Sex Reversal Induced by Dietary Supplementation with 17α-Methyltestosterone during the Critical Period of Sex Differentiation in Oriental River Prawn (Macrobrachium nipponense)

Simple Summary The steroid 17α-methyltestosterone (MT) inhibits ovarian function and is often used to induce sex reversal artificially in vertebrates. In oriental river prawn, sex reversal through vertebrate sex hormones can be observed. Neo-males (sex-reversed female prawns) were maintained by exogenous androgen, and over-reliance led to slow testis growth, small body size, and low growth rate, but sperm was still produced. In female prawns, MT inhibited ovary development and promoted growth. Abstract The steroid 17α-methyltestosterone (MT) inhibits ovarian function and is often used to induce sex reversal artificially in vertebrates. In the present study, different concentrations of MT were added as dietary supplementation, and the effects on sex ratio, growth, and gonadal development were examined. After 40 days, the sex ratio (male:female) in each group increased at different degrees with 50 (1.36:1), 100 (1.57:1), and 200 (2.61:1) mg/kg MT, and neo-males with testis–ovary coexistence were observed in the 200 mg/kg MT group. Furthermore, 50 and 100 mg/kg MT could induce female reversion in neo-males. Histologically, the development of the testes in experimental groups was slower, but the ovaries of the experimental and control groups had similar developmental rates. The expression levels of DMRT11E, Foxl2, and SoxE1 in males at 200 mg/kg MT were 8.65-, 3.75-, and 3.45-fold greater than those of the control group. In crustaceans, sex reversal through vertebrate sex hormones can be observed. Neo-males (sex-reversed female prawns) were maintained by exogenous androgen, and over-reliance led to slow testis growth, small body size, and low growth rate, but sperm was still produced. In female prawns, MT inhibited ovary development and promoted growth.


Introduction
The oriental river prawn (Macrobrachium nipponense; Crustacea; Decapoda; Palaemonidae) is farmed in China on a large scale [1]. Although the annual aquaculture production was about 224,413 tons in 2021 and the output value was more than 20 billion, some problems exist in its development. The male has a faster growth rate and better disease resistance than the female [2]. Therefore, studies on the mechanisms of sex differentiation and reliable monosex cultivation techniques have important practical value. Artificially induced sex reversal is a first step, and its success depends on the critical period of sex differentiation [3].
The method of dissolving MT into the diets is described as follows [30]: MT was dissolved in 95% ethanol to prepare a stock solution at a concentration of 50 mg/mL and then diluted into concentrations of 5, 10, and 20 mg/mL. Different concentrations of ethanol were then evenly sprayed on the feed (1 mL ethanol per 10 g diet) and stirred with a glass stick for at least 3 min. The diets were then placed under a ventilated laboratory hood and left in the shade for 15 min. The treated diets were added to 15 mL test tubes and placed in a refrigerator at 0 • C to evaporate the remaining alcohol naturally. The diets were left until the color was the same as normal diets and without the smell of alcohol. The control diet was prepared in the same manner using 95% ethanol without the MT. The diets were stored at 4 • C until feeding.

Experimental Design
At 25 days after metamorphosis, the juvenile oriental river prawns were fed on diets with different concentrations of MT for 40 days. The experimental design is shown in Figure 1. The start of the MT treatment was set as day 0 (PL25), and subsequent sampling stages were denoted as days 10, 20, 30, and 40. until the color was the same as normal diets and without the smell of alcohol. The control diet was prepared in the same manner using 95% ethanol without the MT. The diets were stored at 4 °C until feeding.

Experimental Design
At 25 days after metamorphosis, the juvenile oriental river prawns were fed on diets with different concentrations of MT for 40 days. The experimental design is shown in Figure 1. The start of the MT treatment was set as day 0 (PL25), and subsequent sampling stages were denoted as days 10, 20, 30, and 40. Figure 1. Experimental design. Yellow triangles represent the sex ratio statistic stage, red triangles represent the histological sampling stage, and blue triangles represent the growth data collection stage. The gray circle indicates that secondary sexual characteristics have not appeared and the green circles indicate that secondary sexual characteristics have appeared.

