Cyclic nucleotides regulate oocyte meiotic maturation and quality in mammals
Mammalian oocytes are arrested at diplotene stage of first meiotic prophase for a long time in ovarian follicle. The achievement of meiotic competency starts with resumption from diplotene arrest, passes through metaphase I (M-I) to metaphase II (M-II), and ends with the extrusion of first polar body (PB-I) at the time of ovulation.[2,3] Hence, progression of meiotic cell cycle from diplotene arrest to M-II stage and successful extrusion of PB-I enables oocyte to get converted into haploid female gamete needed for successful fertilization and early embryonic development.[4-6] Thus, the meiotic maturation of oocyte is an important event that determines its quality and directly affects reproductive outcome in mammals including human.[4-6]
The oocyte meiotic maturation is mainly regulated by cyclic adenosine 3’, 5’-monophosphate (cAMP) and cyclic guanosine 3’, 5’-monophosphate (cGMP). These nucleotides are either received from encircling granulosa cells or generated by oocyte itself in mammals.[7-10] Synthesis of cAMP as well as cGMP is regulated by adenylyl cyclase (AC) as well as guanylyl cyclase (GC), while their degradation occur by cyclic nucleotide phosphodiesterases (PDEs) both in encircling somatic cells and oocyte within the follicular microenvironment.[10-12]
Pituitary gonadotropins regulate these enzymatic pathways in the granulosa cells and oocyte to reduce their levels in follicular oocyte.[8-12] The decrease of cAMP as well as cGMP levels initiates downstream signaling pathways to phosphorylation of cyclin-dependent kinase 1 (Cdk1) and synthesis/degradation of cyclin B1. Thus, changes in Cdk1 phosphorylation status and cyclin B1 level destabilize maturation-promoting factor (MPF) in the oocyte. MPF destabilization overcomes diplotene arrest and oocyte undergoes meiotic resumption, progression from M-I to M-II, and extrudes first PB-I to become female gamete just before ovulation in mammalian oocytes.[8-12]
cAMP SIGNALING AND REGULATION OF OOCYTE MEIOTIC MATURATION
The cAMP is an important regulator of meiotic maturation in mammalian oocytes. It is continuously generated in the encircling granulosa cells and gets transferred to the oocyte through gap junctions to maintain diplotene arrest within the follicular microenvironment [Figure 1].[5,12,13] Oocyte also generates cAMP sufficient enough to maintain meiotic arrest[1,7,10] suggesting that the sustained high level of cAMP is associated with the maintenance of meiotic arrest in vivo.
In mammalian oocyte, AC is responsible for conversion of adenosine triphosphate (ATP) into cAMP and results in the sustained high level of cAMP level within the oocyte.[7,10] A closely related nine transmembrane bound (AC 1–9) genes with sequence homology and structural similarities have recently been reviewed in the human genome. Out of which, AC3 has been detected in both mouse and rat oocytes, which is widely expressed intracellular source of cAMP in mammal. Further, AC activators such as forskolin increase intraoocyte cAMP level and inhibit spontaneous resumption from diplotene arrest in mice, rats, bovine, and human oocytes cultured in vitro.
It is well established that AC signaling pathway generates intrinsic cAMP in mammalian oocytes, cAMP- phosphodiesterase (cAMP-PDE) is required to be inactivated to prevent degradation of cAMP so that the elevated cAMP level could be maintained to maintain meiotic arrest in mammalian oocyte. This notion is further strengthened by observations that PDE inhibitors increase intraoocyte cAMP level and maintains diplotene arrest in rat, mouse, bovine, pig, and human oocytes cultured in vitro.
