Glycoprotein hormones have a common α-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 α-subunit of these glycoprotein hormones, a tethered-molecule was constructed by fusing the NH2-terminus of the α-subunit to the COOH-terminus of the β-subunit of equine chorionic gonadotropin (eCG). The following deletion mutants were created by PCR; Ile was inserted at position 96 to form Δ96, Lys was substituted at position 95 to form Δ95, His was inserted at position 93 to form Δ93 and Tyr was substituted at position 87 to form Δ87. Each mutant was transfected into CHO-K1 cells. Tethered-wt eCG, and Δ96, Δ95, and Δ93 mutants were efficiently secreted into the medium but the Δ87 mutant was not secreted. Interestingly, the RT-PCR, real-time PCR, and northern blot analyses confirmed that the RNA was transcribed in the Δ87 mutant. However, the Δ87 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 Δ95 and Δ93 mutants were completely inactive in both the LH- and FSH-like activity assays. The Δ96 mutant showed slight activity in the LH-like activity assay. In comparison to the wild type, the activity of the Δ96 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 α-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 α-subunit is very important for the secretion and functioning of this hormone.

Equine chorionic gonadotropin (eCG) is a heavily glycosylated glycoprotein composed of non-covalently linked α- and β-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 (Δ1, Δ2, Δ3, Δ4, and Δ5) 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 (Δ1, Δ4, and Δ5) were transcripted, but not translated into proteins. Rec-eCG Δ2 was secreted in much lower amounts than the wild type. Only the rec-eCG Δ3 (β-subunit: Gln94-Ile95-Lys96→Ala94-Ala95-Ala96) was efficiently secreted. Although activity is low, its LH-like activity was similar to that of tethered eCGβα. However, the FSH-like activity of rec-eCGβαΔ3 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 β-subunit appear to be of utmost importance for signal transduction of the FSH receptor.

The miniature pig is considered to be a better organ donor breed for xenotransplantation than other pig breeds because the size of the organs of the miniature pig is similar to that of humans. In this study, we aimed at identifying differentially expressed genes in the miniature pig ovary during pregnancy. For this, we used the miniature pig ovary model, annealing control primer‐based reverse transcription polymerase chain reaction (PCR), quantitative real‐time PCR (qRT‐PCR), and northern blotting analysis. We identified 13 genes showing differential expression on the based of pregnancy status and validated 8 genes using qRT‐PCR. We also sequenced the full‐length cDNA of ephrin receptor A4 (EphA4), which had a significant difference in expression level, and validated it by northern blotting. These genes may provide a better understanding of the cellular and molecular mechanisms during pregnancy in miniature pig ovary.

The glycoprotein hormone family represents a class of heterodimers, that includes the placental hormone equine chorionic gonadotropin (eCG) and the anterior pituitary hormones‐ follitropin (FSH), lutropin (LH), and thyrotropin (TSH). The 4 hormones are heterodimers, with a common α‐subunit and unique β‐subunits. eCG is the most heavily glycosylated of the known pituitary and placental glycoprotein hormones. Recent observations using single chain glycoprotein hormone analogs in which, the β‐and α‐subunits are linked, implied that heterodimeric‐like quaternary configuration is not a prerequisite for receptor/signal transduction. To study the function and signal transduction of tethered rec‐eCG, a single chain eCG molecule was constructed and rec‐eCG protein was produced. Molecular mass of the single chain is about 45 kDa. All mice were ovulated by tethered rec‐eCG treatment. The dual activity of tethered rec‐eCG was determined in receptor cell lines of nonequid species; in fact, this dual activity was proven in species other than horse. Tethered rec‐eCG in equids does not bind to FSH receptors, suggesting that eCG is primarily an LH‐like hormone in the horse. Taken together, these data suggest that tethered rec‐eCG has dual activity in nonequid species in vitro. However, it has only LH‐like activity in equid species in vitro.

Placenta is the main nutrition source for the fetus during pregnancy. Thus, it has a pivotal function in the pregnant process. Many functions of the placenta have been elucidated. An abnormal placenta is associated with a high rate of pregnancy failure in somatic cloned bovine. Differentially expressed genes (DEGs) were examined in a comparison between normal and cloned bovine placenta using annealing control primer (ACP)-based GeneFishing PCR. Using 120 ACPs, nearly 80 genes were identified and the fragments of 42 DEGs were sequenced. 38 of these genes were known genes and four were unknown. To determine the DEGs result, six target clones expressing on one-side of a normal and a clone placenta were selected. Through an analysis of the target genes using the real-time PCR, the expressing pattern was found to be somewhat different from the DEGs. Additionally, several genes appeared with the same expression pattern. Taken together, this suggests that the target genes would be essential for research into what influences the placental formative mechanisms during fetal development.

This study was conducted to investigate the specific expression genes in the cloned bovine tissues. Donor cells, cloned tissues were analysed by RAPD-RFLP method. The results were detected three genes (CH-U7B, CH-U7M and CH-U7P) in the cloned fetus. It was found a single copy genes by southern hybridization. Sequence analysis of CH-U7M gene was shown 99% homology to a previously reported EST from a cloned bovine fetus. The putative ORF was encode a protein of hydrophobicity index 0.03. Semi-quantitative RT-PCR by using the CH-LS001 specific primer was remarkably detected in the lung tissue of cloned fetus. Further investigation of these genes may provide one of the key information to explain the early death, abnormal fetus, large off-spring and the low pregnancy rate in the production of cloned bovine.

