Survival of mouse embryos frozen to -196 deg and -269 deg C

This study was undertaken to develop novel markers for chicken primordial germ cells (PGCs), which are of potentially enormous value in transgenic research. Gonadal cells collected from 5.5-day-old chicken embryos were cultured in a Dulbecco's minimal essential medium and the PGC colonies formed during the primary culture period were subcultured three times. Characterization of the PGCs with the candidate marker reagents was performed on the mixed cell population 2 hours after seeding, after the primary culture period (day 10), and after the third passage (day 40). Mouse embryonic stem (ES) cells were used as controls. The cytochemical reagents investigated included periodic acid-Schiff (PAS) stain, antibodies to stage-specific embryonic antigens (SSEA-1, SSEA-3, and SSEA-4), antibody to epithelial membrane antigen (EMA)-1, antibodies to integrins alpha6 and beta1, several lectins (Solanum tuberosum agglutinin [STA], Dolichos biflorus agglutinin [DBA], concanavalin A agglutinin [ConA], and wheat germ agglutinin [WGA]), and double staining with antibodies to SSEA-1, SSEA-3, SSEA-4, integrin alpha6, or integrin beta1 and then with the lectin STA. Densitometric quantification was used to identify PGC-specific markers. The results showed that chicken PGCs were stained selectively by PAS and by antibodies to SSEA-1, SSEA-3, SSEA-4, EMA-1, integrin alpha6, and integrin beta1. The control mouse ES cells reacted with PAS, anti-SSEA-1, and anti-EMA-1 antibodies, as well as with antibodies to integrins alpha6 and beta1, but not with antibodies to SSEA-3 and SSEA-4. Chicken PGCs reacted with the lectins STA and DBA, but mouse ES cells reacted with STA and WGA. The results of double staining of PGC colonies subcultured three times showed that the intensity of staining was not altered by concomitant use of the marker reagents. This study demonstrated that, in addition to PAS and antibodies to SSEA-1 and EMA-1, new specific markers of chicken PGCs are recognized by the lectins STA and DBA and by antibodies to SSEA-3 and SSEA-4 and integrins alpha6 and beta1. Double staining using these newly developed markers might be the method of choice for rapid characterization of chicken PGCs.

Development of Novel Markers for the Characterization of Chicken Primordial Germ Cells

Alpha 2 macroglobulin (A2M; also known as ovostatin), a homotetrameric protein with four disulfide-linked subunits, has the unique feature of inactivating/inhibiting most known proteases including serine-, threonine-, cysteine-, aspartic- and metalloproteases. In chickens, A2M has been identified and characterized biochemically, but little is known of its functional role(s) in the oviduct, hormonal regulation of expression or its expression in ovarian carcinomas in chickens. Therefore, we investigated estrogen regulation of A2M gene expression during development of the chicken oviduct, and its expression in normal and cancerous ovaries from chickens. To determine tissue-specific expression of A2M in chickens, we collected various organs from male and female chickens and performed RT-PCR analyses. To examine A2M gene expression in the oviduct of 1-week-old female chicks that received a subcutaneous implant of 15 mg DES in the abdominal region for 20 days, we performed RT-PCR, qPCR and in situ hybridization analyses using cDNAs from control- (n = 5) and DES-treated oviducts (n = 5), and then each segment of the oviduct from DES-treated chicks. To determine if A2M is a biomarker of ovarian cancer in hens, we collected cancerous (n = 10) ovaries from a total of 136 chickens which had completely stopped egg-laying and performed RT-PCR and in situ hybridization analyses. We found that A2M is most abundant in the chicken oviduct, specifically luminal (LE) and glandular epithelia (GE), but it was not detected in any other tissues of either sex. We then determined that DES (dietylstilbestrol, a synthetic nonsteroidal estrogen) increased A2M mRNA only in LE and GE of the oviduct of chicks. Further, expression of A2M was most abundant in GE of endometrioid adenocarcinoma of cancerous, but not normal ovaries of hens. Collectively, results of the present study indicate that A2M is novel estrogen-stimulated gene expressed in LE and GE of the chicken oviduct and may be used for monitoring effects of therapies for ovarian cancer in laying hens.

