Expression profiling and CRISPR/Cas9 mediated editing of deleted in azoospermia like gene in chicken

Deivendran Rengaraj,Hyung Chul Lee,Soo Taek Han,You Yun Kim,Kelly Chungu,Jae Yong Han 한국가금학회 2018 한국가금학회 정기총회 및 학술발표회 Vol.2018 No.10

DAZL gene, germ cells, and chicken early embryonic development

Deivendran Rengaraj,Hyung Chul Lee,Hong Jo Lee,Jae Yong Han 한국수정란이식학회 2018 한국수정란이식학회 학술대회 Vol.2018 No.11

The deleted in azoospermia like (DAZL) gene has been identified in many vertebrate species. DAZL shows high homology with deleted in azoospermia (DAZ) genes that identified only in humans, great apes and Old World monkeys, and boule homolog (BOLL) that identified in many vertebrate species. These genes encode RNA binding proteins (RBP), which regulate the post-transcriptional functions of several genes. In humans, DAZ copies are linked to Y chromosome, while DAZL and BOLL are linked to chromosomes 3 and 2, respectively. DAZ copies has been reported to express in prenatal and postnatal germ cells, particularly in the premeiotic spermatogonia. BOLL has been reported to express during the meiotic G2/M transition in germ cells. DAZL has been reported to express in all stages of germ cells. Compared to humans and mice, the detailed functionalities of DAZL is not clear in many vertebrate species. In our studies, we use chickens as an animal model to examine the expression profiling of DAZL gene in germ cells right from the early embryonic development to the adult. Also, we are studying the effects of small interfering RNA (siRNA) mediated knockdown of DAZL and Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR associated protein 9 (CRISPR/Cas9) mediated knockout of DAZL in the chicken primordial germ cells (PGCs). In the chicken, DAZL is linked to chromosome 2 (2p1.3-p1.2), and encodes a 289 amino acids protein. By in situ hybridization, we detected a strong expression of DAZL in the germ plasm of chicken oocytes. Later, the expression of DAZL was strongly detected in all stages of intrauterine development and post-ovipositional development especially in the PGC specifying cells. Moreover, the expression of DAZL was strong and constant in the male and female germ cells until adult stage. The siRNA mediated knockdown of DAZL significantly reduced the PGCs proliferation and increased the apoptosis in vitro. We examined the knockout efficiency of DAZL using CRISPR/Cas9 technique in chicken DF1 fibroblast cell line, prior to test in the PGCs. The results of T7 endonuclease I (T7E1) assay and subsequent sequencing indicates clear mutations on the DAZL gene in DF1 cells, and the method could be applicable to cause mutations on the DAZL gene in PGCs. In conclusion, chicken DAZL express in all stages of germ cells as a germ line marker, and alteration in the gene expression causes germ cells impairment.

Annotation of the Y chromosome-encoded proteins in human, chimpanzee, and mouse

Deivendran Rengaraj 한국동물생명공학회(구 한국동물번식학회) 2016 발생공학 국제심포지엄 및 학술대회 Vol.2016 No.10

Expression and knockdown analysis of glucose phosphate isomerase in chicken primordial germ cells.

Rengaraj, Deivendran,Lee, Sang In,Yoo, Min,Kim, Tae Hyun,Song, Gwonhwa,Han, Jae Yong Society for the Study of Reproduction [etc.] 2012 BIOLOGY OF REPRODUCTION Vol.87 No.3

