• Journal of Animal Reproduction and Biotechnology
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• v.34 no.2
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• pp.117-122
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• 2019

The objective of this study was to establish an in vitro culture system for ovarian preantral follicles of B6D2F1. First, we optimized the in vitro preantral-follicle culture by culture duration, follicle stimulating hormone (FSH) type, and activin A concentration. Duration of in vitro culture for 9, 11, and 13 days was sufficient for the normal development of preantral follicles to antral follicles. Formation of cumulus cell-oocyte complex (COC) was induced by treatment with human chorionic gonadotropin (hCG; 2.5 IU/mL) and epidermal growth factor (EGF; 5 ng/mL). In addition, metaphase II (MII) oocytes formed during this in vitro culture of preantral follicles. In vitro preantralfollicle culture for 9 days showed higher rates of growth and maturation, thus yielding a greater number of antral follicles, and there were significant differences (p < 0.05) in the number of MII oocytes (that formed from these preantral follicles via differentiation) between the 9-day culture and 11-day or 13-day culture. The follicles cultured for 9 days contained a tightly packed well-defined COC, whereas in follicles cultured for 11 days, the COC was not well defined (spreading was observed in the culture dish); the follicles cultured for 13 days disintegrated and released the oocyte. Second, we compared the growth of the preantral follicles in vitro in the presence of various FSH types. There were no significant differences in the growth and maturation rates and in differentiation into MII oocytes during in vitro culture between preantral follicles supplemented with FSH from Merck and those supplemented with FSH from Sigma. To increase the efficiency of MII oocyte formation, the preantral follicles were cultured at different activin A concentrations (0 to 200 ng/mL). The control follicles, which were not treated with activin A, showed the highest rate of differentiation into antral follicles and into MII oocytes among all the groups (0 to 200 ng/mL). Therefore, activin A (50 to 200 ng/mL) had a negative effect on oocyte maturation. Thus, in this study, we propose an in vitro system of preantral-follicle culture that can serve as a therapeutic strategy for fertility preservation of human oocytes for assisted reproductive medicine, for conservation of endangered species, and for creation of superior breeds.

• The Korean Journal of Zoology
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• v.31 no.2
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• pp.87-94
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• 1988

Korean frogs (R. dybowskii and R. nigromaculara) were collected from chonnam area and their oocyte maturation was induced by using in ultro follicle culture system. Follicles were isolated from the frog ovary and cultured for 24 hr in (amphibian Ringer's soluion AR) at 22 C in the presence or absence of hormones. Follicular cocytes of R. dybowskii were induced to mature (germinal vesicle breakdown, GVBD) by the presence of progesterone, 0.1 $\mu$g/2 ml and that of R. nigromaculata by 1 $\mu$g/2 ml of progesterone. Follicles of the frogs were also responded to (frog pituitary homogenate FPH) in terms of their cocyte maturation. Follicular cocytes of R. dybowskii were induced to mature by FPH at concentration of 0.01 pituitary equivalent/2 ml and that of R.nigromaculata at 0.1 pit equiv./2 ml. The culture time required for the maturation of bath frog follicles was 915 hr. The responsiveness of the follicles of korean frogs to hormones (progesterone or FPH) was nearly the same as that of R. pipiens which are most commonly used amphibians. Particularly, follicular cocytes of R. dybowskii used from February matured spontaneously without stimulation of hormones during in vitro culture. Furthermore, those cocytes were spontaneous- ly ovulted when the ovarian fragments were cultured in a flask.

• Development and Reproduction
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• v.9 no.2
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• pp.123-134
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• 2005

Germ cell development during oogenesis, ovarian maturation and first sexual maturity in female Ruditapes philippinarum were investigated by cytological and histological observations. R. philippinarum is dioecious. During vitellogenesis, the Golgi complex, glycogen particles, and mitochondria were involved in the formation of lipid droplets and lipid granules in the cytoplasm of the early vitellogenic oocyte. In the late vitellogenic oocyte, cortical granules, the endoplasmic reticulum, and mitochondria were involved in the formation of proteid yolk granules in the cytoplasm. At this time, exogenous lipid granular substance and glycogen particles in the germinal epithelium passed into the oocyte through the microvilli of the vitelline envelope. The spawning period was once a year between early June and early October, and the main spawning occurred between July and August when seawater temperature was approximately $20^{\circ}C$. The reproductive cycle of this species can be categorized into five successive stages: early active stage(January to March), late active stage(Februaryto May), ripe stage(April to August), partially spawned stage(May to October), and spent/inactive stage (August to February). Percentages of female clams at first sexual maturity of $15.1{\sim}20.0mm$ in shell length were 52.6%(50% of the rate of group maturity was 17.83mm in length), and 100% for the clams >25.1mm.

