2.1. Animals and Sampling

Ricefield eels used in this study were purchased from the Wuhan Baishazhou Aquatic Products Wholesale Market. All the treatments involving ricefield eels in this study were reviewed and approved by the Animal Ethics Committee of the Yangtze River Fisheries Research Institute, Chinese Academy of Fisheries Sciences (Wuhan, China). Sampling was conducted in accordance with the guiding principles for the Protection and Utilization of Experimental Animals. Three female ricefield eels were randomly selected for tissue collection. The body length (49.8 ± 2.46 cm) and body weight (95.91 ± 6.91 g) of those adult individuals were measured. The eels were first anesthetized by a 0.05% solution of 3-aminobenzoic acid ethyl ester methanesulfonate-222 (MS-222) (Sigma, USA) before dissection. The following tissue were collected: heart, liver, spleen, kidney, muscle, gonad, intestine, skin, telencephalon, midbrain, and cerebellum. Each tissue sample was collected in triplicate. These tissues were preserved in RNAlater (Biological Industries, Israel) and subsequently stored at −80°C. Additionally, ricefield eels at different developmental stages (unfertilized egg, fertilized egg, gastrula, eye capsule, hatching stage, 3 days post-hatching [dph], 8 dph, 16 dph, 30 dph, ovarian stages II–IV, and intersexual gonad stages [early, metaphase, late], as well as testis stages II–V) were collected and frozen directly at −80°C. Each sample from these stages was collected in triplicate. Meanwhile, a portion of the gonadal tissues was fixed overnight in Bouin’s solution at 4°C and then transferred to 70% ethanol for immunohistochemistry experiments.

2.2. Histological Sections of the Gonads

After fixation with Bouin’s solution, gonadal tissue samples at various developmental stages were dehydrated in a graded series of ethanol (70%, 80%, 90%, 95%, and 100%) and subsequently embedded in paraffin. Sections measuring 4.5 µm were cut and utilized for hematoxylin–eosin (HE) staining and immunofluorescence analysis, respectively.

2.3. Quantitative Real-Time PCR

Total RNA extraction was performed from embryos at various developmental stages and different tissues of the rice field eel using the RNeasy Plus Mini Kit (Qiagen, Düsseldorf, Germany) according to the manufacturer’s instructions. The integrity and concentration of the RNA were assessed using a Nanodrop-2000 spectrophotometer (Thermo Fisher Scientific, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).

cDNA synthesis from total RNA was performed using the PrimeScript RT Reagent Kit with gDNA Eraser (Takara, Japan). Ricefield eels Piwil1 (GenBank number KF663555) and reference gene β-actin (GenBank number AY647143.1) sequences were acquired from the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/). Primers for each gene were designed by Primer 3 (https://primer3.ut.ee/). Primer sequences for β-actin were 5-GTCGCCCCTGAAGAGCACCCT-3 and 5-TCACACCATCACCGGAGTCCA-3′. Primers for Piwil1 were 5-ACCCGTCGTAACAGCACCGAA-3′ and 5-CTTGACACCGCTGTACTTGTCC-3. qRT-PCR was conducted on a QuantStudio6 Flex real-time PCR system (Thermo Fisher Scientific) with TBPremix Ex Taq TM Ⅱ kit as instructed, using a reaction volume of 20 μL containing 1 µL cDNA sample, 10 µL TB Green Premix Ex Taq II, 0.40 μL Rox Reference Dye II, 0.40 µL of each primer, and 7.80 µL ddH₂O. Relative Piwil1 mRNA levels were calculated based on the 2^−ΔΔCT method using the PCR R package [32]. Data were presented as means ± SD with three samples in each group. The significant differences between the means were analyzed using the one-way analysis of variance (ANOVA). All statistical differences were considered significant when p < 0.05.

2.4. Western Blot Analysis

The polyclonal antibody specific to Piwil1 of ricefield eel was produced against the peptide of 15 amino acids (KLGERGGRRQDFHDC). The peptide was conjugated to the KLH peptide (GenScript Company), and then the peptide-KLH conjugate was used to immunize the rabbit to get the polyclonal antibody.

Tissues were lysed using RIPA buffer (Beyotime, Shanghai) supplemented with a protease inhibitor cocktail (Roche, Shanghai, China). The extracted proteins were separated using a 12% SDS-PAGE gel and subsequently transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk in phosphorus-buffered saline (PBS, pH 7.4) at room temperature for 1 h. They were then incubated overnight at 4°C with polyclonal antibodies against PIWIL1 or β-Actin (Biosharp, Wuhan), followed by incubation with horseradish peroxidase-labeled secondary antibodies (Biosharp, Wuhan) at room temperature for 1 h. Finally, the signals were visualized by enhanced chemiluminescence (ECL) Western-blotting Analysis Kits (Pierce Co., Rochford, USA).

2.5. Immunofluorescence Analysis

Immunofluorescence analysis was conducted using the following method: paraffin sections of tissues were prepared, dewaxed, and subjected to antigen retrieval using Histo VT One solution (Nacalai Tesque, Kyoto, Japan). After blocking with 5% bovine serum albumin (BSA) at room temperature for 1 h, the samples were incubated overnight at 4°C with PIWIL1 antibody. Following three washes with phosphate-buffered saline with tween 20 (PBST), the samples were incubated for 1 h at room temperature with the fluorescein isothiocyanate (FITC)-conjugated goat antirabbit secondary antiserum (Thermo). Nuclei were stained with 500 μL DAPI staining solution (10 µg/mL) for 40 min in the dark. After washing with PBS again, the sections were sealed with SlowFade Gold antifade reagent (Invitrogen). Images were taken under a fluorescent microscope (BX51-34 FL, Olympus) with a digital camera (DP-73, Olympus).

