MOLECULAR BASIS OF SEX DIFFERENTIATION

It is clear from the foregoing discussion that AGH controls the sex differentiation process in the malacostracan crustaceans. However, molecular studies using genetic markers to trace the germline differentiation of the gonad in crustaceans are limited. Genetic information is transmitted from generation to generation by highly specified germline cells that are segregated as primordial germ cells, from somatic lineages during embryogenesis. Traditional cytological observations have failed to identify immature germ cells as they are morphologically indistinguishable from somatic cells.

In vertebrates, several genes have been identified during the course of gonadal differentiation. Recent gene expression studies relating to sex differentiation in crustaceans include mainly vasa gene. Vasa gene is a member of the DEAD-box gene family and has a restricted expression exclusively to the germ cell lineage in most animals (Mich et al., 2009). Zygotic expression of vasa mRNA is specific to germ cell lineage throughout the development of the embryo and the adult gonad, without any expression in the somatic tissue (Li et al., 2010).

In Crustacea, the vasa gene expression was investigated in Daphnia magna (Cladocera), Parhyale hawaiensis (Amphipoda), and L. vannamei, Fenne- ropenaeus chinensis, M. japonicus (Decapoda) (Sagawa et al.,, 2005; Extavour, 2005; Ozhan-Kizil et al., 2009; Aflalo et al., 2007; Sellars et al., 2007). In M. japonicus, Mjpl10 vasa gene expression was observable from the 2-cell embryonic stage onward (30 min postspawning), suggesting that it is maternally expressed. Mjpl10 expressed continually at low levels throughout the embryo- genesis. Mjpl10 expression increases significantly in the first 25 h after hatching (nauplii IV) and decreases gradually over the next 52-day period. The transcript levels of Mjpl10 are also detected in the ovary and testes of mature adults. These results suggested that vasa gene control germ cell migration toward somatic gonad tissue sometime between larval stages nauplii I and IV (Sellars et al., 2007). This is in agreement with the previous histological studies on M. japoni- cus and L. vannamei that sex differentiation occurred in these shrimps early during postlarval development. (Nakamura et al., 1992; Campos-Romos et al., 2006; Garza-Torres et al., 2009; Zhao et al., 2009). In the Pacific white shrimp,

L. vannamei, Lv-vasa gene is specifically expressed in male and female gonads. In the adult females, Lv-vasa gene is expressed during late oogenesis, suggesting a possible maternal contribution to the embryo (Aflalo et al., 2007). In the Chinese shrimp, F. chinensis, vasa gene expression occurs during the migration of primordial germ cells to the genital ridge (Feng et al., 2011). Fc-vasa-like mRNA was first localized in the cytoplasm of oocytes before and after spawning, and in the embryo, it was uniformly distributed in the early embryos from the 2-cell embryo to the blastula, suggesting that vasa mRNA was of maternal origin. Then, the expression of the Fc-vasa gene centralizes gradually and is restricted to the 5-cell clusters as precursor cells of primordial germ cells at the limb bud stage. These 5-cell clusters are located at the base of the antennules and antenna, as well as the cephalic lobe. Later on, these cells migrate to a dorsolateral position in the naupliar and zoel stages and gradually enter the genital ridge at the mysis 1 stage. The histological appearance of gonads in the PL1 is common to F. chinensis, L. vannamei, and Penaeus japonicus. Furthermore,

M. rosenbergii vasa gene (Mrvlg) transcripts were detected in the cytoplasm of oogonia, previtellogenic, and vitellogenic oocytes and were also detected in the nucleoplasm of mature oocytes. In the testis, the Mrvlg transcript was detected in the cytoplasm of spermatogonia and primary spermatocytes but was detected in the nuclei of secondary spermatocytes and sperm (Nakkrasae and Damrongphol, 2007). In D. magna, vasa protein was first visualized in one of the blastomeres at the 8-cell stage; as cleavage progresses, the number of vasa- positive blastomeres increased. These blastomeres divided further and moved toward the site of future gonad (Sagawa et al., 2005). Similar expression of vasa gene was reported in P. hawaiensis (Ozhan-Kizil et al., 2009) and its expression is schematically plotted in Fig. 2.7. Vasa-like gene expression in all the above penaeid shrimps, the cladoceran, D. magna and the amphipod, P. hawaiensis, as well as several insect species point to the possible phylogenetic linkage between insect and crustacean (Table 2.3)

Among the brachyuran crabs, vasa gene was characterized from the Chinese mitten crab Eriocheir sinensis, in which, the mRNA expression was specific to the gonad and its temporal expression in the ovary and testes were significantly different in various developmental periods. Especially, Es-vasa mRNA transcripts were at the highest levels during periods of rapid development in the gonads (stage III-2 in ovaries and spermatocyte stage in testes) and gradually decreased as the gonads matured (Wang et al., 2012). Similarly, in the green mud crab Scylla paramamosain, the Sp-vasa expression was higher in the growth stage of ovary than in the maturation stage, and in stages I and II of testis, the expression level of SP-vasa was higher than in stage III (Wang

Model of RNA localization and protein expression in the germ cells of P

FIGURE 2.7 Model of RNA localization and protein expression in the germ cells of P. hawaiensis. Schematic drawings of Parhyale embryos at the 8-cell stage, the 64-cell stage, and the germ band stage. At the 8-cell stage, maternal Ph-vasa RNA are ubiquitous. At the 64-cell stage, maternal RNA becomes localized to the germ cells by its differential stabilization in that lineage. At this time, zygotic transcription starts to contribute to RNA levels. At the germ band stage, RNA of Ph-vasa is detected in the germ cell cluster together with zygotic Vasa protein. Reproduced from Ozhan-Kizil, G., Havemann, J., Gerberding, M., 2009. Germ cells in the crustacean Parhyale hawaiensis depend on Vasa protein for their maintenance but not for their formation.Dev. Biol. 327, 230-239, Elsevier, Fig. 7, 237.

TABLE 2.3 Full-Length Vasa Ortholog cDNAs

Group

Common

Name

Gene

Name

Polypeptide

Length

Gene Bank Accession

Parhyale

hawaiensis

Amphipod

crustacean

Vasa

676

ABX76969

Daphnia magna

Water flea

DmaVas

779

BAE00180

Litopenaeus

vannamei

Pacific white shrimp

Vasa-like

protein

703

AAY89069

Fenneropenaeus

chinensis

Chinese white shrimp

Vasa

712

ABQ00071

Macrobrachium

rosenbergii

Giant

freshwater

prawn

Mrvlg

710

ABC872 71

Moina

macrocop

Water flea

MmVas

843

BAD99524

Artemia

franciscana

Brine shrimp

AfVas

726

BAD99523

Palaemon

carinicauda

Ridgetail

prawn

Vasa

600

AGF90963

Palaemon

modestus

Siberian

prawn

Vasa

602

AID54678.1

et al., 2012). To sum up, vasa gene expression location during the progression of embryogenesis in several crustaceans has shown the utility of this gene as a marker gene for germ cell specification and to the accurate tracking of PGCs during gonadogenesis.

 
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