Adult Ciona intestinalis Type A (Ciona robusta) were cultivated in sterile artificial sea water (ASW) at 18°C at the Maizuru Fisheries Research Station at Kyoto University or Misaki Marine Biological Station at the University at Tokyo.

The 20-amino acid fragment of PEP51 (CSTGNKIYWNKFEQIKSHLY-NH₂, at amino acid sequence 22–41) was conjugated with keyhole limpet hemocyanin and used as an antigen (Sigma-Aldrich Japan, Tokyo, Japan). Antigen affinity-purified anti-PEP51 antibody raised in rabbits was obtained from Sigma-Genosys (Sigma-Aldrich Japan, Japan). Western blotting analysis confirmed that the anti-PEP51 antibody recognized endogenous PEP51 in the Ciona follicles and synthetic PEP51 peptide (Eurofin Genomics, Tokyo, Japan).

The Full-length sequence of PEP51 peptide (MLSIKSLVSICMVVQSILKNICSTGNKIYWNKFEQIKSHLYNLINQPNKLC) was chemically synthesized by Eurofin Genomics (Tokyo, Japan) with a purity of 99.06%.

Ciona ovaries were fixed in Bouin’s solution, embedded in paraffin, and cut into 7-μm sections. Preparation and immunostaining of the ovary sections were performed as previously described (15, 16). Immunoreactivity was visualized using the anti-PEP51 antibody (1:1000) and an alkaline phosphatase (AP) conjugated secondary antibody (1:2000) with BCIP-NBT (Nacalai Tesque, Kyoto, Japan) as a chromogen. No specific immunostaining was observed using a secondary antibody alone or the preabsorbed anti-PEP51 antibody. The preabsorbed anti-PEP51 antibody (1:1000) was prepared by incubation with the antigen peptide (CSTGNKIYWNKFEQIKSHLY-NH₂), which was used to generate the antibody, at a final concentration of 10^-6 M for overnight at 4 ˚C. All of the immunoreactivity experiments were performed in triplicate.

Isolated Ciona ovaries were washed with ASW, cut into several pieces, and enzymatically disaggregated with 0.2% (w/v) collagenase in ASW for 15 min at room temperature with orbital shaking. The disaggregated follicles from the ovaries were fractionated using a series of stainless-steel sieves of different mesh sizes (150, 90, 63, 38, and 20 μm; TOKYO SCREEN CO. LTD., Tokyo, Japan) as described previously (6, 7).

Proteins in the Ciona follicles were extracted using Proteoextract complete proteome extraction kit (Merck Millipore, Burlington, MA, USA) and quantified using the BCA protein assay kit. A 30-μg aliquot of soluble protein or 10-ng synthetic PEP51 peptide were separated by 16.5% tricine-SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) and transferred to PVDF membranes. The membranes were blocked with 5% skimmed milk in Tris-buffered saline with Tween 20, TBS-T (20mM Tris–HCl, 150mM NaCl, 0.1% Tween 20) at 4°C, and subsequently incubated overnight with rabbit anti-PEP51 antibody (diluted 1:2000) at 4°C. After washing three times with TBS-T, the blots were incubated with an AP-conjugated anti-rabbit IgG secondary antibody (diluted 1:50,000) for 1 h at room temperature. Blots were visualized by BCIP-NBT (Nacalai Tesque) according to the manufacture’s instruction.

Ciona ovaries from five adults were collected and washed with ASW, and the follicles were fractionated as described above. Isolated stage III follicles were enzymatically disaggregated (0.1% trypsin, 0.5% collagenase, and 0.2% actinase E in ASW) for 15 min at room temperature by orbital shaking to obtain single-cell suspensions. After disaggregation, 5% fetal bovine serum (BSA) was added, and cells were passed through a 20-μm cell strainer (Celltrix, Sysmex, Kobe, Japan) and were washed with ASW. Cells were then centrifuged (390 ×g for 2 min at 4°C) and resuspended in a staining solution (1% BSA in ASW) for 5 min. The anti-PEP51 antibody (1:300) and Alexa Fluor 488-conjugated secondary antibody (1:500) in 1% dimethyl sulfoxide (DMSO) containing the staining solution was then added and incubated for 30 min on ice, respectively. After washing with ASW, PEP51-positive (PEP51(+)) cells were sorted using a fluorescence-activated cell sorting (FACS) system (FACSMelody; BD Biosciences, NJ, USA). Subsequently, endogenous peptides in the FACS-sorted cells were extracted in 50% acetonitrile (ACN) containing 0.1% trifluoroacetic acid (TFA) by pipetting. The evaporated extract was treated with reverse phase C8 ZipTip for de-salting (Merck Millipore, Carrigtwohill, Ireland), and the elute was evaporated and concentrated. The peptides on an anchorchip were measured using Bruker rapifleX, matrix-assisted laser desorption/ionization (MALDI) time of flight (TOF) instrument (Bruker Daltonics, Bremen, Germany), and the resultant mass spectrometric data were analyzed using MASCOT database search (Matrix Science, Tokyo, Japan). In addition, In-gel tryptic digestion of the band corresponding to a size of 6kDa on tricine-SDS-PAGE of the stage III follicle lysate was analyzed for protein identification using a nanoLC-ESI Q-TOF MS instrument (QSTAR XL, Applied Biosystems, Waltham, MA, USA). The tandem MS (MS/MS) spectrometric data were analyzed using a MASCOT database search (Matrix Science). In-gel tryptic digestion and nanoLC-ESI Q-TOF MS/MS analysis was conducted by Japan Proteomics, Inc. (Miyagi, Japan).

