• Clinical and Experimental Reproductive Medicine
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• v.47 no.2
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• pp.94-100
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• 2020

The inverse correlation between maternal age and pregnancy rate represents a major challenge for reproductive endocrinology. The high embryo ploidy error rate in failed in vitro fertilization (IVF) cycles reflects genetic misfires accumulated by older oocytes over time. Despite the application of different follicular recruitment protocols during IVF, gonadotropin modifications are generally futile in addressing such damage. Even when additional oocytes are retrieved, quality is frequently poor. Older oocytes with serious cytoplasmic and/or chromosomal errors are often harvested from poorly perfused follicles, and ovarian vascularity and follicular oxygenation impact embryonic chromosomal competency. Because stimulation regimens exert their effects briefly and immediately before ovulation, gonadotropins alone are an ineffective antidote to long-term hypoxic pathology. In contrast, the tissue repair properties (and particularly the angiogenic effects) of platelet-rich plasma (PRP) are well known, with applications in other clinical contexts. Injection of conventional PRP and/or its components (e.g., isolated platelet-derived growth factors as a cell-free substrate) into ovarian tissue prior to IVF has been reported to improve reproductive outcomes. Any derivative neovascularity may modulate oocyte competence by increasing cellular oxygenation and/or lowering concentrations of intraovarian reactive oxygen species. We propose a mechanism to support intrastromal angiogenesis, improved follicular perfusion, and, crucially, embryo ploidy rescue. This last effect may be explained by mRNA upregulation coordinated by PRP-associated molecular signaling, as in other tissue systems. Additionally, we outline an intraovarian injection technique for platelet-derived growth factors and present this method to help minimize reliance on donor oocytes and conventional hormone replacement therapy.

• Korean Journal of Ichthyology
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• v.13 no.4
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• pp.254-260
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• 2001

In the goby Micropercops swinhonis, the follicular layer of full-grown oocytes consists of an outer layer (theca cell) and an inner layer (granulosa cell). As the oocyte grows, columnar cells of inner granulosa layer secrete mucin to their cytoplasm and then surround the oocyte. Such granulosa cells appear to be cuboidal cells in the early vitellogenesis, yolk vesicle stage, to be replaced by columnar cell secreting mucins (adhesive materials) in the middle vitellogenesis, yolk granule stage. The enveloping layer of the oocyte has a muco- follicle layer filled with mucins. The mucins are an amorphous and electron-dense substance. Interestingly, the oocyte enveloping layer becomes thickened towards the animal pole as vitelogenesis proceeds. A zona radiata of about $7.8{\sim}11.5\;{\mu}m$ thick is present below the muco-follicle layer. The zona radiata is composed of an one-layered electron-dense externa and a three to five-layered electron-less interna.

• Clinical and Experimental Reproductive Medicine
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• v.21 no.3
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• pp.285-296
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• 1994

Follicular oocytes were released from the graafian follicles of ovaries from 3-4 weeks old mice. The spontaenous maturation of these follicular oocyes was inhibited by the treatment of dbcAMP and progesterone and these oocytes were cultured for 2-8hr in the Modified Hank's balanced salt solution(MHBS). Ethylenediaminetetraaceticacid(EDTA) and calmoudulin antagonist, trifluoperazine (TFP) were treated to the culture medium in order to investigate whether these chemical agents inhibit calcium uptake into the oocyte and oocyte maturation. $^{45}Ca^{2+}$, 10-${\mu}$Ci/ml was added to the culture medium during the culture period. $^{45}Ca^{2+}$uptake into the oocytes was examined whether and when various kind of oocyte maturation inhibiting agents inhibit or stimulate the influx of calcium into oocytes. Dibutyryl cAMP and progesterone decrease $^{45}Ca^{2+}$uptake into the oocytes and synergistic inhibiting effect of dbcAMP and progesterone was prominent at much lower dosages. Calcium uptake into oocytes seems to be higher during first 2 hour culture period rather than next 4hr culture. After 8hr culture, calcium uptake level of the oocytes which GVBD already took place gradually approached to the level of those which were maintained at GV by the treatment of dbcAMP and progesterone. However, $^{45}Ca^{2+}$uptake into the GV maintained oocytes did not change at all even after 8hr culture period. In addition, calcium chelating agent, EDTA inhibited calcium uptake into oocytes as well as nuclear maturation of oocytes. Lower dosage used in the present study did not inhibit calcium uptake as well as oocyte maturation.

