• Asian-Australasian Journal of Animal Sciences
• /
• v.33 no.4
• /
• pp.547-555
• /
• 2020

Objective: Apoptosis of ovarian granulosa cells (GCs) affects mammalian follicular development and fecundity. This study aimed to explore the regulatory relationship between microRNA-26a (miR-26a) and the 3β-hydroxysteroid-Δ24-reductase gene (DHCR24) gene in porcine follicular granular cells (pGCs), and to provide empirical data for the development of methods to improve the reproductive capacity of pigs. Methods: The pGCs were transfected with miR-26a mimic, miR-26a inhibitor and DHCR24-siRNA in vitro. The cell apoptosis rate of pGCs was detected by the flow cytometry. The secretion levels of estradiol (E2) and progesterone (P) in pGCs were detected by enzyme-linked immunosorbent assay. Double luciferase validation system was used to detect the binding sites between miR-26a and DHCR24 3'-UTR region. Qualitative real-time polymerase chain reaction and Western blotting were used to verify the DHCR24 mRNA and protein expression in pGCs, respectively, after transfecting with miR-26a mimic and miR-26a inhibitor. Results: Results showed that enhancement of miR-26a promoted apoptosis, and inhibited E2 and P secretion in pGCs. Meanwhile, inhibition of DHCR24 also upregulated the Caspase-3 expression, reduced the BCL-2 expression, promoted pGCs apoptosis, and inhibited E2 and P secretion in pGCs. There were the binding sites of miR-26a located within DHCR24 3'-UTR. Up-regulation of miR-26a inhibited DHCR24 mRNA and protein expression in pGCs. Conclusion: This study demonstrates that miR-26a can promote cell apoptosis and inhibit E2 and P secretion by inhibiting the expression of DHCR24 in pGCs.

• Clinical and Experimental Reproductive Medicine
• /
• v.30 no.4
• /
• pp.333-340
• /
• 2003

Objective: This study was performed to examine the maturation and the development to the blastocyst stage of immature oocytes collected from patients with high risk of ovarian hyperstimulation syndrome (OHSS). Materials and Methods: Cumulus-oocyte complexes (COCs) were collected following only HCGpriming for non stimulated IVF-ET cycles of the patients. At the time of oocyte collection, COCs were classified into three groups in accordance with their appearance (Group I: oocytes with dispersed cumulus cells; Group II: oocytes with compacted cumulus cells; Group III: oocytes with sparse cumulus cells). The in vitro maturation and blastocyst development rates of the COCs were compared among these groups. From August 2001 to June 2002, 48 IVM/IVF-ET cycles from 42 patients (mean age: $32.4{\pm}3.8$ years) were performed. To prevent the occurrence of OHSS, the patients were primed with 10, 000 IU HCG alone 36 h before oocyte collection without gonadotropin stimulation. Oocytes were aspirated on cycle days from 7 to 13. The normal COCs were classified into three groups according to their appearance. The aspirated immature oocytes were cultured in YS maturation medium containing 30% (v/v) human follicular fluid (HFF), 1 IU/ml FSH, 10 IU/ml HCG and 10 ng/ml rhEGF. Fertilization was induced by intracytoplasmic sperm injection (ICSI). All zygotes were co-cultured with cumulus cells in $10{\mu}l$ YS medium containing 10% HFF until day 7 after oocyte collection. Blastocyst transfer was performed on day 5 after ICSI. Results: Th e mean number of oocytes cultured in the IVM/IVF cycles was $24.7{\pm}10.6$. Of 1185 COCs, those assigned to Group I, II and III were 470 (39.7%), 414 (35.0%) and 301 (25.4%), respectively. The maturation rate (94.5%, 444/470, p<0.05) in Group I was significantly higher than those of Group II (62.8%, 260/414) and Group III (73.1%, 220/301). Especially, 30.9% of COCs in Group I (145/470) was matured on the day of oocyte aspiration. There were no differences in the rates of fertilization and cleavage among the three groups. The development rate to the blastocyst stage in Group I (54.6%, 206/377, p<0.05) was also significantly higher than those in Group II (33.0%, 68/206) and Group III (30.1%, 52/173). Twenty-four clinical pregnancies (50.0%) was obtained and 22 pregnancies (45.8%) are ongoing. Implantation rate in the present study was 24.6%. Conclusion: These results suggest that there is a positive correlation between the appearance of COCs and the developmental competence of the immature oocytes in non stimulated IVM/IVF cycles.

