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From the Departments of * Biochemistry, Cell
Biology, and Histology, Faculty of Veterinary Medicine; and
Cell Biology, University Medical Centre
Utrecht, Utrecht University, Utrecht, The Netherlands
| Correspondence to: Dr A.M.M. van Pelt, Room HP G02.525, Department of Cell Biology, UMC Utrecht, location AZU, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. |
| Received for publication August 2, 2001; accepted for publication December 11, 2001. |
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Abstract |
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isoform) of protein
phosphatase 2A. No clues regarding the nature of the remaining four clones
could be found. Hence, using differential screening on both constructed cDNA
libraries, we were able to select several genes that are interesting
candidates for studying the molecular mechanisms of normal gonocyte
development.
Key words: cDNA library, differential screening, in situ hybridization, A spermatogonia, testis, rat
In humans, it has been recognized that the development of these gonocytes is a critical step in the establishment of the male germ line. Growing evidence indicates that several types of germ cell tumors that occur in adult men originate from impaired gonocyte development (Skakkebaek et al, 1998; Dieckmann and Skakkebaek, 1999). However, knowledge about the factors and genes involved in gonocyte development is still scarce.
In rats, testicular germ cell development starts around Day 13 postcoitum (Kemper and Peters, 1987). Primordial germ cells (PGCs) proliferate and migrate toward the genital ridges to become enclosed by Sertoli cells. These germ cells are then called gonocytes (Clermont and Perey, 1957; Sapsford, 1962; Huckins and Clermont, 1968). The proliferative activity of gonocytes is different from that of PGCs in that after division, the daughter cells remain connected by an intercellular bridge. Moreover, gonocytes differ structurally from PGCs. Around Day 18 postcoitum in rats, the gonocytes cease their mitotic activity and remain quiescent until a few days after birth, when they resume proliferation, and spermatogenesis starts (Sapsford, 1962; Huckins and Clermont, 1968; Novi and Saba, 1968; Bellve et al, 1977).
Gonocytes and A spermatogonia cannot be discriminated by their morphology. Hence, the germ cells at the start of spermatogenesis are often named gonocytes, but most likely they have developed further and represent a mixed population of As, Apr, and Aal spermatogonia, and the more differentiated A1 spermatogonia (Van Haaster and de Rooij, 1993; De Rooij, 1998). Van Dissel-Emiliani et al (1993) developed a monoclonal antibody that recognized a marker that is specifically expressed by the quiescent gonocytes until the day of birth. Hence, it may be assumed that gonocytes are specific to fetal testes, whereas As, Apr, and Aal spermatogonia of adult testes arise shortly after birth. The development of gonocytes toward As, Apr, and Aal spermatogonia then takes place during the quiescent period of germ cells, around the time of birth.
Until now, the study of genes involved in the development of gonocytes has been hampered by their very low numbers per testis and the lack of an isolation method that allows sufficient purification of gonocytes to perform such studies. Previously, we developed methods to obtain highly purified populations of quiescent gonocytes from fetal rats and adult As, Apr, and Aal spermatogonia (Van Pelt et al, 1996; Van den Ham et al, 1997). In this study, we used these methods to isolate sufficient gonocytes and early spermatogonia to allow the construction of a complementary DNA (cDNA) library from each cell type. Then, 500 clones from the gonocyte library were differentially screened using both the gonocyte and the spermatogonia library. Dot blot analyses of selected clones were used to verify the differential screening results. Seven clones consistently appeared to be more abundant in the gonocyte library compared to their ration in the spermatogonia library. Also, in situ hybridization confirmed that the isolated genes are highly expressed in gonocytes, and either are not expressed or hardly expressed in As, Apr, and Aal spermatogonia. Hence, the identified genes are interesting candidates for investigating the process of normal gonocyte development.
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Materials and Methods |
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Cell Isolation
Gonocytes were isolated during their quiescent period at 18, 19, and 20
days postcoitum by using an immunomagnetic isolation procedure as described by
van den Ham et al (1997).
