Long Non-Coding RNAs in Fish Reproduction: Emerging Regulators from Gonadal Fate to Fertility

1. Introduction

Long noncoding RNAs (lncRNAs) are traditionally defined as transcripts longer than 200 nucleotides that lack protein-coding potential. For a long time, these RNAs were considered “junk transcripts,” however, it is now well established that they play important roles in regulating gene expression in eukaryotic organisms. LncRNAs influence multiple cellular processes, including chromatin organization, RNA translation, processing and stability, and protein regulation – thereby contributing to the fine-tuning of molecular programs at several levels [1,2,3].

Research on lncRNAs began in the early 1990s with the characterization of Xist (X-inactive specific transcript) and H19 (maternally expressed imprinted transcript), the first lncRNAs for which biological functions were described. Xist is a key regulatory lncRNA responsible for X-chromosome inactivation in female mammals. It is transcribed from the X chromosome and acts in cis by spreading across the chromosome from which it is expressed and recruiting chromatin-remodeling complexes that establish transcriptional silencing [4]. H19, another developmentally regulated lncRNA, is highly expressed during embryogenesis and has been implicated in the modulation of genes associated with growth, proliferation, and cell differentiation. Dysregulated H19 expression has also been reported in several types of cancer, where it promotes epithelial–mesenchymal transition and contributes to tumor progression and aggressiveness [5,6].

Despite their significance, these pioneering discoveries initially attracted limited scientific attention because they were regarded as isolated observations. Interest in lncRNAs increased substantially with the emergence of high-throughput sequencing technologies in the early 2010s. The ENCODE (Encyclopedia of DNA Elements) project revealed that more than 80% of the human genome is transcribed, whereas less than 2% encodes proteins. These findings led to the identification of thousands of long transcripts that exhibited tissue-specific, developmental stage-specific, and disease-associated expression patterns. Importantly, many of these transcripts were found to possess regulatory functions, challenging the traditional protein-centric view of genome activity [7,8,9].

The biogenesis of lncRNAs closely resembles that of canonical messenger RNAs (mRNAs). Most lncRNAs are transcribed by RNA polymerase II and undergo post-transcriptional processing, including 5′ capping, 3′ polyadenylation, and, in many cases, splicing. However, some lncRNAs are transcribed by RNA polymerase III and may not undergo extensive processing, highlighting the diversity of lncRNA biogenesis pathways [2,10,11,12].

Despite some exceptions, most lncRNAs are expressed at relatively low levels compared with mRNAs and often display highly restricted, cell type-specific expression patterns. Their expression is frequently linked to cellular identity, developmental stage, and physiological state [2,11]. Although lncRNAs are predominantly localized in the nucleus, many are also found in the cytoplasm and mitochondria. Importantly, their low abundance does not indicate limited functional significance – on the contrary, accumulating evidence suggests that low expression levels may enhance the specificity of lncRNA-mediated regulatory interactions and constitute an intrinsic feature of their mechanisms of action [13].

LncRNAs are usually categorized according to their genomic position and orientation relative to protein-coding genes [14,15] (Figure 1A). Within this classification, sense lncRNAs are transcribed from the same strand as a protein-coding gene, and may partially or completely overlap its exons or introns. In contrast, antisense lncRNAs originate from the opposite strand of a coding locus. Bidirectional lncRNAs are transcribed in the opposite direction from promoters located adjacent to protein-coding genes. Intronic lncRNAs originate entirely from the intronic region of a protein-coding gene, without overlapping exons, whereas intergenic lncRNAs (lincRNAs) are located between protein-coding genes and do not overlap any annotated coding regions.

LncRNA in Gene Regulation: Mechanisms and Functions

LncRNAs interact with DNA, RNA, and proteins to coordinate chromatin organization and transcriptional activity at both local and distant genomic loci. Depending on their mode of action, lncRNAs can regulate neighboring genes (cis-acting lncRNAs) or influence the expression of genes located elsewhere in the genome (trans-acting lncRNAs). Beyond transcriptional regulation, lncRNAs modulate multiple aspects of RNA metabolism, including splicing, stability, localization, and translation. They have also been implicated in the organization and function of organelles and cellular compartments, highlighting their broad regulatory potential across diverse biological processes [2,16].

The regulatory functions of lncRNAs are mediated through a variety of molecular mechanisms (Figure 1B). One important mechanism involves direct interactions with DNA through the formation of RNA-DNA-DNA triplexes or R-loops, which can alter chromatin accessibility and thereby activate or repress gene expression. A well-characterized example is the lncRNA TARID, which forms an R-loop at CG-rich sequences near the transcription start site of the tumor suppressor gene TCF21. This structure recruits TET1 (ten-eleven translocation 1), leading to promoter demethylation and a chromatin state permissive for gene expression [17].

LncRNAs also regulate chromatin organization through interactions with chromatin-modifying and -remodeling complexes that are recruited to specific genomic regions, either promoting or inhibiting gene expression. For example, the lncRNA ANRIL recruits Polycomb group protein complexes to the promoters of its neighbour genes CDKN2A and CDKN2B, thereby contributing to the regulation of cellular senescence [18]. Conversely, some lncRNAs function as molecular decoys by preventing histone or chromatin modifiers from binding to their target genomic regions. The lncRNA lncPRESS1, for instance, maintains the pluripotent state in human embryonic stem cells by sequestering the chromatin-bound histone deacetylase SIRT6. This interaction prevents the deacetylation of the promoters of several pluripotency genes, thereby inhibiting differentiation and preserving the undifferentiated cellular state [19].

Beyond local chromatin regulation, certain lncRNAs exert chromosome-scale effects on genome organization. The archetypal example is XIST, the lncRNA responsible for X-chromosome inactivation in female mammals. XIST spreads along one X chromosome, coating it and recruiting chromatin-remodeling complexes that promote extensive heterochromatin formation and transcriptional silencing across the chromosome. Through this mechanism, XIST establishes dosage compensation between males and females by ensuring that only one X chromosome remains transcriptionally active [20].

Another major class of lncRNA functions involves their role as molecular scaffolds. In this capacity, lncRNAs facilitate the assembly of multiprotein complexes by bringing together proteins that may not otherwise interact efficiently. A well-characterized example is the long isoform of the lncRNA NEAT1, which serves as a structural scaffold for the formation and maintenance of paraspeckles – membraneless subnuclear bodies that participate in RNA processing, nuclear retention of transcripts, and cellular responses to stress. Through the recruitment and spatial organization of multiple nuclear proteins, NEAT1 plays an essential role in paraspeckle assembly and function [21].

