• 수산해양교육연구
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• 제28권5호
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• pp.1231-1243
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• 2016

The ultrastructural study on oocyt development and the process of vitellogensis in the oocytes during oogenesis in female Pampus echinogaster were investigated by electron microscope observations. In the previtellogenic phase, in particular, several intermitochondrial cements appear in the cytoplasms of the chromatin nucleleolus oocyte and perinuclear oocyte. The number of intermitochondrial cements are associated with the multiplication of the number of mitochondria in the early developmental stage. In the early vitellogenic phase, the Golgi complex in the cytoplasm of the yolk vesicle oocyte is involved in the formation of yolk vesicles containing carbohydrate yolks. At this time, many pinocytotic vesicles containing yolk precursors (exogenous substances) by pinocytosis are observed in the cytoplasm near the region of initial formation of the zona pellucida. In the late vitellogenic phase, two morphological different bodies, which formed by the modified mitochondria, appeared remarkably in the yolked oocytes. The one is the multivesicular bodies and another is yolk precursors. The multivesicular bodies were transformed into the primary yolk globules, while yolk precursors were connected with exogeneous pinocytotic vesicles near the zona pellucida. After the pinocytotic vesicles were taken into yolk precursors, the yolk precursors were transformed into the primary yolk globules. Thereafter, primary yolk globules mixed with each other, eventually, they developed into secondary and tertiary yolk globules. In this study, vitellogenesis of this species occurred by way of endogenous autosynthesis and exogenous heteogenesis. Vitellogenesis occurred through the processes of endogeneous autosynthesis, involving the combined activity of the Golgi complex, mitochondria and multivesicular bodies formed by modified mitochondria. However, the process of heterosynthesis involved pinocytotic incorporation of extraovarian precursors (such as vitellogenin in the liver) into the zona pellucida (by way of granulosa cells and thecal cells) of vitellogenic oocytes.

• 대한한방부인과학회지
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• 제24권1호
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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.

• Clinical and Experimental Reproductive Medicine
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• 제33권1호
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• pp.15-23
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• 2006

목 적: 본 연구는 흰쥐 난소를 실험모델로 하여 미성숙 전동 난포의 성장에 대한 pituitary adenylate cyclase-activating polypeptide (PACAP)의 영향을 얄아보고자 하였다. 연구방법: 미성숙 전동 난포를 생후 21일된 흰쥐로부터 분리하여 PACAP을 첨가하거나 첨가하지 않은 무혈청 배양액에서 3일 동안 배양하고, 푸로게스테론 호르몬의 생성, 난포의 성장, 과립막세포의 증식 및 유전자의 동태 등을 관찰하였다. 증식의 정도는 thymidine incorporation 방법으로 검색하고 유전자의 변동은 Northern 분석을 이용하였다. 결 과: PACAP으로 처리한 군은 난포의 직경이 75% 증가한 반면 난포자극호르몬인 FSH로 처리한 군은 65% 증가하였고, PACAP 처리는 과립막 세포의 증식을 강화시켰다. FSH와 PACAP 공히 배양된 흰쥐 난포의 과립막 세포와 FSH에 반응하는 세포주인 GFSHR-17에서의 프로게스테론 생성을 촉진시켰고, PACAP이 FSH의 작용을 증진시켜 SF-1과 아로마타제 유전자 발현을 촉진시켰다. 결 론: 본 연구는 PACAP이 과립막증식과 스테로이드합성을 통하여 전동 난포의 성장을 촉진함을 시사하였고, 또한, SF-1, 아로마타제 등에 대한 FSH의 작용을 도와주는 역할을 PACAP이 담당하므로 PACAP은 초기 난포성장에 필요한 난소국소인자임을 유추할 수 있었다.

• 한국동물학회지
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• 제33권4호
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• pp.435-445
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• 1990

시상하부에서 합성, 분비되는 gonadotropin releasing horrnone (GnRH)의 면역반응성이 생식소를 비롯한 여러 부위에서도 검출됨이 알려졌으나, 이 펩타이트가 과연 생식소에서 국부적으로 합성되는 지에 관해서는 아직 밝혀지지 않았다. 본 연구에서는 흰쥐 생식소에서 GnRH유전자발현을 연구하기 위하여 GnRH-like mRNA와 GnRH펩타이트의 발현과 세포내 분포 양상을 조사하였다. GnRH 방사면역측정법과 GnRH를 크로마토그라피 방법으로 분리한 결과,시상하부에서 합성되는 GnRH와 유사한 GnRH 면역반은이 흰쥐 생식소 추출물에서 상당량 검출되었다. GnRH-면역반응이 흰쥐 난소의 다양한 세포군에서 나타냄에 반하여, GnRH-like mRNA는 granulsa,theca 그리고 luteal 세포에서만 주로 발현되었다. 또한 흰쥐 정소에서 GnRH면역반응성은 원시정세포, Sertoli,Leydig 세포에서만 검출된 반면에, GnRH-like mRNA는 정세관내의 Seertoli세포에서만 발현되었다. 따라서 이 연구는 생식소에 존재하는 GnRH는 생식소 내에서 국부적으로 합성, 발현되는 결과라고 사료되며, 생식소 내에서 생성된 GnRH는 생식소내 세포군간의 정보교환의 매개자로서 역활을 수행하고 있다고 추정된다.