The cAMP as intraoocyte regulator of meiotic maturation is further strengthened by in vitro studies that membrane permeable analogs such as db-cAMP or 8-bromo-cAMP increase intracellular cAMP and maintain diplotene arrest in rat, mouse, rabbit, goat, and porcine oocytes. Measurement of intracellular cAMP further suggests that high intraoocyte cAMP level maintains diplotene as well as M-II arrest, while transient decrease in its level induces meiotic resumption from diplotene as well as M-II arrest in rat oocytes cultured in vitro.[18,26]
Gonadotropin surge increases intracellular cAMP level in the encircling granulosa cells of preovulatory follicles through adenylate cyclase pathway.[27,28] through the activation of mainly G stimulatory protein-coupled cell surface receptors. The increased cAMP level disrupts gap junctions among encircling somatic cells and between granulosa cells and oocyte that results in the interruption in the transfer of cAMP to the oocyte. In addition, hydrolysis through various cAMP-PDEs further reduces cAMP level leading to meiotic resumption from diplotene arrest in follicular oocytes.[7,29-31]
Physical removal of encircling granulosa cells disrupts gap junctions and culture of denuded oocytes in appropriate medium allows oocyte to undergo spontaneous resumption from diplotene arrest under in vitro culture conditions.[27-31] Thus, a transient decrease occurs in the oocyte that initiates downstream signaling pathway to induce resumption from diplotene arrest in mammalian oocytes. Taken together, these studies suggest that sustained high level of cAMP maintains diplotene arrest, while reduction in its level signals downstream pathway to induce meiotic resumption from diplotene arrest.[29,32] Indeed, cAMP is an intraoocyte regulator of meiotic maturation in mammalian oocytes.
cGMP SIGNALING AND REGULATION OF OOCYTE MEIOTIC MATURATION
The cGMP is another important signal molecule produced by the membrane GC natriuretic peptide receptor 2 (NPR2, also called guanylyl cyclase-B or GC-B) in the granulosa cells of follicular oocytes.[34-36] The GC-B is expressed in mural as well as cumulus granulosa cells, while its expression was not reported in oocyte or theca cells of the follicle.[34-36] The mural granulosa cells produce C-type natriuretic peptide (CNP or natriuretic peptide C or NPPC) and activates NPR2.[34,35] Unlike cAMP, cGMP is generated in the granulosa cells only and transferred to the oocyte through gap junctions to regulate the meiotic cell cycle.
The cGMP is hydrolyzed by specific PDEs that regulate cGMP level in the granulosa cells as well as in oocyte.[35,37] This possibility is further supported by in vitro studies that various PDE inhibitors increase intraoocyte cGMP level and responsible for diplotene arrest in rat, mice, and porcine oocytes cultured in vitro. A decrease of intraoocyte cGMP increases PDE3A activity that results in the decrease of cAMP level. The decrease of cGMP triggers resumption from diplotene arrest in rat oocytes cultured in vitro. In addition, pituitary gonadotropins decrease cGMP level and induce meiotic exit from diplotene arrest in mouse[40-42] and pig oocytes, while high level of cGMP maintains diplotene as well as M-II arrest in rat oocytes cultured in vitro.[8,11,33] Taken together, these observations suggest that increase of cGMP prevents spontaneous exit from diplotene arrest, while reduction in its level initiates downstream pathway to induce exit from diplotene arrest in mammalian oocytes. Indeed after cAMP, cGMP is another intraoocyte regulator of oocyte meiotic maturation in mammals.
CYCLIC NUCLEOTIDES REGULATE MPF
Changes in cAMP as well as cGMP levels regulate oocyte meiotic competency either by changing the activity of MPF or regulating its destabilization/stabilization process. Increase of cAMP level results in the activation of protein kinase A (PKA), which, in turn, phosphorylates several proteins and stabilizes MPF to maintain meiotic arrest.[13,16] The cGMP from granulosa cell origin inhibits the activity of PDE 3A, which is responsible for the hydrolysis of cAMP and elevates intraoocyte cAMP level in the oocyte [Figure 1]. Increased level of cAMP promotes the phosphorylation of Cdk1 at threonine-161 and stabilizes MPF.[42,44] Thus, meiotic arrest in diplotene as well as M-II stage is maintained by sustained high level of stabilized MPF in oocytes. Indeed, high level of cAMP as well as cGMP maintains stabilized MPF and thereby meiotic arrest at diplotene as well as M-II stage in vivo as well as in vitro.[8,11] The non-specific as well as specific PDE inhibitors prevent spontaneous exit from diplotene arrest under in vitro culture conditions probably by increasing cAMP and cGMP level. PDE inhibitors reduce fertilization rate, blastocyst formation and also induce pregnancy loss without disturbing reproductive cyclicity and ovulation process in mammals.
