Introduction

Spermatogenesis is a highly ordered process, during which the spermatogenic stem cell develops into spermatozoa. After mitotic proliferation and meiotic division, spermiogenesis dramatically takes place with three subsequent significant events happening in spermatids: nucleus reshaping, acrosome biogenesis and flagellum formation [1, 2]. Contrary to our more substantial morphological knowledge about spermatogenesis, the functions of motor proteins during morphological rearrangements are still less understood. On account of the similarity in sperm structure morphological changes during spermiogenesis compared to mammals, we have used an octopod cephalopod Octopus tankahkeei as a suitable model system for the study of molecular mechanisms of spermatogenesis [3–7]. Kinesin II motor protein is typically a heterotrimeric complex composed of KIF3A/KIF3B or KIF3A/KIF3C heterodimers and a scaffold or adaptor non-motor protein KAP3 [8–15]. As originally found in the neuronal axon, KIF3 was used to drive fast anterograde transport, which is a microtubule plus-end directed activity pivotal for neurite elongation [10, 16–19] and neuronal polarity [20, 21]. There is also abundant evidence that KIF3 is responsible for transporting melanosomes and endosomes, the Golgi-ER secretary pathway, and also KIF3 takes part in mitosis [15, 22–25]. In addition, KIF3 is involved in Hedgehog signaling pathways on account of its contribution to the primary cilium, a specialized immotile cilium that has been implicated in intracellular signaling as the cell’s antenna [26]. KIF3 also determines embryonic left–right asymmetry [27, 28]. Furthermore, KIF3 subunits are involved in intraflagellar transport (IFT), a bidirectional movement critical for the maintenance and assembly of motile cilia and flagella [29–33]. KIF3 function is particularly intriguing with respect to spermiogenesis because of the massive reorganization and development of both the axonemal tail and the specialized membrane components that occur during this process.

In a previous study, we found a homologue of rat C-terminal kinesin KIFC1 which participates in octopus sperm nucleus shaping via the microtubular structure manchette [34, 35]. We also isolated an N-terminal kinesin gene encoding a homologue of KIF3A which possibly takes part in intraflagellar and intramanchette transport in O. tankahkeei [36]. As we know, KIF3A and KIF3B always interact with each other, forming a stable heterodimer in intracellular transport. To date, nobody knows whether KIF3B participates in spermatogenesis as KIF3A does and its relationship with KIF3A during spermatogenesis in O. tankahkeei. Based on the observation of kif3a expression pattern and its association with kif3b [36], we hypothesize that the octopus homologue of kif3b shares a similar expression pattern with ot-kif3a, and the putative Kinesin II complex plays a key role in the flagellum elongation, intraflagellar transportation and intramanchette transportation.

For this paper, we cloned a cDNA encoding a highly conserved homologue of KIF3B for the first time and detected its distribution profile in various tissues of O. tankahkeei. In addition, we analyzed the expression pattern of ot-kif3b mRNA and compared it with the expression pattern of ot-kif3a, along with a corresponding discussion on the motor’s potential functions during spermiogenesis in O. tankahkeei.

Materials and methods

Animals

Specimens of O. tankahkeei were purchased from Ningbo Aquatic Products Market from July, 2009 to November, 2010. Forty adult male individuals were selected, half of which were transported to the Sperm Laboratory at Zhejiang University in sea water tanks with aeration facility. Following temporary maintenance, the animals were anesthetized on ice and dissected to obtain the testis, heart, gill, muscle, stomach, liver and pancreas. The tissues were then swiftly placed and preserved in liquid nitrogen for RNA extraction. Testes and seminal vesicles from eight different animals were also subjected to fixation with 4% PFA-PBS (ph 7.4) for in situ hybridization. Meanwhile, the other twenty individuals were carried to the Faculty of Life Science and Bioengineering at Ningbo University and sampled in the same way for histological analysis and transmission electron microscopy, respectively.

