Biol Reprod Email Content Delivery
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print September 26, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.064667
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
78/1/151    most recent
biolreprod.107.064667v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yano, A.
Right arrow Articles by Yoshizaki, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yano, A.
Right arrow Articles by Yoshizaki, G.
Agricola
Right arrow Articles by Yano, A.
Right arrow Articles by Yoshizaki, G.
BIOLOGY OF REPRODUCTION 78, 151–158 (2008)
DOI: 10.1095/biolreprod.107.064667
© 2008 by the Society for the Study of Reproduction, Inc.

Testis; 

Flow-Cytometric Isolation of Testicular Germ Cells from Rainbow Trout (Oncorhynchus mykiss) Carrying the Green Fluorescent Protein Gene Driven by Trout vasa Regulatory Regions

Ayaka Yano 2, Kensuke Suzuki 2, and Goro Yoshizaki 1 2 3

Department of Marine Biosciences,2 Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan Solution Oriented Research for Science and Technology (SORST),3 Japan Science and Technology Agency, Saitama 332-0012, Japan

ABSTRACT

There is a need to isolate different populations of spermatogenic cells to investigate the molecular events that occur during spermatogenesis. Here we developed a new method to identify and purify testicular germ cells from rainbow trout (Oncorhynchus mykiss) carrying the green fluorescent protein gene driven by trout vasa regulatory regions (pvasa-GFP) at various stages of spermatogenesis. Rainbow trout piwi-like (rtili), rainbow trout scp3 (rt-scp3), and rainbow trout shippo1 (rt-shippo1) were identified as molecular markers for spermatogonia, spermatocytes, and spermatids, respectively. The testicular cells were separated into five fractions (A–E) by flow cytometry (FCM) according to their GFP intensities. Based on the molecular markers, fractions A and B were found to contain spermatogonia, while fractions C and D contained spermatocytes, and fraction E contained spermatids. We also classified the spermatogonia into type A, which contained spermatogonial stem cells (SSCs), and type B, which did not. As none of the molecular markers tested could distinguish between the two types of spermatogonia, we subjected them to a transplantation assay. The results indicated that cells with strong GFP fluorescence (fraction A) colonized the recipient gonads, while cells with weaker GFP fluorescence (fraction B) did not. As only SSCs could colonize the recipient gonads, this indicated that fraction A and fraction B contained mainly type A and type B spermatogonia, respectively. These findings confirmed that our system could identify and isolate various populations of testicular cells from rainbow trout using a combination of GFP-dependent FCM and a transplantation assay.

flow cytometry, GFP, spermatogenesis, spermatogonial transplantation, vasa

INTRODUCTION

Spermatogenesis involves the mitotic proliferation of spermatogonia, followed by the meiotic division of spermatocytes and spermiogenesis, in which the spermatids develop into spermatozoa. During this process, numerous hormones and gene products regulate the proliferation, differentiation, and survival of testicular cells. In teleost fish, spermatogenesis occurs synchronously within cysts, and most species have an annual reproductive cycle [1–3]. Studying the regulatory mechanism of spermatogenesis in fish testes can therefore provide unique opportunities to improve our understanding of the process. However, investigations of fish spermatogenesis have been performed mainly from the point of view of morphology and endocrinology [1–6], and the underlying molecular mechanisms have remained unclear.

Purifying different types of testicular germ cells would aid the study of the molecular events that occur during spermatogenesis. In fish, cell separation is generally performed by density-gradient centrifugation [7–9]; however, it is difficult to obtain highly pure stage-specific cells without contamination using this approach. The panning technique has also been used to separate adhesive and floating cells in fish [10–12]; this approach is useful for collecting Sertoli cells and spermatogonia from cell cultures but is not suitable for isolating different types of testicular germ cells. Although these conventional methods are simple and convenient to use, they cannot yield highly pure populations of testicular germ cells. Hence, there is a need to develop a novel method for isolating stage-specific germ cells in order to study the molecular events that occur during spermatogenesis.

Recently, pure primordial germ cells (PGCs) were isolated by green fluorescent protein (GFP)-dependent flow cytometry (FCM) from the genital ridges of transgenic rainbow trout (Oncorhynchus mykiss) carrying the GFP gene driven by trout vasa-gene regulatory sequences (pvasa-GFP) [13]. The vasa gene in fish was shown to be expressed in PGCs and cells undergoing gametogenesis, which decrease their expression level during spermatogenesis [14–19]. FCM measures the fluorescence intensity of each cell and could therefore be a powerful method to isolate cells at different stages of spermatogenesis from pvasa-GFP transgenic fish, which show dramatic changes in their GFP intensity during the process.

There is a need to establish an objective technique to identify fish spermatogenic cells at different stages. A histological method based on cell size, nuclear shape, and cyst structure has generally been used to identify testicular cell types [3]; however, this approach is time consuming, and it is difficult to determine the spermatogenic stage of the isolated cells.

In the current study, we developed a novel method to identify and purify trout testicular germ cells. The first aim of this study was to characterize stage-specific molecular markers in order to identify isolated cells objectively. The second aim was to purify stage-specific cells from pvasa-GFP transgenic rainbow trout testes using FCM. We initially cloned rainbow trout homologs of piwi, Scp3, and Shippo1 as candidate molecular marker genes. Drosophila PIWI is essential for maintaining germ-line stem cells [20]. The members of the PIWI family, which contain PAZ and PIWI domains [21], have been highly conserved during the evolution of species ranging from insects to mammals and play essential roles in the self renewal of germ-line stem cells and spermatogenesis [20, 22–24]. We therefore isolated rainbow trout homologs of the PIWI family as candidate spermatogonial markers. The synaptonemal complex (SC) is located along the paired meiotic chromosomes [25]. Three meiosis-specific components of the SC have been characterized in mammals: SCP1, SCP2, and SCP3 [26–29]. SCP3 is a component of the axial/lateral element of the SC and is expressed in spermatocytes from the leptotene to the late meiotic stages [27, 28]. We therefore cloned the SCP3 rainbow trout homolog as a candidate spermatocyte marker. SHIPPO1 (also known as outer dense fiber 3 or ODF3), which encodes a highly hydrophobic protein and exists in the cytoplasm of spermatids and along the entire length of the tail of spermatozoa, was also cloned as a candidate spermatid marker [30].