• Korean Journal of Ichthyology
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• v.23 no.3
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• pp.179-186
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• 2011

Slime flounder, Microstomus achne is distributed in the coastal waters of Korea, west sea of Japan, BoHai, Yellow sea and East china sea. They are mainly caught by bottom trawl net during winter, from December to March. Sexual maturation of slime flounder were investigated using samples collected from commercial catch in the southern coast of Korea from November, 2006 to March, 2007. The ovary of the slime flounder is a conical bag in shape and is bilateral structure develops lengthily from the posterior of the abdomen to the end of the anal fin. The testis also is bilateral in structure, usually located in small size in the abdomen. In females, the gonadosomatic index (GSI) were peaked in January (12.46), then decreased rapidly thereafter. Female GSI values plummeted to 2.72 in March just after spawning. Male GSI values were peaked in December (2.46) before in the spawning season, then decreased slowly thereafter. The reproductive cycle would be classified into three successive developmental stages : maturation stage (November to January), ripe and spawning stage (December to February), degenerative and resting stage (February to March). Relationships between the fish sizes in total length (TL) and the number of ovarian eggs (F), the body weights (BW) and the number of ovarian eggs were indicated by the exponential equation respectively: F=29.027TL-767.8 (r$^2$=0.7686), F=0.3998BW+24.288 (r$^2$=0.8919).

• Development and Reproduction
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• v.20 no.4
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• pp.315-329
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• 2016

Fas ligand (FasL) and its receptor Fas have been implicated in granulosa cell apoptosis during follicular atresia. Although interferon-gamma (IFN-${\gamma}$) is believed to be involved in the regulation Fas expression in differentiated granulosa or granulosa-luteal cells, the expression of this cytokine and its role in the regulation of the granulosa cell Fas/FasL system and apoptosis during follicular maturation have not been thoroughly investigated. In the present study, we have examined the presence of IFN-${\gamma}$ in ovarian follicles at different stage of development by immunohistochemistry and related their relative intensities with follicular expression of Fas and FasL, and with differences in granulosa cell sensitivity to Fas activation by exogenous agonistic Anti-Fas monoclonal antibody (Fas mAb). Although IFN-${\gamma}$ immunostaining was detectable in oocyte and granulosa cells in antral follicles, most intense immunoreactivity for the cytokine was observed in these cells of preantral follicles. Intense immunoreactivity for IFN-${\gamma}$ was most evident in granulosa cells of atretic early antral follicles where increased Fas and FasL expression and apoptosis were also observed. Whereas low concentrations of IFN-${\gamma}$ (10-100 U/mL) significantly increased Fas expression in undifferentiated granulosa cells (from preantral or very early antral follicles) in vitro, very higher concentrations (${\geq}1,000U/mL$) were required to up-regulate of Fas in differentiated cells isolated from eCG-primed (antral) follicles. Addition of agonistic Fas mAb to cultures of granulosa cells at the two stages of differentiation and pretreated with IFN-${\gamma}$ (100 U/mL) elicited morphological and biochemical apoptotic features which were more prominent in cells not previously exposed to the gonadotropin in vivo. These findings suggested that IFN-${\gamma}$ is an important physiologic intra-ovarian regulator of follicular atresia and plays a pivotal role in regulation of expression of Fas receptor and subsequent apoptotic response in undifferentiated (or poorly differentiated) granulosa cells at an early (penultimate) stage of follicular development.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.30-30
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• 2001

The culture of preantral follicles has important biotechnological implications through its potential to produce the large quantity of oocytes for embryo production, transgenesis research, conservation of rare breed, and a potential source of ovarian genetic material. The present study was conducted to establish the optimal conditions of in vitro culture for intact bovine preantral follicles; and to examine the developmental ability of oocytes derived from the in vitro-grown preantral follicles; and to investigate the effects of various concentrations of FSH and LH on these processes. Bovine preantral follicles (150 $\pm$ 1.2${\mu}{\textrm}{m}$), surrounded by theca cell, were isolated enzymetically and mechanically from ovarian cortical slides in Leibovitz L-15 medium containing 1 mg/$m\ell$ collagens and 0.2 mg/$m\ell$ DNase I and cultured for 25 days in the presence of different concentrations of bovine FSH and LH in $\alpha$MEM medium with insulin, transferrin, and selenite. The survival was tested by frypan Blue and Hematoxylin. The survival and growth rates of follicles were higher in FSH treatment groups than these in control (P<0.001), but there were no significant differences between the LH treatment groups and the control. In 25 days, the survival and growth rates of follicles in FSH and LH treatment group (50%, 300$\pm$1.0${\mu}{\textrm}{m}$) were higher than in FSH treatment group (40%, 244$\pm$0.5${\mu}{\textrm}{m}$) and the control group (25%, 160$\pm$ 1.0${\mu}{\textrm}{m}$). Fifty-five percent of healthy antral follicles were obtained, and 60% of the oocytes complete meiotic maturation to the metaphase II stage. Twenty-two percent of the mature oocytes underwent cleavage, and 9% developed to the blastocyst stage. In this study, in vitro-grown oocytes (111 $\pm$ $1.5mutextrm{m}$), under our culture conditions, were not equivalent in size to the in vivo-grown oocytes (130$\pm$1.3${\mu}{\textrm}{m}$). Therefore, these results suggest that bovine preantral follicles with intact theca cell can grow to the antral stage in 25days, and that oocytes from those follicles can acquire the meiotic competence and normally undergo fertilization and development to the blastocyst stage. However, the developmental capacity of in vitro-grown oocytes is presumably not comparable to those of the in vivo counterparts.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.33 no.3
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• pp.219-224
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• 2000

