROS act as signal molecules and modulate various cellular functions, including meiotic cell cycle resumption, arrest, and apoptosis in oocytes of several mammalian species. A moderate increase of ROS triggers meiotic resumption from diplotene- as well as M-II arrest, while levels of ROS beyond the physiological range could induce MPF destabilization, reduce survival factors, and trigger mitochondria-mediated oocyte apoptosis. Hence, ROS act as a double-edged sword in oocyte biology, which, on the one side, could be beneficial at moderate levels and, on the other side, detrimental when their levels go beyond the physiological range.
This work was partly funded by Department of Science and Technology, Ministry of Science and Technology, Government of India.
- Agarwal A, Gupta S, Sharma R. Oxidative stress and its implications in female infertility: a clinician’s perspective. Reprod Biomed Online 2005; 11(5):641–50.
- Fujii J, Iuchi Y, Okada F. Fundamental roles of reactive oxygen species and protective mechanisms in the female reproductive system. Reprod Biol Endocrinol 2005; 3:43. doi: 10.1186/1477-7827-3-43.
- Tripathi A, Kumar KV, Chaube SK. Meiotic cell cycle arrest in mammalian oocytes. J Cell Physiol 2010; 223(3):592–600. doi: 10.1002/jcp.22108.
- Pandey AN, Tripathi A, Premkumar KV, Shrivastav TG, Chaube SK. Reactive oxygen and nitrogen species during meiotic resumption from diplotene arrest in mammalian oocytes. J Cell Biochem 2010; 111(3):521–8. doi: 10.1002/jcb.22736.
- Goud AP, Goud PT, Diamond MP, Gonik B, Abu-Soud HM. Reactive oxygen species and oocyte aging: role of superoxide, hydrogen peroxide, and hypochlorous acid. Free Radic Biol Med 2008; 44(7):1295–304. doi: 10.1016/j.freeradbiomed.2007.11.014.
- Attaran M, Pasqualotto E, Falcone T, Goldberg JM, Miller KF, Agarwal A, et al. The effect of follicular fluid reactive oxygen species on the outcome of in vitro fertilization. Int J Fertil Womens Med 2000; 45(5):314–20.
- Orrenius S, Gogvadze V, Zhivotovsky B. Mitochondrial oxidative stress: implications for cell death. Annu Rev Pharmacol Toxicol 2007; 47:143–83. doi: 10.1146/annurev.pharmtox.47.120505.105122.
- Forman HJ, Maiorino M, Ursini F. Signaling functions of reactive oxygen species. Biochemistry 2010; 49(5):835–42. doi: 10.1021/bi9020378.
- Kehrer JP, Klotz LO. Free radicals and related reactive species as mediators of tissue injury and disease: implications for health. Crit Rev Toxicol 2015; 45(9):765–98. doi: 10.3109/10408444.2015.1074159.
- Chaube SK, Prasad PV, Thakur SC, Shrivastav TG. Hydrogen peroxide modulates meiotic cell cycle and induces morphological features characteristic of apoptosis in rat oocytes cultured in vitro. Apoptosis 2005; 10(4):863–74. doi: 10.1007/s10495-005-0367-8.
- Tripathi A, Khatun S, Pandey AN, Mishra SK, Chaube R, Shrivastav TG, et al. Intracellular levels of hydrogen peroxide and nitric oxide in oocytes at various stages of meiotic cell cycle and apoptosis. Free Radic Res 2009; 43(3):287–94. doi: 10.1080/10715760802695985.
- Tripathi A, PremKumar KV, Pandey AN, Khatun S, Mishra SK, Shrivastav TG, et al. Melatonin protects against clomiphene citrate-induced generation of hydrogen peroxide and morphological apoptotic changes in rat eggs. Eur J Pharmacol 2011; 667(1–3):419–24. doi: 10.1016/j.ejphar.2011.06.005.
- Agarwal A, Durairajanayagam D, du Plessis SS. Utility of antioxidants during assisted reproductive techniques: an evidence based review. Reprod Biol Endocrinol 2014; 12:112. doi: 10.1186/1477-7827-12-112.
- Tripathi A, Chaube SK. High cytosolic free calcium level signals apoptosis through mitochondria-caspase mediated pathway in rat eggs cultured in vitro. Apoptosis 2012; 17(5):439–48. doi: 10.1007/s10495-012-0702-9.
- Tripathi A, Chaube SK. Reduction of phosphorylated Thr-161 Cdk1 level participates in roscovitine-induced Fas ligand-mediated apoptosis in rat eggs cultured in vitro. In Vitro Cell Dev Biol Anim 2015; 51(2):174–82. doi: 10.1007/s11626-014-9812-8.
