In the present study, we investigated the role of binding immunoglobulin protein/glucose-regulated protein, 78-kDa (BIP/GRP78)-regulated endoplasmic reticulum (ER)-stress on meiotic maturation and cumulus cells expansion in porcine cumulus-oocyte complexes (COCs). Previously, it has been demonstrated that unfolded protein response (UPR)- related genes, such as molecules involved in ER-stress defense mechanisms, were expressed in matured oocytes and cumulus cells during in vitro maturation (IVM) of porcine oocytes. However, BIP/GRP78-mediated regulation of ER stress in porcine oocytes has not been reported. Firstly, we observed the effects of knockdown of BIP/GRP78 (an UPR initiation marker) using porcine-specific siRNAs (#909, #693, and #1570) on oocyte maturation. Among all siRNAs, siRNA #693 significantly reduced the protein levels of UPR marker proteins (BIP/GRP78, ATF4, and P90ATF6) in porcine COCs observed by Western blotting and immunofluorescence analysis. We also observed that the reduction of BIP/GRP78 levels by siRNA#693 significantly inhibited the meiotic maturation of oocytes (siRNA #693: 32.5±10.1% vs control: 77.8±5.3%). In addition, we also checked the effect of ER-stress inhibitors, tauroursodeoxycholic acid (TUDCA, 200 μM) and melatonin (0.1 μM), in BIP/ GRP78-knockdown oocytes. TUDCA and melatonin treatment could restore the expression levels of ER-stress marker proteins (BIP/GRP78, p-eIF2α, eIF2α, ATF4, and P90ATF6) in siRNA #693-transfected matured COCs. In conclusion, these results demonstrated that BIP/GRP78-mediated regulation of UPR signaling and ER stress plays an important role in in vitro maturation of porcine oocytes.

Purine metabolism is known factor for nuclear maturation of oocytes through both follicle cells and oocyte itself. However, it is largely unknown the roles of purine metabolism in the oocyte competence for fertilization and early development. In this study, the effects of adenosine in oocyte competence for development were examined using adenosine and its synthetic inhibitors. Adenosine treatment from GV intact stage for 18 hr (fGV) caused of decrease the fertilization rate but of increase the cleavage rate compared from the other stage treatment groups. Hadacidin did not effect on fertilization rate but increased cleavage rate without stage specificity. Adenosine did not block the effects of hadacidin with the exception of fGV group. By the inhibition of purine synthetic pathways the fertilization rate was decreased in the fGV and fGVB groups but increased in the fMII group. Exogenous adenosine caused of decrease fertilization rate in the fGVB group but increase in the fMII group. Cleavage rate was dramatically increased in the adenosine treatment with synthetic inhibitors. It means that the metabolism of purine has stage specific effects on fertilization and cleavage. Exogenous adenosine had only can improve oocyte developmental competence when it treated at GV intact stage. On the other hand, endogenous synthesis in all maturation stage caused of increase the cleavage rate without effects on fertilization. These data suggest that adenosine at GV stage as a paracrine fashion and inhibitions of endogenous adenosine in all stage improve oocyte developmental competence..

To overcome the risk of the ovarian hyperstimulation syndrome (OHSS) in patients have polycystic ovarian syndrome (PCOS) and to prepare emergency fertility preservation in patients undergoing anticancer treatment, several researchers have reported IVM of oocytes retrieved from ovaries exposed by only hCG priming. However, the maturation rate and the developmental potential of embryos from IVM oocytes are significantly lower than those of oocytes matured in vivo. Here, we investigated the optimal time point for immature oocyte collection at post hCG only injection for in vitro maturation, in vitro fertilization and blastocyst formation. Immature GV oocytes were collected from 25 days old B6D2F1 female mouse at 12 hr, 14 hr, 16 hr or 24 hr post hCG injection. Oocytes were collected from antral or late secondary follicle by puncturing with 26 G needle. Collected oocytes were cultured in G2 medium with 10% FBS, FSH, estradiol, and hCG for 16 hr in vitro and subjected in vitro fertilization and further embryonic development. To examine follicular maturation, we estimated the numbers of primordial, primary, secondary follicle and antral follicle on ovaries of each time point post hCG. To confirm the optimal time point post hCG injection for collecting immature oocytes, we recovered the oocytes from each time point. There is no difference in the number of oocytes per mice. Oocytes collected at 14 hr post hCG injection were shown higher maturation rate to MII stage and blastocyst formation compare to other three groups (p<0.01). However, there is no difference in the maturation rate on the other three groups. Also, apoptotic signal with TUNEL assay or anti-PARP staining was not change in ovaries from all experimental groups. Granulosa cell proliferation test with anti Ki-67 or anti AMH was not show any difference. According to these results, there are no significant differences in four different time points at 12 hr, 14 hr, 16 hr or 24 hr of collection of immature oocytes in hCG primed mouse. However, oocytes from 14 hr post hCG injection showed higher percentages of maturation rate, in vitro fertilization rate, blastocyst formation.

