A combination of bovine serum albumin with insulin-transferrin-sodium selenite and/or epidermal growth factor as alternatives to fetal bovine serum in culture medium improves bovine embryo quality and trophoblast invasion by induction of matrix metallopr

Mesalam, Ayman,Lee, Kyeong-Lim,Khan, Imran,Chowdhury, M. M. R.,Zhang, Shimin,Song, Seok-Hwan,Joo, Myeong-Don,Lee, Jae-Hoon,Jin, Jong-In,Kong, Il-Keun CSIRO Publishing 2019 Reproduction, fertility, and development Vol.31 No.2

<P> This study investigated the use of bovine serum albumin (BSA) plus insulin-transferrin-sodium selenite (ITS) and/or epidermal growth factor (EGF) as alternatives to fetal bovine serum (FBS) in embryo culture medium. The developmental ability and quality of bovine embryos were determined by assessing their cell number, lipid content, gene expression and cryotolerance, as well as the invasion ability of trophoblasts. The percentage of embryos that underwent cleavage and formed a blastocyst was higher (P&lt;0.01) in medium containing ITS plus EGF and BSA than in medium containing FBS. Culture with ITS plus EGF and BSA also increased the hatching ability of blastocysts and the total cell number per blastocyst. Furthermore, the beneficial effects of BAS plus ITS and EGF on embryos were associated with a significantly reduced intracellular lipid content, which increased their cryotolerance. An invasion assay confirmed that culture with ITS plus EGF and BSA significantly improved the invasion ability of trophoblasts. Real-time quantitative polymerase chain reaction analysis showed that the mRNA levels of matrix metalloproteinase-2 (MMP2) and MMP9, acyl-CoA synthetase long-chain family member 3, acyl-coenzyme A dehydrogenase long-chain and hydroxymethylglutaryl-CoA reductase significantly increased upon culture with ITS plus EGF and BSA. Moreover, protein expression levels of matrix metalloproteinase-2 and -9 increased (P&lt;0.01) in medium supplemented with ITS plus EGF and BSA compared with medium supplemented with FBS. Taken together, these data suggest that supplementation of medium with ITS plus EGF and BSA improves invitro bovine embryo production, cryotolerance and invasion ability of trophoblasts. </P>

Effects of Serum Type in Maturation Medium on In Vitro Development of Bovine Embryos

Ayman Mesalam,Shimin Zhang,Lianguang Xu,Myeong-Don Joo,Ji-Yoon Hwang,Kyeong-Lim Lee,Il-Keun Kong 한국동물생명공학회(구 한국동물번식학회) 2018 발생공학 국제심포지엄 및 학술대회 Vol.2018 No.06

This study investigated the effect of Charcoal-Dextran Stripped fetal bovine serum (CDS FBS) and heat-inactivated FBS (HI FBS) in maturation medium on their ability to support in vitro oocyte maturation, cumulus cell-oocyte gap junctional communication, and development of bovine embryos. Oocytes were cultured in TCM-199 supplemented with either 8% BSA, 10% CDS FBS, or 10% HI FBS and 1 μg/mL estradiol-17β, 10 μg/mL FSH, 10 ng/mL EGF, 0.6 mM cysteine, 0.2 mM sodium pyruvate, and followed by in vitro fertilization and the zygotes were cultured in SOF-BE1 medium. The developmental ability and quality of bovine embryos were determined by assessing their cell number, lipid content, mitochondrial activity, gene expression, immunocytochemistry, and cryo-tolerance. The differences in embryo development between experimental groups were analyzed by one-way ANOVA. We have shown that CDS FBS had a higher significant (p<0.05) effect on the rate of blastocyst formation comparing to HI FBS and BSA (45.2±0.7% vs. 37.4±1.5% and 31.1±3.9%, respectively, six replicates were performed). Culture of oocytes with CDS FBS increased (p<0.05) the expression of gape junction proteins, CX37 and CX43, at both transcriptional and translation levels. We also found that CDS FBS significantly increased total cell number and decreased the apoptotic index in day-8 blastocyst comparing to BSA group. Furthermore, the beneficial effects of CDS FBS on embryos were associated with significantly reduced intracellular lipid content and increased mitochondrial activity in both oocytes and blastocyst. Taken together, these data suggest that supplementation of maturation medium with CDS FBS affect cumulus cell-oocyte gap junctional communication, and subsequently improved in vitro developmental competence of bovine oocytes and embryos.

