Sexual size dimorphism is a phenomenon observed in certain aquatic species, in which there is a substantial weight disparity between male and female individuals. For instance, male Nile tilapia (Oreochromis niloticus) exhibits a growth rate up to 1.4-times that of its female counterparts [17]. The individual size of female Chinese tongue sole (Cynoglossus semilaevis) can be up to four times larger than that of males [18, 19]. In this study, although females exhibited significantly faster growth rates compared with male largemouth bass, the disparity in weight was not as pronounced as that observed in other aquatic species. During the development and maturation of gonads, the volume and weight of ovaries became considerably larger than those of testes, resulting in a greater proportion of gonad weight relative to body weight in female fish. The difference in gonad weight between male and female largemouth bass is one of the key factors contributing to their divergent growth patterns. Therefore, investigating the mechanisms of gonad development by studying mature gonads can provide insights into dimorphic growth of male and female largemouth bass.
In this study, the KEGG database was utilized to cluster the DEGs in mature gonads between male and female largemouth bass. These DEGs were associated with ‘signal transduction’, ‘lipid metabolism’, and ‘endocrine system’ pathways. In our study, HE staining revealed that the selected male and female largemouth bass were in the early stages of sperm release and ovulation, respectively. At this stage, a large number of DEGs were enriched in the ‘neuroactive ligand-receptor interaction’, ‘MAPK signaling’, ‘purine metabolism’, and ‘cell adhesion molecules signaling’ pathways. These findings suggest that cell proliferation, differentiation, apoptosis, and information transmission play crucial roles in the gonad development of largemouth bass.
After reaching stage Ⅲ, oocytes initiate the synthesis of many lipid droplets within the cell, followed by the synthesis of yolk particles upon reaching stage Ⅳ. The accumulation of these lipid droplets and yolk particles contributes to the increase in oocyte volume [3, 20]. These observations align with the results of the histological analysis of largemouth bass ovaries in this study. Lipid droplets consist of phospholipid-coated triglycerides are important for the storage of lipids produced by lipid metabolism [21].
Several genes showed particularly high transcript levels in the ovaries compared with the testes. Among them, acsl5 and cidec are pivotal regulatory genes involved in lipid metabolism. ascl5 encodes acyl-CoA synthetase long chain family member 5, a member of the acyl-CoA synthetase (ACS) family, whose members contribute to the accumulation of long chain fatty acids through catalyzing the synthesis of fatty acyl-CoAs [22]. Excess fatty acids in cells undergo catalysis by ACSs and combine with triglycerides to form lipid droplets, which are subsequently deposited within the cells [23, 24]. Cidec is a protein associated with lipid droplet synthesis that acts as a lipolysis inhibitor, facilitating the accumulation of triglycerides within the cells [25]. In this study, the high transcript levels of acsl5 and cidec in oocytes may have contributed to the accumulation of lipid droplets. Specifically, acsl5 promotes triglyceride accumulation, whereas cidec inhibits triglyceride decomposition and utilization, thereby providing conducive conditions for lipid droplet formation. Vitellogenin (Vtg) is a yolk precursor that is synthesized primarily in the liver and transported to the ovaries via the bloodstream. It is subsequently internalized into oocytes through endocytosis facilitated by vitellogenin receptors (Vtgrs) [26, 27]. Notably, Lrp13 (low-density lipoprotein receptor-related protein 13) functions as a Vtgr in aquatic animals [28]. The expression of lrp13 effectively enhances yolk particle accumulation within cells. Therefore, acsl5, cidec, and lrp13 are potential regulatory genes involved in lipid droplet formation and yolk accumulation in oocytes, processes that are essential for oocyte growth and ovarian development. elovl1a encodes a protein that is involved in fatty acid chain elongation and makes a significant contribution to phospholipid synthesis. Notably, elovl1a expression in zebrafish tissues has been shown to result in lipid droplet accumulation [29] Our results indicate that elovl1a may also participate in lipid droplet synthesis in the ovary of largemouth bass.
