Knockdown of DEAD-box helicase 4 (DDX4) decreases the number of germ cells in male and female chicken embryonic gonads
Nana Aduma A , Hiroe Izumi B , Shusei Mizushima A B and Asato Kuroiwa
A B C
+ Author Affiliations
- Author Affiliations
A Biosystems Science Course, Graduate School of Life Science, Hokkaido University, Kita 10 Nishi 8, Kita-ku, Sapporo, Hokkaido 060-0810, Japan.
B Division of Reproductive and Developmental Biology, Department of Biological Sciences, Faculty of Science, Hokkaido University, Kita 10 Nishi 8, Kita-ku, Sapporo, Hokkaido 060-0810, Japan.
C Corresponding author. Email: asatok@sci.hokudai.ac.jp
Reproduction, Fertility and Development 31(5) 847-854 https://doi.org/10.1071/RD18266
Submitted: 13 July 2018 Accepted: 27 November 2018 Published: 17 December 2018
Abstract
DEAD-box helicase 4 (DDX4; also known as vasa) is essential for the proper formation and maintenance of germ cells. Although DDX4 is conserved in a variety of vertebrates and invertebrates, its roles differ between species. This study investigated the function of DDX4 in chicken embryos by knocking down its expression using retroviral vectors that encoded DDX4-targeting microRNAs. DDX4 was effectively depleted in vitro and in vivo via this approach. Male and female gonads of DDX4-knockdown embryos contained a decreased number of primordial germ cells, indicating that DDX4 is essential to maintain a normal level of these cells in chicken embryos of both sexes. Expression of doublesex and mab-3 related transcription factor 1 (DMRT1) and sex determining region Y-box 9 (SOX9), which are involved in testis determination and differentiation, was normal in male gonads of DDX4-knockdown embryos. In contrast, expression of cytochrome P450 family 19 subfamily A member 1 (CYP19A1), which encodes aromatase and is essential for ovary development, was significantly decreased in female gonads of DDX4-knockdown embryos. Expression of forkhead box L2 (FOXL2), which plays an important role in ovary differentiation, was also slightly reduced in DDX4-knockdown embryos, but not significantly. Based on several pieces of evidence FOXL2 was hypothesised to regulate aromatase expression. The results of this study indicate that aromatase expression is also regulated by several additional pathways.
Additional keywords: ovary, primordial germ cells, sex differentiation, testis.
References
Aramaki, S., Kubota, K., Soh, T., Yamauchi, N., and Hattori, M.-A. (2009). Chicken dead end homologue protein is a nucleoprotein of germ cells including primordial germ cells. J. Reprod. Dev. 55, 214–218.| Chicken dead end homologue protein is a nucleoprotein of germ cells including primordial germ cells.Crossref | GoogleScholarGoogle Scholar |
Braat, A. K., van de Water, S., Korving, J., and Zivkovic, D. A. (2001). Zebrafish vasa morphant abolishes vasa protein but does not affect the establishment of the germline. Genesis 30, 183–185.
| Zebrafish vasa morphant abolishes vasa protein but does not affect the establishment of the germline.Crossref | GoogleScholarGoogle Scholar |
Carrera, P., Johnstone, O., Nakamura, A., Casanova, J., Jäckle, H., and Lasko, P. (2000). VASA mediates translation through interaction with a Drosophila yIF2 homolog. Mol. Cell 5, 181–187.
| VASA mediates translation through interaction with a Drosophila yIF2 homolog.Crossref | GoogleScholarGoogle Scholar |
Chue, J., and Smith, C. A. (2011). Sex determination and sexual differentiation in the avian model. FEBS J. 278, 1027–1034.
| Sex determination and sexual differentiation in the avian model.Crossref | GoogleScholarGoogle Scholar |
Das, R. M., Van Hateren, N. J., Howell, G. R., Farrell, E. R., Bangs, F. K., Porteous, V. C., Manning, E. M., McGrew, M. J., Ohyama, K., Sacco, M. A., Halley, P. A., Sang, H. M., Storey, K. G., Placzek, M., Tickle, C., Nair, V. K., and Wilson, S. A. (2006). A robust system for RNA interference in the chicken using a modified microRNA operon. Dev. Biol. 294, 554–563.
| A robust system for RNA interference in the chicken using a modified microRNA operon.Crossref | GoogleScholarGoogle Scholar |
Elbrecht, A., and Smith, R. G. (1992). Aromatase enzyme activity and sex determination in chickens. Science 255, 467–470.
| Aromatase enzyme activity and sex determination in chickens.Crossref | GoogleScholarGoogle Scholar |
Ewen-Campen, B., Donoughe, S., Clarke, D. N., and Extavour, C. G. (2013). Germ cell specification requires zygotic mechanisms rather than germ plasm in a basally branching insect. Curr. Biol. 23, 835–842.
| Germ cell specification requires zygotic mechanisms rather than germ plasm in a basally branching insect.Crossref | GoogleScholarGoogle Scholar |
Fridolfsson, A. K., and Ellegren, H. (1999). A simple and universal method for molecular sexing of non-ratite birds. J. Avian Biol. 30, 116–121.
