Denuded Oocytes Research Articles

Inhibitory Role of the Mud Crab Short Neuropeptide F in Vitellogenesis and Oocyte Maturation via Autocrine/Paracrine Signaling.

Neuropeptides, in addition to their classical role in the nervous system, act on intraovarian factors to regulate reproductive functions in vertebrates. However, this function of neuropeptides has not been characterized in crustaceans. Short neuropeptide F (sNPF), a highly conserved invertebrate neuropeptide, has been reported to be involved in feeding, metabolism, and in differentiation processes including reproduction. Although sNPF and its receptor (sNPFR) have been detected in the ovary in different species, ovarian colocalization of sNPF/sNPFR has not been investigated. In this study, we identified Scylla paramamosain (mud crab) sNPF (Sp-sNPF) as an endogenous ligand for the S. paramamosain orphan G protein-coupled receptor NPY2R in mammalian cell line HEK293T. We designated this receptor as Sp-sNPFR. RNA in situ hybridization in pre-vitellogenic ovary and reverse transcription-PCR on isolated denuded oocytes and follicle layers showed that Sp-sNPF was exclusively localized to the follicle cells, whereas Sp-sNPFR was detected in both follicle cells and oocytes. We also found that Sp-sNPF partly suppressed spontaneous maturation of denuded oocytes and caused intracellular cAMP accumulation and Ca2+ mobilization. Moreover, injection of synthetic Sp-sNPF peptides inhibited the expression of vitellogenin and vitellogenin receptor genes in vivo. These combined results suggest for the first time that Sp-sNPF may have inhibitory functions in vitellogenesis and oocyte maturation possibly via the autocrine/paracrine pathway.

N-octanoylated ghrelin peptide inhibits bovine oocyte meiotic resumption

BackgroundStudies have shown that ghrelin plays an important role in the mammalian reproductive system, including the central, gonadal levels, and also during in vitro maturation of oocytes; however, the functions of ghrelin in bovine oocyte meiosis require further investigation. ObjectiveWe aimed to evaluate the effects of an n-octanoylated ghrelin peptide on oocyte meiotic resumption and the developmental competence of mature oocytes in vitro. Experimentaldesign: The expression of GHRL (encoding ghrelin) mRNA and its receptor (the growth hormone secretagogue receptor, GHSR) in the cumulus-oocyte complex (COCs), denuded oocytes (DOs), and cumulus cells (CCs) was assessed using quantitative real-time reverse transcription PCR (qRT-PCR), and the effects of the n-octanoylated ghrelin peptide on meiotic resumption were studied at four different doses (0, 10, 50, and 100 ng/mL) in a 6 h culture system. ResultsqRT-PCR analysis showed that GHRL and GHSR mRNAs were expressed in all tested samples; however, GHRL was predominantly expressed in DOs, and GHSR was predominantly expressed in CCs. Germinal vesicle breakdown was inhibited significantly by 50 ng/mL ghrelin compared with that in the negative control (P < 0.05). Further studies showed that n-octanoylated ghrelin increased the levels of cAMP and cGMP in the CCs and DOs, which inhibited the meiotic resumption of bovine oocytes. And the inhibitory role in the developmental competence of mature oocytes were also included, ghrelin could significantly improve the cleavage rate (P < 0.05) and blastocyst rate (P < 0.05). ConclusionN-octanoylated ghrelin maintained bovine oocytes meiotic arrest and further improved their developmental competence; therefore, n-octanoylated ghrelin could be considered as a potential pharmaceutical inhibitor of meiosis for the in vitro maturation of bovine oocytes.

Distinct Heat Stress Responses of Porcine Cumulus Cells and The Oocyte Is Associated With Disrupted Intercellular Communication