GDF9, a member of the TGF-β family, is an essential growth factor in the ovary [30]. It facilitates the initial stage of follicle development and growth while stimulating granulosa cell proliferation. Moreover, it promotes granulosa cell differentiation induced by follicle-stimulating hormone [31]. The loss of gdf9 expression was shown to result in arrested follicular development at the primary stage [32]. The protein CCNB1 serves as a regulator of the cell cycle. It forms a complex with cyclin-dependent kinase 1 (CDC2) known as maturation-promoting factor, which plays a role in regulating oocyte meiosis initiation and progression [33]. The expression of ccnb1 is essential for the normal division and proliferation of germ cells [34]. The results of our study suggest that cth1 is also involved in the regulation of oocyte meiosis, highlighting its significance as a regulatory gene for oocyte meiosis. These genes play a direct role in promoting oocyte proliferation and differentiation, thereby ensuring optimal gonad development.
Sex steroid hormones are critical for sex differentiation, gonad development, and the manifestation of secondary sexual characteristics in fish. ldlrap1a, srd5a1, and hsd17b12a encode proteins involved in sex steroid hormone synthesis, and are thus important for promoting ovarian development and growth through regulation of steroid hormone metabolism. We found that lfng, which belongs to the β1–3 N-acetyltransferase family, was highly expressed in the ovary of largemouth bass. Its product is known to modify Notch receptors, which facilitate communication between germ cells and extracellular follicular cells, thereby effectively maintaining material transport and facilitating ovarian development [35]. Notably, we observed that some genes expressed at high levels in the ovary were also expressed in the testis to some extent, suggesting that they also play a role in testis development. For instance, ccnb1 was expressed at high levels in the ovary and at moderately high levels in the testis of largemouth bass. Previous research has demonstrated ccnb1’s role as a regulatory gene involved in spermatocyte meiosis [34]. Therefore, some genes are required for both testis and ovary development; however, their high expression levels in the ovary suggests that they play a more important role in ovary growth and development.
Unlike oocytes during development, spermatocytes undergo a gradual reduction in size during meiosis and experience a progressive loss of organelles. During sperm maturation, only the nucleus and acrosome structure are retained, resulting in a smaller volume of testes than of ovaries. In this study, many DEGs were upregulated in the testes compared with the ovaries, and these DEGs were enriched in processes related to spermatogenesis and flagella formation. Flagella and cilia are organelles that form through the assembly of microtubule structures [36]. Tektins (encoded by tekt1) are filamentous proteins that co-assemble with tubulins to form axonemal microtubules, basal bodies, and centrioles in cilia and flagella. Previous studies have demonstrated that tekt1 is expressed in spermatocytes and sperm, and that it is involved in sperm flagella formation [37]. The meiosis-specific nuclear structural protein 1 (mns1) is expressed in sperm cells and localizes to sperm flagella; this detergent-resistant protein is a crucial component of flagella [38]. Thus, mns1 plays a significant role as one of the key regulatory genes involved in flagella formation. lrguk encodes a protein that participates in various aspects of sperm assembly, including acrosome attachment, sperm head formation, initiation of axon growth, and flagellar development. Studies have demonstrated that lrguk is also involved in the regulation of microtubule structure formation within the flagella [39]. The involvement of spag6, spef2, and nme5 genes in sperm flagella formation has been well-established, highlighting their crucial role in flagellar motility [40–42]. Additionally, it has been demonstrated that tdrd12 exhibits predominant expression in the acrosome, and plays a role in acrosome development [43]. SYCP3 is a component of the synaptic complex that governs homologous recombination during meiosis, and it is also involved in meiotic regulation [44]. The cytoplasmic ribonucleoprotein granule TDRD5 is involved in RNA processing during spermatogenesis [45]. The results of the present study highlight the significance of both tdrd5 and star as regulatory genes in spermatogenesis.