As, Apr, and Aal spermatogonia from VAD rats
were isolated by centrifugation on a discontinuous Percoll (Pharmacia Biothech
AB, Uppsala, Sweden) gradient, as described by van Pelt et al
(1996).
Gonocytes and A spermatogonia were identified using a Nomarski microscope based on morphological criteria as previously described (Van Dissel-Emiliani et al, 1989). After isolation, the cells were lysed in guanidinium isothiocyanate solution (Ausubel et al, 1993; 1 x 106 cells/100 µL) and stored at -80°C until use.
Poly A+ RNA Isolation
Shortly before use, the lysed cells were thawed and diluted with extraction
buffer (400 µL total) from the Quickprep micro mRNA Purification Kit
(Pharmacia Biotech, Milwaukee, Wis). poly A+ RNA was then isolated
according to the manufacturer's protocol followed by extraction and
precipitation according to standard protocols
(Ausubel et al, 1993). Five
micrograms of this poly A+ RNA were subsequently used to construct
the cDNA libraries.
Complement DNA Library Construction
A primary cDNA library of gonocytes and the population of As,
Apr, and Aal spermatogonia was constructed using the ZAP
Express cDNA Gigapack II Gold Cloning Kit, as described by the manufacturer
(Stratagene, La Jolla, Calif). These primary libraries were then amplified to
obtain a large, stable quantity of hightiter phage stock of the libraries. To
prevent underrepresentation of slow-growing clones, only one amplification
step was performed, as recommended by the manufacturer. To test the
heterogeneity of the clones within the libraries, 10 randomly isolated clones
from each library were digested using EcoRI and XhoI
endonucleases (both from Boehringer-Mannheim, Roche Molecular Biochemicals,
Mannheim, Germany). Digestion products were then separated by agarose gel
electrophoresis and analyzed.
Polymerase Chain Reaction
The polymerase chain reactions (PCRs) described in this paper were
performed using the following protocol. About 500 ng of purified plasmid DNA
was amplified in 50 µL of 1 x Goldstar reaction buffer, Goldstar
taq polymerase (2.5 U), forward primer
(5'-GCTCTAGAAGTACTCTCGAG-3'; 5 pmol), reverse primer
(5'-GATCCAAAGAATTCGGCACG-3'; 5 pmol) (all from Eurogentec,
Seraing, Belgium), dATP, dGTP, dCTP, dTTP (5 nmol each, Boehringer-Mannheim),
MgCl2 (1.5 mM), and 5% dimethyl sulfoxide. Amplification of the
inserts was performed using a Techne Dri-Block cycler (Techne Ltd, Cambridge,
United Kingdom) and the following scheme: 10 minutes of denaturation at
94°C and 5 minutes of primer annealing at 55°C; and 30 cycles at
72°C (3-minute extension), 94°C (1-minute denaturation), and 55°C
(1-minute annealing). This was followed by primer extension at 72°C for 10
minutes and cooling the reaction down to 4°C.
Synthesis of cDNA and RNA Probes
Nonradioactive digoxigenin (DIG) labeled cDNA probes representing either
the gonocyte library (gonocyte probe) or the spermatogonia library
(spermatogonia probe) were made as follows. Excision of plasmids from the
gonocyte and spermatogonia phage library was performed according to the
manufacturer's mass excision protocol (Stratagene). For this, 10 times the
quantity of the primary libraries was used to obtain plasmid mixtures that
correctly represented either library. These plasmid mixtures were further
purified according to standard protocols
(Ausubel et al, 1993). A PCR to
label and obtain all full-length inserts from the mixture of plasmids from
each library was then performed as described above, replacing 1.75 nmol dTTP
with an equal amount of DIG-labeled dUTP (Boehringer-Mannheim). PCR products
were then purified in phenol:chloroform (1:1) and precipitated in
acetate:ethanol.
