Histological Observation and Functional Gene Expression Analysis of Gonadal Development in Ruditapes philippinarum Under Artificial Culture Conditions

TL;DR

This study shows how artificial conditions can speed up the reproductive maturity of Manila clams and identifies specific genes involved in the process.

Contribution

The study reveals sex-specific gene expression patterns linked to accelerated gonadal maturation in clams under artificial culture conditions.

Findings

Manila clams reached full gonadal maturity within 28 days under controlled food and temperature conditions.

Sex-specific gene expression patterns were observed, with genes like GnRH, ERR, 17β-HSD, CYP17, and Dmrt4-like showing distinct temporal activity.

Gene expression changes closely matched histological changes in reproductive tissue development.

Abstract

This study investigated how to improve the reproductive efficiency of the Manila clam, a popular seafood species, for aquaculture purposes. Controlling reproduction timing is key to improving yield. To induce gonadal maturation, we maintained the clams in an artificial system with regulated food and temperature. This treatment prompted them to reach full reproductive maturity within 28 days. To understand how gonadal maturation was accelerated under these artificial conditions, we monitored the activity of key reproductive genes. We found clear, sex-specific patterns: some genes were active early in males, while others peaked later in females, and these genetic changes matched the physical development of the reproductive tissue. This work helps explain the biological process behind accelerated clam maturation and provides a scientific basis for future methods to make clam farming more stable and productive.

This study employed an artificial greenhouse-based system to enhance gonadal development in the Manila clam, Ruditapes philippinarum. The dynamics of gonadal development and expression patterns of five regulatory genes were analyzed through histological sections and quantitative PCR. Under the controlled conditions (Chlorophyll a: 18.61 ± 1.36 μg/L; temperature: 27.3 ± 2.0 °C), the clams’ gonads reached full maturity within approximately one month, underscoring the critical importance of appropriate food availability and temperature. Gene expression analysis revealed sex- and stage-specific patterns. The gonadotropin-releasing hormone (GnRH) gene was more expressed in males at day 7, reaching a significant peak at day 7. In contrast, females exhibited synchronized expression peaks for both estrogen-related receptor (ERR) and 17β-hydroxysteroid dehydrogenase (17β-HSD) at day 14 (p < 0.05). Cytochrome P450 17 (CYP17) exhibited a dynamic, sex-specific profile, with significantly higher levels in males at day 7 but elevated expression in females at day 14 and day 21. Furthermore, doublesex and mab-3-related transcription factor 4-like (Dmrt4-like) showed clear sex-dependent temporal patterns: it peaked early during maturation in males, while in females, the peak occurred significantly later, at day 21 (p < 0.05). These dynamic changes in gene expression were closely synchronized with histological alterations, providing mechanistic insights into the regulation of gonadal maturation in R. philippinarum.

Figures

Full text

1. Introduction

Gonadal development is a fundamental process regulating the reproductive cycle of bivalves. This process is influenced by the interaction between intrinsic genetic regulation and extrinsic environmental factors. On one hand, genetic determinants involve the coordinated expression of genes located on sex chromosomes and autosomes. On the other hand, key environmental drivers include water temperature and food availability [1]. The concentration of Chlorophyll a (Chl a) exhibits seasonal fluctuations and serves as an important indicator of phytoplankton abundance, thereby reflecting food availability for bivalves [2]. A notable example comes from the coast of Huelva, Spain, where a significant increase in Chl a concentration in March coincided with the spawning period of the wedge clam (Donax trunculus), thereby supporting gametogenesis. A secondary peak in Chl a levels observed in September facilitated post-spawning recovery, further emphasizing the critical role of food availability in reproductive activities [3]. This phenomenon is widespread in coastal ecosystems and has been reported in the temperate Bohai Sea of China [4]. Likewise, in a subtropical lagoon in Mexico, seasonal food availability is closely associated with energy reserves and gonad development in the oyster Crassostrea corteziensis [5]. These findings suggest that seasonal peaks in phytoplankton biomass play a common role in regulating bivalve reproductive cycles.

