Cargando…

Characteristics of Cyp11a during Gonad Differentiation of the Olive Flounder Paralichthys olivaceus

The P450 side-chain cleavage enzyme, P450scc (Cyp11a) catalyzes the first enzymatic step for the synthesis of all steroid hormones in fish. To study its roles in gonads of the olive flounder Paralichthys olivaceus, an important maricultured fish species, we isolated the cyp11a genomic DNA sequence o...

Descripción completa

Detalles Bibliográficos
Autores principales: Liang, Dongdong, Fan, Zhaofei, Zou, Yuxia, Tan, Xungang, Wu, Zhihao, Jiao, Shuang, Li, Jun, Zhang, Peijun, You, Feng
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6164156/
https://www.ncbi.nlm.nih.gov/pubmed/30200601
http://dx.doi.org/10.3390/ijms19092641
_version_ 1783359533067796480
author Liang, Dongdong
Fan, Zhaofei
Zou, Yuxia
Tan, Xungang
Wu, Zhihao
Jiao, Shuang
Li, Jun
Zhang, Peijun
You, Feng
author_facet Liang, Dongdong
Fan, Zhaofei
Zou, Yuxia
Tan, Xungang
Wu, Zhihao
Jiao, Shuang
Li, Jun
Zhang, Peijun
You, Feng
author_sort Liang, Dongdong
collection PubMed
description The P450 side-chain cleavage enzyme, P450scc (Cyp11a) catalyzes the first enzymatic step for the synthesis of all steroid hormones in fish. To study its roles in gonads of the olive flounder Paralichthys olivaceus, an important maricultured fish species, we isolated the cyp11a genomic DNA sequence of 1396 bp, which consists of 5 exons and 4 introns. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) results indicated that the flounder cyp11a was exclusively expressed in gonad and head kidney tissues. Its expression level in the testis was higher than that in the ovary. According to the in situ hybridization patterns, cyp11a was mainly expressed in the Leydig cells of the testis, and the thecal cells of the ovary. Immunofluorescence analysis showed that Cyp11a was located in the cytoplasm of the cultured flounder testis cells. Further quantitative real-time PCR results presented the cyp11a differential expression patterns during gonad differentiation. Among different sampling points of the 17β-estradiol (E2, 5 ppm) treatment group, cyp11a expression levels were relatively high in the differentiating ovary (30 and 40 mm total length, TL), and then significantly decreased in the differentiated ovary (80, 100 and 120 mm TL, p < 0.05). The pregnenolone level also dropped in the differentiated ovary. In the high temperature treatment group (HT group, 28 ± 0.5 °C), the cyp11a expression level fluctuated remarkably in the differentiating testis (60 mm TL), and then decreased in the differentiated testis (80, 100 mm TL, p < 0.05). In the testosterone (T, 5 ppm) treatment group, the cyp11a was expressed highly in undifferentiated gonads and the differentiating testis, and then dropped in the differentiated testis. Moreover, the levels of cholesterol and pregnenolone of the differentiating testis in the HT and T groups increased. The expression level of cyp11a was significantly down-regulated after the cultured flounder testis cells were treated with 75 and 150 μM cyclic adenosine monophosphate (cAMP), respectively (p < 0.05), and significantly up-regulated after treatment with 300 μM cAMP (p < 0.05). Both nuclear receptors NR5a2 and NR0b1 could significantly up-regulate the cyp11a gene expression in a dosage dependent way in the testis cells detected by cell transfection analysis (p < 0.05). The above data provides evidence that cyp11a would be involved in the flounder gonad differentiation and development.
format Online
Article
Text
id pubmed-6164156
institution National Center for Biotechnology Information
language English
publishDate 2018