• The Korean Journal of Zoology
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• v.14 no.4
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• pp.181-191
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• 1971

In Diplonichus esakii each ovary comprises five telotrophic ovarioles. In the fourth instar, the anterior part of the germarium consists of undifferentiated cells. The middle part contains a spherical trophic and the posterior part comprises young oocytes, followed by the upside downbell shape prefollicular tissue. The bell form is the standard characteristic in this instar larva, and the nutritive cord is found although somewhat indistinctly. In the fifth instar larva, the trophic core is elliptical form, and the oocyte is found at the base of the core. The oocytes are connected with the ocre by the nutritive cord. In the prefollicular tissue are also found some oocytes. In the adult, the vitellarium is filled by the developmental oocytes. The yolk granules inside each oocyte migrate from the base of the follicular epithelial cells to the center of the oocyte. Finally, the ooplasm of the oocyte becomes completely homogeneous. Therefore, according to the advancing of instars the nutritive cord developes completely before the oocyte has chorion and the follicular epithelial cell binucleates. The upper part of the ovariole consists of unchorionated oocytes, and the proximal part comprises chorionated oocytes in the adult.

• Journal of Veterinary Clinics
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• v.27 no.1
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• pp.46-49
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• 2010

In this study, we investigated the number of follicles, oocyte recovery rate and oocyte competence after in vitro maturation according to the size of follicle. And equine oocyte competence after in vitro maturation was investigated in terms of the diameter of follicle with criteria of maturation: nuclear stage after Hoechst staining. The average number of follicles per ovary with middle size (11-20 mm, 2.68) was higher than those of small (5-10 mm, 0.74) and large size follicle (> 21 mm, 1.63), therefore medium follicle (53.1%) had higher proportion than other size of follicles. The average numbers of follicle per ovary was 5.05. The rate of oocyte recovery in small (54.5%) and middle follicle (50%) was higher than that in large follicle (40.9%). After culture for 48 h in Medium 199, 50%, 45.5%, and 44.4% of oocytes from the follicles with diameters of 5-10, 11-20, > 21 mm, respectively reached the metaphase II stage. This is the first report showing number of follicle, oocyte recovery rate according to follicular size, and in vitro oocyte maturation in Jeju mare in Korea. To fulfill in vitro equine embryo production, further studies such as the seasonal effect, in vitro fertilization etc is need.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.5
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• pp.693-696
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• 2001

The effect of replacement of in vitro maturation medium completely with the buffalo follicular fluid (buFF) on in vitro oocyte maturation of buffalo oocytes was studied. 5 to 8 buffalo cumulus oocyte complexes were cultured in a single drop with each of the eight media studied i.e., M199+steer serum (10% v/v), M199+steer serum (10% v/v)+PMSG, M199+buFF (10% v/v), M199+buFF (10% v/v)+PMSG, M199+buFF (50% v/v), M199+buFF (50% v/v)+ PMSG, buFF (100%) and buFF+PMSG at $39^{\circ}C$ and 5% $CO_2$ in air for 24 h. Supplementation of M199 with Steer serum alone resulted in IVM rate of 35% only. When the above medium was supplemented with PMSG, the maturation rate rallied to 82%. Significant increase in the maturation rates were observed when M199 was supplemented with increasing levels of buFF. A further increase in the maturation rate was also obtained when PMSG was incorporated into the medium of M199 supplemented with buFF. The rate of maturation was to the tune of 91% when oocytes were matured in buFF alone which was increased non significantly on the addition of PMSG. Highest maturation rate (97%) obtained with M199+buFF (50%v/v)+PMSG did not differ significantly from that obtained by either M199+buFF (10%v/v)+PMSG or buFF+PMSG. It is suggested that buFF alone without any supplementation can form the effective in vitro maturation medium for buffalo oocytes.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.3
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• pp.205-211
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• 2019

In this study, we investigated the possibility of using mouse embryonic stem cell conditioned medium (ESCM) and embryonic stem cell medium (ESM) for in vitro maturation in the efficient in vitro production of blastocysts from porcine follicular oocyte. Depending on the concentration of supplement of ESCM added to the NCSU-23 solution did not affect 2-cell development rates and blastocysts development. However, in particular, the survival rate (10 days of culture) of blastocyst was significantly higher than that of the control group as the additive concentration (30%) increased (p < 0.05). The survival rate of blastocysts showed a similar tendency even with addition of ESM (30%) alone. On the other hand, the duration of the addition of these additives during IVM (0-44 h) was that the IVM I period (0-22 h) were more effective than the IVM II period (22-44 h). Thus, the effect of these additives is probably due to the combination of the various physiologically active substances of ESCM or the appropriate amino acids and vitamins of ESM. In particular, these additives were more effective during the first half (IVM I) of in vitro maturation. In summary, optimization of ESCM or ESM supplementation may improve in vitro maturation of porcine oocyte and affect developmental competency. Therefore, if more efficient methods of adding ESCM or ESM to basal culture medium can be developed during in vitro maturation of porcine follicle oocytes, high quality blastocysts will be developed from low porcine follicular oocyte compared to other domestic animals.