LncRNAs can also regulate gene expression at the post-transcriptional level by functioning as competing endogenous RNAs (ceRNAs), commonly referred to as molecular sponges. In this mechanism, lncRNAs bind microRNAs (miRNAs) through complementary sequence interactions, thereby reducing the availability of these miRNAs to bind their target messenger RNAs. As a result, repression of the target transcripts is alleviated, leading to increased gene expression. A representative example is LncND, a primate-specific lncRNA expressed in neural progenitor cells. LncND sequesters miR-143-3p, a microRNA that negatively regulates the neurodevelopmental genes NOTCH1 and NOTCH2. By limiting miR-143-3p availability, LncND promotes Notch signaling and contributes to neuronal differentiation and development [22].

Altogether, these examples illustrate the remarkable versatility of lncRNAs as regulators of gene expression. Through their ability to interact with DNA, RNA, and proteins, lncRNAs influence transcriptional, epigenetic, and post-transcriptional processes across multiple biological contexts. Their diverse mechanisms of action underscore their importance as key components of gene regulatory networks and provide a foundation for understanding their roles in development, physiology, and disease.

2. LncRNAs in Fish Reproduction

LncRNAs as Regulators of Gametogenesis and Spermatogenesis

Among the diverse biological processes influenced by lncRNAs, mammalian gametogenesis represents a highly regulated and extensively studied example (Robles et al., 2019). In fish, RNA-Seq studies have identified thousands of lncRNAs with reproductive organ–specific expression, strongly associated with reproductive processes and their maintenance [23,24]. In the flounder Paralichthys olivaceus, differentially expressed lncRNAs (DElncRNAs) have been implicated in both cis and trans regulation of reproduction-related transcripts, including genes such as foxo1, foxm1, smad3, foxr1, camk4, ar, and tgfb3, which participate in classical gonadal development pathways such as insulin, MAPK, and calcium signaling [25]. Enrichment analyses revealed that positively regulated cis targets were associated with fertilization and steroid biosynthesis, whereas negatively regulated ones were linked to steroid binding, microtubule binding, sexual differentiation, gonadal development, and MAPK signaling. Conversely, in trans regulation – based on co-expression with mRNAs – DElncRNAs were associated with hormonal regulation, dynein binding, calcium signaling, and ovarian steroidogenesis, as well as MAPK, Wnt, and TGF-β pathways. Expression profiles varied among ovarian, intersex, and testicular stages, often showing higher abundance in ovaries and intersex gonads, suggesting roles in sexual differentiation and gametogenesis. Moreover, experimental work involving the administration of follicle-stimulating hormone (Fsh) and human chorionic gonadotropin (hCG) confirmed the hormone-responsive expression of specific lncRNAs [26], reinforcing their participation in regulatory networks controlling both gamete formation and sex reversal. Taken together, these findings demonstrate that lncRNAs are key regulators of gametogenesis in fish, acting through epigenetic, transcriptional, and post-transcriptional mechanisms to modulate major developmental pathways during gonadal differentiation [25].

Functional studies in model teleost species have provided additional evidence for the regulatory potential of lncRNAs in reproduction. In medaka (Oryzias latipes), lnc4c was identified as a gonad-specific lncRNA and proposed as the primary microRNA transcript (pri-miRNA) for ola-miR-202 [24]. Using single-molecule fluorescence in situ hybridization, the authors demonstrated that pre-miR-202 is highly expressed in the nuclei of somatic testicular cells, including Sertoli and Leydig cells, whereas lnc4c is expressed at relatively low levels. Co-expression of lnc4c and pre-miR-202 was observed exclusively in Sertoli cells and involved only a limited number of transcript molecules. Importantly, deletion of the lnc4c locus resulted in reduced testis size, decreased germ cell abundance, and impaired sperm viability. These defects were accompanied by increased production of hormones and signaling molecules by somatic testicular cells, as well as accelerated differentiation of male germ cells. Therefore, these findings indicate that lnc4c plays a critical role in maintaining normal testicular development and spermatogenesis in medaka [24].

Additional evidence for the involvement of lncRNAs in germ cell development has been reported in zebrafish (Danio rerio) [27]. The lncRNA lnc172 is highly expressed in the cytoplasm of spermatogonia, spermatocytes, and myoid cells in the testes, while in the ovaries, it was detected in ovarian somatic cells [27]. In contrast, lnc304 was localized to both the nucleus and cytoplasm of spermatogonia and spermatocytes, while in females, it was localized in the nucleus of the previtellogenic oocyte. Both lncRNAs exhibited co-localization with the germ cell marker ddx4 (aka vasa) in spermatogonia. Functional analyses involving knockdown and overexpression experiments demonstrated that elevated lnc172 expression disrupted normal primordial germ cells (PGC) migration, causing ectopic localization of germ cells in the tail, somites, eyes, and brain. These results highlight a crucial role for lnc172 in guiding PGCs toward the genital ridge during early development [27].

Further insights into lncRNA-mediated regulation of fertility have been obtained from studies of rainbow trout (Oncorhynchus mykiss). Comprehensive analyses of the interactions among miRNAs, mRNAs, and lncRNAs during gonadal development in triploid fish at different developmental stages (65 days post-fertilization (dpf), 180 dpf, and 600 dpf) highlight the importance of lncRNAs involved in the regulation of gonadal fertility [28]. Their study identified 22 differentially expressed genes (DEGs) associated with meiosis and fertility, including sycp2, sycp3, dmc1, and ccne1. In zebrafish, sycp2 has been shown to be essential for homologous recombination and pairing during the early stages of meiosis [29]. Similarly, dmc1, which is associated with homologous recombination and expressed exclusively during meiosis, plays key roles in gametogenesis and gonadal development across several species [30,31,32]. In addition, ccne1, a member of the cyclin family, is important for homologous pairing and DNA repair during meiosis [33]. The authors reported reduced ccne1 expression in triploid trout (180 and 600 dpf), while dre-miR-15a-5p_R+1, a predicted negative regulator of ccne1, was increased. Moreover, three novel lncRNAs (MSTRG.74687.3, MSTRG.76205.1, and MSTRG.30919.1) showed inversed correlation with this miRNA. These findings suggest a potential lncRNA–miRNA–mRNA regulatory network in which these lncRNAs may modulate ccne1 expression indirectly through miRNA-mediated regulation, thereby potentially influencing meiotic progression and gonadal development in trout [28].