Additionally, in order to separate spermatogonia, spermatocytes, and spermatids from rainbow trout testes, testicular cells from pvasa-GFP rainbow trout were fractionated by FCM on the basis of their GFP intensities.

MATERIALS AND METHODS

RNA Isolation and cDNA Synthesis

Testes were isolated from a 12-mo-old rainbow trout, with a gonad-somatic index (GSI) of 0.029%, which had been reared under a natural photoperiod at 10°C. Total RNA was extracted from the testes using Isogen (Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions. The quality of the isolated RNA was examined by spectrophotometry and denaturing gel electrophoresis. An aliquot (2 µg) of total RNA was used for first-strand cDNA synthesis. Reverse transcription (RT) was performed using Ready-To-Go You-Prime First Strand-Beads (GE Healthcare, NJ) with the adapter-oligo (dT) primer 5'-CTGATCTAGAGGTACCGGATCCTATAGGGCACGCGTGGT18-3', according to the manufacturer's instructions. All procedures described herein were conducted in accordance with the International Guiding Principles for Biomedical Research Involving Animals as promulgated by the Society for the Study of Reproduction.

Cloning and Sequencing of Rainbow Trout piwi-like (rtili), Rainbow Trout scp3 (rt-scp3), and Rainbow Trout shippo1 (rt-shippo1)

The polymerase chain reaction (PCR) was performed using 1 µM of each primer, 1 µl of cDNA from the rainbow trout testes, 400 µM dNTP, 2.5 µM MgCl2, and 2.5 µM La Taq Buffer (Takara Bio Inc., Shiga, Japan) with 0.5 units of Takara La Taq (Takara Bio Inc.) in a total volume of 10 µl.

rtili. Two partial cDNA fragments of the rainbow trout Mili homolog (Fig. 1a, fragments 1 and 2) were initially amplified with the degenerate PCR primers mili F1 5'-TATCACSCGCTACAACAAYCGYACCTA-3', mili R1 5'-TCAGGGATYCCYGTCATGAAGGASAGCTC-3', mili F2 5'-TGGTGTACCGRAYGGMGTGTC-3', and mili R2 5'-TGRATGGTMCCAGGCCARTTCCAGTACA-3', designed from the conserved regions of Mili (mouse Piwil2; AB032605 in GenBank: http://www.ncbi.nlm.nih.gov) and the Fugu rubripes genome sequence, which showed high similarity to Mili (Scaffold_10505 and Scaffold_14933 in the Joint Genome Institute Fugu Genome Database: http://genome.jgi-psf.org/fugu6/fugu6.home.html). The degenerate PCR conditions are shown in Table 1. RT-PCR with mili F3 5'-TCATAACCGGCGAGATCCTGCTTCT-3' and mili R3 5'-TCATACAGCTCCACAGTCTTCAGCT-3' primers was used to amplify the DNA fragment between 1 and 2 (Fig. 1a, fragment 3). The RT-PCR conditions are shown in Table 1. After determining the DNA sequence of the partial cDNA fragments (Fig. 1a, fragments 1–3), 3'-rapid amplification of cDNA ends (3'-RACE) PCR and 5'-RACE PCR were performed to isolate a full-length cDNA. Two specific primers, 3'-rtili F1 5'-TCAGTCTACAACACAGCAAACCTCT-3' and 3'-rtili F2 5'-TAGGCTGACCTTCAAAATGTGCCACAT-3', were synthesized as forward primers for 3'-RACE PCR. The adapter primers AP1 5'-CTGATCTAGAGGTACCGGATCC-3' and AP2 5'-CTATAGGGCACGCGTGGT-3' were used as the reverse primers for 3'-RACE PCR (Fig. 1a, fragment 4). The first and nested PCR conditions are shown in Table 1. 5'-RACE PCR was performed using a Gene Racer kit (Invitrogen, CA) according to the manufacturer's protocol, with GR1 5'-CGACTGGAGCACGAGGACACTGA-3' and 5'-rtili R1 5'-TCGTTGAGCCGTCGGCCATGGTAGAA-3' primers (Fig. 1a, fragment 5). The PCR conditions are shown in Table 1.