- Tripathi A, Chaube SK. Roscovitine inhibits extrusion of second polar body and induces apoptosis in rat eggs cultured in vitro. Pharmacol Rep 2015; 67(5):866–74. doi: 10.1016/j.pharep.2015.01.011.
- Tripathi A, Shrivastav TG, Chaube SK. An increase of granulosa cell apoptosis mediates aqueous neem (Azadirachta indica) leaf extract-induced oocyte apoptosis in rat. Int J Appl Basic Med Res 2013; 3(1):27–36. doi: 10.4103/2229-516X.112238.
- Tripathi A, Shrivastav TG, Chaube SK. Aqueous extract of Azadirachta indica (neem) leaf induces generation of reactive oxygen species and mitochondria-mediated apoptosis in rat oocytes. J Assist Reprod Genet 2012; 29(1):15–23. doi: 10.1007/s10815-011-9671-0.
- Laloraya M, Kumar GP, Laloraya MM. Histochemical study of superoxide dismutase in the ovary of the rat during the oestrous cycle. J Reprod Fertil 1989; 86(2):583–7.
- Laloraya M, Pradeep KG, Laloraya MM. Changes in the levels of superoxide anion radical and superoxide dismutase during the estrous cycle of Rattus norvegicus and induction of superoxide dismutase in rat ovary by lutropin. Biochem Biophys Res Commun 1988; 157(1):146–53.
- Basini G, Simona B, Santini SE, Grasselli F. Reactive oxygen species and anti-oxidant defences in swine follicular fluids. Reprod Fertil Dev 2008; 20(2):269–74.
- Rizzo A, Minoia G, Trisolini C, Mutinati M, Spedicato M, Jirillo F, et al. Reactive oxygen species (ROS): involvement in bovine follicular cysts etiopathogenesis. Immunopharmacol Immunotoxicol 2009; 31(4):631–5. doi: 10.3109/08923970902932962.
- Behrman HR, Kodaman PH, Preston SL, Gao S. Oxidative stress and the ovary. J Soc Gynecol Investig 2001; 8(1 Suppl Proceedings):S40–2.
- Pasqualotto EB, Agarwal A, Sharma RK, Izzo VM, Pinotti JA, Joshi NJ, et al. Effect of oxidative stress in follicular fluid on the outcome of assisted reproductive procedures. Fertil Steril 2004; 81(4):973–6. doi: 10.1016/j.fertnstert.2003.11.021.
- Wiener-Megnazi Z, Vardi L, Lissak A, Shnizer S, Reznick AZ, Ishai D, et al. Oxidative stress indices in follicular fluid as measured by the thermochemiluminescence assay correlate with outcome parameters in in vitro fertilization. Fertil Steril 2004; 82(Suppl 3):1171–6. doi: 10.1016/j.fertnstert.2004.06.013.
- Combelles CM, Gupta S, Agarwal A. Could oxidative stress influence the in-vitro maturation of oocytes? Reprod Biomed Online 2009; 18(6):864–80.
- Tiwari M Chaube SK. A moderate increase of reactive oxygen species triggers meiotic resumption in rat follicular oocytes. J Obstet Gynaecol Res 2016; (in press).
- Oyawoye O, Abdel Gadir A, Garner A, Constantinovici N, Perrett C, Hardiman P. Antioxidants and reactive oxygen species in follicular fluid of women undergoing IVF: relationship to outcome. Hum Reprod 2003; 18(11):2270–4.
- Pasqualotto FF, Pasqualotto EB. Reactive oxygen species and oocyte fertilization. Hum Reprod 2007; 22(3):901; author reply -2. doi: 10.1093/humrep/del437.
- Baka S, Malamitsi-Puchner A. Novel follicular fluid factors influencing oocyte developmental potential in IVF: a review. Reprod Biomed Online 2006; 12(4):500–6.
- Matos L, Stevenson D, Gomes F, Silva-Carvalho JL, Almeida H. Superoxide dismutase expression in human cumulus oophorus cells. Mol Hum Reprod 2009; 15(7):411–9. doi: 10.1093/molehr/gap034.
- Chaube SK, Prasad PV, Tripathi V, Shrivastav TG. Clomiphene citrate inhibits gonadotropin-induced ovulation by reducing cyclic adenosine 3′,5′-cyclic monophosphate and prostaglandin E2 levels in rat ovary. Fertil Steril 2006; 86(4 Suppl):1106–11. doi: 10.1016/j.fertnstert.2006.03.027.