Turning to intrinsic factors, genetic regulation forms the molecular foundation of gonadal development. This regulatory process encompasses genes that are responsible for neuropeptide hormones, sexual differentiation, and steroid pathways [6,7]. The gonadotropin-releasing hormone (GnRH), which is predominantly expressed in neural and gonadal tissues, regulates reproductive development through autocrine and paracrine mechanisms. Notably, its expression levels correlate with those of steroid hormones [6,8]. Estrogen-related receptors (ERRs) function in a ligand-independent manner and exhibit gonad-specific expression patterns, indicating their constitutive roles in reproduction. However, the specific signaling mechanisms of ERRs in mollusks remain largely uninvestigated [9]. Similarly, the Doublesex and mab-3-related transcription factor (Dmrt) family, encoding DNA-binding DM domains, serves as crucial regulators of sexual differentiation. These factors display testis-biased expression and dynamically interact with other regulatory elements such as FOXL2 [7,10]. Key steroidogenic enzymes, including the Cytochrome P450 (CYP) and Hydroxysteroid dehydrogenase (HSD) families, catalyze the conversion of cholesterol into sex steroids. Notably, enzymes such as CYP17 and 17β-HSD exhibit expression patterns that are modulated by both developmental stage and tissue type, making them essential for maintaining hormonal homeostasis [11,12,13,14].

The Manila clam, Ruditapes philippinarum, is an economically significant shellfish species in China. However, its seed production encounters challenge due to a prolonged natural reproductive cycle. For instance, the population in Jiaozhou Bay, Shandong, China, requires approximately three months from gonadal development to the conclusion of spawning. Furthermore, both the timing and frequency of spawning vary considerably among geographical populations, influenced primarily by water temperature and food availability [15,16]. Research indicates that the optimal natural reproductive temperature for the population in Xinghua Bay, Fujian, China, ranges from 23 to 26 °C [16], while low temperatures directly inhibit gonadal development [17]. Similarly, phytoplankton abundance plays a crucial role in regulating gonadal maturation [3]. Consequently, low spring temperatures combined with inadequate food availability represent the main limiting factors for natural reproduction.

Therefore, the artificial regulation of temperature and feeding represents a viable strategy to address this bottleneck. However, the regulatory network and interactions among these five key reproductive genes remain poorly characterized. This study therefore uses histological analysis and quantitative PCR (qPCR) to investigate gonadal development and the expression of essential reproductive genes under these artificially regulated conditions, aiming to provide a molecular foundation for optimizing maturation techniques in R. philippinarum.

2. Materials and Methods

2.1. Clams and Experiment Design

In April 2024, a total of 40 kg (approximately 6600 individuals) of wild R. philippinarum (with an average shell length of 31.56 ± 1.88 mm and an average weight of 6.01 ± 0.96 g) were collected from Xiangshan Bay in Zhejiang Province, China, and subsequently transported to the laboratory at the Zhejiang Ninghai Experimental Base of East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences. The clams were acclimatized for one week in a large reservoir (70 m × 70 m × 1.5 m) supplied with natural seawater pumped from the adjacent sea (Sanmen Bay, Zhejiang, China), with temperature maintained at approximately 22 °C and salinity at 16. Pre-screening of 20 individuals confirmed that their gonads were in a resting state.

For the experiment, a pond measuring 20 m × 10 m within a plastic greenhouse was utilized. Daily management of the plastic film surrounding the greenhouse was used to regulate temperature, light, and ventilation. During the experimental period, seawater was pumped from the reservoir and filtered through a sieve with a pore size of 0.30 mm. The depth of seawater in the pond was maintained at 1.5 m, and continuous aeration of the seawater was implemented. One week prior to placing the clams into the pond, a total of 1200 L of algal inoculum (scaled up from laboratory cultures), specifically Chaetoceros muelleri (with an approximate concentration of 1 × 10^7^ cells/mL), was added to establish the target phytoplankton biomass in the pond. Concurrently, algal nutrients were supplemented, including urea (2.25 kg), potassium dihydrogen phosphate (375 g), sodium silicate (375 g), and ferric citrate (300 g). Previous studies have confirmed that this methodology effectively establishes a stable algal culture system. Before the experiment was initiated, algal growth was monitored by Chl a determination. A threshold of 10 µg/L was defined as the quantitative criterion, based on prior studies and natural background data [5]. The clams were evenly distributed among ten floating baskets, with approximately 4 kg of clams per basket.