A subsequent study conducted by the same research group further explored the roles of ccne1, ccne2, and the associated lncRNAs during oogenesis and spermatogenesis in rainbow trout [34]. The authors demonstrated that ccne1 positively regulated the expression of lncRNA MSTRG.74687, whereas silencing ccne2 did not significantly alter its expression. These findings suggest that MSTRG.74687 may participate in regulatory pathways controlling gonadal development. Functional analyses further supported distinct biological roles for ccne1 and ccne2 in coordinating cell proliferation and reproductive gene expression. Together, these studies revealed a regulatory axis involving lncRNAs, omy-miR-15a-5p, and ccne1 that contributes to the control of meiosis and reproductive development in rainbow trout [34].

Gene expression analyses also identified miR-210 as an additional regulator of reproductive processes, influencing the prioritization and expression of key meiotic genes [35,36]. The resulting lncRNA–miRNA–mRNA interaction networks appear to play central roles in either promoting or repressing genes required for meiosis and gonadal development. These findings contribute to a growing understanding of the epigenetic mechanisms underlying fertility and sterility in fish and may ultimately provide valuable targets for improving reproductive management and selective breeding programs in aquaculture.

Therefore, these studies demonstrate that lncRNAs are integral components of the regulatory networks governing gametogenesis in fish. Through interactions with protein-coding genes, miRNAs, and endocrine signaling pathways, lncRNAs contribute to germ cell development, meiotic progression, gonadal differentiation, and reproductive function. Despite these advances, the specific molecular mechanisms through which many lncRNAs exert their effects remain poorly understood. Future studies integrating functional genomics, gene-editing approaches, and single-cell technologies will be essential for elucidating the precise biological roles of lncRNAs during fish reproduction.

LncRNAs in Response to Sex Hormones

Advances in RNA sequencing technologies and the discovery of lncRNAs in recent decades have demonstrated that gametogenesis and fertility do not depend exclusively on protein-coding genes. This group of transcripts has been associated with crucial roles in various well-documented biological processes [14,37,38,39,40]. In the reproductive system, lncRNAs have been implicated in sex hormone responses [41], oocyte meiosis [42], ovulation [43], and sexual differentiation [44].

In mammals, lncRNAs regulate reproduction mainly by modulating sex steroid signaling and steroidogenesis pathways. In males, the lncRNAs PRNCR1 and PCGEM1 interact with androgen receptor signaling in males [41]. In females, several lncRNAs contribute to ovarian physiology by regulating steroidogenesis, follicular development, and hormone production. SRA and Neat1 are involved in steroid hormone synthesis and corpus luteum function [45,46], while H19 modulates steroidogenic pathways and follicular development through post-transcriptional mechanisms [47,48]. Together, these findings demonstrate that lncRNAs act at multiple levels in reproductive endocrine pathways. However, compared with mammals, the functional roles of lncRNAs in hormonal regulation and gonadal development remain poorly understood in non-mammalian vertebrates.

In vertebrates, reproduction is regulated by a complex network of interactions involving multiple organs, signaling pathways, and gene regulatory mechanisms [49,50]. Successful gamete production requires precise coordination among transcription factors, growth factors, and sex steroids – including estrogens, progesterone, and androgens – which govern gonadal development, gametogenesis, and reproductive function [51,52]. At the systemic level, this process is regulated by the hypothalamic-pituitary-gonadal (HPG) axis, which integrates endocrine signals to regulate gonadal activity throughout the reproductive cycle [49,53].

Within the HPG axis, gonadotropin-releasing hormone (GnRH) secreted by the hypothalamus stimulates the pituitary gland to release follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) [54]. In females, these gonadotropins promote ovarian steroidogenesis through activation of their respective receptors, leading to the production of estrogens and progestogens that regulate oogenesis, follicular growth, meiotic progression, and ovulation [53]. In males, Fsh and Lh act primarily on Sertoli and Leydig cells, stimulating androgen and growth factor production and supporting spermatogenesis [49].

In teleosts, oogenesis is orchestrated by coordinated endocrine and paracrine signaling events that drive the progression of oogonia into fully mature oocytes. This dynamic process encompasses oogonia proliferation, primary growth, vitellogenesis, final maturation, and ovulation [53]. Conversely, the testicular functions center on androgen biosynthesis and the continuous maintenance of spermatogenesis [49]. Male germ cell development proceeds through successive phases of mitosis, meiosis, and spermiogenesis within a specialized cystic organization in which Sertoli cells provide structural, nutritional, and endocrine support to developing germ cells [49,55]. The interstitial compartment contains blood vessels, immune cells, and steroidogenic Leydig cells that contribute to endocrine regulation of reproduction.

Unlike mammals, the interaction between gonadotropins and their receptors does not appear to be specific in fish [56]. Notably, Fsh is considered more potent than Lh in driving androgen synthesis due to the expression of Fsh receptors on Leydig cells [57], a mechanism well documented in zebrafish [58]. Furthermore, Fsh promotes spermatogonial proliferation and differentiation by stimulating Sertoli cells to produce insulin-like growth factor 3 (Igf3), while concurrently downregulating the expression of anti-Müllerian hormone (Amh), a recognized inhibitor of spermatogonial differentiation [59,60]. Therefore, Fsh acts as a master regulator of the spermatogenic process, finely orchestrating the balance of stimulatory and inhibitory molecular pathways required for proper germ cell progression [61].

While the systemic control exerted by this classical network that governs gametogenesis, particularly in non-mammalian vertebrates, remains poorly understood, any disruption of these highly coordinated dynamics severely compromises reproductive success. Interest in the role of lncRNAs in fish reproductive endocrinology has increased considerably in recent years [25]. Studies in this area are promising for understanding, at the molecular level, the dynamics of the regulation of gametogenesis and sex hormones involved in gonadal development, as well as topics related to maturation, sexual differentiation, reproductive health, and fertility. These studies also aim to identify interventional targets and biomarkers essential for advancing reproductive biotechnologies and improving the management of species of ecological and economic interest. This relationship between hormonal regulation of gonadal development and lncRNAs has already been demonstrated in fish [25,40,62].