<P>Glucose is an important monosaccharide required to generate energy in all cells. After entry into cells, glucose is phosphorylated to glucose-6-phosphate and then transformed into glycogen or metabolized to produce energy. Glucose phosphate isomerase (GPI) catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. Without GPI activity or fructose-6-phosphate, many steps of glucose metabolism would not occur. The requirement for GPI activity for normal functioning of primordial germ cells (PGCs) needs to be identified. In this study, we first examined the expression of chicken GPI during early embryonic development and germ cell development. GPI expression was strongly and ubiquitously detected in chicken early embryos and embryonic tissues at Embryonic Day 6.5 (E6.5). Continuous GPI expression was detected in PGCs and germ cells of both sexes during gonadal development. Specifically, GPI expression was stronger in male germ cells than in female germ cells during embryonic development and the majority of post-hatching development. Then, we used siRNA-1499 to knock down GPI expression in PGCs. siRNA-1499 caused an 85% knockdown in GPI, and PGC proliferation was also affected 48 h after transfection. We further examined the knockdown effects on 28 genes related to the glycolysis/gluconeogenesis pathway and the endogenous glucose level in chicken PGCs. Among genes related to glycolysis/gluconeogenesis, 20 genes showed approximately 3-fold lower expression, 4 showed approximately 10-fold lower, and 2 showed approximately 100-fold lower expression in knockdown PGCs. The endogenous glucose level was significantly reduced in knockdown PGCs. We conclude that the GPI gene is crucial for maintaining glycolysis and supplying energy to developing PGCs.</P>

Annotation of the Y chromosome-encoded proteins in human, chimpanzee, and mouse

Deivendran Rengaraj 한국수정란이식학회 2016 한국수정란이식학회 학술대회 Vol.2016 No.10

The Y chromosome is a type of sex chromosome existing primarily in male mammalian species. The Y chromosome passes through the male gamete and determines male sex in humans, non-human primates, and other mammals. The mammalian Y chromosome varies from the X chromosome and the rest of the chromosomes primarily by size and its male sex-determining/spermatogenesis function. In the Y chromosome, male sex-determining function is exclusively located on the short arm, while the spermatogenesis function is distributed widely on the short and long arm. Deletions or mutations particularly in the male-specific region of Y chromosome (MSY) may cause male infertility. During the last few decades, researchers put forth an enormous effort to discover Y chromosome specific genes, and their encoded RNAs and proteins in humans, primates, and rodents. As a result, most of the genes and encoded proteins responsible for male-sex determination, testis development, and spermatogenesis have been discovered in humans, however not well established in non-human primates and rodents. Also, there might be a percent of proteins missing in human Y chromosome. The aim of this study is to annotate the proteins that encoded on the Y chromosome of humans, chimpanzee, and mouse using extensive bioinformatics tools. The human (annotation release 107), chimpanzee (annotation release 103), and mouse (annotation release 105) proteins were first retrieved from the National Center for Biotechnology Information (NCBI) eukaryotic genome annotation resource database. Then, the annotated human proteins (66 proteins) were compared with the core databases of human proteome project such as neXtProt, PeptideAtlas, and the Human Protein Atlas. The X-homologous of human Y chromosome-encoded proteins were searched using the NCBI Protein BLAST program. The cellular pathways and protein-protein interactions involving human Y chromosome-encoded proteins were searched using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway mapping database, the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database, and the Pathway Studio software. Finally, the human Y chromosome-encoded protein homologs/orthologs in chimpanzee and mouse were analyzed using the NCBI bl2seq program. This analysis resulted a significant number of homologous/orthologous proteins between human, chimpanzee and mouse. Our findings provide the scientific community with updated information on the Y chromosome-encoded proteins in humans, chimpanzee, and mouse.

Effects of Dietary Vitamin E on Fertility Functions in Poultry Species

Rengaraj, Deivendran,Hong, Yeong Ho MDPI 2015 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.16 No.5

<P>Vitamin E is found in high quantities in vegetable oils. Although vitamin E has multiple functions in humans and animals, its key function is protecting cells from oxidative damage. Since its discovery, several studies have demonstrated that vitamin E deficiency causes impaired fertility in humans and lab animals. However, the effects of vitamin E deficiency or of its supplementation on the fertility of farm animals, particularly on poultry, are less well studied. Therefore, a comprehensive review of the effects of dietary vitamin E on the fertility of poultry species is needed in order to understand the beneficial role of vitamin E in the maintenance of sperm and egg qualities. Based on the observations reviewed here, we found that a moderate amount of vitamin E in poultry diet significantly protects semen/sperm qualities in male birds and egg qualities in female birds via decreasing the lipid peroxidation in semen/sperms and eggs. This review provides an overall understanding of the effects of dietary vitamin E on fertility functions in poultry species.</P>

Regulation of glucose phosphate isomerase by the 3'UTR-specific miRNAs miR-302b and miR-17-5p in chicken primordial germ cells.