To perform dot blot analysis, 32P (ICN, Costa Mesa, Calif) labeled cDNA probes were produced. PCR amplification was performed to obtain all full-length inserts from the mixture of plasmids from each library. These inserts were then used as templates in 32P labeling reactions using the Prime-it II Random primer labeling kit (Stratagene).
For in situ hybridization, DIG labeled RNA probes were produced from the isolated clones that were identified as containing differentially expressed genes. For this, the plasmids were linearized and RNA probes were synthesized using the DIG RNA labeling kit (Boehringer-Mannheim). T3 RNA polymerase was used for sense, and T7 RNA polymerase for antisense probe production.
Differential Screening of the Gonocyte cDNA Library
About 500 plaque forming units (pfus) from the gonocyte library were plated
with XL1 Blue MRF' cells and transferred to duplicate membranes (Hybond
N+ nylon membranes, Boehringer-Mannheim). These membranes were then
hybridized overnight at 68°C with 50 ng/mL of DIG labeled gonocyte or
spermatogonia probe (differential hybridization) according to the
manufacturer's instructions (DIG Detection Kit, Boehringer-Mannheim). After
stringent washes (2 x 5 minutes at room temperature with 2x
saline-sodium citrate [SSC]/0.1% sodium dodecyl sulfate [SDS] and 2 x 15
minutes at 68°C with 0.1x SSC/0.1% SDS), detection of the
hybridization signal was performed using the DIG Detection Kit
(Boehringer-Mannheim) and exposure of the membranes to x-ray film.
Clones that showed a stronger hybridization signal when hybridized to the gonocyte library compared to the hybridization signal when hybridized to the spermatogonia library were isolated and stored at 4°C in 1 mL SM buffer (0.1 M NaCl, 10 mM MgSO4 · 7H2O, 50 mM Tris-HCl, and 0.01% gelatin [w/v]) with 20 µL of chloroform until further analysis.
Southern Blotting to Rescreen and Purify the Isolated cDNA
Clones
Plasmids were excised from the isolated phages using the manufacturer's
single clone excision protocol (Stratagene). Subsequently, these cDNAs were
rescreened and purified from contaminating cDNAs using differential
hybridization analysis of Southern blots. Complementary DNAs that showed
consistent results with the differential screening during these procedures
were then selected for further analyses
(Figure 1).
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Dot Blot Analysis to Verify the Differential Abundance of the
Selected Clones in the Gonocyte Library
The differential abundance of the selected clones in the gonocyte library
was verified by dot blot analysis. For this, the clones, as well as a clone
containing human ß-actin, were spotted in triplicate on duplicate Hybond
N+ nylon membranes and hybridized overnight with 32P
labeled gonocyte or spermatogonia probes. After performing stringent washes,
the membranes were exposed to x-ray film. Densitometric analysis of this x-ray
film to normalize the results to ß-actin was performed using a Biorad
GS-700 Imaging Densitometer (BioRad, Hercules, Calif). The dot blot analysis
was repeated in 3 independent experiments.
Sequencing and Sequence Analysis
Single-run sequencing of the isolated clones was performed by Eurogentec
(Seraing, Belgium) using T7 primers. Sequences were then analyzed by alignment
to submitted GenBank sequences and the database of expressed sequence tags
(dbest) in Heidelberg, Germany (Altschul et
al, 1997).
