To the best of our knowledge, this is the first comprehensive study of the M. americanum male reproductive tract transcriptome. In this study, the transcriptome profile was used to identify genes related to male reproduction. The tree tissues combined generated 1,059,447 unigenes, with the highest ExN50 value at 100, indicating that the assembled transcripts had high coverage (Bryant et al.,2017). Based on Blastx results against Swiss-prot database, the top hits were H. sapiens, D. melanogaster, and M. musculus, which is comparable to the transcriptomic analysis of M. ronsenbergii postlarvae in response to nodavirus infection (Pasookhush et al., 2019), where they mention that those results were expected, since the majority of the database subjects were obtained from well-studied model organisms. Not surprising, when comparing against nr-Macrobrachium sp database, the majority of M. americanum unigenes matched with M. nipponense, for which a genome is available (Jin et al., 2021).
According to GO annotation, about 20% of unigenes were involved in a reproductive process or reproduction within the category of biological processes. The results generated in this study showed an important series of transcripts homologous to known genes involved with sex determination and differentiation, spermatogenesis, spermiogenesis, fertilization, maturation of testicular cells, neuropeptides, hormones, hormone receptors, and/or embryogenesis processes. Most of these transcripts have been identified in crustaceans.
DMX-like and heat shock proteins (HSPs) transcripts, were found in the ovary and testis of two crab species and were upregulated in female organisms, so it is thought that it could be related to sex determination (Zhang et al., 2021).
Sex-lethal (Slx), doublesex and mab-3 related transcription factor (Dmrt), Sox, and Vasa, in several studies exhibit male-biased expression patterns and play crucial roles in testis development and spermatogenesis (Farhadi et al., 2021). It is suggested that the sex-lethal homologs play important roles in the embryonic development and sex differentiation of M. nipponense (Zhang et al., 2013). Several Dmrt genes (MroiDmrt1A MroiDmrt1b, MroDmrt11E MroiDmrt1c, MroiDmrt1d) have been identified in M. rosenbergii. It is hypothesized that these genes are involved in male and female gonad development and male determination by direct or indirect upstream of IAG (Abayed et al., 2019). Furthermore, it has been demonstrated that Dmrta2 might act as an activator of cdkn2c during spermatogenesis in zebrafish and that the dmrt-like gene was specifically expressed in males’ crabs (Xu et al., 2013; Zhang et al., 2021). The Sox (Sry-related high-mobility group (HMG) box) family of transcription factors are involved in diverse developmental processes, including sex determination (Farhadi et al., 2021; Liu et al., 2015). The Sox gene has been identified in several crustacean species, for example, the MnSoxE1 gene expression has been found in M. nipponense in embryonic development, post-larval stages, oocytes, spermatocytes and Sertoli cells, implicating its involvement in tissue cell differentiation and formation, in gonadal differentiation and development and in the maintenance of testis in mature prawns (Hu et al., 2020). In M. rosenbergii, vasa gene was detected in the cytoplasm of spermatogonia and primary spermatocytes and in the nuclei of secondary spermatocytes and sperm (Nakkrasae et al., 2007)
DNA polymerase zeta catalytic subunit (Rev3) and Male reproductive-related LIM protein (MRLIM) expression, have been related with sex determination and differentiation. Rev3 was identified in M. nipponense and it had positive effects on male sexual differentiation and development (Jin et al., 2019). While, MRLIM was up-regulated in three species of male crabs (Zhang et al., 2021), was expressed in testis and ovaries of M. nipponense with a strong expression in the testis and is a male-specific protein in M. rosenbergii (Qiao et al., 2015).
Interestingly, we also found a high expression pattern of ADAM 29 (Disintegrin and metalloproteinase domain-containing protein 29) and expression of ADAM 10 in all the tissues evaluated. The ADAM family plays an important role in the fertilization process, it is involved in sperm and egg adhesion, fusion, sperm quality and transduction of cellular signals related to maturation of testicular cells. Es-ADAM10 and Es-ADAM17 are related to the apoptosis of spermatocytes in E. sinensis crab, mainly in the regulation of sperm quality (Li et al., 2015). ADAM29 may play a certain role in the signal transduction during the maturation of testis-associated cells, it is exclusively expressed in the human testis in different stages of spermatogenesis and interstitial cells (Wang et al., 2001).
