Spontaneous Exit from Diplotene Arrest in Mammalian Oocytes in vitro: Is It ROS-Mediated?
Ashutosh N. Pandey, Meenakshi Tiwari, Anumegha Gupta, Alka Sharma, Shilpa Prasad, Pramod K. Yadav, Anil K. Yadav, Devendra K. Pandey, and Shail K. Chaube
Cell Physiology Laboratory, Department of Zoology, Institute of Science, Banaras Hindu University, Varanasi-221005, UP, India
Correspondence: email@example.com (S.K.C.)
Pandey AN et al. Reactive Oxygen Species 10(28):166–170, 2020; ©2020 Cell Med Press
(Received: January 21, 2020; Revised: March 10, 2020; Accepted: March 24, 2020)
ABSTRACT | The diplotene arrest is maintained for a long period within the follicular microenvironment of mammalian ovary. Normally, resumption from diplotene arrest occurs in response to pituitary gonadotrophin surge at puberty. However, gonadotrophin-independent meiotic exit from diplotene arrest was reported in rabbit oocytes cultured in vitro and thereafter in several mammalian species. Since no obvious stimulus is required, scientists consider it as spontaneous exit from diplotene arrest that initiates oocyte maturation in vitro. Emerging evidence suggests that in vitro culture conditions do not meet to the follicular microenvironment and result in generation of reactive oxygen species (ROS) in oocytes. A moderate increase of ROS modulates 3′,5′-cyclic adenosine monophosphate (cAMP) as well as Ca2+ levels and initiates downstream pathways to destabilize maturation promoting factor (MPF). MPF destabilization results in meiotic exit from diplotene arrest under in vitro culture conditions. Thus, spontaneous exit from diplotene arrest under in vitro culture conditions in mammalian oocyte is ROS-mediated.
KEYWORDS | Mammalian oocytes; Reactive oxygen species; Redox signaling; Spontaneous meiotic resumption
ABBREVIATIONS | cAMP, 3′,5′-cyclic adenosine monophosphate; M-I, metaphase-I; MPF, maturation promoting factor; ROS, reactive oxygen species
- Involvement of ROS
- ROS Signaling
Unlike other vertebrates, oocyte meiosis in mammals is unique and involves several stop/go channels in order to produce right female gamete. The majority of oocytes are arrested at diplotene stage of first meiotic prophase during fetal life and remain arrested for a long time until pituitary gonadotrophin surge at puberty [1, 2]. The gonadotrophin-independent meiotic resumption from diplotene arrest was reported for the first time in rabbit cumulus-enclosed oocytes cultured in vitro . This discovery led reproductive biologists to test unique feature of meiotic resumption from diplotene arrest without any obvious stimulation, that is the so called spontaneous maturation of oocyte under in vitro culture conditions, in several mammalian species including the human .
Among mammals, rat is an interesting animal model for the study of oocyte meiosis due to several peculiarities. For instance, diplotene-arrested oocytes undergo spontaneous exit from diplotene arrest and reach metaphase-I (M-I) stage but they are unable to extrude first polar body and remain arrested at M-I stage of meiotic cell cycle under in vitroculture conditions (Figure 1) [1, 4, 5]. During final stages of folliculogenesis, these somatic cells play an important role in oocyte growth, development maturation, and ovulation. Within the follicular microenvironment, oocytes are encircled by several layers of granulosa cells (mural and cumulus cells). The diplotene arrest from birth to puberty in mammalian oocytes is due to synthesis and transfer of several meiosis inhibitory factors, growth factors, survival factors, and signal molecules from encircling granulosa cells to the oocyte. The rate of spontaneous meiotic resumption from diplotene arrest is much high if the encircling granulosa cells are removed and denuded oocytes are cultured in vitro . Denudation causes disruption of gap junctions and dissociation of granulosa cells from oocyte and removal of inhibitory factors and cyclic nucleotides from encircling granulosa cells to the oocyte via gap junctions. Scientists believe that the spontaneous exit from diplotene arrest in vitro could be due to removal of inhibitory factors from follicular origin . However, emerging evidence suggests that the generation of ROS due to in vitro culture conditions could be associated with spontaneous resumption of meiosis in mammalian oocytes.
