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KIFC1 and myosin Va: two motors for acrosomal biogenesis and nuclear shaping during spermiogenesis of Portunus trituberculatus

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Abstract

To investigate the molecular mechanisms underlying the spermiogenesis of the swimming crab Portunus trituberculatus, full lengths of motor proteins KIFC1 and myosin Va were cloned by rapid-amplification of cDNA ends from P. trituberculatus testes cDNA, and their respective probes and specific antibodies were used to track their localization during sperm maturation. Antisense probes were designed from the gene sequences and used to detect the mRNA levels of each gene. According to the results of fluorescence in situ hybridization (FISH), the transcription of kifc1 and myosin Va began at the mid-stage of spermatids, with the kifc1 mRNA being most active at the location where the acrosome cap was formed and the myosin Va was more concentrated in the acrosome complex. Immunofluorescence results showed that KIFC1 and myosin Va were highly expressed in each stage of spermigenesis. In the early spermatids, they were randomly dispersed in the cytoplasm together with cytoskeletons. At the mid-stage, the motors were gathered above one side of the nucleus where the acrosome would later form. In the late spermatids and mature sperm, the KIFC1 was closely distributed in the perinuclear region, indicating its role in nucleus deformation. Myosin Va was distributed in the acrosome complex until sperm maturity. This suggests myosin Va’s potential role in material transportation during acrosome formation and maturation. The above results provide a preliminary illustration of the essential roles of KIFC1 and myosin Va in the spermiogenesis of the swimming crab P. trituberculatus.

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Acknowledgements

The authors are grateful to all members of the Sperm Laboratory in Zhejiang University for their valuable assistance on the present work. The authors also thank Chris Wood for his effort in grammar checking and linguistic polishing of this manuscript. This project was supported by the Natural Science Foundation of China (Nos. 31572603 and 41276151) and the Zhejiang Provincial Natural Science Foundation of China (No. LY15H040003).

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Authors and Affiliations

  1. The Sperm Laboratory, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, China

    Dan-Dan Ma, Meng-Ying Pan & Wan-Xi Yang

  2. School of Marine Sciences, Ningbo University, Ningbo, Zhejiang, 315211, People’s Republic of China

    Cong-Cong Hou

  3. The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, 310003, China

    Fu-Qing Tan

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  1. Dan-Dan Ma
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Correspondence to Wan-Xi Yang.

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Funding

This project was supported by the Natural Science Foundation of China (Nos. 31,572,603 and 41,276,151) and the Zhejiang Provincial Natural Science Foundation of China (No. LY15H040003).

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Fig. S1

Full-length cDNA of the kifc1 in P. trituberculatus. The deduced amino acid sequence is shown below the nucleotide sequence. The 2638-bp full length kifc1 cDNA consists of a 129-bp 5′ untranslated region, a 385-bp 3′ untranslated region and a 2124-bp open reading frame encoding 708 amino acids. (DOC 104 kb)

Fig. S2

Full-length cDNA of the myosin va in P. trituberculatus. The deduced amino acid sequence is shown below the nucleotide sequence. The 5561-bp full length myosin va cDNA consists of a 133-bp 5′ untranslated region, a 142-bp 3′ untranslated region and a 5286-bp open reading frame that encodes 1761 amino acids. (DOCX 39 kb)

Fig. S3

Induced expression of recombinant protein Pet 28a–MVa CBD. 1–4 Induction at 16 °C for 12 h; 5–8 induction at 37 °C for 12 h. 1, 5 control supernatant; 2, 6 control precipitate; 3, 7 Pet 28a–CBD supernatant; 4, 8; Pet 28a–Mva CBD precipitate. The recombinant protein expression was induced at both 37 °C and 16 °C, highly expressed in the precipitate as inclusion body protein (at about 37 KD, blue box). (GIF 349 kb)

High resolution image (TIFF 737 kb)

Fig. S4

KIFC1 overexpression in GC1 spg cell lines. KIFC1 is dispersed in both the nucleus and the cytoplasm but with the higher accumulation in the nucleus. 20 h KIFC1 modulates microtubule structure and changes cell morphology. 48 h the nucleus was seen to be elongated. Bar 20 μm). (GIF 513 kb)

High resolution image (TIFF 877 kb)

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Ma, DD., Pan, MY., Hou, CC. et al. KIFC1 and myosin Va: two motors for acrosomal biogenesis and nuclear shaping during spermiogenesis of Portunus trituberculatus . Cell Tissue Res 369, 625–640 (2017). https://doi.org/10.1007/s00441-017-2638-4

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