| A simple and universal method for molecular sexing of non-ratite birds.Crossref | GoogleScholarGoogle Scholar |
Govoroun, M. S., Pannetier, M., Pailhoux, E., Cocquet, J., Brillard, J. P., Couty, I., Batellier, F., and Cotinot, C. (2004). Isolation of chicken homolog of the FOXL2 gene and comparison of its expression patterns with those of aromatase during ovarian development. Dev. Dyn. 231, 859–870.
| Isolation of chicken homolog of the FOXL2 gene and comparison of its expression patterns with those of aromatase during ovarian development.Crossref | GoogleScholarGoogle Scholar |
Gruidl, M. E., Smith, P. A., Kuznicki, K. A., McCrone, J. S., Kirchner, J., Roussell, D. L., Strome, S., and Bennett, K. L. (1996). Multiple potential germ-line helicases are components of the germ-line-specific P granules of Caenorhabditis elegans. Proc. Natl Acad. Sci. USA 93, 13837–13842.
| Multiple potential germ-line helicases are components of the germ-line-specific P granules of Caenorhabditis elegans.Crossref | GoogleScholarGoogle Scholar |
Hamburger, V., and Hamilton, H. L. (1992). A series of normal stages in the development of the chick embryo. Dev. Dyn. 195, 231–272.
| A series of normal stages in the development of the chick embryo.Crossref | GoogleScholarGoogle Scholar |
Hudson, Q. J., Smith, C. A., and Sinclair, A. H. (2005). Aromatase inhibition reduces expression of FOXL2 in the embryonic chicken ovary. Dev. Dyn. 233, 1052–1055.
| Aromatase inhibition reduces expression of FOXL2 in the embryonic chicken ovary.Crossref | GoogleScholarGoogle Scholar |
Hughes, S. H., Greenhouse, J. J., Petropoulos, C. J., and Sutrave, P. (1987). Adaptor plasmids simplify the insertion of foreign DNA into helper-independent retroviral vectors. J. Virol. 61, 3004–3012.
Kawasaki, I., Shim, Y. H., Kirchner, J., Kaminker, J., Wood, W. B., and Strome, S. (1998). PGL-1, a predicted RNA-binding component of germ granules, is essential for fertility in C. elegans. Cell 94, 635–645.
| PGL-1, a predicted RNA-binding component of germ granules, is essential for fertility in C. elegans.Crossref | GoogleScholarGoogle Scholar |
Kito, G., Aramaki, S., Tanaka, K., Soh, T., Yamauchi, N., and Hattori, M. A. (2010). Temporal and spatial differential expression of chicken germline-specific proteins cDAZL, CDH and CVH during gametogenesis. J. Reprod. Dev. 56, 341–346.
| Temporal and spatial differential expression of chicken germline-specific proteins cDAZL, CDH and CVH during gametogenesis.Crossref | GoogleScholarGoogle Scholar |
Kuramochi-Miyagawa, S., Watanabe, T., Gotoh, K., Takamatsu, K., Chuma, S., Kojima-Kita, K., Shiromoto, Y., Asada, N., Toyoda, A., Fujiyama, A., Totoki, Y., Shibata, T., Kimura, T., Nakatsuji, N., Noce, T., Sasaki, H., and Nakano, T. (2010). MVH in piRNA processing and gene silencing of retrotransposons. Genes Dev. 24, 887–892.
| MVH in piRNA processing and gene silencing of retrotransposons.Crossref | GoogleScholarGoogle Scholar |
Kuznicki, K. A., Smith, P. A., Leung-Chiu, W. M., Estevez, A. O., Scott, H. C., and Bennett, K. L. (2000). Combinatorial RNA interference indicates GLH-4 can compensate for GLH-1; these two P granule components are critical for fertility in C. elegans. Development 127, 2907–2916.
Lambeth, L. S., Cummins, D. M., Doran, T. J., Sinclair, A. H., and Smith, C. A. (2013). Overexpression of aromatase alone is sufficient for ovarian development in genetically male chicken embryos. PLoS One 8, e68362.
| Overexpression of aromatase alone is sufficient for ovarian development in genetically male chicken embryos.Crossref | GoogleScholarGoogle Scholar |
Loffler, K. A., Zarkower, D., and Koopman, P. (2003). Etiology of ovarian failure in blepharophimosis ptosis epicanthus inversus syndrome: FOXL2 is a conserved, early-acting gene in vertebrate ovarian development. Endocrinology 144, 3237–3243.
| Etiology of ovarian failure in blepharophimosis ptosis epicanthus inversus syndrome: FOXL2 is a conserved, early-acting gene in vertebrate ovarian development.Crossref | GoogleScholarGoogle Scholar |
Nakata, T., Ishiguro, M., Aduma, N., Izumi, H., and Kuroiwa, A. (2013). Chicken hemogen homolog is involved in the chicken-specific sex-determining mechanism. Proc. Natl Acad. Sci. USA 110, 3417–3422.