During the experiment, the seawater temperature in the pond was measured daily using a mercury thermometer. Salinity and pH were also monitored daily with a portable salinity meter (AR8212, Xima Instrument, Shenzhen, China) and a portable pH meter (PH828, Xima Instrument, Shenzhen, China), respectively. Seawater samples were collected beginning on day 0 (the start of the experiment) and continuing at 7-day intervals for Chl a concentration determination. Clam sampling was following the same schedule, and the sex of the clams was first determined microscopically at 40× magnification. Then, three female and three male clams were dissected; their gonads were excised, fixed in Bouin’s solution, and stored at 4 °C. Additionally, the gonads of another three female and three male clams were preserved in RNA stabilization solution (#R0118, Beyotime Biotechnology, Shanghai, China) and stored at −80 °C [18].

2.2. Determination of Chlorophyll a Concentration

For the determination of Chl a concentration, 500 mL water samples were collected using a water sampler and subsequently filtered through 0.45 μm cellulose ester (aqueous) filter membranes (Shanghai Xingya Purification Material Factory, Shanghai, China). The filter membranes were frozen at −20 °C for one hour, then thawed at room temperature for 20 min; this freeze–thaw cycle was repeated three times under light-protected conditions to ensure complete cell rupture. Following this process, the filter membrane was placed in a centrifuge tube containing 10 mL of methanol and subjected to dark extraction at 4 °C for 24 h.

After extraction, the sample was vortexed and centrifuged at 3500 rpm for ten minutes to facilitate the extraction of Chl a. The clear supernatant was transferred to a 1 cm pathlength quartz cuvette for spectrophotometric analysis. Absorbance measurements were conducted using a visible light spectrophotometer (Model: 721N, Shanghai Yidian Analytical Instrument Co., Ltd., Shanghai China). Prior to each set of measurements at a given wavelength, the instrument was zeroed (blanked) using a cuvette filled with pure methanol. For each extract, the absorbance (E) was recorded sequentially at 750 nm, 665 nm, and 652 nm. Three replicates were prepared for each sampling. This study builds upon the methanol-based Chl a extraction and quantification system established by Porra [19], with particular emphasis on addressing interference caused by chlorophyll degradation products (e.g., pheophorbide a) present in culturing ponds through optimization of absorption coefficient parameters. The Chl a concentration was calculated using the following formula:

$$[eqn]$$

where E750, E665, and E652 represent the absorbance values of the extract solution at wavelengths 750 nm, 665 nm, and 652 nm, respectively, V_methanol_ refers to the volume (mL) of the methanol extract solution, V_sample_ refers to the volume (mL) of the filtered water sample and δ represents the optical path length (cm) of the cuvette.

2.3. Histological Sectioning and Observation

The gonad samples were removed from the fixative and rinsed with distilled water, followed by immersion in 70% ethanol. Dehydration was performed by sequential immersion in 75% ethanol for 1 h, 85% ethanol for 1 h, 90% ethanol for 1 h, 95% ethanol for 30 min, and 100% ethanol for 30 min. Clearing treatment was then conducted by immersion in an equal-proportion mixture of ethanol and xylene for 30 min, followed by pure xylene for 30 min. Paraffin infiltration at 57 °C involved first immersing the tissues in an equal-proportion mixture of xylene and paraffin for 30 min, then rapidly transferring them to pure paraffin for 1.5 h. Finally, the tissues were embedded and sectioned to achieve thicknesses ranging from 4 to 7 μm. The sections were stained with hematoxylin-eosin (H-E), mounted and scanned under a digital slice scanner (KFBIO KF-PRO-120-HI). The resulting images were analyzed using KFSlideOS software. Gonadal developmental stages were determined individually for each specimen based on histological examination.

