Apoptosis In Cumulus-oocyte Complexes Research Articles

A Redesigned Method for CNP-Synchronized In Vitro Maturation Inhibits Oxidative Stress and Apoptosis in Cumulus-Oocyte Complexes and Improves the Developmental Potential of Porcine Oocytes.

In vitro embryo production depends on high-quality oocytes. Compared with in vivo matured oocytes, in vitro oocytes undergo precocious meiotic resumption, thus compromising oocyte quality. C-type natriuretic peptide (CNP) is a follicular factor maintaining meiotic arrest. Thus, CNP-pretreatment has been widely used to improve the in vitro maturation (IVM) of oocytes in many species. However, the efficacy of this strategy has remained unsatisfactory in porcine oocytes. Here, by determining the functional concentration and dynamics of CNP in inhibiting spontaneous meiotic resumption, we improved the current IVM system of porcine oocytes. Our results indicate that although the beneficial effect of the CNP pre-IVM strategy is common among species, the detailed method may be largely divergent among them and needs to be redesigned specifically for each one. Focusing on the overlooked role of cumulus cells surrounding the oocytes, we also explore the mechanisms relevant to their beneficial effect. In addition to oocytes per se, the enhanced anti-apoptotic and anti-oxidative gene expression in cumulus cells may contribute considerably to improved oocyte quality. These findings not only emphasize the importance of screening the technical parameters of the CNP pre-IVM strategy for specific species, but also highlight the critical supporting role of cumulus cells in this promising strategy.

Iloprost affects in vitro maturation and developmental competence of bovine oocytes

Prostacyclin (PGI2) is synthesised in oviductal fluid and enhance the embryo development during the preimplantation period. The objective of the present study was to determine the effect of an analogue of prostacyclin (iloprost) on the in vitro maturation (IVM) and the developmental competence of bovine oocytes. Cumulus oocyte complexes (COCs) were cultured in maturation medium with iloprost (0.5 μM) for 24 h. We found that iloprost assisted maturation rates and cumulus cell expansion of bovine oocytes, and it increased the mRNA expression of genes related to cumulus expansion: ADAM17, AREG, and TNFAIP6 and cathepsin genes (CTSK and CTSS). Moreover, iloprost reduced the occurrence of apoptosis in COCs and promoted an antiapoptotic balance in the transcription of genes involved in apoptosis (BAX and BCL2). COCs treatment with iloprost during IVM also reduced intracellular reactive oxygen species (ROS) levels, while glutathione (GSH) levels and the mRNA expression of antioxidant genes CAT and GPx4 were markedly increased. We also showed that an analogue of PGI2 influenced the mitochondrial status via distribution rates of mitochondria and mitochondrial membrane potential in oocytes. Although, iloprost-enhanced maturation had no direct effect on number of embryos cleaved, it increased blastocyst rates of bovine embryos as well as proportion of expanded blastocysts. These results indicate that the supplementation of maturation medium with iloprost is beneficial for the maturation efficiency and developmental competence of bovine oocytes.

Low oxygen concentrations improve yak oocyte maturation and enhance the developmental competence of preimplantation embryos

Mammalian oocyte maturation and early embryo development are highly sensitive to the in vitro culture environment, and oxygen concentration is one of the important factors. In the present study, we aimed to explore the effects of different oxygen concentrations (20%, 10%, 5% or 1% O2) on yak oocyte maturation, in vitro fertilization (IVF), and embryo development competence, as well as its effects on the oxidative response, metabolism, and apoptosis in cumulus-oocyte complexes (COCs) and the embryo. The results revealed that the maturation rate of oocytes, blastocysts rate and hatched blastocysts rate in the group with 5% oxygen concentration were significantly higher (P < 0.05) than other groups, but the cleavage rate with 5% oxygen concentration was significantly lower (P < 0.05) than the 20% and 10% oxygen concentrations. The maturation rate of oocytes, the cleavage rate, blastocysts rate and hatched blastocysts rate with the 1% oxygen concentration were the lowest. The blastocyst cultured with 5% oxygen concentration had significantly greater (P < 0.05) numbers of total cells, inner cell mass (ICM) cells and trophectoderm (TE) cells compared to the other groups. Analysis of the apoptosis index of oocytes and blastocyst cells by transferase dUTP nick end labeling (TUNEL) showed that the number of apoptotic cells significantly reduced (P < 0.05) with 5% oxygen concentration, but increased significantly (P < 0.05) in the 1% oxygen concentration group. Also, the qRT-PCR and western immunoblotting analysis confirmed that the transcription levels of the metabolism genes, antioxidant response genes, apoptosis genes, oocyte competence genes and embryonic developmental markers showed significant differences (P < 0.05) in the COCs or blastocysts matured in 5% oxygen concentration group compared to the other groups. In summary, our findings demonstrate that 5% oxygen concentration improves oocyte maturation and blastocyst development in the yak, increases blastocyst cell numbers, reduces apoptosis rate in the oocyte and blastocyst as well as reduces embryo cleavage rate.

