The objective of this study was to compare of different isolation method of mouse preantral follicles, and to examine in vitro development of mouse preantral follicles isolated by different method. Preantral follicles were mechanically or enzymatically extracted from mouse ovaries. Mechanical isolation method used fine gauge needles and enzymatic method of isolating follicles used collagenase. The recovered preantral follicles were cultured for 10 days in alpha-minimal essential medium (-MEM) + 5% FBS + Insulin-Transferrin-Selenium (ITS) + 100 mIU/ml FSH. The collected primary follicles by enzymatic treatment were higher than mechanical method. Others stage preantral follicle by mechanical isolation were higher than enzymatic method. After 10 days of culture, no statistical differences were shown in survival rates of preantral follicle among the 2 culture groups. The metaphase II rates of the oocytes were significantly higher (p<0.05) in mechanical method (17.8%) than in enzymatic method (5.1%). These results suggest that the isolation method of choice depends on the target stage preantral follicles and mechanical isolation is an optimal method of preantral folliclesin a culture of mouse preantral follicle.

Culture of preantral follicles has important biotechnological implications through its potential to produce large quantities of oocytes for embryo production and transfer. The objective of this study was to determine the comparison of different isolation method of mouse preantral follicles, and to examine in vitro development of mouse preantral follicles isolated by different method. Preantral follicles were mechanically or enzymatically extracted from mouse ovaries. Mechanical isolation method used fine gauge needles and enzymatic method of isolating follicles used 1 mg/ml collagenage (Type IA) and 0.2 mg/ml DNase Ⅰ in Leibovitz L-15 medium. The solution containing Leibovitz L-15 medium, enzyme and ovary fragments was incubated at 37℃ for 30 min. The selection criteria are as follows: primary follicle of 75 to 99 μm, early secondary follicle of 100 to 125 μm and late secondary follicle of 126 to 150 μm in diameter. The recovered preantral follicles were cultured for 10 days in alpha-minimal essential medium (α-MEM) + 5% FBS + ITS + 100 mIU/ ml FSH. The collected primary follicles by enzymatic treatment were higher than mechanical method. Others stage preantral follicle by mechanical isolation were higher than enzymatic method. After 10 days of culture, no statistical differences were shown in survival rates of preantral follicle among the 2 culture groups. The metaphase Ⅱ rates of the oocytes were significantly higher (p<0.05) in mechanical method (17.8%) than in enzymatic method (5.1%). These results suggest that the isolation method of choice depends on the target stage preantral follicles and mechanical isolation is an optimal method of preantral follicles in a culture of mouse preantral follicle.

The objective of this study was to examine the effect of eCG and various concentrations (20, 40, and 80 ) of porcine FSH on nuclear maturation and intracellular glutathione (GSH) level of oocytes, and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in pigs. Immature pig oocytes were matured in TCM-199 supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones (10 IU/ml hCG and 10 IU/ml eCG or FSH) for the first 22 h and then further cultured in hormone-tree medium for an additional 22 h. Nuclear maturation of oocytes () was not influencem foreCG and various concentrations FSH. Embryonic development to the cleavage stage () and mean number of cells in blastocyst ( cells) after PA were not altered but blastocyst formation e-treignificaddlor(p<0.05) improvem forthe supplementation eith 80 FSHr(64%) compared to 47%, io8%, iand 47% in oocytes that were treated with eCG, 20,i and 40 FSH,i numectivelo. In SCNT, fusion () of cell-cytoplast couplets and siosequent embryo cleavage () were not influencem fordifferent gonadotropins but blastocyst formation tended to increase forthe supplementation eith 80 FSHr(25% vs. ). Our nuults demonstrated that oocyte maturation and embryonic development after PA and SCNT e-frinfluencem fortype of gcem fortype of gits concentration. In this study, supplementation of maturation medium eith 80 FSHrimproved preimplantation development of PA and SCNT pig embryos, probably by increasing intracellular GSH concentration of matured oocytes.