Throughout their meiotic maturation in most mammals, oocytes are arrested twice, prophase I and metaphase II. Being released from these arrests, transient or oscillation of intracellular Ca2+ concentration is observed in the ooplasm, which is not answered in relation to the specific role in the resumption of meiotic arrest. Recently, Ca2+/calmodulin-dependent protein kinase II (CaM KII) has been known as a Ca2+ oscillation decoder from the in vitro experiment. CaM KII is multifunctional serine/threonine kinase observed in most cells. Present studies were performed to investigate the role of CaM KII during resumption of meiotic arrest and activation in vitro of mouse oocytes. It was questioned whether CaM KII might be involved in the meiotic resumption of mouse oocytes. Compared to the control, both of CaM KII inhibitors, KN-93 and KN-62, significantly inhibited germinal vesicle breakdown (GVBD) of mouse oocytes in a dose-dependent manner. As the concentration of KN-93 increased, concomitant decrease of intracellular Ca2+ concentration ([Ca2+]i) was also observed using confocal laser scanning microscope (CLSM) and an intracellular Ca2+ indicator, fluo 3-AM. When GVBD oocytes were treated with 6% ethanol, small [Ca2+]i transient was observed in oocytes bathed with Ca2+-free medium and large increase was observed in oocytes bathed with Ca2+-containing medium, suggesting that [Ca2+]i transient could happen from intracellular Ca2+ store as well as Ca2+ influx through Ca2+-channel on the oolemma. However, KN-93 inhibited the [Ca2+]i transient of GVBD oocytes in both cases. Using monoclonal antibodies against α-subunit of CaM KII, tubulin and microtubule-assocaited proteins (MAPs), CaM KII has been colocalized on the spindle with tubulin and MAPs. The present study also demonstrated the presence of α-subunit of CaM KII in heart, kidney, testes, ovary as well as in brain of the mouse. In ovarian follicles, CaM KII was expressed in granulosa cells and oocytes. Based on overall the above results, followings are suggested. First, CaM KII might be involved in the regulatory mechanism of meiotic resumption. Second, CaM KII might play a regulatory role in the stabilization of microtubule.

This study was performed to evaluate the effects of fibroblast co-culture on in vitro maturation (IVM) of prepubertal mouse preantral follicles. The intact preantral follicles were obtained from the ovaries of 12－14 day old mice and these were cultured individually in α-minimal essential medium (α-MEM) supplemented with 5% fetal bovine serum (FBS), 100 mIU/㎖ recombinant follicle stimulating hormone (rFSH), 1% insulin-transferrin-selenium, 100 μg/ml penicillin and 50 ㎍/㎖ streptomycin as base medium for 12 days. A total of 200 follicles were cultured in base medium co-cultured with mouse embryonic fibroblast (MEF) (MEF group) (n=100) or only base medium as control group (n=100). Survival rate of follicles on day 12 of culture were significantly higher in the MEF group of 90.0%, compared with 77.0% of the control group (p=0.021). Follicle diameters on day 6 and 8 of the culture period were significantly larger in the MEF group than those in the control group (p=0.021, p=0.007, respectively). Estradiol levels in culture media on day 4, 6, 8, 10 and 12 of the culture period were significantly higher in the MEF group (p=0.043, p=0.021, p=0.006, p<0.001 and p=0.008, retrospectively). Our data suggest that MEF cell co-culture on IVM of mouse preantral follicle increases survival rate and promotes follicular growth and steroid production.