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-61641562018-10-10 Characteristics of Cyp11a during Gonad Differentiation of the Olive Flounder Paralichthys olivaceus Liang, Dongdong Fan, Zhaofei Zou, Yuxia Tan, Xungang Wu, Zhihao Jiao, Shuang Li, Jun Zhang, Peijun You, Feng Int J Mol Sci Article The P450 side-chain cleavage enzyme, P450scc (Cyp11a) catalyzes the first enzymatic step for the synthesis of all steroid hormones in fish. To study its roles in gonads of the olive flounder Paralichthys olivaceus, an important maricultured fish species, we isolated the cyp11a genomic DNA sequence of 1396 bp, which consists of 5 exons and 4 introns. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) results indicated that the flounder cyp11a was exclusively expressed in gonad and head kidney tissues. Its expression level in the testis was higher than that in the ovary. According to the in situ hybridization patterns, cyp11a was mainly expressed in the Leydig cells of the testis, and the thecal cells of the ovary. Immunofluorescence analysis showed that Cyp11a was located in the cytoplasm of the cultured flounder testis cells. Further quantitative real-time PCR results presented the cyp11a differential expression patterns during gonad differentiation. Among different sampling points of the 17β-estradiol (E2, 5 ppm) treatment group, cyp11a expression levels were relatively high in the differentiating ovary (30 and 40 mm total length, TL), and then significantly decreased in the differentiated ovary (80, 100 and 120 mm TL, p < 0.05). The pregnenolone level also dropped in the differentiated ovary. In the high temperature treatment group (HT group, 28 ± 0.5 °C), the cyp11a expression level fluctuated remarkably in the differentiating testis (60 mm TL), and then decreased in the differentiated testis (80, 100 mm TL, p < 0.05). In the testosterone (T, 5 ppm) treatment group, the cyp11a was expressed highly in undifferentiated gonads and the differentiating testis, and then dropped in the differentiated testis. Moreover, the levels of cholesterol and pregnenolone of the differentiating testis in the HT and T groups increased. The expression level of cyp11a was significantly down-regulated after the cultured flounder testis cells were treated with 75 and 150 μM cyclic adenosine monophosphate (cAMP), respectively (p < 0.05), and significantly up-regulated after treatment with 300 μM cAMP (p < 0.05). Both nuclear receptors NR5a2 and NR0b1 could significantly up-regulate the cyp11a gene expression in a dosage dependent way in the testis cells detected by cell transfection analysis (p < 0.05). The above data provides evidence that cyp11a would be involved in the flounder gonad differentiation and development. MDPI 2018-09-06 /pmc/articles/PMC6164156/ /pubmed/30200601 http://dx.doi.org/10.3390/ijms19092641 Text en © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Liang, Dongdong
Fan, Zhaofei
Zou, Yuxia
Tan, Xungang
Wu, Zhihao
Jiao, Shuang
Li, Jun
Zhang, Peijun
You, Feng
Characteristics of Cyp11a during Gonad Differentiation of the Olive Flounder Paralichthys olivaceus
title Characteristics of Cyp11a during Gonad Differentiation of the Olive Flounder Paralichthys olivaceus
title_full Characteristics of Cyp11a during Gonad Differentiation of the Olive Flounder Paralichthys olivaceus
title_fullStr Characteristics of Cyp11a during Gonad Differentiation of the Olive Flounder Paralichthys olivaceus
title_full_unstemmed Characteristics of Cyp11a during Gonad Differentiation of the Olive Flounder Paralichthys olivaceus
title_short Characteristics of Cyp11a during Gonad Differentiation of the Olive Flounder Paralichthys olivaceus
title_sort characteristics of cyp11a during gonad differentiation of the olive flounder paralichthys olivaceus
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6164156/
https://www.ncbi.nlm.nih.gov/pubmed/30200601
http://dx.doi.org/10.3390/ijms19092641
work_keys_str_mv AT liangdongdong characteristicsofcyp11aduringgonaddifferentiationoftheoliveflounderparalichthysolivaceus
AT fanzhaofei characteristicsofcyp11aduringgonaddifferentiationoftheoliveflounderparalichthysolivaceus
AT zouyuxia characteristicsofcyp11aduringgonaddifferentiationoftheoliveflounderparalichthysolivaceus
AT tanxungang characteristicsofcyp11aduringgonaddifferentiationoftheoliveflounderparalichthysolivaceus
AT wuzhihao characteristicsofcyp11aduringgonaddifferentiationoftheoliveflounderparalichthysolivaceus
AT jiaoshuang characteristicsofcyp11aduringgonaddifferentiationoftheoliveflounderparalichthysolivaceus
AT lijun characteristicsofcyp11aduringgonaddifferentiationoftheoliveflounderparalichthysolivaceus
AT zhangpeijun characteristicsofcyp11aduringgonaddifferentiationoftheoliveflounderparalichthysolivaceus
AT youfeng characteristicsofcyp11aduringgonaddifferentiationoftheoliveflounderparalichthysolivaceus