• 한국양식학회지
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• 제11권4호
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• pp.475-485
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• 1998

동사리, Odontobutis platycephala 수컷의 명확한 생식주기를 밝히고자 1995년 12월부터 1997년 11월까지 생식소 중량지수(gonadosomatic index, GSI), 정소의 발달양상 및 혈중 성스테로이드 호르몬 변화양상을 조사하였다. 정소의 모양은 7월부터 10월까지는 콩깍지 모양이었는데, 11월부터는 후반부가 비대해져 올챙이와 유사한 형태였다. GSI는 7월부터 9월까지 0.14~0.18였으며, 10월에 $0.43{\pm}0.04$로 증가하여 2월까지 유의한 변화를 보이지 않았으나, 3월에는 $0.52{\pm}0.09$로 증가하였으며, 5월까지는 비슷한 수준에 머물렀다가, 6월에는 $0.28{\pm}0.05$로 낮아졌다. 생식주기에 따른 정소의 생식세포 발달은 전반부, 경계부, 후반부에서 다른 양상을 보였다. 7월의 대부분 개체의 정소는 세정관이 없었으며, 정자형성과정이 아직 시작되지 않은 상태로 정원세포들만을 볼 수 있었다. 전반부에서 정자형성단계의 세정관들은 8월에 출현하여 9월부터 12월까지는 80% 이상이었으며 1월부터 3월까지는 점차적으로, 4월에는 급격히 감소하여 6월에는 나타나지 않았다. 정자완성단계 세정관은 12월부터 출현하여 3월까지 점차적으로, 4월에 80%로 급격히 증가한 후 6월에 90%로 년 중 최고 수준에 이르렀다. 그러나 정자방출후 세정관은 나타나지 않았으며, 정원세포들은 7월에 100%, 8월에 65% 였으나 나머지 기간에는 20%이하로 년 중 존재하였다. 경계부에서도 8월부터 정자형성단계 세정관이 출현한 후 매월 증가하여 11월에 82%에 이르렀으며 12월부터 감소하여 3월부터는 나타나지 않았다. 정자완성단계 세정관은 11월에 출현하여 2월까지는 18% 이하였으나, 3월부터 6월까지 29%~57%의 수준을 보였다. 정자방출후 세정관은 3월부터 6월까지 12%~25%로 나타났다. 정원세포는 8월에 85%, 9월에 67%, 10월에 35%, 11월에 10%로 감소하였으며, 1월부터는 점차적으로 증가하여 4월에 50%를 보인후 6월까지 다시 점차적으로 감소하였다. 후반부에서는 일부의 개체에서 수개의 정원세포를 지닌 세정관이 7월부터 출현하였다. 정자형성단계에 세정관은 8월에 80%, 9월에 85%였으나 10월부터는 급격히 감소하여 11월에서 2월까지는 10%이하였으며, 3월부터는 나타나지 않았다. 정자완성단계 세정관은 8월에 출현하여 10월부터 급격히 증가해 11월에는 75%에 이르렀고 12월에는 15%로 급격히 감소하여 3월까지 유의한 변화를 보이지 않았으며 4월부터는 나타나지 않았다. 정자방출후 세정관은 11월에 출현하여 12월에 82%로 급격히 증가한 후 6월까지 85%~95%로 높은 수준을 유지하였다. 정원세포들은 8월에 15%로 감소하였으며 10월부터 3월까지는 나타나지 않았다. 그러나 4월부터 다시 출현하여 6월까지 10%이하에 머물렀다. 혈중 testosterone (T)의 농도는 8월부터 완만하게 증가하여 11월에 $0.61{\pm}0.09 ng/m\ell$ 였으며, 12월부터는 $3.99{\pm}1.22 ng/m\ell$로 급격히 상승하여 3월까지 높은 수준을 유지했다. 그러나 4월에 $0.52{\pm}0.14ng/m\ell$로 감소하기 시작하였으며, 5월에 $0.17{\pm}0.13 ng/m\ell$로 낮아진 후 7월까지 유의한 변화는 보이지 않았다(P<0.05). $17\alpha$,$20\beta$-dihyd-roxy-4-pregnen-3-one ($17\alpha$,$20\beta$-P)은 12월에 $0.19{\pm}0.01 ng/m\ell$로 약간 감소하였으며, 4월까지는 유의한 변화는 보이지 않고, 5월에 $0.38{\pm}0.03 ng/m\ell$로 급격히 증가하여 peak에 달하였다. 6월에는 $0.19{\pm}0.04 ng/m\ell$로 낮아졌으며, 11월까지 유의한 변화는 보이지 않았다(P<0.05). 이상의 결과를 종합하면 동사리는 정소의 부위에 따라 각각 다른 발달양상을 보였다. 즉 전반부에는 7월이 정원세포증식기, 8월~11월은 성숙초기, 12월~3월은 성숙중기, 4월~5월은 성숙후기, 6월은 기능적 성숙기로 구분되었다. 경계부에서는 7월이 정원세포 증식기, 8월~10월은 성숙초기, 11월~2월은 성숙중기였으며, 3월~6월은 성숙후기, 기능적 성숙기, 후번식기의 특징이 동시에 출현하였다. 후반부에서는 7월이 정원세포증식기, 8월~9월은 성숙중기, 10월은 성숙후기, 11월은 기능적 성숙기, 12월~6월은 후번식기로 구분되었다. 이러한 생식주기의 변화는 성스테로이드 호르몬의 변화양상과 밀접한 관련이 있는 것으로 생각된다.