• 한국발생생물학회지:발생과생식
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• 제21권2호
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• pp.111-120
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• 2017

Equine chorionic gonadotropin (eCG) is a unique molecule that elicits the response characteristics of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in other species. Previous studies from this laboratory had demonstrated that recombinant eCG (rec-eCG) from Chinese hamster ovary (CHO-K1) cells exhibited both FSH- and LH-like activity in rat granulosa and Leydig cells. In this study, we analyzed receptor internalization through rec-eCGs, wild type eCG ($eCG{\beta}/{\alpha}$) and mutant eCG ($eCG{\beta}/{\alpha}{\Delta}56$) with an N-linked oligosaccharide at $Asn^{56}$ of the ${\alpha}-subunit$. Both the rec-eCGs were obtained from CHO-K1 cells. The agonist activation of receptors was analyzed by measuring stimulation time and concentrations of rec-eCGs. Internalization values in the stably selected rat follicle-stimulating hormone receptor (rFSHR) and rat luteinizing/chorionic gonadotropin receptor (rLH/CGR) were highest at 50 min after stimulation with 10 ng of $rec-eCG{\beta}/{\alpha}$. The dose-dependent response was highest when 10 ng of $rec-eCG{\beta}/{\alpha}$ was used. The deglycosylated $eCG{\beta}/{\alpha}{\Delta}56$ mutant did not enhance the agonist-stimulated internalization. We concluded that the state of activation of rFSHR and rLH/CGR could be modulated through agonist-stimulated internalization. Our results suggested that the eLH/CGRs are mostly internalized within 60 min by agonist-stimulation by rec-eCG. We also suggested that the lack of responsiveness of the deglycosylated $eCG{\beta}/{\alpha}{\Delta}56$ was likely because the site of glycosylation played a pivotal role in agonist-stimulated internalization in cells expressing rFSHR and rLH/CGR.

• 한국발생생물학회지:발생과생식
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• 제2권2호
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• pp.173-178
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• 1998

비만유전자 산물인 leptin은 지방 조직에서 생성되어 혈액으로 분비되며, 신진대사, 식욕, 체열 등을 조절하여 비만의 억제 조절 물질로 작용하는 것으로 알려져 있다. 또한 leptin은 비만 뿐만 아니라 생식 생리와도 관련이 있는 것으로 보이며, 이러한 leptin의 작용이 난소에 직접적인지 혹은 시상하부나 뇌하수체를 매개로 하는지는 아직 정확하게 밝혀지지 않고 있으며, 난소에서의 leptin 및 leptin 수용체의 발현 양상에 대한 연구 또한 미진한 상태에 있다. 따라서 본 연구는 생후 3주령과 8주령의 흰쥐 난소에서 leptin과 leptin 수용체의 발현 양상을 면역조직화학방법과 RT-PCR 방법으로 조사하였다. 면역조직화학방법 결과 3주령과 8주령 흰쥐 모두에서 leptin은 협막세포와 폐쇄 난포의 일부 과립세포에 염색되었고, leptin수용체는 협막세포, 간질세포와 난포강이 형성되지 않은 난포의 난자에 염색되었다 RT-PCR 결과 3주 및 8주 흰쥐 난소에서 leptin mRNA는 모두 발현되지 않은 반면, leptin 수용체 mRNA는 모두 발현되었다. 결론적으로 leptin mRNA가 난소에서 발현되지는 않지만, 면역조직화학방법으로 leptin의 발현을 확인하였고, leptin 수용체는 난소에서 RT-PCR 방법과 면역조직화학방법으로 모두 확인할 수 있었다. 이러한 결과로 보아 혈액에서 난소 내로 유입된 leptin이 협막세포, 간질세포와 난자의 leptin 수용체에 결합하여 난소의 생리적 기능을 조절할 수 있는 것으로 사료된다.

