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Distribution and migration of immature germ cells in the pearl oyster Pinctada fucata with the expression pattern of the vasa ortholog by in situ hybridization.

ABSTRACT The vasa orthologs have been used as a specific germ line molecular marker in many animal species. In this study, the expression of the vasa ortholog (povlg1) in adult and juvenile pearl oysters (Pinctada fucata) was observed by in situ hybridization. The in situ hybridization with pov1gl made it possible to detect the immature germ cells, which could not be detected by hematoxylin and eosin staining. During the reproductive season (May to July), spermatogonia, spermatocytes, oogonia, and oocytes showed povlgl expression in the gonads of mature adult pearl oysters. By contrast, in spent pearl oysters in the nonreproductive season (September and October), only small ovoid (8.9 X 5.2 [micro]m) povlg1-positive cells were observed in the base of acini. In juvenile (1 mo old) pearl oysters, a clump of germ cells first formed from several cells that were distributed symmetrically and lateral to the visceral mass. These cells then migrated to the ventrolateral periphery of the visceral mass in 2-mo-old oysters. The cells then migrated posteriorly along the periphery of the visceral mass with increasing cell numbers and size in 4-mo-old oysters. This observation of immature germ cell distribution and migration provides useful information for the control of gametogenesis and about pearl quality.

KEY WORDS: pearl oyster, Pinctada fucata, vasa, polvg1, germ cell, bivalve, mollusc

INTRODUCTION

The pearl oyster (Pinctada fucata) is the most important bivalve mollusc for pearl production in Japan. In pearl culture, before insertion of a shell bead and mantle allograft, it is necessary to eliminate the germ cells from the gonad by spawning induction and/or by suppression of gonad development during the prespawning season (Wada 1991). Wada (1999) suggested that inclusion of germ cells between the inserted mantle allograft, the shell bead, and agranular hemocyte layer induced abnormal secretion of dark organic materials onto the shell beads.

To produce high-quality pearls, the control of gametogenesis is required. To control gametogenesis in the pearl oyster, it is necessary to understand germ line formation. There are, however, few reports about the formation of the germ line in the pearl oyster and other bivalves. In a study of the blue mussel (Mytilus galloprovincialis), proliferation and migration of immature germ cells was observed by in situ hybridization (ISH) using the vasa ortholog (Myvlg) (Obata et al. 2010). The ISH with Myvlg made it possible to recognize immature germ cells. In blue mussels, the immature germ cells could not be distinguished from somatic cells, such as hemocytes and connective tissue, in sections stained with hematoxylin and eosin (H&E). Because immature germ cells also could not be detected by the H&E stain in the pearl oyster, detection of them by ISH with the vasa ortholog was attempted to determine their distribution in this oyster.

The vasa orthologs have been used as a specific germ line molecular marker in many animal species. The vasa was first identified in Drosophila as a maternally supplied factor required for germ line development (Hay et al. 1988). The vasa encodes a protein member of the DEAD-box family. It is suggested that vasa acts as a translational regulator in the oocytes (Saffman & Lasko 1999). The expression pattern is restricted to the germ line cells. A vasa ortholog has been isolated and used as a molecular marker to detect primordial germ cells (PGC) and other germ line cells from many animals, e.g., in Caenorhabditis elegans (Roussel & Bennett 1993), Xenopus laevis (Komiya et al. 1994), and Danio rerio (Olsen et al. 1997). In bivalves, vasa orthologs have been isolated in oysters (Crassostrea gigas) to observe distribution of PGC (Fabioux et al. 2004), in the Japanese spiny oyster (Saccostrea kegaki) as a gene marker in early development (Kakoi et al. 2008), and in the blue mussel (Mytilus galloprovincialis) (Obata et al. 2010).

Matsumoto et al. (2013) reported that the vasa ortholog was detected in Pinctada fucata gene models (http://marinegenomics. oist.jp/pinctada_fucata), which is a draft genome released by Takeuchi et al. (2012). Miura et al. (2013) reported three vasa ortholog sequences--povlg1, povlgl, and povlg4--and induced sterilization by using an RNAi approach. There are. however, no reports about immature germ cell proliferation and migration. In Miura et al. (2013), povlg1 showed the highest transcript level among three vasa orthologs in the immature ovary and mature testis. Therefore, the povlg1 sequence was used for the detection of immature germ cells by ISH in the pearl oyster.