Figure 01
View larger version (17K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 1 Structures of full-length rtili (a), rt-scp3 (b), and rt-shippo1 (c) cDNAs and amplified regions using degenerate and specific primers. a) Fragment 1 corresponding to nucleotides 1559–1821 and fragment 2 corresponding to nucleotides 2739–3123 of the rtili sequence were amplified by degenerate primers (mili F1 and mili R1 for fragment 1 and mili F2 and mili R2 for fragment 2). Fragment 3 corresponding to nucleotides 1764–2793 of rtili was amplified using specific primers (mili F3 and mili R3) designed from the sequence data of fragments 1 and 2. 3'-RACE PCR was performed using 3'-rtili F1 and AP1, 3'-rtili F2 and AP2 primers to amplify fragment 4 corresponding to nucleotides 3071–4205. The 5'-rtili R1 and GR1 primers were used to amplify fragment 5 corresponding to nucleotides 1–1651 of rtili. b) scp3 F1 and scp3 R1 primers were used to amplify fragment 1 corresponding to nucleotides 602–996 of the rt-scp3 sequence. 3'-RACE PCR was performed using 3'-rtscp3 F1 and AP1, 3'-rtscp3 F2 and AP2 primers to amplify fragment 2 corresponding to nucleotides 908–1312. 5'-RACE PCR using 5'-rtscp3 R1 and GR1 primers was used to amplify fragment 3 corresponding to nucleotides 1–655 of rt-scp3. c) shippo F1 and shippo R1 primers were used to amplify fragment 1 corresponding to nucleotides 213–889 of the rt-shippo1 sequence. 3'-RACE PCR using 3'-rtshippo1 F1 and AP2 primers was used to amplify fragment 2, which corresponded to nucleotides 834–1058 of rt-shippo1. 5'-RACE PCR using 5'-rtshippo1 R1 and GR1 primers was used to amplify fragment 3 corresponding to nucleotides 1–268 of rt-shippo1.


View this table:
[in this window]
[in a new window]
[Download PPT slide]
  		TABLE 1 The reaction conditions for degenerate PCR, RACE-PCR, and RT-PCR.

rt-scp3. The highly conserved region of mouse Scp3 (NM_011517 in GenBank) and zebrafish partial cDNA (BQ479743 and BQ479518 in GenBank), which showed high similarity to mouse Scp3, were used to design primers scp3 F1 5'-TCAGAAAMTKGARCARATKTGGAA-3' and scp3 R1 5'-TAYARCATGGAYTGAAGAGACTT-3' (Fig. 1b, fragment 1). After determining the DNA sequence of a partial cDNA fragment, 3'-RACE PCR and 5'-RACE PCR were used to isolate a full-length cDNA. Primers 3'-rtscp3 F1 5'-TGAGATGGCAGTGTTGCAGAAGA-3' and 3'-rtscp3 F2 5'-TCATGTTAAGGAATAAATACAGGATGCA-3' were designed for 3'-RACE PCR (Fig. 1b, fragment 2), and primer 5'-rtscp3 R1 5'-TCAGCTTCTGCCTTTGATGCATACTG-3' was designed for 5'-RACE PCR (Fig. 1b, fragment 3). The degenerate, 3'-RACE, and 5'-RACE PCR conditions are shown in Table 1.

rt-shippo1. A partial rainbow trout Shippo1 homolog cDNA fragment (Fig. 1c, fragment 1) was amplified from the cDNA of the rainbow trout testes with shippo F1 5'-TCTACAGCAGCCCTGGACCCAAGT-3' and shippo R1 5'-TGAACTCAGAATGACGAATTCCGA-3' primers designed from the rainbow trout expressed sequence tag (TC47547 in the Institute for Genomic Research Sequence Database: http://compbio.dfci.harvard.edu/tgi) that showed high similarity to mouse Shippo1. 3'-RACE PCR was carried out using 3'-rtshippo1 F1 5'-TTCACCAGGATCAACCTCCAA-3' and AP2 primers (Fig. 1c, fragment 2). 5'-RACE PCR was carried out using 5'-rtshippo1 R1 5'-TAGTTCAGACCTGTCGCTCCAGGCA-3' and GR1 primers (Fig. 1c, fragment 3). The degenerate, 3'-RACE, and 5'-RACE PCR conditions are shown in Table 1.

Northern Blot Analysis

Total RNAs were extracted from the brain, gill, heart, liver, spleen, gut, kidney, and muscle of a 3-yr-old male rainbow trout and from the testes (GSI = 0.039% or 0.459%) or ovaries (GSI = 0.457%) of 12-mo-old rainbow trout using the method described previously. A 30-µg sample of each total RNA was used for Northern blotting. The full-length cDNAs of rtili, rt-scp3, and rt-shippo1 were labeled with [{alpha}-32P] dCTP (MP Biomedicals, CA) by the random-priming method using an oligo labeling kit (GE Healthcare). The blotting and hybridization procedures are described elsewhere [31].

Semiquantitative PCR of Maturating Testes

In order to determine the expression patterns of rtili, rt-scp3, and rt-shippo1 at different stages of maturity, RT-PCR was performed using specific primers. cDNAs from testes containing only type A spermatogonia (GSI = 0.080%), those with lobules containing both type A and type B spermatogonia (GSI = 0.100%), those containing spermatocytes together with type A and type B spermatogonia (GSI = 0.350%), and those containing spermatids and spermatozoa in addition to type A spermatogonia (GSI = 3.200%) were synthesized from 16-mo-old male fish. In order to obtain semiquantitative results, we performed 25 PCR cycles using real rtili-Fw 5'-TTCTGCCAGAGCTCTCCTTTACG-3' and real rtili-Rv 5'-AGCTGCTTCAGGGAGTGAGTGTG-3' primers for rtili expression, real scp3-Fw 5'-CAGTATGCATCAAAGGCAGAAGC-3' and real scp3-Rv 5'-ACCACTCTAGCCTGCTGGAACAG-3' primers for rt-scp3 expression, real shippo1-Fw 5'-TCTACAGCCCTGGACCCAAGT-3' and real shippo1-Rv 5'-AGCAGTCTGAGCTGAATTGGCGA-3' primers for rt-shippo1 expression, and real actin-Fw 5'-ACTACCTCATGAAGATCCTG-3' and real actin-Rv 5'-ATCTCCTGCTCAAAGTCCAGC-3' primers for beta-actin expression. The resultant PCR products were electrophoresed on a 2% agarose gel.

In Situ Hybridization

In situ hybridization of rtili, rt-scp3, and rt-shippo1 was performed as described previously by Sawatari and colleagues [32]. After colorimetric reactions had been performed, the slides were counterstained with methyl green (Division Chroma, Muenster, Germany).