Cumulus cells (CCs) play critical roles in oocytes maturation through intercellular communication. The adverse effect of heat stress (HS) on oocyte maturation has been well documented, whereas the HS responses of CCs and the oocytes in association with cumulusoocyte communication remain unclear. In this study, cumulus‐oocyte complexes (COCs) were subjected to HS at 42°C for 24 h during in vitro maturation. Cumulus expansion was impaired and oocyte quality was reduced with lower survival rate, polar body extrusion rate and early embryo developmental potentials. Oocytes and cumulus cells isolated from COCs demonstrated distinct responses to HS. In oocytes, mitochondrial DNA (mtDNA)‐encoded genes and heat shock protein (HSP)‐related genes were significantly down‐regulated, with significantly lower ATP content yet higher mitochondria reactive oxygen species (mROS) and caspase 3 activity. In contrast, CCs isolated from heat‐exposed COCs showed significant activation of mtDNAencoded genes and HSP‐related genes, which was accompanied by increased ATP content and caspase 3 activity yet unaltered mROS content. When denuded oocytes and isolated CCs were subjected to HS separately, ATP content in denuded oocytes decreased more extensively than that in oocytes isolated from heat‐exposed COCs. Also, mROS was significantly increased in CCs but not in denuded oocytes, which coincided with less pronounced increase of caspase 3 in denuded oocytes. Moreover, F‐actin, a marker of trans‐zonal projections, and occludin, a tight junction protein, and their co‐localization, were significantly reduced in heat‐exposed COCs. These results indicate that cumulus cells and oocytes show distinct HS responses, indicating disrupted cumulus‐oocytes communication in heat‐exposed porcine oocytes.Support or Funding InformationThis work was supported by the National Key Research and Development Program of China (2016YFD0500502), the National Basic Research Program of China (2014CB138502), the Priority Academic Program Development of Jiangsu Higher Education Institutions, and Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality and Safety Control.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Relative importance of phosphatidylinositol-3 kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK3/1) signaling during maturational steroid-induced meiotic G2-M1 transition in zebrafish oocytes.

Participation and relative importance of phosphatidylinositol-3 kinase (PI3K) and mitogen-activated protein kinase (MAPK) signalling, either alone or in combination, have been investigated during 17α,20β-dihydroxy-4-pregnen-3-one (DHP)-induced meiotic G2-M1 transition in denuded zebrafish oocyte. Results demonstrate that concomitant with rapid phosphorylation (activation) of Akt (Ser473) and MAPK (ERK1/2) at as early as 15 min of incubation, DHP stimulation promotes enhanced an GVBD response and histone H1 kinase activation between 1 and 5 h in full-grown oocytes in vitro. While p-Akt reaches its peak at 60 to 90 min and undergoes downregulation to the basal level by 240 min, ERK1/2 phosphorylation (activation) increases gradually until 120 min and remains high thereafter. Although, priming with MEK1/2 inhibitor U0126 is without effect, PI3K inhibitors, wortmannin or LY294002, delay the GVBD response significantly (P < 0.001) until 3 h but not at 5 h of incubation. Interestingly, blocking PI3K and MEK function together could abrogate steroid-induced oocyte maturation at all time points tested. While DHP stimulation promotes phospho-PKA catalytic (p-PKAc) dephosphorylation (inactivation) between 30-120 min of incubation, simultaneous inhibition of PI3K and MEK1/2 kinases abrogates DHP action. Conversely, elevated intra-oocyte cAMP, through priming with either adenylyl cyclase (AC) activator forskolin (FK) or dibutyryl cAMP (db-cAMP), abrogates steroid-induced Akt and ERK1/2 phosphorylation. Taken together, these results suggest that DHP-induced Akt and ERK activation precedes the onset of meiosis (GVBD response) in a cAMP-sensitive manner and PI3K/Akt and MEK/MAPK pathways together have a pivotal influence in the downregulation of PKA and resumption of meiotic maturation in zebrafish oocytes in vitro.

