• Journal of Animal Reproduction and Biotechnology
• /
• v.37 no.1
• /
• pp.34-41
• /
• 2022

In vivo oocytes grow and mature in ovarian follicles whereas oocytes are matured in vitro in plastic culture dishes with a hard surface. In vivo oocytes show a superior developmental ability to in vitro counterparts, indicating suboptimal environments of in vitro culture. This study aimed to evaluate the influence of an agarose matrix as a culture substrate during in vitro maturation (IVM) on the development of pig oocytes derived from small antral follicles (SAFs). Cumulus-oocyte complexes (COCs) retrieved from SAFs were grown in a plastic culture dish without an agarose matrix and then cultured for maturation in a plastic dish coated without (control) or with a 1% or 2% (w/v) agarose hydrogel. Then, the effect of the soft agarose matrix on oocyte maturation and embryonic development was assessed by analyzing intra-oocyte contents of glutathione (GSH) and reactive oxygen species (ROS), expression of VEGFA, HIF1A, and PFKP genes, and blastocyst formation after parthenogenesis. IVM of pig COCs on a 1% (w/v) agarose matrix showed a significantly higher blastocyst formation, intra-oocyte GSH contents, and transcript abundance of VEGFA. Moreover, a significantly lower intra-oocyte ROS content was detected in oocytes matured on the 1% and 2% (w/v) agarose matrices than in control. Our results demonstrated that IVM of SAFs-derived pig oocytes on a soft agarose matrix enhanced developmental ability by improving the cytoplasmic maturation of oocytes through redox balancing and regulation of gene expression.

• Animal Bioscience
• /
• v.37 no.6
• /
• pp.1007-1020
• /
• 2024

Objective: We increased the nuclear maturation rate of antral follicle derived oocytes by using a pre-in vitro maturation (IVM) culture system and improved the developmental potential of these porcine pathenotes by supplementing with melatonin. Furthermore, we investigated the expression patterns of genes involved in cumulus expansion (HAS2, PTGS2, TNFAIP6, and PTX3) derived from small and medium antral follicles before and after oocyte maturation. Methods: Only the cumulus oocyte-complexes (COCs) derived from small antral follicles were induced with [Pre-SF(+)hCG] or without [Pre-SF(-)hCG] the addition of human chorionic gonadotropin (hCG) during the last 7 h of the pre-IVM period before undergoing the regular culture system. The mature oocytes were investigated on embryonic development after parthenogenetic activation (PA). Melatonin (10^-7 M) was supplemented during in vitro culture (IVC) to improve the developmental potential of these porcine pathenotes. Results: A pre-IVM culture system with hCG added during the last 7 h of the pre-IVM period [Pre-SF(+)hCG] effectively supported small antral follicle-derived oocytes and increased their nuclear maturation rate. The oocytes derived from medium antral follicles exhibited the highest nuclear maturation rate in a regular culture system. Compared with oocytes cultured in a regular culture system, those cultured in the pre-IVM culture system exhibited considerable overexpression of HAS2, PTGS2, and TNFAIP6. Porcine embryos treated with melatonin during IVC exhibited markedly improved quality and developmental competence after PA. Notably, melatonin supplementation during the IVM period can reduce and increase the levels of intracellular reactive oxygen species (ROS) and glutathione (GSH), respectively. Conclusion: Our findings indicate that the Pre-SF(+)hCG culture system increases the nuclear maturation rate of small antral follicle-derived oocytes and the expression of genes involved in cumulus expansion. Melatonin supplementation during IVC may improve the quality and increase the blastocyst formation rate of porcine embryos. In addition, it can reduce and increase the levels of ROS and GSH, respectively, in mature oocytes, thus affecting subsequent embryos.