| Chicken hemogen homolog is involved in the chicken-specific sex-determining mechanism.Crossref | GoogleScholarGoogle Scholar |
Pisarska, M. D., Barlow, G., and Kuo, F. T. (2011). Minireview: roles of the forkhead transcription factor FOXL2 in granulosa cell biology and pathology. Endocrinology 152, 1199–1208.
| Minireview: roles of the forkhead transcription factor FOXL2 in granulosa cell biology and pathology.Crossref | GoogleScholarGoogle Scholar |
Raymond, C. S., Kettlewell, J. R., Hirsch, B., Bardwell, V. J., and Zarkower, D. (1999). Expression of Dmrt1 in the genital ridge of mouse and chicken embryos suggests a role in vertebrate sexual development. Dev. Biol. 215, 208–220.
| Expression of Dmrt1 in the genital ridge of mouse and chicken embryos suggests a role in vertebrate sexual development.Crossref | GoogleScholarGoogle Scholar |
Roussell, D. L., and Bennett, K. L. (1993). glh-1, a germ-line putative RNA helicase from Caenorhabditis, has four zinc fingers. Proc. Natl Acad. Sci. USA 90, 9300–9304.
| glh-1, a germ-line putative RNA helicase from Caenorhabditis, has four zinc fingers.Crossref | GoogleScholarGoogle Scholar |
Smith, C. A., McClive, P. J., Western, P. S., Reed, K. J., and Sinclair, A. H. (1999a). Conservation of a sex-determining gene. Nature 402, 601–602.
| Conservation of a sex-determining gene.Crossref | GoogleScholarGoogle Scholar |
Smith, C. A., Smith, M. J., and Sinclair, A. H. (1999b). Gene expression during gonadogenesis in the chicken embryo. Gene 234, 395–402.
| Gene expression during gonadogenesis in the chicken embryo.Crossref | GoogleScholarGoogle Scholar |
Smith, C. A., McClive, P. J., Hudson, Q., and Sinclair, A. H. (2005). Male-specific cell migration into the developing gonad is a conserved process involving PDGF signalling. Dev. Biol. 284, 337–350.
| Male-specific cell migration into the developing gonad is a conserved process involving PDGF signalling.Crossref | GoogleScholarGoogle Scholar |
Spike, C., Meyer, N., Racen, E., Orsborn, A., Kirchner, J., Kuznicki, K., Yee, C., Bennett, K., and Strome, S. (2008). Genetic analysis of the Caenorhabditis elegans GLH family of P-granule proteins. Genetics 178, 1973–1987.
| Genetic analysis of the Caenorhabditis elegans GLH family of P-granule proteins.Crossref | GoogleScholarGoogle Scholar |
Styhler, S., Nakamura, A., Swan, A., Suter, B., and Lasko, P. (1998). vasa is required for GURKEN accumulation in the oocyte, and is involved in oocyte differentiation and germline cyst development. Development 125, 1569–1578.
Tanaka, S. S., Toyooka, Y., Akasu, R., Katoh-Fukui, Y., Nakahara, Y., Suzuki, R., Yokoyama, M., and Noce, T. (2000). The mouse homolog of Drosophila Vasa is required for the development of male germ cells. Genes Dev. 14, 841–853.
Tanaka, R., Izumi, H., and Kuroiwa, A. (2017). Androgens and androgen receptor signaling contribute to ovarian development in the chicken embryo. Mol. Cell. Endocrinol. 443, 114–120.
| Androgens and androgen receptor signaling contribute to ovarian development in the chicken embryo.Crossref | GoogleScholarGoogle Scholar |
Taylor, L., Carlson, D. F., Nandi, S., Sherman, A., Fahrenkrug, S. C., and McGrew, M. J. (2017). Efficient TALEN-mediated gene targeting of chicken primordial germ cells. Development 144, 928–934.
| Efficient TALEN-mediated gene targeting of chicken primordial germ cells.Crossref | GoogleScholarGoogle Scholar |
Tsunekawa, N., Naito, M., Sakai, Y., Nishida, T., and Noce, T. (2000). Isolation of chicken vasa homolog gene and tracing the origin of primordial germ cells. Development 127, 2741–2750.
Wang, J., and Gong, Y. (2017). Transcription of CYP19A1 is directly regulated by SF-1 in the theca cells of ovary follicles in chicken. Gen. Comp. Endocrinol. 247, 1–7.
| Transcription of CYP19A1 is directly regulated by SF-1 in the theca cells of ovary follicles in chicken.Crossref | GoogleScholarGoogle Scholar |
Wang, D. S., Kobayashi, T., Zhou, L. Y., Paul-Prasanth, B., Ijiri, S., Sakai, F., Okubo, K., Morohashi, K., and Nagahama, Y. (2007). Foxl2 up-regulates aromatase gene transcription in a female-specific manner by binding to the promoter as well as interacting with ad4 binding protein/steroidogenic factor 1. Mol. Endocrinol. 21, 712–725.
| Foxl2 up-regulates aromatase gene transcription in a female-specific manner by binding to the promoter as well as interacting with ad4 binding protein/steroidogenic factor 1.Crossref | GoogleScholarGoogle Scholar |