• 한국수정란이식학회지
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• 제24권3호
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• pp.199-205
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• 2009

This study was carried out to investigate the effective genetic resources preservation system using the frozen boar semen. The porcine oocytes were matured for 44 hours in NCSU-23 medium with or without 10% Porcine Follicle Fluid (PFF), 0.5 ${\mu}g/ml$ porcine FSH, 0.5 ${\mu}g/ml$ equine LH, 1.0 ${\mu}g/ml$ 17 $\beta$-estradiol ($E_2$) and 10 ng/ml Epidermal Growth Factor (EGF) under mineral oil at $38.5^{\circ}C$ in humidified atmosphere of 5% $CO_2$ in air. After 44 h of culture, the oocytes were inseminated with frozen-thawed semen and fresh semen prepared with mTBM medium for 6 h. Later, set of 50 presumptive zygotes were transferred into 4-well dish (500 ${\mu}l$) of IVC medium. for embryos freezing, slow-freezing and vitrification methods were used as a cryopreservation. Differences among treatments were analyzed using General Linear Model Procedure by SAS Package (version 6.12) differences were considered significant when p<0.05. Following IVF and IVC, the rates of cleavage and blastocysts formation were significantly higher (p<0.05) in hormone supplemented group than that of hormone-free group (25.7 vs, 12.1). The development rates to cleavage and blastocysts were significantly higher in PZM-5 group than NCSU-23 group (60.3%, 46.6% vs 27.4%, 11.1%). Further improvement was achieved when PZM-5 was supplemented with FBS. Cleavage rates was significantly higher in fresh semen source group than frozen semen (66.7% vs 43.7%). However in blastocysts rates was similar two groups. Post-thaw survival rates of embryos were 1.2% and 2.2% in slow-frezing and vitrification groups, respectively. The results of our study suggest that it is still possible to improve the culture conditions and boar semen cryopreservation for enhance reproductive technology and animal genetic resources conservation.

• Asian-Australasian Journal of Animal Sciences
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• 제23권3호
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• pp.292-301
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• 2010