Effect of charcoal:dextran stripped fetal bovine serum on <i>in vitro</i> development of bovine embryos

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>

Improvement of in vitro produced bovine embryo quality using Charcoal:Dextran Stripped Fetal Bovine Serum on culture media

Mesalam Ayman,Lianguang Xu,Byung-Hyun Choi,Beon-Young Park,Rami Kong,Mi-Ju Son,Jong-In Jin,Sung Woo Kim,Il-Keun Kong 한국수정란이식학회 2017 한국수정란이식학회 학술대회 Vol.2017 No.05

This study investigated the effect of Charcoal:Dextran Stripped fetal bovine serum (CDS FBS) and heat-inactivated FBS (HI FBS) in embryo culture medium on their ability to support in vitro 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 percentages of embryos that underwent cleavage and formed a blastocyst were significantly (P<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 a significantly reduced intracellular lipid content, as identified by Nile red staining, which increased their cryo-tolerance. The post-thaw survival rate of blastocysts was significantly (P<0.05) higher in the CDS FBS than in the HI FBS group (85.33 ± 4.84% vs. 68.67 ± 1.20%). Quantitative real-time 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 increased upon culture with CDS FBS. Moreover, the mRNA levels of sirtuin 1, superoxide dismutase 2, and anti-apoptotic associated gene B-cell lymphoma 2 in frozen-thawed blastocysts were significantly (P<0.05) higher in the CDS FBS group than in the HI FBS group, however, the mRNA level of the pro-apoptotic gene BCL2-associated X protein was significantly reduced. Taken together, these data suggest that supplementation of medium with CDS FBS improves in vitro bovine embryo developmental competence and cryo-tolerance.

Replacement of FBS with BSA in the IVM medium: Effects on maturation and subsequent development of bovine oocytes in vitro

Ayman Mesalam,Muhammed Idress,Lianguang Xu,Myeong-Don Joo,Ji-Yoon Hwang,Kyeong-Lim Lee,Marwa El Sheikh,Il-Keun Kong 한국수정란이식학회 2018 한국수정란이식학회 학술대회 Vol.2018 No.11

This study investigated the use of bovine serum albumin (BSA) as alternatives to fetal bovine serum (FBS) in in vitro maturation medium. The oocyte maturation, cumulus cell-oocyte gap junctional communication, and development of bovine embryos were determined by assessing their cell number, lipid content, mitochondrial activity, gene expression and cryo-tolerance. Oocytes were cultured in TCM-199 supplemented with 1 μg/ml estradiol-17ß, 10 μg/ml FSH, 10 ng/ml EGF, 0.6 mM cysteine, 0.2 mM sodium pyruvate and either 8% BSA (BSA group), 10% FBS (FBS group), or neither BSA nor FBS (TCM group), and followed by in vitro fertilization and the zygotes were cultured in SOF-BE1 medium. The differences in embryo development between experimental groups were analyzed by one-way ANOVA. We have shown that the percentages of embryos that underwent cleavage and formed a blastocyst were non significantly different among all experimental groups (37.4 ± 1.5% for FBS group vs. 31.1 ± 3.9% for BSA group and 34.5 ± 1.6% for TCM group, six replicates were performed). Furthermore, there was no significant difference between the percentage of MII oocyte between FBS (71.8 ± 1.9%) and BSA groups (69.3 ± 2.3%). However, culture of oocytes with FBS increased (P < 0.05) the cumulus cell expansion as well as expression of gape junction proteins, CX37 and CX43, at both transcriptional and translation levels. We also found that FBS significantly increased total cell number and decreased the apoptotic index in day-8 blastocyst comparing to BSA group. The beneficial effects of BSA on embryos were associated with significantly reduced intracellular lipid content and increased mitochondrial activity in both oocytes and blastocyst. Taken together, these data suggest that supplementation of maturation medium with BSA, as alternatives to FBS, can be used as defined medium that support consistently the development of IVP bovine embryos.