• Journal of Embryo Transfer
• /
• v.24 no.3
• /
• pp.151-157
• /
• 2009

Serial ultrasonography was conducted on Miniature Schnauzer bitches, on purpose to observe the ultrasonographic appearance of normal ovaries and ovarian structures during the estrous cycle. The size of ovaries was increased from $76.8{\pm}7.5mm^2(Mean{\pm}S.D)$ on Day-12 (Day-0=ovulation day) to $114.4{\pm}5.5mm^2$ on Day-8 and there was no significantly different between both ovaries. The ovaries were recognized by its proximity to the caudal renal pole and appeared moderately echogenic oval shape with a smooth contour. The size of follicles was increased from $8.1{\pm}4.5mm^2$ on Day-12 to $114.4{\pm}5.5mm^2$ on Day-0 and there was no significantly different between both ovaries. The number of follicles was increased from $2.8{\pm}0.7$ on Day-12 to $1.1{\pm}0.1$ on Day-0 and there was no significantly different between both ovaries. The follicles were small anechoic fluid-filled structures in early of proestrus, more increased, and indistinguished from each follicles in late of proestrus. The size of corpora lutea was increased from $19.3{\pm}2.1mm^2$ on Day-0 to $26.4{\pm}8.1mm^2$ on Day-8 and there was no significantly different between both ovaries. The number of corpora lutea was increased from $1.4{\pm}0.6$ on Day-0 to $2.9{\pm}0.4$ on Day-38 and there was no significantly different between both ovaries. The corpora lutea were small anechoic cavity and thin hyperechoic wall in early of diestrus, became more hyperechoic, and increased homogenous structures. The results of this study would be useful for differential diagnosis between normal and abnormal structures of ovaries.

• Asian-Australasian Journal of Animal Sciences
• /
• v.11 no.5
• /
• pp.480-485
• /
• 1998

Application of trans rectal ultrasonography to buffaloes (n=6) to follow the growth of large antral follicles individually, on each day of one interovulatory interval revealed that follicular turnover during oestrous cycle occured in waves. There was a predominance of a two-wave pattern (5/6 animals) compared to that of a three-wave pattern (1/6 animals). For two-wave pattern, the first wave emerged at Day $0.20{\pm}0.19$ (Day 0 = day of ovulation) and was marked by development of a dominant anovulatory follicle which grew in size from $5.40{\pm}0.24mm$ at the day of detection to a maximum diameter of $12.40{\pm}0.81mm$ on Day $8.60{\pm}1.57$, with a growth rate of $0.88{\pm}0.17mm/day$ and then regressed, with a mean persistence of $19.40{\pm}1.54$ days. The second wave emerged at Day $9.20{\pm}1.06$ and was marked by development of a dominant ovulatory follicle which grew in size from $4.20{\pm}0.37mm$ at the day of detection to a maximum diameter of $13.80{\pm}0.37mm$ on Day $21.00{\pm}1.38$, with a growth rate of $0.66{\pm}0.12mm/day$ and then ovulated on Day $21.60{\pm}1.25$, with a mean persistence of $11.80{\pm}1.39$ days. The maximum diameters attained and the growth rates of dominant anovulatory and dominant ovulatory follicles, and the mean number of follicles ${\geq}3mm$ diameter detected at the time of emergence of first and second waves ($11.80{\pm}1.74$ and $9.00{\pm}2.81$, respectively) were not significantly different. In the animal which showed a three-wave pattern, the first, second and third waves emerged on Days 1, 10 and 19, respectively. All animals, except one had at least one subordinate follicle in the first or second or both waves. The subordinate follicles increased in diameter over a few days and then regressed. The results indicate that in buffaloes, the follicular turnover during oestrous cycle occurs predominantly in a two-wave pattern.

• Asian-Australasian Journal of Animal Sciences
• /
• v.11 no.4
• /
• pp.404-409
• /
• 1998

Nine cows were superovulated by administration of 8 injections of Folltropin each (2.5 ml/injection, 1.75 mg/ml) i.m spread over 4 days, beginning on Day 10 of oestrous cycle, and 30 and 20 mg prostaglandin $F_{2{\alpha}}$ was given along with the 5th and 6th injections of Folltropin, respectively, to induce luteolysis. The animals were artificially inseminated 48, 60 and 72 h after the first prostaglandin $F_{2{\alpha}}$ injection. The number of corpora lutea was recorded by palpation per rectum and by ultrasonography on Day 6 (Day 0 = day of oestrus). The ovaries were examined daily by ultrasonography on Days 3-9 of the oestrous cycle for following the growth and regression of the largest follicle, which was considered the morphologically dominant follicle. The animals were classified into two groups depending upon the presence (n = 4) and absence of a dominant follicle (n = 5). There was a high correlation (r = 0.97, p < 0.001) between the number of corpora lutea observed by palpation per rectum and that determined by ultrasonography. Mean (${\pm}SEM$) number of corpora lutea determined by ultrasonography ($11.20{\pm}3.71$ vs $3.25{\pm}0.75$) and by palpation per rectum ($10.40{\pm}3.91$ vs $2.25{\pm}0.75$) was significantly higher (p < 0.05) in the nondominant group compared to that in the dominant group. There was no difference in the numbers of follicles 2-3 mm ($13.80{\pm}4.49$ vs $8.00{\pm}1.08$), 4-6 mm ($7.00{\pm}1.87$ vs $3.50{\pm}1.33$), and the total number of follicles ${\geq}2mm$ ($22.00{\pm}5.95$ vs $12.50{\pm}1.26$) between the two groups, one day prior to initiation of superovulation. There was, however, a significant (p<0.01) positive correlation between the number of corpora lutea with the numbers of follicles 2-3 mm (r = 0.83), 4-6 mm (r = 0.80) and the total number of follicles ${\geq}2mm$ (r = 0.89) observed one day prior to initiation of superovulation. The results of this study indicate that the presence of a dominant follicle adversely affects the superovulatory response in cattle.