Evidence linking lncRNAs to hormonal regulation in fish has already emerged from several species. In the rice-field eel (Monopterus albus), Fsh promotes oocyte growth and facilitates the transition from primary to secondary follicles, a critical step during oocyte maturation [63]. In the Japanese eel (Anguilla japonica), human chorionic gonadotropin (hCG) stimulates gonadal maturation and regulates the expression of the lncRNA HAS2-AS1, which in turn promotes the expression of HAS2, a gene associated with cumulus expansion and cellular migration [64]. Further evidence for hormone-responsive lncRNA expression was provided by He and colleagues (2022) [26] , who investigated ovarian lncRNAs in M. albus. The authors demonstrated that Fsh and hCG induced the expression of the lncRNA LOC109960696, associated with Smad2, in both time- and dose-dependent manners. In addition, five differentially expressed lncRNAs (LOC109952131, LOC109953466, LOC109954337, LOC109954360, and LOC109958454) exhibited significantly higher expression levels in ovaries and intersex gonads than in testes. Exposure to Fsh or hCG further increased the expression of these transcripts, indicating that gonadotropin signaling directly influences lncRNA expression profiles during gonadal development.

The role of lncRNAs in hormone-dependent reproductive regulation has also been investigated in the gynogenetic Japanese flounder (Paralichthys olivaceus) [25]. Transcriptomic analyses identified 2,284 lncRNAs differentially expressed between testes and ovaries, including 1,870 testis-biased and 414 ovary-biased transcripts. Interestingly, the proportion of sex-biased lncRNAs exceeded that observed for both mRNAs and miRNAs, suggesting an enhanced contribution of non-coding RNAs to gonadal regulation. Functional analyses revealed that these DElncRNAs participate in steroidogenesis, gonadal development, fertilization, and sex differentiation through both cis- and trans-regulatory mechanisms. Moreover, several lncRNAs functioned as competing endogenous RNAs within complex lncRNA–miRNA–mRNA interaction networks. Through interactions with miRNAs such as let-7 and miR-20a, these lncRNAs influenced the expression of dnah1, a gene encoding an axonemal dynein heavy chain required for flagellar assembly and sperm motility. Given that mutations in dynein-related genes are associated with male infertility in multiple vertebrate species, these findings provide compelling evidence that lncRNAs contribute directly to reproductive performance and sexual development [25,65].

Collectively, current evidence indicates that lncRNAs represent important regulatory components of endocrine pathways controlling fish reproduction. By integrating hormonal signals with transcriptional, post-transcriptional, and epigenetic regulatory mechanisms, lncRNAs influence steroidogenesis, gametogenesis, gonadal development, and fertility. Nevertheless, the majority of available studies remain descriptive, and the precise molecular mechanisms underlying lncRNA-mediated hormonal regulation are still poorly understood. Future research combining functional genomics, endocrine manipulation, and gene-editing approaches will be essential for elucidating how lncRNAs coordinate hormonal signaling networks and reproductive physiology in teleost fish.

3. Regulatory Roles of lncRNAs in Sex Determination, Sexual Dimorphism and Sex Differentiation

LncRNAs in Sexual Size Dimorphism

Sexual size dimorphism is defined as a pronounced difference in body size between males and females within a species [66,67,68]. Evolutionary studies have shown that this characteristic is commonly observed in many teleost fish species [68,69,70,71,72,73,74]. In fish, sexual size dimorphism is associated with sexual selection and is linked to territoriality in males and fecundity in females [66]. Notably, this phenomenon is observed in several fish species of commercial importance to aquaculture, such as the olive flounder (Paralichthys olivaceus) and the Nile tilapia (Oreochromis niloticus) [75,76].

Two interesting studies in fish have identified potential lncRNAs involved in the establishment of female-biased sexual size dimorphism in fish. In Chinese tongue sole (Cynoglossus semilaevis), highlighted genes were found together with their respective lncRNAs and miRNAs, forming regulatory networks [77]. Analysis of these networks suggested that miR-460-x may regulate two genes, cdk2 and bub3, both of which play critical roles in the cell cycle and cell proliferation [46,78,79]. Interestingly, miR-460-x was downregulated in females, whereas several associated lncRNAs (MSTRG.24810.1, MSTRG.3094.1, MSTRG.5569.1, MSTRG.9796.1, MSTRG.23232.1, and MSTRG.7061.1) were upregulated. This pattern suggests that these lncRNAs may be involved in competition with miRNAs for binding to target genes (consistent with the competing endogenous RNA – ceRNA – hypothesis), thereby regulating sexual size dimorphism. However, the precise molecular mechanisms through which these ncRNA influence growth-related pathways remain to be elucidated.

A similar pattern was reported in olive flounder (Paralichthys olivaceus), a marine fish with high commercial value in East Asia due to its flesh quality and rapid growth [80,81]. In this species, females exhibit faster growth rates than males, resulting in female-biased sexual size dimorphism [81]. By integrating miRNA–target anticorrelation analyses, lncRNA–mRNA co-expression data, and statistical testing, the authors constructed a ceRNA regulatory network, with the strongest signals detected in gonadal and muscle tissues. Functional enrichment analyses revealed pathways associated with growth and cell proliferation, including those involving miR-460-x and its target genes, as well as a novel miRNA (novel-m0387-3p) linked to apoptosis and other regulatory processes. Additionally, several lncRNAs were predicted to exert trans-regulatory effects on genes located at distant genomic loci, further supporting their potential role in growth regulation.

Furthermore, transcriptomic analyses of brain and pituitary tissues revealed significant sex-specific differences in gene expression and identified several enriched KEGG pathways, including ovarian steroidogenesis, prolactin signaling pathway, arachidonic acid metabolism, Hippo signaling pathway, and cytokine–cytokine receptor interaction, among others. When integrated with mRNA–miRNA–lncRNA regulatory networks, these findings suggest that non-coding RNA-mediated regulatory mechanisms play crucial roles in the establishment and maintenance of female-biased sexual size dimorphism in olive flounder.