Sex Ratio Statistics
The prawns in the control and experimental groups were randomly selected from more than 90 individuals at 10, 20, 30, and 40 days to determine the sex ratio. Each group had at least three replicates. The distinctions between the secondary sexual characteristics of male and female prawns are shown in Table 2 [31]. Yellow triangles represent the sex ratio statistic stage, red triangles represent the histological sampling stage, and blue triangles represent the growth data collection stage. The gray circle indicates that secondary sexual characteristics have not appeared and the green circles indicate that secondary sexual characteristics have appeared.

Sex Ratio Statistics
The prawns in the control and experimental groups were randomly selected from more than 90 individuals at 10, 20, 30, and 40 days to determine the sex ratio. Each group had at least three replicates. The distinctions between the secondary sexual characteristics of male and female prawns are shown in Table 2 [31]. Table 2. Male and female appearance characteristics of oriental river prawn.

Characteristic Male Female
Physique Big Small The second pereiopod About 1.5 times body length No longer than body length Width of the fifth pereiopod Narrower, equidistant Greater than width of the fourth pereiopod The second ventral extremity With a rod-like projection None Genital pore Located at the base of the fifth pereiopod Located medial to the base of the third pereiopod Note: The difference between male and female oriental river prawns is distinguished mainly from the physique, second pereiopod, width of the fifth pereiopod, second ventral extremity, and genital pore.

Measurement of the Growth Traits
The average body weights of juvenile oriental river prawns were determined at the beginning of the experiment on day 0 (initial mean weight) and 40 days (final mean weight) after the MT treatment. The body weights of 50 randomly selected prawns from each replicate were measured.
The weight growth rate (WGR) was determined as follows: The specific growth rate (SGR) was determined as follows: where Wt is the average body weight 40 days after treatment with MT, W0 is the average body weight before treatment with MT, and t is the total days of culture [32].

Histological Observations of Testicular Development
Male and female M. nipponense were separated after being treated with different concentrations of MT and stained with hematoxylin and eosin (HE) to study the histological changes in the testis and ovary. After 40 days, samples of male and female prawns from the control and 200 mg/kg MT group were mounted on slides and stained with HE, and prepared as described in previous studies [33]. Observations took place using a stereo microscope (SZX16; Olympus Corporation, Tokyo, Japan). Comparative labeling was performed with various cell types based on cell morphology [7].

The qPCR Analysis
After treatment with different concentrations of MT, qPCR analysis was used to measure the expression levels of eukaryotic translation initiation factor 5A (EIF, GenBank Juvenile male and female prawns treated with different concentrations of MT were separated and immediately frozen in liquid nitrogen to prevent RNA degradation. Three male and three female prawns in each replicate were randomly selected for RNA extraction from the whole sample. RNA was extracted using RNAiso Plus Reagent (Takara Bio. Inc., Shiga, Japan). The concentration of RNA was quantified by using a Bio-Photometer (Eppendorf, Hamburg, Germany) and 1 µg of total RNA from each sample was reversetranscribed into cDNA using a Prime Script RT reagent Kit (Takara Bio. Inc.) following the manufacturers' protocols. The expression levels of the genes tested in each sample Animals 2023, 13, 1369 6 of 16 type were identified by qPCR using Ultra SYBR Mixture (CWBio, Beijing, China). The qPCR amplifications were carried out in a total volume of 25 µL, containing 1 µL cDNA (50 ng), 10 µL SsoFast EvaGreen Supermix (Bio-Rad, Washington, USA), 0.5 µL of genespecific forward and reverse primers (listed in Table 3), and 13 µL of DEPC water. The reaction mixture was initially incubated at 95 • C for 30 s to activate the Hot Start Taq DNA polymerase, followed by 40 cycles at 95 • C for 10 s and 60 • C for 1 s. A melting curve was conducted at the end of the qPCR reaction at 65-95 • C (in 0.5 • C increments) for 10 s. At least three replicate qPCRs were performed per sample, in each concentration gradient of female prawns n ≥ 3 and male prawns n ≥ 3, and EIF was used as the reference gene [34]. The relative copy number of genes between different concentrations was calculated using the 2 −∆∆CT method [35].