In this study, the povlg1 expression pattern in adult pearl oysters during the reproductive and nonreproductive seasons, and the proliferation and migration of germ cells in juvenile pearl oysters by ISH were observed.

MATERIALS AND METHODS

The Pearl Oysters

The pearl oysters were produced in the hatchery at the Mie Prefecture Fish Farming Center and cultured in Ago Bay in 2010.

Section Preparation for ISH

Five adult pearl oysters (22-28 mo old) were collected from May to July (reproductive season) and 15 adult pearl oysters from September to October (nonreproductive season). The soft parts of the adult and juvenile pearl oysters with shells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS).

Five 1-mo-old, five 2-mo-old, and fifteen 4-mo-old pearl oysters were collected. The shell height ranges of the 1-, 2- and 4-mo-old oysters were 2-5, 8-15, and 28-36 mm, respectively. The juvenile pearl oysters with shells were fixed with 4% paraformaldehyde in PBS. After fixation, the shell was removed with tweezers.

Fixed samples were dehydrated in an ethanol series, embedded in paraffin wax and sectioned at six micrometers thickness. Transverse serial sections of whole juvenile pearl oysters were prepared. Adjacent sections of 4-mo-old samples were stained with H&E to observe the vasa ortholog-positive cells.

The In Situ Hybridization

Complementary DNA (cDNA)-coded open reading frame of povlg 1 (accession No. AB695267) was synthesized from the total RNA of mature male and female gonads with the primer sets, forward: 5'-TCGACATGGGATTTGGTCCAG- 3', reverse: 5'-TCCCTTTGAGATGTTCCCTTC-3', using the First strand cDNA synthesis Kit ReverTra Ace-a- (Toyobo, Osaka. Japan). These PCR products were cloned into pGEM-T Easy Vector (Promega) and labeled with digoxigenin using a DIG RNA labeling kit with T7 RNA polymerase (Roche. Indianapolis, IN).

The ISH was performed according to the method described by Wilkinson and Nieto (1993) with the following modification. Proteinase K treatment (1 [micro]g/ml) was carried out for 15 min at 37[degrees]C. Hybridization was performed at 65[degrees]C overnight with the probes (1 [micro]g/ml) in hybridization buffer. Blocking was performed with Blocking Reagent (Roche) before the antibody reaction. A mixture of 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium was used for color development of the antiDigoxigenin-AP Fab fragments (Roche). After ISH, each sample was counterstained with Nuclear Fast Red (VECTOR. Burlingame, CA) and observed under a Nikon E600 (Nikon, Tokyo, Japan) light microscope. The number of povlg1-positive cells was counted in transverse serial sections of whole juvenile pearl oysters.

The size of the long and short axes of the povlg1-positive cells was collected from 20 cells in each individual from the nonreproductive season and the 1-, 2-, and 4-mo-old juvenile pearl oysters.

RESULTS

Germ Cell-Specific Expression of povlg1 in Adult Pearl Oysters by ISH

In the March to July samples (reproductive season, n = 5), gametogenesis in the connective tissue of the visceral mass of mature adult pearl oysters was observed. There was povlg1 signal in the sections of male and female gonads stained with the antisense probe (Fig. IA, B. D, E).

In the male gonad, spermatogonia, spermatocytes, spermatids, and sperm were located in series from the basal membrane of the acinus to its center (Fig. 1A). Spermatogonia that were attached to the basal membrane of the acinus were larger than the spermatocytes. Spermatids and spermatozoa (which had condensed nuclei) were smaller than spermatocytes. Povlg1-expression was restricted to spermatogonia and spermatocytes. Spermatogonia had a stronger signal than spermatocytes. The povlg1 signal was not detected in spermatids and sperm (Fig. IA. B).

In the female gonad, flattened oogonia were gathered adjacent to each other along the acinus wall (Fig. ID). Early oocytes, each with an obvious nucleus and nucleolus, were also located along the acinus wall. Mature oocytes, larger than oogonia and oocytes, were partly attached to the acinus wall. The povlgl signal was stronger in oogonia and early oocytes than the mature oocytes (Fig. ID, E). There was no povlg1 signal in the sections of male and female gonads stained with the sense probe (Fig. 1C, F).