E3 ligases have been shown to play a role in spermiogenesis and condensation of the sperm nucleus; the regulation of junctional complexes between cells of the seminiferous epithelium; the process of meiosis in spermatocytes; and the regulation of germ cell apoptosis (Richburg et.al., 2014). E3 ubiquitin-protein ligase siah-1 (SIAH1) and ubiquitin carboxyl-terminal hydrolase FAF-X (USP9X) were found in M. nipponense testes (Quiao et.al.,2012). Alternatively, ubiquitin carboxyl-terminal hydrolases remove ubiquitin from peptides or small C-terminal ubiquitin adducts. Ubiquitin carboxyl-terminal hydrolase L3 (uch-l3) and ubiquitin carboxyl-terminal hydrolase L5 (uch-l5) are highly expressed in testis, ovary, spermatocytes and spermatids of M. nipponense and function in the meiotic differentiation of spermatocytes into spermatids (Quiao et.al.,2012).
Cyclin-dependent kinases (CDKs), cell division control protein 42 and cyclin-CDK inhibitors (CKIs) are involved in the pathway through which the cell cycle is regulate In mammals, CDKs play an essential role in the meiosis of spermatocytes and oocytes and several studies indicate that they have the conservative function in the germ cells meiosis during gametogenesis in crustaceans, so it was not surprising to notice the expression of this gene in the reproductive tract of M. americanum males. In M. rosenbergii, Cdk2 transcript is low in spermatogonia, high in spermatocytes, but reduced in spermatids and sperm, suggesting that cdk2 might play an important role in the regulation of meiotic maturation of spermatocyte and oocyte (Chen et al., 2013). Additionally, we identified high expression of Cell division control protein 42 homolog (Cdc42) in M. americanum reproductive tract. Little is known about the functions in mitosis Cdc42, however, an invitro study in Hela cells suggested that Cdc42 works in metaphase by regulating bi-orient attachment of spindle microtubules to kinetochores (Oceguera-Yanez et al., 2005).
Also, we identified other genes related with spermatogenesis and spermiogenesis (Tables 4 and 5). Male germ cell-associated kinase (Serine/threonine-protein kinase MAK) expression in M. americanum is consistent with other studies in mice, where this gene was found in testis and is supposed to be involved in the phase around meiotic cell division. (Koji et al., 1992). ATP-dependent RNA helicase spindle-E and Cytoplasmic polyadenylation element-binding protein 2 (CPEB2) is needed for spermatogenesis promotion in Drosophila (Kotov et al., 2014). MAPKs are important in spermatogonia stem cell renewal, germ cell cycle, meiosis, spermiogenesis, and spermiation (Li et al., 2009).
Genes related to chromatin remodeling were identified. Lysine-specific demethylase 3A and chromodomain-helicase-DNA binding proteins (CHD5 and CHD7) have been found in the testes of Eriocheri sinensis (Li and Qian et al., 2017). The former is a protease related to modification of histones, and regulates the expression of target genes, which are required for packaging and condensation of sperm chromatin in mice (Wilson et al., 2017). The latter is a protein that regulates the histone-to-protamine chromatin remodeling process, its deficiency causes defective sperm chromatin compaction and male infertility in mice (Li et al., 2014)
In this research, we found KIFC that in testes of M.nipponense, works together with lamellar complex and acroframosome to drive acrosome formation and cellular transformation (Wang et al., 2012). We also identified insulin-like androgenic gland hormone (IAG), which has been detected in androgenic gland and terminal ampulla of M. rosenbergii, (Piyaviriyakul and Darawiroj, 2014). IAG has a key role in male sexual differentiation and is a viable target for biotechnologies based on sexual manipulation. For example, in Macrobrachium rosenbergii, the silencing of Mr-IAG prior to the appearance of male sexual characteristics caused the full and functional sex reversal of males into neo-females. Successfully mated with untreated males, the neo-females produced