Follicle-stimulating hormone (FSH) is a pituitary glycoprotein hormone that is encoded by separate alpha- and betasubunit genes. It plays a key role in stimulating and regulating ovarian follicular development and egg production in chicken. FSH signal transduction is mediated by the FSH receptor (FSHR) that exclusively interacts with the beta-subunit of FSH, but characterization of prokaryotic expression of the FSHb gene and its effect on the expression of the FSHR gene in local chickens have received very little attention. In the current study, the cDNA fragment of the FSHb gene from Dagu chicken was amplified using reverse transcription polymerase chain reaction (RT-PCR), and inserted into the pET-28a (+) vector to construct the pET-28a-FSHb plasmid. After expression of the plasmid in E. coli BL21 (DE3) under inducing conditions, the recombination protein, $FSH{\beta}$ subunit, was purified and injected into the experimental hens and the effect on the mRNA expression levels of the FSHR gene was investigated. Sequence comparison showed that the coding region of the FSHb gene in the local chicken shared 99%-100% homology to published nucleotides in chickens; only one synonymous nucleotide substitution was detected in the region. The encoded amino acids were completely identical with the reported sequence, which confirmed that the sequences of the chicken FSHb gene and the peptides of the $FSH{\beta}$ subunit are highly conserved. This may be due to the critical role of the normal function of the FSHb gene in hormonal specificity and regulation of reproduction. The results of gene expression revealed that a recombinant protein with a molecular weight of about 19 kDa was efficiently expressed and it was identified by Western blotting analysis. After administration of the purified $FSH{\beta}$ protein, significantly higher expression levels were demonstrated in uterus, ovary and oviduct samples (p<0.05). These observations suggested that the expressed $FSH{\beta}$ protein possesses biological activity, and has a potential role in regulation of reproductive physiology in chickens.

• 생명과학회지
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• 제25권10호
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• pp.1184-1195
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• 2015

새로운 실험 동물로 대두되고 있는 제브라피쉬는 척추동물 생식생물학 연구에서도 중요한 역할을 한다. 제브라피쉬의 난자 성숙은 maturation inducing hormone (MIH, 17α,20β-Dihydroxy-4-pregnen-3-one)에 의해 촉발된다. 대부분의 동물의 난자성숙에는 cdc2 kinase와 cyclinB 단백질 복합체인 MPF의 활성화가 필요하다. 발톱개구리와 생쥐에서는 MPF 활성이 두 가지 기작에 의해 조절되는데, 하나는 cyclinB 결합이고 또 다른 하나는 Wee1과 Cdc25에 의한 T14/Y15 잔기의 억제성인산화와 탈인산화이다. 발톱개구리나 생쥐와 달리 제브라피쉬를 포함한 대부분의 진골어류(teleost)는 GV 난자에 pre-MPF complex가 존재하지 않으므로 MPF 활성화는 전적으로 cyclinB 단백질의 de novo synthesis에 의존한다. 다른 종과 마찬가지로 제브라피쉬의 모계유래 mRNA도 CPEB, Dazl, Pum1/Pum2, insulin-like growth factor2 mRNA-binding protein 3 등 다양한 RNA binding protein (RBP)의 결합에 의해 번역이 조절된다. 그러나 제브라피쉬 난자에서 단백질 번역 조절에 관여하는 자세한 작용 기작은 확실하게 규명되지 않았다. 그러므로 제브라피쉬 난자의 성숙과정을 연구하는 것은 척추동물 난자 초기 성숙과정에서 단백질 번역 조절의 역할을 규명할 수 있는 새로운 정보를 제공할 것이다.

• 한국임상수의학회지
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• 제27권6호
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• pp.693-697
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• 2010

본 연구는 총 4두의 삵에서 발정기와 비발정기, 임신기와 비임신기 동안의 분변에서 steroid metabolic materials를 추출한 후, TR-FIA kit를 이용하여 estradiol과 progesterone의 농도를 측정하였다. 발정기간 (2월) 중 estradiol 농도의 최저는 평균 $4.02{\pm}1.9$ng/g 이었고, 최고는 평균 $86.01{\pm}35.2$ng/g (dry fecal weight) 이었다. 비발정기(11월)의 최저는 평균 $4.42{\pm}1.32$ng/g 이었고, 최고는 평균 $15.62{\pm}6.84$ng/g 이었다. 임신기 (3월) 중 progesterone농도의 최저는 평균 $427{\pm}24.49$ng/g 었고, 최고는 평균 $1490{\pm}265.27$ng/g 이었다. 비임신기 (11월)의 progesterone의 최저는 평균 $71.25{\pm}29.61$ng/g 이었고, 최고는 평균 $291.75{\pm}90.30$ng/g 이었다. 위의 결과에 따라 삵에서 TR-FIA에 의한 분변내 steroid hormone 의 측정은 발정과 임신에 관련된 난소활동을 비 침습적으로 평가하기 위한 적절한 방법으로 판단되었다. 본 연구는 삵과 같이 접근이 어렵고 멸종위기에 처한 야생동물의 사육관리의 효율화와 번식계획의 수립에 도움이 될 것이다.

