• Clinical and Experimental Reproductive Medicine
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• v.29 no.1
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• pp.37-44
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• 2002

Objective : Heat shock protein family is related to protective mechanism of cells by environmental changes. This study was performed to evaluate the effect of cryopreservation on the heat shock protein 90 (Hsp90) expression in mouse ovarian tissue. Methods : Cryopreservation of mouse ovarian tissue was carried out by slow freezing method. The mRNA level of Hsp90 expression in both fresh and cryopreserved mouse ovarian tissue was analyzed by RT-PCR. The protein expression of Hsp90 was evaluated by Western blot analysis and immunohistochemistry. Results: The mRNA and protein of Hsp90 were expressed in both fresh and cryopreserved mouse ovarian tissue. The amount of Hsp90 mRNA was increased in cryopreserved ovarian tissue after 60 and 90 minutes after thawing and incubation. The amount of Hsp90 protein was increased in the cryopreserved ovarian tissue after 6 hours of the incubation in Western blot analysis. In immunohistochemical study, Hsp90 protein was localized in cytoplasm of oocytes and granulosa cells. Significant level of immunoreactive Hsp90 protein was detected in theca cells contrast to the weak expression in ovarian epithelial cells. Conclusion: This results showed the increase of Hsp90 expression in both mRNA and protein level in the cryopreserved mouse ovarian tissue. It can be suggested that Hsp90 may play a role in the protective or recovery mechanism against the cell damage during cryopreservaion.

• Journal of Life Science
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• v.11 no.2
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• pp.103-107
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• 2001

The equine chorionic gonadotropin (eCG) subunits $\alpha$ and ${\beta}$ are transcribed from different genes and associate noncovalently to form the bioactive eCG heterodimer. Dimerization is rate limiting for eCG secretion, and dissociation leads to hormone inactivation. The correct conformation of the heterodimer is alto important for efficient secretion, hormone-specific post-translational modifications, receptor binding and signal transduction. To determine whether ${\alpha}$ and ${\beta}$ subunits can be synthesized as a single polypeptide chain (tethered-eCG) and also display biological activity, the tethered-eCG molecule by fusing the carboxyl terminus of the eCG ${\beta}$-subunit to the amino terminus of the af-subunit was construe-ted and transfected into chinese hamster ovary (CHO-Kl) cells. LH- and FSH-like activities were assayed in terms of testosterone production and aromatase activity in primary cultured rat Leydig cells and granulosa cells, respectively. The tethered-eCG was efficiently secreted and showed similar LH-like activity to the dimeric eCG ${\alpha}$/${\beta}$ and native eCG. FSH-like activity of the tethered-eCG was also shown similarly in comparison with the native and wild type eCG ${\alpha}$/${\beta}$. Our data for the first time suggest that the tethered-eCG can be expressed efficiently and the produced product by the CHO-K1 cells is fully LH- and FSH-like activities in rat in vitro bioassay system. Our results also suggest that this molecular can imply particular models of FSH-like activity not LH-like activity in the eCG. Taken together, these data indicate that the constructs of tethered molecule will be useful in the study of mutants that affect subunit association and/or secretion.

• Asian-Australasian Journal of Animal Sciences
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• v.33 no.6
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• pp.879-887
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• 2020

Objective: Numerous studies have indicated that the apoptosis and proliferation of granulosa cells (GCs) are closely related to the normal growth and development of follicles and ovaries. Previous evidence has suggested that miR-126-3p might get involved in the apoptosis and proliferation of GCs, and phosphatidylinositol 3-kinase regulatory subunit 2 (PIK3R2) gene has been predicted as one target of miR-126-3p. However, the molecular regulation of miR-126-3p on PIK3R2 and the effects of PIK3R2 on porcine GCs apoptosis and proliferation remain virtually unexplored. Methods: In this study, using porcine GCs as a cellular model, luciferase report assay, mutation and deletion were applied to verify the targeting relationship between miR-126-3p and PIK3R2. Annexin-V/PI staining and 5-ethynyl-2'-deoxyuridine assay were applied to explore the effect of PIK3R2 on GCs apoptosis and proliferation, respectively. Real-time quantitative polymerase chain reaction and Western Blot were applied to explore the regulation of miR-126-3p on PIK3R2 expression. Results: We found that miR-126-3p targeted at PIK3R2 and inhibited its mRNA and protein expression. Knockdown of PIK3R2 significantly inhibited the apoptosis and promoted the proliferation of porcine GCs, and significantly down-regulated the mRNA expression of several key genes of PI3K pathway such as insulin-like growth factor 1 receptor (IGF1R), insulin receptor (INSR), pyruvate dehydrogenase kinase 1 (PDK1), and serine/threonine kinase 1 (AKT1). Conclusion: MiR-126-3p might target and inhibit the mRNA and protein expressions of PIK3R2, thereby inhibiting GC apoptosis and promoting GC proliferation by down-regulating several key genes of the PI3K pathway, IGF1R, INSR, PDK1, and AKT1. These findings would provide great insight into further exploring the molecular regulation of miR-126-3p and PIK3R2 on the functions of GCs during the folliculogenesis in female mammals.