2.4. Gene Expression Analysis

Total RNA was extracted from the gonad tissue of R. philippinarum using the TransZol Up Plus RNA Kit (TransGen Biotech Co., Ltd., Beijing, China). The purity and concentration of the RNA were assessed with a NanoDrop UV spectrophotometer (Thermo Scientific, Waltham, MA, USA).

Reverse transcription was performed using the ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo, Shanghai, China) according to the manufacturer’s protocol, with the reaction scaled up to a final volume of 20 μL. Briefly, gDNA was removed by incubating 4 μL of 4× DN Master Mix (pre-mixed with gDNA Remover) with an appropriate volume of RNA template containing 500 ng total RNA. Nuclease-free water was then added to adjust the volume to 16 μL, and the mixture was incubated at 37 °C for 5 min. Subsequently, 4 μL of 5× RT Master Mix II was added to bring the final volume to 20 μL. The reverse transcription program consisted of 37 °C for 15 min, and a final enzyme inactivation at 98 °C for 5 min. The resulting cDNA was stored at −20 °C until use.

Specific primer sequences for GnRH, 17β-HSD, CYP17, ERR, and Dmrt genes were obtained based on published studies regarding reproductive regulatory genes in R. philippinarum [20,21,22].

The reference gene β-actin sequence was sourced from the NCBI database (https://www.ncbi.nlm.nih.gov/). The reference gene β-actin was selected based on its validated expression stability in related studies of R. philippinarum [23], supported by its established application as a reliable reference in prior research on this species [24]. Primers were designed using Primer Premier 5 software and synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) (Table 1). Real-time quantitative PCR was performed using the RT-qPCR EasyTM (One Step)-SYBR Green I kit (Forgene, Chengdu, China). The reaction system comprised a total volume of 20 µL, which included: 10 µL 2 × Real PCR Easy^TM^ Mix-SYBR, 0.8 µL each of forward and reverse primer (10 µM stock, final concentration 400 nM each), 1 µL cDNA template, 0.4 µL 50 × ROX Reference Dye, and 7 µL ddH_2_O. The thermal cycling conditions were as follows: 95 °C pre-denaturation for 3 min; PCR Stage: 95 °C for 5 s, 60 °C for 25 s (40 cycles); Melt Curve Stage: 95 °C for 15 s, 60 °C for 60 s. Relative gene expression levels were calculated using the 2^−ΔΔCT^ method [25] and are presented relative to the expression level in the male control group at 0 d.

In accordance with the MIQE guidelines [26], the amplification specificity and efficiency of all primer pairs were rigorously validated. Specificity was confirmed by a single peak in the melting curve analysis for each gene. Amplification efficiency and the corresponding correlation coefficient (R^2^) were determined using a standard curve generated from a serial dilution of a pooled cDNA template. The amplicon length, amplification efficiency, R^2^ value, and final primer concentration for each gene are comprehensively summarized in Table 1. It is noted that for the Dmrt4-like gene, the calculated suboptimal amplification efficiency is likely due to stable secondary structure within the amplicon. Given that this effect is systematic, highly consistent across all samples, and characterized by a singular melting peak, the data remain fully acceptable for relative quantification.

2.5. Statistical Analysis

All statistical analyses were conducted using IBM SPSS Statistics 26. Prior to these parametric tests, potential outliers were screened using the Grubbs test [27], and none were detected. Subsequently, the homogeneity of variances was assessed using Levene’s test (based on the median), and normality was evaluated with the Shapiro–Wilk test.

