Seminal vesicle protein caCA12 in Corydoras aeneus inhibits sperm motility for sperm drinking

Abstract

Seminal vesicle (SV) secretions enhance fertilization by regulating sperm motility and fertilization capacity, and by forming plugs that prevent mating with other males. Although SVs are rare in teleosts, certain species, such as Corydoras spp., do possess them. In Corydoras spp. and other species that exhibit sperm drinking or related behaviors, females attach their mouths to the males' genital pore to ingest semen, a reproductive behavior known as sperm drinking. However, the major proteins and functions of seminal vesicle fluid (SVF) in Corydoras remain unidentified. This study aimed to identify the SVF proteins in Corydoras aeneus and clarify the functions of the identified major SVF proteins. The SVF of this species was found to be highly viscous with a high protein concentration. Sperm motility was strongly suppressed in the presence of the SVF. We identified three SVF proteins - alpha-2-macroglobulin (A2M), carbonic anhydrase 12 (CA12) and lymphocyte antigen 6 (Ly6) - through RNA sequencing (RNA-Seq), LC-MS/MS and amino acid sequencing. Additionally, we found that the identified CA12, termed 'caCA12,' was degraded into about 10 kDa and 33 kDa polypeptides containing the CA domain. The 33 kDa polypeptide with the CA domain was found to inhibit sperm motility. The identified SVF proteins, including caCA12, may play a role in keeping sperm in an immotile state until they are close to the female ova, facilitating the remarkable sperm drinking reproductive process observed in C. aeneus.

Keywords: Corydoras; Carbonic anhydrase; Inhibition of sperm motility; Seminal vesicle fluid; Sperm drinking.

Fig. 1.

Male reproductive organs related to reproduction and the function of seminal vesicle fluid (SVF) in Corydoras aeneus. (A) Photograph of an adult male C. aeneus. Scale bar: 1 cm. (B) Illustration of sperm drinking, a reproductive behavior in Corydoras spp. The white duct in the female is the gastrointestinal tract, and the upper yellow sac is the ovary. In sperm drinking, Corydoras sperm migrate through the female gastrointestinal duct and fertilize the eggs at an external place between the pelvic fins. (C) Morphology of the male reproductive organ of Corydoras. Photograph (left) and diagram (right) of the male reproductive organ. Dashed lines on the diagram indicate the histological location of the testis and seminal vesicle (SV). Scale bar: 2 mm. (D) Hematoxylin and eosin (HE) staining of the testis. Scale bar: 200 µm. Yellow arrowhead indicates spermatozoa in a cyst. (E) HE staining of the seminal vesicles. Scale bar: 200 µm. White arrowhead indicates secretory epithelium and asterisk indicates eosin-positive SVF inside the epithelium. (F) Corydoras sperm motility in Hepes buffer, PBS buffer or SVF at different dilutions. Sperm motility values were normalized to the number of motile spermatozoa in the control condition using distilled water (DW). Independent experiments were performed using spermatozoa collected from different Corydoras males. PBS solution was undiluted (100% PBS), or diluted to 75% PBS and 50% PBS with DW; 50% SVF in DW was undiluted (50% SVF), or diluted to 5% SVF, 0.5% SVF, 0.05% SVF, 0.005% SVF and 0.0005% SVF. Data are means+s.d. Numbers in parentheses are number of male C. aeneus examined. Unpaired two-tailed Student's t-test was conducted (*P<0.05, **P<0.01, ***P<0.001 versus DW).

Fig. 2.

Experimental workflow used to identify the SVF proteins in this study.

Fig. 3.

Identification of proteins in the SVF. (A) SDS-PAGE of SVF. Yellow arrows indicate bands of each candidate protein with molecular weight and protein names identified. Black arrows indicate bands that could not be identified because of low concentration. (B) Expression of candidate genes I, II and III in SV tissues. Black arrowheads indicate RT-PCR bands of each candidate gene.

Fig. 4.

Structure and function of C. aeneus CA12. (A) 2D structure of C. aeneus carbonic anhydrase 12 (caCA12). The three trypsin cleavage sites (314R, 438K and 539K) and regions used for antibody generation (CA12-33 and CA12-10) are indicated. (B) 3D structure of caCA12 as predicted by Alphafold2. Left: color gradient showing the orientation of the protein from the N-terminus (blue) to the C-terminus (red). Right: location of the CA domain (blue); predicted digestion sites (314R, 438 K and 539 K; black); and C-terminal region including the repeated alignment (pink). (C) Schematic diagram of caCA12 secretion from the SV epithelium. (D) Immunoblotting of caCA12 collected from SV tissue (tissue) and SVF (fluid). Two antibodies, anti-caCA12-33 against the CA domain and anti-caCA12-10 against the repeated amino acid domain, were used to detect caCA12. Dark blue arrowheads indicate a band at about 35 kDa corresponding to the CA domain of caCA12. The pink arrowhead indicates a band at about 10 kDa corresponding to the repeat domain of caCA12. Light blue arrowheads indicate bands of about 60 and 70 kDa corresponding to undigested caCA12 in the SV tissue. (E) Motility of C. aeneus sperm mixed with SVF, recombinant CA12 proteins or NaHCO₃ (HCO₃⁻). Sperm motility was normalized to the number of motile spermatozoa in the control condition using distilled water (DW). Independent experiments were performed using spermatozoa collected from different C. aeneus males. All additive proteins were adjusted to 41 µmol l⁻¹ as the physiological condition. bCA, bovine CA; FL, full-length caCA12; 33 kDa, N-terminal peptide of caCA domain; RP, repeat domain (TAYPTIQPFAKLPIQP) of caCA12. Data are means+s.d. Numbers in parentheses are number of male C. aeneus examined. Unpaired two-tailed Student's t-test was conducted (***P<0.001 versus DW).

Fig. 5.

Mechanism showing the role of SV proteins in sperm drinking. In SVF, caCA12 inhibits sperm motility immediately after male ejaculation and within the gastrointestinal tract, preventing sperm from expending energy prematurely. The viscosity of SVF, combined with the enzymatic activity of caCA12, acts as a barrier, preventing sperm from diffusing into the gastrointestinal tract and so protecting sperm from digestion. After passing through the gastrointestinal tract, caCA12 is digested or removed from the sperm surface, allowing sperm to regain motility and move towards the eggs. A2M, alpha-2-macroglobulin; Ly6/uPAR, lymphocyte antigen 6/urokinase-type plasminogen activator receptor.