• 한국수정란이식학회지
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• 제19권3호
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• pp.245-255
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• 2004

포유동물 난자의 체외수정은 외래유전자 도입에 의한 형질전환동물 생산과 우수한 형질을 가진 개체의 보존, 인간의 불임연구 등과 같은 수정란이식 기술로서 널리 이용되고 있다. 돼지 난포란을 이용한 체외수정란의 생산은 초기단계인 체외성숙 기술의 미확립, 그로인한 체외수정 시 높은 다정자 침입율과 불완전한 웅성전핵 형성 몇 체외발달능 정지현상(cell blocking) 등 어려움 때문에 아직도 다른 가축보다 양질의 수정란을 생산하기가 어려운 것으로 알려져 있다. 이와 같은 것을 해결하기 위하여 많은 연구자들은 배양액내에 hormon, growth factor, antioxdants 등과 같은 외인성 인자들을 첨가하고 있다. 이들 인자 중 antioxdant는 free radical을 소거하고 과산화물 생성을 억제하여 난자를 산화적 스트레스로부터 보호한다. 따라서 본 연구는 돼지 난포란으로부터 체외수정란의 생산에 있어서 배양액내 cysteine, catalase 및 glutathione의 첨가가 체외성숙, 체외수정 및 체외배양에 어떤 영향을 미치는지를 검토하였다. 실험 1은 체외성숙용 배양액인 TCM-199 용액에 catalase(100, 200, 500U/$m\ell$)와 glutathione(0.5, 1.0, 1.5mM/$m\ell$)의 첨가, 실험 2는 성숙된 난자의 체외수정용 배양액인 mTBM 용액에 cysteine(0.1, 1.6, 1.0mM/$m\ell$), catalase(100, 200, 500U/$m\ell$)와 glutathione(0.5, 1.0, 1.5mM/$m\ell$)의 첨가, 실험 3은 체외성숙 및 체외수정된 난자의 체외배양용 배양액인 NCSU-23 용액에 cysteine(0.1, 1.6, 1.0mM/$m\ell$), catalase(100, 200, 500U/$m\ell$)와 glutathione(0.5, 1.0, 1.5mM/$m\ell$)을 첨가하여 배반포로의 배 발달율을 관찰하였던 결과는 다음과 같다. 1. 체외성숙시 catalase의 경우는 500U 첨가군의 27.2%로 무첨가군의 15.4%보다 유의하게 높았다(p<0.05). 한편 glutathione의 경우 배반 포로의 배 발달율은 무첨가군과의 차이는 없었다. 그러나 1.0mM 첨가군에서 상실배까지의 배 발달율인 72%는 무첨가군의 53.9%보다 유의하게 높았다(p<0.05). 2. 체외수정시 여러 종류의 항산화제 첨가는 첨가하는 농도와 관계없이 배반포로의 배 발달율은 무첨가군과 차이가 없었다. 3. 체외배양시 여러 종류의 항산화제 첨가는 첨가하는 농도와 관계없이 배반포로의 배 발달율은 무첨가군과 차이가 없었다. 이상의 결과를 종합적으로 하면 돼지 난포란을 이용한 배반포의 체외생산에 있어서 배양액내 항산화제의 첨가는 체외성숙단계에서만 효과적이었다. 이것은 아마도 항산화제가 체외성숙 시 난포란 내에서 일어나는 여러 가지 생화학 반응의 처리시간과 관련하여 활성화시킴으로써 난포란의 생존력을 높인 것이라고 사료되기 때문에 앞으로는 돼지 난포란의 효율적인 체외성숙에 대해서 배양액내 첨가물질은 물론 나아가서 방법론적인 측면에서 더욱 연구되어져야 할 것이다.

• 한국가축번식학회지
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• 제26권1호
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• pp.41-51
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• 2002