• Korean Journal of Veterinary Research
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• v.63 no.4
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• pp.40.1-40.7
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• 2023

To optimize the most efficient method for porcine in vitro maturation (IVM), we compared the effects of supplementing extracellular vesicles (EVs) derived from porcine follicular fluid (pFF). The cumulus oocyte complexes were grouped into 4 groups with different supplementations as following: pFF (G1), pFF-depleted EVs (G2), EVs (G3) and control (G4) groups. After IVM with different supplementations, maturation rates and the developmental competences of porcine oocytes and blastocyst development were investigated. Additionally, glutathione (GSH) and reactive oxygen species (ROS) levels were measured in mature oocytes. The EVs were isolated and characterized with cryo-TEM and nanoparticle tracking analysis. The pFF significantly affected the maturation rate, whereas the presence of EVs did not show notable difference in the maturation rates. Although there were numerical increases in the measured parameters in EV and pFF-depleted EVs groups, no significant differences were observed between them. The EV group showed similar oocyte maturation rate for both positive and negative control groups. The GSH was not different among the groups, but ROS levels were significantly lower in pFF-supplemented group when compared with other groups with the highest level in the control group. G2 group wasn't significantly different G1 and G3 group. G3 group wasn't significantly different from G2 and G4 group. This suggests that EVs in IVM medium which probably effected partially to protect against oxidative stress and potentially enhance the quality of oocytes. This study indicates that the EVs in pFF play a significant role in improving the efficiency of oocyte maturation in porcine.

• Korean Journal of Animal Reproduction
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• v.16 no.4
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• pp.289-299
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• 1993

The polypeptide patterns of cellular and follicular components were analysed by SDS-PAGE and two dimensional(2-D)electrophoresis combined with isoelectric focusing (IEF) to establish protein profiles in each of the components in porcine follicles. Oocyte-cumulus complexes were cultured in M16+FCS+Gn at 39 in an atmosphere of 5% CO$_2$, in air for 35 h. At the end of the culture, the zona-free oocyte, ZP alone and cumulus cells were prepared and analysed either on 10% SDS-PAGE for the protein profile at the first dimensional gel or 2-D protein pattern. The amounts of each samples were determined for the visualization with Coomasie brilliant blue (CBB) or silver staining, thus giving useful information for the identification of specific proteins in the components or appropriate amount of samples for proper visualization. Oocyte showed 25 and 114 kd major protein band. Other minor components were additionally visualized with CBB on the same gel after silver staining procedure. Cumulus cells also showed specific proteins which is not present in the oocytes. The number of cumulus cell was proper to give major bands with CBB and additional minor bands with silver staining. To establish the degree of contamination from the remnant of the corona radiata to the ZP, zonae were differently prepared or analysed by SDS-PAGE.The preparation of the ZP in this study did not showed any contamination judged by the protein profile of the components. Also follicular fluid showed its specific protein profile without any significant differences among the different sizes of follicles. The established protein profile of each follicular component should be helpful for the identification and elimination of contaminated components, i. e., antigen preparation or immunological studies. The results also suggest that the preparation of each components in the study was appropriate and can be used for a further sensitive biochemical analysis in mammalian oocytes and early embryos.

• Reproductive and Developmental Biology
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• v.30 no.2
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• pp.99-105
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• 2006

The growing oocytes become progressively capable of resuming meiosis, and full meiotic competence appear when they are about 80% of the size of fully grown oocytes. As hormonal influences vary at different stages of reproductive cycle, the size of oocytes may vary according to the reproductive stages. The present study was designed to compare the diameter between the ovulated and freshly collected immature canine oocytes. The ovulated oocytes were collected 72 hr after ovulation by oviductal tube flushing by laparotomy under general anesthesia. Immature oocytes were collected by ovarian slicing method. Diameter of all oocytes was measured directly using epiflurescence microscope with a calibrated micro-eyepiece micrometer at ${\times}200$ magnification. The thickness of zona pellucida and diameter of cytoplasm were measured separately and recorded. A total of 2209 zona intact oocytes were collected, among them 628 from anestrus, 675 from follicular, 838 from luteal and 68 by fallopian tubes flushing methods. The average number of oocytes was 104.7, 168.8, 119.7 and 11.3 for anestrus, follicular, luteal and fallopian tubes flushing methods, respectively. The average diameters of the ooplasm and oocyte were significantly varied in different reproductive stages as well as with ovulated oocytes (P<0.05). The average diameter of ooplasm and oocyte was 115.6 and 127.7, 143.0 and 162.0, 134.6 and 150.6, 159.6 and 185.6 for anestrus, follicular, luteal and ovulated oocytes, respectively. Highest number of oocytes with larger diameter could be collected from the follicular and luteal stages. In conclusion, the follicular and luteal ovaries are the best sources of oocytes for canine IVM.