Immunohistochemistry

Prior to sorting the stage-specific spermatogenic cells from the pvasa-GFP transgenic fish using GFP-dependent FCM, it was important to identify the GFP expression levels at the different stages. We thus performed an immunohistochemical analysis of the pvasa-GFP transgenic fish testes with a GFP antibody (Roche Diagnostic, Manheim, Germany). Tissues from the pvasa-GFP transgenic rainbow trout testes were fixed for 16 h in Bouin fluid, dehydrated, and embedded in paraffin. For the immunohistochemical localization of GFP, 5-µm sections were mounted on 3-aminopropyltriethoxy silane (APS)-coated slides (Matsunami Glass, Tokyo, Japan), deparaffinized, and rehydrated. The immunoreactions were performed using a Vectastain Elite ABC kit (Vector Laboratories, CA), and visualized using a 3,3'-diaminobenzidine (DAB) substrate kit (Vector Laboratories) according to the manufacturer's protocol. Anti-GFP (Roche Diagnostic) was used at a final dilution of 1:500 in PBS as the primary antiserum. Subsequently, the slides were washed in distilled water and counterstained with hematoxylin.

Isolation of Testicular Cells by FCM

GFP-dependent cell sorting was used to isolate the testicular cells from 16-mo-old pvasa-GFP transgenic fish, according to the method of Kobayashi and colleagues [13]. The testicular cells were separated into five fractions. A sample of 1000 cells from each fraction was resuspended in PBS containing 5% FBS to analyze the morphology, and 2000 cells were resuspended in Isogen to isolate RNAs for real-time PCR.

Real-Time PCR

We analyzed isolated testicular cells using real-time PCR in order to identify the spermatogenic stage based on the molecular markers rtili, rt-scp3, and rt-shippo1. We also identified populations containing somatic cells using the cell marker gsdf [32]. The amplification of cDNA samples was performed using the iQ SYBR Green Supermix RT-PCR kit (Bio-Rad Laboratories, CA) in an iCycler real-time detection system (Bio-Rad Laboratories) under the following conditions: 95°C for 3 min, followed by 40 cycles of 20 sec at 95°C, 20 sec at 62°C, and 20 sec at 72°C. The primer sequences were as follows: real rtili-Fw and real rtili-Rv for rtili, real scp3-Fw and real scp3-Rv for rt-scp3, real shippo1-Fw and real shippo1-Rv for rt-shippo1, real gsdf-Fw 5'-ATCTTTTGTCCTCCAGTCCTCGGA-3' and real gsdf-Rv 5'-CTGCAGGTTGAGGGCTCCAATAA-3' for gsds, and real actin-Fw and real actin-Rv for beta-actin. The beta-actin gene expression was analyzed as an internal control of equal RNA loading with the primers real actin-Fw and real actin-Rv as described previously. Data were analyzed using the comparative cycle-threshold method [33].

Germ-Cell Transplantation Assay

Spermatogonia can be classified into two subtypes: type A, which contain spermatogonial stem cells (SSCs), and type B, which are committed to differentiate into spermatocytes. Recently, the capabilities of rainbow trout SSCs to self-renew and differentiate into gametes of both sexes have been demonstrated by the transplantation of type A spermatogonia [34]. Moreover, the colonization efficiency and percentages of recipients containing mature gametes were shown to be similar in this previous study [34]. These facts suggest that most germ cells that colonize recipient gonads behave like SSCs and produce large numbers of mature gametes. The detection of colonization activity through a transplantation experiment thus appears to be a good indicator of the type A spermatogonia fraction containing SSCs. Donor cells were therefore initially prepared from cell fractions A and B, which were isolated by FCM. Approximately 3000 cells from each fraction were then transplanted into recipient fish according to the method of Takeuchi and colleagues [35]. The dissociated testicular cells from 7-mo-old immature testes, which contained approximately 3000 type A spermatogonia, were then transplanted into recipient fish as a control.

RESULTS

Cloning of Candidate Marker Genes

The marker rtili (AB331649 in GenBank) encodes a protein of 1055 amino acids, which has PAZ and PIWI domains. Its predicted amino-acid sequence is 61.0% identical and 87.5% similar to that of mouse MILI. Phylogenetic trees based on the amino-acid sequences of PIWI family members revealed that Rtili belonged to the MILI branch (Fig. 2a).


Figure 02
View larger version (20K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 2 Phylogenetic analysis of Rtili (a), Rt-scp3 (b), and Rt-shippo1 (c). Trees were constructed by the unweighted pair-group method with arithmetic mean (UPGMA) by GENETYX ver. 7.0.3 (Genetyx Corp., Tokyo, Japan). Horizontal distances are proportional to evolutionary divergence expressed as substitutions per site. The GenBank accession numbers of the aligned amino-acid sequences were as follows. a) Drosophila PIWI; AAD08705, MIWI (mouse piwil1); BAA93705, MILI (mouse piwil2); BAA93706, Ziwi (zebrafish piwil1); NP_899181, Zili (zebrafish piwil2); ABM46842. b) Medaka Scp1; BAE45256, medaka Scp3; BAD36840, mouse SCP1; NP_035646, mouse SCP2; NP_796165, mouse SCP3; NP_035647, rat SCP1; NP_036942, rat SCP2; NP_57009, rat SCP3; CAA53430. c) Human ODF1; NP_077721, human ODF2; AAD22544, human ODF3; NP_444510, mouse ODF1; NP_032783, mouse ODF2; AAB87525, mouse ODF3; NP_081295, Xenopus ODF3; CAJ83130.

