• 한국가축번식학회지
• /
• 제19권4호
• /
• pp.307-322
• /
• 1996

The corpus luteum (CL) is formed by the action of a surge of luteinizing hormone (LH) on the pre-ovulatory follicle. Luteal cells derived from granulosa and theca interna cells continue to secrete progesterone for about two weeks. LH in domestic animals is essential for the normal secretion of progesterone at all stages of the luteal phase. For this process in the rodents, 20$\alpha$-hydroxysteroid dehydrogenase (20$\alpha$-HSD) is indispensable. 20$\alpha$-HSD is an enzyme to be a biologically inactive steroid. This enzyme plays a critical role in the regulation of the rat luteal function and reported to be present in steroid-producing tissues such as the testis and adrenal gland. We have purified 20$\alpha$-HSD and found two distinct 20$\alpha$-HSD molecules (HSD-1 and HSD-2). Their molecular weights are both estimated to be 33kd.The amino acid compositions of HSD-1 and HSD-2 are mostly similar, but there is a slight difference in the content of lysine. We demonstrated that 1) CL of previous generations contribute more to whole ovarian 20$\alpha$-HSD activity, 2) newly formed corpora lutea contain only 20$\alpha$-HSD-1 activity, and 3) old CL express activities of each HSD isozyme as shown in the luteal tissue of cycling rats on the day of diestrus where only degenerating old CL exist. The increase in 20$\alpha$-HSD activity identified seems to be related to the increase in the numbers of 20$\alpha$-HSD-positive cells. Interestingly, 20$\alpha$-HSD-1 activities were strongly found in the follicle fluids and theca interna cells by immunohistochemical study. Thus, the activity of 20$\alpha$-HSD may be related to a survival mechanism of those luteal cells and follicles remaining in the ovaries. Luteal cells arise from two sources. The small luteal cells are all of theca cell origin, while the large luteal cells are mainly of granulosa cell origin. CL of Korean Native Cattle, as those of other animal species, contains two morphologycally and functionally distinct luteal cell populations, such as small and large luteal cells as well as nonluteal cells. In all reproductive states except in the late luteal phase, the bovine CL also contained more small luteal cells than large luteal cells. Luteal tissue secretes a variety of growth factors (proteins) and the pattern of secretion changes during all stages of the luteal phase. These growth factors could be important in regulating the function of the bovine corpus luteum and may act in a potential endocrine autocrine and paracrine mechanisms. Therefore, further work has to be done to elucidate the role of growth factors in the ovary, especially in the corpus luterum. Interest should be focussed on interaction of these growth factors in the regulation of luteal cell and the localization of cytokine synthesis in differnet luteal cells.

• 한국발생생물학회지:발생과생식
• /
• 제8권1호
• /
• pp.27-33
• /
• 2004

생쥐 신생자 시기에 생식 세포의 사멸 기전을 알아보기 위하여 세포 사멸을 보이는 생식 세포에서 세포 사멸 관련 단백질인 caspase-3, caspase-activated DNase(CAD), Bax 및 Fasnas ligand의 발현을 조사하였다. 활성화된 caspase-3와 CAD의 면역화학적 염색 결과 일차 및 이차 난포내 TUNEL 양성을 보이는 세포 사멸 난자에서 발현되는 것을 관찰하였다. CAD 또한 TUNEL 양성을 보이는 세포 사멸 난자에서 염색되었다. 활성화된 caspase-3와 CAB에 양성을 보이는 대부분의 난자들은 폐쇄의 형태학적 특징이라고 할 수 있는 세포질 내 공포가 관찰되었다. Bax는 세포질 내 공포가 존재하는 세포 사멸된 난자에서 염색되었다. Fas ligand 또한 TUNEL 양성을 보이는 난자에서 염색되었다. 이러한 TUNEL 양성을 보이는 생식 세포에서 활성화된 caspase-3와 CAD가 염색된 결과는 생쥐 난포 발달 초기 단계에 caspase-3와 CAD가 생식 세포의 세포사멸에 중요한 역할을 할 수 있다는 것을 보여주고 있다. 또한 TUNEL 양성을 보이는 난자에서 Bax와 Fas ligand가 발현된 결과는 이러한 단백질이 난자의 세포 사멸을 조절하는 요소로 작용할 수 있을 것으로 사료된다.

