In order to examine the effects of four different light spectra (white, red, green, and blue) on the oocyte maturation, the change of reproductive parameters, via brain-pituitary-gonad (BPG) axis in grass puffer, were investigated. After exposure four different light spectra for 7 weeks, the abundance of gonadotropin-releasing hormone (GnRH) mRNA which is a type of seabream (sbGnRH) and two different subunit of gonadotropin hormones mRNAs, follicle-stimulating hormone (fshβ) mRNA and luteinizing hormone (lhβ) mRNA, were analyzed in the brain and pituitary. Histological analysis showed that the mature oocyte ratio in the green spectrum was higher than other light spectra-exposed groups. Gonadosomatic index (GSI) and oocyte developmental stage were also investigated in the gonad based on histological observations. GSI value with the presence of yolk stage oocytes was significantly increased in the green spectrum-exposed group when compared to that of the other light-exposed groups (white, red, and blue) (p˂0.05). The abundances of sbGnRH mRNA and fshβ mRNA in the green spectrum-exposed group were also significant higher than those of the other light spectra-exposed groups (p˂0.05). These results indicate that the maturation of oocyte in grass puffer can be accelerated by exposure to the spectrum of green. To better understand the molecular mechanism for the maturation of oocyte in grass puffer, further study examining the relationship between oocyte development and its related genes is required.

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.

In order to examine the effects of four different light spectra (i.e., white, red, green, blue) on the oocyte maturation in grass puffer, the change of reproductive parameters via brain-pituitary-gonad (BPG) axis were investigated in this study. After exposure to four different light spectra for 7 weeks, the abundance of gonadotropin-releasing hormone (GnRH) mRNA which is a type of seabream (sbGnRH) and two different of subunit of gonadotropin hormones mRNAs, follicle-stimulating hormone (fshβ) mRNA and luteinizing hormone (lhβ) mRNA, were analyzed in the brain and pituitary. Gonadosomatic index (GSI) and oocyte developmental stage were also investigated in the gonad based on anatomical- and histological observations. GSI value was significantly increased in the green spectrum-exposed group when compared to that of the other light-exposed groups (i.e., white, red, blue), with the presence of yolk stage oocytes (p˂0.05). The abundances of sbGnRHmRNA and fshβ mRNA in the green spectrum-exposed group were also significant higher than those of the other light spectra-exposed groups(p˂0.05). However, there was no significant difference between the abundances of lhβmRNA in all light spectra-exposed groups. These results indicate that the maturation of oocyte in grass puffercan be accelerated by exposure to the spectrum of green. The sbGnRHmRNA and fshβ mRNA may play an important reproductive parameters role in the initiation of maturation of oocyte. To better understand the molecular mechanism for the maturation of oocytein grass puffer, further study examining the relationship between oocyte development and its related genes (e.g., sbGnRHmRNA and fshβmRNA) is required.

The aim of this study was to determine the effect of additional alpha-linolenic acid (ALA) supplementation during in vitro maturation (IVM) and culture (IVC) on nucleic maturation and embryo development of pigs. Cumulus-oocyte complexes (COCs) were incubated in IVM medium containing different concentration of ALA (25, 50 and 100 μM) for 44 h. After in vitro maturation, nuclear maturation of oocytes were evaluated by aceto-orcein stain. Mature oocytes with 50 μM ALA were fertilized and cultured in IVC medium with ALA (25, 50 and 100 μM) during early-embryogenesis (48 hours after fertilization). Then, embryos were cultured with 25 μM ALA during early embryogenesis and/or late embryogenesis (120 hours after early-embryogenesis). In results, oocyte maturation were significantly increased by 50 μM ALA treatment groups compared with control groups (p<0.05). Treatment of 25 μM ALA during early-embryogenesis enhanced cleavage rate of embryo compared with other groups (p<0.05), whereas formation and total cell number of blastocyst had no significant difference. Similarly, cleavage rate of embryos were increased by 25 μM ALA supplement during early- or late-embryogenesis than ALA treatment both stage of embryogenesis (p<0.05), but did not influence to blastocyst formation. Interestingly, total cell number of blastocyst were enhanced in ALA treatment group during early-embryogenesis. These findings indicated that ALA supplement enhance the nuclear maturation of oocyte and embryo development, however, excessive ALA could negatively influence. Therefore, we suggest that ALA is used for improvement of in vitro production of mammalian embryo and further study regarding with functional mechanism of ALA is needed.

