Mn-XRN1 Has an Inhibitory Effect on Ovarian Reproduction in Macrobrachium nipponense

XRN1 is an exoribonuclease that degrades mRNA in the cytoplasm along the 5′–3′ direction. A previous study indicated that it may be involved in the reproduction of Macrobrachium nipponense. Quantitative real-time PCR was used to detect the spatiotemporal expression pattern of Mn-XRN1. At the tissue level, Mn-XRN1 was significantly expressed in the ovary. During development, Mn-XRN1 was significantly expressed at the CS stage of the embryo, on the 10th day post-larval and in the O2 stage of ovarian reproduction. The in situ hybridization results showed the location of Mn-XRN1 in the ovary. The expression of Mn-VASA was significantly increased after in vivo injection of Mn-XRN1 dsRNA. This suggests that Mn-XRN1 negatively regulates the expression of Mn-VASA. Furthermore, we counted the number of M. nipponense at various stages of ovarian reproduction on different days after RNAi. The results showed that ovarian development was significantly accelerated. In general, the results of the present study indicate that Mn-XRN1 has an inhibitory effect on the ovarian maturation of M. nipponense. The inhibitory effect might be through negative regulation of Mn-VASA.


Introduction
M. nipponense is an economically important freshwater aquaculture species in China due to the advantages of good stress resistance and rapid growth [1]. However, the rapid sexual maturity of M. nipponense has been a constraint on increased production. Female prawns have a short maturation cycle and can generally lay eggs two to three times per year [2]. Faster sexual maturity allows females to spend most of their energy on ovarian development, resulting in smaller individuals [3]. Furthermore, rapid sexual maturation leads to inbreeding and multigenerational coexistence, which in turn affects the germplasm [4,5]. The sex regulation mechanism of M. nipponense is currently unclear. Only by understanding this mechanism can we regulate the speed of sexual maturity in M. nipponense, thereby solving the problems of yield and germplasm caused by rapid sexual maturity. Therefore, we analyzed the gonadal transcriptome of M. nipponense and screened for a significantly expressed gene, Mn-XRN1, in the RNA degradation and ribosomal pathways.
XRN1 is an important and conserved ribonuclease that degrades RNA in the 5 -3 direction in the cytoplasm [6]. It is well-known that eukaryotic messenger RNA (mRNA) has two complete stability determinants, 5 7-methylguanosine cap and 3 poly(A)tail, that protect transcripts from exonucleases and enhance translational initiation. Therefore, in order for degradation to begin, either of these two structures must be destroyed, or the mRNA must undergo internal cleavage by intranuclear dissolution attack [7]. This cides with the three pathways by which XRN1 degrades mRNA in eukaryotes (Figure (1) Deenylation-dependent degradation occurs. The mRNA degradation process invo several steps. First, the mRNA is dealkenylated by the CCR4-CAF1-NOT1 complex PARN enzyme to eliminate most of the 3′poly(A) [8,9]. Then, the 5′ cap is hydrolyzed a cap-removing complex containing DCP2, and, finally, the mRNA is degraded by XR in the 5′-3′ direction [7,[10][11][12][13]. (2) Independent of deenylated degradation, such as n sense-mediated decline (NMD), occurs. NMD targets and certain long noncoding RN can bypass deenylation, directly remove the 5′ cap, and then be degraded by XRN1 in 5′-3′ direction [14][15][16]. (3) Nuclear cracking-dependent degradation also occurs. mRNA is lysed internally, producing unprotected 5′ and 3′ fragments. The 3′ fragme are degraded by XRN1 [17][18][19]. In addition, XRN1 is involved in various aspects of R metabolism, such as RNA silencing, rRNA maturation, and transcription termina [11,20]. XRN1 has been well studied in terms of degradation mechanisms. However, the production-related functions of XRN1 have been poorly studied. In yeast, mutation XRN1 lead to larger cells, increased doubling time, and defective spore production [21, Knocking down the XRN1 gene in Caenorhabditis elegans results in a failure of abdom closure, eventually leading to death of the embryo in the twofold stage of developm [23]. Pacman is the homologue of XRN1 in Drosophila. Mutations of Pacman in Drosop can lead to a variety of developmental phenotypic defects, including decreased ferti dull wings, and