Fibroblast growth factor 2 regulates cumulus differentiation under the control of the oocyte.

We first assessed regulation of FGF2 expression in cumulus cells by FSH and oocyte-secreted factors during in vitro maturation (IVM). Then, we tested the hypothesis that FGF2 regulates meiotic progression, cumulus expansion, and apoptosis in cumulus-oocyte complexes (COC) undergoing IVM. In vitro maturation of bovine COC was utilized as a model to assess regulation of FGF2 expression by FSH and oocyte-secreted factors (via microsurgical removal of the oocyte), as well as effects of graded doses of FGF2 on meiotic progression, degree of cumulus expansion, dissociation of cumulus cells, and cumulus cells apoptosis. Expression of genes regulating functional endpoints altered by FGF2 treatment was assessed in cumulus cells by real-time PCR. Cultures were replicated 4-5 times and effects of treatments were tested by ANOVA. FGF2 mRNA expression was increased by FSH and oocyte-secreted factors during IVM. Addition of FGF2 to the IVM medium advanced meiosis resumption, decreased the ease with which cumulus cells were dissociated, and inhibited cumulus cells apoptosis. Decreased cumulus dissociation was accompanied by decreased expression of TNFAIP6. This is the first study showing that FGF2 expression is regulated by the oocyte in cumulus cells. Moreover, we report novel effects of FGF2 on cumulus cell survival and extracellular matrix (ECM) quality during IVM that may favor acquisition of developmental competence and suggest physiological roles during the final steps of COC differentiation.

Dose-dependent effects of luteinizing hormone and follicle stimulating hormone on &lt;i&gt;in vitro&lt;/i&gt; maturation, apoptosis, secretion function and expression of follicle stimulating hormone receptor and luteinizing hormone receptor of sheep oocytes

TThe present study compared the effects of follicle stimulating hormone (FSH) and luteinizing hormone (LH) on in vitro maturation (IVM), apoptosis, and secretion function in sheep oocytes, as well as gene expressions of the receptors (FSHR, LHR, and GnRHR) in cumulus-oocyte complexes (COCs). The COCs were recovered from sheep ovaries and pooled in groups. The COCs were cultured for 24 hours in IVM medium supplemented with various concentrations of LH (5–30 μg/mL) and FSH (5–30 IU/mL). They were allocated to LH-1 (5 µg/mL), LH-2 (10 µg/mL), LH-3 (20 µg/mL), and LH-4 (30 µg/mL) groups, and FSH-1 (5 IU/mL), FSH-2 (10 IU/mL), FSH-3 (20 IU/mL), and FSH-4 (30IU/mL) groups. The apoptosis of COCs was assessed by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). The maturation rates of oocytes improved gradually as LH and FSH concentration increased from 0 to 10 μg/mL(IU/mL), reaching a peak value of 44.1% of LH-2 and 48.5% of FSH-2 group. Oocyte apoptosis rates of LH-2 and FSH-2 groups were the lowest among LH- and FSH-treated groups, respectively. The germinal vesicle breakdown (GVBD) rate of the FSH-2 group was higher than that of the control group (CG) and FSH-4 groups. The GVBD rate of LH-2 group also increased in comparison with the CG group. FSH concentration of the FSH-2 group was greater than that of CG. Expression levels of FSHR, LHR, and GnRHR mRNAs of FSH-2, LH-3, and LH-3 group, respectively, were higher than CG. Levels of FSHR proteins in FSH-2 and FSH-3 groups were greater than CG. Levels of GnRHR proteins were increased with a maximum increment of FSH-4. The FSH and LH supplemented into the IVM medium could promote the maturation rate, reduce the apoptosis rate of sheep oocytes, and increase FSH concentrations in IVM medium fluid. Additionally, FSH and LH enhanced expression levels of FSHR, LHR, and GnRHR mRNAs of sheep COCs. Keywords: Apoptosis, cumulus-oocyte complexes, germinal vesicle breakdown, protein expression, receptor