FIGURE 1. Representative photomicrographs showing spontaneous exit diplotene arrest in rat oocytes cultured in vitro. (A) Diplotene-arrested oocytes showing germinal vesicle and nucleolus (arrows). (B) Meiotic resumption from diplotene arrest as evidenced by the absence of germinal vesicle and nucleolus. (C) Diplotene-arrested oocytes showing diploid set of chromosomes at the time of in vitro culture (arrow). (D) Resumption of meiosis as evidenced by the formation of metaphase-I plate after 3 h of culture in vitro (arrow). Bar = 20 µm. The photomicrographs are adopted from Pandey AN and Chaube SK. BioResearch Open Access 2014; 3:183-91.
2. INVOLVEMENT OF ROS
In mammalian ovary, increased metabolism during final stages of folliculogenesis may generate ROS within follicular microenvironment. This is supported by the observations that follicular fluid contain high physiological level of ROS during final stages of folliculogenesis further strengthening the possibility of involvement of ROS during spontaneous resumption from diplotene arrest of mammalian oocytes [7–14] In addition, an increased level of ROS may result in the decrease of enzymatic antioxidant activity. This notion is supported by the observations that reduced catalase activity and increased level of ROS in the follicle [9, 15] are associated spontaneous meiotic resumption from diplotene arrest and ovulation in rat follicular oocytes [11, 13, 14]. The beneficial role of ROS during meiotic maturation, fertilization, and developmental competency of oocytes have been reported in the human [16–18].
Normally, diplotene-arrested follicular oocytes are isolated from ovary and cultured for extended period in appropriate medium under in vitro conditions to induce maturation process in several mammalian species during various assisted reproductive technology programs. Several efforts have been made to improve in vitro culture conditions for mammalian oocytes in tissue culture laboratories worldwide. These artificial tissue culture media as well as in vitro conditions never meet the requirement of follicular microenvironment of ovary in terms of pH, osmolarity, nutrients, and growth factors, among others. Thus, minor changes in physical as well as biological conditions may induce cellular stress and thereby ROS generation in oocytes cultured in vitro. This possibility is supported by the observations that culture of rat oocytes for 3 h in vitro resulted in the generation of H2O2 [5, 19]. Further, exogenous supplementation of a low level of H2O2 induced meiotic resumption from diplotene arrest in rat oocytes cultured in vitro [1, 5]. However, supplementation of nonenzymatic antioxidants prevented spontaneous exit from diplotene arrest [5, 20], suggesting the involvement of ROS during spontaneous resumption from diplotene arrest in mammalian oocytes cultured in vitro [2, 5, 14].
3. ROS SIGNALING
A growing body of evidence suggests that ROS act as signal molecules and modulate the physiology of mammalian oocytes . A moderate increase of ROS interacts with other major signal molecules, such as 3′,5′-cyclic adenosine monophosphate (cAMP) and Ca2+ of the oocyte cultured in vitro [14, 19]. Recent studies suggest that ROS reduced the cAMP level in rat oocytes that underwent spontaneous exit from diplotene arrest under in vitro culture conditions [5, 19]. On the other hand, ROS increased the cytosolic free Ca2+ level during spontaneous exit from diplotene arrest in rat oocytes cultured in vitro [5, 19]. The ROS-mediated increase of the cytosolic free Ca2+ level could be due to changes in the mitochondria membrane potential of oocytes . The interaction of ROS with cAMP and Ca2+ initiates downstream pathways to modulate phosphorylation/dephosphorylation of Cdk1, a catalytic unit of maturation promoting factor (MPF) as well as cyclin B1 level, a regulatory unit of MPF. ROS-mediated changes in Cdk1 phosphorylation status and cyclin B1 level result in MPF destabilization [19, 22]. Finally, MPF destabilization causes spontaneous exit from diplotene arrest in most of the mammalian species, including the human.