• Korean Journal of Clinical Laboratory Science
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• v.48 no.1
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• pp.8-14
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• 2016

Granulosa cells surround the oocyte within the ovarian follicle and play an essential role in creating conditions required for oocyte as well as follicular development. The current study was conducted to examine the gene expression profile of mouse ovaries during the primordial to primary follicle transition process. Total RNAs from mouse ovaries on day 5 and day 12 were synthesized cDNA using annealing control primers. The DEGs were cloned and their identities were analyzed by BLAST search. The Plekha5 and Anxa11 were highly expressed in primary follicle stage. By contrast, their expression was increased in granulosa cells at the primary follicle stage. We have successfully discovered a list of genes expressed in day 5 and day 12 ovaries and confirmed that some of them are differentially expressed in PMF and/or PRI. This is a spatial-temporal regulatory mechanism on the ovarian folliculogenesis through membrane fusion. The gene expression profile from the current study would provide insight for future study on the mechanism(s) involved in primordial-primary follicular transition. This will provide information for identification of the mechanism of ovarian dysfunction.

• Development and Reproduction
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• v.2 no.1
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• pp.1-8
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• 1998

In mammalian ovary, major portion(>99%) of ovarian follicles undergo atresia. Recent studies have shown that this phenomenon is mediated via GC apoptosis. Ceramide, a product of sphingomyelin hydrolysis, has been proposed as a novel lipid second messenger with specific roles in mediating antiproliferative responses including apoptosis and cell cycle arrest. In the present study, we have examined the effect of ceramide on apoptotic cell death of GC in vitro. GCs were harvested by squeezing the antral follicles from the immature mice (3-4 weeks) and cultured in MEM medium with 10% fetal bovine serum. The cells were treated with various concentrations of ceramide (0 to 50 \mu M)and cultured up to 24 h.Cell death was determined by MTT cell viability assay and apoptosis was examined by acridine orange staining, in situ 3'-end labeling(TUNEL), and flow cytometry. Ceramid treatment induced apoptotic cell death of GC in a time- and a dose-dependent manner. Results of flow cytometric analysis showed that creamide-induced cell death was mostly confined to the $G_{0}$/$G_{1}$ cells. these results provide an evidence for ceramide as a lipid second messenger of apoptosis in mouse GC.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.2
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• pp.174-183
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• 2001