RNA extraction and degenerate primer design

Total RNA was extracted following established protocols in our laboratory [34]. Protein sequences of KIF3B homologues from various species were aligned by Vector NTI advanced version.10 (Invitrogen) and a number of conserved blocks of amino acid residues were detected. Degenerate primers, F1, R1, R2 and F3 (Supplementary Table.S1) were designed using the online software CODEHOP (http://bioinformatics.weizmann.ac.il/blocks/codehop.html) based on the amino acid residues of selected regions.

cDNA synthesis and full-length ot-kif3b cDNA cloning

First-strand cDNA synthesis was conducted by HiFi-MMLV cDNA kit (CWBIO) according to the manufacture’s instruction. Degenerate primers and a touchdown PCR strategy was used to obtain a cDNA fragment of ot-kif3b. The program was set as: 94°C for 180 s; 16 cycles of 94°C for 30 s, 55°C (which was reduced by 0.5°C each cycle) for 30 s and 72°C for 30 s; 20 cycles of 94°C for 30 s, 47°C for 30 s and 72°C for 40 s; 72°C for 10 min. Eventually, the PCR product was gel-excised, ligated to PMD-18T vector (Takara), transformed into DH5α Competent cells (Takara) and finally sequenced by Shanghai Sangon Company.

After a segment of the target cDNA was obtained, both 5′ and 3′ rapid-amplification of cDNA ends (RACE) were performed for the full-length ot-kif3b cDNA by utilizing Smart RACE cDNA amplification Kit (CloneTech) and 3′ Full RACE Amplification Kit (Takara) according to the Kit instructions. Gene specific primers (F4, F5, and R4) (Supplementary Table.S1) designed from the obtained sequence and adaptor primers supplied in the RACE Kit were used for the RACE-PCR reaction, of which the system and cycling conditions were recommended in the manual book. For the products purification, transformation and sequencing, see above.

Sequence analysis, multiple sequences alignment, and phylogenetic analysis

The sequence analysis of ot-kif3b cDNA and multiple sequences alignment was performed by the Vector NTI advanced version.10 (Invitrogen). Sequence comparison between ot-kif3a and ot-kif3b was operated by the BLAST program at the National Center for Biotechnology Information (http://blast.ncbi.nlm.nih.gov/). The phylogenetic analysis and statistical neighbor-joining bootstrap tests of the phylogenies were carried out by using the MEGA version 4.0 software. The secondary structure of ot-KIF3A and ot-KIF3B was predicted with Jpred 3 (http://www.compbio.dundee.ac.uk/www-jpred/).

Tissue expression pattern analysis

In order to examine tissue expression pattern of ot-kif3b, RNA extracted from the heart, gill, muscle, stomach, liver and pancreas of adult males underwent RT-PCR, respectively, with a pair of gene specific primers (TF and TR) (Supplementary Table.S1) resulting in a 233 bp product. PCR was running as follows: initial incubation at 94°C for 5 min; 30 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 30 s; 72°C for 10 min. Primers of octopus β-actin (Actin-F and Actin-R) were used as control.

Histological analysis

Samples for histological analysis were fixed in Bouin’s fixative, and dehydrated in graded alcohols followed by infiltration with Histoclear (xylene) and paraffin. Then the samples were sectioned at 7 μm, stained with haematoxylin/eosin and observed under the light field of a Nikon Eclipse E80i fluorescence microscope.

Transmission electron microscopy

Ten samples for TEM were fixed in 2.5% glutaraldehyde (0.2 M sodium cacodylate buffer, pH 7.4) for 1–2 h at 4°C, then postfixed in 1% osmium tetroxide under the same condition. Samples were subsequently dehydrated and embedded in Spurr’s resin. Thin sections were cut on a LKB-III ultramicrotome, double stained with uranium acetate and lead citrate, and then observed and photographed using a JEM-1200EX transmission electron microscope.

In situ hybridization

In situ hybridization was performed according to the methods used in our previous study [34]. Briefly, a 501 bp fragment of ot-kif3b was obtained by PCR using two gene specific primers (Probe-F1, Probe-R1) (Supplementary Table.S1) and ligated to PGEM-T EASY Vector (Promega). After that, DIG-11-UTP incorporated riboprobe complementary in a segment of ot-kif3a mRNA and was transcribed in vitro using related reagents and linearized plasmid as a template. Then sections from testes and seminal vesicle were prepared for the prehybridization and hybridization. The chromogenic reaction was performed using NBT and BCIP (Promega) after the sections were incubated with anti-DIG-Ap-conjugated Fab fragment (Roche Diagnostics). Finally the hybridized sections were observed under the bright field of a Nikon Eclipse E80i fluorescence microscope.