Structural Changes of Zona Pellucida Surface of Immature, In vivo and In vitro Matured Canine Oocytes Using Scanning Electron Microscopy

최병현,Ayman Mesalam,송석환,주명돈,황지윤,오선화,이경림,공일근 사단법인 한국동물생명공학회 2018 한국동물생명공학회지 Vol.33 No.4

Zona pellucida (ZP), a primarily representative coat of mammalian egg and embryo, has an extremely heterogeneous morphology during different developmental stages. The objective of the present study was to compare the morphological changes of the ZP surface of immature, in vitro and in vivo matured canine oocytes by using scanning electron microscopy (SEM). Canine ovaries were collected from local veterinary hospitals to recover immature oocytes. The ovaries were sliced and the released cumulus oocyte complexes (COCs) were washed with TL-HEPES. The selected COCs were randomly divided into two groups, first group was processed immediately at immature state and the second group was processed 72 h after in vitro maturation, and compared with in vivo derived oocytes. Oocytes were fixed, critical point dried and examined under SEM. The diameters of oocyte and outer holes of the ZP were measured on a total of 249 oocytes; the results were analyzed using One-way ANOVA. Our results showed that, the diameter of immature oocytes significantly differed (p < 0.05) from that of in vivo matured oocytes (79.60 ± 0.77 μm vs. 101.46 ± 1.07 μm, respectively). Similarly, a significant difference (p < 0.05) in the diameters between those of in vitro and in vivo matured oocytes were found (79.51 ± 2.36 μm vs. 101.46 ± 1.07 μm, respectively). Moreover, the diameters of the outer holes of the ZP were significantly (p < 0.05) larger in in vivo matured (1.48 ± 0.42 μm) than in vitro matured for 72 and immature oocytes (1.10 ± 0.16 and 0.43 ± 0.12 μm, respectively). Taken together, these data indicates that the ZP surface is related to oocyte maturity in canine.

Effect of Predator Stress on the Reproductive Performance of Female Mice after Non-surgical Embryo Transfer

Shimin Zhang,Ayman Mesalam,Kyeong-Lim Lee,Seok-Hwan Song,Lianguang Xu,Yuguo Yuan,Il-Keun Kong 한국동물생명공학회(구 한국동물번식학회) 2018 발생공학 국제심포지엄 및 학술대회 Vol.2018 No.06

Predator stress can exert detrimental effects on female mammals, leading to be morphological or behavioral and ultimately come at the cost of survival, growth, body condition, or reproduction. Although there have many studies about predator stress on reproductive output of rodents, however, few talk about the effect of visual or auditory stress to the pregnant female. In this study, we investigated the possible effect of predator stress, visual or visual plus auditory, on the reproduction performance of female mice after non-surgical embryo transfer. The reproductive performance has been assessed for pregnancy rate, implantation rate, gestation length, live pups rate and neonatal birth weight. Moreover, female serum cortisol and progesterone level were measured by using Electrochemiluminescence Immunoassay. Surprisingly, the exposure to predator stress did not lead to a significant change in pregnancy rates of the tested mice. However, the stressed mice showed significant (p<0.05) decrease in implantation rate compared to the control group. Similarly, the live pups rate and neonatal birth weight were significantly lower in the group exposed to cat stress than in the control group. Furthermore, mice exposed to visual plus auditory stress showed significant reduction in gestation length compared to the control mice. Our data showed that predator stress in form of visual plus auditory combined stimuli significantly increased the serum cortisol level. On the other hand, progesterone level did not show any significant variation among different experimental groups. Taken together, our findings imply that predator stress adversely affect the reproductive efficiency of pregnant mice by decreasing the implantation rate, live birth rates, neonatal birth weight and prolong the gestation length. These data will help in identify the reasons behind the effects of prenatal predator stress on reproductive failure.