본 연구는 체외성숙/체외수정 유래의 돼지 난자를 이용하여 체외발달시 배양액의 종류나 교체에 따른 영향을 구명하고자 수행하였다. mNCSU-23에서 체외성숙시킨 다음 mTBM에서 체외수정시킨 난자를 목적에 따라 두 가지로 나누어 실험한 결과는 다음과 같다. 1. 체외성숙/체외수정란을 NCSU-23에서 배양액 교체없이 7일 동안 배양하거나 CZB에서 4일 배양한 다음 Pig-MEM으로 옮겨서 나머지 3일간 배양한 결과, 난분할율은 배양액간 차이를 보이지 않

본 연구는 mNCSU-23에서 체외성숙시킨 난자를 이용하여 돼지 체외수정시 적절한 배양액과 정자농도를 구명하고자 수행하였다. 정액은 액상정액을 이용하였고 체외수정배양액으로 mTBM과 mTLP-PVA, 정자농도는 운동정자수 5, 1 , 5 sperm/로 체외수정 한 다음 NCSU-23에서 체외발달을 유도한 결과는 다음과 같다. 1. 돼지 체외성숙란을 mTBM 또는 mTLP-PVA에서 운동정자수 1 sperm/로 체외수정시킨 다음 체외발달시킨 결과, 정자침

본 연구는 pFF, cysteine, -mercaptoethanol, 성선자극호르몬 등 여러 가지 체외성숙 촉진 물질이 첨가딘 성숙배양액에 EGF 첨가가 돼지 미성숙 난포란의 체외성숙에 효과적인지 또한 그 효가는 배양소적당 COC의 수에 영향을 받는지을 구명하고자 EGF의 첨가 유무와 배양소적당 COC수(50개 또는 15개)를 조합한 요인시험을 실시했다. 도축돼지의 난소에서 채취한 COCs를 각 처리별로 mNCSU-23 에서 성숙배양하고 mTBM에서 운

본 연구는 보다 안정된 돼지 체외수정란 생산시스템 확립을 목적으로 체외성숙배양액 mNCSU-37, mNCSU-23 및 TCM-199 가 각각 미성숙 난포란의 체외성숙, 체외수정, 체외발달에 미치는 영향을 구명하기 위하여 수행하였다. 도축돼지의 난소에서 채취한 COCs를 10% pFF가 포함된 각각의 성숙배양액에서 최종동도가 의 농도로 체외수정 시킨 다음, NCSU-23 에서 체외발달을 유도한 결과는 다음과 같다. 1. TCM-199에서 성숙시킨 난자가

Contemporary systems for in vitro culture of ovarian follicles do not recapitulate the mechanical heterogeneity in mammalian ovary. Here we report microfluidic generation of biomimetic ovarian microtissue for miniaturized three-dimensional (3D) culture of early secondary preantral follicles by using alginate (harder) and collagen (softer) to fabricate the ovarian cortical and medullary tissues, respectively. This biomimetic configuration greatly facilitates follicle development to antral stage. Moreover, it enables in vitro ovulation of cumulus-oocyte complex (COC) from the antral follicles in the absence of luteinizing hormone (LH) and epidermal growth factor (EGF) that are well accepted to be responsible for ovulation in contemporary literature. These data reveal the crucial role of mechanical heterogeneity in the mammalian ovary in regulating follicle development and ovulation. The biomimetic ovarian microtissue and the microfluidic technology developed in this study are valuable for improving in vitro culture of follicles to preserve fertility and for understanding the mechanism of follicle development and ovulation to facilitate the search of cures to infertility due to ovarian disorders.

Ultrastructural studies on oocyte development and vitellogenesis in oocytes, and the functions of follicle cells during oogenesis and oocyte degeneration were investigated to clarifyb the reproductive mechanism on vitellogenesis of Scapharca subcrenata using electron microscope observations. In this study, vitellogenesis during oogenesis in the oocytes occured by way of autosynthesis and heterosynthesis. Of two processes of vitellogenesis during oogenesis, the process of endogenous autosynthesis involved the combined activity of the Golgi complex, mitochondria and rough endoplasmic reticulum. However, the process of exogenous heterosynthesis involved endocytotic incorporation of extraovarian precursors at the basal region of the oolema of the early vitellogenic oocytes before the formation of the vitelline coat. In this study, follicle cells, which attached to the previtellogenic and vitellogenic oocytes, were easily found. In particular, the follicle cells were involved in the development of previtellogenic oocytes by the supply of nutrients, and vitellogenesis in the early and late vitellogenic oocytes by endocytosis of yolk precursors. Based on observations of follicle cells attached to degenerating oocytes after spawning, follicles of this species are involved in lysosomal induction of oocyte degeneration for the resorption phagosomes (phagolysosomes) in the cytoplasm for nutrient storage, as seen in other bivalves. In this study, the functions of follicle cells can accumulate reserves of lipid granules and glycogen particles for vitellogenesis from degenerating oocytes after spawning.