During the rapid phase of gonadal development of the freshwater teleost, the catfish (Silurus asotus), the influence of hCG upon the inducement of final oocyte maturation and spawning was investigated electrophoretically and ultrastructurally. The electrophoretic patterns obtained were different in the presence and absence of some of the major or minor zones, because of the hormone level in catfish. The vitellogenin of hormone-treated fish was stained more intensively than that of sham-treated fish. These proteins showed some minor or main bands of egg extracts which migrated at positions corresponding to molecular weights of approximately 90,000. However, the thickness of electrophoretic band in molecular weight for hCG-treated fish was slightly lower than that for saline control. It seemed the plasma protein with molecular weight of approximately 45,000 in hCG-treated fish disappeared. In contrast to the control fish, the ovaries in the catfish treated with hCG shows a marked ultrastructural change under the electron microscope. No dilated profiles were seen in the granulosa cells of the mature oocyte before ovulation. After germinal vesicle breakdown (GVBD), the zona radiata interna (ZRI) becomes more compact, and there is a loss of all the processes from the pore canals. There is a wide space between the vitelline membrane and zona radiata. Also, during final maturation, the microvillar processes from the oocyte are seen no longer to penetrate deeply into the extracellular spaces of the overlying granulosa cells, and the reticulate patterns of the zona radiata interna becomes occluded, giving the zona radiata a more solid appearance. It has been possible to initiate 100% oocyte maturation in yolk granules and follicles in vivo by treatment with hCG and a high water temperature ($27^{\circ}C$). In hCG-treated fish, the percentages of successful artificial fertilization and hatching were maximal at 15 h after a single injection. It seems clear that a long acting preparation containing hCG can be successfully used in prespawning fish to advance the final events of gonadal maturation and initiate spawning. Further studies are necessary to evaluate the potential of hCG to either stimulate or inhibit the reproductive development of fish at other stages of the seasonal reproductive cycle.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.2
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• pp.207-216
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• 1998

Certain types of somatic cells, as well as follicular cumulus cells associating with mammalian oocytes, are known to produce beneficial effects on in vitro fertilization and pre implantation development of mammalian eggs when they are present in oocyte culture medium. To investigate the nature of the effects of somatic cells, the resistance of mouse oocytes against chymotrypsin treatment was examined after culture within various cell conditioned media. When mouse oocytes matured for 17-18 hr in the presence of cumulus cells were treated with 1 % chymotrypsin, half of them remained still alive even after 240 min $(t_{50}>240.0)$. In contrast half of mouse oocytes cultured without cumulus cells underwent degeneration within 65.0 min $(t_{50}=65.0{\pm}13.2min)$ of the same treatment. To see if the effects were duc to the secretory products of cumulus cells, mouse cumulus cells were cultured for 20 hr in medium containing 0.4% BSA and the supernatant of culture medium (conditioned medium) was taken. After maturation in the cumulus cell conditioned medium, mouse oocytes exhibited $t_{50}=190.0{\pm}10.8$ min upon chymotrypsin treatment whereas half of oocytes cultured without conditioned medium degenerated within 25.5 min. Human granulosa cell conditioned medium gave similar effects such that oocytes matured in conditioned medium exhibited $t_{50}=183.3{\pm}19.1$ min while $t_50$ of control group oocytes was $60.0{\pm}6.8$ min, Oocytes matured in vero cell conditioned medium exhibited $t_{50}=196.7{\pm}8.8$ min. On the other hand, amniotic cell conditioned medium resulted in the chymotrypsin resistance of $t_{50}=80.0{\pm}8.4$ min which was not statistically different from the control value of $t_{50}=48.0{\pm}13.2$ min. Based upon these results, it is suggested that certain somatic cell types including cumulus cells might change the biochemical properties of mouse oocyte membrane during meiotic maturation as revealed by the enhanced resistance against chymotrypsin treatment. Such effects of somatic cells appear to be mediated via the secretory products rather than direct communication between somatic cells and oocytes.

• Korean Journal of Animal Reproduction
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• v.21 no.2
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• pp.111-116
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• 1997

We determined the effects of follicular fluid fractions in the maturation medium on bovine oocyte maturation, fertilization and subsequent development, as well as on number of cells in blastocysts following culture. Follicular fluid and oocytes from bovine follicles less than 5 mm in diameter were collected from the ovaries of slaughtered cows. Follicular fluid was separated into different molecular weight fractions by untrafiltration through a membrane using a centrifuge at 500$\times$g, for 2h. For the maturation medium, follicular fluid fractions (30%, v/v), whole fluid (30%) or PVP(3mg/ml) were added to TCM 199(0.1$\mu\textrm{g}$/ml estradiol-17$\beta$, 100IU hCG). After maturation for 24h, oocytes were fertilized in vitro with bull frozen-thawed spermatozoa and cultured on a monolayer of granulosa cells for 9 days after fertilization. There were no differences in maturation rates or fertilization rates among any maturation conditions. The rates of development to >2-cell stage of the oocytes were significantly decreased when fraction of follicular fluid below 10,000 MW were added into maturation medium, compared with control and fraction above 10,000 MW(26.0% vs 40.8% to 64.0%, respectveily. p<0.01). Likewise, the rates of development to blastocysts of fertilized oocytes were significantly decreased in maturation medium containing fraction of follicular fluid (<10,000 MW). The average cell number of blastocysts derived from oocytes that matured in the fraction(>10,000 MW) of follicular fluid was 154.7$\pm$13.7. These embryos contained more cells than those matured in whole follicular fluid, or the fraction(<10, 000 MW) of follicular fluid or control(107.0$\pm$8.4, 91.8$\pm$11.8 and 95.8$\pm$6.2, respectively). In conclusion, we found that fractions of follicular fluid contained factors stimulating or inhibiting oocyte cytoplasmic matruation. These suggest that a factor(s) inducing cytoplasmic maturation of oocytes may exist in >10,000 MW fraction of follicular fluid.