The cloned cDNA that we named rt-scp3 (AY601674 in GenBank) encoded 239 amino acids and showed 56.0% identity and 74.0% similarity to mouse SCP3. The phylogenetic trees based on amino-acid sequences revealed that Rt-scp3 was strongly associated with SCP3 (Fig. 2b).

The marker rt-shippo1 (AY601673 in GenBank) encoded 249 amino acids, which included Cys-Gly-Pro repeats at the carboxy (C)-terminal end, and showed 50.0% and 49.0% identity with mouse and human SHIPPO1, respectively. The phylogenetic trees based on amino-acid sequences revealed that the isolated gene was an ortholog of Shippo1 (Fig. 2c).

Expression of rtili, rt-scp3, and rt-shippo1

The Northern blot analysis showed that rtili, rt-scp3, and rt-shippo1 were expressed specifically in the gonads (Fig. 3). The length of rtili was estimated to be 4 kbp, and its expression was detected in the testes and strongly in the ovaries (Fig. 3). An rt-scp3 transcript of about 1.5 kbp was present in both the testes and the ovaries, and an rt-shippo1 transcript of about 1 kbp was detected in the testes and weakly in the ovaries (Fig. 3).


Figure 03
View larger version (34K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 3 Northern blot analysis of rtili (top), rt-scp3 (middle), and rt-shippo1 (bottom) in various adult rainbow trout tissues (brain, gill, heart, liver, spleen, gut, kidney, muscle, ovary, and testis). The expression levels of rtili (top), rt-scp3 (middle), and rt-shippo1 (bottom) were restricted to gonadal tissue.

Semiquantitative RT-PCR was used to identify the mRNA levels in the maturating testes at each stage: those containing only type A spermatogonia (Fig. 4, testis 1), those with lobules containing both type A and type B spermatogonia (Fig. 4, testis 2), those containing spermatocytes together with type A and type B spermatogonia (Fig. 4, testis 3), and those containing spermatids and spermatozoa in addition to type A spermatogonia (Fig. 4, testis 4). As shown in Figure 4, rtili was detected at all stages and was strongly expressed in testes with lobules containing both type A and type B spermatogonia (testes 2 and 3). By contrast, rt-scp3 was not expressed in testes that only contained spermatogonia (testes 1 and 2) and was expressed in testes with spermatocytes (testis 3). Furthermore, the expression of rt-shippo1 was observed only in testes containing spermatids and spermatozoa (testis 4).


Figure 04
View larger version (63K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 4 Expression of rtili, rt-scp3, and rt-shippo1 during testis maturation. PCR amplification was performed by each specific primer. The cDNA from testes containing only type A spermatogonia (1), those with lobules containing both type A and B spermatogonia (2), those containing spermatocytes together with type A and B spermatogonia (3), and those containing spermatids and spermatozoa in addition to type A spermatogonia (4) from 16-mo-old individuals were used for PCR. rtili (top), rt-scp3 (middle), and rt-shippo1 (bottom) were expressed predominantly in testes with type B spermatogonia (testes 2 and 3), those with spermatocytes (testis 3), and those with spermatids and spermatozoa (testis 4), respectively. beta-actin was used as an internal control for the PCR. Bar = 50 µm.

In situ hybridization of adult testes was used to examine the cell-type-specific expression of three candidate marker genes. Cells positive for rtili were detected only in type A and type B spermatogonia (Fig. 5b). Moreover, rt-scp3 and rt-shippo1 were expressed specifically in primary spermatocytes (Fig. 5d) and spermatids (Fig. 5f), respectively.


Figure 05
View larger version (146K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 5 Expression of rtili (a, b), rt-scp3 (c, d), and rt-shippo1 (e, f) in 16-mo-old testes. rtili, rt-scp3, and rt-shippo1 expression as revealed by section in situ hybridization (b, d, and f). Sequential sections of b, d, and f were stained with hematoxylin and eosin (a, c, and e, respectively). rtili, rt-scp3, and rt-shippo1 were expressed mainly in type A and B spermatogonia (a), primary spermatocytes (d), and spermatids (f), respectively. SG-A, Type A spermatogonia; SG-B, type B spermatogonia; SC, spermatocyte; ST, spermatid; SZ, spermatozoa. Bar = 50 µm.

GFP Expression in pvasa-GFP Transgenic Rainbow Trout Testes

An immunohistochemical analysis with a GFP-specific antibody was used to identify GFP-positive cells in the transgenic pvasa-GFP rainbow trout testes (Fig. 6). Strong staining was detected in the cytoplasm of type A spermatogonia adjacent to the lobule boundary wall (Fig. 6). The GFP protein expression level dramatically decreased in type B spermatogonia and that of spermatocytes were even lower (Fig. 6). The GFP protein was not detected in spermatids and spermatozoa (Fig. 6).


Figure 06
View larger version (182K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 6 Identification of GFP expression levels in spermatogenic cells in pvasa-GFP rainbow trout testes at various stages. Immunohistochemistry was carried out using a GFP antibody. Strong staining was detected in the cytoplasm of type A spermatogonia. The GFP protein expression levels dramatically decreased in type B spermatogonia, and those of spermatocyte were even lower. SG-A, Type A spermatogonia; SG-B, type B spermatogonia; SC, spermatocyte; ST, spermatid. Bar = 50 µm.