• 한국발생생물학회지:발생과생식
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• 제13권2호
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• pp.79-87
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• 2009

출산과 수유 과정에서의 잘 알려진 기능 때문에, 옥시토신(oxytocin, OT)은 '여성 뇌하수체후엽 호르몬(female nerohypophyseal hormone)'으로 알려져 왔다. 그러나 최신 연구들에 따르면 OT가 중추신경계와 말초조직 수준에서 수컷 생식을 조절하는 국부적인 기능을 가짐이 알려졌다. 일부 실험용 설치류에서, OT는 사회적인 자극들에 반응하여 뇌의 특정 지역으로 분비되는데, 이 뇌 OT와 그 수용체(OTR)를 매개로한 작용들은 수컷의 다양한 행동, 특히 교미 관련 행동의 조절에 깊이 관여한다. 최근에 개발된 OT와 OTR knockout 생쥐 모델들의 사회적, 성적 행도으이 분자 조절 기작들에 대한 중요한 실마리들을 제공하다. OT는 또한 설치류의 정소, 부정소, 전립선에서도 합성되며, 이들 조직에서도 OTR이 발현됨이 보고되어왔다. OT는 말초적으로 testosterone(T)을 dihydrotestosterone(DHT)로 전환시키는 5alpha-reductase의 활성조절을 통해 정소의 스테로이드호르몬 합성과 분비에 관여한다. 안드로겐 전환을 유도하는 OT 작용들이 부정소와 전립선에서도 발견되는데, 이는 OT가 이들 안드로겐 의존적인 생식도관의 기능들, 예를 들어 평활근 수축 유도와 같은 기능을 조절함을 시사한다. 이러한 맥락에서, 수컷의 뇌와 생싯기간 중추신경계와 생식기관에서의 OT의 기능들에 대한 향후 연구들은 복잡한 사회적, 성적 행동에 대한 이해 증진과 심리적 혹은 남성과학적 이상에 대한 치료법 개발에 대한 기반을 제공할 수 있을 것이다.

• 한국동물생명공학회지
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• 제34권2호
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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.

• Reproductive and Developmental Biology
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• 제34권1호
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• pp.33-40
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• 2010

Equine chorionic gonadotropin (eCG) is a heavily glycosylated glycoprotein composed of non-covalently linked $\alpha$- and $\beta$-subunits. To study the function and signal transduction of tethered recombinant-eCG (rec-eCG), a single chain eCG molecule was constructed, and the rec-eCG protein was prepared. In this study, we constructed 5 mutants (${\Delta}1$, ${\Delta}2$, ${\Delta}3$, ${\Delta}4$, and ${\Delta}5$) of rec-eCG using data about known glycoprotein hormones to analyze the role of specific follicle stimulating homone (FSH)-like activity. Three amino acids of certain specific sites were replaced with alanine. The expression vectors were transfected into CHO cells and subjected to G418 selection for 2~3 weeks. The media were collected and the quantity of secreted tethered rec-eCGs was quantified by ELISA. The LH- and FSH-like activities were assayed in terms of cAMP production by rat LH/CG and rat FSH receptors. Then, the metabolic clearance rate analyzed by the injection of rec-eCG (5 IU) into the tail vein was analyzed. The mutant eCGs (${\Delta}l$, ${\Delta}4$, and ${\Delta}5$) were transcripted, but not translated into proteins. Rec-eCG A2 was secreted in much lower amounts than the wild type. Only the rec-eCG ${\Delta}3$ ($\beta$-subunit: $Gln^{94}-Ile^{95}-Lys^{96}{\rightarrow}Ala^{94}-Ala^{95}-Ala^{96}$) was efficiently secreted. Although activity is low, its LH-like activity was similar to that of tethered $eCG{\beta\alpha}$. However, the FSH-like activity of rec-$eCG{\beta\alpha\Delta}3$ was completely flat. The result of the analysis of the metabolic clearance rate shoed the persistence of the mutant in the blood until 4 hours after the injection. After then, it almost disappeared at 8 hours. Taken together, these data suggest that 94~96 amino acid sequences in eCG $\beta$-subunit appear to be of utmost importance for signal transduction of the FSH receptor.