all-male progenies (Jiang et al., 2020). The researchers found that silencing of Mr-IAG led to the cessation of testicular spermatogenesis and spermatogenic cell development in the terminal ampulla, accompanied by hypertrophy and hyperplasia of the androgenic gland (AG) (Yang et al., 2020). There are several factors that are involved in IAG signaling such as insulin-like androgenic gland hormone-binding protein (IAGBP), insulin-like growth factors (IGFs) and insulin-like receptor (IR), which were distinguished in the present study. The former was also expressed in the heart, testis, eyestalk, nerve cord, muscle, androgenic gland, hepatopancreas, and brain of M. nipponense and the injection of IAGBP double-stranded RNA (dsRNA) significantly reduced the transcription of IAG in M. nipponense and M. rosenbergii (Li et al., 2015; Yang et al., 2020). Insulin-like growth factors (IGFs) belong to the insulin-like peptide superfamily. In vertebrates, IGFs are bound in biological fluids to the IGF-binding proteins (IGFBPs), IGFBPs carry IGFs to release at the target site. In vitro, it has been shown that IGFs stimulate DNA synthesis of trout male germ cells by interacting directly with these cells through one IGF receptor. (Li et al., 2015; Loir and Gac et al., 1994). IR, was detected in the androgenic gland cells, spermatocytes and terminal ampullae of M. rosenbergii, and IR knockdown, induced sex reversal of this prawn (Tan et al., 2020)
We also found farnesoic acid O-methyltransferase (FAMeT) in all the tissues. FAMet is the enzyme that catalyzes methylation of farnesoic acid (FA) to produce methyl farnesoate (MF). The mechanism of MF induced testicular or ovarian maturation is found through ecdysteroid synthesis in Y-organs (Chandra-Nagaraju, 2011). The injection of MF in M. malcholumsonii caused and increase in the testicular index (Chandra-Nagaraiu, 2011). Meanwhile, the knockdown of MrFAMet in the muscle of M. rosenbergii males and females, downregulated the expression of ecdysone receptor gene (MrEcR) and silence of MrEcR decresed the expression of MrFAMeT as well (Qian and Liu et al., 2019). Furthermore, MF acts as a ligand for retinoid-X-receptor (RXR) and synergize with ecdysteroids to stimulate RXR-EcR heterodimer complex for expression of combined regulatory genes of these two hormones (Chandra-Nagaraiu, 2011).
Moreover, we found several neuromodulators receptors like: serotonin, tyramine/octopamine, dopamine receptors. In invertebrates, tyramine (TA) is the synthetic precursor of Octopamine (OA) and Dopamine (DA) and is involved in several processes independent from OA and DA including muscle contraction and excretion. In reproduction, OA is related with the modification of aggressive behavior and gonadal development in crustacean. (Reyes-Colón et al., 2010). On the other hand, DA has inhibitory effects on the androgenic gland (AG) cell proliferation and IAG production in M. rosenbergii, while serotonin has stimulatory effects on the testicular development, AG cell proliferation, IAG production, spermatogenesis and Gonadotropin-releasing hormone (Siangcham et al., 2013).
In this study, we found that FMRF-Amide neuropeptide presents a high expression pattern in the terminal ampulla. In M. nipponense females, FMRF amide receptor have been identified in the central nervous system, eyestalk and ovaries (Suwansa-ard et al., 2015). Meanwhile, in Octopus vulgaris FMRFamide-like protein was found in the epithelium of the seminal vesicle in the male and around the alveoli of the outer glandular component of the oviducal gland in the female, so they suggested that this peptide can modify the activity of the reproductive tract by affecting gamete transport (seminal vesicle contraction is essential for the transport of gametes) or by regulating secretory products like mucus and mucilaginous substance (di Cristo et al., 2002). So further investigations of the molecular mechanism of FMRF amide-like protein in terminal ampulla need to be undertaken for practical application in M. americanum reproduction.