bristle defects [24,25]. Saccharomyces cerevisiae cells lacking Xrn1p exh increased chromosome loss, nucleosome defects, and impaired spindle isolation in ce lar processes associated with microtubule function [26]. Furthermore, XRN1 may be sential for proper bone formation in humans. Mutations in XRN1 can lead to osteo coma, which is produced by mesenchymal-derived cells that are unable to different correctly to produce an unmineralized portion of the bone matrix called osteoids [27,2 The present study focused on female M. nipponense, to determine the potential fu tion of Mn-XRN1 in the reproduction of M. nipponense, and to identify the regulatory lationship with other genes. We used real-time fluorescence quantitative PCR to anal the expression of Mn-XRN1 in different tissues, different developmental stages, XRN1 has been well studied in terms of degradation mechanisms. However, the reproduction-related functions of XRN1 have been poorly studied. In yeast, mutations in XRN1 lead to larger cells, increased doubling time, and defective spore production [21,22]. Knocking down the XRN1 gene in Caenorhabditis elegans results in a failure of abdominal closure, eventually leading to death of the embryo in the twofold stage of development [23]. Pacman is the homologue of XRN1 in Drosophila. Mutations of Pacman in Drosophila can lead to a variety of developmental phenotypic defects, including decreased fertility, dull wings, and bristle defects [24,25]. Saccharomyces cerevisiae cells lacking Xrn1p exhibit increased chromosome loss, nucleosome defects, and impaired spindle isolation in cellular processes associated with microtubule function [26]. Furthermore, XRN1 may be essential for proper bone formation in humans. Mutations in XRN1 can lead to osteosarcoma, which is produced by mesenchymal-derived cells that are unable to differentiate correctly to produce an unmineralized portion of the bone matrix called osteoids [27,28].
The present study focused on female M. nipponense, to determine the potential function of Mn-XRN1 in the reproduction of M. nipponense, and to identify the regulatory relationship with other genes. We used real-time fluorescence quantitative PCR to analyze the expression of Mn-XRN1 in different tissues, different developmental stages, and different ovarian reproduction stages of M. nipponense. In situ hybridization (ISH) was used to determine the location of the Mn-XRN1 gene. The expression of the Mn-XRN1 gene was knocked-down using RNA interference (RNAi) technology. Following RNAi, we counted the number of each ovarian stage in M. nipponense to observe the effect on ovarian development after Mn-XRN1 knockdown.

Animals
Healthy female M. nipponense of consistent weight required for the present study were obtained from the Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences (120 • 13 44 E, 31 • 28 22 N). They were cultured in tanks with circulating water and fed with snail meat twice per day.

Bioinformatics Analysis
The cDNA fragment of the target gene Mn-XRN1 was obtained from the M. nipponense transcriptome cDNA library in our laboratory. The ORF Finder (https://www.ncbi.nlm.nih. gov/orffinder/ (accessed on 20 June 2022)) was used to predict the Mn-XRN1 open reading frame and the BLAST tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi#alnHdr_317467911 (accessed on 23 June 2022)) was used for comparison to analyze sequence. Based on the known cDNA fragment, we used the Primer-BLAST (https://www.ncbi.nlm.nih.gov/ tools/primer-blast/ (accessed on 12 July 2022)) to design the specific primers which were used to verify Mn-XRN1. The specific primers are listed in Table 1 and were sent to the Shanghai Exsyn-bio Technology Company for sequencing. The phylogenetic tree was constructed based on amino acid sequence using MEGA5.1 software and the online website iTOL (https://itol.embl.de/ (accessed on 15 August 2022). Multiple sequence alignment of Mn-XRN1 amino acids was performed by DNAMAN 6.0 software. The three-dimensional protein structure of Mn-XRN1 was constructed by the online website (https://swissmodel.expasy.org/ (accessed on 17 August 2022). The online program Expasy (https://web.expasy.org/protparam/ (accessed on 20 August 2022)) was used to calculate the protein molecular weight and isoelectric point. The conserved domain of the protein was analyzed by InterpPro (https://www.ebi.ac.uk/interpro/ (accessed on 25 August 2022)).