Morphological and Molecular Characterization of Isolated Cells

The GFP intensities of the various testicular cells isolated from the pvasa-GFP transgenic rainbow trout were analyzed by FCM (Fig. 7a). Fraction A contained cells showing strong fluorescence, with diameters of approximately 15 µm and heterochromatin clumps (Fig. 7b, arrowheads). Fraction B contained cells showing weaker fluorescence, with diameters of approximately 10 µm and large nuclear bodies (Fig. 7b). Extremely weak fluorescence was also detected in the cells of fraction C (Fig. 7b); this contained cells with diameters of approximately 8 µm and smaller nuclear bodies than the cells in fraction B (Fig. 7b). The GFP-negative fractions D and E contained cells that were smaller than 5 µm (Fig. 7b). In addition, a few cells with diameters of approximately 7 µm were observed in fraction E (data not shown).


Figure 07
View larger version (36K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 7 Morphological and molecular characterization of isolated cells. a) Fluorescent intensity pattern of total testicular cells, which were separated into five fractions (A–E). b) Bright and fluorescent views of isolated cells. Arrowheads indicate heterochromatin clumps. Bar = 10 µm. c) Characterization of isolated cells using the molecular markers rtili, rt-scp3, rt-shippo1, and gsdf by real-time PCR. The isolated cells from fractions A and B expressed mainly rtili. The cells from fractions C and D expressed mainly rt-scp3. The cells from fraction E expressed rt-shippo1 and gsdf. Error bars represent mean ± SEM from triplicate reactions.

We further characterized the fractionated testicular germ cells using real-time PCR with the somatic marker gsdf and the germ-cell markers described previously (Fig. 7c). The spermatogonia marker rtili was expressed mainly in fractions A and B (Fig. 7c). By contrast, the spermatocyte marker rt-scp3 was expressed mainly in fractions C and D (Fig. 7c). The spermatid marker rt-shippo1 and the somatic-cell marker gsdf were detectable only in fraction E (Fig. 7c). Notably, rtili was more strongly expressed in fraction B than fraction A, and rt-scp3 was more strongly expressed in fraction C than fraction D (Fig. 7c).

Identification of Stem-Cell Populations by Transplantation Assay

Observations of the recipient embryos at 60 days posttransplantation revealed that the donor cells from GFP-positive fraction A were incorporated into the genital ridges of the recipients (51.0% ± 9.0%), producing colonies containing 10.1 ± 3.8 cells (Fig. 8). This value was not significantly different from the control (Fig. 8; colonization efficiency = 53.6% ± 11.3%; cell number per colony = 11.3 ± 3.8). By contrast, the donor cells from fraction B were not incorporated into the recipient gonads (Fig. 8).


Figure 08
View larger version (48K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 8 Identification of the type A spermatogonia fraction by germ-cell transplantation. a) The frequency of production of GFP-positive germ-cell colonies after transplantation of the cell populations from fraction A or B into the recipient gonads. Unsorted testicular cells were also transplanted as a control. Data represents the means ± standard error (SE) from at least five independent experiments. At least 55 recipients were used in each transplantation experiment. b) Incorporation of GFP-labeled donor germ cells into a recipient gonad at 60 days posttransplantation in a fluorescent view. The cells from fraction A were incorporated into the genital ridge of the recipient gonad and produced colonies of 10.1 ± 3.8 cells. Bar = 100 µm.

DISCUSSION

We initially established a method to identify fish testicular germ cells at various stages of spermatogenesis. In situ hybridization showed that rtili, rt-scp3, and rt-shippo1 could be used as markers of spermatogonia, spermatocytes, and spermatids, respectively. Semiquantitative PCR showed that the spermatogonia marker rtili was expressed more strongly in testes that contained type B spermatogonia than testes containing only type A spermatogonia. These results suggest that rtili is more strongly expressed in type B spermatogonia than in type A spermatogonia. Comparing the rtili levels in the spermatogonia cell fraction can therefore help to identify the two types of spermatogonia.

Next, we isolated different types of spermatogenic cells from pvasa-GFP rainbow trout by GFP-dependent FCM. Fractions A and B contained large cells with large nuclear bodies, which were similar to those of the spermatogonia observed in the tissue section. Real-time PCR showed that rtili was more strongly expressed in fraction B than in fraction A, suggesting that they contained mainly type B and type A spermatogonia, respectively. The immunohistochemical observations showed that single type A spermatogonia adjacent to the lobule boundary wall expressed GFP more strongly than type B spermatogonia. In order to confirm this point, we performed a transplantation assay, which is an established technique for use with rainbow trout [34]. The transplantation experiment showed that fraction A could colonize the recipient gonads, while fraction B could not. In the mouse, differentiating spermatogonia are known to be incapable of colonization after transplantation [36]. These facts suggested that the weaker GFP-positive cells (fraction B) contained mainly type B spermatogonia, while the stronger GFP-positive cells (fraction A) contained mainly type A spermatogonia—that is, the fractions showing strong vasa gene expression contained mainly type A spermatogonia. Fractions C and D, which contained cells with diameters of 5–7 µm, expressed the spermatocyte marker rt-scp3. Furthermore, the isolated cells were similar in size to the spermatocytes observed in the tissue section. These results indicated that fractions C and D contained spermatocytes. The fact that the GFP-negative cells in fraction E expressed the spermatid marker rt-shippo1 and the somatic-cell marker gsdf suggested that this fraction contained spermatids and somatic cells. The majority of the isolated cells from fraction E had diameters less than 5 µm, indicating that the fraction contained mainly spermatids, although some had diameters of approximately 7 µm and were predicted to be somatic cells.