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Differential expression of alpha 2 macroglobulin in response to dietylstilbestrol and in ovarian carcinomas in chickens

Pleiotrophin (PTN) is a developmentally-regulated growth factor which is widely distributed in various tissues and also detected in many kinds of carcinomas. However, little is known about the PTN gene in chickens. In the present study, we found chicken PTN to be highly conserved with respect to mammalian PTN genes (91–92.6%) and its mRNA was most abundant in brain, heart and oviduct. This study focused on the PTN gene in the oviduct where it was detected in the glandular (GE) and luminal (LE) epithelial cells. Treatment of young chicks with diethylstilbesterol induced PTN mRNA and protein in GE and LE, but not in other cell types of the oviduct. Further, several microRNAs, specifically miR-499 and miR-1709 were discovered to influence PTN expression via its 3′-UTR which suggests that post-transcriptional regulation influences PTN expression in chickens. We also compared expression patterns and CpG methylation status of the PTN gene in normal and cancerous ovaries from chickens. Our results indicated that PTN is most abundant in the GE of adenocarcinoma of cancerous, but not normal ovaries of hens. Bisulfite sequencing revealed that 30- and 40% of −1311 and −1339 CpG sites are demethylated in ovarian cancer cells, respectively. Collectively, these results indicate that chicken PTN is a novel estrogen-induced gene expressed mainly in the oviductal epithelia implicating PTN regulation of oviduct development and egg formation, and also suggest that PTN is a biomarker for epithelial ovarian carcinoma that could be used for diagnosis and monitoring effects of therapies for the disease.

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Chicken pleiotrophin: regulation of tissue specific expression by estrogen in the oviduct and distinct expression pattern in the ovarian carcinomas.

S-adenosylhomocysteine hydrolase-like protein 1 (AHCYL1), also known as IP3 receptor-binding protein released with IP3 (IRBIT), regulates IP3-induced Ca2+ release into the cytoplasm of cells. AHCYL1 is a critical regulator of early developmental stages in zebrafish, but little is known about the function of AHCYL1 or hormonal regulation of expression of the AHCYL1 gene in avian species. Therefore, we investigated differential expression profiles of the AHCYL1 gene in various adult organs and in oviducts from estrogen-treated chickens. Chicken AHCYL1 encodes for a protein of 540 amino acids that is highly conserved and has considerable homology to mammalian AHCYL1 proteins (>94% identity). AHCYL1 mRNA was expressed abundantly in various organs of chickens. Further, the synthetic estrogen agonist induced AHCYL1 mRNA and protein predominantly in luminal and glandular epithelial cells of the chick oviduct. In addition, estrogen activated AHCYL1 through the ERK1/2 signal transduction cascade and that activated expression of AHCYL1 regulated genes affecting oviduct development in chicks as well as calcium release in epithelial cells of the oviduct. Also, microRNAs, miR-124a, miR-1669, miR-1710 and miR-1782 influenced AHCYL1 expression in vitro via its 3′-UTR which suggests that post-transcriptional events are involved in the regulation of AHCYL1 expression in the chick oviduct. In conclusion, these results indicate that AHCYL1 is a novel estrogen-stimulated gene expressed in epithelial cells of the chicken oviduct that likely affects growth, development and calcium metabolism of the mature oviduct of hens via an estrogen-mediated ERK1/2 MAPK cell signaling pathway.

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AHCYL1 Is Mediated by Estrogen-Induced ERK1/2 MAPK Cell Signaling and MicroRNA Regulation to Effect Functional Aspects of the Avian Oviduct