Additionally, we distinguish the expression of ecdysone receptor (EcR) and Retinoid X receptor (RXR) in the male reproductive tract. In M. nipponense, it was shown that EcR variants have sex-specific expression patterns, where MnEcR-S1 and -S2 predominate in testes, while MnEcR-L1 and -L2 are prevail in the ovary, suggesting different roles for isoforms of MnEcR in male and female organisms (Shen et al., 2013). Exposure to 20-hydroxyecdysone induced spermatogenesis in the lobster and ecdysteroids have been shown to enhance DNA synthesis in the testes of M. rosenbergii (Chandra-Nagaraiu, 2011
In vertebrates, the gonadotropins (GTHs) follicle-stimulating hormone (FSH) and the luteinizing hormone (LH) play an important role in spermatogenesis and oogenesis. The function of FSH and LH is known relatively well in mammals, but is nearly unknown in crustaceans. Here we identify FSH and gonadotropin-releasing hormone receptors expression in the reproductive tract of M. americanum. FSH was also perceived in other crustaceans like P. trituberculatus and Marsupenaeus japonicus, where it is correlated with the developmental stage of ovaries and ovarian maturation (Huang et al., 2008; Haihui et al., 2011). Gonadotropin-releasing hormone increases the size of androgen gland and testes, induces insulin-like androgen gland hormone production and spermatogenesis in M. rosenbergii (Siangcham et al., 2013)
In this study, we found several protease inhibitors (Table 8). Protease inhibitors like serine-type protease inhibitors or serpins, modulate the function of proteases, and have a role in sperm maturation of crustaceans, protecting immature sperm from premature capacitation and/or acrosome reaction before they reach female thelycum. (Chotwiwatthanakun et al., 2018). Male reproduction-related kazal-type peptidase inhibitor (MRPINK) was expressed in testis, vas deferens, and terminal ampullae of M. rosenbergii and has an inhibitory effect on the gelatinolytic activity of M. rosenbergii and on the proteolytic activity of sperm extracts to vitelline coat components. (Kao et al., 2007, Li et al., 2008). Furthermore, other study revelas that two male reproductive tract-derived Kazal-type protease inhibitors (KPIs) can completely inhibit the gelatinolytic activities of sperm proteases from their own species (M. nipponense and the Chinese mitten crab Eriocheir sinensis) (Qian et al., 2012). Otherwise, we identified serine proteases. In marine animals, serine proteases are important for fertilization as they play key roles in sperm maturation and sperm-egg interactions. For example, Penaeus monodon sperm require trypsin-like activity for a complete acrosome reaction, which occurs in sperm residing in the female thelycum (Chotwiwatthanakun et al., 2018).
Also, we detected the expression of Male reproductive-related serum amyloid A and male reproductive-related protein A. Jiang et al, (2019) found them in the testes of M. rosenbergii and reveal that the former was a male-biased gene and the last was a testis-specific gene. So, these genes could be considered as promising sex-related candidates (Jiang et al., 2019).
In this study we found PL10A in testes and vas deferens. It was suggested that in M. nipponense, PL10 might be involved in the early differentiation/development of an oocyte (Qiu et al., 2013).
Seminal vesicle secretory protein 2 (SVS2), Cathepsin L and Angiotensin-converting enzyme (ACE) were identified in the reproductive tract of M. americanum. SVS2 was firstly found in mice and is associated with semen-coagulating protein and sperm decapacitation factor. (Chang et al., 2010). Cathepsin L has been reported to control development of the ovary in L. vannamei and M. nsis (Qiao et al., 2012) and is involved in transforming immature spermatozoa in to mature and motile sperm in the epididymis of the rat Kathryn and Bernard, 2001). Angiotensin-converting enzyme (ACE) was demonstrated to be an important protease among the peptide-degrading enzymes present in the female spermatophore and it is hypothesized that ACE could provide dipeptides or amino acids that are necessary for different metabolic pathways in the crayfish A. leptodactylus (Simunic et al., 2009).
Also, we distinguished female sterile homeotic protein (fsh) and innexin inx 3 gene expression in vas deferens and testes respectively. In M. nipponense, it was demonstrated that fish is related to histodifferentiation and organogenesis during the embryonic developmental process. While inex 3, that was described in D. melanogaster, has a role in embryogenesis (Zhang et al., 2015)
KEGG analysis revealed that Necroptosis, NOD-like receptor signaling pathway and apoptosis were the most abundant metabolic pathways in the reproductive tract of M. americanum. Necroptosis is a form of regulated necrotic cell death. In a study, it was detected specifically in spermatogonia stem cells in the testes of old mice and proven to promote aging-associated deterioration of the male reproductive system. Interesting, suppressing necroptosis seems to be an effective way to delay the age-related changes in reproductive organ of mice (Xie et al., 2020). NOD-like receptors (NLRs) are recognized as pattern recognition receptors (PRRs) during innate immune regulation. However, it was reported that Nlrp14 and NLR are specifically expressed in testis and ovary of mice. In the male mice, Nlrp14 knockout strongly compromised the differentiation of spermatogonial stem cells and meiosis (Yin et al., 2020). Further studies should be done in M. americanum to know if necroptosis is involved in the deterioration of male reproductive system and to understand the function of NLRs in male spermatogenesis. On the other hand, apoptosis occurs at a high rate in the testes, this process of regulated cell death serves several important functions in the testis, such as: maintaining appropriate germ cell to Sertoli cells ratio, removing defective germ cells and maintenance of overall quality control in sperm production (Shukla et. Al., 2012).