• Journal of Animal Reproduction and Biotechnology
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• v.38 no.2
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• pp.47-53
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• 2023

Despite numerous advances in in-vitro embryo production (IVP), many documented factors have been shown to influence the development of mammalian preimplantation embryos and the success of IVP. In this sense, elevated levels of reactive oxygen species (ROS) correlate with poor outcomes in assisted reproductive technologies (ART) due to oxidative stress (OS), which results from an imbalance between ROS production and neutralization. Indeed, excessive production of ROS compromises the structural and functional integrity of gametes and embryos both in vivo and in vitro. In particular, OS damages proteins, lipids, and DNA and accelerates cell apoptosis. Several in-vivo and in-vitro studies report an improvement in qualityrelevant parameters after the use of various antioxidants. In this review, we focus on OS and the source of free radicals and their effects on oocytes, sperm, and the embryo during IVP. In addition, antioxidants and their important role in IVP, supplementation during oocyte in vitro maturation (IVM), in vitro culture (IVC), and semen extenders were discussed. Nevertheless, various methods for determining the level of ROS in germ cells have been briefly described. Still, it is crucial to develop standardized antioxidant supplement systems to improve overall IVP success. Further studies should explore the safety, efficacy, mechanism of action, and combination of different antioxidants to improve IVP outcomes.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.7
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• pp.969-976
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• 2009

Use of oocytes from prepubertal animals for in vitro embryo production holds potential application for reducing generation intervals and increasing genetic progress through embryo transfer. The objective of these studies was to compare the effect of three sperm pretreatments (prior to in vitro fertilization) and seven embryo culture protocols on fertilization rate and (or) subsequent development of in vitro fertilized embryos derived from oocytes harvested from ovaries of 1-6 month old prepubertal Boer goats in China. Cleavage rates were highest for embryos fertilized with heparin-treated versus calcium ionophore- or caffeine-treated sperm. Similar rates of blastocyst development were observed using heparin- and ionophore-treated sperm, which were higher than obtained with caffeine-treated sperm. No differences in cleavage or blastocyst rates were observed following embryo culture in basal medias (synthetic oviductal fluid (SOF), Charles Rosenkrans 1 (CR1) or tissue culture medium-199 (TCM-199)) containing 10% fetal bovine serum (FBS). Cumulus or oviductal cell co-culture did not enhance cleavage or blastocyst rates relative to culture in SOF+10% FBS. Replacement of FBS in SOF medium with 0.3% BSA increased cleavage rates, but did not increase rates of blastocyst development. Sequential culture in SOF+0.3% BSA followed by SOF+10% FBS increased blastocyst yield versus continuous culture in SOF+10% FBS and tended to increase blastocyst yield versus continuous culture in SOF+0.3% BSA. These results demonstrate a pronounced effects of sperm pretreatments and in vitro embryo culture systems on rates of blastocyst development and provide a potential protocol (sperm pretreatment with heparin and sequential embryo culture in SOF+0.3% BSA followed by SOF+10% FBS) for generation of the significant numbers of in vitro produced blastocysts from oocytes of prepubertal Boer goats necessary for application of embryo transfer in rural regions of China for distribution of Boer goat genetics.

• Journal of Embryo Transfer
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• v.33 no.4
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• pp.281-286
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• 2018

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.

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.10
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• pp.1358-1364
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• 2011

The sex-determining region on the Y (SRY) gene is important in mammalian sex determination and differentiation. We report a study of the abundance of SRY gene products in bovine ejaculate. RT-PCR experiments using RNA extracted from bovine spermatozoa with SRY-specific primers yielded a 456 bp product, but the amount of SRY mRNA in sperm was lower than that in the testes (p<0.01). A protein of approximately 27 KDa was detected by western blotting. The SRY transcript was detected in the midpiece of approximately half the spermatozoa by in situ hybridization, and the SRY protein was detected in the heads of half the spermatozoa by immunofluorescence, indicating that SRY mRNA and protein may only be present in Y-bearing spermatozoa. These results suggest that the SRY transcript and protein are present in bovine ejaculated Y-sperm. The roles of the SRY gene in spermatogenesis, sperm motility, and the sperm-oocyte interaction merit further investigation.

• Development and Reproduction
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• v.12 no.1
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• pp.19-30
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• 2008