본 연구는 돼지 난포란의 체외배양액과 배발달 배양액에 저분자량의 황화합물인 cysteamine을 각자 0, 25, 50 및 100$\mu$M 첨가배양함으로서 cysteamine이 돼지 난포란의 체외성숙과 체외배발달에 미치는 영향을 규명하고자 실시하였다. 본 연구에서 얻어진 결과를 요약하면 다음과 같다. 1. 돼지 난포란을 체외성숙배양액인 0.9mM CySH가 함유된 NCSU-23 배양액에 cysteamine을 각각 0, 25, 50 및 100$\mu$M 첨가배양하여 성숙을 유기한 다음, 체외수정을 실시한 결과, 모든 처리구에서 핵성숙률, 정자침투율, 웅성전핵형성률, 다정자침입률 및 평균침입정자수에서 유의적(P〉0.05)인 차이가 인정되지 않았다. 2. 체외수정을 실시한 후, 배발달배양액인 NCSU-23에 7일간 배양한 결과, 배반포형성률은 각각 17.9$\pm$6.1, 17.4$\pm$6.3, 24.2$\pm$l.9 및 16.9$\pm$2.0%로서 50$\mu$M의 cysteamine 처리구가 유의적(P＜0.05)으로 높은 결과를 나타냈고, 총세포수에 있어서도 각각 30.7$\pm$2.4, 34.9$\pm$2.8, 39.6$\pm$2.3 및 36.8$\pm$3.6개로서 50$\mu$M의 cysteamine 처리구가 유의적(P＜0.05)으로 높은 결과를 나타냈다. 3. 작성된 배반포기 수정란의 총세포수에 대한 내부세포괴세포(ICM/total cells)가 20~40%범주에 드는 비율은 각각 20.5, 41.6, 19.5 및 31.8로서 25$\mu$M 처리구가 가장 높은 성적을 나타냈다. 4. 체외에서 생산된 초기 수정란을 배발달배양액인 NCSU-23에 cysteamine을 각각 0, 25, 50 및 100$\mu$M 첨가하여 7일간 배양한 결과, 배반포기 도달률은 각각 16.0$\pm$0.2, 13.6$\pm$1.7, 25.0$\pm$0.8 및 15.7$\pm$4.5%로서 50$\mu$M의 cysteamine 처리구가 유의적(P＜0.05)으로 높은 결과를 나타냈고, 총세포수에 있어서는 각각 27.0$\pm$3.7, 36.1$\pm$4.8, 34.0$\pm$3.8 및 25.2$\pm$4.4개로서 25$\mu$M의 cysteamine 처리구가 유의적(P＜0.05)으로 높은 결과를 나타냈다. 5. 작성된 배반포기 수정란의 총세포수에 대한 내부세포괴세포(ICM/total cells)가 20~40% 범주에 드는 비율은 처리구가 대조구보다 낮은 결과를 나타냈다. 결론적으로 돼지난포란을 이용하여 체외성숙을 유기할 때 효과적인 cysteamine의 농도는 50$\mu$M이 적당하며, 초기배발달을 유기할 때의 효과적인 cysteamine의 농도는 25~50$\mu$M인 것으로 판단된다.

• Journal of Animal Science and Technology
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• 제55권4호
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• pp.249-255
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• 2013

Intercellular interactions are important for the proper development and regulation of tissue function. This is especially necessary in the epididymis, a part of male reproductive tract where sperm become mature and acquire their fertilizing capacity. The caput region of the epididymis consists of several types of cells, including principal, basal, and apical cells. Direct intercellular communication is thus required to precisely regulate the functions of the caput epididymis. In this regard, connexin (Cx) is a molecule that forms channels, which allow the direct exchange of small molecules between cells, enabling intercellular communication. In this study, the expression of Cx isoforms in the caput epididymis at different postnatal ages was determined by using quantitative real-time polymerase chain reaction analysis. Nine of 13 Cx isoforms were detected. The transcript levels of Cx30.3, 31, 31.1, 32, and 40 were highest at 45 days of age, while the expression of Cx43 and 45 gradually decreased with age. A substantial fluctuation of Cx26 expression was detected, with significant decreases before and during puberty, followed by a transient increase at adult-hood and rapid decreases at an old age. A significant increase in Cx37 transcript was observed at 25 days of age, followed by gradual decreases at adult and old ages. These results indicate the significant differential expression of various Cx isoforms in the caput epididymis during postnatal development. It further suggests that the functional regulation and developmental maturation of the caput epididymis are highly related to the postnatal age-related differential expression of Cx isoforms.

