Developmental Expression of HSP60 and HSP10 in the Coilia nasus Testis during Upstream Spawning Migration

Abstract

Heat shock protein 60 (HSP60) and heat shock protein 10 (HSP10) are important chaperones, which have been proven to have essential roles in mediating the correct folding of nuclear encoded proteins imported to mitochondria. Mitochondria are known as the power house of the cell, with which it produces energy and respires aerobically. In this regard, the obtained HSP60 and HSP10 have typical characteristics of the HSP60/10 family signature. Their mRNA transcripts detected were highest during the developmental phase (in April), while the lowest levels were found in the resting phase (after spawning in late July). Additionally, the strongest immunolabeling positive signals were found in the primary spermatocyte, with lower positive staining in secondary sperm cells, and a weak or absent level in the mature sperm. At the electron microscopic level, immunogold particles were localized in the mitochondrial matrix. Data indicated that HSP10 and HSP60 were inducible and functional in the Coilia nasus testis development and migration process, suggesting their essential roles in this process. The results also indicated that HSP60 may be one indicator of properly working mitochondrial import and refolding in the fish testis. This study also provides an expanded perspective on the role of heat shock protein families in spawning migration biology.

Keywords: Coilia nasus; Heat shock protein 10; Heat shock protein 60; Spawning Migration.

Figure 1

Nucleotide and deduced amino acid sequences of Cn-HSP60 and Cn-HSP10.The deduced amino acid sequence is shown upon the nucleotide sequence. The termination codon is marked by an asterisk. (A) is shown the Cn-HSP60: RGD Cell attachment sequence is shown in bold line. The conserved domain (26-571 aa, 60 kDa chaperonin (groEL) ) is in bold (Cpn60); Amidation site (193–196aa); ASN Glycosylation site (103–106 aa, 380–383aa, 426–429 aa); CAMP phosphorylation site (156–159 aa, 249–252 aa, 369–372 aa); CK2 phosphorylation site (105–108 aa, 163–166 aa, 200–203 aa, 206–209 aa, 351–354 aa, 381–384 aa, 410–413 aa, 540–543aa), Myristyl (77–82 aa, 112–117 aa, 143–148 aa, 183–188 aa, 322–327 aa, 435–440 aa, 484–489 aa, 556–561 aa, 563–568 aa, 569–574 aa); PKC phosphorylation site (70–72 aa, 200–202 aa, 231–233 aa, 247–249 aa) are underlined. The C-terminal Gly-Gly-Met (GGM) repeat is in italic. (B) is shown the Cn-HSP10: The putative mitochondrial targeting sequence is in italic. The conserved domain (GroES domain) is shaded in bold; the predicted mobile loop is underlined.

Figure 2

Phylogenetic trees of HSP60 and HSP10 family members. Phylogenetic tree constructed by the MEGA 4.0 program by the neighbor-joining distance method. (A) is for HSP60s and (B) for HSP10s. The statistical robustness of the tree was estimated by bootstrapping with 1000 replicates. Bootstrap values were indicated by genetic distance.

Figure 2

Phylogenetic trees of HSP60 and HSP10 family members. Phylogenetic tree constructed by the MEGA 4.0 program by the neighbor-joining distance method. (A) is for HSP60s and (B) for HSP10s. The statistical robustness of the tree was estimated by bootstrapping with 1000 replicates. Bootstrap values were indicated by genetic distance.

Figure 3

HSP60 and HSP10 mRNA Expression Patterns. (A,B) are for different organs by RT-PCR and RT-qPCR method, respectively; (C) is for different migration phase. Data were expressed as the mean fold difference (mean ± SE, pooled RNA, n = 6, one in each section, total n = 3 × 6 = 18). Expression values were normalized to those of 18sRNA. Values with the different superscript letters are significantly different (p < 0.05, a < b < c < d < e). In (A), Mar: molecular marker, 1: blood, 2: brain, 3: gill, 4: liver, 5: stomach, 6: intestine, 7: testis and 8: ovary. The different migration phases of the fish: onset phase (Chongming section in March), developmental phase (Nantong section in March to April), multiplication phase (Jingjiang section in April to May), mature phase (Zhenjiang section in May), mature later phase (Dangtu section in late May to early June), and resting phase (Anqing section in mid to late June).

Figure 4

HSP60 and HSP10 protein expression patterns in different migration phases. Three fish in the different developmental phases were used for western blot (WB). The crude protein extract of adult Coilia nasus testes were pooled and then the WB was done as described. Marker; I: onset phase; II: developmental phase; III: multiplication phase; IV: mature phase; V: mature later phase; VI: resting phase; Nc: Negative Control. (A): HSP60 and HSP10 protein expression patterns in different migration phase; (B): The results were semi-quantitated analyzed by ImageJ2x 2.1 program. The expression level of HSP60 was considerately higher than the level of HSP10. HSP60 level reached the peak in the developmental phase. With the testes maturing, both proteins presented declining.

Figure 5

Localization of HSP60/HSP10 in the mature testis. Immunohistochemical (IHC) positive signals of HSP60/HSP10 immunolabeling are shown in brown. (T1): the whole testis section stained with H&E; (T2): negative control (NC); (T3): different part and developmental phase of testis for IHC with anti-HSP60; (T4): magnify for the IHC with anti-HSP60; (T5): different part and developmental phase of testis for IHC with anti-HSP10; (T6): magnify for the IHC with anti-HSP10, respectively. pSp: primary spermatocytes, sSp: secondary spermatocyte, and Sp: spermatids. Scale bar = 100 um.

Figure 6

Colloidal gold immunocytochemical detection of HSP60/HSP10 in the sperm mitochondria. Colloidal gold particles are mainly present in the sperm mitochondrial inner membranes. The black dots show the immunolabeled mitochondria matrix. The little box shows the magnified figure of the immunolabeled mitochondria. N: nucleus. (C1): secondary spermatocyte incubated with anti-HSP60 (×5600); (C2): secondary spermatocyte incubated with anti-HSP60 (×5600); (C3): secondary spermatocyte incubated with anti-HSP10 and anti-HSP60 (×5600); (C4): secondary spermatocyte for the negative control with omission of the first antibody (×6600).