In September and October samples (nonreproductive season. n = 15), gametes were almost spawned and a few sperm or degenerated oocytes remained in the acini of 10 of the 15 individuals. In these samples, degenerate oocytes had the same level of povlg1 signal as in the reproductive season (Fig. 2A). The other five individuals were spent, and there were no cells that had the characteristic of germ cells in the acini; however, small ovoid povlg1-positive cells were observed along the base of the acini in these individuals (Fig. 2B). The size of the long and short axes of the povlg1 -positive cells was 8.9 [+ or -] 1.5 [micro]m and 5.2 [+ or -] 1.1 |tm, respectively.

There was no signal in hemocytes and connective tissue of the gonads (Figs. 1 and 2).

Detection of Povlg1-Positive Cells in Juvenile Pearl Oysters

The dotted line in Figure 3A indicates the position of the histological sections shown in Figure 3B and C. The povlg1-positive cells were observed symmetrically on the sections at the point of connection of both pedal retractor muscles in the shells of 1-mo-old oysters (2-5 mm shell height, n = 5). These cells formed two clusters, which were in contact with the visceral mass (Fig. 3B, C). Figure 3C is a higher magnification of the positive cells. The number of povlg1-positive cells was 16. 8 [+ or -] 3.4 (n = 5). The size of the long and short axes of the positive cells was 8.1 [+ or -] 1.3 [micro]m and 5.2 [+ or -] 1.1 [micro]m, respectively.

In 2-mo-old oysters (8-15 mm shell height, n = 5), povlg1-positive cells were observed at the ventrolateral periphery of the visceral mass of the section at the same cutting plane point (dotted line I in Fig. 4A) as in 1-mo-old pearl oysters (dotted line in Fig. 3A) (Fig. 4B, C). The positive cells were not observed in the same position as in the 1-mo-old samples. The povlg1-positive cells were also observed in the sections in the region of dotted line II in Figure 4A. There were no positive cells in this plane in 1-mo-old samples. These cells were observed on both sides of the point of connection of the pedal retractor muscle and foot (Fig. 4D. E). The number of povlg 1positive cells was 156.0 [+ or -] 37.3. The size of the long and short axes of the positive cells was 8.1 [+ or -] 1.3 |0.m and 5.3 [+ or -] 0.9 [micro]m, respectively.

In 4-mo-old pearl oysters (28-36 mm shell height, n = 15), the povlg1-positive cells were observed on the sections at the same location as in 2-mo-old pearl oysters. In this location, the acini were formed in five of 15 individuals. The povlg1-positive cells were also observed on the sections at the point of separation of the pedal retractor muscles (Fig. 5B, C). The dotted line in Figure 5A indicated this point. The number of povlg1 -positive cells in a pearl oyster was 2849.5 (n = 2). The size of the long and short axes of the positive cells was 8.5 [+ or -] 1.3 pm and 5.5 [+ or -] 0.9 pm in individuals that did not have acini. In the individuals with acini, the povlg1 -positive cells in the acini had a nucleus weakly and unclearly stained with hematoxylin in H&E-stained adjacent sections (Fig. 5E, D). The size of the long and short axes of the positive cells was 12.8 [+ or -] 2.1 (im and 6.5 [+ or -] 1.1 pm in individuals that had acini.

DISCUSSION

The ISH with povlgl probes revealed that povlg1 was specifically expressed in gametes. No expression was detected in somatic tissue as described in Miura et al. (2013). In this study of pearl oyster, povlg1 expression decreased as spermatogenesis and oogenesis progressed (Fig. 1), a finding similar to that in the blue mussel (Obata et al. 2010). Immature germ cells could be detected by ISH with povlg1 in nonreproductive season adult and juvenile pearl oysters.

The povlg1-positive cells were observed as small ovoid cells in the base of the acini of adult pearl oysters in the nonreproductive season (Fig. 2B). Choi and Chang (2003) reported that the primary oogonia and spermatogonia of the pearl oyster were 2-5 and 3 [micro]m in diameter, respectively. The size of the povlg1-positive cells observed in the nonreproductive season was 8.9 [+ or -] 1.5 pm and 5.2 [+ or -] 1.1 [micro]m (Fig. 2B), which was bigger than oogonia and spermatogonia. From the size of these cells, it was suggested that these povlgl-positive cells are different stages of the germ line from spermatogonia or oogonia.