This study was conducted to investigate stimulatory effect of epidermal growth factor (EGF) on nuclear maturation and the expression level of EGF-receptor (EGFR), GM-130 (a marker of Golgi apparatus), transport protein Sec61 subunit beta (Sec61β), and coatomer protein complex subunit gamma 2 (COPG2) in porcine oocytes. The cumulus-oocyte complexes were collected from follicle with 3-6 mm in diameter. They were incubated in medium with/without EGF for 22 h (IVMⅠ) and subsequently incubated hormone-free medium with/without EGF for 22 h (IVMⅡ). Nuclear maturation state was checked by aceto-orcein stain. Protein expression of EGFR, GM-130, Sec61β, and COPG2 were measured by immunofluorescence. In results, nuclear maturation of oocytes in EGF non-treated oocytes were significantly lower than EGF-treated groups at IVMⅠ or IVMⅡ stage (P<0.05), whereas maturational rate in EGF treatment groups at both of IVM stage was higher in among the all treatment groups (P<0.05). EGFR, GM-130, Sec61β and COPG2 were expressed in the cytoplasm of oocytes. Especially, GM-130 and EGFR were strongly expressed, but Sec61β and COPG2 were weakly expressed in cortical area of cytoplasm. The protein level of GM-130, Sec61β, and COPG2 were significantly higher in the EGF-treated groups (P<0.05). However EGFR was no difference between non EGF-treated groups and control. In conclusion, EGF plays an important role in the systems for oocyte maturation with endoplasmic reticulum and Golgi apparatus. In addition, the protein levels of Sec61β and COPG2 could be changed by EGF in the porcine oocytes during maturation.

Fas ligand (FasL) and its receptor Fas have been implicated in granulosa cell apoptosis during follicular atresia. Although interferon-gamma (IFN-γ) is believed to be involved in the regulation Fas expression in differentiated granulosa or granulosa-luteal cells, the expression of this cytokine and its role in the regulation of the granulosa cell Fas/FasL system and apoptosis during follicular maturation have not been thoroughly investigated. In the present study, we have examined the presence of IFN-γ in ovarian follicles at different stage of development by immunohistochemistry and related their relative intensities with follicular expression of Fas and FasL, and with differences in granulosa cell sensitivity to Fas activation by exogenous agonistic Anti-Fas monoclonal antibody (Fas mAb). Although IFN-γ immunostaining was detectable in oocyte and granulosa cells in antral follicles, most intense immunoreactivity for the cytokine was observed in these cells of preantral follicles. Intense immunoreactivity for IFN-γ was most evident in granulosa cells of atretic early antral follicles where increased Fas and FasL expression and apoptosis were also observed. Whereas low concentrations of IFN-γ (10-100 U/mL) significantly increased Fas expression in undifferentiated granulosa cells (from preantral or very early antral follicles) in vitro, very higher concentrations (≥ 1,000 U/mL) were required to up-regulate of Fas in differentiated cells isolated from eCGprimed (antral) follicles. Addition of agonistic Fas mAb to cultures of granulosa cells at the two stages of differentiation and pretreated with IFN-γ (100 U/mL) elicited morphological and biochemical apoptotic features which were more prominent in cells not previously exposed to the gonadotropin in vivo. These findings suggested that IFN-γ is an important physiologic intra-ovarian regulator of follicular atresia and plays a pivotal role in regulation of expression of Fas receptor and subsequent apoptotic response in undifferentiated (or poorly differentiated) granulosa cells at an early (penultimate) stage of follicular development.

The marine medaka, Oryzias dancena is a suitable sample as a laboratory animal because it has a small size and clearly distinguishes between female and male. Data on the growth and maturity of the diploid and triploid sea cucurbit species suitable for laboratory animals are very useful for studying other species. Triploidy was induced in the marine medaka by cold shock treatment (0°C) of fertilized eggs for 45 min, applied two minutes after fertilization. The diploid and triploid male fish were larger than their female counterparts (P<0.05), and the concentrations of thyroid stimulating hormone (TSH) and thyroxine (T4) were higher in the induced triploids over 1 year (P<0.05). In both the diploid and tri-ploid groups the concentrations of TSH and T4 were higher in the male fish than in the females (P<0.05), while the testo-sterone and estradiol-17ß concentrations in the induced triploids were lower than in the diploids (P<0.05). The gonadosomatic index (GSI) of the triploid fish was lower than that for the diploids, and the GSI for females in each ploidy group were higher than that for the males. For both groups the GSI was highest at 4 months of age, and decreased thereafter to 12 months. Analysis of the gonads of one-year-old triploid fish suggested that the induction of triploidy probably causes sterility in this species; this effect was more apparent in females than in males.

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..

