| Follicular atresia is to activate apoptosis in cumulus cells and oocytes. Immature oocytes with different developmental potencial in vitro maybe come from the follicles in various atresia processes, and oocytes are destined to apoptosis in atresia follicles in vivo. In present study, several detection approaches of apoptosis were applied to detect incidence of apoptosis in cumulus-oocyte complex, and the apoptosis effect on oocytes developmental potencial was observed. Meanwhile, FasL was added to in vitro maturation media to induce oocytes apoptosis, so as to further realize the relationship between bovine oocytes apoptosis and development.1. The morphological criterion on oocytes with different developmental competenceCumulus-oocyte complexes (COCs) were selected and classified into 4 groups according to oocyte cytoplasm and cumulus status: GI (COCs with granular cytoplasm and more than five layers of cumulus cells); GII ( COCs with homogeneous cytoplasm and more than five layers of cumulus cells); GUI (COCs with granular cytoplasm and between two and five layers of cumulus cells) and GIV (COCs with homogeneous cytoplasm and two to five layers of cumulus cells) . Developmental competence was evaluated by nuclear maturation (MII) after in vitro maturation and development rates in different stages following in vitro fertilization. Results showed that the rates of nuclear maturation and cleavage were 71.8%, 57.8%, 84.2%, 69.2% and 66.4%, 57.5%, 85.0%, 75.0%, respectively; the rates of blastocysts and expanded blastocyst formation were 22.7%, 15.7%, 38.9%, 26.4% and 5.9%, 3.9%, 15.9%, 8.6%, respectively. The results indicated that developmental competence within COC groups demonstrated that the highest rates of nuclear maturation, cleavage, blastocyst and hatching blastocyst were in GIII-COCs, and the lowest rates were found in GII-COCs (P < 0.05). Previous findings suggested that COCs showing early signs of atresia (e.g. slight expansion of the cumulus and slight granulation of the cytoplasm) had higher development potential than those considered to be healthy. So, in present study, those oocytes with various developmental potential could come from the follicles in different atresia grades.2. The detection of apoptosis in cumulus cells from different COCsResults of Annexin-V staining showed that the rates of early apoptosis in cumulus cells were 77.01%, 65.85%, 83.20%, 76.01% in four COC groups. Cumulus cells in GIII displayed a significantly higher ratio of Annexin-V staining positive cells than those in the other three groups (P < 0.05). Results of TUNEL staining showed rates of late apoptosis in cumulus cells were 2.01%, 0.78%, 6.35%, 2.08%. Cumulus cells in GIII displayed a significantly higher ratio of TUNEL staining positive cells than those in the other three groups (P < 0.05). Results indicated that cumulus cells from GIII with higher development competence displayed higher apoptosis degree. This further validated COCs in GIII should come from those follicles in higher atresia signals.3. The detection of apoptosis in oocytes from different COCs3.1 TUNEL detection of oocytes in COCs from four groupsNo TUNEL positive signal was detected in any oocyte, regardless of cytoplasm and cumulus status. The result indicated that late apoptosis should not be associated with developmental potential in oocytes.3.2 The detection of Bax and Bcl-2 protein in oocytesThe results showed that expression of Bax and Bcl-2 was detected in immatured oocytes. Bax was mainly detected in the cytoplasm, close to nuclei;Bcl-2 mainly located nuclear membrane and close to nuclei.3.3 The detection of Annexin-V and real-time RT-PCR in all groups of oocytesEarly apoptosis (Annexin-V staining positive) was detected in many oocytes of each group (ranging from 14.4 to 46.7%). Oocytes in GUI displayed a significantly higher ratio of Annexin-V staining positive cells than those in the other three groups (P < 0.05). The lowest early apoptosis ratio existed in GII-COCs. The transcript of Bax demonstrated correspondence with Annexin-V staining of oocytes, while the transcription of Bcl-2 showed the contrary pattern in different groups. Results indicated early apoptosis seems to be positively correlated to developmental competence in immature oocytes. Better developmental competence coupled with higher early apoptosis degree could be attributed to finer cytoplasm maturation. 4. The impact of FasL on oocytes apoptosis and embryos development4.1 The detection of Fas and FasL proteinsThe results showed that expression of Fas protein was detected in immatured oocytes, while FasL was not. Fas protein maily located in cytoplasm membrane, close to nucleus and several parts of cytoplasm.4.2 Effects of FasL on oocytes in vitro maturationOocytes were cultured in maturation medium supplemented with different concentration of FasL. Results showed that oocytes treated with 2ng/ml, 10ng/ml and 50ng/ml FasL displayed significantly higher ratios of Annexin-V positive cells (55.3%, 51.2% and 62.4%) than those in negative control(0ng/ml). TUNEL staining showed that no TUNEL positive oocytes was found in control group; the rates of TUNEL positive oocytes were 20.8%, 41.6% and 48.4% in 2ng, 10ng and 50ng treatments, respectively. In addition oocytes in 10ng and 50ng treatments displayed a significantly higher ratio of late apoptosis than those in control (P < 0.05). From above all, it was concluded that FasL enhanced the incidence of oocytes apoptosis after maturation, and concentration of FasL was positively correlated to ratio of apoptosis cells.3. Effects of FasL on oocytes in vitro maturation and developmentAfter 22h in vitro maturation, rates of nuclear maturation in 0ng, 2ng treatments and polar bodies exclusion rates in all groups were evaluated. Oocytes matured in 2ng treatments displayed a significantly higher ratio of Mil-phase and polar bodies exclusion rates than those in control (P < 0.05). The lower rates existed in 10ng and 50ng treatments as compared to control (P<0.05)o There was no significant difference between the rates of blastocyst of 2ng treatments and control. Oocytes matured in 10ng and 50ng treatments displayed significantly lower ratio of blastocyst than that in 2ng and control treatments (P < 0.05). It was concluded that heavy apoptosis happening to oocytes induced by FasL resulted in its developmental potencial decreased, development competence of oocytes seems to be not influenced by the slightly apoptosis in those from 2ng group.The present study systemically investigated the relationships among follicles atresia, cumulus cell apoptosis, oocyte apoptosis and the subsequent developmental potencial. For the first time, it was proposed that the immature oocytes could have encountered early apoptosis process preceding to "central regulatory checkpoint in apoptosis", and early apoptosis effect on oocytes developmental potencial was surveyed. Meanwhile, biology protein can induce apoptosis in oocytes. The mechanism of regulating apoptosis in oocytes was briefly discussed. Results indicated that efficiency of embryos production was related to apoptosis. There are important significance in development and application of fertilizating in vitro and transgenic animal in present study. |
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