Reproductive biology of the naked-headed goby, Faronigobius gymnauchen was investigated by means of histological methods. The ovary was consisted of several ovarian lamellae and the oogonia originated from the inner surface of the ovarian lamella. The testis was seminiferous tubule One in internal structure. Seminiferous tubule was consisted of many testicular cysts which contained numerous germ cells in a same developmental stage. The size of group maturity was 4.5 cm intotal length. Gonadosomatic index(GSI) of the female and male was the highest in June and July, respectively. Reproductive cycle could be classified into the growing ($January{\~}March$), maturation ($April{\~}May$), ripe and spent (June{\~}July$), and recovery and resting ($August{\~}December$). Oocyte development was group-synchronous, and yolk nucleus was observed in the early growing oocyte.

• The Journal of Korean Obstetrics and Gynecology
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• v.24 no.1
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• pp.27-41
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• 2011

Objectives: This study was designed to investigate the effect of Ojeok-san(OJS) on the estradiol valerate(EV)-induced polycystic ovaries(PCO) in rats. Methods: PCO was induced by single intramuscular injection with estradiol valerate(EV)(4mg) in female rats. Normal group(n=8) were injected with sesame oil and orally administrated distilled water for sixty days. PCO control group (n=8) were injected with EV and orally administrated distilled water for sixty days. OJS treated group(n=8) were injected with EV and orally administrated OJS for sixty days. Then, we measured weights of body, ovaries, adrenal glands and uterus of rats. The histomorphometrical changes of ovaries were also evaluated. And we observed the NGF and CRF expression by immunohistochemistry. Results: The results were as follows - The weights(mg) of ovaries in OJS treated group($57.4{\pm}9.4$) were significantly increased(p < 0.05) compared with PCO control group($42.3{\pm}8.5$). - The numbers of mature follicles in OJS treated group($9.3{\pm}2.5$) were significantly increased(p < 0.05) compared with PCO control group($6.1{\pm}2.1$). - The expressions of NGF-immunoreactive cells in the ovarian granulosa cells in OJS treated group were weaker than PCO control group. Conclusion: From the these results, we concluded that Ojeok-san(OJS) has inhibitory effect on the development of EV-induced polycystic ovary. And it's effect maybe related with decreased NGF activities in the ovary.

• Journal of Veterinary Science
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• v.23 no.1
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• pp.3.1-3.12
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• 2022

Background: Domestic yaks are the most important livestock species on the Qinghai-Tibetan Plateau. Adult female yaks normally breed in the warm season (July to September) and enter anestrous in the cold season (November to April). Nevertheless, it is unclear how ovarian activity is regulated at the molecular level. Objectives: The peculiarities of yak reproduction were assessed to explore the molecular mechanism of postpartum anestrus ovaries in yaks after pregnancy and parturition. Methods: Sixty female yaks with calves were observed under natural grazing in Haiyan County, Qinghai Province. Three yak ovaries in pregnancy and postpartum anestrus were collected. RNA sequencing and quantitative proteomics were employed to analyze the pregnancy and postpartum ovaries after hypothermia to identify the genes and proteins related to the postpartum ovarian cycle. Results: The results revealed 841 differentially expressed genes during the postpartum hypoestrus cycle; 347 were up-regulated and 494 genes were down-regulated. Fifty-seven differential proteins were screened: 38 were up-regulated and 19 were down-regulated. The differential genes and proteins were related to the yak reproduction process, rhythm process, progesterone-mediated oocyte maturation, PI3K/AKT signaling pathway, and MAPK signaling pathway categories. Conclusions: Transcriptome and proteomic sequencing approaches were used to investigate postpartum anestrus and pregnancy ovaries in yaks. The results confirmed that BHLHE40, SF1IX1, FBPX1, HSPCA, LHCGR, BMP15, and ET-1R could affect postpartum hypoestrus and control the state of estrus.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.42 no.1
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• pp.41-47
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• 2009

The seasonal reproductive cycle of Gomphina melanaegis collected in the coastal area of Jumunjin, between April 2006 and March 2007, was analyzed. Maturation cycle parameters such as the gonad index (GI), ovarian egg diameter, frequency of developmental stages, protein content, and RNA/DNA variation in the gonads were analyzed monthly for the 40 samples. According to the indices from histological sections, the frequency of gonad developmental stages, and the oocyte diameter, this clam has a long-term partial spawning pattern from March to October. However, GI and nucleic acid values showed that the mature stage is from March to July and that the main spawning season is August. The peak RNA and DNA contents were good indicators of sexual maturation in females and males, respectively. The variation in protein content corresponded with the RNA/DNA ratios.