This study describes results on sexual maturation and characteristics of natural spawned eggs to develop a method for the production of stable, healthy fertilized eggs from captive-reared yellowtail kingfish, Seriola lalandi. A total of 59 yellowtail kingfish were captured off the coast of Jeju Island, after which the broodstock was cultured in indoor culture tank (100 m3) until they were 6.1–14.9 kg in body weight. As part of the rearing management for induced sex maturation, the intensity of illumination was maintained at 130 lux. The photoperiod (light/dark; L/D) was set to a 12 L/12 D from October 2013 to January 2014, and 15 L/9 D from February 2014 to June 2014. Feeds comprised mainly EP (Extruded Pellets), with squid cuttlefish added for improvement of egg quality, and was given from April to June 2014. The first spawning of yellowtail kingfish occurred in May 3, 2014, at a water temperature of 17.0°C. Spawning continued until June 12, 2014, with the water temperature set at 20.5°C. Time of spawning was 26 times at this period. The total number of eggs that spawned during the spawning period was 4,449×103. The buoyant rate of spawning eggs and fertilization rate of buoyant eggs during the spawned period were 76.1% and 100%, respectively. The diameters of the egg and oil globule were 1.388 ± 0.041 mm and 0.378 ± 0.029 mm, respectively, which was higher in early eggs than in those from late during the spawned period.

Cytokinesis is the final event in the cell division. After cytokinesis, one parent cell divided into two symmetric daughter cells. Unlike somatic cell which is symmetrically divided, oocyte meiotic maturation is highly asymmetric division, producing mature ovum and polar body. Class III phosphoinositide 3-kinase (PI3K) has been known as a key molecular component that regulates cell cycle progression, autophagy and endosomal trafficking. However, emerging evidences suggest that class III PI3K and its interactors are involved in midbody abscission during cytokinesis. Here we showed that beclin-1, a key component of PI3K is required to regulate midbody abscission during oocyte asymmetric division. Beclin-1 was widely distributed during meiotic maturation forming small vesicles. However, these vesicles were not colocalized with autophagosomal marker LC3. Instead, beclin-1 was detectable at the midbody ring during cytokinesis. Depletion of beclin-1 showed various defects including the failure of cytokinetic abscission, spindle separation and chromosome decondensation. Similar phenotype was observed when class III PI3K activity was inhibited. Therefore, our results demonstrate that PI3K is essential for cytokinesis but not autophagy during oocyte meiosis.

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.

Primary oocytes that are arrested in first meiotic prophase for years enter maturation process to meet a critical precondition for successful fertilization. During maturation, oocyte finishes meiosis I and progresses to the metaphase II stage, achieving meiotic maturity. Although importance of oocyte maturation for oocyte quality has been recognized, it is not fully understood for molecular mechanisms underlying oocyte maturation. Here, we found that dexamethasone-induced Ras-related protein 1 (RASD1), a member of RAS superfamily of small GTPases, was expressed in the mouse ovary. Immunohistochemical analysis revealed that Rasd1 expression was dominant in oocyte cytoplasm. Real-time PCR and RT-PCR analyses showed that Rasd1 mRNA was steadily expressed in germinal vesicle (GV), germinal vesicle break down (GVBD), metaphase I (MI) oocytes, but decreased in metaphase II(MII) oocytes during oocyte maturation. Konckdown of Rasd1 using RNAi system in the GV oocytes suppressed oocyte maturation through disruption of meiotic spindle and formation of misarranged chromosomes. Taken together, Rasd1 is a critical factor for MI-MII transition of oocyte and is involved in the regulation of spindle formation during oocyte maturation. Further study is needed to examine relationship between Rasd1 and spindle formation in MI-MII transition.

Dynamic reorganization of actin filaments is essential for various stages of mammalian oocyte maturation, including spindle migration, actin cap formation, polar body extrusion, and cytokinesis. Various actin binding proteins (ABPs) have been known to be involved in the regulation of actin filament remodeling. We elucidate roles of three different actin binding proteins in mouse oocyte maturation. The heterodimeric actin-capping protein (CP) binds to the fast-growing(barbed) ends of actin filaments and plays essential roles in various actin-mediated cellular processes. When CP is knockdowned or inhibitory component was overexpressed, asymmetric division of oocyte have been compromised. It turns out that knockdown or inhibition of CP deplete cytoplasmic actin mesh level, which have been known to be essential for maintain cytoplasmic actin mesh. Another actin binding proteins, tropomodulin 3 (Tmod3), binds to the slow-growing end of actin filaments and knockdown or expression deletion mutant of Tmod3 also decrease actin mesh level in maturing oocyte and it severely ablated asymmetric division of oocyte. Finally, tropomyosin 3, actin filament binding proteins protect actin filament from depolymerization, is also important to maintain cortex integrity in maturing oocyte. Taken together, these finding showed the essential roles of actin binding proteins in remodeling of actin filaments in mammalian oocyte development.