• Korean Journal of Veterinary Research
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• v.33 no.1
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• pp.179-188
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• 1993

The developmental capacity of bovine oocytes under three different culture systems was investigated in this experiment ; One was culture in TCM199 with bovine oviductal epithelial cells(BOEC) for in vitro culture, another was culture in TCM199 with BOEC for 2 days and then transfer of 4~8cell embryos to rabbit oviduct(RO) and the other was transfer of 1 or 2cell embryos to RO for in vivo culture. And the other concern of this experiment was to investigate the effect of culture period and transfer site on recovery. Immature bovine oocytes were cultured in TCM199 with granulosa cells for 22-24hrs and then fertilized in vitro using frozen-thawed semen treated with BO-caffine and BO-BSA. Fifteen to 18hrs after in vitro fertilization oocytes were cultured in TCM199 with BOEC or transferred to RO for 5 days. The rate of development to the morula or blastocyst was higher in transfer of 1 or 2cell embryos to RO(23.1%) than culture in TCM199 with BOEC(11.7%). But, there was no difference between transfer of 1 or 2cell embryos and transfer of 4~8cell embryos to RO(12.8%). Recovery under different culture periods in RO was significantly higher in 90~95hrs(70.1%) than 122~125hrs(50.9%, p<0.05) and recovery significantly increased when oocytes were transferred deeper in RO(2.5cm>, 47.7% ; 2.5~4.5cm, 63.9% ; 4.5cm<, 77.3%, p<0.05). The results show that transfer of 1 or 2cell embryos to RO is an effective means of supporting the further development of in vitro matured and fertilized bovine oocytes than culture in TCM199 with BOEC or transfer of 4~8cell embryos to RO, and recovery from RO increases when oocytes are transferred deeper and incubated shorter in RO.

• Korean Journal of Animal Reproduction
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• v.17 no.1
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• pp.57-68
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• 1993

This stduy was carried out to find a reliable method for the production of in vitro fertilized embryos having more excellent development capacity and freezability in the rabbit and cattle. The greatest number of rabbit oocytes was recovered 6hrs after HCG injection(P<0.05). The maturation rate in vitro was slightly higher in the oocytes(6-h-oocytes) from 6h than those (8-h-oocytes)from 8 hrs after HCG injection and the beneficial effect of FSH during oocyte maturation was significantly great in the oocytes from large follicles. The cleavage rate into 2-to-6-cell stage was not differ between the 6-h-oocytes and 8h-oocytes, but the cleavage of these oocytes was greatly promoted by FSH addition to maturation medium and the cleavge of 8-h-oocytes matured without FSH was significantly low. The embryo development into 16-cell to morula was not promoted by the co-culture with rabbit oviduct epithelial cells. The freezability by embryo stages was ovidusly high at 4-cell and morula stage in 6-h-oocytes and the viability of 16-cell embryos from 8h-oocytes was similar to that of morula stage. The implantation sites after surgical tranfer of fresh rabbit embryos were not implanted. In bovine experiment, the in vitro development into 16-cell and morula after in vitro maturation and fertilization in the follicular oocytes was slightly improved by the co-culture with granulosa cells compared to that with oviduct epithelial cells and the frozen-thawed viability rate of these embryos ranged from 14 to 40%. The excellent fresh embryos were transferred nonsurgically to 6 recipients, but were not pregnant.