Proper development of fertilized oocyte to blastocyst is a key step in mammalian development to implantation. During development of preimplantation embryos, the mammalian embryo needs supply the energy substrate for keep viability. Usually mammalian oocyte get substrate especially energy substrate from oviduct and uterus, because it does not store much substrate into cytoplasm during oogenesis. Carbohydrates are known as a main energy substrate for preimplantation stage embryos. Glucose, lactate and pyruvate are essential component in preimplantation embryo culture media and there are stage specific preferences to them. Glucose transporter and $H^+$-monocarboxylate cotransporter are a main mediator for carbohydrate transport and those expression levels are primarily under the control of intrinsic or extrinsic factors like insulin and glucose. Other organic substances, amino acids, lipids and nucleotides are used as energy substance and cellular regulation factor. Though since 1960s, successful development of fertilized embryo to blastocyst has been accomplished with chemically defined medium for example BWW and give rise to normal offspring in mammals, the role of metabolites and the regulation of intermediary metabolism are still poorly understood. Glucose may permit expression of metabolic enzymes and transporters in compacting morula, capable of generating the energy required for blastocyst formation. In addition, it has been suggested that the cytokines can modulate the metabolic rate of carbohydrate in embryos and regulate the preimplantation embryonic development through control the metabolic rate. Recently we showed that lactate can be used as a mediator for preimplantation embryonic development. Those observations indicate that metabolites of carbohydrate are required by the early embryo, not only as an energy source, but also as a key substrate for other regulatory and biosynthetic pathways. In addition metabolites of carbohydrate may involve in cellular activity during development of preimplantation embryos. It is suggested that through these regulation and with other regulation mechanisms, embryo and uterus can prepare the embryo implantation and further development, properly.

• Journal of Embryo Transfer
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• v.32 no.1
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• pp.17-24
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• 2017

Ganglioside GD1a is specifically formed by the addition of sialic acid to ganglioside GM1a by ST3 ${\beta}$-galactoside ${\alpha}$-2,3-sialyltransferase 2 (ST3GAL2). Above all, GD1a are known to be related with the functional regulation of several growth factor receptors, including activation and dimerization of epidermal growth factor receptor (EGFR) in tumor cells. The activity of EGF and EGFR is known to be a very important factor for meiotic and cytoplasmic maturation during in vitro maturation (IVM) of mammalian oocytes. However, the role of gangliosides GD1a for EGFR-related signaling pathways in porcine oocyte is not yet clearly understood. Here, we investigated that the effect of ST3GAL2 as synthesizing enzyme GD1a for EGFR activation and phosphorylation during meiotic maturation. To investigate the expression of ST3GAL2 according to the EGF treatment (0, 10 and 50 ng/ml), we observed the patterns of ST3GAL2 genes expression by immunofluorescence staining in denuded oocyte (DO) and cumulus cell-oocyte-complex (COC) during IVM process (22 and 44 h), respectively. Expression levels of ST3GAL2 significantly decreased (p<0.01) in an EGF concentration (10 and 50 ng/ml) dependent manner. And fluorescence expression of ST3GAL2 increased (p<0.01) in the matured COCs for 44 h. Under high EGF concentration (50 ng/ml), ST3GAL2 protein levels was decreased (p<0.01), and their shown opposite expression pattern of phosphorylation-EGFR in COCs of 44 h. Phosphorylation of EGFR significantly increased (p<0.01) in matured COCs treated with GD1a for 44 h. In addition, ST3GAL2 protein levels significantly decreased (p<0.01) in GD1a ($10{\mu}M$) treated COCs without reference to EGF pre-treatment. These results suggest that treatment of exogenous ganglioside GD1a may play an important role such as EGF in EGFR-related activation and phosphorylation in porcine oocyte maturation of in vitro.

• Development and Reproduction
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• v.18 no.3
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• pp.153-160
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• 2014

Adaptive development of early stage embryo is well established and recently it is explored that the mammalian embryos also have adaptive ability to the stressful environment. However, the mechanisms are largely unknown. In this study, to evaluate the possible role of aquaporin in early embryo developmental adaptation, the expression of aquaporin (AQP) 5 gene which is detected during early development were examined by the environmental condition. To compare expression patterns between in vivo and in vitro, we conducted quantitative RT-PCR and analyzed localization of the AQP5 by whole mount immunofluorescence. At in vivo condition, Aqp5 expressed in oocyte and in all the stages of preimplantation embryo. It showed peak at 2-cell stage and decreased continuously until morula stage. At in vitro condition, Aqp5 expression pattern was similar with in vivo embryos. It expressed both at embryonic genome activation phase and second mid-preimplantation gene activation phase, but the fold changes were modified between in vivo embryos and in vitro embryos. During in vivo development, AQP5 was mainly localized in apical membrane of blastomeres of 4-cell and 8-cell stage embryos, and then it was localized in cytoplasm. However, the main localization area of AQP5 was dramatically shifted after 8-cell stage from cytoplasm to nucleus by in vitro development. Those results explore the modification of Aqp5 expression levels and location of its final products by in vitro culture. It suggests that expression of Aqp5 and the roles of AQP5 in homeostasis can be modulated by in vitro culture, and that early stage embryos can develop successfully by themselves adapting to their condition through modulation of the specific gene expression and localization.