3.1. Histological Analysis of Ricefield Eel Gonads

Ricefield eel is a protogynous hermaphrodite with a natural ability for sex change. The histological characterization of different gonadal stages in the ricefield eel was analyzed. Immature females exhibited ovaries containing oocytes in the previtellogenic stage, which is the initial stage of oocyte growth. In stage Ⅱ ovaries, oocytes of varying sizes were observed, including primary oocytes with a small spherical shape (phase Ⅰ), and larger cortical–alveolar oocytes, which displayed cortical alveoli at the peripheral zone of the ooplasm (phase Ⅱ) (Figure 1A,A’). Vitellogenic oocytes, characterized by eosinophilic yolk granules and oil vesicles accumulated in the cytoplasm, were the primary marker in stage Ⅲ of the ovary (Figure 1B,B’). Subsequently, the nuclear area began to shrink and gradually migrated to the peripheral region. In stage Ⅳ of the ovary, characterized by the presence of yolk and large oocytes, the oocytes reached their maximum sizes (Figure 1C,C’).

In the gonads of rice field eels at the intersexual stage, testicular lobules and oocytes were observed simultaneously. In the early stage, vitellogenic oocytes, along with spermatogonia and primary spermatocytes, which are characteristic of typical testicular cells, were present (Figure 1D,D’). As the gonads continued to develop, the oocytes gradually degenerated, while the number of primary spermatocytes increased, accompanied by the emergence of secondary spermatocytes and spermatids. This was considered to be the middle period of the intersexual stage (Figure 1E,E’). In the late stage of intersexual development, the gonads were predominantly filled with testicular cells, including spermatogonia, primary spermatocytes, secondary spermatocytes, and spermatids, with very few oocytes remaining (Figure 1F,F’).

The testes primarily consist of testicular lobules and spermatogenic cells. In stage II of testis, cleft cavities were observed within individual seminiferous tubules, and a significant number of spermatogonia and primary spermatocytes were dispersed along the lobule walls (Figure 1G,G’). When testis developed to stage III, secondary spermatocytes began to differentiate (Figure 1H,H’). In the subsequently developed testis at stage IV, secondary spermatocytes and spermatids were the predominant types of differentiated cells, while primary spermatocytes were scarcely observed (Figure 1I,I’). Finally, in testis at stage V, the lobular lumen was filled with a substantial number of mature sperm (Figure 1J,J’).

3.2. Transcription Profile of Piwil1 in Ricefield Eel

Tissue distribution analysis by qRT-PCR showed that Piwil1 was predominantly expressed in the gonad, followed by the telencephalon, midbrain, and skin. No expression was detected in the cerebellum, heart, liver, spleen, kidney, intestines, and muscle (Figure 2A). The time-dependent expression profiles of Piwil1 were detected during embryonic and gonadal development. In the early embryo developmental stages, Piwil1 transcript signals were only found in unfertilized egg and fertilized egg, whereas no transcription was detected in the following developmental stages of gastrula, eye capsule, and hatching stages (Figure 2B). During the various developmental stages of gonad, Piwil1 transcription levels increased from stage II to stage III of the ovary, and then decreased in stage IV. When the ricefield eel entered the sex change period, Piwil1 transcription levels increased ~4-fold in the early stage of intersexual gonad compared to stage IV of the ovary. In the middle and late stages of intersexual gonad, Piwil1 transcription levels showed a slight decrease but remained higher than all stages of the ovary. The expression level of Piwil1 significantly increased during testis development, peaking in stage III, followed by stages II and IV, before sharply decreasing in stage V (Figure 2B).

3.3. Western Blot Analysis of the PIWIL1 Protein in Ricefield Eel

The specificity of the polyclonal antibody against PIWIL1 (~100 kDa) was confirmed by western blot analysis (Figure 3A). The tissue expression pattern of PIWIL1 was further detected with the specific polyclonal antibody. As shown in Figure 3B, PIWIL1 protein was mainly expressed in the ovary, intersexual gland, and testis; however, the expression of PIWIL1 protein was undetected in other tissues, including the heart, liver, spleen, kidney, intestine, and muscle.

3.4. Subcellular Localization of PIWIL1 in Gonads

The subcellular localization of PIWIL1 at different stages of gonadal development was analyzed using immunofluorescent (Figure 4). The results indicated that PIWIL1 was primarily localized in germ cells. In the ovary, PIWIL1 protein was concentrated in the cytoplasm of oocytes at stages II and III (Figure 4A–C). At stage IV of oocytes, the expression signal of PIWIL1 protein decreased and shifted to the surrounding granulosa cells (Figure 4E–G). In the intersexual gonads, strong green fluorescent signals were observed in the cytoplasm of oocytes, spermatogonia, and primary spermatocytes, together with weaker signals present in secondary spermatocytes (Figure 4I–K). In the testis, PIWIL1 protein expression was detected in the cytoplasm of spermatogonia and primary spermatocytes, but it weakened in the cytoplasm of secondary spermatocytes and was not detected in spermatozoa (Figure 4M–O). No positive signals were observed in the negative control (Figure 4D,H,L, and P).