• Development and Reproduction
• /
• v.12 no.3
• /
• pp.215-220
• /
• 2008

Sex differentiation process of the spotted Black Bullhead, Pseudobagrus koreanus, was investigated using fish samples of different age after hatching. The primordial germ cells appeared separately under air bladder in 1-day larva (total length: $6.63{\sim}6.95\;mm$). The primordial gonad with a genital ridge developed in 5-day prelarva ($7.50{\sim}9.36\;mm$). The ovarian differentiation started in about 25-day juvenile ($11.58{\sim}13.21\;mm$). The somatic tissues enlongated in the tip of one end of undifferentiated gonad and fused each other. Thus a small ovarian cavity appeared. The testicular differentiation was initiated in 30-day juvenile ($12.19{\sim}13.72\;mm$). The rudiment of sperm duct was appeared in the lower part of the undifferentiated gonad. In 50-day juvenile ($16.28{\sim}17.06\;mm$), the ovary started to fill with peri-nucleolus oocytes, and the spermatogonia started to develop. In 250-day juvenile ($35.49{\sim}51.12\;mm$), the ovary became bigger and filled with oocytes, and the number of spermatogonia started to increase. Considering these results, the spotted Black Bullhead could be a differentiated type in sex differentiation and gonochorism in sexuality.

• The Korean Journal of Zoology
• /
• v.39 no.3
• /
• pp.257-265
• /
• 1996

Previously, we demonstrated that estradiol (E$_2$) was produced by medium sized follicles of Rana nigromaculata and Rana dybowskii in vitro. Present experiments were carried out to determine when the growing follicles have obtained the ability to produce E$_2$. Follicles in different growth stages were isolated and cultured for 6 hours in the presence or absence of frog pituitary homogenates (FPH, 0.1 pituitary/2m1) or various steroid precursors (200 ng/2m1). levels of progesterone (P$_4$), 17 -hydroxyprogesterone (17$\alpha$-OHP), androstenedione (AD), testosterone Cr) or E$_2$ in the medium were measured by RIA. The smallest follicles failed to produce steroids, whereas the smaller follicles produced considerable amounts of steroids (211 pg/follicle), and the medium sized follicles produced a large amounts of steroids (1653 pg/follicle) in response to FPH. Addition of pregnenolone (P5) resulted in a marked increase in P$_4$ but not in other steroids by the smallest follicles whereas the treatment resulted in a marked increase in P$_4$, 17$\alpha$-OHP, AD, T and E$_2$by the smaller and medium follicles. When the amounts of steroids are calculated on the basis of unit surface area (pg/mm$_2$), the ability of the smallest follicles to produce P$_4$ from P5 was similar to those of smaller and medium sized follicles. However, the smallest follicles failed to metabolize P$_4$ to other steroids whereas the smaller and medium follicles did. Taken together, the data suggest that the smallest follicles do not response to FPH in terms of steroid production but they have capacity to convert P5 to P$_4$ and that the smaller follicles have potential to produce E$_2$ although much less efficient than medium sized follicles.

• Clinical and Experimental Reproductive Medicine
• /
• v.31 no.2
• /
• pp.111-117
• /
• 2004

Objectives: To investigate the effect of LH receptor in folliculogenesis, we confirm the expression level of LH receptor (LH-R) mRNA in human granulosa cells (GCs) and its expression levels were analyzed by comparison to embryo developmental rate and pregnancy rate. Materials and Methods: GCs were obtained at the time of oocyte retrieval from the patients undergoing IVF-ET program. The patients were divided into two groups: Group I (n=20) is poor responder (retrieved oocyte(s)$\leq$3ea), Group II (n=80) is normal responder (retrieved oocytes>3ea). After the extraction of total RNA, semiquantitative RT-PCR was performed and the expression level of LH-R mRNA was normalized by $\beta$-actin. Statistical analysis was performed by using $X^2$ test, Student's t-test and Pearson correlation. Results: In Group II, the relative values of LH-R mRNA (0.680 vs. 0.463, p<0.005) and pregnancy rate (54.7% vs. 23.1%, p<0.05) were significantly higher than in Group I. Number of retrieved oocyte(s) was gradually increased when the expression of LH-R mRNA was increased (p<0.05). But the quality of retrieved oocyte and transferred embryo were not related with the expression of LH-R mRNA. When the pregnancy rate was compared with FSH only group and FSH combined with hMG group in the ovarian stimulation protocol, FSH combined with hMG group was significantly higher than FSH only group in Group I (37.5% vs. 0%), and the expression of LH-R mRNA was significantly higher in hMG combined group than FSH only group (p<0.05). Conclusion: Expression level of LH-R mRNA has important role in ovarian function related with the response to gonadotrophin in human folliculogenesis. Furthermore these data might provide the evidence that additional use of hMG is helpful to poor responders.