RNA Extraction and cDNA Synthesis
An RNAiso Plus kit (Takara, Otsu City, Shiga Prefecture, Japan) was used to extract RNA from the whole tissues of the prawns. An electrophoresis apparatus (Bio-Rad, Hercules, CA, USA) was used to detect the quality of the RNA. Reverse transcriptase M-MLV kits (Takara) were used to synthesize the first strand of cDNA.

The qPCR Analysis
The Bio-Rad iCycler iQ5 Real-Time PCR System (Bio-Rad, Hercules, CA, USA) was used to detect the expression levels of genes in different tissues. The reaction system and procedures are referred to in a previous study [29]. All primers used for qRT-PCR are listed in Table 1. The 2 − Ct method was used to calculate the expression level of all genes required for this study. EIF (eukaryotic translation initiation factor 5A) was used as the reference gene [30].

In Situ Hybridization (ISH)
M. nipponense ovarian tissues at five different stages of reproduction were collected. Probes were designed according to known sequences. Three ISH experiments were performed on each tissue to analyze the mRNA locations of Mn-XRN1. The detailed steps are described in previous studies [5,29].

RNA Interference (RNAi)
The potential function of Mn-XRN1 was explored using RNAi. The specific RNAi primers, which are shown in Table 1, were designed by the snap Dragon programs (https: //www.flyrnai.org/snapdragon (accessed on 5 September 2022)). The Transcript AidTM T7 High Yield Transcription kit (Fermentas, Inc., Waltham, USA) was used to synthesize the Mn-XRN1 dsRNA. The Mn-XRN1 dsRNA was injected into the pericardial cavity. Each prawn was injected with 12 µg/g Mn-XRN1 dsRNA [31]. On the 5th and 10th day after injection, supplementary injection was performed to maintain the interfering effect [31]. A total of 300 female prawns in the second period (O2 period) of ovarian development were carefully selected and divided into six groups. These groups were divided into three experimental groups and three control groups. The GFP dsRNA was injected into control groups. Table 1. Primers designed in this study.

Sequence Analysis of Mn-XNR1
The full-length cDNA sequence of Mn-XRN1 was 12,128 base pairs (bp) long. Its ORF was 4872 bp long and encoded 1623 amino acids. The lengths of the 5 untranslated region (UTR) and the 3 −UTR were 125 bp and 7131 bp, respectively. The molecular weight and theoretical isoelectric point of the protein were 185.60176 kDa and 6.33, respectively. According to the prediction results, Mn-XRN1 had two highly conserved regions: CR1 and CR2. The residue orders were 1−354 and 425−594, respectively. Following the CR2 domain, this domain was called D. The D domain was divided into D1, D2, D3, and D4. The results of amino acid sequence comparison between M. nipponense and other crustaceans showed that M. nipponense had 55.30%, 54.67%, 55.11%, and 53.85% homology with P. monodon, F. chinensis, L. vannamei, and P. trituberculatus, respectively ( Figure 3).
The phylogenetic tree was divided into two main branches. One was an insect, the other was a crustacean, and there was a separate mollusk. M. nipponense was one of the crustacean clades ( Figure 4).
The three-dimensional protein structure of Mn-XRN1 compared with the three-dimensional structure of L. vannamei, P. monodon, and F. chinensis is shown in Figure 5. The results showed that M. nipponense and other species shared the same CR1 and CR2 domains. The threedimensional spatial structure of proteins had high similarity. The CR1 and CR2 domains were connected by a CR1-CR2 linker structure.