Rengaraj, Deivendran,Park, Tae Sub,Lee, Sang In,Lee, Bo Ram,Han, Beom Ku,Song, Gwonhwa,Han, Jae Yong Society for the Study of Reproduction [etc.] 2013 BIOLOGY OF REPRODUCTION Vol.89 No.2

<P>Glucose phosphate isomerase (GPI) involves in the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate in glucose pathways. Because glucose metabolism is crucial for the proliferation and differentiation of embryonic stem and germ cells, reducing GPI expression may affect the characteristic features of these cells. MicroRNAs (miRNAs) have been shown to regulate genes. In the present study, we investigated the regulation of chicken GPI by its predicted miRNAs. We determined the expression patterns of seven GPI 3'-untranslated region (3'UTR)-targeting miRNAs, including the gga-miR-302 cluster, gga-miR-106, gga-miR-17-5p, and gga-miR-20 cluster in chicken primordial germ cells (PGCs), compared with GPI mRNA. Among the miRNAs, gga-miR-302b, gga-miR-302d, and gga-miR-17-5p were expressed at lower levels than GPI mRNA. The remaining four miRNAs-gga-miR-302c, gga-miR-106, gga-miR-20a, and gga-miR-20b-were expressed at higher levels than the expression of GPI mRNA. Next, we cotransfected four candidate miRNAs-gga-miR-302b, gga-miR-106, gga-miR-17-5p, and gga-miR-20a-with GPI 3'UTR into 293FT cells by dual fluorescence reporter assay. Overexpression of gga-miR-302b and gga-miR-17-5p miRNAs in 293FT cells significantly downregulated GPI expression, whereas the other two miRNAs had no effect. Then, knockdown and overexpression of these four candidate miRNAs were performed by RNA interference assay to regulate GPI in PGCs. In the RNA interference assay, the expression of GPI was greatly regulated by gga-miR-302b and gga-miR-17-5p. Finally, we examined the effects of GPI regulation on PGC proliferation and migration. Our results suggested that the regulation of GPI by gga-miR-302b and gga-miR-17-5p affected PGCs proliferation. However, regulation of GPI using these two miRNAs did not affect the migration of PGCs into embryonic gonads.</P>

Bioinformatics Annotation of Human Y Chromosome-Encoded Protein Pathways and Interactions

Rengaraj, Deivendran,Kwon, Woo-Sung,Pang, Myung-Geol American Chemical Society 2015 JOURNAL OF PROTEOME RESEARCH Vol.14 No.9

<P>We performed a comprehensive analysis of human Y chromosome-encoded proteins, their pathways, and their interactions using bioinformatics tools. From the NCBI annotation release 107 of human genome, we retrieved a total of 66 proteins encoded on Y chromosome. Most of the retrieved proteins were also matched with the proteins listed in the core databases of the Human Proteome Project including neXtProt, PeptideAtlas, and the Human Protein Atlas. When we examined the pathways of human Y-encoded proteins through KEGG database and Pathway Studio software, many of proteins fall into the categories related to cell signaling pathways. Using the STRING program, we found a total of 49 human Y-encoded proteins showing strong/medium interaction with each other. While using the Pathway studio software, we found that a total of 16 proteins interact with other chromosome-encoded proteins. In particular, the SRY protein interacted with 17 proteins encoded on other chromosomes. Additionally, we aligned the sequences of human Y-encoded proteins with the sequences of chimpanzee and mouse Y-encoded proteins using the NCBI BLAST program. This analysis resulted in a significant number of orthologous proteins between human, chimpanzee, and mouse. Collectively, our findings provide the scientific community with additional information on the human Y chromosome-encoded proteins.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jprobs/2015/jprobs.2015.14.issue-9/acs.jproteome.5b00491/production/images/medium/pr-2015-00491g_0007.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/pr5b00491'>ACS Electronic Supporting Info</A></P>