The decreased level of cGMP relieves inhibition of PDE3A in the oocyte[40,41] and active enzyme reduces intraoocyte cAMP level. Hence, reduced intraoocyte cAMP level results in the inactivation of PKA and thereby MPF destabilization and triggers meiotic exit from diplotene and M-II arrest.[33,44] Further, denudation process by physical removal of encircling granulosa cells interrupts transfer of cAMP as well as cGMP from the granulosa cells to the oocyte and spontaneous hydrolysis by PDEs results in the transient decrease of their levels in the oocyte.[8,9] The decrease of these cyclic nucleotides drives downstream signaling pathways to induce meiotic exit from diplotene arrest in vitro. Similarly, pituitary gonadotropins surge disrupt gap junctions among encircling granulosa cells and between encircling cumulus granulosa cells to the oocyte. Disruption of gap junctions affects the transfer of cAMP as well as cGMP from granulosa cells to the oocyte,[11,14,15] thus causing a transient decrease of their levels in the oocyte. The reduced intraoocyte cAMP level modulates phosphorylation status of Cdk1 and triggers cyclin B1 degradation that destabilizes MPF and/or increases Cdk1 activity.[9,12] The meiotic competency is initiated due to destabilized MPF and/or increased Cdk1 activity that leads to meiotic exit from diplotene arrest in pre-ovulatory follicles of several mammalian species.
CLINICAL AND COMMERCIAL USE OF CYCLIC NUCLEOTIDES
The in vitro maturation (IVM) is clinically attractive reproductive technology, wherein immature germinal vesicle stage oocytes are collected and cultured for IVM until it reaches M-II stage possessing PB-I.[47-49] The artificial elevation of cAMP in under in vitro culture conditions has shown potential to improve pregnancy rates. To increase cellular cAMP, COCs are exposed to AC activators and PDE inhibitors have been used during pre-IVM phase.[50-54] This type of cAMP modulating system has been shown to increase cAMP level in COC substantially that mimics to some extent the in vivo spike of cAMP caused by gonadotropin surge. Studies suggest that artificial modulating system of cAMP significantly improves oocyte quality, thereby blastocyst development, blastocyst quality, and pregnancy rates.[56-58] Thus, cAMP modulating system during pre-IVM stage could be a potential approach to bridge gap between IVM and IVF and has clinical as well as commercial relevance.
The cAMP as well as cGMP are produced in encircling granulosa cells and transferred to follicular oocyte through gap junction. Oocyte is also capable of generating cAMP good enough to maintain meiotic arrest at diplotene as well as M-II stages. Thus, high sustained levels of these cyclic nucleotides do not permit oocyte to complete meiotic maturation and affect oocyte quality. On the other hand, gonadotropin surge or removal of granulosa cells disrupts the transfer of these two cyclic nucleotides to the oocyte. As a result, intraoocyte cAMP as well as cGMP levels decrease that further increase their hydrolysis by oocyte-specific PDEs. Thus, a transient decrease of cyclic nucleotides triggers downstream pathway to destabilize MPF and/or its activity. The MPF destabilization drives spontaneous exit from diplotene arrest and oocyte achieve meiotic competency by reaching at M-II stage and extruding PB-I. The M-II arrested oocytes with PB-I are the right choice for successful fertilization and various assisted reproductive technology (ART) programs to optimize the reproductive outcome in several mammalian species including human. Indeed, cyclic nucleotides play an important role in the regulation of oocyte meiotic maturation and quality that could be used to improve ART outcome in several mammalian species including human.
Declaration of patient consentPatient’s consent not required as there are no patients in this study.
Financial support and sponsorshipThis study was financially supported by the Department of Science and Technology, Ministry of Science and Technology, Government of India (Grant no. EMR/2014/000702).
Conflicts of interestThere are no conflicts of interest.
- Biotech Mol Biol Rev. 2011;6:155-65.Developmental competence of in vivo and in vitro matured oocytes: A review.
- [Google Scholar]
- Health Popul Perspect Issues. 2001;24:218-31.Role of meiotic maturation regulatory factors in the meiotic competence of mammalian oocytes.
- [Google Scholar]
- Health Popul Perspect Issues. 2002;25:74-85.Does cAMP act as a regulator for oocyte meiotic resumption in mammal?
- [Google Scholar]
- J Mol Cell Biochem. 2018;2:1-4.Role of phosphodiesterase 3a in regulation of diplotene arrest of mammalian oocytes.
- [Google Scholar]
- Pharmazie. 2012;67:5-13.Forskolin and derivatives as tools for studying the role of cAMP.
- [Google Scholar]