Spatial-Temporal Expression Analysis of Mn-XRN1
At the organizational level, we analyzed the expression of Mn-XRN1 in different tissues of M. nipponense ( Figure 6A). The highest expression of Mn-XRN1 occurred in the ovaries, followed by the heart. Mn-XRN1 was least expressed in muscle. The expression of Mn-XRN1 in the ovaries and muscles differed by 18-fold (p < 0.05).
Next, the expression modes of Mn-XRN1 at different embryonic and metamorphic developmental stages were examined. The embryonic period starts from early oogenesis to the pre-hatch stage. During embryonic development, maximum expression of Mn-XRN1 in the CS stage was observed ( Figure 6B). Mn-XRN1 mRNA levels then decreased significantly after the CS stage and remained at a low level (p < 0.05).
The metamorphic developmental period begins at the L1 stage and ends at the PL25 stage. The expression of Mn-XRN1 showed a downward trend from L1 to L10 stages ( Figure 6C). At the L15 stage, the expression increased suddenly and significantly. In PL10 to PL25 stages, the expression of Mn-XRN1 was higher than that of the larval development stages and reached a maximum in the PL10 stage (p < 0.05).
The mRNA expression level of Mn-XRN1 in the ovary showed a trend of first increasing and then decreasing, and reached a maximum in O2 stage ( Figure 6D). The mRNA expression of Mn-XRN1 in the O1, O3, and O4 stages was not much different, but it decreased significantly in the O5 stage (p < 0.05).

Spatial-Temporal Expression Analysis of Mn-XRN1
At the organizational level, we analyzed the expression of Mn-XRN1 in different tissues of M. nipponense ( Figure 6A). The highest expression of Mn-XRN1 occurred in the ovaries, followed by the heart. Mn-XRN1 was least expressed in muscle. The expression  . L1: the 1st-day after hatching, PL1: the 1st-day after larvae, and so on. The mRNA expression level of Mn-XRN1 in the different reproduction periods of the ovaries (D). O1: undeveloped period, O2: developing period, O3: nearly ripe period, O4: ripe period, O5: spent period. Statistical analysis was carried out using the one-way ANOVA method. Data are shown as mean ± SEM(n = 6). Bars marked with different letters represent significant differences in data (p < 0.05).

Localization of Mn-XRN1 in the Ovaries
In situ hybridization was used to detect the location of Mn-XRN1 mRNA in different stages of the ovary (Figure 7). The ISH results showed that the signal of Mn-XRN1 was stronger in the yolk granules and follicular cells. More signals were gathered in the nucleus in the O2 period. Mn-XRN1 signals were also detected in the cytoplasmic membrane and nucleus of oocytes. . L1: the 1st-day after hatching, PL1: the 1st-day after larvae, and so on. The mRNA expression level of Mn-XRN1 in the different reproduction periods of the ovaries (D). O1: undeveloped period, O2: developing period, O3: nearly ripe period, O4: ripe period, O5: spent period. Statistical analysis was carried out using the one-way ANOVA method. Data are shown as mean ± SEM(n = 6). Bars marked with different letters represent significant differences in data (p < 0.05).

Localization of Mn-XRN1 in the Ovaries
In situ hybridization was used to detect the location of Mn-XRN1 mRNA in different stages of the ovary (Figure 7). The ISH results showed that the signal of Mn-XRN1 was stronger in the yolk granules and follicular cells. More signals were gathered in the nucleus in the O2 period. Mn-XRN1 signals were also detected in the cytoplasmic membrane and nucleus of oocytes.