Our study demonstrates that GFP-dependent FCM using pvasa-GFP rainbow trout testes is a powerful method to isolate different types of spermatogenic cells. Although this technique is restricted to transgenic fish carrying pvasa-GFP, it has the advantage that the dissociated testicular cells can be directly subjected to FCM without the need for any complex preparation. The simplicity of this process is valuable when isolating cells for in vitro culture and transplantation. One potential concern when using germ cells isolated by FCM is the potential for the target cells to sustain physical damage during the sorting procedure. However, our transplantation experiments revealed no differences in colonization activity between the FCM-purified testicular cells and intact germ cells (data not shown). Thus, the isolated germ cells can be used for in vitro culture and transplantation without significant damage. In addition, isolating spermatogenic cells based on vasa expression (GFP intensity), using cell size (forward scatter) and granularity (side scatter), might improve the purity. Moreover, traditional FCM analysis of testicular tissues provides histograms of cells according to their DNA content using staining DNA fluorescence, such as Hoechst 33342. The stained cells are discriminated into three main fractions: haploid (spermatids, spermatozoa, and spermatocytes), diploid (spermatogonia and somatic cells), and tetraploid (spermatocytes). By using a combination of this method of DNA content quantification and GFP-dependent methods, it will be possible to isolate even more purified populations of each type of germ cell.

Isolated testicular cells can be used to elucidate the molecular and physiological systems of spermatogenesis through a range of methods, including transcriptome analysis, proteome analysis, in vitro culture, and transplantation assay. In particular, the in vitro culture of specific cell types will be important to identify the critical factors that control proliferation and development during spermatogenesis. Additionally, isolating stage-specific cell populations, especially pure spermatogonia, could help to establish spermatogenic cell lines in rainbow trout.

In this study, the spermatogonia from 16-mo-old meiotic testes (fraction A) showed similar colonization activity to those from 7-mo-old immature testes containing only type A spermatogonia (control). These data suggested that the SSCs in meiotic testes did not alter their transplantation ability without being affected by secretion factors and the microenvironment in maturating testis. As fish testes have a unique mechanism for spermatogenesis compared with mammals [2, 6], it will be of particular interest to investigate SSCs and their environment in fish using GFP-dependent FCM and transplantation experiments.

Correspondence: 1Goro Yoshizaki, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan. FAX: 81 3 5463 0558; e-mail: goro{at}kaiyodai.ac.jp

Received: 30 July 2007.

First decision: 23 August 2007.

Accepted: 11 September 2007.