• 한국수정란이식학회지
• /
• 제15권3호
• /
• pp.231-236
• /
• 2000

This study was conducted to examine the effects of exogenous gonadotropins (PMSG+hCG) and an antioxidant (cysteine) on in vitro maturation of bovine follicular oocytes. Cumulus-oocyte complexes (COCs) aspirated from 2 to 5 mm ovarian follicles were cultured for 22 to 24 hours in a modified bovine embryo culture medium (mBECM) supplemented with 3 mg/mL bovine serum albumin, to which PMSG (10 IU/mL) + hCG (10 IU/mL) and/or cysteine (0.6 mM) were added. When examined the expansion of cumulus ce1ls at the end of maturation culture, greater (p<0.05) expansion was found after addition of PMSG+hCG (79 to 96%) to mBECM than after no addition (0%), regardless of the presence or absence of cysteine in the medium. The addition of cysteine did not stimulate cumulus expansion, but a high proportion (92%) of expansion was achieved when COCs were cultured after the addition of PMSG+hCG and cysteine to the medium. No difference in the proportion of oocytes underwent germinal vesicle breakdown (initiation of maturation) was found after the addition of PMSG+hCG and/or cysteine to mBECM. However, nuclear maturation (development to the metaphase-II stage) of oocytes was significantly stimulated by the combined addition of PMSG+hCG and cysteine, compared with no addition. In conclusion, both exogenous gonadotropins and an antioxidant are important for nuclear maturation of bovine immature oocytes and these factors have a cell-specific stimulatory action.

• Clinical and Experimental Reproductive Medicine
• /
• 제44권1호
• /
• pp.33-39
• /
• 2017

Objective: The aim of this study was to assess the changes of follicular fluid (FF) and serum levels of cerebellin precursor protein 1 (cbln1) and betatrophin in patients with polycystic ovary syndrome (PCOS) undergoing in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) with a gonadotropin-releasing hormone (GnRH) antagonist protocol. Methods: Twenty infertile women with PCOS and 20 control women diagnosed as poor responders undergoing ovarian stimulation with a GnRH antagonist were included. Blood samples were obtained during ovum pick-up. Follicular fluid from a dominant follicle was collected from the subjects. Using enzyme-linked immunosorbent assays, FF and serum levels of cbln1 and betatrophin were measured in both groups of participants. Metabolic and hormonal parameters were also determined and correlated with each other. Results: Both groups of women had similar serum and FF betatrophin levels ($55.0{\pm}8.9ng/mL$ vs. $53.1{\pm}10.3ng/mL$, p=0.11). The serum and FF betatrophin levels of poor responders were found to be similar ($49.9{\pm}5.9ng/mL$ vs. $48.9{\pm}10.7ng/mL$, p=0.22). Conversely, the FF cbln1 levels of PCOS women were found to be significantly higher than the serum cbln1 levels ($589.1{\pm}147.6ng/L$ vs. $531.7{\pm}74.3ng/L$, p<0.02). The FF cbln1 levels of control participants without PCOS were significantly higher than their serum cbln1 levels ($599.3{\pm}211.5ng/L$ vs. $525.3{\pm}87.0ng/L$, p=0.01). Positive correlations were detected among body mass index, insulin resistance, serum insulin, total testosterone, and betatrophin levels in the PCOS group. Conclusion: Follicular fluid betatrophin and cbln1 concentrations may play a pivotal role on follicular growth in PCOS subjects undergoing IVF/ICSI with an antagonist protocol.

• 한국패류학회지
• /
• 제25권2호
• /
• pp.145-151
• /
• 2009

담수 복족류인 다슬기, Semisulcospira libertina libertina의 난자형성과정 동안 생식세포의 미세구조 변화를 광학현미경과 전자현미경으로 관찰하였다. 난소는 나탑 후방부의 간췌장의 표면에 위치하며, 성숙시기에는 녹색을 나타내었다. 난소는 다수의 난자형성소낭들로 이루어져 있었다. 난자형성과정은 5단계로, 난원세포기, 난황형성전기, 난황형성개시기, 난황형성활성기, 성숙기로 구분하였다. 난원세포는 직경 $4-6\;{\mu}m$의 원형으로 커다란 핵을 가진다. 난황형성전기의 난모세포는 직경이 약 $20\;{\mu}m$이며, H-E 염색에서 세포질은 호염기성을 나타냈다. 난황형성개시기의 난모세포는 직경 $60-80\;{\mu}m$ 내외로 세포질에는 전자밀도가 낮은 난황과립들을 가지며, 난병에 의하여 난자형성소낭에 연결되어 있었다. 난황형성 활성기의 난모세포는 직경 $100-120\;{\mu}m$로 난황과립의 전자밀도, 크기, 양이 증가하였으며, 세포질에는 골지체, 미토콘드리아, 조면소포체들이 발달하였다. 성숙기 난모세포의 세포질 대부분은 전자밀도가 높은 단백질성의 난황과립들로 채워져 있었으며, 난막에서는 길이 약 $1.1\;{\mu}m$의 미세융모가 관찰되었다.

