Role of Intracellular Cyclic Adenosine 3′,5′-Monophosphate Concentration and Oocyte-Cumulus Cells Communications on the Acquisition of the Developmental Competence During In Vitro Maturation of Bovine Oocyte1

Abstract

The present study was designed to address the physiological role played by cAMP on gap junction (GJ) mediated communications between oocyte and cumulus cells during in vitro maturation. Cyclic AMP was stimulated by different collection and maturation media known to induce different rates of nuclear maturation and developmental competence as well as different levels of cumulus expansion. Cumulus-oocyte complexes (COCs) were matured for 0, 3, 7, 12, 18, and 24 h in the absence of stimulation or in the presence of serum and gonadotropins (fetal bovine serum+human menopausal gonadotropins [FCS+hMG]) or 0.01 microg/ml of invasive adenylate cyclase (iAC). For each time point, intracellular cAMP concentration ([cAMP]i) was determined either in the whole COC or oocyte after cumulus cell removal. GJ functional status was analyzed by microinjection of Lucifer yellow fluorescent dye in cumulus-enclosed oocytes and by immunohistochemical localization of connexin 43 (Cx43). In the absence of stimulation, [cAMP]i in COC and oocyte was lower than in other groups, and communications declined after 3 h of culture. In the FCS+hMG group, [cAMP]i increased significantly in COC, with a peak between 3 and 7 h that was temporally correlated with the beginning of the cumulus expansion process, which occurred only in this group and with the termination of the communications. COC matured in the presence of iAC showed a moderate increase of [cAMP]i during all of the maturation times as well as a prolongation of oocyte-cumulus cell communications. The immunohistochemical localization of Cx43 confirmed the delay in connexons protein turnover in iAC-treated COCs. Our results show that cumulus expansion and oocyte developmental competence are induced by different levels of cAMP and that its intracellular concentration may affect cell coupling between oocyte and cumulus cells. We hypothesize that the higher developmental competence of COCs matured in the presence of iAC could be achieved through a moderate increase of intracellular cAMP, which in turn determines a prolongation of communications between the two cell types.

Regulation of sheep oocyte maturation using cAMP modulators

“…Furthermore, meiotic resumption occurs in a rapid and uncontrolled manner [25]. However, the SPOM and related IVM models using elevated cAMP concentrations attenuated spontaneous meiosis, thereby resulting in a more gradual cessation of oocyte transcription and transition into the M-phase and promoting and prolonging oocyte-cumulus cell gap junction communication (GJC) [13,[32][33][34][35]. Part of the rationale for these approaches were substantial increases in cAMP occurred in vivo following LH-induction of the ovulatory cascade in porcine [36] and murine COCs [13,37].…”

“…Part of the rationale for these approaches were substantial increases in cAMP occurred in vivo following LH-induction of the ovulatory cascade in porcine [36] and murine COCs [13,37]. In contrast, liberation of the oocyte from the follicle for spontaneous IVM drastically decreases COC cAMP [13,34,38]. In the current study, the 1.5 to 2 h pre-IVM treatment with cAMP modulators, FSK and IBMX in the SPOM system, resulted in a > 10-fold increase in COC cAMP concentrations compared to COCs exposed to pre-IVM conditions in the absence of FSK and IBMX.…”

Role of gap junction-mediated communications in regulating large-scale chromatin configuration remodeling and embryonic developmental competence acquisition in fully grown bovine oocyte

“…As previously described [23], COCs were retrieved from midsized antral follicles (2-6 mm) with a 16-gauge needle mounted on an aspiration pump (COOK-IVF, Brisbane QLD, Australia) in M199 supplemented with 20 mM of HEPES, 1790 units/L of Heparin and 0.4 % of BSA (M199-H). For in vitro meiotic arrest (IVA) experiments, COCs were aspirated in M199-H supplemented with the non-selective PDE inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) at the final concentration of 0.5 mM [12,14]. After examination under a stereomicroscope, only COCs medium-brown in color, with five or more complete layers of cumulus cells enclosing an oocyte with finely granulated homogenous ooplasm were used.…”

“…Intercellular communications between oocytes and cumulus cells were assessed by Lucifer yellow (LY) microinjection as previously described [14]. Briefly, a 3 % solution of LY in 5 mM lithium chloride was pressure injected into the oocytes and the spread of the dye into surrounding cumulus cells was monitored with an inverted fluorescence microscope (Nikon Diaphot; Nikon Corp., Tokyo, Japan).…”

“…In assisted reproductive technologies (ART), the physical removal of the cumulus-oocyte complex (COC) from middle size antral follicles initiate oocyte's meiotic resumption before the journey of the oocyte development has been completed and causes asynchrony between cytoplasmic and nuclear maturation, thus decreasing oocyte developmental competence (Reviewed in [9][10][11]). Spontaneous resumption of meiosis is mainly due to a decrease in the concentration of intraoocyte cyclic adenosine monophosphate (cAMP), which has a major role in controlling mammalian oocyte maturation [12][13][14].…”