Maturation rates of oocytes and levels of FSHR, LHR and GnRHR of COCs response to FSH concentrations in IVM media for sheep

This study investigated the FSH influence on maturation rates of oocytes in vitro maturation (IVM), and expression levels of the follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR) and gonadotropin releasing hormone receptor (GnRHR) of cumulus-oocyte complexes (COCs) response to FSH treatment. 1686 COCs were harvested from 1063 ovaries of sheep. COCs were cultured 26h at 38.5°C and 5.0% CO2 in IVM media supplemented with 0, 5, 10, 20 and 30 IU/ml FSH. They were allocated in to FSH-1 (basal line), FSH-2, FSH-3, FSH-4 and FSH-5 groups. The apoptosis of COCs was assessed by Tunel assay. Expression levels of mRNA and protein for FSHR, LHR and GnRHR in sheep COCs were detected using real time RT-PCR and Western blotting respectively. The results showed that the maturation rates of oocytes were improved gradually when FSH supplement increased from 0 to 10μg/ml. FSH-3 group showed the highest maturation rate. Apoptosis rates of FSH-treated groups were less than that of FSH-1 group with a minimum of FSH-3 group. Expression levels of FSHR and LHR mRNAs in FSH-3 and FSH-4 were significantly higher than in FSH-1. Expression level of GnRHR mRNA in FSH-3 was higher than in FSH-1 (P<0.05). Expression levels of FSHR proteins in FSH-3 and FSH-4 groups were higher than that of FSH-1 group. Expression levels of GnRHR proteins increased gradually with a maximal increment of FSH-5. Maturation rates of COCs had significant positive correlations with mRNA and protein levels of FSHR, LHR and GnRHR. In conclusion, FSH could accelerate the maturation rate of sheep oocytes and reduce their apoptosis rate, also increase the expression levels of FSHR, LHR and GnRHR mRNAs, and strengthen expressions of FSHR and GnRHR proteins. 10 IU/ml FSH additions were the optimal dose for IVM of sheep oocytes.

FSHR and LHR Expression and Signaling as Well as Maturation and Apoptosis of Cumulus-Oocyte Complexes Following Treatment with FSH Receptor Binding Inhibitor in Sheep

Background/Aims: Currently, it remains unknown whether FSH receptor binding inhibitor (FRBI) influences follicular development and reproduction functions in humans and animals. The present study aimed to investigate FRBI effects on in vitro maturation (IVM) and apoptosis of cumulus-oocyte complexes (COCs) of sheep, to determine the effect of FRBI on mRNA and protein levels of FSHR and LHR in COCs, and to elucidate the signal pathway of FRBI effects. Methods: COCs were in vitro cultured for 24h in the IVM media supplemented with varying concentrations of FRBI (0, 10, 20, 30 and 40µg/mL) and FSH (10IU/mL). The harvested COCs were observed under an inverted microscope and maturation rates of COCs were determined. Real time RT-PCR and Western blotting were utilized to detect mRNA and protein levels of FSHR and LHR. The concentrations of FSH, LH and caspase-3 were determined using especial ELISA kits for sheep, respectively. Results: Maturation rates of COCs decreased gradually as FRBI concentrations increased from 0 to 40µg/mL, reaching a bottom value of 23.76% of the FRBI-4 group. The maximal apoptosis rate was detected in the FRBI-4 group. IP3 contents of FRBI-3 and FRBI-4 groups were reduced as compared to control group (CG) and FSH groups (P<0.05). Levels of FSHR protein of FRBI-3 and FRBI-4 groups as well as LHR protein of FRBI-4 group were significantly less than that of CG and FSH group. FSH contents of four FRBI treatment groups were gradually decreased along with the supplementation doses of FRBI. Caspase-3 contents of FRBI groups were reduced with a maximum reduction of the FRBI-2 group. Conclusion: Our results revealed supplement of FRBI into IVM media could dose-dependently decrease the maturation rate and increase apoptosis rate of sheep COCs. A lower dose of FRBI treatment slightly promoted IP3 production, but a higher dose of FRBI reduced IP3 production. FRBI suppressed the mRNA and protein expression levels of FSHR and LHR in sheep COCs. Our study will help to therapy effectively ovarian diseases, improve ovarian and follicular functions, and further to promote fertility of humans and animals.

Presence of encircling granulosa cells protects against oxidative stress-induced apoptosis in rat eggs cultured in vitro.