• Reproductive and Developmental Biology
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• 제34권1호
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• pp.47-53
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• 2010

Glycoprotein hormones have a common $\alpha$-subunit that is involved in the signaling pathway together with G protein, adenylcyclase and cAMP induction; however, it is an unclear how this common structure is related to hormonal action. To determine the biological functions of the COOH-terminal amino acids in the $\alpha$-subunit of these glycoprotein hormones, a tethered-molecule was constructed by fusing the $NH_2$-terminus of the $\alpha$-subunit to the COOH-terminus of the $\beta$-subunit of equine chorionic gonadotropin (eCG). The following deletion mutants were created by PCR; Ile was inserted at position 96 to form ${\Delta}96$, Lys was substituted at position 95 to form ${\Delta}95$, His was inserted at position 93 to form ${\Delta}93$ and Tyr was substituted at position 87 to form ${\Delta}87$. Each mutant was transfected into CHO-K1 cells. Tethered-wt eCG, and ${\Delta}96$, ${\Delta}95$, and ${\Delta}93$ mutants were efficiently secreted into the medium but the ${\Delta}87$ mutant was not secreted. Interestingly, the RT-PCR, real-time PCR, and northern blot analyses confirmed that the RNA was transcribed in the ${\Delta}87$ mutant. However, the ${\Delta}87$ mutant protein was not detected in the medium or the intracellular fraction of the cell lysates. The LH- and FSH-like activities of the recombinant proteins were assayed in terms of cAMP production using rat LH/CG and rat FSH receptors. The metabolic clearance rate (MCR) was determined by injecting rec-eCG (2 IU) into the tail vein. The ${\Delta}95$ and ${\Delta}93$ mutants were completely inactive in both the LH- and FSH-like activity assays. The ${\Delta}96$ mutant showed slight activity in the LH-like activity assay. In comparison to the wild type, the activity of the ${\Delta}96$ mutant in the FSH-like activity assay was the highest among all the mutants. The MCR assay in which rec-eCG was injected showed a peak at 10 min in all the treatment groups, which disappeared 4 h after injection. These results imply a direct interaction between the receptor and the COOH-terminal region of the a-subunit. The data also reveal a significant difference in the mechanism by which the eCG hormone interacts with the rLH and rFSH receptors. The COOH-terminal region of the $\alpha$-subunit is very important for the secretion and functioning of this hormone.

• 농업과학연구
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• 제37권3호
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• pp.393-398
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• 2010

Early pregnancy diagnosis of bovine is an essential component for efficient reproductive plan in farms because long term of non-pregnancy results in economic losses by failure of offspring production and low milk yield in dairy cattle. The major steroid hormones related with reproduction are known to be progesterone and estrogen in bovine pregnancy. To evaluate detection level of hormones in milk, plasma and milk progestrone and estrogen of Holstein cows was analyzed during artificial insemination (AI) and embryo transfer (ET). Progesterone concentration at 21 days postestrus was significantly different in plasma and milk between pregnant and non-pregnant cows. Estrogen concentration at estrus was higher in pregnant recipients than that in non-pregnant recipients. To analyze correlation between hormone levels and conception rates in Holstein, the conception and return rates were checked following AI, and the returned cows were on the track of pregnancy after consecutive AI. Pregnant cows following first AI were considered as high conception group while pregnant cows following third AI were rated as low conception group. Proportion of high and low conception groups in this study was 78.2% and 9.1%, respectively. Hormone analysis indicated that high conception group had higher estrogen level during estrus than low conception group ($26.45{\pm}3.32$ vs $19.017{\pm}2.97$). Progesterone level was not different between high and low conception groups during estrus but increased significantly after 21 days postestrus (21 day: $4.95{\pm}1.12$ vs $0.95{\pm}0.23$, 35 day: $12.47{\pm}3.82$ vs $2.41{\pm}1.21$). In conclusion, the pattern of progesterone and estrogen secretion in Holstein milk samples could be a good candidate for early pregnancy detection and selection of recipients during ET.