The povlgl1-positive cells in juvenile pearl oysters were almost the same size as the positive cells observed in the nonreproductive season (~8 X 5 [micro]m). These povlg1-positive cells in juvenile pearl oysters had similar characteristics to somatic cells and hemocytes in connective tissue when stained with H&E. Therefore, these immature germ cells could be distinguished from somatic cells by ISH only. Because the povlg1-positive cells in the acini of 4-mo-old juvenile pearl oysters had a nucleus that was weakly and unclearly stained with hematoxylin (Fig. 5E), and a bigger size (~13 X 7 [micro]m), these cells were distinguished from other somatic cells.

The number of the povlg1-positive cells in juveniles increased with age, 16.5 in 1-mo-old, 156.0 in 2-mo-old, and 2849.5 in 4-mo-old oysters. Their sizes increased in 4-mo-old oysters, especially in those with acini. It is suggested that the povlg1-positive cells increase in number and size in 4-mo-old oysters for maturity to the next stage of gametogenesis. This observation can be applied to the behavior of PGC in mice described by Lin (1997) as follows. Primordial germ cells migrate to the urogenital ridge accompanied by proliferation. By 11.5 days postcoitum, they reach the gonadal primordium in the genital ridges where they continue to proliferate. Meanwhile, they undergo substantial growth in size. These gametogenic cells were characterized as germ line stem cells (GSC). Germ line stem cells are the self-renewing population of germ cells that serve as the source for gametogenesis. In most animals, GSC are established during preadult gonadogenesis following the proliferation and migration of embryonic PGC (Lin 1997). Therefore, it is suggested that the povlg1-positive cells in 1- to 4-mo-old juvenile pearl oysters were GSC.

The process of migration of immature germ cells of 1- to 4-mo-old juvenile pearl oysters is summarized in a schematic drawing from observations of povlg1-positive cells (Fig. 6). First, the clumps of germ cells formed from several cells are distributed symmetrically and lateral to the paired visceral mass of 1-mo-old oysters (Fig. 6A, C). Next, these cells migrate to the ventrolateral periphery of the visceral mass in 2-mo-old specimens (Fig. 6B, C). Then, they migrate posteriorly along the periphery of the visceral mass with increasing cell numbers in 4-mo-old oysters (Fig. 6B, D). In immature blue mussels, the immature germ cells are distributed symmetrically and lateral to a pair of nephric tubules (Obata et al. 2010). The first distribution of the immature germ cells was almost similar in this study; however, the migration pattern was different between the blue mussels and the pearl oyster. In the pearl oyster, the germ cells migrate ventrally along the periphery of the visceral mass, whereas they migrate anteriorly along the mantle basement in the blue mussel. This difference is explained by the difference in position of the gonad in the two species. The gonad is formed in connective tissue of the visceral mass in the pearl oyster. In the blue mussel, the gonad forms in the connective tissue of the visceral mass and mantle.

In pearl production, because of the inclusion of germ cells between the inserted mantle allograft, the shell bead and agranular hemocyte layer induce abnormal pearl formation. Therefore, control of gametogenesis is required to produce high-quality pearls. The ISH with povlg1 made it possible to detect the immature germ cells that could not be detected by H&E staining. The observations of immature germ cells in this study are very useful for research to control gametogenesis in pearl oysters.

ACKNOWLEDGMENT

We thank the Mie Prefecture Fish Farming Center for providing pearl oysters.

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NATSUMI SANO, * SHUNSUKE KIMATA, MAYU OBATA AND AKIRA KOMARU

Graduate School of Bioresources, Mie University, 1577 Kurimamachiya, Tsu, Mie 514-8507, Japan

* Corresponding author. E-mail: natsumi5@bio.mie-u.ac.jp

DOI: 10.2983/035.034.0309
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Author:Sano, Natsumi; Kimata, Shunsuke; Obata, Mayu; Komaru, Akira
Publication:Journal of Shellfish Research
Article Type:Report
Geographic Code:9JAPA
Date:Dec 1, 2015
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