Ciona ovaries were fixed in 4% paraformaldehyde (PFA) and 0.1% glutaraldehyde (GA) (Distilled EM grade, Electron Microscopy Sciences, Hatfield, PA, USA) in 0.1M phosphate buffer (PB) pH 7.4 at 4 °C for 1h. After washing, the samples were dehydrated and infiltrated with a 50:50 mixture of ethanol and resin, transferred to a fresh 100% resin (LR white, London Resin, Berkshire, UK), and polymerized by an ultraviolet polymerizer. Ultra-thin sections of the polymerized resins were mounted on nickel grids and incubated with anti-PEP51 antibody, followed by 15nm gold particle-labelled secondary antibody. The grids were placed in 2% GA in 0.1 M PB and dried, then stained with 2% uranyl acetate for 15 min and a Lead stain solution (Sigma-Aldrich, Tokyo, Japan). The samples were observed under a transmission electron microscope (JEM-1400Plus, JEOL, Tokyo, Japan) at 100 kV. Digital images were obtained with a CCD camera (EM-14830RUBY2, JEOL). Immunoelectron microscopy was carried out by Tokai Electron Microscopy, Inc. (Nagoya, Japan).

Total RNA (0.5 μg) extracted from Ciona stage III follicles was reverse-transcribed to the template cDNA at 55 °C for 60 min using the gene-specific primer (5’-GGTTCTAACATGTGTGGTCTTG-3’, identical to nucleotides 1009–1030), which was designed according to the sequence of the Pep51 gene (Ensembl gene ID: ENSCING00000021415) on the Ensembl web browser (http://asia.ensembl.org/Ciona_intestinalis/Gene/). The template cDNA was amplified by PCR using the primers 5’- TTCTACACCCTGCAAACACTGTAAC-3’, identical to nucleotides 310–334, and 5’-CTGATTGTGCTTCGTATCCTG-3’, complementary to nucleotides 988–1008. The PCR product was cloned into the TArget clone vector (Toyobo, Osaka, Japan). Subcloned inserts were sequenced on an Applied Biosystems 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA), using a Big-Dye sequencing kit (Applied Biosystems) and universal primers (T3 and T7 primers).

An open reading frame of Pep51 was amplified using gene-specific primers with extensions to vector ends (Fw; 5’-CCGGAATTCGCCATGTTGAGCATAAAATC-3’, and Rv: 5’- AAAGCGGCCGCTTAGCACAGTTTATTTG-3’, including stop codon), which contain an EcoRI site and NotI site, respectively. The amplified PCR product and the expression vector pcDNA4/V5 (Thermo Fisher Scientific, Waltham, MA, USA) were digested with EcoRI and NotI restriction enzymes, followed by ligation using Takara Ligation Mighty Mix (Takara, Shiga, Japan) according to the manufacturer’s instructions. The expression vector, empty pcDNA4 vector (0.1 μg), or transfection medium alone was transiently transfected into 5×10⁴ African green monkey kidney fibroblast cells (COS-7 line) or genetically engineered human embryonic kidney cells (HEK293MSR line) in 9.5-mm wells of multi-well glass-bottom dish (Matsunami, Osaka, Japan) with Lipofectamine 2000 (Thermo Fisher Scientific), according to the manufacturer’s instructions. COS-7 cells and HEK293MSR cells were grown under 5% CO₂ at 37°C in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated FBS, and DMEM supplemented with 10% heat-inactivated FBS and 1% non-essential amino acids, respectively. Transfection efficiency (approximately 60-70%) was evaluated by an immunocytochemistry as described below.