To evaluate the effects of benzo[a]pyrene (B[a]P), one of polycyclic aromatic hydrocarbons (PAHs), on in vitro oocyte maturation (GVBD) and sex steroid hormone production, maturing oocytes (oocyte diameters=0.74, 0.88 and 0.93 mm) of the longchin goby, Chasmichthys dolichognathus were incubated with B[a]P (1, 10 and 100 ng/mL) for 24 hours. After incubation, the oocytes were fixed with clearing solution (ethanol:formalin:glacial acrtic acid=6:3:1). The location of the germinal vesicle was observed under low-power magnification using a dissecting microscope. Steroids in aliquots of the incubation media were extracted twice using five volumes of ethylacetate:cyclohexane (1:1). Then, the T, E2 and 17α20βP levels were measured by RIA. In oocytes 0.74 mm diameter (vitellogenic oocytes), B[a]P had no significant effect on GVBD at the concentrations tested. In oocytes 0.88 mm diameter (fully vitellogenic oocytes), B[a]P inhibited GVBD significantly at 1 and 100 ng/mL. T production was decreased and the ratio of E2/T was increased significantly at 1 and 10 ng/mL compared with control. In 0.93 mm diameter oocytes (germinal vesicle located near the center of oocytes), B[a]P induced GVBD significantly at 10 and 100 ng/mL and decreased the ratio of E2/T significantly at 1 and 10 ng/mL compared with control. These findings suggest that B[a]P has different sensitivity to the oocyte maturation according to the oocyte diameters.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants derived from incomplete combustion of carbons and crude oil. In this study, we investigated the effects of benzo[a]pyrene (B[a]P), a representative PAHs on in vitro sex steroid hormone production and germinal vesicle breakdown (GVBD) using isolated oocytes of longchin goby (Chasmichthys dolichognathus) and chameleon goby (Tridentiger trigonocephalus). Oocytes in diameters of 0.8-0.9 (end vitellogenic stage) and 0.9-1.0 mm (germinal vesicle migratory stage) from longchin goby and 0.5 mm (fully vitellogenic stage) from chameleon goby were used. In GVBD assay, B[a]P at 10 nM stimulated GVBD in the oocytes of 0.8-0.9 mm from longchin goby. B[a]P at 1 nM stimulated GVBD in the oocytes with diameter 0.5 mm from chameleon goby. In steroid production from oocytes of longchin goby, B[a]P at 100 nM decreased testosterone (T) production, B[a]P at 1,000 nM increased estraiol-17 (J (E2) production and 10 and 100 nM increased -dihydroxy-4-pregnen-3-one () production in the oocytes with diameter 0.8-0.9 mm. B[a]P at 1,000 nM increased E2 production, 100 and 1,000 nM increased production in the oocytes with diameter 0.9-1.0 mm. In steroid production of oocytes from chameleon goby, B[a]P at 1,000 nM increased production. B[a]P at 10 nM increased production. In the ratio of to T (/T), B[a]P at 100 and 1,000 nM increased /T in the oocytes of longchin goby. B[a]P at 100 nM also increased /T in the oocytes of chameleon goby. Taken together, these results suggest that B[a]P have not only weak estrogenic effects but progestogenic effects on oocyte maturation.

멜라토닌(N-acetyl-5-methoxytryptamine)은 포유동물의 뇌의 송과선에서 분비되는 호르몬으로 수면과 생체 리듬 등을 조절하고 난소 기능과 번식에도 영향을 미친다. 또한, 강력한 scavenger로서 항산화제의 역할을 한다. 이 연구의 목적은 멜라토닌이 생쥐 난구세포-핵낭(germinal vesicle, GV) 시기 난자 복합체의 체외성숙에 미치는 영향을 알아보는 것이다. 3주령의 ICR 암컷 생쥐의 난소에서 회수된 난자-난구세포 복합체