• Development and Reproduction
• /
• v.5 no.2
• /
• pp.137-144
• /
• 2001

Gametogenesis and gonadal development of the sea squirt Halocythia roretzi, which is two years old were investigated by histological study. The specimens were collected in Guryong-po coastal area Kyoungsangbuk-do, Korea from May 1996 to April 1997. The sea squirt is hermaphrodite and oviparous. The ovary is located in the inner wall of the tunic year-round, but the testis can be distinguished from in June. The ovary is composed of 6∼8 gonoducts at the left side and 8∼10 ones at the right side, the testis consists of the complex gonad having irregular sacular structures. Oogonia in the ovarian sac were 11.7∼15.6 ${\mu}m$ in diameter. The early developing oocytes were 39.6∼47.6 ${\mu}m$ and nucleus 10.0∼25.0 ${\mu}m$ in diameter. Oocytes in the ovarian sacs during vitellogenesis were 158.6∼210.0 ${\mu}m$, and fully ripe oocytes which were to 210.0∼230.9 ${\mu}m$ in diameter had several test cells in the cortical parts showing a characteristic of vertebrate. The testis showed a general spermatogenesis as in the marine animals. The three-year old sea squirt occurred the first spawning between January to February under 10$^{\circ}C$

• Reproductive and Developmental Biology
• /
• v.31 no.3
• /
• pp.181-186
• /
• 2007

Inhibitor of DNA binding protein or inhibitor of differentiation(Id) is largely considered as positive and/or negative regulators of proliferation, differentiation, angiogeneisis, and apoptosis. The four Id genes(Id1, Id2, Id3, and Id4) were known in mammals. However, little is known about the expression and function of these genes in reproductive physiology. Among them, this study was conducted to analyze the expression pattern of Id3 mRNA on folliculogenesis in rat ovary. After PMSG administration, the ovaries were obtained at 3, 6, 12, 24, 36, and 48hrs, fixed, dehydrated, and paraffin embedded. For in situ hybridization, anti-sense and sense Id3 cRNA probes were prepared and applied to the ovarian section. The ovarian sections were coated with NTB-2 emulsion. After that, the slides were developed and counterstained with hematoxylin and eosin staining. The hybridization signal was estimated on a scale of 1+ to 4+. In oocyte, the intensity of Id3 mRNA in primordial and primary follicles was scored at ${\geq}2+$, but the intensity was less than 1+ in secondary, dominant, and preovulatory follicles. In granulosa cells, the Id3 mRNA was strongly expressed(3+ or 4+) in dominant and preovulatory follicles. Taken together, Id3 mRNA was expressed specifically at follicle stages and follicular tissue and might be closely related with follicle development.

• The Korean Journal of Malacology
• /
• v.20 no.1
• /
• pp.55-63
• /
• 2004

Gonadosomatic index, condition index and reproductive cycle with the gonadal development of the female Octopus ocellatus were investigated by histological observations and morphometric data, from January to December, 2000. And changes in biochemical components of the ovary and the trunk tissues including the digestive organ associated with gonadal development were studied by biochemical analysis from January to October, 2001. The specimens were collected at the coastal waters of Buan, Jeollabuk-do, Korea, from January 2000 to October 2001. O. ocellatus is a dioecious organism. The gonad of O. ocellatus locates medially in posterior region of the body. Morphology of the ovary shows round and oval in shape, the average diameter and external colour of ripe ovary was 32 mm and semitransparent light brown in colour. As the ovary was getting mature, transparent elongated eggs covered with chorion were present in the ovarian cavity. Monthly changes in the gonadosomatic index (GSI) showed a similar pattern with those of the condition index. The GSI and condition index began to increase in March and reached the maximum in April. And then, their values decreased from May and reached the minimum in September. Reproductive cycle of O. ocellatus can be categorized into five successive stages: early developing stage (September to December), late developing stage (November to March), ripe stage (March to May), partially spawned stage (April to June), and degenerative/resting stage (June to October). Follicle cells attached to an oocyte were involved in vitellogenesis in the cytoplasm of the vitellogeneic oocyte and formation of chorion (secondary egg membrane) of the ovarian eggs. Spawning occurred between April and June. The spawning period was once a year and the peak took place between May and June. This species belongs to semelparity. According to changes in biochemical contents of the ovary and the digestive organ, monthly variations of moisture, total protein, total lipid and glycogen contents (%) in the ovary showed a negative correlationship with those of the trunk tissues including the digestive organ. Accordingly, it is assumed that the ovary only may be received nutrient supply (total lipid content) for gonadal development from the trunk tissues including the digestive organ (r = -0.55, p < 0.05).