Increased metabolism in mammalian ovary during final stages of folliculogenesis or minor changes in physical and biochemical culture conditions in vitro may cause cellular stress and induce generation of ROS. A moderate increase of ROS modulates cAMP and Ca2+ levels and initiate downstream pathways in order to destabilize MPF. The MPF destabilization results in spontaneous exit from diplotene arrest under in vitro culture conditions. Indeed, spontaneous resumption of meiosis from diplotene arrest may not be spontaneous and could be associated with ROS generated due to in vitro culture conditions.
CONFLICTS OF INTEREST STATEMENT
The authors declare no conflict of interest.
- 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.
- 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.
- Pincus G, Enzmann EV. The comparative behavior of mammalian eggs in vivo and in vitro : I. The activation of ovarian eggs. J Exp Med 1935; 62(5):665–75. doi: 10.1084/jem.62.5.665.
- 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.
- 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.
- Gupta A, Tiwari M, Prasad S, Chaube SK. Role of cyclic nucleotide phosphodiesterases during meiotic resumption from diplotene arrest in mammalian oocytes. J Cell Biochem 2017; 118(3):446–52. doi: 10.1002/jcb.25748.
- Cheon YP, Kim SW, Kim SJ, Yeom YI, Cheong C, Ha KS. The role of RhoA in the germinal vesicle breakdown of mouse oocytes. Biochem Biophys Res Commun 2000; 273(3):997–1002. doi: 10.1006/bbrc.2000.3052.
- Sugino N. Reactive oxygen species in ovarian physiology. Reprod Med Biol 2005; 4(1):31–44. doi: 10.1111/j.1447-0578.2005.00086.x
- 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. doi: 10.1071/rd07147.
- Revelli A, Delle Piane L, Casano S, Molinari E, Massobrio M, Rinaudo P. Follicular fluid content and oocyte quality: from single biochemical markers to metabolomics. Reprod Biol Endocrinol 2009; 7:40. doi: 10.1186/1477-7827-7-40.
- Combelles CM, Gupta S, Agarwal A. Could oxidative stress influence the in-vitro maturation of oocytes? Reprod Biomed Online 2009; 18(6):864–80. doi: 10.1016/s1472-6483(10)60038-7.
- Tiwari M, Prasad S, Tripathi A, Pandey AN, Singh AK, Shrivastav TG, et al. Involvement of reactive oxygen species in meiotic cell cycle regulation and apoptosis in mammalian oocytes. React Oxyg Species (Apex) 2016; 1(2):110–16. doi: 10.20455/ros.2016.817.
- Tiwari M, Chaube SK. Moderate increase of reactive oxygen species triggers meiotic resumption in rat follicular oocytes. J Obstet Gynaecol Res 2016; 42(5):536–46. doi: 10.1111/jog.12938.
- Tiwari M, Chaube SK. Increase of reactive oxygen species associates with the achievement of meiotic competency in rat oocytes cultured in vitro. React Oxyg Species (Apex) 2017; 4(11):320–35. doi: 10.20455/ros.2017.853.
- Singh D, Pandey RS. Changes in catalase activity and hydrogen peroxide level in rat ovary during estrous cycle and induction of catalase in rat ovary by estradiol-17 beta. Indian J Exp Biol 1998; 36(4):421–3.
- 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. doi: 10.1093/humrep/deg450.
- 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. doi: 10.1016/s1472-6483(10)62005-6.
- Pasqualotto FF, Pasqualotto EB. Reactive oxygen species and oocyte fertilization. Hum Reprod 2007; 22(3):901. doi: 10.1093/humrep/del437.
- 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.
- Takami M, Preston SL, Toyloy VA, Behrman HR. Antioxidants reversibly inhibit the spontaneous resumption of meiosis. Am J Physiol 1999; 276(4):E684–8. doi: 10.1152/ajpendo.1999.276.4.E684.
- Premkumar KV, Chaube SK. Increased level of reactive oxygen species persuades postovulatory aging-mediated spontaneous egg activation in rat eggs cultured in vitro. In Vitro Cell Dev Biol Anim 2016; 52(5):576–88. doi: 10.1007/s11626-016-0007-3.
- Oh JS, Susor A, Conti M. Protein tyrosine kinase Wee1B is essential for metaphase II exit in mouse oocytes. Science 2011; 332(6028):462–5. doi: 10.1126/science.1199211.