• 한국수정란이식학회지
• /
• 제10권2호
• /
• pp.105-113
• /
• 1995

This study was conducted to examine the efficiency of enucleation and blastomere isolation from recipient oocytes and donor embryos, respectively and to determine the effect of oocyte age and electric voltage on the fusion rate and in vitro development of the fused oocytes in rabbit nuclear transplantation. Immature oocytes collected from ovarian follicles were matured in vivo for 12 h in TCM-199 containing FCS and hormones and in vivo matured oocytes were collected 17 to 18 h post-HCG. The fresh and frozen donor embryos of 8- to 16-cell stage were collected from the oviduct of superovulated does. The proportion of successfully enucleated oocytes was greatly lower in in vitro matured oocytes (42.3%) than that (62.7%) in in vivo matured oocytes The level of cytochalasin B for in vivo matured oocytes did not affect the efficiency of enuleation, but 7.5 $\mu$g /mL cytochalasin B for in vitro matured oocytes showed a high enucleation rate significantly. The isolation efficiency of a single blastomere nucleus did not differ between 8- and 16-cell stage embryos. The percentage of single blastomeres isolated from 16-cell stage fresh embryos after 0.5% pronase treatment was greatly higher at 16-min treatment (94.4%) than at 8-min(78. 1%) and the blastomeres(61.5%) isolated from frozen-thawed embryos after 16-min pronase were significantly fewer than those of fresh embryos. The age of recipient oocytes affected nuclear fusion rate. The reconstituted oocytes fused at 24-h age showed slightly higher fusion rate (77.8%) than those (65.0%)fused at 18-h age. The fusion rate of in vitro and in vivo matured oocytes inserted with fresh blastomere did not differ among electric voltages, but the cleavage rate and development to morula-blastocysts of in vitro matured oocytes was more higher under 0.6 kV/cm than under 0.8 to 1.2 kV/cm, while the cleavage rate and development of in vivo matured oocytes was higher under 0.8 to 1.0 kV/cm than under 1.2 kV/cm. The fusion and cleavage rate fol1owing insertion with frozen-thawed blastomere was not different between the in vitro and in vivo matured oocytes and was similar to those from fresh blastomere insertion.

• 생약학회지
• /
• 제28권3호
• /
• pp.104-111
• /
• 1997

The traditional herbal medicine, Jackyakgamcho-tang(JGT), was reported to decrease serum testosterone levels and make pregnancy possibel in anovulatory woman and rat. JGT contains Paeoniae Radix(PR) and Glycyrrhizae Radix(GR) in equal amount. This study was designed to investigate the effect of JGT and its components(PR, GR, paeoniflorin and glycyrrhizin) on uterine and ovarian responses, follicular development, and estrogen secretion in the immature rat. The samples(water extracts of JGT, PR, GR; pure compound of paeoniflorin and glycyrrhizin) were administered orally to rats from the 21th day of age to the 28th or 30th days of age for 7 or 9 days. JGT(400mg/kg) and PR(100mg/kg, 200mg/kg) treatments significantly increased serum estradiol above levels in control rats, but both GR and glycyrrhizin had no effect on this parameter. Gross observation and histological analysis revealed that an increased number of growing follicules was observed in the ovaries of JGT and PR treated rat. However the lutenized follicles and ova present in the oviducts were not observed in all rats except one treated with estrogen as a positive control. These results indicate that JGT stimulates the estrogen production and follicular maturation in the immature rat and PR is the main component to induce such reaction.

• 한국가축번식학회지
• /
• 제24권3호
• /
• pp.225-230
• /
• 2000

본 연구는 소 수정란의 동결에 있어서 완만 및 급속동결과 vitrification동결 후의 생존성을 비교하고 이울러, 내동제의 종류 및 배 발달단계가 vitrification동결 보존 후의 발생율을 조사하고자 수행 하였다. 1. 소 수정란의 vitrification 동결보존 후의 발생율과 부화율은 67.5%와 27.5%로서 급속동결 및 완만동결 운해 후의 발생율과 부화율 42.5% 와 20.0% 및 52.5%와 25.0%에 비해 높게 나타났으나 대조군(82.5%, 37.5%)에 비해서 낮게 나타났다. 2. EFS와 EDS내동제의 처리에 따른 소 수정란의 vitrification 동결보존 후의 발생율과 부화율은 47.5%와 22.5% 및 52.5%와 27.5%로서 대조군의 82.5%와 37.5%에 비해 낮게 나타났다. 3. 소 수정란의 2 cell, 8 cell, morulae, blastocyst stage별 vitrification 동결보존 후의 발생율과 부화율은 25.0%와 15.0%, 32.5%와 20.0%, 37.5%와 20.0%, 52.5%와 27.5% 및 47.5%와 25.0%로서 대조군(82.5%와 37.5%)에 비해 낮게 나타났다.

