• Animal Bioscience
• /
• 제37권6호
• /
• 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.

• Asian-Australasian Journal of Animal Sciences
• /
• 제25권5호
• /
• pp.629-634
• /
• 2012

This study was conducted to improve the yield of mature oocytes from in vitro-culture of ovarian primary follicles by optimizing follicle retrieval from neonatal mice of different ages. Primary follicles of 75 to $99{\mu}m$ in diameter were collected daily from 7- to 14-day-old neonatal mice, and subsequently cultured in ${\alpha}$-MEM medium. Number of primary follicles isolated, growth of the follicle during in vitro-culture and maturation of intrafollicular oocytes were monitored. Overall, mean number of preantral follicles per animal was improved from 10.7 to 88.7 as the age of follicle donors was increased from 7 to 14-day-old. Number of primary follicles was increased gradually up to 11-day-old (35.7 follicle per an animal), then reduced to 29 in 14-day-old (p = 0.0013). More follicles retrieved from 10-day-old or 11-day-old females maintained their morphological normality at the end of primary culture than the follicles retrieved from 9-day-old. Of those cultured, primary follicles retrieved from 11-day-old mice yielded largest larger number of early secondary follicles than the follicles retrieved from in the other ages (39 vs. 13 to 29%). More than 3.3-times increase (0.86 to 2.86; p<0.05) in an average number of mature oocytes per animal was observed in the group of 11-day-old, compared with 9-day-old. However, no difference was found in the percentage of primary follicles developing into the pseudoantral stage (21 to 30%; p = 0.5222) and in the percentage of oocytes mucified (32 to 39%; p = 0.5792). In conclusion, a positive correlation between retrieval time and follicle growth was detected, which influences the efficiency to derive mature oocytes by follicle culture.

• 한국수산과학회지
• /
• 제28권6호
• /
• pp.736-752
• /
• 1995

Fine structural changes of germ cell during the gametogenesis of Korean rockfish, Sebastes schlegeli sampled in west coast of Korea were investigated from September 1993 to August 1994. In a layer of microvilli of oocyte with active yolk duplication, many pinocytotic vesicles containing protein granules regarded as yolk precursors were observed. The multivesicular bodies were formed by gathered mitochondria. They are participated in formation of the primary yolk globules homogeneously filled with high dense particles and enclosed within a limiting membrane. The precursors of yolk globule appeared to be formed by modification of mitochondria and they developed into the primary yolk globules with participation of large and dense pinocytotic vesicles. Yolk globules in mature oocyte were consisted of three components: the crystalline type main body, the superficial layer with dense and fine granules, and the limiting membrane. Steroid hormone secreting cells were recognized in the interstitial cells of growing testis. Numerous endoplasmic reticula and large mitochondria with well developed tubular cristae appeared in their cytoplasms. The axoneme in the tail flagellum of spermatozoon consisted of nine pairs of microtubules at the periphery and one pair at the center, and they were covered with doublet microtubules.

• 한국가축번식학회지
• /
• 제15권3호
• /
• pp.207-220
• /
• 1991

This study was carried out to investigate the ultrastructural changes of ooplasm and follicular membrane of oocytes, obtained from 150 of 3-year-old female rainbow trout(Oncorhynchus mykiss). All data were collected from March in 1989 to February in 1990, and from August to October in 1991. The size of the nucleoli and number of the yolk granules increased as the oocyte growed. Yolk granules were deposited in the oocyte as crystalline granules. Due to the presence of large early and late maturing oocytes, their ovaires were enlarged, transparent and granular. The lattice was broken down at hydration, leaving the egg transparent. As thepercentages of fish in LPO and EMO stage increased from September to October, Mean GSI values increased. Follicle cells such as granulosa cell and thecal cell change a squamous into cuboid shape in LPO and EMO stage. Seasonal changes in the microscopic appearance of the ovaries were well correlated with those in bothgonadosomatic index and macroscopic apearance. Under the natural conditions,t he ovarian follicle influences the histological development and periodical secretion of the hormones, sufficient for a oogenesis and gonadal steroid production. The electrophoretic pattern of major band in mature stage was much thicker(70∼110k dalton) than that in previtellogenic phase.

• 한국동물학회지
• /
• 제31권2호
• /
• pp.87-94
• /
• 1988

전라남도 일대에서 서식하는 북방상개구리(R. dybowskii)와 참개구리(R. nigromaculara)를 채집하는 생체외배양에 의한 여포난자의 성숙을 유도하였다. 북방산개구리의 여포난자는 배양액(amphibian Ringer's soluion AR)에 첨가한 progesterone, 0.1 $\mu$g/2 ml에 의해 난자의 성숙(핵붕괴)이 유도 되었으며 참개구리의 난자는 1 $\mu$g/2 ml (frog pituitary homogenate FPH)을 얻어서 그 효과를 조사해본 결과 북방산개구리에서는 0.01 pituitary equivalent/2 ml에서, 참개구리는 0.1 pit equiv./2 ml에서부터 여포난자의 성숙이 일어났다. 난자의 성숙에 요하는 시간은 두 개구리에서 모두 9-15시간이었으며 호르몬에 대한 반응성, 성숙기간 등은 개구리 재료로 가장 많이 사용되는 법개구리(R. pipiens)와 거의 일치하였다. 특이하게 2개월 이후에 사용한 북방산개구리의 여포난자는 호르몬의 도움없이도 성숙이 일어났으며 성숙기간도 3시간으로 매우 빨라졌다. 난소조각을 배양했을 때 자발적으로 성숙을 일으키는 여포들은 자발적인 배란까지도 일으키는 것을 발견하였다.