Statistical Analysis
The statistical analyses were all conducted using IBM SPSS Statistics for Windows, version 23.0. (IBM Corporation, Armonk, NY, USA). The significant differences between groups were determined by one-way ANOVA, followed by the least significant difference and Tukey's test. In addition, to determine if the effect was linear and/or quadratic, a follow-up trend analysis using orthogonal polynomial contrasts was performed [36]. Quantitative data are expressed as mean ± standard error of the mean (SEM). Probability (p) values < 0.05 were considered statistically significant.

Effects of MT Concentration on the Sex Ratio of Juvenile Prawns
The results for the sex ratio (male:female) at four different concentrations of MT over various lengths of time are presented in Figure 2. As shown in Figure 2A, throughout the culture phase the sex ratio of the control group was maintained around 1. In Figure 2B, the sex ratio gradually decreases with increasing culture time. Compared with the control group, the ratio was significantly higher at 10 and 20 days (p < 0.05). However, the sex ratio at 30 and 40 days were 1.44 and 1.36. respectively, and were not significantly different. The sex ratio showed a similar trend of decline in Figure 2C, at 40 days it was 1.57. Compared to the 50 and 100 mg/kg MT experimental groups ( Figure 2D), the sex ratio was high and stable in the 200 mg/kg MT experimental group, with significant differences between the four concentrations (p < 0.05).
Animals 2023, 13, x FOR PEER REVIEW high and stable in the 200 mg/kg MT experimental group, with significant differenc tween the four concentrations (p < 0.05).

Histological Observations of the Testis and Ovary
As shown in Figure 3A, after 40 days of culture, the normal developing male p was in the spermatid phase, with primary and second spermatocytes in the testis large number of sperm observed in the vas deferens. Normally developed sperm observed, as shown in Figure 3E, but some male prawns had fewer sperm compa the control shown in Figure 3C. Figure 3B,D show that the ovaries of the control an perimental groups are at the same stage of development, with many oogonia and pr oocytes observed and no yolk granule accumulation. The testis-ovary results are s in Figure 3E. Follicle cells and cytoplasmic membrane can be seen in the ovarian c The testes are filled with primary and secondary spermatocytes, but no mature spe

Histological Observations of the Testis and Ovary
As shown in Figure 3A, after 40 days of culture, the normal developing male prawn was in the spermatid phase, with primary and second spermatocytes in the testis and a large number of sperm observed in the vas deferens. Normally developed sperm were observed, as shown in Figure 3E, but some male prawns had fewer sperm compared to the control shown in Figure 3C. Figure 3B,D show that the ovaries of the control and experimental groups are at the same stage of development, with many oogonia and primary oocytes observed and no yolk granule accumulation. The testis-ovary results are shown in Figure 3E. Follicle cells and cytoplasmic membrane can be seen in the ovarian cavity. The testes are filled with primary and secondary spermatocytes, but no mature sperm.

Effects of MT Concentration on Growth Traits of Juvenile Prawns
Effects of different concentrations of MT on growth traits of male and female juvenile prawns are described in Table 4. In males (Table 4), after 40 days of treatment with the gradual increase in MT concentration, the WGR and SGR of males decreased gradually, and there were significant differences between the 50, 100, and 200 mg/kg MT groups and the control group (p < 0.05), but there was no difference within the three treatment groups.