Effect of MMP-2 & -9 on the Improvement of Mouse Pregnancy Potential

Shimin Zhang,Ayman Mesalam,Myeong-don Joo,Ji-Yoon Hwang,Seon-hwa Oh,Muhammad Idress,Hongyu Liu,Il-Keun Kong 한국동물생명공학회(구 한국동물번식학회) 2018 발생공학 국제심포지엄 및 학술대회 Vol.2018 No.06

Matrix metalloproteinases (MMPs) comprise a family of zinc-dependent proteinases. Proteins of the MMPs family are involved in the breakdown of extracellular matrix in normal physiological processes, such as embryonic development. Successful implantation is closely linked to the expression of MMPs which greatly influences ability of an embryo to degrade the basement membrane of the uterine epithelium, mainly composed of type IV collagen, and invade the uterine stroma. The purpose of this study was to determine the MMP-2 & -9, also known as type IV collagenase, concentrations in mouse uterine fluid during implantation window, investigate the roles of MMP-2 & -9 on mouse trophoblastic cells invasion in vitro and study the effect of MMP-2 & -9 cotransfer with mouse embryos on reproductive performances. Protein level was detected by Elisa kit and invasiveness was assessed by an invasion assay. Optimal concentrations of MMP-2 and -9 was co-transferred to 2.5 dpc recipients using the non-surgical embryo transfer. Our results showed that MMP-2 & -9 protein levels in mouse uterine fluid were significantly increased at 2.5 dpc. Moreover, in vitro treatment significantly promoted both spreading and invasion of mouse trophoblastic cells as compared with the non-treated. Embryo transfer results showed that MMP-9 cotransfer enhanced mouse implantation and pregnancy rate compared with the control and MMP-2 cotransfered groups, however, that was not significant. Taken together, our findings imply that MMP-9 cotransfer with embryos can regulate embryo invasion during preimplantation, which may have serious consequence on embryo implantation and can be applied in other mammals so far as human being.

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.

Structural Changes of Zona Pellucida Surface of Immature, In vivo and In vitro Matured Canine Oocytes Using Scanning Electron Microscopy

Choi, Byung-Hyun,Mesalam, Ayman,Song, Seok-Hwan,Joo, Myeong-don,Hwang, Ji-Yoon,Oh, Seon-Hwa,Lee, Kyeong-Lim,Kong, Il-Keun The Korean Society of Embryo Transfer 2018 한국동물생명공학회지 Vol.33 No.4

Zona pellucida (ZP), a primarily representative coat of mammalian egg and embryo, has an extremely heterogeneous morphology during different developmental stages. The objective of the present study was to compare the morphological changes of the ZP surface of immature, in vitro and in vivo matured canine oocytes by using scanning electron microscopy (SEM). Canine ovaries were collected from local veterinary hospitals to recover immature oocytes. The ovaries were sliced and the released cumulus oocyte complexes (COCs) were washed with TL-HEPES. The selected COCs were randomly divided into two groups, first group was processed immediately at immature state and the second group was processed 72 h after in vitro maturation, and compared with in vivo derived oocytes. Oocytes were fixed, critical point dried and examined under SEM. The diameters of oocyte and outer holes of the ZP were measured on a total of 249 oocytes; the results were analyzed using One-way ANOVA. Our results showed that, the diameter of immature oocytes significantly differed (p < 0.05) from that of in vivo matured oocytes ($79.60{\pm}0.77{\mu}m$ vs. $101.46{\pm}1.07{\mu}m$, respectively). Similarly, a significant difference (p < 0.05) in the diameters between those of in vitro and in vivo matured oocytes were found ($79.51{\pm}2.36{\mu}m$ vs. $101.46{\pm}1.07{\mu}m$, respectively). Moreover, the diameters of the outer holes of the ZP were significantly (p < 0.05) larger in in vivo matured ($1.48{\pm}0.42{\mu}m$) than in vitro matured for 72 and immature oocytes ($1.10{\pm}0.16$ and $0.43{\pm}0.12{\mu}m$, respectively). Taken together, these data indicates that the ZP surface is related to oocyte maturity in canine.