In the present study, different expression of protein from Taekwang was revealed by 2-DE, and expressions of protein on each week after flowering was investigated. After analysis of expression of protein, MALDI-TOF was executed to identify expected protein function. Results revealed that there were three patterns of expression of protein during the maturing. The first pattern was that proteins were gradually expressed as up-regulation from 1 week to 6 week. The second pattern was that proteins were expressed gradually from 1 week to 5 week and then it started down-regulation in 6 week. The last pattern was that proteins were gradually as up-regulation from 1 week to 3 week and then down-regulation until 6 week. This phenomenon suggests that young stage has more protein related to correspondence mechanism against disease and growth and then maturing stage has more expression of protein related to storage protein. In MALDI-TOF analysis, p24 oleosin isoform A protein was identified that relates oleosin which is synthetic product in oil body. This protein spot increased gradually until 5 week and then decreased after 5 week. It explained that the protein is active until maturing stage to protect oil in seed and then its activity has gradually degraded. This result may be expected that a protein, related to growth of a seed has increased until maturing and then a seed fills up with a storage protein

Rearing experiments were conducted using several regimes combined with different photoperiods and water temperatures to investigate the role of temperature and photoperiod as environmental cues regulating reproduction in red marbled rockfish, Sebastiscus tertius. The initial gonadosomatic index (GSI) was 1.62±0.32% and that in the 15˚C control was 7.01±0.46% at the end of the experiment. The highest GSI was 9.10±0.35% in the 12L:12D photoperiod-treated fish. The highest GSI at 19˚C was 15.03±0.86% in the 12L:12D photoperiod treatment. The initial hepatosomatic index (HSI) was 3.09±0.72% and that in the 15˚C control was 3.88±0.45%. The highest HSI was 4.00±0.56% in the 15L:9D treated fish. The highest HSI at 19˚C was 4.05±0.63% in the 12L:12D treated fish.

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.

Spatial- and temporal-specific expression patterns are primarily regulated at the transcriptional level by the promoter. Therefore, it is important to determine the binding motifs of transcription factors to understand the networks associated with embryogenesis. Here, we used a protein-binding microarray (PBM) to determine the binding motif of OsSMF1, which is a basic leucine zipper transcription factor that is involved in the regulation of rice seed maturation. OsSMF1 (previously called RISBZ1) is known to interact with GCN4 motifs (TGA(G/C)TCA) to regulate seed storage proteins (SSPs). In addition, OsSMF1 (also known as OsbZIP58) functions as a key regulator of starch synthesis in the rice seed. Quadruple 9-mer-based PBM (Q9-PBM) and electrophoretic mobility shift assay (EMSA) experiments revealed that OsSMF1 binds to the ACGT (CCACGT(C/G)), GCN4 (TGA(G/C)TCA), and GCN4-like (GGATGAC) motifs with Kd values of 0.3353 μM, 0.6458 μM, and 1.117 μM, respectively. We also identified 60 putative OsSMF1 target genes using a combination of data from expression microarrays and RiceArrayNet (RAN) analysis. Of these OsSMF1 target genes, 20, 22, and 17 genes contained ACGT, GCN4, and GCN4-like motifs within the 2-kb promoter region, respectively. In addition to known target genes, we also identified 35 potential OsSMF1 target genes that have not been previously described in immature seeds. We also confirmed that OsSMF1 directly regulates Os03g0168500 (thioredoxin-related protein), RPBF, NAC6, and two hypothetical proteins (Os12g0621600 and Os11g0582400) in vivo. This study suggests that OsSMF1 functions in a wide range of seed development processes with specific binding affinities for three DNA binding motifs

전통 재래식 간장에 표고버섯 및 천마를 첨가하여 일정기간 숙성시킨 후 분리하여 이화학적 성질 등의 변화를 측정하였다. NaCl의 함량은 대조구에 비해 실험구가 높게 나타났으며, 다른 재래식 방법으로 제조한 간장에 비해 전체적으로 염도가 낮았다. pH의 변화는 숙성이 될 수록 감소 하였으며 총당과 환원당의 함량 또한 대조구에 비해 실험군이 높은 값을 보였는데 표고버섯이나 천마로부터 유래된 당의 영향으로 인해 높아진 것으로 판단된다. Protease 활성은 실험구가 6~8배 이상 높은 값을 보였고 항산화 활성에서는 천마간장이 총 폴레페놀 함량 90.23 mg/mL, 총 플라보노이드 함량 404.44 mg/mL로 가장 높은 값을 보였으며 전자공여능 측정에서 버섯 간장이 72.71%를 나타냈다.