171 Protective Effect of Sodium Pyruvate Against H2O2-Induced Oxidative Stress in Bovine and Swine Oocytes

A previous study from our laboratory (Ramos-Ibeas et al. 2017 Mol. Cell. Biochem. 429, 137-150, 10.1007/s11010-017-2942-z) described the protective effect of sodium pyruvate (SP) against H2O2-induced oxidative stress in somatic and embryonic stem cells. In this study, we tested the same experimental conditions on bovine and swine oocytes during in vitro maturation (IVM). To this aim, cumulus-enclosed oocytes (COC) recovered from bovine and swine ovaries were treated for 1 h with different concentrations of H2O2 and then matured in serum-free TCM-199 with and without SP supplementation. All experiments were done in 3 replicates using 12 to 20 oocytes per experimental group; data were analysed by two-way ANOVA. As a first step, we determined the dose-response curve of H2O2 treatment during IVM, using the reaching of metaphase 2 as endpoint. We found that swine COC were more sensitive to H2O2 damage than bovine COC: indeed, they tolerated a range of concentrations from 128 to 512 μM, whereas the range for bovine COC was between 1024 and 4096 μM. The COC from both species were treated for 1 h with 128 μM (swine) or 2048 μM (bovine) H2O2 and then matured with or without different concentrations of SP. Control COC, unexposed to H2O2, were also matured with and without SP supplementation. We found that the addition of 25 mM SP to swine COC reduced the maturation rate both in control and H2O2-treated groups. Instead, reducing the addition of SP to 10 mM we observed a beneficial effect because the metaphase II level in treated oocytes increased from 38% (15/40) to 67% (24/36) and the difference was statistically significant. In bovine COC, the addition of 25 mM SP did not affect control oocytes during IVM but decreased maturation rate in treated oocytes, whereas 10 mM SP had no effect on the control group and showed a tendency to increase maturation rate in treated oocytes. In a second set of experiments, to determine if cumulus cells acted as a barrier for SP to reach the oocyte, the COC were denuded after H2O2 treatment and matured with or without SP. In swine denuded oocytes (DO) matured with SP, the rate of metaphase II increased significantly both in control [from 71% (34/48) to 92% (44/48)] and in treated groups [from 48% (23/48) to 72% (36/50)]. In bovine DO, the supplementation of SP did not increase the maturation rate significantly (25/41 v. 29/41 in control groups, 12/36 v. 18/40 in treated groups). In conclusion, we found that toxic H2O2 concentrations were approximately 10-fold higher for bovine than for swine, indicating that bovine COC were more resistant to oxidative stress. This could explain why SP supplementation had a significant protective effect in swine but only a minor effect in bovine. Moreover, the difference in SP effect between bovine and swine COC could be due to their morphology because bovine cumulus is often larger and more compact than swine cumulus and this difference could influence how SP reaches the inner oocyte and how it is metabolized. This work was funded by projects EpiHealth FP7 n. 278418, EpiHealthNet FP7-PEOPLE-2012-ITN n. 317146 and Fecund FP7 n. 312097.

POSSIBILITY FOR IMPROVING OVARIAN ACTIVITY, CYTOPLASMIC MATURATION, FERTILIZATION AND DEVELOPMENTAL COMPETENCE IN VITRO OF OOCYTES BY ADMINISTRATION OF DOE RABBITS WITH GREEN TEA EXTRACT

The aim of this study was to evaluate the effect of oral administration of doe rabbits with green tea extract polyphenols (GTPs) at different levels on ovarian activity, oocyte quality, in vitro maturation, fertilization and development of oocytes. Total of 12 mature NZW doe rabbits (5-6 months of age, 2.5-3.20 kg LBW) were used in this study. Does were divided into 3 similar groups, (n=4). The 1st group (G1) was control, while does in the 2nd (G2) and 3th (G3) groups were given daily for one month oral 1 ml distilled water containing 200 and 400 mg/kg LBW of GTPs, respectively. At the end of treatment period, does in each group were slaughtered and immediately ovaries were removed and oocytes were collected by slicing technique for evaluation. Only compact-cumulus oocytes (COCs) were in vitro matured and fertilized. Results showed that ovarian weight relative to LBW, oocyte number/doe and recovery rate of oocyte were higher (P<0.05) in G2 and G3 than in G1. Number of visible follicles/doe was not affected by treatment. Frequency distribution was higher (P<0.05) for COCs and lower (P<0.05) for denuded oocytes in G2 and G3 as compared to G1. Frequency distribution of both expanded and partial denuded oocytes were not affected by treatment. Percentage of full expanded oocytes was the highest (P<0.05) in G2, followed by G3, while G1 showed the lowest (P<0.05) values. Percentage of partial expansion oocytes was higher (P<0.05) in G3 than in G1 and G2. Fertilization rate was higher (P<0.05) in G2, followed by G3, while G1 showed the lowest (P<0.05) values. Percentage of blastocysts was higher (P<0.05) in G2 and G1 than in G3. Percentage of morulae, and degenerated and un-cleaved embryos were not affected by GTPs treatment. In conclusion, treatment of doe rabbits with green tea extract at a level of 200 mg/kg LBW as daily oral dose for one month is recommended to enhance ovarian activity, oocyte quality, cytoplasmic maturation and in vitro embryo production.

Functional role of Forskolin and PD166285 in the development of denuded mouse oocytes.