Two-way ANOVA was used to assess the effects of sex, time point, and their interaction on gene expression. A significant interaction was detected for all genes. Therefore, simple effects were further analyzed for sex at each time point and for time points within each sex, with both sets of these comparisons analyzed using Tukey’s HSD post hoc test. The significance level for all tests was set at p < 0.05.

3. Results

3.1. Variations in Key Water Parameters

The variations in key environmental parameters are shown in Figure 1. Water temperature and salinity (Figure 1A) ranged from 24.2 to 31.5 °C (27.3 ± 2.0 °C) and 19.2 to 22.5 (20.66 ± 0.78), respectively. Concomitantly, pH and Chl a concentration (Figure 1B) were maintained within stable ranges of 8.0–8.7 (8.30 ± 0.18) and 16.19–20.4 μg/L (18.61 ± 1.36 μg/L), respectively.

3.2. Histological Staging of Gonadal Development

Gonadal samples of R. philippinarum were initially examined macroscopically, followed by paraffin sectioning and staining procedures. Microscopic observations were conducted to characterize the features of testicular tissue, ovarian structures, and germ cells. Based on histological analyses [17], clams from Xiangshan Bay were classified into five distinct phases, as illustrated in Figure 2.

Resting Stage (Day 0 of maturation induction): Gonads were barely visible. Follicles were atrophic, containing sparse oogonia in females and scattered spermatogonia in males.

Proliferative Stage (Days 7–14): Gonadal structures began to form. Follicle walls thickened and were lined with early germ cells—oogonia and primary oocytes in females, and spermatogonia and primary spermatocytes in males. In females, the primary oocytes commenced vitellogenesis, marking the onset of yolk accumulation [28].

Growth Stage (Days 14–21): Follicles expanded in size and density. Oocytes showed primary oocytes in females, while males exhibited active spermatocyte development and early spermatid presence.

Maturation Stage (Days 21–28): Gonads became fully developed, filling the visceral mass. Follicles were densely packed with mature oocytes in females and spermatozoa arranged in radial clusters in males.

Spawning Stage (Day 28): Post-spawning gonads regressed, showing follicle rupture and residual germ cells.

3.3. Expression of Genes Related to Gonadal Development in R. philippinarum

The expression profile of GnRH in clams is shown in Figure 3A. In females, GnRH expression remained high at day 0, day 14 and day 21, but declined significantly at day 7 and day 28 compared to initial levels (p < 0.05) (Table S1). Males exhibited a distinct pattern: expression peaked at day 7 before dropping below initial levels after day 21 (Table 2). At day 0 and day 7, male expression exceeded female levels, with a significant difference at day 7 (p < 0.01) (Table S2).

The dynamics of ERR expression are illustrated in Figure 3B. In females, expression levels gradually increased during early gonadal development, peaking at day 14 (p < 0.05), then declined rapidly to baseline by day 21–28. Conversely, males maintained relatively stable levels from day 0 to day 21, followed by a decline at day 21. Notably, expression was significantly higher in females compared to males at day 14–21.

Sex-specific expression was observed for 17β-HSD (Figure 3C). Females maintained low expression during day 0–7, followed by significant upregulation at day 14 (p < 0.05). Males reached peak expression at day 7. Comparative analysis showed significantly higher expression in females than males at day 14 and 21 (p < 0.01).

CYP17 expression was synchronized with gonadal developmental stages (Figure 3D). Females showed fluctuating patterns with significant upregulation only at day 14. Males peaked at day 7, returned to baseline at day 14, and declined thereafter. Expression was significantly higher in males than females at day 7 (p < 0.01) but reversed at day 14 and 21.

Dmrt4-like expression is shown in Figure 3E. Females exhibited unimodal expression, progressively increasing to peak at day 21 before rapid attenuation to baseline. Males showed significant upregulation at day 7 (p < 0.05), followed by a gradual decrease. Significant sex-based differences occurred specifically from day 7 to day 21.