생쥐의 난자는 특별한 첨가물이나 난구세포 없이 체외 배양해도 성숙율이 높은 반면 돼지 난자의 체외성숙율은 매우 낮다. 본 연구는 이러한 차이의 원인을 연구하기 위하여 생쥐와 돼지 난자의 에너지 생성에 관여하는 유전자인 malate dehydrogenase(Mor2)의 기능을 RNAi를 이용하여 비교 분석하였다. 생쥐와 돼지 각각의 Mor2 double-stranded RNA(dsRNA)를 제작하고, 생쥐는 난구세포를 제거한 미성숙(GV) 난자의 세포질에

본 연구는 소의 미성숙 난포란의 체외성숙시 의 첨가가 체외성숙, 체외수정 후 웅성전핵의 형성 및 세포질 내의 GSH 수준에 미치는 영향을 알아보고자 실시하였다. 체외성숙시 과 의 를 첨가한 경우 대조구에 비하여 성숙율이 증가하는 것으로 나타났으며(p<0.05), 모든 실험구에 있어서 12시간 체외성숙보다 24시간 체외성숙에서 높은 성숙율을 나타냈다(p<0.05). 체외수정 후 웅성전핵 형성에 있어서는 와 농도의 첨가구에서 대조구에 비하여 높게 나타났으나

본 실험은 생쥐 난자의 성숙과 생존에 미치는 selenium의 영향을 알아보고자 수행하였다. 난자의 성숙은 현미경을 통해 관찰하였으며, 핵막 붕괴(germinal vesicle breakdown, GVBD)와 극체 형성(polar body formation, PB)은 체외 배양 시작 후 각각 2.5, 13시간에 확인하였다. 난자의 생존은 72 시간동안 체외 배양하면서 형태학적 차이로 정상 난자와 비정상 난자를 판별하였다. 또한 각 단계별로 수집된 난자의

소 체외성숙시 난포란에 영향을 주는 과립막세포와 난자의 핵성숙을 촉진하는 물질이 확인되지 않은 혈청내의 물질 뿐만 아니라 호르몬이나 생리활성인자 등에 의해 촉진됨이 밝혀졌다. 이에 따라 체외성숙 및 체외발달에 사용되는 배양액의 조성도 복합배양액에서 단순배양액으로 전환을 시도하고 있으며, 체내의 조건에 보다 더 접근하고자 하는 시도들이 수행되고있다. 본 연구는 한우 난포란의 체외성숙율 및 체외수정 후 배발달율을 향상시키기 위하여 TGF-의 첨가가 미치는

This study was conducted to determine the ability of nuclear development of canine oocytes depend on the kind of maturation media and addition of serum sources. Ovaries were collected from a bitches at various stages of estrus cycle by an ovariohysterectomy. Oocytes were collected of cumulus oocytes complexes after slicing of ovaries with blade. The maturation medium was containing 0.6 mM/ml cysteine, 0.2 mM pyruvic acid, 20 ng/ml and 1 rbST Exp. 1, the oocytes were matured in four different maturation medium as follows: 1) TCM-199, 2) DMEM, 3) NCSU37 and 4) modified-NCSU37 with 10% FBS. Exp. 2: the oocytes were matured in mNCSU37 supplemented with different protein sources (10% FBS, 10% EDS, 0.3% BSA and 0.1% PVA) to select the optimal one. Oocytes were matured in a humidified atmosphere containing 5% at for 72 hrs. The maturation rate were analyzed by Duncan's multiple range test using General Linear Models procedure in SAS. The rates of meiotic resumption to MI-MII depend on different culture media were achieved with TCM-199 (5.2%), DMEM (5.0%), NCSU37 (7.2%) and m-NCSU37 (5.9%), respectively. The rates of meiotic resumption to MI-MII according to addition of protein source were 10% FBS (13.3%), 10% EDS (25.0%), 0.3% BSA (25.0%) and 0.1% PVA (15.4%), respectively. In conclusion, the results obtained showed that in vitro maturation media and protein supplement to m-NCSU37 culture medium tested did not promote the final steps of IVM in canine oocytes.