• Applied Biological Chemistry
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• v.49 no.4
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• pp.293-297
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• 2006

Angiotensin converting enzyme(ACE) is a zinc-containing dipeptidase widely distributed in mammalian tissues and is thought to play a significant role in blood pressure regulation by hydrolyzing angiotensin I to the potent vasoconstrictor, angiotensin II. Recently, the presence of ACE in pig ovary was reported and the ACE from pig kidney was isolated and characterized. However no nucleotide sequence of the ACE gene from pig is yet known. We report here the cloning of the ACE cDNA from pig kidney by using the reverse transcriptase-polymerase chain reaction. The complete amino acid sequence deduced from the cDNA contains 1309 residues with a molecular mass of 150 kDa, beginning with a signal peptide of 33 amino acids. Amino acid sequence analysis showed that pig kidney ACE is also probably anchored by a short transmembrane domain located near the C-terminus. This protein contains a tandem duplication of the two homologous amino acid peptidase domain. Each of these two domains bears a putative metal-binding site (His-Glu-Met-Gly-His) identified in mammalian somatic ACE. The alignment of pig ACE amino acid sequence with human, rabbit, and mouse reveals that both two domains have been highly conserved during evolution.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.1
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• pp.23-30
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• 2019

Objective: Recent studies have demonstrated that lin-28 homolog B (LIN28B)/miRNA let-7 (let-7) plays a role in the regulation of pubertal onset in mammals. However, the role of LIN28B/let-7 in the onset of ovine puberty remains unknown. We cloned the Duolang sheep Lin28B cDNA sequence, detected the expression change of LIN28B, let-7a and let-7g in hypothalamus, pituitary and ovary tissues at three different pubertal stages. Methods: The reverse transcriptase polymerase chain reaction (RT-PCR) was used to clone the cDNA sequence of LIN28B gene from Duolang sheep and the bioinformatics methods were applied to analyze the amino acid sequence of LIN28B protein. The mRNA expression levels of the LIN28B gene at different pubertal stages were examined by real time RT-PCR. Results: LIN28B cDNA of Duolang sheep was cloned, and two transcripts were obtained. The amino acid sequence of transcript 1 shares 99.60%, 98.78%, and 94.80% identity with those of goat, wild yak and pig, respectively. Strong LIN28B mRNA expression was detected in the hypothalamus, pituitary, ovary, oviduct and uterus, while moderate expression was found in the liver, kidney, spleen and heart, weak expression was observed in the heart. No expression was found in the lungs. Quantitative real-time PCR (QPCR) and western-blot analysis revealed that the LIN28B was highly expressed in the hypothalamus and ovary at prepuberty stages, and this expression significantly decreased from the prepuberty to puberty stages (p<0.05). Markedly increased levels of mRNA expression were detected in the pituitary from prepuberty to puberty (p<0.05) and then significantly decreased from puberty to post-puberty (p<0.05). The expression levels of let-7a and let-7g showed no significant changes among different pubertal stages (p>0.05). Conclusion: These results provided a foundation for determining the functions of LIN28B/let-7 and their role in the onset of sheep puberty.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.43-43
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• 2003

This study was conducted to produce transgenic pig harboring human tissue plasminogene activator (tPA) gene. Two different tPA genes containing bovine $\beta$-casein promoter and mouse uroplakin promoter were prepared for microinjection and confirmed the expression level of tPA protein from the CHO (Chinese hamster ovary) cell lines by gene transfection. Concentration of tPA expression from the six cell lines (all of CHO cells) were average 212.4 ng/ml. Reconstructed DNA to used the CHO cell were microinjected into the pronuclei of in vivo embryos The total of 2,307 zygotes were collected from 95 donors and 1,851 embryos were in 1-cell stage which were visualized the pronuclei for DNA microinjection. The concentration of linear DNA was 2.0 ng per microliter and injected into zygotes with two pronuclei on an inverted Nikon microscope equipped with narishige micromanipulator and modulation contrast optics. The 541 embryos injected with bovine $\beta$-casein promoter-tPA were transferred to 22 recipients. The 1,154 embryos injected with mouse uroplakin promoter-tPA were transferred to 51 recipients. Sixty nine offspring from 9 delivered sows were produced. We analysed the transgenes with PCR methods from 69 offsprings, but could not detect the PCR product from piglet tails DNA.