4. Discussion

4.1. Gonadal Development of Clams Under Artificial Culture Conditions

Water temperature is a critical factor influencing energy allocation in bivalves. Under optimal thermal conditions, energy is primarily directed towards gametogenesis; conversely, at lower temperatures, it is redirected to basal metabolism and acclimation processes. Notably, when water temperatures drop below the biological zero for clams, the onset of gamete development is significantly hindered [29]. For R. philippinarum, natural reproductive temperatures typically range from 23 to 26 °C; however, springtime temperatures in areas such as Jiaozhou Bay often remain below the spawning threshold (about 14 °C), thereby suppressing gonadal development [15,16,17]. This study addressed these limitations through temperature regulation which provided a gradually warming environment that reduced energy loss and enabled gonadal maturation during spring.

A stable food supply is equally critical for the reproductive success of filter-feeding bivalves whose reproduction directly relies on natural phytoplankton biomass. Although short-term fluctuations in Chl a concentration were observed during the experiment, these did not introduce significant sample heterogeneity, as bivalves can derive reproductive energy both from immediate feeding and from the mobilization of internal reserves [5], implying that their energy allocation system possesses an inherent buffering capacity against short-term variations in food intake. In natural environments, Chl a concentration typically ranges from 1 to 4 μg/L [30]. In contrast, our artificial system maintained an average Chl a concentration of 18.61 μg/L. Thus, this high and stable food supply not only ensures continuous energy intake, promotes the conversion of glycogen into reproductive energy, and prevents gonadal developmental delays commonly observed under low-food conditions [31,32]. Consequently, the combined regulation of temperature and food availability in our artificial system effectively shortened the reproductive cycle.

The gonadal development of bivalves is generally characterized by a series of well-defined histological stages, and accurate stage identification is essential for describing gametogenic progression. Histological studies in geographically closer regions, such as Incheon Bay, Korea, provide a direct reference for the present study [33]. From a cyclical perspective, natural populations of R. philippinarum require significantly longer maturation periods [15,34]. In contrast, under the controlled conditions in the present study, the entire gonadal development cycle was completed within 28 days. This duration is shorter than the several months required in natural populations, highlighting the efficiency of artificial regulation.

4.2. Expression of Gonadal Development Regulatory Genes

In bivalve mollusks, the GnRH signaling pathway connects the neuroendocrine system with gonadal development, coordinating reproductive processes through the regulation of steroid hormone synthesis and energy allocation. Our study observed a marked sexual variation in the expression of GnRH in clams: expression increased progressively in males throughout reproductive maturation, whereas it declined in females during the critical vitellogenic stage (proliferative stage). This dynamic is conserved with the expression pattern of Cg-GnRH in the Pacific oyster (Crassostrea gigas) [35], suggesting that GnRH commonly regulates sex-specific resource allocation in bivalves. In males, high GnRH expression likely activates the GnRH–GnRHR–GPB5–LGR/LGR5L signaling pathway [36], upregulating steroidogenic enzymes such as CYP17A, HSD17B12, and HSD3B1, thereby enhancing testosterone synthesis and driving spermatogenesis. Given that vitellogenesis is an energy-demanding process [37], the downregulation of GnRH expression in females during the proliferative stage could be an adaptive mechanism to prevent premature energy expenditure. This would effectively prioritize metabolic resources for vitellogenesis and oocyte maturation. Furthermore, analogous to the local expression of IGnRH-III in the ovary of the donkey’s ear abalone (Haliotis asinine) [6], we speculate that GnRH in clams may also act directly on early oocytes via paracrine or autocrine mechanisms to modulate their development. Thus, our results not only confirm the conserved role of GnRH in bivalve reproduction but also reveal its molecular basis for implementing sex-specific reproductive strategies through dual pathways: the neuro-gonadal axis and local regulation.