Effects of MT Concentration on Growth Traits of Juvenile Prawns
Effects of different concentrations of MT on growth traits of male and female juvenile prawns are described in Table 4. In males (Table 4), after 40 days of treatment with the gradual increase in MT concentration, the WGR and SGR of males decreased gradually, and there were significant differences between the 50, 100, and 200 mg/kg MT groups and the control group (p < 0.05), but there was no difference within the three treatment groups. The WGR and SGR of 200 mg/kg MT were 1628.87 ± 77.43 and 5.86 ± 0.05, respectively, which were less than 25% of the values of the control group. The FMW, WGR, and SGR of male prawns significantly decreased linearly with an increase in dietary MT levels (p < 0.05). In females (Table 4), the WGR and SGR increased gradually with the increase in MT concentration after 40 days of treatment. The WGR and SGR of the 200 mg/kg MT group were 1549.79 ± 72.32 and 5.59 ± 0.09, respectively, which was more than 30% greater than that of the control group (p < 0.05). The FMW, WGR, and SGR of female prawns significantly increased linearly with the increase in dietary MT levels (p < 0.05). Moreover, 40 days after 200 mg/kg MT was administered to juvenile prawns, the WGR and SGR of females and males tended to be the same and were not significantly different in SPSS analysis.

Analysis of Male-Specific and Female-Specific Genes by qRT-PCR
All the samples measured were whole prawns that came from three separate individuals in the same group. As shown in Figure 4 Figure 6 shows MniDMRT11E, MnSoxE1, and MnFoxl2 mRNA levels in males and females measured at 40 days after different concentrations of MT. All the samples measured were whole prawns from three separate individuals in the same group. In male prawns, the MniDMRT11E, MnFoxl2, and MnSoxE1 expression levels of 200 mg/kg MT were 8.65-, 3.75-, and 3.45-fold greater than those of the control group (p < 0.05), respectively. The expression levels of these three genes in females were different from those in males. In female prawns, the expression levels of MniDMRT11E and MnFoxl2 gradually decreased with the increase in MT concentration, and at 200 mg/kg MT, they were 2.56and 3.40-fold less than those in the control group (p < 0.05), respectively. The MnSoxE1 expression level at 200 mg/kg MT was not significantly different from that of the control group.     Figure 6 shows MniDMRT11E, MnSoxE1, and MnFoxl2 mRNA levels in males and females measured at 40 days after different concentrations of MT. All the samples measured were whole prawns from three separate individuals in the same group. In male prawns, the MniDMRT11E, MnFoxl2, and MnSoxE1 expression levels of 200 mg/kg MT were 8.65-, 3.75-, and 3.45-fold greater than those of the control group (p < 0.05), respectively. The expression levels of these three genes in females were different from those in males. In female prawns, the expression levels of MniDMRT11E and MnFoxl2 gradually decreased with the increase in MT concentration, and at 200 mg/kg MT, they were 2.56and 3.40-fold less than those in the control group (p < 0.05), respectively. The MnSoxE1 expression level at 200 mg/kg MT was not significantly different from that of the control group.  Figure 6 shows MniDMRT11E, MnSoxE1, and MnFoxl2 mRNA levels in males and females measured at 40 days after different concentrations of MT. All the samples measured were whole prawns from three separate individuals in the same group. In male prawns, the MniDMRT11E, MnFoxl2, and MnSoxE1 expression levels of 200 mg/kg MT were 8.65-, 3.75-, and 3.45-fold greater than those of the control group (p < 0.05), respectively. The expression levels of these three genes in females were different from those in males. In female prawns, the expression levels of MniDMRT11E and MnFoxl2 gradually decreased with the increase in MT concentration, and at 200 mg/kg MT, they were 2.56-and 3.40-fold less than those in the control group (p < 0.05), respectively. The MnSoxE1 expression level at 200 mg/kg MT was not significantly different from that of the control group.