LncRNAs in Sex Determination Mechanisms

Fish are considered one of the most species-diverse groups of vertebrates [82], and this diversity may be associated with the wide range of reproductive strategies observed within the group [83]. Sex determination in fish refers to the mechanisms that direct sexual differentiation, whereas sexual differentiation is the process through which testes or ovaries develop from an initially undifferentiated gonad [84]. These mechanisms may be genetic or environmental in nature and play a fundamental role in reproductive biology [83,85].

The turbot (Scophthalmus maximus) is widely cultivated in Europe and China [86,87](. In this species, females exhibit higher growth rates than males [87,88], making the production of all-female stocks highly desirable for aquaculture. Transcriptome analysis of males and females turbot were performed to identify and better understand the regulatory mechanisms associated with the species’ primary sex-determining gene. A lncRNA of 78,309 bp was identified within the genomic region containing the sex-determining gene sox2 (2,557 bp). This lncRNA, named sox2ot, spans the sex-determination region and contains the sox2 gene within one of its introns. Furthermore, it exhibits complete complementarity to the 3′ untranslated region (3′UTR) of sox2. These findings suggest that sox2ot may participate in post-transcriptional regulatory mechanisms controlling the activity of this sex-determining gene [87]. Expression analyses revealed female-specific upregulation of sox2 during the critical period of gonadal differentiation (50–55 dpf). In contrast, the lncRNA sox2ot did not exhibit differential expression between sexes, supporting the hypothesis that its role may be limited to the regulation of sox2 rather than acting as a sex-specific transcript itself. Together, these findings suggest that sox2ot may contribute to the post-transcriptional regulation of sox2 during sex determination in this species.

LncRNA-Mediated Regulation of Sex Differentiation and Sex Reversal

In teleosts, endocrine sex reversal has been demonstrated in both gonochoristic species and hermaphrodites. Hormonal sex reversal provides a valuable tool for producing monosex populations in aquaculture and for advancing our understanding of the mechanisms underlying sexual differentiation [89,90].

Previous studies in teleost fish have shown that treatment with mifepristone (RU485), a progesterone antagonist, altered the expression of steroid hormone receptor genes, thereby influencing the reproductive process and sexual differentiation in these animals [91,92]. During the process of induced masculinization (RU486-500 µg/g diet) in Takifugu rubripes, thereabout hundreds of DElncRNAs were identified across sex and treatment. A key finding was that the predicted targets of such differentially expressed lncRNAs are enriched in canonical regulators of sex determination/differentiation, as well as genes involved in steroid hormone pathways. Specifically, lncRNAs targeting genes of the steroidogenic axis, including dmrt1, dmrt3, gsdf, amhr2, and nanos2, as well as ovarian pathway genes such as foxl2, cyp19a1a, and hsd17b1, were significantly enriched [93]. Additionally, enrichment of RU486-associated DElncRNAs was observed in pathways related to ovarian steroidogenesis, steroid hormone biosynthesis, calcium signaling, and cortisol synthesis and secretion. These findings suggest that RU486 promotes masculinization through stress- and endocrine-mediated mechanisms, involving extensive regulatory reprogramming by non-coding RNAs. In this context, lncRNAs appear to participate in both co-expression interactions and competitive endogenous RNA (ceRNA) networks [93].

In Takifugu rubripes, masculinization induced by the aromatase inhibitor (AI) letrozole was also evaluated [94]. Transcriptomic analysis revealed differential expression of lncRNAs between genetic sexes and between control XX gonads and AI-treated XX gonads. The results indicate that letrozole treatment triggers substantial ncRNA remodeling during masculinization. Notably, the authors identified DElncRNAs and a ceRNA regulatory network comprising 180 mRNAs, 30 miRNAs, 314 lncRNAs, and 6 circRNAs, which were implicated in the regulation of cell fate determination, cell motility, and hormonal signaling during induced masculinization. Functional enrichment analysis of targets associated with this network highlighted pathways such as TGF-β signaling and ion transport-related processes, which may contribute to the cellular signaling transitions underlying steroid-induced sex reversal [94]. The authors also identified gsdf (a member of the TGF-β superfamily essential for sex determination/differentiation in teleosts) as a predicted target of lncRNA000338. Moreover, gsdf expression was elevated in undifferentiated male gonads, providing a compelling example of a potential lncRNA–sex gene regulatory pair acting during the earliest stages of sexual differentiation [94].

Also in T. rubripes, early sexual differentiation was assessed by analyzing undifferentiated (bipotential) gonads approximately 40 days post-hatching. Sex-biased lncRNA expression is already detectable at this developmental stage. This finding supports the hypothesis that lncRNAs contribute to the early divergence of male and female developmental pathways prior to overt gonadal differentiation [95]. Together, these studies indicate that lncRNAs in T. rubripes are associated with key genes involved in sex determination/differentiation, such as dmrt1, gsdf, amhr2, and nanos2, as well as with endocrine pathways governing estrogen production and broader steroid/stress physiology (e.g., cyp19a1a and hsd17b1 for estrogen synthesis; cyp11c1 for cortisol synthesis and secretion). These pathways correspond precisely to the biological mechanisms targeted in models of sex reversal induced by steroidal/endocrine disruptors.

Another interesting hermaphroditic fish is Monopterus albus, which undergoes sex reversal from ovary to testis through an ovotestis stage, characterized by the presence of both male and female gonadal tissue within the same individual. Gonadal tissue development occurs through the processes of sex determination and differentiation [96,97,98,99]. This process is known to occur cyclically: during the initial developmental stage, the gonadal arrangement is ovarian; after spawning, the development of male germ cells begins; upon reaching sexual maturity, both ovarian and testicular structures can be observed; and finally, exclusively testicular tissue is formed [97,98,99,100].

Qiaomu Hu et al. (2023) [101] investigated the epigenetic and regulatory mechanisms of lncRNAs during gonadal differentiation and sex reversal in Monopterus albus. Differential expression of lncRNAs was observed when comparing the different gonadal stages (ovary, ovotestis, and testis). Following an integrated analysis of lncRNA and mRNA localization and co-expression data, two relevant lncRNAs and their respective target genes were selected: MSTRG.38036/psmβ8 and MSTRG.12998/ptk2β. Expression of both transcript pairs increased from the ovary to the ovotestis stage and subsequently declined during the transition from ovotestis to testis, suggesting dynamic regulation throughout gonadal development. Based on the expression profiles of genes previously implicated in sexual differentiation [102,103], the authors proposed that these transcripts may contribute to the sex-reversal process.