Results

Sequence analysis of ot-kif3b and comparison with ot-kif3a

The complete ot-kif3b cDNA (GenBank accession number HQ689699) (Supplementary Fig.S1.) we finally obtained consisted of a 102 bp 5′ untranslated region (UTR), a 2,208 bp open reading frame (ORF), and a 55 bp 3′ UTR. The ORF encoded a putative protein of 736 amino acids with a predicted molecular weight of 84 kDa and a theoretical isoelectric point of 8.75.

Alignment among predicted Ot-KIF3B and its homologues from vertebrates and invertebrates was shown in Supplementary Fig.S2. The result indicated that sequence of Ot-KIF3B shared 68, 68, 69, 68, and 67% identity with KIF3B from Homo sapiens, Mus musculus, Gallus gallus, Danio rerio, and Xenopus laevis, respectively. The putative ATP-binding domain including the VVVRCRP, NGTIFA, GQTGTGKT and DGENHIRVGKLNLVDLAGSERQ sequences and the microtubule-binding motif, HIPYRDSKLTRLL, were highly conserved among all the species. A phylogenic tree was constructed based on the amino acid sequences of KIF3B homologues from various organisms. The putative protein was closely related to KIF3B homologues from invertebrates to vertebrates (Supplementary Fig.S3.).

We previously reported a member of Kinesin II superfamily in O. tankahkeei, which was homologous to KIF3A and named Ot-KIF3A [36]. In order to elucidate the fundamental relationship between the two subunits, we compared their primary and secondary structures. A diagonal dot matrix comparison between Ot-KIF3A and Ot-KIF3B showed the regions of high identities (52% in overall length) expanding from the motor domain to the latter half of the stalk region (Fig. 1a). According to secondary structure prediction analysis, Ot-KIF3B has an NH2-terminal motor domain, a coiled-coil stalk region and a COOH-terminal divergent tail domain (Fig. 1b).

Fig. 1
figure 1

Comparison of sequence and secondary structure of Ot-KIF3A and Ot-KIF3B. a Diagonal dot matrix view was performed for pairwise identities of Ot-KIF3A and Ot-KIF3B by Vector NTI advanced version.10 (Invitrogen) software using a window size of 8 and stringency of 60%. The Ot-KIF3A query sequence is represented on the X-axis and the numbers represent the bases/residues of the query. The Ot-KIF3B subject is represented on the Y-axis and again the numbers represent the bases/residues of the subject. Alignments are shown in the plot as lines. b Diagram showing the secondary structure predictions of Ot-KIF3A and Ot-KIF3B. Both contained an N-terminal highly conserved motor domain which may form a globular head, the coiled-coil stalk region, and a C-terminal tail region that was significantly different

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Tissue distribution of ot-kif3b

To investigate the distribution pattern of ot-kif3b in different tissues from O. tankahkeei, RT-PCR with a pair of gene specific primer (TF1, TR1) (Supplementary Table.S1) were utilized to amplify a 233 bp cDNA fragment. The result showed that ot-kif3b was expressed in all the tissues above, but the expression level varies with different tissues (Fig. 2). Its expression was high in the gill, pancreas, and muscle, paralleled in the testis, heart and the stomach, and slightly lower in the liver.

Fig. 2
figure 2

The distribution of ot-kif3b by RT-PCR in various tissues of the O. tankahkeei. β-actin was used as a positive control (lower panel). H heart, G gill, S stomach, L liver, P pancreas, M muscle, T testis

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Histological and morphogenesis changes in the testis and the spatial–temporal expression pattern of ot-kif3b during spermiogenesis