ObjectivecAMP and mature promoting factor (MPF) play critical roles during the maturation of mammalian oocytes. The aim of this study was to produce the offspring from denuded oocytes (DOs) in mice by regulating cAMP and MPF.MethodsIn this study, we used DOs at the germinal vesicle (GV) stage in mice and regulated levels of cAMP and MPF in DOs by adding Forskolin and PD166285 during in vitro maturation without follicle stimulating hormone and luteinizing hormone, respectively.ResultsCombined use of 50 μM Forskolin for 3 h and 2.5 μM PD166285 for additional 21 h enhanced the developmental competence of DOs, maturation rate of DOs was 76.71%± 4.11%, blastocyst rate was 18.33%±4.44% after parthenogenetic activation (PA). The DOs could successfully be fertilized with sperm in vitro, cleavage rate was 17.02%±5.82% and blastocyst rate was 5.65%±3.10%. Besides, 2-cell in vitro fertilization embryos from DOs produced 4 normal live offspring (4/34).ConclusionThe results confirmed that the combination of Forskolin and PD166285 can induce DOs to complete meiosis process and produce normal offspring.

Differential effects of mitochondrial inhibitors on porcine granulosa cells and oocytes

Oocytes and granulosa cells rely primarily on mitochondrial respiration and glycolysis for energy production, respectively. The present study examined the effect of mitochondrial inhibitors on the ATP contents of oocytes and granulosa cells. Cumulus cell-oocyte complexes (COCs) and granulosa cells (GCs) were collected from the antral follicles of porcine ovaries. Treatment of denuded oocytes with either carbonyl cyanide m-chlorophenyl hydrazine (CCCP), antimycin, or oligomycin significantly reduced ATP content to very low levels (CCCP, 0.12 pM; antimycin, 0.07 pM; and oligomycin, 0.25 pM; P < 0.05), whereas treatment with a glycolysis inhibitor (bromopyruvic acid, BA) had no effect. Conversely, the ATP content of granulosa cells was significantly reduced by treatment with the glycolysis inhibitor but was not affected by the mitochondrial inhibitors (ATP/10,000 cells; control, 1.78 pM and BA, 0.32 pM; P < 0.05). Reactive oxygen species (ROS) generation after CCCP treatment was greater in oocytes (1.6-fold) than that seen in granulosa cells (1.08-fold). Oocytes surrounded by granulosa cells had higher ATP levels than denuded oocytes. Treatment of COCs with CCCP reduced, but did not completely abolish, ATP content in oocytes (control, 3.15 pM and CCCP, 0.52 pM; P < 0.05), whereas treatment with CCCP plus a gap junction inhibitor, 18α-glycyrrhetinic acid, and CCCP decreased the ATP content to even lower levels (0.29 pM; P < 0.05). These results suggest that granulosa cells are dependent on glycolysis and provide energy to oocytes through gap junctions, even after treatment with CCCP.

Ganglioside GM3 induces cumulus cell apoptosis through inhibition of epidermal growth factor receptor-mediated PI3K/AKT signaling pathways during in vitro maturation of pig oocytes.

Gangliosides are components of the mammalian plasma membrane that help regulate receptor signaling. Ganglioside GM3, for example, plays an important role in initiating apoptosis in cancer cells; however, physiological roles for GM3 in normal processes, such as during pig oocyte maturation, are not clear. The aim of this study was to investigate the functional link between GM3 and cellular apoptosis in porcine cumulus-oocyte-complexes (COCs) during in vitro maturation. Our results indicated that denuded oocytes possess less ST3GAL5, a GM3-synthesizing enzyme, than cumulus cells or COCs after 44 hr of in vitro maturation. GM3 also affected the meiotic maturation of cultured pig oocytes, as evaluated by orcein staining. In vitro treatment of COCs with exogenous GM3 also reduced cumulus cell expansion, the proportion of meiotic maturation, and increased cumulus cell transcription of PTX3, TNFAIP6, and HAS2. Interestingly, GM3 treatment reduced the expression of Epidermal growth factor receptor (EGFR)-mediated Phosphoinositide 3-kinase/AKT signaling proteins in COCs in a concentration-dependent manner, instead increasing the abundance of pro-apoptotic factors such as AIF, activated Caspase 9, cleaved PARP1, and Caspase 3 were. Thus, GM3 might affect porcine oocyte maturation via suppression of EGFR-mediated PI3K/AKT signaling and/or induction of apoptosis during in vitro maturation.

Impact of oocyte-secreted factors on its developmental competence in buffalo.