Estrogen is a key regulator in the development of the female reproductive system. It also stimulates oviduct development in immature chicks. We identified candidate genes and pathways associated with the development of chicken oviducts. A pellet containing the synthetic estrogen analog diethylstilbestrol (DES) was implanted subcutaneously in 1-wk-old female chicks for 10 days. The pellet was removed from half the group for 10 days and an additional dose was given for a further 10 days. Total RNA was extracted from the oviducts of DES-treated and untreated chicks and subjected to an Affymetrix chicken GeneChip analysis. We found differential expression of 2,290 and 1,745 transcripts from the oviducts that were treated with DES once and twice, respectively. We also found a twofold or greater change in the expression of 77 and 390 transcripts between the two control and DES-treated time points, respectively, whilst the expression of 10 transcripts changed that were common to all groups. Analyses of real-time PCR and in situ hybridization of selected genes confirmed the validity of the gene expression patterns observed in the microarray analysis. In particular, CCRN4L, FAM26F, HAS2, NELF, and NTM were up-regulated in the DES-treated chicken oviducts. High-throughput analysis revealed that the differentially expressed genes were related to tubular formation, epithelial differentiation, hormone interactions, nerve development, and tissue remodeling in the chicken oviduct. This study provides novel insights into candidate genes regulating oviduct development and differentiation via estrogen. The identified genes may serve as biomarkers of reproductive tract development in chicks.

Discovery of Candidate Genes and Pathways Regulating Oviduct Development in Chickens Short title: DES-regulated genes during early chick oviduct development Summary sentence: High-throughput analysis revealed that differentially expressed genes were related to tubular formation, epithelial differentiation, hormone interactions, nerve development, and tissue remodeling in the chicken oviduct. Keywords: chicken, oviduct, estrogen, diethylstilbestrol, microarray analysis

이종간 카이메라 연구에 응용하기 위하여 닭, 꿩, 메추리, 칠면조의 cytochorome b 유전자의 상동성 검증을 통하여 종 특이적인 DNA 염기서열 변이를 발견하였다. 이러한 염기서열 차이를 이용하여 꿩을 특이적으로 구별할 수 있는 한쌍의 프라이머를 제작하여 species-specific PCR을 수행하였다. 실험결과 꿩에서만 특이적으로 DNA절편이 증폭됨을 확인하였다. 본 종구별법은 멸종위기 조류의 복원을 위한 이종간 카이메라 연구에 매우 유용하게 이용될 수 있을 것이다.

Avian Species Identification using Species-specific DNA Polymorphisms

Department of Animal Science and Biotechnology, Chungnam National University, Daejeon 305-764, KoreaABSTRACT Tyrosinase (TYR) gene is located on chromosome 1 in chicken and it is composed of five exons and four introns. TYR gene is described as a key enzyme in melanin biosynthesis. Most examples of complete albinism in chicken have been due to defects in the tyrosinase gene. The association of feather color and sequence polymorphism in the Tyrosinase (TYR) gene was investigated using Korean Native chicken H breed (H_PL), Korean Native chicken L/W breed(L/W_PL) and ‘Woorimatdag’ commercial chickens (Woorimatdag_CC). From L_PL and W_PL breed analyses, 4 synonymous SNPs (locus G33A, G116A, C217T and C247T) and 2 SNPs (G838A and G958A) were detected in 4th exon and 4th intron of TYR gene respectively. The genotype frequencies for 6 SNPs were compared between L_PL and W_PL and W_PL represented homozygous SNP types in all the analyzed SNP positions while L_PL displayed various SNP types.(Key words : Korean native chickens, feather color, TYR, SNP)

http://koreascience.or.kr:80/article/JAKO201416060569850.pdf

Genetic Variations of Chicken TYR Gene and Associations with Feather Color of Korean Native Chicken (KNC)

The transgenic approach and precise editing of specific loci in the genome have diverse practical uses in animal biotechnology. Recent advances in genome-editing technology, including clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) have helped to generate highly valuable and quality-improved poultry. The production of transgenic and genome-edited birds mainly depends on primordial germ cells (PGCs), which are the progenitor cells of gametes, due to the unique system that is quite different from the mammalian system. This chapter introduces the basic physiology of avian PGCs and the latest PGC-mediated methodologies in transgenesis and genome editing of birds. Based on these techniques, future applications of precisely genome-modulated poultry are discussed to provide opportunities and benefits for humans.

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Jae Yong Han

Papers

Avian Species Identification using Species-specific DNA Polymorphisms

Genetic Variations of Chicken TYR Gene and Associations with Feather Color of Korean Native Chicken (KNC)

Transgenesis and Genome Editing in Poultry

Estrogen-induced AHCYL1 activates mitogen-activated protein kinase cell signaling pathways and stimulates oviduct development in chickens