To explore the spatial and temporal expression pattern of ot-kif3b transcript during testis development and to determine the testicular cell types expressing this gene, we performed in situ hybridization and histological and transmission electron microscopy analysis. Our data showed that in early spermatid the nucleus was nearly round with mass-like chromatin condensed gradually. Mitochondria were well-distributed in the cytoplasm near the Golgi apparatus (Fig. 3a, b). The mRNA signals were already detectable but weak in cytoplasm (Fig. 3c, d, arrow). As the flagellum emerged from the caudal end of the intermediate spermatid, the nucleus became eclipse-shaped and the chromatin existed as fibrosis. The posterior nuclear pocket invaginated deeply with the centriole located near the fossa of the pocket where axoneme of the tail originated (Fig. 3e, f). In this period, the message was expressed all over the cell and especially concentrated at the tail region (Fig. 3g, arrow). In late spermatid, the nucleus elongated into a long column and the chromatin compressed into stratification. It seemed clear that mitochondria aggregated to the posterior region of the spermatid (Fig. 3i, j). Obviously, the signal intensity increased to an extent reaching the maximum (Fig. 3h, k, arrow). Gradually, the kif3b transcripts accumulated around the nucleus and in the back of the spermatid, along with the growing tail (Fig. 3l). After the remodeled spermatid underwent significant morphological change, the final spermatid had a slim columned nucleus, with a highly condensed chromatin inside (Fig. 3m, n). In mature spermatozoa, the acrosomal vesicle became transferred into a helicoidal acrosomal cone (Fig. 3o), which was unique to the sperm of the octopod cephalopods [37]. The transcripts were abundant mostly around the nucleus and in the midpiece of the tail (Fig. 3p, arrow). This expression pattern was in accordance with that of ot-kif3a during spermiogenesis of O. tankahkeei [36], suggesting they may maintain a close relationship in their functions. The general ot-kif3a/b expression pattern during spermiogenesis is illustrated using a schematic picture (Fig. 4).

Fig. 3
figure 3

Histological and morphogenesis changes in the testis and the spatial–temporal expression pattern of ot-kif3b during spermiogenesis. a, b In early spermatid, the nucleus was nearly round within mass like chromatin gradually condensed. The posterior of the nucleus depressed into a pocket while mitochondria were well-distributed in the cytoplasm near the Golgi apparatus. c, d In early spermatid, the signal is detected (arrow); e, f In intermediate spermatid, the nucleus shaped ellipse and the chromatin existed as fibrosis. The posterior nuclear pocket invaginated deeply. g, h The message expressed all over the cell, especially concentrating at the tail region (arrow). i, j In the late spermatid undergoing dramatic lateral compression and elongation, the acrosomal vesicle is long cystiform in shape at the apical tip while in the opposite part, endonuclear channel was formed. Mitochondria aggregated to the posterior region of the spermatid. k, l The message is abundant around the nuclear, enriching at the tail region (arrow). m–o The final spermatid had a slim columned nucleus in shape, with a highly condensed chromatin inside. The arosomal vesicle became into a helicoidal acrosomal cone. The flagellum is clear. h In final spermatid, the signal is mostly around the nucleus with the most intensity in the midpiece of the tail (arrow). q In mature sperm, the expression pattern is similar to that of the final spermatid (arrow). a, e, i, m, histological analysis, by HE staining. Scale bar, 2 μm; b, f, j, n, o Transmission Electron Microscopy analysis. Scale bar, 0.5 μm. c, d, g, h, k, l, p, q in situ hybridization analysis. Arrows indicated ot-kif3b transcripts in corresponding stages. Scale bar, 1 μm. AV acrosomal vesicle, M mitochondria, N nucleus, PNP posterior nuclear pocket, EC endonuclear channel, MP midpiece, F flagellum, AC acrosomal cone

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Fig. 4
figure 4

A schematic model produced on the basis of the previous exhibition and representing the spatial and temporal expression profile of ot-kif3b during spermiogenesis of O. tankahkeei. The blue spherical granule indicates the mRNA transcripts with a corresponding density. a In early spermatid, the signal is already appeared with a low level. b In intermediate spermatid, the transcripts gradually increase as well as emerge in the growing tail. c In drastically remodeling spermatid, the messages distribute along the longitudinal elongation and get the maximum. d In late spermatid, the signal is still abundant especially around the nucleus and in the midpiece and the tail. e In mature spermatid, the transcripts are still visible. N nucleus, AV acrosomal vesicle, PNP posterior nuclear pocket, C centrioles, F flagellum, AC acrosomal cone. EC endonuclear channel, MP midpiece, T tail