Increased oxidative stress (OS) due to in vitro culture conditions can affect the quality of denuded eggs during various assisted reproductive technologies (ARTs). Presence of intact granulosa cells may protect eggs from OS damage under in vitro culture conditions. The present study was aimed to investigate whether encircling granulosa cells could protect against hydrogen peroxide (H2O2)-induced egg apoptosis in ovulated cumulus oocyte complexes (COCs) cultured in vitro. The OS was induced by exposing COCs as well as denuded eggs with various concentrations of H2O2 for 3h in vitro. The morphological changes, total reactive oxygen species (ROS) as well as catalase expression, Bax/Bcl-2, cytochrome c levels and DNA fragmentation were analysed in COCs as well as denuded eggs. Our results suggest that H2O2 treatment induced morphological apoptotic features in a concentration-dependent manner in denuded eggs cultured in vitro. The 20µM of H2O2 treatment induced OS by elevating total ROS level, reduced catalase and Bcl-2 expression levels with overexpression of Bax and cytochrome c and induced DNA fragmentation in denuded eggs cultured in vitro. The presence of encircling granulosa cells protected H2O2-induced morphological apoptotic features by preventing the increase of Bax, cytochrome c expression levels and DNA fragmentation in associated egg. However, 20µM of H2O2 was sufficient to induce peripheral granulosa cell apoptosis in COCs and degeneration in few denuded eggs cultured in vitro. Taken together our data suggest that the presence of encircling granulosa cells could be beneficial to protect ovulated eggs from OS damage under in vitro culture conditions during various ART programs.

Effects of Aroclor 1254 on in vivo oocyte maturation in the mouse.

Polychlorinated biphenyls (PCBs) are stable, lipophilic compounds that accumulate in the environment and in the food chain. Though some studies provided evidence that PCBs had adverse effects on reproductive function, most of these results were from in vitro models. Therefore we investigated the effect of Aroclor 1254 (a commercial PCBs mixture) treatments on in vivo maturation and developmental potential of mouse oocytes. In the present study, female ICR mice were treated with different doses (12.5, 25 and 50 mg/kg) of Aroclor 1254 (a commercial PCB mixture) once every 72 hours by intraperitoneal injection for 9 days. After three treatments of Aroclor 1254, the mice were superovulated to collect oocytes one day after the last exposure. The effects of Aroclor 1254 on oocyte maturation, fertilization, and preimplantation embryonic development were investigated. Immunofluorescence-stained oocytes were observed under a confocal microscope to assess the effects of Aroclor 1254 on spindle morphology. Parthenogenic activation and the incidence of cumulus apoptosis in cumulus-oocyte complexes were observed as well. Oocytes exposed to different doses of Aroclor 1254 in vivo were associated with a significant decrease in outgrowth potential, abnormal spindle configurations, and the inhibition of parthenogenetic activation of ovulated oocytes. Furthermore, the incidence of apoptosis in cumulus cells was increased after exposed to Aroclor 1254. These results may provide reference for the treatment of reproductive diseases such as infertility or miscarriage caused by environmental contaminants.

Cathepsin B activity has a crucial role in the developmental competence of bovine cumulus–oocyte complexes exposed to heat shock during in vitro maturation

Cathepsin B was found to be correlated inversely with the quality of bovine oocytes and embryos. The aims of this study were to evaluate i) the relationship between heat shock during in vitro maturation (IVM) of bovine cumulus-oocyte complexes (COCs) and cathepsin B activity in relation to apoptosis and ii) the effect of supplementation of cathepsin B inhibitor (E-64) during IVM of heat-shocked COCs on embryonic development. After IVM at 38.5 °C for 22 h (control group) or at 38.5 °C for 5 h followed by 41 °C for 17 h (heat shock group) either with or without 1 μM E-64, activities and protein expression of cathepsin B and caspase 3 were evaluated as well as TUNEL staining. After IVF, developmental rate, total cell number, and the percentage of apoptotic cells in blastocysts were evaluated on day 8 (day 0, IVF day). Heat-shocked IVM COCs showed significantly high activities and expressions of both cathepsin B, and caspase 3 accompanied by a significant increase in number of TUNEL-positive cells. Addition of E-64 significantly decreased the activities of cathepsin B and caspase 3, and TUNEL-positive cells in heat-shocked IVM COCs. Moreover, addition of 1 μM E-64 during IVM under heat shock conditions significantly improved both developmental competence and quality of the produced embryos. These results indicate that heat shock induction of cathepsin B is associated with apoptosis of COCs, and inhibition of cathepsin B activity can improve the developmental competence of heat-shocked COCs during IVM.