• The Korean Journal of Zoology
• /
• v.32 no.3
• /
• pp.281-289
• /
• 1989

The present study was disigned to determine the concentration of bioavailable steroid hormones in the atretic follicular fluid (FF). The concentradons of progesterone (P), testosterone (T), estradiol (E), androstenedione (A), and 5-$\alpha$ dihydrotestosterone (DIlT) were determined by the established methods of luminescent immunoassay (LIA) or radioimmunoassay (RIA). Concentrations of T, A and Diff in human FF from smail (< 6 mm). medium (8-15 mm), and large (> 15 mm) atretic follicles were significandy higher than those of normal ones (p < 0.01). However, the levels of T, A and DHT in smail atretic foflicle were significandy lower than those found in normal one. The concentrations of P in atretic FF from porcine small (< 3 mm), medium (4-6 mm), and large (> 7 mm) follicles were not different from that of normal ones. However, the concentration of E in atretic forncles of each group was significantly lower than that of normal group (p < 0.001 in each group). On the other hand, the percentages of bioavailable T (BI) in human FF were significandy (p <0.001) higher than those in normal groups. The BT in normal or atretic FF was more than 90 % of total T. The present result demonstrates that the bioavailable androgen, but not E levels in atretic follicles is higher than that of normal one, and that the atretic mechanism might be dependent on the ovarian forncle size in the developmental stage and on the animal model system. Moreover, the present study suggests that the steroids found in the FF are the bioavailable forms and the concentration of BT in FF could be used as one of the valuable criteria classifying the ovarian atretic follicle.

• The Journal of Korean Obstetrics and Gynecology
• /
• v.24 no.3
• /
• pp.48-72
• /
• 2011

Objectives: This study was designed to investigate the effects of Yongdamsagantang on the polycystic ovary(PCO) induced by estradiol valerate(EV) in rats. Methods: After administrating Yongdamsagan-tang to PCO induced rats, we measured the weight of body, ovaries, adrenal glands, and uterus of rats. The observation through naked eye and histopathological observation of ovaries were evaluated. Also, the number of follicle and corpora lutea and content of androstenedione(ADD) and total estrogen were evaluated. The expressions of nerve growth factor(NGF) and corticotropin releasing factor(CRF) were analyzed by immunohistochemistry. The breeding rate and number of implantation with normal male rats were evaluated. Results: - The weight(mg) of ovaries in YST treated group($73.8{\pm}7.6$) was significantly increased(p<0.001) compared with control group($54.3{\pm}4.5$). - The number of mature follicles in YST treated group($7.3{\pm}2.4$) was significantly increased(p<0.01) compared with control group($3.5{\pm}1.2$). - The number of atretic follicles in YST treated group($9.0{\pm}1.5$) was significantly decreased(p<0.01) compared with control group($13.4{\pm}3.8$). - The number of cystic follicles in YST treated group($3.1{\pm}1.1$) was significantly decreased(p<0.01) compared with control group($6.0{\pm}2.0$). - The number of corpora lutea in YST treated group($3.8{\pm}2.1$) was significantly increased(p<0.001) compared with control group($0.3{\pm}0.7$). - The expression of NGF-immunoreactive cells in the ovarian granulosa cells in YST treated group was lesser observed than control group. - The expression of NGF-immunoreactive cells in the adrenal cortex in YST treated group was lesser observed than control group. - The breeding rate in YST treated group(100 %) was significantly increased (p<0.05) compared with control group(50 %). - The number of implantation in YST treated group($6.4{\pm}4.7$) was significantly increased(p<0.05) compared with control group($1.4{\pm}2.6$). Conclusions: We concluded that Yongdamsagan-tang activates the maturation of follicles, normal ovulation, breeding rate and number of implantation. Therefore, this may be effective for the treatment of anovulation, amenorrhea and sterility of PCOS patients.