• 한국발생생물학회지:발생과생식
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• 제6권2호
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• pp.123-129
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• 2002

비만유전자 산물인 leptin은 비만뿐만 아니라 여성의 생식 생리와 관련이 있는 것으로 보이나, 아직 이러한 leptin이 난소에 직접적으로 작용하는지 정확하게 밝혀지지 않고 있다. 따라서 본 연구에서는 흰쥐 난소에서 leptin과 leptin 수용체의 발현을 면역조직화학방법으로 확인하고 발정주기에 따른 leptin과 leptin 수용체의 발현 양상을 RT-PCR 방법으로 조사하고자 하였다. 면역조직화학적 염색방법 결과 흰쥐 난소내에서 leptin은 협막세포와 폐쇄 난포의 일부 과립세포에 염색되었고, leptin 수용체는 협막세포, 간질세포와 난포강이 형성되지 않은 난포의 난자에 염색되었다. 특히 폐쇄 난포에서는 leptin과 leptin 수용체가 정상 난포에 비해 강하게 염색되었다. 흰쥐의 발정주기 동안 혈청내 estradiol, progesterone leptin의 농도는 ELISA 방법으로 측정하였고, 난소내 leptin과 leptin 수용체의 mRNA 발현 양상은 RT-PCR 방법으로 조사하였다. 혈중 leptin 농도를 측정한 결과 estrous 시기에 비하여 metestrous 시기에 유의하게 증가하였고, 이 시기에 progesterone 농도가 함께 증가하는 것을 관찰할 수 있었다. Leptin mRNA는 모든 발정주기에서 발현되지 않았지만 leptin 수용체 mRNA는 diestrous 시기를 제외한 다른 발정주기에 모두 발현되었다. 이러한 결과는 leptin이 흰쥐 난소의 기능을 조절하는데 직접적으로 관여할 수 있다는 것을 제시하고 있다.

• 대한한방부인과학회지
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• 제24권3호
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• pp.48-72
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• 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.

• 한국가축번식학회지
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• 제17권2호
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• pp.75-83
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• 1993

Purine has been identified in the preparation of follicular fluid and shown an activity in maintaining oocyte meiotic arrest. Therefore this study was performed to examine the inhibitory effect of purine on germinal vesicle break down(GVBD) in the presence and absence of human fetal cord serum(HFCS) or human mature follicular fluid(HMFF), as a protein source, in vitro culture. Immature oocytes(GV stage) were collected from ovaries of 21∼28 days old ICR mice by puncturing the antral follicles with a fine needle, at 48 hrs after PMSG injection. Some of the oocytes were denuded by drawing the cumulus-enclosed(complex) oocytes in and out of a pasteur pipet. Complex oocytes and denuded oocytes were cultured 3 hrs. in T6 media containing 0.75mM adenosine or/and 4mM hypoxanthine, with HFCS or HMFF. Their GVBD rates were observed at every 1 hr. during the culture time. Both adenosine and hypoxanthine have shown a time-dependent inhibitory effect on GVBD in complex and denuded oocytes and the inhibitory effect was maximized in culture medium containing hypoxanthine and adenosine. HFCS and HMFF increased the GVBD rates in the presence of the purines, thus HFCS and HMFF may contain a factor that could reverse the inhibitory effect of purines. Also complex oocytes were more sensitive to not only the inhibitory effect of purines but the promoting action of HMFF on GVBD than denuded oocytes. Therefore it was reconfirmed that granulosa cells play an important part in meiotic arrest and resumption.

• Asian-Australasian Journal of Animal Sciences
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• 제20권6호
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• pp.842-849
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• 2007

The purpose of the present study was to produce live offspring from in vitro fertilized goat embryos. Oocytes were collected from abattoir ovaries and kept in oocyte collection medium. Oocytes were washed 4-5 times with maturation medium containing medium-199 with 5 ${\mu}g/ml$ FSH, 100 ${\mu}g/ml$ LH, 1 ${\mu}g/ml$ estradiol-$17{\beta}$ 50 ${\mu}g/ml$ gentamycin, 10% inactivated estrus goat serum, and 3% BSA (fatty acid free). Oocytes were placed in 100 ${\mu}l$ drops of maturation medium containing granulosa cell monolayer and incubated in a 5% $CO_2$ incubator at $38.5^{\circ}C$ for 27 h. For capacitation of spermatozoa fresh semen was processed and mixed in 3 ml fertilization TALP medium containing 50 ${\mu}g/ml$ heparin and kept in the above incubator for 2 h. The capacitated spermatozoa were coincubated with matured oocytes for fertilization. Cleaved embryos were separated and cultured in embryo development medium with oviductal cells and 494 embryos were produced. Recipient goats were synchronized with two injections of 15 mg $PGF_{{2}{\alpha}}$/goat 10 days apart. Eighty early stage embryos were transferred into the uterotubal junction of 14 surrogate mothers using laparoscopy techniques. One recipient delivered twin kids, whereas another two recipients each.delivered a single kid The parentage of these kids was evaluated using highly polymorphic co-dominant microsatellites markers. From the present study, it was concluded that live goat kids can be produced from in vitro matured and fertilized goat embryos, to the best of our knowledge for the first time in India.