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Discussion

In eukaryotic cells, cytoskeleton, which forms a network, contributes to the cellular integrity. This network provides a highway for transporting and re-localizing of cellular components via the activity of molecular motors [38, 39]. Microtubule-dependent kinesin motors are essential for intracellular transport, carrying different cargoes, such as vesicles, membranous organelles, synthesized proteins, and mRNAs to specific destinations [40–44]. The developed delivery system determines the unique morphology and functions of differentiated cells, not only in neuron cells [43, 44] and somatic cells [45, 46] but also in germ cells [34–36, 47–50]. Kinesins have been demonstrated to play significant roles in spermiogenesis not only in mammals [51], but also in invertebrates [34–36, 50]. For instance, KIF2Aβ is distributed in the perinuclear area during spermatogenesis interacting with translin associated factor-X (TRAX) in mouse [51]. In intraflagellar transport (IFT), KIF17, interacting with the nuclear import protein (importin-β2), is proposed to be regulated by a ciliary localization signal (CLS) and inhibited by Ran GTPase to obtain a passport into the ciliary compartment [52]. A carboxyl-terminal (C-terminal) motor protein KIFC1 is believed to participate in a Golgi-acrosome bidirectional pathway and thus contribute to spermatid nuclear reshaping and acrosome morphogenesis in mammals [49, 53] as well as in crustaceans [50, 54]. However, the functions of kinesin motors during spermiogenesis in mollusks such as Cephalopoda remain unclear.

To date, the characterization of kif3b gene and its expression pattern during octopod spermiogenesis is reported in this work for the first time. The amino acids of Ot-KIF3B shared common features of the Kinesin II superfamily, including an amino-terminal motor domain, a coiled-coil stalk and a carboxy-terminal tail region. Multiple sequence alignments showed that sequences of the putative protein and KIF3B homologues from vertebrates were highly conserved in spite of the taxonomic distance between octopod and vertebrates, which is similar to the situation of Ot-KIF3A [36]. Based on the accepted argument that KIF3A and KIF3B always form a heterodimer at the α-helical rod domain, associating with the adaptor KAP3 [10, 12, 16, 18, 55], we compared the fundamental structures of Ot-KIF3A and Ot-KIF3B. Ot-KIF3B showed 53% identity in full length with Ot-KIF3A and they were extremely similar at the motor domain. Such identities suggested that the putative Ot-KIF3A and Ot-KIF3B may be alternative spliced variants of kif3.

Tissue distribution pattern of ot-kif3b revealed its ubiquitous expression in all the tissues examined at gene level was consistent with that of ot-kif3a [36]. This result was in agreement with reports proposing KIF3 was one of the most abundantly expressed KIFs, not only functioning in the best known intraflagellar transport but also in neural, melanosome, Golgi-ER transport, mitosis, and tumor suppression [12, 23, 24, 43]. We supposed that the function of this motor protein may not be restricted to testis, but was tissue specific relying on different microenvironments. In situ hybridization directly demonstrated that expression of ot-kif3b mRNA was gradually high throughout the developing spermatid. The expression profile of ot-kif3b was quite similar to that of ot-kif3a, but unlike ot-kifc1 which was not expressed at mature stages [34].

Such extraordinary similarity of expression pattern between ot-kif3a and ot-kfi3b, together with the homologous fundamental structure, gave us strong reason to believe that the putative Ot-KIF3A and Ot-KIF3B may form a conventional heterodimeric complex, of which both coiled-coil stalks interacted stably with each other, just like in many vertebrates and invertebrates [10, 12, 13, 43].

From intermediate to mature spermatids, the expression of ot-kif3a/b is abundant in the midpiece and flagellum of the tail. These observations agree with a previous study claiming that the heterodimeric kinesin II is associated with the midpiece and flagellum of spermatic cells in sea urchins [56] and rats [38]. Further studies proposed that the complex was essentially involved in intraflagellar transport (IFT) [30, 57]. Kinesin II transports the rafts along microtubules to the tip of the axoneme beneath the flagellum membrane [30, 57–59]. Considering IFT transport is evolutionarily conserved [29] and the striking similar structure of the sperm flagella between O. tankahkeei and mammals, we assume that Ot-KIF3B with its close partner Ot-KIF3A, may take part in the maintenance and assembly of the sperm flagellar axoneme.