• 한국발생생물학회지:발생과생식
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• 제17권3호
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• pp.289-297
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• 2013

Bisphenol A (BPA) is an estrogenic endocrine disrupter. However, depending on a way of treatment, the harmful effects of BPA have not been confirmed. Also, trans-generational effects of BPA on male reproduction are still controversial. Because the reabsorption of testicular fluid in the efferent ductules (ED) and initial segment (IS) is important for sperm maturation, the present study was designed to determine trans-generational effect of BPA administrated orally on expression of water transport-related molecules in the mouse ED and IS. Ethanol-dissolved BPA was diluted in water to be 100 ng (low), $10{\mu}g$ (medium), and $1mg/m{\ell}$ water (high). BPA-containing water was provided for two generations. Expression of ion transporters and water channels in the ED and IS were measured by relative real-time PCR analysis. In the ED, BPA treatment caused expressional increases of carbonic anhydrase II, cystic fibrosis transmembrane regulator, $Na^+/K^+$ ATPase ${\alpha}1$ subunit, and aquaporin (AQP) 1. No change of $Na^+/H^+$ exchange (NHE) 3 expression was detected. BPA treatment at medium dose resulted in an increase of AQP9 expression. In the IS, the highest expressional levels of all molecules tested were observed in medium-dose BPA treatment. Generally, high-dose BPA treatment resulted in a decrease or no change of gene expression. Fluctuation of NHE3 gene expression by BPA treatment at different concentrations was detected. These findings suggest that trans-generational exposure to BPA, even at low dose, could affect gene expression of water-transport related molecules. However, such effects of BPA would be differentially occurred in the ED and IS.

• Journal of Animal Science and Technology
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• 제50권1호
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• pp.45-56
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• 2008

본 연구는 생후 발달 과정 동안 monocarboxylate transporter(MCT) isoform과 MCT의 발현 조절 단백질로 알려진 basigin(Bsg)과 embigin의 mRNA 발현을 흰쥐의 부정소에서 부위별로 real-time PCR 방법을 사용하여 알아보았으며, 에스트로젠과 에스트로젠 수용체 α의 작용에 의해 MCT1 발현이 조절되는지를 알아보기 위해 estrogen receptor α knockout(αERKO) 마우스를 이용하여 immunohistochemistry 방법을 통해 탐구하였다. 본 연구 결과는 다양한 MCT isoform(MCT1, 2, 3, 4와 8), Bsg과 embigin의 mRNA 발현이 부정소의 부위별로 연령에 따라 다르게 나타나며, 부정소에서 MCT1 단백질 발현은 corpus와 caudal 부위에서 apical 지역에 한정되어 나타나는 것을 보여 주었다. 또한 부정소에서 MCT1 단백질 발현은 에스트로젠 수용체 α의 존재 여부와 상관 없음이 보여졌다. 따라서, 본 연구는 MCT가 남성 생식기관인 부정소에서 정자 성숙과 저장을 위한 적절한 환경을 형성함으로써 남성 생식력의 유지에 관여 할 수 있음을 시사한다. (색인어 : Epididymis, Monocarboxylate transporter, Basigin, Embigin)

• Reproductive and Developmental Biology
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• 제33권1호
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• pp.55-61
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• 2009

The epididymis in the male reproductive tract is the site where spermatozoa produced from the testis become mature. The epididymis is divided into 4 different segments, initial segment and caput, corpus, and caudal epididymis, depending upon functional and morphological features. Aquaporins (Aqps) are water channel molecules, which are present in the epididymis and play a major role in removal of epididymal water, resulting in creation of microenvironment for sperm maturation and concentration of sperms. Nandrolone decanoate (ND) is a synthetic anabolic-androgenic steroid, which is used to treat clinical diseases and improve physical ability and appearance. Even though it is well determined that the ND causes the male infertility by affecting the testis, little is known the effect of the ND on the epididymis. The present study was focused to examine the effect of ND at different treatment doses and periods on expression of Aqp1 and Aqp9 genes in the epididymis of pubertal rats. Results showed that mRNA expression of Aqp1 and Aqp9 genes among the parts of the epididymis was differentially regulated by ND treatment doses. In addition, treatment periods of ND caused differential expression of Aqp1 and Aqp9 mRNAs among segments of the epididymis. Therefore, it is believed that male infertility induced by ND could be resulted not only from malfunction of the testis but also from aberrant gene expression of Aqp1 and Aqp9 in the epididymis.