• Journal of Embryo Transfer
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• v.5 no.1
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• pp.1-20
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• 1990

Follicular atresia is a universal and characteristic phenomenon of both non-mammalian and mammalian vertebrates. Generally it is estimated that greater than 99% of follicles become atretic in higher domestic animals and human. The number of selected follicles developing to the preovulatory stage are thus fewer. Follicles can become atretic at any stage of development. The previous studies emphasized on descriptive and retrospect aspects of a limited population of the fully grown preovulatory follicle. The main efforts in ovarian physilogical researches are focused on follicular development culminating in ovulation but recent advances have resulted in a better understanding of atresia. Nowadays, recent studies are concentrated on the induction of atresia in a selected population of follicles and of the associated cellular, endocrine, biochemical and molecular changes. The factors initiating atresia and follicle selections are worthy of investigations. Another intriguing question is whether one can predict when a follicle will become atretic, i.e., what biochemical markers indicate that a follicle is destined for atresia. It is generally agreed that atretic process may vary even in antral follicles at different stages of their differentiations and among species. The dicisive factors are follicular responsiveness and the hormonal milieu. Some generalizations can be made on the basis of experimental induction of atresia. Alteration of the pattern of follicular steroid production is associated with the initiation stage of atretic process. Atresia appears to be a process unfolding gradually and affecting progressively in increasing number of functions and components of the follicle. The oocyte may be the latest to be afflicted in the atretic process. The high steroidogenic activity of atretic follicles lends support to the notion that atresia is not necessarily a degenerative process and that atretic follicles may play an essential role in ovarian physiology. The simultaneous occurence of growth and atretic processes may render the search for regulatory mechanisms involved in atresia difficult extremely. The questions such as how follicles are selected to undergo ovulation rather than atresia or what the mechanism of atresia is remain unanswered. However, the factors regulating or modifying ovarian hormonal milieu for the initiation of follicular growth and maturation or of atresia are being elucidated.

• Reproductive and Developmental Biology
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• v.41 no.1
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• pp.17-23
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• 2017

The oocyte undergoes various events during in vitro maturation (IVM) and subsequence development. One of the events is production of reactive oxygen species (ROS) that is a normal process of cell metabolism. But imbalances between ROS production and antioxidant systems induce oxidative stress that negatively affect to mammalian reproductive process. In vitro environments, in vitro matured oocytes have many problems, such as excessive production of ROS and imperfect cytoplasmic maturation. Therefore, in vitro matured oocytes still have lower maturation rates and developmental competence than in vivo matured oocytes. In order to improve the IVM and in vitro culture (IVC) system, antioxidants, vitamins were added to the IVM, IVC medium. Antioxidant supplementation was effective in controlling the production of ROS and it continues to be explored as a potential strategy to overcome mammalian reproductive disorders. Based on these studies, we expect that the use of antioxidants in porcine oocytes could improved maturation and development rates.

• Journal of Embryo Transfer
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• v.16 no.3
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• pp.223-231
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• 2001

The success of nuclear transplantation with mammalian oocytes depends critically on the potential of oocytes activation, which mainly caused to prevent the re-accumulation of maturation promoting factor (MPF). This study was conducted to compare the effect of combined treatment of lonomycin with a Hl-histone kinase inhibitor (dimethylaminopurine, DMAP) or cdc2 kinase inhibitor (sodium pyrophosphate, SPP) on activation of bovine oocytes. In vitro matured bovine oocytes with the first polar body (PB) and dense cytoplasm were assigned to 3 experimental groups. For activation treatment, oocytcs were exposed to 5 $\mu$M lonomycin for 5 min (Group 1), and followed by 1.9 mM dimethylaminopurine (DMAP) for 3 h (Group 2) or followed by 2 mM sodium pyrophosphate (SPP) for 3 h (Group 3). The activation effects in the three treatments and the control group (untreated) were judged by the extrusion of the second PB and formation of a pronucleus (PN). Differences among groups were analysed using one-way ANOVA after arc-sine transformation of proportional data. All three treatments led to high activation rates (90% to 95%), with significant difference from the control. However, the extrusion of the second PB and the rate of PN formation differed remarkably among treatments. In Group I and 3, about 95% of the oocytes had extruded the second polar body, but one PN had formed in a higher proportion of oocytes in Group 3 than in Group 1 (90% vs. 5%). In experiment 2, the rates of cleavage and development into blastocysts in Group 1 were significantly lower than those of Group 2 and 3 (8.7% and 0% vs. 50.5% and 11.6%, and 44.6% and 7.2%, respectively, P<0.05). In experiment 3, ~80% of parthenotes in Group 1 were developed with haploid chromosomal sets. However, when ionomycin was followed immediately by DMAP (Group 2). only 20% of parthenotes were haploid. In Group 3, combined treatment with ionomycin and SPP, the appearance of abnormal chromosomal tracts was significantly (P〈0.05) reduced and the proportion of haploid parthenotes was increased to 85% (17/20) than in Group 2. These results demonstrate that SPP acted as a cdc2 kinase inhibitor and formed the haploidy in oocyte activation. Thus, the present study suggests that cdc2 kinase inhibitor, such as sodium pyrophosphate, may have an effective role in oocyte activation for the production of cloned embryos/animals by nuclear transplantation.