Oocyte-secreted factors (OSFs) play an important role in the acquisition of oocyte developmental competence through bidirectional cross-talk between oocyte and cumulus cells via gap junctions. Thus, the present study was designed to investigate the effect of two OSFs, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), on the developmental competence of buffalo oocytes derived from two different follicle sizes. Cumulus-oocyte complexes (COCs) from large follicles (LF, >6 mm) or small follicles (SF, 0.05) between DOs and combination groups. Relative mRNA analysis revealed significantly higher (P > 0.05) expression of the cumulus cell marker genes EGFR, HAS2, and CD44 in LF-derived than SF-derived oocyte; the expression of these markers was significantly higher (P > 0.05) in DOs and combination groups, irrespective of the follicle size. These results suggested that LF-derived oocytes have a higher developmental competence than SF-derived oocytes and that supplementation of GDF9 and BMP15 modulates the developmental competence of buffalo oocytes by increasing the relative abundance of cumulus-enabling factors and thereby increasing cleavage and the quality of blastocyst production.

MALDI mass spectrometry reveals that cumulus cells modulate the lipid profile of in vitro-matured bovine oocytes

ABSTRACTThe influence of cumulus cells (CC) on the lipid profile of bovine oocytes matured in two different lipid sources was investigated. Cumulus-oocyte complexes (COC) or denuded oocytes (DO) were matured in tissue culture medium (TCM) supplemented with fetal bovine serum (FBS) or serum substitute supplement (SSS). Lipid profiles of TCM, serum supplements, immature CC and oocyte (IO), and in vitro-matured oocytes from COC and DO were then analyzed by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) and submitted to partial least squares-discriminant analysis (PLS-DA). The developmental competence of such oocytes was also assessed. Differences in lipid composition were observed between two types of sera and distinctly influenced the lipid profile of CC. As revealed by PLS-DA, the abundance of specific ions corresponding to triacylglycerols (TAG) or phospholipids (PL) were higher in COC compared to DO both supplemented with FBS or SSS and to some extent affected the subsequent DO in vitro embryo development. DO exposed to SSS had however a marked diminished ability to develop to the blastocyst stage. These results indicate a modulation by CC of the oocyte TAG and PL profiles associated with a specific cell response to the serum supplement used for in vitro maturation.

Inhibition of calcineurin by FK506 stimulates germinal vesicle breakdown of mouse oocytes in hypoxanthine-supplemented medium.

Calcineurin (CN) is a serine/threonine phosphatase which plays important roles in meiosis maturation in invertebrate oocytes; however, the role of CN in mouse oocytes is relatively unexplored. In this study, we examined the expression, localization and functional roles of CN in mouse oocytes and granulosa cells. The RT-PCR results showed that the β isoform of calcineurin A subunit (Cn A) expressed significantly higher than α and γ isoforms, and the expression of Cn Aβ mRNA obviously decreased in oocytes in which germinal vesicle breakdown (GVBD) occurred, while only B1 of calcineurin B subunit (Cn B) was detected in oocytes and stably expressed during oocytes maturation. The following fluorescence experiment showed that Cn A was mainly located in the nucleus of germinal vesicle (GV) stage oocytes and gruanlosa cells, and subsequently dispersed into the entire cytoplasm after GVBD. The decline of Cn A in oocytes suggested that it may play an important role in GVBD. To further clarify the role of calcineurin during meiotic maturation, FK506 (a calcineurin inhibitor) was used in the culture medium contained hypoxanthine (HX) which could keep mouse oocytes staying at GV stage. As expected, FK506 could induce a significant elevation of GVBD rate and increase the MPF level of denuded oocytes (DOs). Furthermore, FK506 could also play an induction role of GVBD of oocytes in COCs and follicles, and the process could be counteracted by MAPK kinase inhibitor (U0126). Above all, the results implied that calcineurin might play a crucial role in development of mouse oocytes and MPF and MAPK pathways are involved in this process.

DNA Double-Strand Breaks Induce the Nuclear Actin Filaments Formation in Cumulus-Enclosed Oocytes but Not in Denuded Oocytes

As a gamete, oocyte needs to maintain its genomic integrity and passes this haploid genome to the next generation. However, fully-grown mouse oocyte cannot respond to DNA double-strand breaks (DSBs) effectively and it is also unable to repair them before the meiosis resumption. To compensate for this disadvantage and control the DNA repair events, oocyte needs the cooperation with its surrounding cumulus cells. Recently, evidences have shown that nuclear actin filament formation plays roles in cellular DNA DSB repair. To explore whether these nuclear actin filaments are formed in the DNA-damaged oocytes, here, we labeled the filament actins in denuded oocytes (DOs) and cumulus-enclosed oocytes (CEOs). We observed that the nuclear actin filaments were formed only in the DNA-damaged CEOs, but not in DOs. Formation of actin filaments in the nucleus was an event downstream to the DNA damage response. Our data also showed that the removal of cumulus cells led to a reduction in the nuclear actin filaments in oocytes. Knocking down of the Adcy1 gene in cumulus cells did not affect the formation of nuclear actin filaments in oocytes. Notably, we also observed that the nuclear actin filaments in CEOs could be induced by inhibition of gap junctions. From our results, it was confirmed that DNA DSBs induce the nuclear actin filament formation in oocyte and which is controlled by the cumulus cells.

