체외 환경에서 생산되는 배아 (Embryo)는 활성산소종 (Reaction oxygen species, ROS) 수준이 일정 수준을 초과함에 따라 산화적인 손상을 받게 된다. 선행연구에 따르면, 항산화제는 ROS를 감소시켜주는 효과를 가지기 때문에 ROS로부터 오는 배아의 단백질, DNA의 손상, 세포 자멸사를 방지하여 배아의 발달률을 향상시킨다. 이전연구에 따르면 항산화제로써 엘라그산 (Ellagic acid, EA)은 ROS를 효과적으로 제거하고, 난자의 산화스트레스를 방지하는 효과를 가지고 있다고 보고되었다. 그리하여, 본 연구를 통해 우리는 소의 수정란 배양체계 중 in vitro culture (IVC) 단계에서 EA의 농도 (0, 5, 10 μM) 별 첨가가 소의 수정란 발달률과, 질적 수준에 미치는 영향을 조사하고자 실험을 진행하였다. 결과적으로, 배반포의 단계별 발달 수준에서 cleavage 형성률은 EA첨가군과 대조군 간의 차이를 발견할 수 없었으나 배반포 형성률에서는 모든 EA 첨가군들이 대조군보다 높았고 EA 첨가군 중에 5 μM 첨가군이 가장 높았다 (p < 0.05). 생산된 배반포의 총 세포 수는 5 μM EA 첨가군이 대조군과 10 μM EA 첨가군 보다 유의적으로 높았으며, 대조군과 10 μM EA 첨가군 사이의 유의적 차이는 없었다 (Control vs. 5 μM vs. 10 μM; 137 ± 7.90 vs. 163.2 ± 7.42 vs. 138.8 ± 6.67, p < 0.05). 세포 자멸사 세포 수는 모든 EA 첨가군들이 대조군보다 유의적으로 낮았다 (Control vs. 5 μM vs. 10 μM; 22.65 ± 4.08, 9.61 ± 1.55, 6.14 ± 0.90, p < 0.05). ROS 수준에서 모든 EA 첨가군들과 대조군 간의 유의적 차이는 없었다 (Control vs. 5 μM vs. 10 μM; 6.81 ± 1.31, 3.86 ± 0.23, 4.11 ± 0.18, p < 0.05). qRT-PCR 실험 결과에서 Nrf2 gene expression은 대조군과, 5 μM 첨가군에서 유의적 차이가 없었으나, 10 μM 첨가군에서는 유의적으로 상향 조절된 것을 관찰하였다. Keap1 gene expression은 5 μM 첨가군에서 유의적으로 하향 조절된 것을 관찰하였다. 하지만 EA의 농도가 10 μM으로 높아짐에 따라 발현 수준이 증가한 것을 관찰할 수 있었다. CAT gene expression은 5 μM 첨가군에서 유의적으로 상향조절 되었으나 10 μM 첨가군에서는 유의적인 차이를 보이지 않았다. SOD1 gene expression은 대조군과 5 μM 첨가군은 유의적인 차이를 보이지 않았으나 10 μM 첨가군에서는 유의적으로 상향 조절된 것을 관찰하였다.

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.

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.

Mammalian fetal ovaries contains numerous primordial germ cells, however fewer ones can yield mature oocytes due to apoptosis and follicle atresia. Successful in vitro reconstitution of primordial germ cells has recently had a significant effect in the field of assisted reproductive technologies. However, the regulatory mechanisms underlying oogenesis remain unknown and recapitulation of oogenesis in vitro remains unachieved. Therefore, development of methods for obtaining mature oocytes by culturing the fetal ovaries in vitro could contribute to clarify these mechanisms. We adapt an in vitro system for culturing mouse fetal ovaries that support successful follicle assembly and improve oocyte growth and maturation. Ovarian tissues from 12.5 days postcoitum (dpc) fetal mice were cultured in vitro and the matured oocytes were differentiated from primordial germ cells after a 31 days culture period. Our results demonstrate that mouse fetal germ cells are able to form primordial follicles with artificial ovarian cells, and that oocytes within the growing follicles are able to mature normally in vitro. Taken together, this in vitro culture system is expected to aid in the development of new strategies to identify the reasons behind failure of follicle assembly and offer a platform for innovative research into preservation of female germ cells and conservation of endangered species.

Previous studies have shown that kisspeptin (Kp-10) is expressed in mammalian ovaries; however, the expression and role of Kp-10 in bovine ovarian granulosa cells are still unclear. In this study, we assessed the expression of Kp-10 and its effects on the proliferation and apoptosis of bovine granulosa cells. Immunohistochemical analysis showed that Kp-10 was expressed in the cytoplasm of bovine ovarian granulosa cells. Moreover, MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2- H-tetrazolium bromide) assays showed that 100 nM Kp-10 significantly inhibited the viability of granulosa cells (P<0.01). Flow cytometry analysis showed that Kp-10 could significantly increase accumulation of cells in the G1 phase, decrease accumulation of cells in the S phase, and promote apoptosis in bovine granulosa cells (P<0.05). Additionally, Kp-10 decreased the mRNA levels of Bcl-2, an anti-apoptotic gene; increased the mRNA levels of caspase-3, a pro-apoptotic gene; and increased the mRNA levels of Fas and Fasl, two membrane surface molecule genes (P<0.05). Thus, our findings demonstrated for the first time that Kp-10 inhibited proliferation and promoted apoptosis in bovine ovarian granulosa cells. These findings provide insights into our understanding of the role of Kp-10 in mediating the proliferation of bovine granulosa cells.

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).

The production of feline induced pluripotent stem cells (iPSCs) can solve the problems that are related with existing unstable supply and demand of eggs as well as ethical aspects about embryonic stem cell at the same time. On the basis of excellent proliferation, it is to facilitate the researches about human disease like FIV and Allergen at the level of cells, not experimental animals. But, a lot of advanced researches are lean too much towards on the transduction using DNA type virus that have the risk of tumorigenesis during reprogramming and on the mLIF-dependent culture condition for the production of feline iPSCs. This being so, this study shows the reprogramming results using Sendai virus vector that is RNA type virus and have no the footprint after transduction. In addition, the feline iPSCs were stably cultured in bFGF-dependent culture condition during the reprogramming step and culture step. In conclusion, we found the bFGF-dependent culture condition in feline iPSCs and suggested the approach using Sendai virus vector as an alternative for reprogramming without concern about tumorigenesis. These methods can be universally applicable to not only the researches about reconstruction and conservation of feline species, but also to a lot of deep studies related with iPSCs or LIF, bFGF to find new approaches.