REFERENCES

  1. Billard R. Spermatogenesis and spermatology of some teleost fish species Reprod Nutr Dev 1986 2877–920
  2. Pudney J. Spermatogenesis in nonmammalian vertebrates Microsc Res Tech 1995 32459–497[CrossRef][Medline]
  3. An Atlas of Fish Histology: Normal and Pathological Features, 2nd ed Patiño R and Takashima F. Chapter 9, Gonads 1995New York Fischer128–153 In:
  4. Schulz RW, Vischer HF, Cavaco JE, Santos EM, Tyler CR, Goos HJ, Bogerd J. Gonadotropins, their receptors, and the regulation of testicular functions in fish Comp Biochem Physiol B Biochem Mol Biol 2001 129407–417[CrossRef][Medline]
  5. Schulz RW and Miura T. Spermatogenesis and its endocrine regulation Fish Physiol Biochem 2002 2643–56[CrossRef]
  6. Viela DAR, Silva MTD, Piexoto HP, Godinho HP, França LR. Spermatogenesis in teleost: insights from the Nile tilapia (Oreochromis niloticus) model Fish Physiol Biochem 2003 28187–190[CrossRef]
  7. van Winkoop A and Timmermans LP. Phenotypic changes in germ cells during gonadal development of the common carp (Cyprinus carpio). An immunohistochemical study with anti-carp spermatogonia monoclonal antibodies Histochemistry 1992 98289–298[CrossRef][Medline]
  8. Saiki A, Tamura M, Matsumoto M, Katowgi J, Watanabe A, Onitake K. Establishment of in vitro spermatogenesis from spermatocytes in the medaka, Oryzias latipes Dev Growth Differ 1997 39337–344[CrossRef][Medline]
  9. Tokalov SV and Gutzeit HO. Spermatogenesis in testis primary cell cultures of the tilapia (Oreochromis niloticus) Dev Dyn 2005 2331238–1247[CrossRef][Medline]
  10. Song M and Gutzeit HO. Primary culture of medaka (Oryzias latipes) testis: a test system for the analysis of cell proliferation and differentiation Cell Tissue Res 2003 313107–115[CrossRef][Medline]
  11. Hong Y, Liu T, Zhao H, Xu H, Wang W, Liu R, Chen T, Deng J, Gui J. Establishment of a normal medakafish spermatogonial cell line capable of sperm production in vitro Proc Natl Acad Sci U S A 2004 1018011–8016[Abstract/Free Full Text]
  12. Miura C, Miura T, Yamashita M, Yamauchi K, Nagahama Y. Hormonal induction of all stages of spermatogenesis in germ-somatic cell cocultures from immature Japanese eel testis Dev Growth Differ 1996 38257–262[CrossRef]
  13. Kobayashi T, Yoshizaki G, Takeuchi Y, Takeuchi T. Isolation of highly pure and viable primordial germ cells from rainbow trout by GFP-dependent flow cytometry Mol Reprod Dev 2004 6791–100[CrossRef][Medline]
  14. Yoon C, Kawakami K, Hopkins N. Zebrafish vasa homologue RNA is localized to the cleavage planes of 2- and 4-cell-stage embryos and is expressed in the primordial germ cells Development 1997 1243157–3165[Abstract]
  15. Knaut H, Pelegri F, Bohmann K, Schwarz H, Nüsslein-Volhard C. Zebrafish vasa RNA but not its protein is a component of the germ plasm and segregates asymmetrically before germline specification J Cell Biol 2000 149875–888[Abstract/Free Full Text]
  16. Kobayashi T, Kajiura-Kobayashi H, Nagahama Y. Differential expression of vasa homologue gene in the germ cells during oogenesis and spermatogenesis in a teleost fish, tilapia, Oreochromis niloticus Mech Dev 2000 99139–142[CrossRef][Medline]
  17. Shinomiya A, Tanaka M, Kobayashi T, Nagahama Y, Hamaguchi S. The vasa-like gene, olvas, identifies the migration path of primordial germ cells during embryonic body formation stage in the medaka, Oryzias latipes Dev Growth Differ 2000 42317–326[CrossRef][Medline]
  18. Yoshizaki G, Sakatani S, Tominaga H, Takeuchi T. Cloning and characterization of a vasa-like gene in rainbow trout and its expression in the germ cell lineage Mol Reprod Dev 2000 55364–371[CrossRef][Medline]
  19. Xu H, Gui J, Hong Y. Differential expression of vasa RNA and protein during spermatogenesis and oogenesis in the gibel carp (Carassius auratus gibelio), a bisexually and gynogenetically reproducing vertebrate Dev Dyn 2005 233872–882[CrossRef][Medline]
  20. Cox DN, Chao A, Baker J, Chang L, Qiao D, Lin H. A novel class of evolutionarily conserved genes defined by piwi are essential for stem cell self-renewal Genes Dev 1998 123715–3727[Abstract/Free Full Text]
  21. Cerutti L, Mian N, Bateman A. Domains in gene silencing and cell differentiation protein: the novel PAZ domain and redefinition of the Piwi domain Trends Biochem Sci 2000 25481–482[CrossRef][Medline]
  22. Deng W and Lin H. miwi, a murine homolog of piwi, encodes a cytoplasmic protein essential for spermatogenesis Dev Cell 2002 2819–830[CrossRef][Medline]
  23. Kuramochi-Miyagawa S, Kimura T, Ijiri TW, Isobe T, Asada N, Fujita Y, Ikawa M, Iwai N, Okabe M, Deng W, Lin H, Matsuda Y, Nakano T. Mili, a mammalian member of piwi family gene, is essential for spermatogenesis Development 2004 31839–849
  24. Houwing S, Kamminga LM, Berezikov E, Cronembold D, Girard A, van den Elst H, Filippov DV, Blaser H, Raz E, Moens CB, Plasterk RH, Hannon GJ, Draper BW, Ketting RF. A role for Piwi and piRNAs in germ cell maintenance and transposon silencing in Zebrafish Cell 2007 12969–82[CrossRef][Medline]
  25. Moses MJ. Synaptonemal complex Annu Rev Genet 1968 2363–412[CrossRef]
  26. Meuwissen RLJ, Offenberg HH, Dietrich AJJ, Riesewijk A, van Iersel M, Heyting C. A coiled-coil related protein specific for synapsed regions of meiotic prophase chromosomes EMBO J 1992 115091–5100[Medline]
  27. Dobson MJ, Pearlman RE, Karaiskakis A, Spyropoulos B, Moens PB. Synaptonemal complex proteins: occurrence, epitope mapping and chromosome disjunction J Cell Sci 1994 1072749–2760[Abstract]
  28. Lammers JHM, Offenberg HH, van Aalderen M, Vink ACG, Dietrich AJJ, Heyting C. The gene encoding a major component of the lateral elements of synaptonemal complexes of the rat is related to X-linked lymphocyte-regulated genes Mol Cell Biol 1994 141137–1146[Abstract/Free Full Text]
  29. Offenberg HH, Schalk JAC, Meuwissen RLJ, van Aalderen M, Kester HA, Dietrich AJJ, Heyting C. SCP2: a major protein component of the axial elements of synaptonemal complexes of the rat Nucleic Acids Res 1998 262572–2579[Abstract/Free Full Text]
  30. de Carvalho C, Tanaka H, Iguchi N, Ventela S, Nojima H, Nishimune Y. Molecular cloning and characterization of a complementary DNA encoding sperm tail protein SHIPPO1 Biol Reprod 2002 66785–795[Abstract/Free Full Text]
  31. Yoshizaki G, Patiño R, Thomas P. Connexin messenger ribonucleic acids in the ovary of Atlantic croaker: molecular cloning and characterization, hormonal control, and correlation with appearance of oocyte maturational competence Biol Reprod 1994 51493–503[Abstract]
  32. Sawatari E, Shikina S, Takeuchi T, Yoshizaki G. A novel transforming growth factor-β superfamily member expressed in gonadal somatic cells enhances primordial germ cell and spermatogonial proliferation in rainbow trout (Oncorhynchus mykiss) Dev Biol 2007 301266–275[CrossRef][Medline]
  33. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR Nucleic Acids Res 2001 292002–2007
  34. Okutsu T, Suzuki K, Takeuchi Y, Takeuchi T, Yoshizaki G. Testicular germ cells can colonize sexually undifferentiated embryonic gonad and produce functional eggs in fish Proc Natl Acad Sci U S A 2006 1032725–2729[Abstract/Free Full Text]
  35. Takeuchi Y, Yoshizaki G, Kobayashi T, Takeuchi T. Mass isolation of primordial germ cells from transgenic rainbow trout carrying the green fluorescent protein gene driven by the vasa gene promoter Biol Reprod 2002 671087–1092[Abstract/Free Full Text]
  36. Shinohara T, Orwig KE, Avarbock MR, Brinster RL. Spermatogonial stem cell enrichment by multiparameter selection of mouse testis cells Proc Natl Acad Sci U S A 2000 978346–8351[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
78/1/151    most recent
biolreprod.107.064667v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yano, A.
Right arrow Articles by Yoshizaki, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yano, A.
Right arrow Articles by Yoshizaki, G.
Agricola
Right arrow Articles by Yano, A.
Right arrow Articles by Yoshizaki, G.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS