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Dynamics of connexin expression in the lycopene treated oocytes of Korean native cow
MMR Chowdhury,Ayman Mesalam,Myeong-Don Joo,Lianguang Xu,Fahmida Afrin,Yeoung-Gyu Ko,Sung Soo Lee,Sung Woo Kim,Il-Keun Kong 한국수정란이식학회 2018 한국수정란이식학회 학술대회 Vol.2018 No.11
Ovarian folliculogenesis and the production of fertilizable oocytes depend on gap junctional intercellular communication within both the developing and the mature follicle. Gap junctions connect oocytes with granulosa cells and granulosa cells with each other. Various nutritional bio-molecules are known to be transferred to the growing oocyte from the granulosa cells via gap junction. Signals that regulate meiotic maturation of fully-grown oocytes pass through the oocyte-granulosa cell gap junctions. Gap junctions also play a critical role in regulating uterine blood flow, contributing to the maternal recognition and also implantation during pregnancy. Due to the challenge of various stressors the in vitro embryo developmental potentials are still suboptimal compared to in vivo. To identify the molecular mechanism of these stressors and to improve the existing embryo developmental potentials, the singlet oxygens quencher lycopene was added to the culture media to counterbalance the oxidative damage caused by ROS. In this study, we have patterned connexin like Cx43, Cx37, Cx32 and Cx26 at protein and transcription level during follicular growth, atresia and blastocyst stage by using immunohistochemistry, conventional PCR and RT-qPCR. Lycopene (0.2 μM) significantly (P < 0.05) increased the gap junctional communication protein (connexin) expression of Cx43, Cx37, Cx32, Cx26 as compared to the control group at both transcription and translation level during follicular growth, atresia and blastocyst stage. Lycopene potentiates ovarian folliculogenesis, provides the production of fertilizable oocytes and improved embryo developmental capabilities by increasing gap junctional intercellular communication.
Heat stress augmented transglutaminase activity in mouse oocyte
MMR Chowdhury,Yeoung-Gyu Ko,Sung Soo Lee,Sung Woo Kim 한국수정란이식학회 2018 한국수정란이식학회 학술대회 Vol.2018 No.11
Transglutaminase (TGM2) belongs to a family of cross-linking enzymes responsible for catalyzing Ca2+-dependent acyl-transfer reactions between the substrate proteins. TGM2 is a cytosolic protein that has also been observed in the nucleus and can be expressed to the cell surface or extracellular matrix. Despite ubiquitous expression, its functions are poorly understood and still need to be elucidated. Moreover, there is no clear data regarding the role of transglutaminase in mammalian oocytes. So, in this study, we have patterned the transglutaminase 2 (TGM2) and anti-N epsilon gamma glutamyl lysine (AB424) activity in heat stressed mouse oocytes. We have collected mouse oocytes from the (6–9 weeks old) mouse and in vitro matured for 20 h. Immunocytochemistry was performed to checked the transglutaminase 2 (TGM2) and anti-N epsilon gamma glutamyl lysine (AB424) activity after 6 h of heat stress (HS) at 39.1 ℃. Both TGM2 and AB424 expression were significantly (P < 0.05) higher compared to control when oocytes were subjected to HS at 6 h of IVM at 39.1 ℃. Our hypothesis is that TGM2 and AB424 activity may be correlated with the cellular regression and also involvement in apoptosis. We hope that, our study will help to elucidate the normal function of mouse oocyte and also identification of the principal proteins as well as the pathogenic mechanism of altered physiology. These results suggest that the nuclear accumulation of the transglutaminase may play an important role in nuclear remodeling during folliculogenesis and early embryonic development
Mesalam, Ayman,Kong, Rami,Khan, Imran,Chowdhury, MMR,Choi, Byung-Hyun,Kim, Sung Woo,Cho, Kyu-Woan,Jin, Jong-In,Kong, Il-Keun Elsevier 2017 Reproductive biology Vol.17 No.4
<P><B>Abstract</B></P> <P>This study investigated the ability of charcoal:dextran stripped fetal bovine serum (CDS FBS) and heat-inactivated fetal bovine serum (HI FBS) to support <I>in vitro</I> development of bovine embryos. The developmental ability and quality of bovine embryos were determined by assessing their cell number, lipid content, mitochondrial activity, gene expression, and cryo-tolerance. The percentage of embryos that formed a blastocyst was significantly (<I>P<</I> 0.05) higher in medium containing CDS FBS than in medium containing HI FBS (42.84±0.78% vs. 36.85±0.89%, respectively). Furthermore, the beneficial effects of CDS FBS on embryos were associated with significantly (<I>P<</I> 0.05) increased mitochondrial activity, as identified by MitoTracker Green, as well as a reduced intracellular lipid content, as identified by Nile red staining, which increased their cryo-tolerance. Quantitative reverse transcription PCR showed that the mRNA levels of acyl-CoA synthetase long-chain family member 3, acyl-coenzyme A dehydrogenase long-chain, hydroxymethylglutaryl-CoA reductase, and insulin-like growth factor 2 receptor were significantly (<I>P<</I> 0.05) increased upon culture with CDS FBS. Moreover, the mRNA levels of sirtuin 1, superoxide dismutase 2, and the anti-apoptotic gene B-cell lymphoma 2 in frozen-thawed blastocysts were significantly (<I>P<</I> 0.05) higher in the CDS FBS-supplemented group than in the HI FBS-supplemented group, whereas that of the pro-apoptotic gene BCL2-associated X protein was significantly lower. Taken together, these data suggest that supplementation of medium with CDS FBS improves the <I>in vitro</I> developmental competence and cryo-tolerance of bovine embryos.</P>
Lianguang Xu,Seok-Hwan Song,Kyeong-Lim Lee,Ayman Mesalam,MMR Chowdhury,Myeong-Don Joo,Shimin Zhang,Imran Khan,Il-Keun Kong 한국수정란이식학회 2017 한국수정란이식학회 학술대회 Vol.2017 No.05
Bovine somatic cell nuclear transfer (bSCNT) embryos can develop to the blastocyst stage at a rate similar to that of embryos produced by in vitro fertilization (IVF). However, the efficiency of somatic cell cloning has remained low, and applications have been limited, irrespective of the nuclear donor species or cell types. One possible explanation is that the reprogramming factors of each oocyte is insufficient or not properly adapted for the receipt of a somatic cell nucleus, because it is naturally prepared only for the receipt of a gamete. Here, we would like to introduce the aggregation method (agSCNT), a new experimental system that enables and increase oocyte volume and examined its subsequent development. Judgement by the blastocyst formation rate or total cell number was significantly higher in the agSCNT group than that in the SCNT group, and was very similar to that in the control IVF group. Moreover, the cleavage formation rate in the agSCNT group (61.5 ± 1.3) was higher than that in the SCNT group (39.7 ± 2.1), while still less than that in the IVF group (75.4 ± 1.3). We also analyzed the epigenetic modifications in bovine IVF, agSCNT, and untreated SCNT embryos. In conclusion, the present study demonstrated that agSCNT improves the in vitro developmental competence and quality of cloned embryos, as evidenced by increased total cell numbers (TC).