Our study showed synchronous, sexually differentiated expression of the key steroidogenic enzyme gene CYP17 and GnRH in clams. This expression pattern is consistent with the finding in the C. gigas that the neuropeptide Cg-GnRH can regulate the CYP gene family [38]. Furthermore, the significant upregulation of multiple CYP members in female gonads observed in that study suggests GnRH may act as an upstream coordinator of a complex steroidogenic enzyme network [38]. Specifically, in males, the synchronous peaking of GnRH and CYP17 expression during the active spermatogenesis phase (day 7) is consistent with the pattern of high cyp17a1 expression during early gonadal development in the Yesso scallop (Mizuhopecten yessoensis) [39]. Furthermore, this co-expression corresponds to the mechanism where CYP17 acts in concert with other steroidogenic enzymes like HSD3B and HSD17B to effectively synthesize androgens. CYP17 may subsequently regulate sperm motility through the synthesis of progestins such as Dihydroprogesterone (DHP) [40], and drive androgen synthesis to support spermatogenesis. Conversely, in females, the co-upregulation of GnRH and CYP17 during the late vitellogenic to maturation stage (day 14) suggests a functional shift in this signaling pathway. The expression of CYP17 at this stage may prioritize catalyzing 17α-hydroxylation reactions, providing precursors for synthesizing progestins that promote final oocyte maturation, rather than being channeled into the estrogen synthesis pathway. Therefore, our results demonstrate the crucial role of CYP17 as a key downstream effector of GnRH signaling in bivalve reproduction.

Following the initial conversion of pregnenolone by CYP17A1 in the steroidogenic pathway, 17β-HSD acts as a crucial downstream regulator, governing the final steps of sex hormone production in mollusks [41]. In our study, the proliferative stage expression peak aligns with the expression pattern and function of 17β-HSD11 in the small abalone (Haliotis diversicolor supertexta) [42]. In this species, the enzyme catalyzes the oxidation of testosterone (T) to androstenedione (4A), and its expression is suppressed during the maturation phase to maintain adequate levels of active androgens. The expression peak observed in clams may serve a similar function, reducing local testosterone concentrations to create a suitable hormonal microenvironment for the finely regulated process of spermatogenesis. In females, studies have shown that 17β-HSD12 and its homologous isoforms in oysters and scallops are highly expressed in the ovary and promote estrogen activation by catalyzing the reduction of estrone (E1) to estradiol (E2) [39,43]. Correspondingly, the pronounced expression burst observed in clams at the mid-developmental stage (day 14) likely signifies a key activation phase, aimed at maximizing the bioactivity of endogenous estradiol to directly drive vitellogenesis.

Beyond the regulation of steroidogenic enzymes, ERR acts as a key transcriptional regulator that mediates sex-specific reproductive strategies by coordinating energy metabolism with gonadal development [9]. In this study we observed that in male clams, ERR expression remained high during early developmental stages but decreased markedly by the spawning stage. Its sustained high expression in the early phase provides a transcriptional foundation for continuous spermatogonia proliferation and the initiation of meiosis, which is consistent with findings in the fruit fly (Drosophila melanogaster), where the loss of ERR leads to disrupted germ cell organization and abnormal spermatogenesis [44]. The sharp decline in expression on day 28 coincides with the initiation of sperm release, suggesting that ERR’s primary role in spermatogenesis is largely complete by this stage. In female clams, a distinct peak in ERR expression during growth stages may temporally activate the gene regulatory network associated with oogenesis. This regulatory pattern finds functional support across marine invertebrates: in the Japanese spineless cuttlefish (Sepiella japonica), ERR has been shown to upregulate key genes such as Vg, CDK1 and Cyclin B, directly driving vitellogenesis and oocyte maturation [45]. Furthermore, tissue localization studies in mussels have revealed that ERR is specifically expressed in oocytes within the ovary [46], suggesting that ERR may control female gamete development by modulating the germ cell microenvironment. Notably, ERR expression in males tended to be higher than in females during the early phase, indicating that it performs distinct functions in their respective reproductive processes: in males, it supports continuous spermatogonia renewal, whereas in females, it is transiently activated to initiate oogenesis.