- Chaube SK, Shrivastav TG, Prasad S, Tiwari M, Tripathi M, Pandey AN, et al. Clomiphene citrate induces ROS-mediated apoptosis in mammalian oocytes. Open J Apoptosis 2014; 3(3):52–8. doi: 10.4236/ojapo.2014.33006.
- Chaube SK, Prasad PV, Khillare B, Shrivastav TG. Extract of Azadirachta indica (neem) leaf induces apoptosis in rat oocytes cultured in vitro. Fertil Steril 2006; 85(Suppl 1):1223–31. doi: 10.1016/j.fertnstert.2005.11.034.
- Chaube SK, Shrivastav TG, Tiwari M, Prasad S, Tripathi A, Pandey AK. Neem (Azadirachta indica L.) leaf extract deteriorates oocyte quality by inducing ROS-mediated apoptosis in mammals. Springerplus 2014; 3:464. doi: 10.1186/2193-1801-3-464.
- Tiwari M, Prasad S, Tripathi A, Pandey AN, Ali I, Singh AK, et al. Apoptosis in mammalian oocytes: a review. Apoptosis 2015; 20(8):1019–25. doi: 10.1007/s10495-015-1136-y.
- Mehlmann LM. Stops and starts in mammalian oocytes: recent advances in understanding the regulation of meiotic arrest and oocyte maturation. Reproduction 2005; 130(6):791–9. doi: 10.1530/rep.1.00793.
- Wassarman PM, Albertini, DF. The mammalian ovum. In: The Physiology of Reproduction (E Knobil, JD Neill), 2nd ed. Raven Press, New York, NY. 1994, p.79–122.
- Mrazek M, Fulka Jr J, Jr. Failure of oocyte maturation: possible mechanisms for oocyte maturation arrest. Hum Reprod 2003; 18(11):2249–52.
- Takami M, Preston SL, Toyloy VA, Behrman HR. Antioxidants reversibly inhibit the spontaneous resumption of meiosis. Am J Physiol 1999; 276(4 Pt 1):E684–8.
- Pandey AN, Chaube SK. A moderate increase of hydrogen peroxide level is beneficial for spontaneous resumption of meiosis from diplotene arrest in rat oocytes cultured in vitro. Biores Open Access 2014; 3(4):183–91. doi: 10.1089/biores.2014.0013.
- Chebotareva T, Taylor J, Mullins JJ, Wilmut I. Rat eggs cannot wait: spontaneous exit from meiotic metaphase-II arrest. Mol Reprod Dev 2011; 78(10–11):795–807. doi: 10.1002/mrd.21385.
- Premkumar KV, Chaube SK. An insufficient increase of cytosolic free calcium level results postovulatory aging-induced abortive spontaneous egg activation in rat. J Assist Reprod Genet 2013; 30(1):117–23. doi: 10.1007/s10815-012-9908-6.
- Premkumar KV, Chaube SK. RyR channel-mediated increase of cytosolic free calcium level signals cyclin B1 degradation during abortive spontaneous egg activation in rat. In Vitro Cell Dev Biol Anim 2014; 50(7):640–7. doi: 10.1007/s11626-014-9749-y.
- Prasad S, Tiwari M, Koch B, Chaube SK. Morphological, cellular and molecular changes during postovulatory egg aging in mammals. J Biomed Sci 2015; 22:36. doi: 10.1186/s12929-015-0143-1.
- Prasad S, Tiwari M, Tripathi A, Pandey AN, Chaube SK. Changes in signal molecules and maturation promoting factor levels associate with spontaneous resumption of meiosis in rat oocytes. Cell Biol Int 2015; 39(6):759–69. doi: 10.1002/cbin.10444.
- Uy B, McGlashan SR, Shaikh SB. Measurement of reactive oxygen species in the culture media using Acridan Lumigen PS-3 assay. J Biomol Tech 2011; 22(3):95–107.
- Chaube SK, Dubey PK, Mishra SK, Shrivastav TG. Verapamil reversibly inhibits spontaneous parthenogenetic activation in aged rat eggs cultured in vitro. Cloning Stem Cells 2007; 9(4):608–17. doi: 10.1089/clo.2007.0001.
- Chaube SK, Tripathi A, Khatun S, Mishra SK, Prasad PV, Shrivastav TG. Extracellular calcium protects against verapamil-induced metaphase-II arrest and initiation of apoptosis in aged rat eggs. Cell Biol Int 2009; 33(3):337–43.
- Chaube SK, Khatun S, Misra SK, Shrivastav TG. Calcium ionophore-induced egg activation and apoptosis are associated with the generation of intracellular hydrogen peroxide. Free Radic Res 2008; 42(3):212–20. doi: 10.1080/10715760701868352.