Effect of Antioxidants (β-mercaptoethanol and Cysteamine) on Assisted Reproductive Technology In vitro.

Oocyte Culture of Germinal Vesicle (GV) and its growth improves Assisted Reproductive Technology (ART) invitro and infertility. Inappropriate culture medium environment, low quality of oocytes, increase in Oxidative Stress (OS) events, Reactive Oxygen Species (ROS) and free radicals production are the main factors that result in unsuccessful Invitro Maturation (IVM) and decrease in reproduction. The present study was conducted with the aim to evaluate the effect of β-mercaptoethanol (BME) and Cysteamine (CYS) on IVM improvement, embryo fertilization and development of blastocyst of mouse immature oocyte. Oocytes were obtained from 4-6 weeks old Naval Medical Research Institute (NMRI) female mice, 48 hours after stimulation with Intraperitoneal (IP) injection of 10 IU Pregnant Mare Serum Gonadotropin (PMSG). GV oocyte with and without cumulus cells were isolated from ovaries and cultured in Tissue Culture Medium (TCM) 199 with availability of 100 μM of antioxidants (BME and CYS). After 24 hours, mature oocyte in metaphase II (MII) were fertilized with sperm in In vitro Fertilization (IVF) medium (T6) and evaluated for fetal development into blastocyst. BME and CYS could significantly (p<0.05) increase the rate of IVM and oocyte evolution, and embryo formation in medium culture. Furthermore, it is demonstrated that existence of Cumulus Oocyte Complexes (COC) significantly showed better IVM, fertilization and evolution trend as compared to oocytes without cumulus cover or Denuded Oocytes (DO), especially in TCM199 plus BME and CYS. So that the change in GV stage oocytes to MII (maturation rate), fertilization rates or 2PN formation, and two cell embryos formation or blastocyst development rate in the treatment group with addition of BME & CYS and COC was statistically significant as compared to the DO group (p-value < 0.0001). Both cellular and environmental factors could be important and involved in ART improvement.

168 THE DEVELOPMENTAL POTENTIAL OF PARTHENOGENETIC EMBRYOS IS AFFECTED BY PROLACTIN DURING THE PROLONGED CULTURE OF BOVINE CUMULUS-ENCLOSED OOCYTES

The technique of somatic cell nuclear transfer includes a delayed activation of reconstituted oocytes (4–6 h after IVM). The prolonged culture of mammalian oocytes is known to be associated with aging of the ova leading to a decline in their quality and developmental capacity. The aim of the present research was to study effects of 2 closely related hormones, prolactin (PRL) and growth hormone (GH), during the prolonged culture of bovine oocytes on their developmental potential after parthenogenetic activation. Bovine cumulus-enclosed oocytes (CEO) were cultured for 16 h in TCM 199 containing 10% FCS, 10 μg mL−1 of porcine FSH, and 10 μg mL−1 of ovine LH. After 16 h of maturation, CEO or denuded oocytes were transferred to the fresh medium consisting of TCM 199 supplemented with 10% FCS and cultured for 12 h in the absence (Control) or in presence of 50 ng mL−1 of bovine PRL or 10 ng mL−1 of recombinant bovine GH or protein kinase inhibitors. The following inhibitors were used: (1) PP2 (an inhibitor of Src-family tyrosine kinases, 10 mM), (2) triciribine (an inhibitor of Akt kinase, 25 μM), and (3) calphostin C (a protein kinase C inhibitor, 0.5 μM). After the prolonged culture for 12 h, oocytes were activated by sequential treatment with ionomycin (5 μM for 5 min) immediately followed by 6-dimethylaminopurine (2 mM for 4 h). Activated oocytes were cultured in CR1aa medium until Day 5 postactivation and then transferred to the same medium supplemented with 5% FCS and cultured up to Day 7. The cleavage and blastocyst rates were assessed at Day 2 and 7, respectively. The data from 4 to 6 replicates (106–169 oocytes per treatment) were analysed by ANOVA. After the prolonged culture of CEO, the cleavage rate did not differ between the control and the hormone-treated groups, varying from 64.3 to 70.1%. Meanwhile, the blastocyst yield in the control group (12.5 ± 1.6%) was lower than in the PRL group (21.5 ± 2.4%, P &lt; 0.05), but was similar to that in the GH group (13.9 ± 1.8%). Furthermore, the developmental capacity of cultured denuded oocytes was unaffected by both GH and PRL. The enhancing effect of PRL on the yield of parthenogenetic blastocysts derived from CEO was also observed in the presence of PP2 (26.8 ± 2.4 v. 15.7 ± 2.0%; P &lt; 0.01). By contrast, calphostin C abolished this effect of PRL, although it did not affect the developmental potential of CEO in the control medium. At the same time triciribine reduced the blastocyst rate both in the control and PRL-treated groups (from 16.5 ± 1.8 to 8.1 ± 0.8% and from 24.3 ± 1.6 to 16.3 ± 2.4%, respectively; P &lt; 0.05). Our data indicate that PRL can support the developmental potential of parthenogenetic embryos by affecting bovine oocytes during the prolonged culture, with the effect being mediated by cumulus cells and achieved via activation of protein kinase C. This research was supported by the Federal Agency for Scientific Organizations and RFBR (project No. 15-08-99473).