In Situ Hybridization
To verify the expression of the isolated genes by gonocytes and A
spermatogonia in vivo, we hybridized these genes to sections of testes from
19-day-postcoital fetuses as well as to adult VAD and adult normal rats. For
this, testes were collected from 19-day-postcoital rats, adult VAD rats, and
adult normal rats. Tissues were then prepared and sectioned for in situ
hybridization as described by Wilkinson and Green
(1990). In situ hybridization
was performed as described by Braissant and Wahli
(1998) with minor
modifications. All aqueous solutions needed for in situ hybridizations were
treated with diethylpyrocarbonate (DEPC) or were made with DEPC-treated
deionized water. Sections of 5 µm were mounted on positively charged glass
slides (Superfrost/plus, Menzel Gläser, Braunschweig, Germany) and
air-dried overnight at 37°C. Before hybridization, sections were
deparaffinized, rehydrated, and postfixed with freshly prepared 4%
paraformaldehyde in PBS (w/v, 10 minutes). Slides were then incubated twice
for 15 minutes in PBS containing 0.1% active DEPC (v/v) under continuous
agitation. This was followed by an incubation in triethanolamine (0.1 M, pH
8.0) containing 0.25% acetic anhydride (v/v, 10 minutes under continuous
agitation) and an incubation in 5x SSC (15 minutes), all at room
temperature. Slides were prehybridized for 2 hours at 60°C with freshly
denatured hybridization mix containing 50% deionized formamide, 5x SSC
final and 1 µg/µL herring sperm DNA (Promega Corp, Madison, Wis).
Thereafter, the slides were incubated for 40 hours at 60°C with freshly
denatured hybridization mix containing either 10 ng/µL of sense or
antisense DIG labeled RNA probe. During prehybridization and hybridization,
slides were covered by a glass coverslip and stored in a box saturated with
50% formamide and 5x SSC. After hybridization, sections were washed in
2x SSC at room temperature (30 minutes), 2x SSC at 60°C (60
minutes), and 0.1x SSC at 60°C (60 minutes). Detection of the DIG
labeled RNA probe was performed as described by van Pelt et al
(1999) using mouse anti-DIG
monoclonal antibody (Boehringer-Mannheim) and diaminobenzidine (DAKO Corp,
Carpintera, Calif) as a substrate. Slides were counterstained with hematoxylin
and mounted.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Fourteen As, Apr, and Aal spermatogonia isolation experiments yielded a total of 10.3 x 106 As, Apr, and Aal spermatogonia from the testes of VAD animals. Two to 3 male VAD rats were used for each isolation experiment. The yield of As, Apr, and Aal spermatogonia per testis was on the average of 1.5 ± 0.67 (x105) As, Apr, and Aal spermatogonia, with a mean purity of 86% ± 3.4% (Table 1).
From these gonocytes and A spermatogonia, 6.1 and 23.5 µg, respectively, of poly A+ RNA was isolated. This poly A+ RNA was subsequently used to construct the cDNA libraries.
Complement DNA Library Construction
For each cDNA library, 5 µg of purified poly A+ RNA was used.
The primary cDNA libraries contained 3.1 x 105 and 4.3
x 105 pfus for the gonocyte and the spermatogonia library,
respectively. No blue plaques (vectors without insert) were detected after
blue/white screening of the libraries using isopropyl-beta-D-thiogalactopy
ranoside (IPTG) and X-Gal. These primary libraries were then amplified once.
Some characteristics of both cDNA libraries are summarized in
Table 1.
To test the heterogeneity of the libraries, 10 randomly isolated clones from each library were digested with XhoI and EcoRI endonucleases and analyzed by agarose gel electrophoresis. These clones all showed different digestion patterns after electrophoresis, varying in size from about 200 to about 3000 base pairs. This indicates that a diversity of cDNAs is present within the libraries.
Differential Screening of the Gonocyte cDNA Library and Rescreening
and Purification of the Isolated cDNA Clones
Five hundred plaques from the gonocyte library were transferred to
duplicate membranes and hybridized with cDNA probes made from either the
gonocyte or the spermatogonia library. Eighteen clones showed a relatively
stronger hybridization signal when hybridized with the gonocyte probe than
with the spermatogonia probe.
During rescreening and purification of these clones using Southern blot analysis, 7 clones consistently showed a stronger hybridization signal when using the gonocyte probe for hybridization. These clones were then selected for further analysis and sequencing.
Dot Blot Analysis to Verify the Differential Abundance of the
Selected Clones in the Gonocyte Library
The relative abundance of the 7 selected clones within our libraries was
verified using dot blot analysis. For this, we used ß-actin as a
standard. Densitometric analysis of the results revealed that after
normalization to ß-actin, the selected clones were again more abundant in
the gonocyte library compared with their presence in the spermatogonia library
(Figure 2).