This study was conducted to compare the expression pattern of the specific factors associated with pregnancy and angiogenesis during early pregnancy in Hanwoo. Synchronized female Hanwoo (4～6 year‐old) were inseminated artificially. After 10 weeks after artificial insemination (AI), the pregnancy was tested by rectal palpation method. Three pregnant and non‐pregnant Hanwoo were used in this experiment, respectively. The plasma progesterone level was measured by ELISA. Western blot analysis was performed to detect the expression of pregnancy associated glycoprotein (PAG) or angiogenic factors (VEGF, B‐FGF, ANP‐1, and TIE‐2). The plasma P4 level was increase gradually in pregnant group and maintained high level. The concentration of PAG was significantly higher from 5th weeks in pregnant group compared to that of non‐pregnant group (p<0.05). The concentrations of the VEGF (p<0.05), B‐FGF (p<0.05), and ANP‐1 (p<0.05) were significantly increased from 6th or 7th week after AI in pregnant group, respectively. And the intensity of TIE‐2, ANP‐1 receptor, was well matched with ANP‐1 (p<0.05). Taken together, it can be postulated that the blood vessels connected with fetus and dam were formed dramatically around 40 days after AI, because the expression levels of the angiogenic factors were increased significantly from this time in pregnant Hanwoo.

본 연구는 한우 체세포를 이용하여 생산된 복제란을 한우 대리모에 이식하여 임신이 확인된 개체에서 임신 기간 중 주요 호르몬의 발현 특성을 인공수정으로 임신된 대리모와 비교 분석하고자 실시하였다. 한우 섬유아세포를 이용하여 생산된 체세포 복제란을 자연발정으로 동기화된 한우 대리모에 이식하여 임신이 확인된 개체를 공시하였으며(n=8), 대조군으로는 인공수정으로 임신된 대리모을 사용하였다(n=5). 발정 관찰 후 60일경에 직장검사로 임신을 확인하였다. 주요 스테로이드 호르몬인 progesterone(P4)와 estradiol-l7 (E2) 농도는 방사선동위원소 면역분석시험(RIA) 방법을 이용하였으며, 혈중 cortisol 농도는 ELISA 방법으로 측정하였다. 인공수정한 대리모의 경우 E2 농도가 분만 시기에 급격하게 증가하였으나, P4 농도는 분만 시기에 급격하게 감소하는 경향을 나타내었다. 이에 반해 복제란 이식우의 혈장 P4 농도는 분만 50일 전부터 분만 10일전까지는 대조군과 유사하게 유지되었으나, 분만예정일에는 전혀 떨어지지 않고 높은 수준으로 유지되었다. 한편, 복제란 이식우에서 분만 때까지 정상적으로 임신이 유지된 대리모들의 경우는 임신 기간 동안 cortisol 농도는 임신 후반기까지 낮게 유지되며 별다른 변화를 나타내지 않았다. 반면에 유산이 일어난 개체의 경우에는 임신 100일경에 cortisol의 농도가 급격하게 증가하는 것을 확인하였다. 이상의 결과를 종합하여 보면, 복제란 이식우의 경우 분만예정일 전 후에 일어나는 급격한 호르몬의 변화가 일어나지 않음을 확인할 수 있었으며, 이러한 현상은 복제란 이식우의 분만 지연과 밀접한 관계가 있는 것으로 사료된다.

본 연구는 돼지에 있어서 정원줄기세포를 포함하는 정소세포를 recipient 돼지의 정소 내로 이식할 수 있는 기법을 개발하기 위하여 시행되었다. 공여세포는 10~14일령의 돼지로부터 채취된 정소에서 효소처리법을 이용하여 회수하였고, recipient의 정소 내로 이식하기 전 형광 마커(PHK26)로 표지하였다. 외과적 수술을 통하여 recipient 돼지부터 정소를 꺼낸 후 초음파 기기와 이식 장치를 이용하여 형광표지된 공여세포를 recipient 정소의 세정관 내로 이식하였다. 14주령의 recipient 정소에 5~7ml 의 공여 세포부유액을 주입하여 정소 내 50% 이상의 세정관 내로 새포부유액의 주입이 가능하였고, 세포부유액이 주입된 세정관 내에서 형광표지된 정소세포들이 고루 이식되어짐이 관찰되었다. 본 연구에서 개발한 이식 기법을 이용하여 효율적인 정소세포의 이식이 가능함에 따라 향후 돼지 정원줄기세포의 연구 및 활용법 개발에 획기적인 돌파구가 마련될 것으로 기대된다.

