Analysis of the function of KIF3A and KIF3B in the spermatogenesis in Boleophthalmus pectinirostris
Spermatogenesis represents one of the most complicated morphological transformation procedures. During this process, the assembly and maintenance of the flagella and intracellular transport of membrane-bound organelles required KIF3A and KIF3B. Our main goal was to test KIF3A and KIF3B location during spermatogenesis of Boleophthalmus pectinirostris. We cloned complete cDNA of KIF3A/3B from the testis of B. pectinirostris by PCR and rapid amplification of cDNA ends (RACE). The predicted secondary and tertiary structures of B. pectinirostris KIF3A/3B contained three domains: (a) the head region, (b) the stalk region, and (c) the tail region. Real-time quantitative PCR (qPCR) results revealed that KIF3A and KIF3B mRNA were presented in all the tissues examined, with the highest expression seen in the testis. In situ hybridization (ISH) showed that KIF3A and KIF3B were distributed in the periphery of the nuclear in the spermatocyte and the early spermatid. In the late spermatid and mature sperm, the KIF3A and KIF3B mRNA were gradually gathered to one side where the flagella formed. Immunofluorescence (IF) showed that KIF3A, tubulin, and mitochondria were co-localized in different stages during spermiogenesis in B. pectinirostris. The temporal and spatial expression dynamics of KIF3A/3B indicate that KIF3A and KIF3B might be involved in flagellar assembly and maintenance at the mRNA and protein levels. Moreover, these proteins may transport the mitochondria resulting in flagellum formation in B. pectinirostris.
KeywordsKIF3A/3B Boleophthalmus pectinirostris Spermiogenesis Flagellar development
Rapid amplification of cDNA ends
Real-time quantitative polymerase chain reaction
Open reading frame
In situ hybridization
Kinesin superfamily proteins
Kinesin-associated protein 3
Phosphate buffered saline
Optimum cutting temperature
Albumin from bovine serum
Transmission electron microscopy
We are indebted to all members of the Fish Reproduction Physiology Laboratory at Ningbo University and the Sperm Laboratory at Zhejiang University for their enlightening discussions.
This project was partially supported by the National Natural Science Foundation of China (No. 31272642), Scientific and Technical Project of Ningbo (No. 2015C110005), Ningbo Natural Science Foundation (No. 2016A610081), Collaborative Innovation Center for Zhejiang Marine High-efficiency and Healthy Aquaculture, K.C. Wong Magna Fund in Ningbo University, and Scientific Research Foundation of Graduate School of Ningbo University.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- Chennathukuzhi V, Morales CR, El-Alfy M, Hecht NB (2003) The kinesin KIF17b and RNA-binding protein TB-RBP transport specific cAMP-responsive element modulator-regulated mRNAs in male germ cells. Proc Natl Acad Sci U S A 100(26):15566–15571. https://doi.org/10.1073/pnas.2536695100 CrossRefPubMedPubMedCentralGoogle Scholar
- Cole DG, Diener DR, Himelblau AL, Beech PL, Fuster JC, Rosenbaum JL (1998) Chlamydomonas kinesin-II-dependent intraflagellar transport (IFT): IFT particles contain proteins required for ciliary assembly in Caenorhabditis elegans sensory neurons. J Cell Biol 141(4):993–1008. https://doi.org/10.1083/jcb.141.4.993 CrossRefPubMedPubMedCentralGoogle Scholar
- Henson JH, Cole DG, Roesener CD, Capuano S, Mendola RJ, Scholey JM (1997) The heterotrimeric motor protein kinesin-II localizes to the midpiece and flagellum of sea urchin and sand dollar sperm. Cell Motil Cytoskeleton 38(1):29–37. https://doi.org/10.1002/(SICI)1097-0169(1997)38:1<29::AID-CM4>3.0.CO;2-C CrossRefPubMedGoogle Scholar
- Hermo L, Pelletier RM, Cyr DG, Smith CE (2010) Surfing the wave, cycle, life history, and genes/proteins expressed by testicular germ cells. Part 1: background to spermatogenesis, spermatogonia, and spermatocytes. Microsc Res Tech 73(4):241–278. https://doi.org/10.1002/jemt.20783 CrossRefPubMedGoogle Scholar
- Hess R, Franca L (2009) Spermatogenesis and cycle of the seminiferous epithelium. In: CY Cheng (ed) Molecular mechanisms in spermatogenesis, pp 1–15. New York: SpringerGoogle Scholar
- Marszalek JR, Ruiz-Lozano P, Roberts E, Chien KR, Goldstein LS (1999) Situs inversus and embryonic ciliary morphogenesis defects in mouse mutants lacking the KIF3A subunit of kinesin-II. Proc Natl Acad Sci U S A 96(9):5043–5048. https://doi.org/10.1073/pnas.96.9.5043 CrossRefPubMedPubMedCentralGoogle Scholar
- Nonaka S, Tanaka Y, Okada Y, Takeda S, Harada A, Kanai Y, Kido M, Hirokawa N (1998) Randomization of left–right asymmetry due to loss of nodal cilia generating leftward flow of extraembryonic fluid in mice lacking KIF3B motor protein. Cell 95(6):829–837. https://doi.org/10.1016/S0092-8674(00)81705-5 CrossRefPubMedGoogle Scholar
- Pan X, Ou G, Civelekoglu-Scholey G, Blacque OE, Endres NF, Tao L, Mogilner A, Leroux MR, Vale RD, Scholey JM (2006) Mechanism of transport of IFT particles in C. elegans cilia by the concerted action of kinesin-II and OSM-3 motors. J Cell Biol 174(7):1035–1045. https://doi.org/10.1083/jcb.200606003 CrossRefPubMedPubMedCentralGoogle Scholar
- Tanaka Y, Kanai Y, Okada Y, Nonaka S, Takeda S, Harada A, Hirokawa N (1998) Targeted disruption of mouse conventional kinesin heavy chain, kif5B, results in abnormal perinuclear clustering of mitochondria. Cell 93(7):1147–1158. https://doi.org/10.1016/S0092-8674(00)81459-2 CrossRefPubMedGoogle Scholar
- tom Dieck S, Altrock WD, Kessels MM, Qualmann B, Regus H, Brauner D, Fejtova A, Bracko O, Gundelfinger ED, Brandstatter JH (2005) Molecular dissection of the photoreceptor ribbon synapse: physical interaction of Bassoon and RIBEYE is essential for the assembly of the ribbon complex. J Cell Biol 168:825–836CrossRefGoogle Scholar