• 대한수의학회지
• /
• 제63권4호
• /
• pp.40.1-40.7
• /
• 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.

• 한국수산과학회지
• /
• 제32권2호
• /
• pp.198-203
• /
• 1999

1996년 4월부터 1997년 4월까지 강원도 주문진 연안에서 채집한 민들조개 (Gomphina melanaegis)의 생식소 발달과정과 생식주기를 조사하였다. 민들조개는 자웅이체로서, 생식소는 소화맹낭과 족부 근육 사이에 위치하였으며, 난소와 정소는 각각 수많은 난소소낭과 정소세관으로 이루어져 있었다. 육중량비는 1994년 8월에 $23.0\%$로 가장 높았으나 9월에 $19.8\%$로 급격히 감소하였으며, 이후 이듬해 3월에 가장 낮은 값을 보인 후 다시 증가하였다. 성숙기의 난모세포 크기는 $50\~60\mu$m로 전자밀도가 높은 인을 가진 핵이 이중의 단위막으로 둘러싸여 있으며 세포질에는 다량의 난황과립과 지방과립 및 미토콘드리아가 분포하였다. 민들조개의 정자는 머리, 중편 및 꼬리로 구성되어 있었다. 치밀한 핵질로 충만한 원추형 머리는 그 선단에 첨체구조를 가지고 있으며, 중편부에는 4개의 미토콘드리아와 원단중심소체가 편모와 연결되어 있었다. 꼬리의 편모는 전형적인 9+2 구조를 나타냈다. 암수의 성비는 1 : 0.79였고, 생물학적 최소형은 각장 25.0 mm였다. 생식주기는 분열증식기 (12$\~$3월), 성장기 (4$\~$5월), 성숙기 (6월), 산란기 (7$\~$8월) 및 휴지기 (9$\~$11월)의 연속적인 5단계로 구분되었다.

• Clinical and Experimental Reproductive Medicine
• /
• 제11권2호
• /
• pp.59-67
• /
• 1984

In vitro fertilization have been performed to know whether the frozen semen has fertilizing ability and can be used clinically. The results of cultured and developed embryos obtained are as follows: 1. The semen was frozen in three media for the good viability. The viability was more than 50% and the motility was also moderate (grade III), 2. As the 33 oocytes were collected from 45 follicles, the oocyte recovery rate was 73.3%. Among them, mature and immature ova were 5% each, and premature ova were 69.7%, When the first polar body was appeared, above ova were inseminated after adequate incubation with activated sperms. 3. The main components of three freezing medium containing egg yolk, glycerol and pyruvate respectively were the best for sperm viability, and Ham's F-10 medium was used for the fertilization and culture of eggs. 4. The results of in vitro fertilization of 33 ova, showed the second polar body developed in 12%, polyspermia in 24%, 1-cell embryo in 21% and 2-cell embryo in 9%. One mature ova developed to blastocyst via 16-cell to 32-cell embryo. The fertilization rate was 66%. 5. Above mentioned results represent that the frozen semen has fertilizing ability and can be used practically in the clinic.

• 한국발생생물학회지:발생과생식
• /
• 제19권1호
• /
• pp.33-41
• /
• 2015

The gonadosomatic index (GSI), gonadal development and changes in hormones in plasma level of the indoor cultured grunt (Hapalogenys nitens) were investigated by histological study from August 2011 to October 2012. The GSI showed similar trends with gonad developmental stages during the culture periods. Changes in plasma level of estradiol-$17{\beta}$ of female H. nitens reached the highest value before the spawning period, and seasonal changes in plasma level of estradiol-$17{\beta}$ were similar in trends of oocyte developments and GSI changes. Testosterone levels of male H. nitens reached the highest value before and after the spent stage. Ovarian developmental stages of H. nitens could be classified into early growing stage, late growing stage, mature stage, ripe and spawning stage, recovery and resting stage. The testicular developmental stages could be divided into growing stage, mature stage, ripe and spent stage, and recovery and resting stage.

• 한국수산과학회지
• /
• 제40권3호
• /
• pp.141-146
• /
• 2007

We investigated the reproductive cycle of the striped eel catfish Plotosus lineatus. Specimens were sampled in the coastal waters of Sungsan and Jocheon, east of Jeju, Korea, from September 2001 to August 2002. The gonadosomatic index (GSI) in females and males were reached a maximum in June $(14.18{\pm}10.38)$ and July $(0.35{\pm}0.12)$, respectively. Based on histological observations and GSI variation, the reproductive cycle was classified into the following successive stages: in females, growing (September-April), mature (April-June), spawning (June-July), and degenerative and recovery (July-September) and in males, growing (August-December), mature (September-May), spent (April-July), and degenerative and recovery (June-August). The total length at 100% maturity exceeded 15 cm and the fecundity ranged from 525 to 1176 eggs. The fecundity was correlated positively with total length and body weight. Histological observations of the gonads suggest that this species is a spring-summer spawner with group-synchronous type oocyte development.