After transfection of the Pep51 expression vector into COS-7 cells and HEK293MSR cells for 2 days at 37°C, a cell-permeable fluorescent active caspase-3/7 probe, FAM-DEVD-FMK (FAM-FLICA Caspase-3/7 assay kit, ImmunoChemistry Technologies, Davis, CA, USA), which detects early stage of apoptosis, was added to PEP51-expressed cells on 9.5mm-wells of multi-well glass-bottom dish for 30 min at 37°C. The stained cells were washed three times with 0.1M PBS pH 7.4, and fixed with 4% PFA in PBS for 30 min at room temperature. Subsequently, the cells were washed three times with TBS-T, blocked with 1% BSA for 30 min, and then treated with the anti-PEP51 antibody conjugated HiLyte Fluor 555 with an Ab-10 rapid fluorescein labeling kit (Dojindo) (diluted to 1:500) in TBS-T for 30 min. The signals of the active caspase-3/7 probe, FAM-DEVD-FMK and immunofluorescence staining were visualized using a confocal laser scanning microscope (Fluoview FV3000, Olympus, Tokyo, Japan) as described previously (15, 16).

According to previous in vivo uptake studies on Ciona follicles (6, 26, 27), follicle cells were enzymatically removed (0.2% actinase E in ASW) from the fractionated follicles for 10 min at room temperature by orbital shaking. After washing, the active caspase-3/7 probe, FAM-DEVD-FMK (FAM-FLICA caspase-3/7 assay kit, ImmunoChemistry Technologies) was added to the defolliculated follicles for 30 min at room temperature. The stained follicles were washed with 0.1M PBS pH 7.4, and fixed with 4% PFA in PBS at 4 °C for 16 hours. The fixed follicles were washed three times with TBS-T, blocked with 1% BSA for 30 min, and then treated with HiLyte Fluor 555-conjugated anti-PEP51 antibody (diluted to 1:200) in TBS-T for 30 min at room temperature. The signals of the active caspase-3/7 probe and immunofluorescence staining were visualized using a confocal laser scanning microscope (Fluoview FV3000, Olympus), as described above. For flow cytometry of test cells, the stained early stage III follicles were enzymatically disaggregated (0.1% trypsin, 0.5% collagenase, and 0.2% actinase E in ASW) for 15 min at room temperature by orbital shaking, and cells were passed through a 20-μm cell strainer (Sysmex) after adding 5% BSA. The stained test cells were fixed with 4% PFA in 0.1M PBS pH 7.4 at room temperature for 30 min. The fixed cells were washed three times with TBS-T, blocked with 1% BSA for 30 min, and then treated with HiLyte Fluor 555-conjugated anti-PEP51 antibody (diluted to 1:500) in TBS-T for 30 min at room temperature. After washing with TBS-T, the stained test cells were analyzed using BD FACSMelody (BD Biosciences).

Results are expressed as means ± standard error for the indicated number of observations. Data were analyzed by one-way analysis of variance with Dunnett’s error protection. Differences were accepted as significant for P <0.05.

We initially searched for the previous transcriptomes of the Ciona intestinalis Type A (accession ID; SRR24652239) and identified a 51-amino acid (51 aa) peptide-encoding gene (Ensembl gene ID: ENSCING00000021415; chromosome 3: 699,794 -700,784). No orthologs of the 51 aa peptide were detected on any database, indicating that this gene, named Pep51, is a Ciona-specific gene. The full-length cDNA was cloned from Ciona ovaries, and the open reading frame was found to encode a 51 aa peptide which completely accorded with that of the aforementioned sequence ( Figure 1 ). To detect the translated PEP51 from the mRNA, we conducted immunohistochemical analysis of Ciona ovary sections using an anti-PEP51 antibody. As shown in Figure 2A , PEP51 was detected in many test cells of early stage III (post-vitellogenic) follicles. No positive signals were observed when antigen-absorbed antibody was applied, confirming the specificity of the immunostaining analysis ( Figure 2B ). In addition, a few PEP51-negative (PEP51(-)) test cells were observed in PEP51-positive (PEP51(+)) test cells of early stage III follicles ( Figure 2C ).

To elucidate the molecular weight of the immunoreactive molecule in stage III follicles, proteins were extracted from follicles of the corresponding stage by fractionating with a series of stainless-steel sieves, followed by western blotting using the anti-PEP51 antibody. A specific band was detected at 6 kDa, which coincided with the size of synthetic PEP51 with the calculated molecular weight of 5897.1175. ( Figure 2D , left panel). Additionally, no bands were detected using the synthetic PEP51-absorbed anti-PEP51 antibody ( Figure 2D , right panel), excluding the possibility of non-specific immunoreaction by the antibody. Additionally, several bands around 18-30 kDa were not detected with the preabsorbed antibody, suggesting the formation of some complexes with PEP51 in stage III follicles ( Figure 2D , left panel). Taken together, these results indicate that PEP51 is expressed in the follicles, and that the amino acid sequence comprises 51 amino acids.