DNA 메틸화는 조직특이적인 유전자 조절에 관여하고, 정상적인 배 발달에 필수적이다. POU5F1은 octamer-binding transcription factor 4 (Oct-4)를 encode하며, 초기 분화에 중요한 전사인자이다. 본 실험에서 소의 Oct-4가 조직특이적이고 발달의존적인 epigenetic 표지 인지를 검토하고자, 착상 전 수정란에서 Oct-4 전사산물과 상류 promoter 영역의 CpGs의 메틸화를 조사하였다. Oct-4 전사산물은 정자 그리고 2-cell에서 8-cell 수정란까지 낮은 수준으로 존재하지만, 상실배와 배반포에서 높게 검출되었다. 이러한 결과는 배 발달 과정의 상실배 단계에서 Oct-4의 de novo 발현이 시작됨을 의미한다. Oct-4 상류 promoter 영역에는 메틸화 가변 영역 (tissue-dependent differentially methylated region, T-DMR)이 존재한다. Oct-4 메틸화 가변 영역의 메틸화 상태는 정자, 성체 체조직과 난자에서 서로 다르고, 수정란으로부터 배반포 단계까지 변화하였는데, 이는 착상 전 초기 배 발달 과정에 active 메틸화와 탈메틸화가 일어남을 의미한다. 이상의 결과, Oct-4 유전자 상류 promoter 영역은 DNA 메틸화의 타깃이고, 그 메틸화 상태는 소 수정란 발달 동안에 다양하게 변화한다.

본 연구는 체세포 복제란 이식우의 분만에 있어서 혈중 스테로이드호르몬, TGF-β1 농도와 분만지연의 상관 관계를 살펴보고자 실시하였다. 대조군으로는 인공수정(AI)을 통하여 임신한 암소(cow)들을 사용하였다(AI-R). 모든 AI-R들은 자연분만(n=5, 임신 284+-0.71일)을 하였다. 분만징후를 보이지 않는 체세포 복제란 이식우(n=5, SCNT-R)들은 분만 예정일보다 10일 정도 지난 임신 292일째에 제왕절개(Caesarean section, C-sec)를 실시하여 분만하였다. 혈액 및 태반 샘플을 분만 전.후에 채취하여 형태 및 중량 등을 측정하였다. 혈장호르몬인 Progesterone(P4)와 Estradiol-17β (E2) 농도는 방사선동위원소 면역 분석 시험(RIA) 방법을 이용하여 측정하였다. 혈장 및 태반분엽의 TGF-β1 농도는 ELISA 방법으로 측정하였다. SCNT-R에서 회수한 태반의 무게는 AI-R의 것과 비교하여 유의적으로 무거웠다(p<0.05). 분만 직전 SCNT-R들의 혈장 내 P4 농도는 AI-R들의 그것과 비교하여 유의하게 높았다(p<0.01). 하지만 SCNT-R들의 혈장 내 E2 농도는 AI-R과 비교하여 상대적으로 낮게 나타났다(p<0.01). 한편, 분만 전.후 SCNT-R들에서 혈장 또는 태반분엽의 TGF-β1 단백질 발현 수준은 AI-R들과 비교하여 각각 유의적으로 높은 수준을 유지하였다(p<0.01). 이상의 결과를 종합하여 보면, 분만 시 P4 및 E2의 이상 발현과 높은 수준의 혈장 및 태반 내 TGF-β1 단백질은 체세포 복제태아의 분만지연을 야기하는 중요한 요인들 중의 하나일 것이라 사료된다.

DNA 결합 단백질 억제(Inhibitor of DNA binding protein) 또는 분화억제(Inhibitor of differentiation) 인자인 Id는 네 종류(Id1-4)가 있으며, 세포의 증식과 분화, 혈관 형성 및 세포자가사멸 등에 정 또는 부의 조절인자로서 널리 알려져 있다. 하지만 아직까지 번식생리 분야에서 이들 유전자들의 발현이나 기능에 관해 알려진 것은 거의 없다. 따라서 본 연구에서는 쥐 난소에서 난포의 발달에 따른 Id3 mRNA의 발현 양상을 살펴보고자 실시하였다. 쥐에게 PMSG 주사 후, 3, 6, 12, 24, 36 및 48시간째에 난소를 회수하여 고정, 탈수 및 파라핀 포매를 실시하였다. In situ hybridization 실험을 위하여 anti-sense와 sense Id3 cRNA 탐침자를 제작한 다음 난소 절편에 반응시켰다. 반응이 끝난 난소 절편은 NTB-2 유광제에 노출시킨 후 현상액에 반응시켰다. 모든 처리가 끝난 슬라이드는 H&E 염색을 실시한 다음 현미경하에서 hybridization 감도를 1+에서 4+로 구분하여 평가하였다. 난자에서는 Id3 mRNA 감도가 원시와 제1차 난포에서 ≥2+으로 확인되었으나, 제2차, 우성 및 배란전 난포에서는 1+ 이하로 관찰되었다. 한편, 과립막세포에서는 우성과 배란 전 난포에서 Id3 mRNA가 3+ 또는 4+로 강하게 발현되는 것을 확인하였다. 이상의 결과를 종합하여 보면, Id3 mRNA는 난포의 발단 단계 또는 난포세포에 따라 특이적으로 발현하는 것으로 보아 난포의 발달과 밀접한 연관이 있을 것으로 사료된다.