Testis-specific novel transcripts in chicken: in situ localization and expression pattern profiling during sexual development.

Rengaraj, Deivendran,Kim, Duk Kyung,Zheng, Ying Hui,Lee, Sang In,Kim, Heebal,Han, Jae Yong Society for the Study of Reproduction [etc.] 2008 BIOLOGY OF REPRODUCTION Vol.79 No.3

<P>Tissue-specific novel transcripts expressed during sexual development were examined by RT-PCR, quantitative RT-PCR (qRT-PCR), and in situ hybridization to provide data for chicken genomics. Public databases for transcript data have been constructed with known and unknown sequences of various tissues from different animals. However, the expression patterns and functions of the transcripts are less known. From the The Institute for Genomics Research Gallus gallus library, we examined 291 tentative consensus (TC) sequences that assembled 100% with transcripts by RT-PCR during male and female sexual development from Embryonic Day 6 to 25 wk of age. We found 85 TC sequences that were specific to testicular development; of these, 43 TC sequences were exclusively upregulated in 25-wk-old testis. Another 52 TC sequences were not specific to one tissue, but occurred in the testis and ovary at different developmental ages. Twelve testis-specific TC sequences upregulated in 25-wk-old testis were randomly selected and further examined with qRT-PCR. For precise localization, these 12 testis-specific TC sequences were examined by in situ hybridization with 25-wk-old adult testis. Six TC sequences were strongly expressed in secondary spermatocytes and haploid spermatids until spermatozoa release. Another six TC sequences were differentially expressed in the adluminal compartment of seminiferous tubules. Among the testis-specific TC sequences, TC120901 is a known gene, phospholipase C, zeta (PLCZ1). Our data provide potential insight into gene expression and genomic information on novel transcripts that are important to avian reproduction.</P>

The distribution of neuron‐specific gene family member 1 in brain and germ cells: Implications for the regulation of germ‐line development by brain

Rengaraj, Deivendran,Lee, Bo Ram,Park, Kyung Je,Lee, Sang In,Kang, Kyung Soo,Choi, Jin Won,Kang, Seok Jin,Song, Gwonhwa,Han, Jae Yong Wiley‐Liss, Inc. 2011 Developmental dynamics Vol.240 No.4

<P><B>Abstract</B></P><P>Vesicular acidification at early endosomes dissociates endocytosed receptor‐ligand complexes. The ligands, receptors, or both are then directed to late endosomes for degradation or recycled back to the plasma membrane. Of neuron‐specific gene (NSG) family members, early endosomal protein neuron‐specific gene family member 1 (NSG1) is the most important in receptor recycling. In this study, we characterized chicken NSG1 (cNSG1). We found several functional sites related to endocytotic machinery in cNSG1 that were highly conserved with most other vertebrate NSG1 proteins. We examined the tissue and duration specificity and the temporal and spatial patterns of c<I>NSG1</I> expression<I>.</I> c<I>NSG1</I> expression was preferentially located in all regions of the brain, neuroendocrine glands, and spinal cord. Unexpectedly, c<I>NSG1</I> expression was strongly detected during male and female germ‐line development. Expression of <I>NSG1</I> in two apparently unrelated cell types such as neurons and germ cells suggests <I>NSG1</I> roles in neurons and germ‐cells chemotaxis and endocytotic machinery. Developmental Dynamics 240:850–861, 2011. © 2011 Wiley‐Liss, Inc.</P>