Cyclic GMP Mediated Inhibition of Spontaneous Germinal Vesicle Breakdown Both with and without Cumulus in Mouse Oocyte.

ABSTRACTIntact germinal vesicle (GV) arrest and release are essential for maintaining the fertility of mammals inducing human. Intact germinal vesicle release, maturation of oocytes is maintained by very complex procedures along with folliculogenesis and is a critical step for embryonic development. Cyclic guanosine monophosphate (cGMP) has been suggested a key factor for meiotic arrest but so far its mechanisms are controversy. In this study we examine the effects of cGMP on germinal vesicle breakdown in cumulus-enclosed oocytes and denuded oocytes. Spontaneous maturation was inhibited by a cGMP agonist, 8-Br-cGMP with concentration dependent manners both in cumulus-enclosed oocytes and denuded oocytes. The inhibitory effect was more severe in denuded oocytes than cumulus-enclosed oocytes. The Rp-8-Br-cGMP and Rp-pCPT-8-Br-cGMP did not severely block GVB compared to 8-Br-cGMP. The spontaneous GVB inhibitory effects were different by the existence of cumulus. Based on them it is suggested that the cumulus modulates the role of cGMP in GV arrest.

IN VITRO FERTILIZATION OF VITRIFIED IMMATURE OR MATURE OOCYTES AS AFFECTED BY MECHANICAL OR NATURAL DENUDATION

The current study aimed to evaluate the role of cumulus-corona radiate complex of immature or in vitro matured bovine oocytes on their vitrification, in vitro fertilization and embryo development. Bovine ovaries were collected from an abattoir and follicular oocytes (3-8 mm in diameter) were aspirated. Oocytes were examined and classified into cumulus oocytes–complexes (COCs), natural denuded oocytes (NDOs) and mechanically denuded oocytes (MDOs). Natural COCs were mechanically denuded by repeated pipetting in phosphate buffer solution (PBS). All types of oocytes were cryopreserved by vitrification as immature or in vitro matured for 24 h prior to vitrification by open-pulled straw cryodevice. After at least 2 weeks of vitrification, all types of vitrified oocytes were evaluated for survival and viability (Normal and abnormal oocytes). Morphologically normal immature oocytes were matured and fertilized in vitro, while morphologically normal mature oocytes were directly fertilized in vitro for determination of cleavage rate (CR). After co-culture of cleaved oocytes for 7 days, rate of morula (MPR) and/or blastocysts (BPR) production was recorded. Results show that survival (SR) and normality (NR) rates were the highest for CCOs (84.47 and 81.73%), followed by MDOs (73.28 and 62.0%), and the lowest for NDOs (60.71 and 54.39%), respectively (P<0.05). SR was higher (P<0.05) for mature than immature oocytes (78.03 vs. 67.60%), while NR was nearly similar for both. CR, MPR and BPR were higher (P<0.05) for CCOs (45.28, 18.05 and 15.28%) than those of MDOs (29.72, 6.06 and 3.03%) and NDOs (25.24, 7.69 and 7.69%), respectively. CR was higher (P<0.05) in mature than in immature oocytes (41.93 vs. 29.36%), while MPR and BPR were nearly similar in both. Based on the obtained results, cumulus cell surrounding cumulus oocytes–complexes play a vital role for survival of bovine oocytes during vitrification and successful in vitro maturation and fertilization as well as embryonic development to morula and blastocyst stages.