• 한국발생생물학회지:발생과생식
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• 제2권1호
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• pp.69-74
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• 1998

부화 후부터 평균전장 0.64 cm를 나타내는 부화 후 150일까지의 버들치, Rhynchocypris oxycephalus를 대상으로 초기생식소 발달과 성분화를 조사하였다. 시언생식세포는 평균전장 0.64cm 자어에서 뚜렷이 나타나\ulcorner. 평균전장 1.91 cm자어에서 시원생식세포는 복강으로돌출되었으며, 평균전장 2.29 cm 자어에서 시원생식세포는 감수분열 난모세포로 전환되었고, 난소로의 분화가 최초 확인되엇따. 평균전장 5.96 cm 자어에서 암컷 생식소는 점진적으로 발달하였으며 성숙단계로 접어드는 핵이동 난모세포를 보였다. 성분화 후 난모세포는 빠르게 증식하는 반면 정소는 평균전장 4.00cm 자어까지는 성장이 중지된 채 증가만 하는 휴지상태이었다. 평규전장 4.00cm 자어에서의 정모세포는 중간기에서 발달이 정지되었으며, 감수분열이 활발하였고, Sertoli-like cell과 정소관이 형성되었다. 본 연구 결과 버들치의 성본화 양상은 분화형 장웅이체 (differentiated gonochorism)인 것으로 나타났다.

• Asian-Australasian Journal of Animal Sciences
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• 제29권12호
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• pp.1702-1709
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• 2016

The reproductive function of G-protein subunit Galphaq (GNAQ), a member of the G protein alpha subunit family, has been extensively studied in humans and rats. However, no data is available on its status in ruminants. The objectives of this study were to evaluate the expression pattern of the GNAQ in the testis and epididymis of sheep by polymerase chain reaction (PCR). The mRNA expression levels were detected by real-time fluorescent quantitative PCR, and cellular localization of GNAQ in the testis and epididymis was examined by immunohistochemistry. Additionally, GNAQ protein was qualitatively evaluated via western blot, with the results indicating that similarities between GNAQ mRNA levels from sheep was highly conserved with those observed in Bos taurus and Sus scrofa. Our results also indicated that GNAQ exists in the caput and cauda epididymis of sheep, while GNAQ in the testis and epididymis was localized to Leydig cells, spermatogonial stem cells, spermatocytes, Sertoli cells, spermatid, principal cells, and epididymis interstitial cells. The concentrations of GNAQ mRNA and protein in the caput and cauda epididymis were significantly greater than those observed in the corpus epididymis (p<0.01) and testis (p<0.05). Our results indicated that GNAQ exists at high concentrations in the caput and cauda epididymis of sheep, suggesting that GNAQ may play an important role in gonad development and sperm maturation.

• BMB Reports
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• 제41권9호
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• pp.664-669
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• 2008

Znf230, the mouse homologue of the human spermatogenesis-related gene, ZNF230, has been cloned by rapid amplification of cDNA ends (RACE). This gene is expressed predominantly in testis, but its expression in different testicular cells and spermatogenic stages has not been previously analyzed in detail. In the present study, the cellular localization of the Znf230 protein in mouse testis and epididymal spermatozoa was determined by RT-PCR, immunoblotting, immunohistochemistry and immunofluorescence. It is primarily expressed in the nuclei of spermatogonia and subsequently in the acrosome system and the entire tail of developing spermatids and spermatozoa. The results indicate that Znf230 may play an important role in mouse spermatogenesis, including spermatogenic cell proliferation and sperm maturation, as well as motility and fertilization.

• 한국발생생물학회지:발생과생식
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• 제18권4호
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• pp.293-300
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• 2014

Direct cell-cell communication through connexin (Cx) complexes is a way to achieve functional accordance of cells within a tissue or an organ. The initial segment (IS), a part of the epididymis, plays important roles in sperm maturation. Steroid hormones influence on expression of a number of genes in the IS of adult animals. However, developmental effect of sex hormones on the gene expression in the IS has not been examined. In this study, estradiol benzoate (EB, an estrogen agonist) or flutamide (Flu, an androgen antagonist) was exogenously administrated at 1 week of postnatal age, and expressional changes of Cx genes in the IS were determined at 4 months of age by a quantitative real-time PCR analysis. Treatment of EB at $0.015{\mu}g/kg$ body weight (BW) increased expression of Cx30.3, 31.1, and 43 genes. However, treatment of 1.5 mg EB/kg BW resulted in expressional decreases of Cx31, 32, and 45 genes and caused increases of Cx30.3 and 43 gene expression. Significant decreases of Cx31, 31.1, 32, 37, and 45 gene expression were detected with a treatment of $500{\mu}g\;Flu/kg$ BW, while expression of Cx43 gene was significantly increased with a treatment of $500{\mu}g\;Flu/kg$ BW. A treatment of $50{\mu}g\;Flu/kg$ BW led to significant increases of Cx30.3, 32, 37, 40, and 43 gene expression. These findings imply that exogenous exposure of steroidal hormones during the early developmental period would result in aberrant expression of Cx genes in the adult IS.