Molecular Characteristic, Protein Distribution and Potential Regulation of HSP90AA1 in the Anadromous Fish Coilia nasus

Abstract

Heat shock proteins play essential roles in basic cellular events. Spawning migration is a complex process, with significant structural and biochemical changes taking place in the adult gonad. To date, the molecular mechanisms underlying migration reproductive biology remain undetermined. In this regard, a full length HSP90AA1 comprising 2608 nucleotides from the anadromous fish Coilia nasus was characterized, encoding 742 amino acid (aa) residues with potential phosphorylation sites. HSP90AA1 mRNA transcripts were detected in all organs, especially in the gonad. Furthermore, the greatest transcript levels were found during the developmental phase, while the lowest levels were found during the resting phase. In addition, the strongest immunolabeling positive signal was found in the primary spermatocyte and oocyte, with lower positive staining in secondary germ cells, and a weak or absent level in the mature sperm and oocyte. Interestingly, HSP90AA1 was mainly located in the cytoplasm of germ cells. These results are important for understanding the molecular mechanism of anadromous migration reproductive biology. In combination with data from other fish species, the result of this present study may facilitate further investigations on the spawning migration mechanism.

Keywords: Coilia nasus; HSP90AA1 expression; anadromous fish; migration mechanism.

Figure 1

Nucleotide and deduced amino acid sequences of HSP90AA1 cDNA. The lower case letters indicate 5′ and 3′ untranslated regions and upper case letters indicate the coding region. The characteristic HSP90 family signatures are boxed. Asparagine _glycosylation sites underlined with wavy lines. PKC_phospho_site is underlined with dotted lines. Tyrosinase_phospho_site is underlined with solid lines. CK2 phospho_site is in dark grey bold and Myristyl site is in light gray. The cytoplasmic C-terminal region EEVD is shown and the stop codon (tga) is indicated with an asterisk, respectively.

Figure 2

Relative expression levels of HSP90AA1 in different tissues from C. nasus. Data were expressed as the mean fold difference (mean ± SE, n = 3). Expression values were normalized to those of 18sRNA. Values with the different superscript letters are significantly different (p > 0.05, a < b < c).

Figure 3

Temporal expression of HSP90AA1 mRNA in the testis and ovary during the spawning phase. The fish different spawning phase [10,27]: onset phase (Chongming section in March), developmental phase (Nantong section in March to April), multiplication phase (Jingjiang/Zhenjiang section in April to May), mature phase (Dangtu/Anqing section in late May to early June), mature later phase (Anqing section in late June to early July), and resting phase (Anqing section in mid to late July). Expression values were normalized to those of 18sRNA. Data were expressed as the mean fold difference (mean ± SE, n = 3). Values with the different superscript letters are of significantly different (p > 0.05, a < b < c).

Figure 4

Localization of HSP90AA1 in the testis and ovary. Immunohistochemical positive signals of anti-HSP90 immunolabeling are shown in brown and immunofluorescence positive signals are shown in green. (T1–T2): whole testes section stained with hematoxylin-eosin; (T3): negative control. T4-T6 and T7-T9 are shown different part and developmental phase of testis for IHC and IF, respectively. pSp: primary spermatocytes, sSp: secondary spermatocyte, and Sp: spermatids. (O1–O2): whole testes section stained with hematoxylin-eosin; (O3): negative control. O4–O6 and O7–O9 are shown different part and developmental phase of ovary for IHC and IF, respectively. pOc: primary oocyte, sOc: secondary oocyte, and Oc: oocyte.

Figure 5

Sampling distribution in the middle and lower reach of the Yangze River. Black dot display the sampling site. AQ: Anqing; DT: Dangtu; ZJ: Zhenjiang; JJ: Jingjiang; NT: Nantong; CM: Chongming.