• Reproductive and Developmental Biology
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• v.33 no.3
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• pp.183-187
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• 2009

This study analyzed change of proteins in granulosa cells during the porcine follicuar development by proteomics techniques. Granulosa cells of the follicles, of which the diameter is $2{\sim}4\;mm$ and $6{\sim}10\;mm$, were collected from ovary of slaughtered pig that each follicle of diameter $1{\sim}4\;mm$ and $6{\sim}10\;mm$. We extracted glanulosa cell proteins by M-PER Mammalian Protein Extraction Reagent. Proteins were refined by clean-up kit and quantified by Bradford method until total protein was $200{\mu}l$. Immobilized pH gradient(IPG) strip used 18 cm, $3{\sim}10\;NL$. SDS-PAGE used 10% acrylamide gel. After silver staining, Melanie 7 and naked eye test were used for spot analyzation. Increasing proteins in glanulosa cell of $6{\sim}10\;mm$ follicle were 7 spots. This spots were analyzed by MALDI-TOF MS and searched on NCBInr. In results, 7 spots were similar to zinc/ling finger protein 3 precursor (RING finger protein 203), angiomotin, heat shock 60 kDa protein 1 (chaperonin) isoform 1 (HSP60), similar to transducin-like enhancer protein 1 (TLE 1), SH3 and PX domains 2A (SH3PXD2A). Those proteins were related with transfer between cells. Increase of proteins has an effect on follicular development.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.3
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• pp.258-264
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• 2020

This study investigated the effect of variation in the number of somatic-cell-cloned embryos and their developmental stage at transfer on pregnancy, as well as the influence of the estrus status of recipient pigs on in vivo development of cloned porcine embryos after embryo transfer. For somatic cell nuclear transfer (SCNT), fibroblast cells were obtained from a male porcine fetus. Recipient oocytes were collected from prepubertal gilts at a local abattoir and then cultured. After SCNT, reconstructed embryos of different numbers and developmental stages were transferred into recipient pigs. The developmental stage of the cloned embryos and the number of transferred embryos per surrogate showed no significant differences in terms of the resulting cloning efficiency. However, the pregnancy rate improved gradually as the number of transferred cloned embryos was increased from 100-150 or 151-200 to 201-300 per recipient. In pre-, peri-, and post-ovulation stages, pregnancy rates of 28.6%, 41.8%, and 67.6% and 16, 52, and 74 offspring were recorded, respectively. The number of cloned embryos and estrus status of the recipient pig at the time of transfer of the cloned embryo affect the efficiency of pig production; therefore, these variables should be particularly considered in order to increase the efficiency of somatic cell pig cloning.

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.196-196
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• 2004

By use of RT-PCR coupled with DHPLC technique (WAVE analysis), pattern of isoforms of porcine cytochrome P450 aromatase gene was investigated. Relatively higher expression of aromatase mRNA was observed in testis than in ovary and this result accounted for the previous findings of higher blood estrogen level in male compared with female in this species. (omitted)

• Journal of Embryo Transfer
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• v.7 no.2
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• pp.111-115
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• 1992

This experiment was carried out to study the behavior of the estrus cycle in sows shortened uterine horns and to see whether the embryos could be recovered nonsurgically. The uteri of sows(n=4) were surgically shortened. It took about 3 hours to surgically remove the middle section of both uterine horns. The lengths of the shortened uterine horns were 18.7 to 29.5cm. After treatment, two sows exhibited natural estrus and the intevals from surgery to estrus were 8 days and 15 days, respectively. But the sows were not successful on synchronization and superovulation with PMSG and PGF$_2$$\alpha$. In the resurgery for confirmative examination, the sows had 6 and 7 corpus lutelin in ovaries, respectively. One sow had a small adhesion between the infundibulum and ovary, and the other sow had unilateral uterine obstruction at the sutured position and purulent materials in the uterus.