• Animal cells and systems
• /
• 제12권4호
• /
• pp.313-319
• /
• 2008

The ultrastructure of oocytes during oogenesis and oocyte degeneration associated with follicle cells in female Sinonovacula constricta(Lamarck, 1818) were investigated by electron microscope observations. Ovarian follicles are surrounded by a matrix of vesicular connective tissue cells(VCT cells). VCT cells contain large quantities of glycogen particles and several lipid droplets in their cytoplasm. It is suggested that VCT cells act as a source of nutrients for vitellogenesis during oogenesis. In early vitellogenic oocytes, several coated vesicles, which appear at the basal region of the oocyte, lead to the formation of membrane-bound vesicles via endocytosis. The uptake of nutritive materials in coated vesicles formed by endocytosis appears through the formation of coated pits on the oolemma during vitellogenesis. During the late stage of oogenesis, yolk precursors(yolk granules), mitochondria and lipid droplets are present in the cytoplasm of late vitellogenic oocytes. In particular, proteinaceous yolk granules containing several different components are intermingles and form immature yolk granules. In the mature oocyte, small immature yolk granules are intermingled and form large mature yolk granules. Vitellogenesis occurs through a process of autosynthesis, involving combined activity of the Golgi complex, mitochondria and rough endoplasmic reticulum in the cytoplasm of vitellogenic oocytes. The process of heterosynthesis is where extraovarian precursors are incorporated into oocytes by endocytosis at the basal region of early vitellogenic oocytes before the formation of the vitelline coat. Follicle cells appear to play an important role in vitellogenesis and oocyte degeneration. The functions of attached follicle cells to the oocyte during oocyte degeneration are phagocytosis and digestion of phagosomes originating from oocyte degeneration. After digestion of phagosomes, it is assumed that the function of follicle cells can permit a transfer of yolk precursors necessary for vitellogenesis and allows for the accumulation of glycogen and lipid during oocyte degeneration, which can be employed by vitellogenic oocytes. Follicle cells of S. constricta may possess a lysosomal system for induction of oocyte breakdown and might resorb phagosomes in the cytoplasm for nutrient accumulation during oocyte degeneration.

• Clinical and Experimental Reproductive Medicine
• /
• 제26권3호
• /
• pp.389-398
• /
• 1999

Objective: This study was designed to evaluate the effects of endogenous LH surge, GnRH agonist (GnRH-a) or human chorionic gonadotropin (hCG) as ovulation trigger on pregnancy rate by intrauterine insemination (IUI). Method: Patients received daily 100 mg of clomiphene citrate (CC) for 5 days starting on the third day of the menstrual cycle followed by human menopausal gonadotropin (hMG) for ovulation induction. Follicles larger than >16 mm in diameter were present in the ovary, frequent LH tests in urine were introduced to detect an endogenous LH surge. Final follicular maturation and ovulation were induced by GnRH-a 0.1 mg (s.c.) or hCG $5,000{\sim}10,000$ IU (i.m.) administration except natural ovulation. Pregnancy was classified as clinical if a gestational sac or fetal cardiac activity was seen on ultrasound. Results: There were no differences in age, duration of infertility and follicle size, but more ampules of hMG were used in GnRH-a group compared to hCG 10,000 IU treated group (p<0.05). Lower level of estradiol ($E_2$) on the day of hCG or GnRH-a injection was observed in hCG 10,000 IU group than other treatment groups (p<0.01). The overall clinical pregnancy rate was 19.8% per cycle (32/162) and 22.2% per patient (32/144). Pregnancy rate was higher in natural-endogenous LH surge group (37.5%, 9/24) than GnRH-a (18.8%) or hCG treated group (20.9% & 13.9%), but this difference was not statistically significant. No patient developed ovarian hyperstimulation. Abortion rate was 22.2% (2/9) in hCG 5,000 IU group. Delivery or ongoing pregnancy rate was 37.5% (9/24), 18.8% (3/16), 16.3% (7/43) and 13.9% (11/79) in endogenous LH surge, GnRH-a, hCG 5,000 IU and hCG 10,000 IU treatment groups, respectively. Conclusion: These results support the concept that use of natural-endogenous LH surge in stimulated cycles may be more effective to obtain pregnancies by IUI than GnRH-a or hCG administration.