Effect of leptin during in vitro maturation of prepubertal calf oocytes: Embryonic development and relative mRNA abundances of genes involved in apoptosis and oocyte competence

During the in vitro maturation of adult bovine oocytes, leptin has beneficial effects on blastocyst development, apoptosis and transcription levels of developmentally important genes. The present study analyzes the differential effects of leptin on prepubertal bovine oocytes and cumulus cells. Effects were determined of leptin treatment during oocyte maturation on their developmental capacity after fertilization (Exp. 1), incidence of apoptosis in cumulus oocyte complexes (COCs) (Exp. 2) or on relative mRNA abundances of genes in cumulus cells and oocytes (Exp. 3). COCs were matured in serum-free medium containing 1 mg/mL polyvinyl alcohol and 0, 10, 100, or 1000 ng/mL leptin (L0, L10, L100, and L1000, respectively), or in medium supplemented with 10% fetal calf serum (FCS) as a positive control. Addition of leptin during oocyte maturation had no effect on cleavage rates after fertilization (FCS, 68.6%; L0, 62.9%; L10, 66.9%; L100, 63.4%; L1000, 60.9%). Similarly, no significant differences in blastocyst rates were observed when oocytes were matured in the presence of L0 (8.4%), L10 (9.3%), L100 (6.7%), L1000 (8.2%), compared to control FCS (9.4%). In Experiment 2, maturation in the presence of 1000 ng/mL of leptin increased the proportion of TUNEL-positive cumulus cell (6.9%) with respect to those matured in the presence of FCS (4.96%), but not at the lower leptin doses. When relative mRNA abundances were examined for seven genes by qRT-PCR, five (TP53, BAX, DNMT3A, PGTS2 and LEPR) showed differences among groups. LEPR expression was significantly higher in the oocytes matured with FCS compared with the other groups and in those matured with PVA (L0) without leptin compared with the three groups of oocytes matured in the presence of leptin. In conclusion, the addition of leptin to the in vitro maturation medium used for prepubertal bovine oocytes does not increase the development potential of the oocytes or reduce the percentage of apoptosis in cumulus cells. Leptin blocks transcription of the leptin receptor (LEPR) probably reflecting selective, differential degradation by doses of leptin.

Relationship between cumulus cell apoptosis, progesterone production and porcine oocyte developmental competence: temporal effects of follicular fluid during IVM

The objective of the present study was to determine the temporal effects of sow follicular fluid (FF) in vitro on cumulus cell viability and function, as well as oocyte nuclear and cytoplasmic maturation. Cumulus-oocyte complexes (COCs) recovered from the ovaries of prepubertal pigs were matured in medium with (+FF) or without (-FF) follicular fluid for the first 22 h of IVM. At 22 h of IVM, each group of COCs was then transferred to medium with or without FF and matured for another 22 h, forming four treatment groups (-FF/-FF, -FF/+FF, +FF/-FF and +FF/+FF). The concentration of progesterone in spent IVM medium and the incidence of cumulus cell apoptosis in individual COCs were determined at 22 and 44 h of IVM. Cumulus expansion was also recorded at 44 h of IVM. Finally, the ability of oocytes to complete meiosis to the MII stage and form blastocysts after IVF and embryo culture was assessed. Maturation with FF for part or the whole of IVM increased cumulus expansion and progesterone production and decreased the incidence of cumulus cell apoptosis compared with the -FF/-FF group (P < 0.05). The changes were greatest for the +FF/+FF group and intermediate for the -FF/+FF and +FF/-FF groups. Regression analysis revealed a negative association between cumulus cell progesterone production and the incidence of cumulus cell apoptosis (P < 0.001). Meiotic maturation was enhanced when FF was present during the first half of IVM. Oocytes matured in the presence of FF during the first and/or second half of IVM displayed an increased ability to form blastocysts compared with the -FF/-FF group (P < 0.05). The extent of the increase was similar for all FF-supplemented groups. The results show that FF exerts several beneficial effects at different times during IVM and suggest that a major role of FF is to provide protection from oxidative stress. We propose that the incidence of cumulus cell apoptosis in COCs must be kept below a certain threshold to ensure adequate functionality, including steroidogenic activity, is maintained for the acquisition of oocyte developmental competence.