The effect of cumulus cells on domestic cat (Felis catus) oocytes during in vitro maturation and fertilization.

The aim of this study was to evaluate the effect of co-culture of denuded oocytes with cumulus cells (CC) or cumulus-oocyte complexes (COCs) on in vitro maturation (IVM) and in vitro fertilization (IVF). Immature oocytes were collected from ovaries of domestic cats following a routine ovariectomy. Oocytes were matured in vitro for 24hr within four groups: (i) denuded oocytes (DO), (ii) DO co-cultured with CC, (iii) DO co-cultured with COC and (iv) COC as a control group. In further experiments, COCs were matured in vitro for 24hr, and then, oocytes were randomly divided into four groups as previously described and fertilized in vitro. Embryos were cultured for up to 7days. At the end of each experiment, oocytes/embryos were stained with Hoechst 33342 solution and observed under an inverted fluorescence microscope. The results of oocyte maturation showed that their meiotic competence decreased significantly in all experimental groups, compared to the control group. The maturation rates were approximately 45%, 24%, 43% and 76% in experiment 1, and 21%, 14%, 33% and 50% in experiment 2 in groups (i), (ii), (iii) and (iv), respectively. Examination of in vitro fertilization revealed that embryos developed up to the morula stage in all experimental groups. DO and oocytes cultured with COC during fertilization showed a lower cleavage rate-36% and 25% as opposed to those co-cultured with loose CC and the control group-43% and 42%, respectively. Results of this study indicate that cumulus cells connected with an oocyte into a cumulus-oocyte complex are irreplaceable for the maturation of domestic cat oocyte, but that the addition of loose CC may be beneficial for IVF.

The nuclear and developmental competence of cumulus-oocyte complexes is enhanced by three-dimensional coculture with conspecific denuded oocytes during in vitro maturation in the domestic cat model.

The objective of the study was to assess the efficacy of coculture with conspecific cumulus-denuded oocytes (CDOs) during in vitro maturation in a three-dimensional system of barium alginate microcapsules on the in vitro embryo development of domestic cat cumulus-oocyte complexes (COCs). In Experiment I, COCs were cocultured with conspecific CDOs or cultured separately in a 3D system for 24hr of in vitro maturation, before assessing the meiotic progression. In Experiment II, the in vitro fertilization of COCs and CDOs was carried out with chilled epididymal spermatozoa and the presumptive zygotes were cultured in vitro separately for 7days in 3D microcapsules before assesment of embryonic development. The results showed that the viability was maintained and that meiosis was resumed in the 3D culture system. The presence of CDOs during in vitro maturation improved the meiotic competence of the COCs, since the proportions of telophase I/metaphase II were higher than that in the groups cultured separately. The enrichment of the maturation system by companion oocytes also enhanced the ability of COCs to develop into embryos, and increased the percentages of morula and blastoycst stages. The COCs cocultured with CDOs developed at higher rates than the COCs cultured separately and the CDOs themselves. The beneficial effects of coculture with conspecific CDOs were presumably due to the paracrine action of some secreted factors that enhanced many molecular patterns related to the complex of cumulus oophorous cells. Further investigations to understand how the 3D microenvironment can influence the features of oocytes and embryos are required.

Sheep oocyte expresses leptin and functional leptin receptor mRNA

ObjectiveTo investigate the expression of leptin and its functional receptor mRNA by reverse transcription-polymerase chain reaction analysis in sheep oocytes. MethodsSheep ovaries were collected in reproductive season from abattoir and immediately were transported to the lab. Cumulus–oocyte complexes were aspirated from antral follicles and were denuded to obtain denuded oocytes. Total RNA of denuded oocytes was extracted, cDNA synthesis was carried out subsequently and cDNA was subjected to PCR amplification using specific designed primers. ResultsGel electrophoresis of PCR products showed the amplification of 162 and 121-bp amplicons for leptin and functional leptin receptor respectively. Sequencing the amplified fragments also confirmed the results. ConclusionsThe result of present study reveals that leptin and its functional receptor (Ob-Rb) mRNA are expressed in sheep oocyte and further studies should investigate the role(s) of leptin on sheep oocyte physiology and embryo development.