• Journal of Animal Science and Technology
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• v.46 no.3
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• pp.315-324
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• 2004

Cytochrome P450 aromatase is the enzyme responsible for biosynthesis of female sex hormone(estrogen) and 19-nortestosterone(nandrolone), a unique steroid hormone endogenously synthesized in the pig. By use of RT-PCR coupled with DHPLC technique (WAVE analysis), expression pattern of isoforms of porcine cytochrome P450 aromatase gene was investigated. Relatively higher expression of aromatase mRNA was observed in testis than in ovary and this result accounted for the previous findings of higher blood estrogen level in male compared with female in this species. The result from the DHPLC demonstrated that PCR amplified DNA fragments of ovary and testis tissues. using unique PCR primers for all three types of aromatase genes, were different from those of type II and ill genes. Further nucleotide sequence analyses of the plasmid clones containing the PCR products revealed that nucleotide sequences of all clones were identical to type I aromatase gene(ovary type). Thus, the result from the present study indicates that the ovary and testis express the same type of aromatase gene. Therefore, the efficacy of DHPLC techniques used for this study helped us to analyze tissue-specific expression of isoform of genes containing the nucleotide sequences with high homology.

• The Korean Journal of Zoology
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• v.33 no.1
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• pp.63-69
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• 1990

To study the follicular atretic mechanism in mammalian ovary, the medium-sized (3.0-6.0mm) follicles of porcine ovary were morphologically classified as nonnal and atretic. Steroid concentrations in the follicular fluid were analyzed by radioimmunoassay, and the proteins in that fluid were electrophoretically separated. Concentrations of progesterone in the atretic follicular fluid of follicular phases were higher than those of normal ones (p < 0.05). Concentrations of testosterone were high in normal luteal and atretic follicular-phases follicles. The concentrations of estradiol remained significanily lower in atretic follicular-phases follicles than normal. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis of follicular fluid proteins, four kinds of proteins (20K, 32K, 33K, 38K) were detected, and those proteins were not present in sera. According to the ovarian cycle, proteins of MW of 112K and 141K were identified. In atretic follicular fludies, MW of 23K and 24K were specifically detected. From the above results, we can conclude that, as ovarian cycle changes, steroid contents and protein composition in atretic follicular fluid are different from the normal developing follicular fluid. To further understand the physiologic roles of the proteins present in the atretic follicular fluids, these proteins need to be characterized and identified.

• Korean Journal of Animal Reproduction
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• v.17 no.2
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• pp.159-164
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• 1993

The tissue levels of ornithine decarboxylase (ODC) during the estrous cycle and pregnancy were investigated in the pig. Sexually mature female cycling pigs were used. One animal was sacrificed on estrous cycle days 3, 10, 17, 18, 19, 20 and during pregnancy on day 11. 12, B. 14, 18, 19, 20, 48, 50 and 52. Tissues from the hypothalamus, pituitary, uterus, ovary and skeletal muscle were removed. They were homogenized in buffer, and supernatants were used for measurement of protein concentration and ODC activity. The release of $^14$CO$_2$ from radiolabeled ornithine was proportional to the amout of protein added over the range of 0.125~4mg and to the incubation time. ODC appered to have some relationship with the biological functions of the pituitary, ovary and uterus during the reproductive period, especially on day 19 of the estrous cycle, while it showed no such activities in hypothalamus and skeletal muscle of mature pigs. Uterine tissues had significantly more ODC activity than other tissues tested(p < 0.05).