Effects of Mn-XRN1 Gene Silencing after RNAi on Ovarian Reproduction
Based on the previous expression pattern of the Mn-XRN1 gene in different tissues, we used RNAi to determine the function of the Mn-XRN1 gene. On the 1st, 5th, and 13th days after injection, the interference efficiency reached 20.24%, 53.51%, and 48.11%, respectively ( Figure 8A). After RNAi, the proportion of ovarian maturity after the O2 phase was greater than that of the control group at the same periods ( Figure 8B). The interference efficiency results were consistent with the observation of ovarian maturation. There were significant differences between the experimental and control groups.  After RNAi silenced Mn-XRN1, we found a significant increase in the expression of Mn-VASA, a member of the Dead Box family, in the ribosomal pathway. Compared with the control group, the differences in Mn-VASA gene expression on days 1, 5, and 13 in the experimental group were 62.78%, 214.99%, and 754.37%, respectively ( Figure 8C). The expression of Mn-VASA in the control group gradually decreased with the reproduction of the ovaries. significant differences between the experimental and control groups.
After RNAi silenced Mn-XRN1, we found a significant increase in the expression of Mn-VASA, a member of the Dead Box family, in the ribosomal pathway. Compared with the control group, the differences in Mn-VASA gene expression on days 1, 5, and 13 in the experimental group were 62.78%, 214.99%, and 754.37%, respectively ( Figure 8C). The expression of Mn-VASA in the control group gradually decreased with the reproduction of the ovaries.

Discussion
XRN1 has a single active site with a narrow inlet that removes secondary structures as the RNA crosses the gap [31]. The 5′ orientation of XRN1 has a series of conservative domains: CR1, CR2, and D. The D Domain can be divided into D1, D2, D3, and D4 [31,32]. These conserved domains have been found in XRN1 of yeast, C. elegans, mice, and other creatures, and have high homology. They play an important role in XRN1's correct degradation of various RNA substrates [33]. Domain CR1 contains seven strictly conserved acidic residues that coordinate metal ions for catalysis; domain CR2 restricts access to the active site, ensuring that XRN1 is the only exoribonuclease; domain D1 is essential for