To further investigate this hypothesis, DNA methylation patterns were examined during sex reversal induced by 17α-methyltestosterone treatment in M. albus larvae. DNA methylation levels were negatively correlated with the expression of MSTRG.38036 and MSTRG.12998, suggesting that methylation may repress the activity of these lncRNAs. The authors related these findings to previous studies demonstrating the role of DNA methylation in regulating gene expression during sex reprogramming [104,105]. Furthermore, dual-luciferase reporter assays revealed that ptk2β regulates cyp19a1 expression through the Ptk2β/EGFR/STAT3 signaling pathway, providing evidence that lncRNAs participate in central molecular pathways controlling gonadal determination and sexual differentiation.

Overall, these studies demonstrate that lncRNAs and their associated mRNAs may contribute to the gonadal remodeling processes underlying sex reversal. This conclusion is supported by the association of these transcripts with genes previously implicated in teleost sexual differentiation and by their integration into regulatory networks involved in gonadal plasticity through the modulation of cellular pathways governing proliferation, differentiation, and tissue reorganization. The findings of Hu and colleagues (2023) [101] broaden our understanding of lncRNA function in fish and suggest that these molecules serve as important molecular mediators of sexual differentiation and gonadal sex reversal, potentially through the integration of epigenetic regulation and transcriptional control.

4. LncRNAs in Gonadal Maturation and Reproductive Regulation

Gonadal maturation refers to the morphological changes through which the male and female gonads undergo to achieve full growth and maturity, leading to the production of viable gametes (sperm or oocytes) during one or more reproductive periods [106]. In teleosts, gonadal maturation is closely linked to the process of puberty, which marks the transition from juvenile status to reproductive capability. According to Okuzawa (2002) [107], the onset and progression of puberty are governed by a complex interplay of neuroendocrine signals, particularly those operating through the HPG axis. The onset of this process occurs with the increased release of GnRH, which stimulates the pituitary to secrete the gonadotropic hormones Fsh and Lh. These hormones promote gonadal growth, germ cell differentiation, and the synthesis of sex steroids, such as estradiol and 11-ketotestosterone, which are crucial for follicular maturation and spermatogenesis, respectively. Environmental factors such as photoperiod and temperature also play crucial roles in regulating puberty and gonadal maturation, with their effects varying among species and reproductive strategies. Furthermore, nutritional status and overall physiological condition can influence HPG-axis sensitivity, thereby affecting the timing of reproductive maturation.

LncRNA-Mediated Regulation of Ovarian Maturation

Ovarian development is a complex biological process involving multiple signaling pathways and gene expression mechanisms [108,109,110]. Successful oogenesis requires the coordinated action of hormones, growth factors, transcription factors, and other molecular regulators [111]. Dong and collaborators (2021) [50] investigated the involvement of lncRNAs in oocyte growth, maturation, and consequently ovulation in Chinese tongue sole (C. semilaevis). High-throughput sequencing was performed on ovaries collected at three developmental stages: stage IV (late vitellogenesis), stage V (maturation), and stage VI (post-ovulation), according to the classification proposed by [112]. Transcriptome sequencing identified 33,562 lncRNAs, including 10,201 previously annotated transcripts and 23,361 novel lncRNAs. The transition from stage V to stage VI exhibited substantially greater transcriptional remodeling than the transition from stage IV to stage V. Specifically, 58 DElncRNAs were identified between stages IV and V, whereas 187 DElncRNAs were detected between stages V and VI, with 13 transcripts shared between both comparisons.

Previous studies have shown that lncRNAs regulate gene expression through antisense, cis-, and trans-regulatory mechanisms and may also influence nearby genes through positional effects [10,113,114]. Consistent with these observations, target-prediction analyses identified numerous putative lncRNA–mRNA regulatory relationships. For example, mmp14 was identified as a cis target of lncRNAs XR_522498.1 and XR_522499.1, whereas col4a1 and crem were predicted cis targets of XR_523405.2 and XR_001761307.1, respectively. Functional enrichment analyses revealed that antisense and cis target genes were associated with pathways including the cell cycle, apoptosis, TGF-β signaling, and Wnt signaling. These pathways regulate a broad range of cellular processes, including proliferation, migration, differentiation, apoptosis, and embryonic development [115,116]. In avian species, TGF-β signaling promotes granulosa-cell proliferation [117], whereas in mice it contributes to the maintenance of oocyte meiotic arrest through mechanisms regulated by Fsh and Lh [118]. Similarly, inhibition of Wnt signaling has been reported to promote oocyte maturation in pigs [119], while in mice it is associated with granulosa-cell cycle arrest [120]. These findings support the hypothesis that lncRNAs may influence ovarian differentiation and maturation by modulating genes involved in these signaling pathways.

Dong and collaborators (2021) [50] further demonstrated that they identified 31 co-expressed lncRNA–mRNA pairs, including 25 positively correlated and 6 negatively correlated interactions in the ovary of Chinese tongue sole (C. semilaevis). Among these, igfbp1, a gene encoding insulin-like growth factor-binding proteins that are highly expressed during the pre-ovulatory period, showed a positive correlation with lncRNA XR_522957. In contrast, cyp17a1, a key steroidogenic enzyme, displayed a negative correlation with lncRNA XR_522278.2. Dual-fluorescence in situ hybridization demonstrated the co-localization of XR_522278.2 and XR_522171.2 with their respective target genes, cyp17a1 and cyp19a1, within follicular cells. To further explore potential reproductive functions, the authors constructed a competing endogenous RNA (ceRNA) network. Several genes directly involved in reproduction, including mmp14, pgr, cyp17a1, cyp19a1, and hsd17b1, were predicted to be regulated by 30 lncRNAs through interactions with 35 miRNAs. Collectively, these findings provide strong evidence that lncRNAs participate in the regulation of gene-expression programs governing ovarian differentiation, maturation, and ovulation.