Furthermore, FACS with the anti-PEP51 antibody and Alexa Fluor 488-conjugated secondary antibody was used to isolate PEP51(+) cells from stage-III follicles. We collected 1,150,000 cells in the PEP51(+) area shown in Figure 3A . Subsequently, endogenous peptides extracted from the sorted PEP51(+) cells were analyzed using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), leading to the detection of a peak corresponding to an MS value of 5898.879 [M + H]⁺ for PEP51 ( Figure 3B ). Moreover, nanoLC Q-TOF MS/MS detected the IYWNKFEQIK sequence in tryptic digests of the peptide corresponding with a size of 6kDa, which completely corresponded with the partial sequence of PEP51 ( Figure 3C ). These analyses confirmed the presence of 51 aa full-length sequence of PEP51 in the test cells of stage III follicles. Moreover, intact endogenous PEP51 was electrophorized in accordance with synthetic PEP51 ( Figure 2D ), and the MS value of endogenous PEP51 (5898.879) ( Figure 3 ) coincides with the calculated molecular weight of 5897.1175, indicating that PEP51 in test cells undergoes no post-translational modifications such as signal peptide cleavage or glycosylation. Collectively, we conclude that PEP51 is a Ciona ovarian intracellular factor in test cells but not peptide hormones or secretory proteins with post-translational modification.

Subsequently, the subcellular localization of PEP51 was analyzed by immunoelectron microscopy using the anti-PEP51 antibody on the Ciona ovaries. Transmission electron micrographs show the characteristic localization of PEP51 in stage III follicles ( Figures 4A–G ). A greater number of anti-PEP51-immunogold particles were observed in test cells forming cellular aggregates embedded at early stage III follicles ( Figures 4A–G ). Higher magnification revealed localization of PEP51 in small cytosolic granules in the test cells ( Figures 4D–F ). Notably, numerous PEP51(+) test cells in early stage III follicles exhibited features typical of apoptosis including apoptotic body-like cellular constituents and chromatin condensation ( Figures 4B, D, F ), as seen in a previous study (26). Furthermore, PEP51(+) test cells were found to surround PEP51(-) (normal) test cells in the cellular aggregates in early stage III ( Figures 4A–C, G ), and such test cell aggregates were not observed in late stage III ( Figure 4H ), supporting the view that PEP51(+) test cells forming the aggregates in early stage III either disappeared or were eliminated at late stage III. Altogether, the localization of anti-PEP51-immunogold particles suggested some roles of PEP51 in the regulation of apoptosis in test cells of early stage III follicles.

Previous studies demonstrated apoptosis of test cells residing within mature Ciona eggs (stage IV follicles) in the oviduct by detection of canonical apoptotic markers, active pan-caspases (26, 27). However, apoptosis of test cells in developing Ciona follicles in the ovary has yet to be investigated. We thus examined the expression of PEP51 and apoptosis of test cells in follicles using the HiLyte Fluor 555-conjugated anti-PEP51 antibody, and a cell-permeable far-red fluorescent 660 dye-labeled probe for the active caspase-3/7, FLICA660-DEVD-FMK. Confocal laser scanning microscopy verified that PEP51 was colocalized with the apoptosis marker in test cells of early stage III follicles (defolliculated follicles of around 130 μm in diameter) ( Figure 5A ). In addition, such an apoptotic signal was not detected in test cells of stage II follicles (defolliculated follicles of around 110 μm in diameter) nor in late stage III (defolliculated follicles of around 150 μm in diameter) to stage IV follicles (defolliculated follicles of around 170 μm in diameter) ( Figure 5B ). These results demonstrated that caspase activation occurred in most PEP51(+) test cells of early stage III follicles ( Table 1 ). To quantitatively analyze this confocal microscope observation, we performed flow cytometry for test cells dissociated from the stained early stage III follicles. FACS analysis revealed that caspase activation occurred in 97.62% of PEP51(+) test cells and 0.81% of PEP51(-) test cells in early stage III follicles, respectively ( Figure 5C ). Taken together, these results suggested the functional role for PEP51 in the induction of apoptosis via activation of caspase-3/7.

To examine whether PEP51 directly induce caspase activation, we evaluated the effect of the PEP51 gene by transfection of Pep51 expression vector into COS-7 cells and HEK293MSR cells and addition of the active caspase-3/7 probe, FLICA660-DEVD-FMK. The apoptosis marker was detected specifically in PEP51-transfected COS-7 cells ( Figure 6A ) at 2 days after transfection. Similar data were obtained using HEK293MSR cells ( Figure 6B ). In contrast, no positive signal was detected in the cells transfected with an empty pcDNA4 vector ( Figures 6A, B ). The ratio of caspase-3/7 signal-positive cells at 2 days after transfection is summarized in Table 2 . These results provided evidence that PEP51 activated caspase-3/7.