Discussion
XRN1 has a single active site with a narrow inlet that removes secondary structures as the RNA crosses the gap [32]. The 5 orientation of XRN1 has a series of conservative domains: CR1, CR2, and D. The D Domain can be divided into D1, D2, D3, and D4 [32,33]. These conserved domains have been found in XRN1 of yeast, C. elegans, mice, and other creatures, and have high homology. They play an important role in XRN1's correct degradation of various RNA substrates [34]. Domain CR1 contains seven strictly conserved acidic residues that coordinate metal ions for catalysis; domain CR2 restricts access to the active site, ensuring that XRN1 is the only exoribonuclease; domain D1 is essential for XRN1 nuclease activity and may play an active role in determining or stabilizing the conformation of the N-terminal fragment. This may be a key factor in XRN1 catalysis; domains D2-D4 may have an impact in ensuring the correct conformation of domain D1, thereby indirectly enhancing the stability of the N-terminal fragment conformation [32]. In the present research, we obtained and verified the sequence of Mn-XRN1 from M. nipponense for the first time, and compared the amino acid sequence with other species. The phylogenetic tree and amino acid sequence alignment results showed that M. nipponense had high homology with crustaceans and was more conserved than other crustaceans. This dovetails with the trend revealed by our previous findings. Compared to other crustaceans, M. nipponense appeared earlier in evolution and had a more conserved genome [35].
We used qRT-PCR to analyze the expression of Mn-XRN1 in M. nipponense. At the tissue level, Mn-XRN1 was significantly expressed in the ovaries. This result showed that Mn-XRN1 might play a key role in the regulation of ovarian reproduction. During the embryonic stage, highly expressed genes directly participate in embryonic development or prepare for future physiology [36]. The expression of Mn-XRN1 peaked at the cleavage stage of embryonic development. This suggested that Mn-XRN1 might be involved in the process of oocyte mitosis. L1 is the first day after hatching, and L15 is the critical day of metamorphosis before the post-larval developmental stages of M. nipponense [37,38]. The relatively high expression of L1 and L15 meant that Mn-XRN1 was associated with post-membrane development and metamorphosis. The post-larval developmental periods have been shown to be a critical period for gonad differentiation and development in M. nipponense. According to histological observations, M. nipponense began to develop gonadal primordium at PL10. After PL10, the gonads began to differentiate and develop [39]. The mRNA expression level of Mn-XRN1 in the PL10-PL20 stage was higher than that in the previous stage and reached a maximum in the PL10 stage, indicating that Mn-XRN1 might be involved in activating and promoting the gonadal differentiation and development in M. nipponense.
Ovarian reproduction in M. nipponense can be divided into five stages: O1, undeveloped stage; O2, developmental stage; O3, near mature stage; O4, mature stage; and O5, declining stage [3]. The mRNA expression of Mn-XRN1 reaches the maximum at O2 stage. These results indicated that Mn-XRN1 may be associated with yolk deposition. In Drosophila, Pacman is also involved in eggogenesis of female nurse cells and within the yolk [40]. On the other hand, the follicular cavity derived from follicular cells is formed in O2 stage [5]. One plausible explanation is that Mn-XRN1 may be involved in activating ovarian development, especially follicle production. ISH results showed detection of Mn-XRN1 signaling in oocytes and follicles of O1, O2, and O5 stages. The ISH result provided a new basis for the viewpoint that Mn-XRN1 may be involved in ovarian maturation and follicle formation in M. nipponense.
RNAi is a technology that inhibits gene expression by using short double-stranded RNA molecules, and is widely used in gene function research in M. nipponense [41][42][43][44][45]. We used RNAi technology to further explore the functionality of Mn-XRN1. On the 1st, 5th, and 13th days after RNAi with Mn-XRN1, the expression of Mn-XRN1 in the ovary of the experimental group decreased significantly compared with the control group. This indicated that Mn-XRN1 dsRNA can effectively inhibit the expression of Mn-XRN1 in this study. Initially, the ovary of M. nipponense in the experimental group and the control group was in the O2 stage. On the 5th, 10th, and 13th days after dsRNA injection, the proportion of ovaries developing to the stage after O2 was greater than that of the control group. The results showed that, after Mn-XRN1 silencing, the ovarian reproduction of M. nipponense was significantly accelerated. Therefore, Mn-XRN1 had an inhibitory effect on ovarian reproduction. In the search for regulatory relationships with other related genes, we found that the gene Mn-VASA was significantly regulated by Mn-XRN1 in the ribosome genesis pathway. After silencing of the Mn-XRN1 gene, the expression of Mn-VASA gene increased significantly, indicating that Mn-VASA was regulated by negative feedback from Mn-XRN1. VASA is thought to be involved in the assembly and transport of vitellin mRNA in follicular cells during secondary yolk genesis [46]. Based on the existing research results, we propose that in M. nipponense, Mn-XRN1 inhibits the production of vitellinogen by inhibiting the expression of the gene Mn-VASA, which is responsible for the production and transport of vitellinogen, thereby controlling development of the ovaries.

Conclusions
In summary, we identified the 5 −3 direction ribonuclease exonuclease gene Mn-XRN1 in M. nipponense. The results of this study indicated that Mn-XRN1 was associated with early physical changes in M. nipponense, such as post-membrane development and metamorphosis. It may also play an important role in gonadal differentiation. In terms of ovarian reproduction, we found that Mn-XRN1 was associated with yolk deposition and follicle production. Further RNAi results showed that Mn-XRN1 inhibited ovarian development. More importantly, we found that the gene Mn-VASA was strongly inhibited by Mn-XRN1. Mn-VASA was associated with the synthesis of vitellinogen. Finally, we proposed a viewpoint that Mn-XRN1 might inhibit the production of vitellinogen by inhibiting the expression of Mn-VASA, thereby controlling ovarian maturation. This study enriched the understanding of molecular mechanism of female sexual maturation during the breeding period of M. nipponense and provided new insights for studying sexual maturity in crustaceans.