• Korean Journal of Veterinary Research
• /
• v.8 no.2
• /
• pp.110-116
• /
• 1968

The mature guinea pigs were grouped as indicated in the table 1. Radio-active iodine(I-131)in dose of 4.5mci, was administered to the experimental groups. The animals were killed for examination in 1, 7, 14, 28, 42, and 55 days after the administration the radio-active iodine. The thyroid and adrenal glands were observed histologically. The results obtained were as follows; 1. One day after the administration, thyroid epithelial cells were abnormally enlarged. After seven days, specimens taken from the middle of the thyroid showed that the follicles and epithelial cells were changing to fibrous tissue, however, some follicles still remained in the verge of the thyroid. Follicles were not observed after fourteen days. After twenty-eight days, the follicles had all changed to fibrous tissue, and had lost their function. 2. The size of the zonas gromerulosa of adrenal cortex epually, in both male and female, showed slight fluctuation in size with no tendency to be changed. 3. Among the zones of the adrenal glands, zona fasciculata showed marked changes. Zona fasciculata was atrophied in Process of time. In females, it was atrophied significantly(P<0.05) after fourteen days, and highly significant (P<0.01) in twenty-eight and fifty-six days after the administration of radioactive iodine. In males, it also decreased significantly(P<0.05) in seventy-eight days and highly significant(P<0.01) in forty-two and fifty-six days after the administration. 4. The size of the Zona reticularis of adrenal cortex in the females increased significantly (P<0.05) in twenty-eight days after the administration. In males, it showed slight fluctuation until twenty-eight days, but it increased significantly(P<0.05) in forty-two and fifty-six days after the administration. 5. The size of the adrenal medulla increased significantly(P<0.05) in twenty-eight and forty-two days in females. It was increased significantly(P<0.05) in fourth-two days and high significantly(P<0.01) in fifty-six days after the administration.

• Development and Reproduction
• /
• v.7 no.2
• /
• pp.127-136
• /
• 2003

The present study was conducted to investigate gene expression profile of mouse ovaries during the primordial-primary follicle transition. We isolated total RNA from mouse ovaries at day1(contains only primordial follicles) and day5(contains both primordial and primary follicles) and synthesized cDNA using annealing control primers(ACP, Seegene, Inc., Seoul, Korea). Using 80 different ACPs for PCR, we cloned, sequenced, and analyzed identities of 41 differentially expressed genes(DEGs). According to BLAST analysis, sequences of 33 clones significantly matched database entries, 4 clones were novel, and 4 clones were ESTs. We selected 8 DEGs with interesting functions, Anx11 and Pepp2-Pending highly expressed in day1 ovary, while Apg3/Autlp-like, BPOZ, Ches1, Kcmf1, NHE3, Nid2, Ninj1, SENP3, Suil-rsl, and TIAP/m-survivin highly expressed in days ovary, and confirmed their different expression between day1 ovaries and days ovaries using semi-quantitative RT-PCR. There was no false positive result. Using in situ hybridization, we found that almost all of genes studied were expressed in the oocyte from primordial follicle stage but expression decreased from primary follicle stage. Meanwhile their expression was increased in cuboidal granulosa cells. Different expression of BPOZ and TIAP/m-survivin between primordial and primary follicles was confirmed by using laser capture microdissection followed by real-time PCR BPOZ and TIAP/m-survivin expressed 4.5 and 3.4 fold higher in primary than primordial follicles, respectively. List of genes obtained from the present study will provide insights for the study of mechanism regulating primordial-primary follicle transition.

• Journal of Radiation Protection and Research
• /
• v.24 no.1
• /
• pp.17-22
• /
• 1999

This study was carried out to evaluate the biochemical and morphological effects of ionizing radiation on mouse ovarian follicles. Immature mice (ICR, 3 week-old) were irradiated with a dose of $LD_{80(30)}$ at KAERI. The ovaries were collected after 6 hours, 12 hours, 1 day, and 2 days post irradiation. With the morphological basis of the histological staining with hematoxylin-eosin, immunohistochemical preparation using in situ 3'-end labeling was evaluated. Flowcytometric evaluation of DNA extracted from the whole ovary was performed. The percentage of $A_0$ (subpopulation of cells with degraded DNA and with lower DNA fluorescence than $G_0/G_1$ cells), apoptotic, cells in the cell cycle was significantly higher in the irradiated group than in the control group. The number of in situ 3'-end labeled follicles increased at 6 hours post irradiation. All the analyses represented that the ionizing radiation-induced follicular atresia was taken place via an apoptotic degeneration. Such a degeneration underwent very fast and acutely. Therefore, it is concluded that the radiation-induced follicular degeneration is, like the spontaneous atresia, mediated by an acute apoptosis of follicular granulosa cells. Flowcytometric evaluation of cell cycles can make the role for quantifying the atretic follicles and understanding the mechanism of the radiation-induced cell death.