LncRNAs in the Regulation of IGF-Induced Ovarian Maturation

Insulin-like growth factors (IGF-1 and IGF-2) constitute an evolutionarily conserved system primarily involved in cellular proliferation across different biological contexts, including growth, proliferation, survival, migration, and differentiation [121,122,123,124,125,126]. In fish, IGFs are primarily synthesized in the liver under growth hormone (GH) stimulation and subsequently released into circulation, where they regulate multiple physiological systems and tissues, including the ovaries, testes, and pituitary gland [127,128,129,130,131,132,133,134,135]. Both igf1 and igf2 are expressed in fish ovaries and have been implicated in ovarian growth and maturation across several species [82,136,137,138]. Their ovarian expression promotes oocyte growth and maturation, while igf1 also plays an important role in regulating ovarian steroidogenesis [139,140,141,142,143].

Ndandala et al. (2025) [23] investigated the involvement of lncRNAs in ovarian growth, development, and maturation in golden pompano (Trachinotus ovatus) by examining stage III ovaries exposed in vitro to IGF1 or IGF2, with the former eliciting a substantially stronger response than the latter. Relative to controls, IGF1 treatment induced the differential expression of 1,494 lncRNAs and 8,728 mRNAs, whereas IGF2 treatment resulted in only 101 DElncRNAs and 373 DEGs. Co-expression and target analyses indicated that candidate lncRNAs and their associated genes were enriched in pathways related to the cell cycle, oocyte meiosis, focal adhesion, and multiple reproductive signaling pathways, including MAPK, PI3K–Akt, GnRH, Wnt, FOXO, and estrogen signaling. Among the identified transcripts, four lncRNAs responsive to IGF1 stimulation (MSTRG.66521.1, MSTRG.49969.1, MSTRG.59923.1, and MSTRG.13767.1) and two responsive to IGF2 stimulation (MSTRG.20896.2 and MSTRG.58123.2) were co-regulated with genes implicated in ovarian development and maturation, including adam23, cyp19a1, cyp17a, slc26a6, htr2b, h2ax, nanos3, krt18, pgr, and inhbb.

Particularly noteworthy was the association with inhbb, whose expression increases sharply in the ovary prior to spawning [144,145]. Furthermore, the trans-target genes identified in the study were enriched in TGF-β signaling pathways involving inhibins and activins, which are known regulators of follicular development and steroidogenesis [145]. Expression profiling revealed that all candidate lncRNAs were highly expressed in the brain, pituitary, liver, and especially in the gonads, suggesting potential roles in coordinating growth and reproductive axis activity. Nevertheless, functional validation through gene knockdown or overexpression experiments remains necessary. Altogether, these findings support the involvement of lncRNAs in IGF-mediated molecular pathways governing ovarian development and oocyte maturation, expanding our understanding of non-coding RNA functions in fish reproduction.

A similar role for lncRNAs has been reported in IGF3-mediated ovarian maturation. The article [77] investigated lncRNA–mRNA regulatory interactions associated with IGF3-induced oocyte maturation in spotted scat (Scatophagus argus). Transcriptomic analyses identified 55 DElncRNAs and 153 DEGs following IGF3 treatment, with particular emphasis placed on two lncRNAs, MSTRG.30211.1 and MSTRG.3260.6.

Co-expression analyses demonstrated that MSTRG.30211.1 was associated with multiple genes involved in key processes required for oocyte maturation. These included ccnd2 and ska2, which regulate cell-cycle progression and meiotic entry; ctsl and kcnma1, involved in oocyte hydration; cdh13, associated with extracellular matrix remodeling; leprot, previously linked to reproductive function in fish; and fyna, which encodes a tyrosine kinase involved in cellular growth, survival, adhesion, motility, and immune responses [146,147,148,149,150,151,152,153,154]. Additional correlated genes included notch2, etnk1, and rsu1. The second candidate lncRNA, MSTRG.3260.6, was associated with several genes involved in signaling, cellular proliferation, and genome maintenance, including pik3ca, styk1b, ndrg3, rap1gap1, adam13, and rpa1. Notably, rpa1 is essential for DNA replication, telomere maintenance, and DNA damage responses during cell division [155,156].

The lncRNA–mRNA network constructed by Wang et al. (2022) [77] indicated that both candidate lncRNAs were connected to a substantial proportion of genes involved in oocyte maturation, suggesting that they may act as central regulators of ovarian developmental processes. Expression analyses further showed that MSTRG.30211.1 was predominantly expressed in ovaries, whereas MSTRG.3260.6 exhibited high expression in both gonads but was more abundant in testes. Together, these findings provide additional evidence that lncRNAs participate in the regulation of IGF-dependent ovarian maturation pathways in teleost fish.

Transcriptomic Regulation of Gonadal Maturation in Males

Studies investigating lncRNAs involved in the regulation of gonadal maturation in male fish remain limited. A recent study byHuang and colleagues (2025) [34] expanded current knowledge regarding the genetic mechanisms underlying sexual maturation in golden pompano (Trachinotus ovatus). Similar to other vertebrates, sexual maturation in salmon is regulated by the HPG axis [157,158]. Salmon has high economic importance for aquaculture; however, when a large proportion of individuals undergo early puberty, somatic growth is reduced due to energy reallocation toward reproduction, compromising production efficiency [159,160,161].

To characterize transcriptomic changes associated with maturation, Huang et al. (2025) analyzed pituitary samples from late-maturing four-year-old males together with 90 gonadal samples from one-year-old fish exhibiting early maturation potential. These gonadal samples included ovaries, immature testes, and mature testes, allowing the identification of maturation-associated transcriptional signatures. Mature testes displayed the highest degree of transcriptomic specialization among the analyzed tissues. Elevated expression of tissue-specific mRNAs, lncRNAs, and lincRNAs was observed, a pattern consistent with previous findings in mammals and birds [98,162]. In contrast, ovaries exhibited comparatively lower transcriptomic specificity. The authors proposed that the extensive transcriptional activity observed in testes reflects the complex gene-expression programs required to support spermatogenesis [163].

Co-expression analyses identified tissue-specific modules whose enriched pathways reflected the biological functions of the corresponding tissues. In immature testes, for example, genes within the module were significantly enriched in the Wnt signaling pathway, which is widely recognized for its role in spermatogonial maintenance and cell fate determination [164,165]. Notably, dysregulation of this pathway has previously been associated with early puberty in Atlantic salmon [166,167], further supporting its potential involvement in male sexual maturation. The same module was also enriched for immune-related processes, which may reflect the establishment of testicular immune privilege, a specialized mechanism that protects developing germ cells from autoimmune responses [168]. Such enrichment may therefore represent an adaptive feature required to maintain germ cell integrity throughout gonadal development. Importantly, the co-expression modules contained both lncRNAs and protein-coding genes, with many candidate interactions involving previously identified lincRNAs. A substantial proportion of these pairs exhibited close genomic proximity and similar expression profiles, supporting the possibility of coordinated regulation through cis-acting mechanisms.