• Clinical and Experimental Reproductive Medicine
• /
• v.31 no.3
• /
• pp.155-168
• /
• 2004

Objective: Melatonin, which is secreted by pineal gland play an important role in the regulation of ovarian function via seasonal rhythm and sleep in most mammals. It also has a role in the protection of cells by removing toxic oxygen free radicals brought about by metabolism. In the present study, effects of melatonin on the mouse oocyte maturation were examined using two different culture conditions provided with 5% or 21% oxygen concentration. Material and Method: Immature mouse oocytes were obtained from the ovarian follicles of $3{\sim}4$ weeks old ICR strain mice intraperitoneally injected with 5 I.U. PMSG 44 hour before. Under stereomicroscope, morphologically healthy oocytes with distinct germinal vesicle (GV) were liberated from the graafian follicles and collected using mouth-controlled micropipette. They were then cultured for 17 hour at $37^{circ}C$, 5% $CO_2$ and 21% $O_2$ (95% air) or 5% $CO_2$, 5% $O_2$ and 90% $N_2$. New modified Hank's balanced salt solution (New MHBS) was used as a culture medium throughout the experiments. Effects of melatonin were examined at a concentration of $0.0001{\mu}M$, $0.01{\mu}M$ or $1.0{\mu}M$. For the prevention of spontaneous maturation of immature oocytes during culture, dibutyryl cyclic AMP (dbcAMP) and/or hypoxanthine were included in the medium. Results: Under 21% oxygen condition, oocytes cultured in the presence of $0.01{\mu}M$ melatonin showed a significantly higher maturation rates, in terms of germinal vesicle breakdown (95.0% vs 89.0%) and polar body formation (88.1% vs 75.4%), compared to those cultured with $0.0001{\mu}M$ or $1.0{\mu}M$ melatonin. However, no difference was observed in oocytes cultured under 5% oxygen whether they were treated with melatonin or not. In the presence of $0.01{\mu}M$ melatonin, oocytes either cultured under 21% or 5% oxygen exhibited no difference in the polar body formation (85.6% vs 86.7%). However, in the absence of melatonin, oocytes cultured under 21% oxygen exhibited lower polar body formation (74.7%). When oocytes were cultured in the presence of dbcAMP alone or with varying concentrations of melatonin, those treated with both compounds always showed better maturation, i.e., germinal vesicle breakdown and polar body formation, compared to those cultured with dbcAMP alone. At the same concentration of melatonin, however, oocytes exposed to 21% oxygen showed poor maturation than those to 5% oxygen. Similar results were obtained from the experiments using hypoxanthine instead of dbcAMP. Conclusion: Based upon these results, it is suggested that melatonin could enhance the meiotic maturation of mouse oocytes under 21% oxygen concentration, and release oocytes from the meiotic arrest by dbcAMP or hypoxanthine regardless of the concentration of oxygen, probably via the removal of oxygen free radicals.

• KSBB Journal
• /
• v.19 no.6 s.89
• /
• pp.433-436
• /
• 2004

Polycystic ovarian syndrome (PCOS) is a very common endocrine disorder in women of reproductive age. There are some evidences that nerve growth factor (NGF) is involved in the pathogenesis of PCOS. In this study, we investigated the effect of Korean red ginseng total saponin (GTS) on the ovarian morphology and NGF expressions in the ovaries, pituitary and hippocampus. The oil control animals were injected with 0.2 ml oil/rat. Animals in estradiol valerate (EV) control group were injected i.m. with 4 mg EV in 0.2 ml oil/rat. The GTS was administered (100 mg/kg) i.p. every other day for 60 days, beginning 1 day after the EV injection. PCO was induced by a single injection of EV (4 mg, i.m.). At day 60, the expressions of NGF were examined by immunohistochemistry. The main findings were as follows; PCO was fully developed with a single i.m. injection of EV, and PCO showed the increased expression of NGF, and GTS administration decreased NGF expressions in the ovaries without affecting pituitary and hippocampus significantly. The present results demonstrate that GTS attenuates PCOS by the stimulation of NGF expression.