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Sequence Analysis
The sequences of the 7 isolated cDNAs were aligned to sequences present in
GenBank and in the dbest. Four sequences showed an alignment to sequences that
are present in GenBank or in dbest that were of an unknown nature.
Furthermore, no structural motifs that might have given clues to their
function were found in these genes. The other clones showed a striking
alignment with ribosomal protein S15a (rat), succinate dehydrogenase (mouse),
and the 65-kd scaffolding subunit ( isoform) of protein phosphatase 2A
(PP2A-A; rat). These results are summarized in
Table 2. The size of the
isolated cDNAs ranged from about 500 to 2000 base pairs.
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In Situ Hybridization
Clones 4 and 9b represent housekeeping genes (ribosomal protein S15a and
succinate dehydrogenase, respectively), and are likely to be abundantly
expressed by all cell types of the testis. Therefore, these clones were not
used for in situ hybridization studies. RNA transcripts of the inserts of
clones 1, 8, 9a, 10, and 12 were hybridized to cross-sections of testes from
19-day postcoitum fetal, adult VAD, and adult normal rats. Using this
technique, we were able to verify the differences in expression of these genes
specifically by gonocytes and A spermatogonia in vivo. When using clones 1,
9a, 10, and 12, a clear hybridization signal was detected in gonocytes at 19
days postcoitum (Figure 3), whereas no signal, or no clear hybridization signal could be detected in adult
As, Apr, and Aal spermatogonia of VAD testes.
To exclude the possibility that these differences in gene expression are the
result of the VAD status of the animals used, we also performed in situ
hybridizations to sections of normal adult testes. Because As,
Apr, and Aal spermatogonia can be reliably recognized
only in adult testes during stages II to VII of the seminiferous epithelium,
these stages are shown in Figure
3. No staining, or hardly any staining of A spermatogonia during
any stage of adult spermatogenesis has been observed.
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In addition to the staining of the gonocytes, the expression of clone 12 was also detected in spermatids and spermatocytes from the pachytene stage and onward in the normal adult testis (Figure 3M). Also, the Sertoli cells at 19 days postcoitum showed a clear staining when using clones 1, 9a, 10, and 12. However, no clear staining was present in the Sertoli cells of adult VAD or normal testes.
The sense probe of clone 10 showed a nuclear staining of unknown nature that was not seen when we used the antisense probe of this clone. When transcripts of clone 8 were used for in situ hybridization studies, no specific hybridization signal was detected in testes of rats at 19 days postcoitum, or the mature VAD testes, or normal testes (results not shown). This is possibly because the sequence of this clone is present in several genes (Table 2).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Extensive analysis of the gene expression of gonocytes has been hampered by the low number of gonocytes per testis (Van Dissel-Emiliani et al, 1989) and the lack of an isolation procedure that yields highly purified populations of gonocytes that allows such studies. Previously, we developed a method to isolate quiescent fetal gonocytes with purities up to about 98% (Van den Ham et al, 1997). In this study, we performed several of these isolation experiments to obtain sufficient gonocytes to extract the poly A+ RNA needed to construct a cDNA library from these cells.
To identify genes that are involved in gonocyte development, we decided to compare the gene expression of the gonocytes with that of their direct descendants, the As, Apr, and Aal spermatogonia. Because it is not possible to isolate the As, Apr, and Aal spermatogonia from normal testes at any age without introducing a profound contamination with the more differentiated A spermatogonia, we isolated highly purified populations of As, Apr, and Aal spermatogonia from VAD animals. Only Sertoli cells, As, Apr, and Aal spermatogonia, and some preleptotene spermatocytes, are present in the seminiferous tubules of these animals (Van Pelt and de Rooij, 1990a). Fourteen isolation experiments yielded sufficient As, Apr, and Aal spermatogonia to extract the poly A+ RNA needed for constructing the spermatogonia cDNA library.