Sex Reversal
This study aimed to determine whether dietary supplementation with different concentrations of MT could induce sex reversal in the oriental river prawn and whether it affected growth and gonadal development. The period before sexual differentiation is known as the period of instability and is usually considered to have a higher degree of sexual plasticity because undifferentiated gonads are more sensitive to exogenous steroids [37]. Previous studies have revealed that the period of unstable sexual differentiation in

Sex Reversal
This study aimed to determine whether dietary supplementation with different concentrations of MT could induce sex reversal in the oriental river prawn and whether it affected growth and gonadal development. The period before sexual differentiation is known as the period of instability and is usually considered to have a higher degree of sexual plasticity because undifferentiated gonads are more sensitive to exogenous steroids [37]. Previous studies have revealed that the period of unstable sexual differentiation in the oriental river prawn was before PL10 and that the gonads were almost fully developed at PL25 [7]. MT has been successfully used to masculinize females in P. olivaceus [38], D. rerio [39], D. labrax [40], O. mykiss [41], and so on. In this experiment, more males were observed after feeding MT to post-larval juvenile oriental river prawns, which was evidence that sex reversal was achieved by using MT as a dietary supplementation after gonadal development. Histological observations also demonstrated that spermatozoa and ovaries coexisted. These results provided an important basis for the regulation of sex differentiation with MT and the establishment of monosex cultures in juvenile oriental river prawns. However, we discovered that the sex ratio did not reach 100% male. This may require further research, such as extending the feed period or increasing the concentration of hormones. Another possible speculation is that the efficiency of sex reversal may be influenced by environmental factors, such as water temperature and climate. Activation of the stress response axis is a key process in environment-induced sex plasticity in fish [42]. Furthermore, recent genetic studies in mice suggest that gonadal sexual reversal requires active maintenance to suppress the opposite sex during adulthood [43]. Therefore, further refinement of feeding times and doses may be needed to make a single feeding sufficient to fully masculinize the population of this species.
In the oriental river prawn, the expression of androgen-related genes is decreased in AG by IAG RNAi [22]. In M. rosenbergii, fully functional sex reversal can be achieved by IAG silencing [6]. Interestingly, in this study, the expression of Mn-IAG in the 100 and 200 mg/kg MT groups was only an eighteenth of that in normal male prawns. Although Mn-SG declined, it remained at a certain level. Salmon (O. kisutch) larvae retain approximately 10% of exogenous steroids 16 days after cessation of treatment [44]. Most rats with a testis removed maintained spermatogenesis in numbers after receiving exogenous hormone treatment. These results showed that the male characteristics of neo-males were maintained by exogenous androgens and that neo-males cannot produce endogenous androgens on their own [45]. Another interesting phenomenon was that the sex ratio decreased with culture at relatively lower concentrations of 50 and 100 mg/kg MT. In sea bass, 200 or more days after MT withdrawal, under the test conditions, the induction of sterility was reversible, and it was observed that a few germ cells at 426 days post-fertilization (DPF) were able to resume mitosis [46], indicating that neo-males retain the possibility of reverting to females. However, a similar phenomenon has not been observed in crustaceans. It has been shown that endogenous hormones produced by the testis are more effective than exogenous hormones in maintaining spermatogenesis at the same concentration. Therefore, a reasonable speculation is that with the growth of prawns, their own endogenous hormones gradually take a dominant role and influence the development of their secondary sexual characteristics.