Overall, our results show that the DEGs in the testis play important roles in the development of spermatocytes into sperm and in the formation of sperm structure, whereas the DEGs in ovaries are involved in the accumulation of lipid droplets and yolk particles in oocytes. The different developmental patterns between spermatocytes and oocytes may be one of the key factors contributing to the significant disparity in gonad tissue volume between male and female largemouth bass. However, the investigation of the aforementioned genes in aquatic organisms remains insufficient. This study relies on the existing gene function database and integrates findings from other disciplines to provide a valuable reference for research on gonad development in aquatic animals.
This study identified potential key genes involved in gonad development of largemouth bass. Notably, some genes may also play a crucial role in sex determination in largemouth bass. Previous studies have demonstrated that foxh1 promotes the expression of cyp19a1a in oocytes, thereby regulating oogenesis. The loss of foxh1 was shown to result in impaired estrogen synthesis, leading to oogenesis arrest [46]. As a member of the fox family, foxh1 also plays a pivotal role in ovariogenesis and sex determination of female largemouth bass [47]. In this study, we identified aldh9a1b, hydin, mlf1, drc1, dynlt5, and odf3b as potential candidate genes involved in male sex determination and spermatogenesis, and zar1l, zar1, hoxb1b, foxh1, and tlr18 as potential candidate genes associated with female sex determination and oocyte development. Similarly, dmrt1 and btg4 may also be involved in both sex determination and gonad development. Unlike the aforementioned genes, both dmrt1 and btg4 exhibited remarkably high Log2(FC) values, with their expression being predominantly restricted to their respective gonad and almost negligible in the other gonad. Hence, these two genes may play indispensable roles in the sex determination and gonad development of largemouth bass.
DMRT1 is a transcription factor that regulates genes involved in sex determination and gonad development in both vertebrate and invertebrate model organisms [48]. It activates the expression of sperm-specific genes, such as sox8 and sox9, while concurrently repressing the expression of ovary-specific genes, such as wnt4. Additionally, it affects sex differentiation in vertebrates by impeding the functionality of the r-spondin-1 signaling protein estrogen receptor [48, 49]. It has been demonstrated that dmrt1 serves as the male sex-determining gene in mandarin fish (Siniperca chuatsi), spotted scat (Scatophagus argus), and other bony fishes [50, 51]. Furthermore, dmrt1 may also play a crucial role in male sex determination of largemouth bass [12], consistent with the inference made in this study. The results of the FISH analysis showed that dmrt1 was highly expressed in spermatogonia and primary spermatocytes, providing further insights into the involvement of dmrt1 in regulating the meiotic process of spermatogonia and primary spermatocytes. DMRT1 inhibits the activity of retinoic acid, which is essential for meiosis, while also suppressing the expression of stra8. Additionally, it activates the transcription of sohlh1, which encodes a spermatogonial differentiation factor, thereby impeding meiosis and regulating mitosis of the spermatogonium cells to promote spermatogonium development [52]. Given its high transcript levels in undifferentiated spermatogonia, dmrt1 may play a role in male sex determination and spermatogenesis in largemouth bass through similar mechanisms.
Different from ccnb1, btg4 exhibited minimal expression in the testis, suggesting that btg4 may serve a specific role in the ovary of largemouth bass. However, no other bgt4 gene has been documented in bony fish species. The FISH analysis revealed robust cytoplasmic expression of the btg4 gene in stage III oocytes, indicating that it may directly participate in regulating cell cycle progression and promoting oocyte cytoplasm development in largemouth bass. Studies have demonstrated that btg4 participates in the regulation of the cell cycle, and its expression can impede cell proliferation and induce cell cycle arrest [53]. Furthermore, btg4 is expressed at high levels during meiosis. The BTG4 protein interacts with CNOT7 (a catalytic subunit of the CCR4-NOT deadenylase) and eIF4E (a pivotal translation initiation factor) [54], and the resulting complex degrades material RNA during the maternal-zygotic transition. If oocytes mature in the absence of btg4, embryonic development is arrested [55]. Therefore, btg4 may play a crucial role in regulating the occurrence of meiosis and in facilitating oocyte cytoplasm maturation, thereby significantly affecting the developmental potential of embryos. However, further research is required to clarify the role of btg4 in sex determination and ovary development.