Several candidate lincRNA–gene pairs were highlighted. Among them, tnfrsf11b, which was preferentially expressed in immature testes, was associated with lincRNA MSTRG.73942 – located only 82 bp upstream in the opposite orientation. This gene has previously been linked to increased sperm production and male fertility [169]. Similarly, fgl1, specifically expressed in mature testes, was associated with lincRNA MSTRG.73143 located 45 bp upstream. FGL1 has been implicated in protecting viable spermatozoa from degenerative processes within seminiferous tubules [170].

Another notable example was lincRNA MSTRG.78700, located downstream of Fam46a, a gene previously reported to be downregulated in Sertoli cells lacking endogenous androgen receptor signaling [171]. In that study, Fam46a exhibited testis-specific expression, particularly in immature testes. The authors also acknowledged methodological limitations, particularly the use of poly(A)-enriched RNA libraries, which restricted the detection of non-polyadenylated lncRNAs. Consequently, they suggested that future studies should employ alternative approaches, such as ribosomal RNA depletion or total RNA sequencing, to improve the identification and characterization of non-coding transcripts. Overall, this study considerably expanded the reproductive transcriptomic landscape of Atlantic salmon by identifying novel protein-coding genes, lincRNAs, and tissue-specific expression patterns. The co-expression networks revealed candidate regulatory pathways and lncRNA–gene interactions potentially involved in sexual maturation. Taken together, these findings highlight the highly specialized transcriptomic profile of mature testes and reinforce the potential regulatory role of lincRNAs in male reproductive development and function.

5. Overview of lncRNAs Associated with Reproductive Processes in Fish

Given the wide range of reproductive processes associated with lncRNA activity in fish, Table 1 provides a comprehensive summary of the principal findings discussed throughout this review. The table highlights the reported involvement of lncRNAs in gonadal development, germ cell maintenance and differentiation, gametogenesis, gonadal maturation, sex determination and differentiation, sex reversal, and sex-specific reproductive regulation, facilitating comparisons across species and reproductive contexts.

6. Conclusions and Future Directions

In this review, we presented the results from multiple transcriptomic datasets focusing on lncRNAs identified as potential regulators of several important aspects of fish reproduction, as well as the potential application of these transcripts in future studies aimed at further elucidating their biological functions and exploring their use in genetic improvement programs for aquaculture productivity. Accumulating evidence indicates that these molecules are tightly regulated during reproduction, exhibiting sex-, tissue-, cell type-, and developmental stage-specific expression patterns across diverse teleost species. From a mechanistic perspective, the field is shifting from simple cataloging approaches toward more regulatory and integrative studies. For example, increasing attention has been directed toward interactions involving competitive endogenous RNAs (ceRNAs), emerging as a related hypothesis explaining how lncRNAs, circRNAs, and miRNAs shape sex determination and sexual differentiation in fish [173,174], as well as hormone-induced sex reversal [101], particularly in hermaphroditic models exposed to gonadotropins (Fsh/hCG) and modulation of the hypothalamic–pituitary–gonadal axis [26].

Despite this progress, important limitations remain. Much of the literature in fish is still dominated by bulk RNA sequencing, differential expression analyses, enrichment approaches, and, more recently, target prediction and ceRNA network analyses, often relying on relatively simple and limited validation strategies. In numerous systems, validation is largely restricted to qPCR and localization assays, creating uncertainty regarding directionality, cellular autonomy, and whether predicted networks actually operate in vivo under physiological conditions [50,95]. There are also technical and bioinformatic limitations that may influence conclusions, such as annotation and mapping success being strongly dependent on reference genome quality and phylogenetic proximity. Many differentially expressed non-coding RNAs (ncRNAs) identified in teleosts remain unannotated, reinforcing the need for more refined, species-adapted approaches, particularly for non-model species [175]. These limitations explain why many studies, despite identifying large numbers of “novel” lncRNAs, cannot generalize their conservation, mechanisms, or biological relevance across taxa [95,176].

Future progress in the field will depend on the adoption of more mechanistically oriented approaches. First, functional validation should become a standard component of lncRNA studies, particularly in tractable model species such as zebrafish, where loss-of-function and gain-of-function experiments have already begun to reveal direct roles for lncRNAs in germ-cell development and reproductive processes [27]. Second, experimental investigation of ceRNA-mediated regulation should be expanded through approaches such as miRNA-binding site mutagenesis, reporter assays, RNA pull-down experiments, and in vivo functional analyses [93,174]. Third, the development of high-quality transcriptomic resources – including improved reference genomes and transcriptomes, spatial transcriptomics, and single-cell sequencing technologies – will be essential for resolving cell-specific lncRNA functions and improving annotation accuracy across fish species.

Several datasets explicitly position ncRNAs as valuable resources for the control of sex differentiation, reproductive performance, and productivity in aquaculture. Furthermore, studies involving endocrine disruption models and hormonal manipulation have demonstrated that ncRNA regulatory networks are highly responsive to exogenous perturbations, highlighting their relevance for both environmental monitoring and reproductive management [93,177]. In Monopterus, the proposed application of gene editing to generate females with enhanced fecundity provides a realistic example of how lncRNA-mediated mechanisms may be translated into practical interventions. However, this prospect also underscores the need for rigorous functional genetic validation before lncRNA-informed biomarkers or molecular targets can be reliably implemented on a large scale [177].

In conclusion, fish reproductive biology is entering a new phase in which lncRNAs can no longer be regarded as passive transcriptional by-products. Instead, they are increasingly recognized as key regulatory components associated with fundamental processes such as gametogenesis, steroidogenesis, and sexual differentiation. Emerging mechanistic evidence further supports competing endogenous RNA (ceRNA) interactions and epigenetic–endocrine coupling as actionable regulatory frameworks underlying reproductive function. Future advances will depend on the integration of robust cross-species annotations and functional validation approaches, ultimately enabling both fundamental insights into the evolution of vertebrate reproduction and practical applications in aquaculture, conservation, and environmental risk assessment.