Gonadal Development
In aquatic species, many studies have reported that MT promoted the maturation of male organs and the formation of secondary sexual characteristics, while inhibiting ovarian development [47,48]. In studies on grass carp (C. idella), it has been proposed that this phenomenon occurs because MT inhibits ovarian development and the energy used for ovarian development is used for growth and development [49]. Furthermore, a previous study on the oriental river prawn revealed that dietary supplementation with MT at concentrations greater than 500 mg/kg delayed ovarian development in juvenile prawns [50]. In this study, the WGR and SGR of female prawns in the 100 and 200 mg/kg MT were significantly increased compared to those of the control group. Meanwhile, the expression levels of Mn-Vg and Mn-Vgr were significantly lower in the experimental group than the control for females at the same developmental stage, indicating that the growth of female prawns was strengthened by inhibited ovarian development.
The WGR and SGR of male prawns decreased after MT supplementation, and no significant difference between male and female prawns occurred in the 200 mg/kg MT group. Histological observation revealed that the testis development of the experimental group was in the spermatocyte phase, and less sperm was found. It is possible that Animals 2023, 13, 1369 13 of 16 the neo-males still had female characteristics such as small body size and slow growth rate. Another suggestion is that this negative growth performance may be the result of energy consumption due to rapid gonadal development [51]. In a study on peacock fish (P. reticulata), high concentrations of MT also caused weight and testis growth rates to decrease dramatically in males [52]. They also caused malformation or even agenesis of the sperm duct system in E. suillus [53]. These results showed that over-reliance on exogenous hormones led to the failure of organs that produce sex hormones.

Sexual Differentiation Genes
DMRT proteins share a distinctive zinc-finger DNA-binding motif termed the DM domain [54,55]. The expression of DMRT is upregulated in association with androgeninduced gonadal masculinization. DMRT was found to be upregulated during sex reversal in rainbow trout [56]. Similarly, in this experiment, DMRT11E was significantly upregulated in neo-males. DMRT11E RNAi in M. nipponense showed negative regulation with the Vg gene and positive regulation with the IAG gene [57]; this result is the same as that found in this study.
Foxl2 is a member of the forkhead/HNF-3-related family of transcription factors that plays an important role in the development and maintenance of ovarian function in vertebrates [58,59]. The significant downregulation of Foxl2 in sex-reversed mandarin fish showed that Foxl2 is involved in sex differentiation [9]. A previous study on RNAi of Foxl2 in the oriental river prawn found considerable evolutionary differences in Foxl2 in crustaceans and fish; Mn-Foxl2 mRNA expression levels were almost 15-fold higher in testes than in ovaries [60].
SoxE subgroup transcription factors are thought to play essential roles in mammalian sex determination and gonadal development [61,62]. In teleost fish, they may only play a role in germline maintenance rather than in sexual determination [63]. In previous studies, SoxE1 was found to be involved in the early differentiation of males in the oriental river prawn and expressed at various stages of gonad development [64].
In the present study, the MniDMRT11E, MnFoxl2, and MnSoxE1 expression levels of 200 mg/kg MT were 8.65-, 3.75-, and 3.45-fold greater than those in the control group males, respectively. These expression levels were negatively correlated with IAG, indicating that these genes are possibly involved in the synthesis and secretion of androgens and are key genes in gonadal androgenization. In female prawns, the expression levels of MniDMRT11E and MnFoxl2 in the experimental group were significantly lower than those in the control, indicating that they are not only involved in the sexual differentiation of males but also key genes for ovarian development. Meanwhile, the expression of SoxE1 was higher at 50 and 100 mg/kg MT than in the control, but not different from the control at 200 mg/kg, suggesting that it was also involved in ovarian development but did not play a major role.

Conclusions
This present study demonstrated that vertebrate sexual hormones could induce sex reversal in crustaceans, and determined vertebrate sexual hormone (MT) concentrations and periods that induced sex reversal in the oriental river prawn. We found that MT at a high concentration (200 mg/kg) induced sex reversal. However, neo-males induced by low concentrations of MT (50 and 100 mg/kg) may revert to females. Neo-males were maintained by exogenous androgens, and over-reliance led to slow testis growth, small body size, and low growth rate, but sperm was still produced. Sex-related genes played a role in male sexual differentiation and female ovarian development. The current research provides an important theoretical basis for identifying the type of chromosomal sex determination in the oriental river prawn and for achieving a monosex culture.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author for scientific purposes.