In bivalves, the Dmrt gene family exhibits remarkable functional divergence across species and genes. Specifically, their expression patterns and functional stages vary considerably among species: in C. gigas, Cg-DMI shows high expression in males during late sexual maturity, suggesting its role in late spermatogenesis [47], whereas in M. yessoensis, my-dmrt2 peaks during early spermatogenesis, indicating a function in the initiation of sperm development [48]. Our study revealed that Dmrt4-like was significantly upregulated during the early stage of male development. This expression pattern aligns more closely with that observed in M. yessoensis but differs from the Akoya pearl oyster (Pinctada fucata), likely reflecting species-specific reproductive strategies that employ distinct temporal activation of Dmrt genes. Although the functional stages of Dmrt genes vary across species, their expression appears to be regulated by a relatively conserved upstream neuroendocrine mechanism mediated by sex steroids and neuropeptides [48]. In our study, the synchronous expression peaks of the GnRH gene and Dmrt4-like in males suggest that GnRH signaling may act as a key upstream event driving Dmrt gene activation and thereby initiating spermatogenesis. This provides correlative evidence that contributes to the evidence for the hypothesis of Nagasawa et al. [48] regarding the involvement of neuroendocrine factors in gonadal development. More direct evidence from Chlamys nobilis demonstrates that Dmrt gene transcription can be specifically induced by both androgens and estrogens [7]. Collectively, these findings outline a potential regulatory pathway in which Dmrt genes may function as downstream effectors of gonadal steroid hormone signaling, participating in the regulation of gametogenesis. In contrast, in females Dmrt4-like exhibited a unique delayed unimodal expression pattern, peaking at day 21. This expression profile resembles that of Pf-Dmrt4 in P. fucata, which shows elevated expression during vitellogenesis [49]. This temporal delay in expression, along with its potential regulation by estrogen, reflects the more complex molecular basis of oogenesis compared to spermatogenesis, revealing fundamental differences in the reproductive strategies between males and females at the molecular level.

In summary, the combination of sustained high food availability and stable temperature elevation provided abundant energy and substrates for gametogenesis. This optimized metabolic environment likely acted as a potent coordinator, synchronizing gene expression to drive an efficient transition through the reproductive stages.

However, the ecological and physiological consequences of this acceleration warrant careful consideration. A primary concern is the conflict between premature energy expenditure and the subsequent natural reproductive window. This conflict could directly disrupt the reproductive neuroendocrine axis, which is highly sensitive to environmental and physiological perturbations. In fish, for example, temperatures beyond the optimal range suppress key BPG-axis genes, halting gametogenesis [50,51]. In this study, spawning was inducted in April–May, thereby depleting energy reserves normally allocated for maintenance and growth. This premature depletion of energy reserves may lead to two main consequences: reduced gamete quality and spawning output, and disruption of endogenous reproductive regulatory rhythms [28]. However, it should be noted that the aim of this artificial conditioning was to shorten the breeding cycle for rapid trait comparison and selection. Once superior families are identified, cultivation can revert to conventional methods, thereby avoiding the long-term risks of sustained intensive conditioning.

5. Conclusions

We investigated gonadal development and key gene expression patterns in R. philippinarum within an artificial cultivation system. The results indicated that optimal food and temperature conditions can facilitate the progression of gonadal development from a resting stage to maturation. Gene expression profiles closely aligned with histologically observed developmental stages. In males, GnRH and CYP17 exhibited synchronized peaks during early spermatogenesis (day 7), with expression significantly surpassing female levels at this stage. Conversely, females demonstrated coordinated upregulation of ERR and 17β-HSD at day 14, corresponding to the initiation of the Growth Stage and active vitellogenesis. Dmrt4-like displayed distinct sex-specific patterns, with an early peak in males at day 7 and a significantly delayed peak in females at day 21. These sex- and stage-specific expression patterns reveal synergistic mechanisms governing gonadal development. These findings contribute to our understanding of gonadal development and provide insights for optimizing artificial maturation techniques to enhance reproductive efficiency.