Sharing a similar molecular mechanism to IFT, intramanchette transport (IMT) also requires Kinesin II heterodimeric homoenzyme [60]. In O. tankahkeei, a microtubule-based perinuclear structure was observed transiently during spermiogenesis [4, 35], which was in agreement with another cephalopod, S. officinalis possessing the same structure originating from the spermatid head [61]. Since the perinuclear structure plays the same as mammalian manchette [4, 35], the abundant distribution of ot-kif3a/b around the perinulear structure shed light on the potential function of this proteins in IMT transport. Likewise, Ot-KIFC1 was co-localized with the manchette-like structure, which was proved to participate in sperm nuclear morphogenesis as its homologue does in rodents [35, 49]. Based on the findings above, however, we are still not sure whether Ot-KIF3A/B takes part in sperm nuclear reshaping through bridging the manchette-like perinuclear microtubules in association with Ot-KIFC1. Even if they did, they must have a distinct mechanism for processes depending on their reverse direction of transportation.

In O. tankahkeei, mitochondria are actively transported posteriorly and eventually form the chondriosomal mantle at mid-piece of the spermatid in biological adaption to internal fertilization. During spermiogenesis, IMT and IFT deliver cargoes for the tail formation, prompting that mitochondria may aggregate in the mid-piece by IMT and IFT [62]. Interestingly, the ot-kif3a/b transcripts expression profile was consistent with the dynamics of mitochondria, making it plausible to speculate the relationship between mitochondria and KIF3A/B in spermatid tail development. In a sense, this assumption supplements a previous suggestion that only KIF1Bα, which belongs to the kinesin 3 family, and KIF5 motors play roles in mitochondrial transport [43, 62, 63]. However, the information available at present doesn’t allow a thorough investigation of the distribution of mitochondria during spermiogenesis of O. tankahkeei. Further studies on the localization of KIF3A/B and mitochondria as well as the machinery between motor proteins and corresponding adaptors will be required before the hypothesis can be advanced to a conclusion regarding the specific transport role in this process [63–65].

Moreover, the timing and synchrony of spermatid differentiation need signaling transduction between spermatogenic cells and between Sertoli cells and Leydig cells [66, 67], which is possibly achieved through participation of the cell’s sensory organelle, or primary cilia. It is recommended that through IFT, primary cilium works as eukaryotic cell’s antenna transducing a multitude of sensory stimuli to coordinate a number of physiological and developmental signaling pathways [58, 68, 69]. Because of its involvement in intraflagellar transport, KIF3 is also implicated in some signaling pathways such as Hedgehog and Wnt pathways [26, 70]. Since ot-kif3a/b transcripts were enriched at the tail region at late and final stages, it’s likely that Ot-KIF3A/B may help the coordination among different cells in testis via regulating the behavior of primary cilia or some signaling factors. Nonetheless, more experiments with direct protein function analysis are still required to ascertain the function of Ot-KIF3A/B.

In conclusion, our results revealed that the octopus homologue of KIF3B firstly detected is in high identities with Ot-KIF3A, reported before by our laboratory, in fundamental structures and shares a similar expression pattern with ot-kif3a, all of which support our hypothesis on their potential interactions in intraspermatid transport during spermiogenesis of O. tankahkeei. The putative heterodimer may be involved in intraflagellar and intramanchette transport by carrying different cargoes to a specific destination, hereby playing key roles in the formation of flagellum and primary cilia. This protein complex may also participate in some signaling transduction pathway and operate the nucleocytoplasmic exchange activities. Furthermore, the heterodimer may in a sense contribute to the aggregation of mitochondria to the midpiece. A schematic model about possible functions of the putative Ot-KIF3A/B heterodimer during spermiogenesis of O. tankahkeei is displayed on Fig. 5.

Fig. 5
figure 5

A schematic model displaying the three possible functions of the putative Ot-KIF3A/B heterodimer during spermiogenesis of O. tankahkeei. a The complex drive different IFT particles to which the scaffolding/adaptor protein KAP3 directly linking, from the distal to the tip of the axon beneath the flagellum membrane, involving in the formation of flagellum and primary cilia, further accomplishing some signaling transduction pathway. b Along the manchette-like perinulear microtubules, Kinesin II may carry some IMT rafts and further operating the nucleocytoplasmic exchange activities. c The heterodimer may in a sense contribute to the aggregation of mitochondria in the midpiece

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