Both constructed primary cDNA libraries contained a large quantity of phages (>2.5 x 105 pfus). Furthermore, no blue plaques were detected after blue/white screening, indicating that clones without inserts were not present or were at least very rare. In addition, digestion of 10 randomly isolated clones from each library showed that large cDNAs with dissimilar digestive patterns were inserted in these clones. This indicates that our libraries are composed of a large variety of genes rather then the result of the insertion of only a few genes. Taken together, we conclude that the gene expression of the gonocytes and the As, Apr, and Aal spermatogonia is appropriately represented by our libraries. To our knowledge, no cDNA libraries from purified fetal spermatogenic stem cells or adult early A spermatogonia have been constructed before. These cDNA libraries now allow us to study the gene expression of two cell types that reside at the very origin of mature spermatogenesis.
Cell development is reflected as well as regulated by alterations in gene
expression. Thus, clones that are more abundant in the gonocyte library
(compared with their abundance in the spermatogonia library) may very well
contain genes that are involved in gonocyte development. In this study, we
compared the gonocyte cDNA library to the spermatogonia cDNA library by
differential screening. Dot blot analysis and normalization of the abundance
of the selected clones to ß-actin confirmed our differential screening
results. Seven out of the 500 screened clones of the gonocyte library were
consistently found to be more abundant in the gonocyte library than in the
spermatogonia library. After sequencing these clones and alignment of the
sequences to GenBank, clones 1, 8, 9a, and 10 appeared to encode genes that
are still of unknown nature. Clone 4 encodes ribosomal protein S15a, whereas
clones 9b and 12 subsequently encode for succinate dehydrogenase and the
isoform of the 65-kd regulatory A subunit of protein phosphatase 2A
(PP2A-A).
The differential expression of the clones was then also verified by in situ
hybridization studies using fetal, VAD, and normal adult testes. The latter
was used to exclude the possibility that the differences in gene expression
were the result of the VAD status of the animals used to construct the
spermatogonia library. Using in situ hybridization, we were able to
investigate the expression levels of the selected genes in individual cells in
vivo. These experiments confirmed that our differential screening results were
not due to differences in the gene expression of the small percentage of
contaminating cells in our libraries. The isolated clones encode genes that
clearly change in expression during the development of the gonocytes. In
addition, these genes are also expressed by fetal Sertoli cells but could not
be detected in the Sertoli cells of adult animals. Finally, clone 12, encoding
PP2A-A is expressed in spermatids and spermatocytes from the pachytene
stage and onward in the normal adult testis.
The result that the housekeeping genes succinate dehydrogenase and ribosomal protein S15a mRNA are more abundant in quiescent gonocytes compared with As, Apr, and Aal spermatogonia may indicate that gonocytes are metabolically more active than As, Apr, and Aal spermatogonia in the VAD testis, although the gonocytes are in mitotic arrest at the time of isolation (Clermont and Perey, 1957; Franchi and Mandl, 1964; Huckins and Clermont, 1968; Hilscher et al, 1974), whereas the As, Apr, and Aal spermatogonia of the VAD testis are slowly proliferating (Van Pelt et al, 1995). Fetal gonocytes may need an enhanced energy metabolism in order to support their development toward A spermatogonia.
Our results also indicate that PP2A-A is differentially expressed by
gonocytes compared with its expression in As, Apr, and
Aal spermatogonia. In vivo, PP2A-A is dimerized to the
catalytic C subunit of PP2A. However, the majority of the PP2A holo-enzyme
exists as a trimer by the recruitment of one of the many regulatory B subunits
(Cohen, 1989;
Kremmer et al, 1997). PP2A has
been described as playing a role in differentiation processes, among others
(Sasaki et al, 1993;
McCright et al, 1996;
Prinetti et al, 1997). For
example, treatment of HL-60 cells with okadaic acid (an inhibitor of PP2A)
renders these cells insensitive to differentiation by retinoic acid
(Morita et al, 1992). Our
results now indicate that PP2A may also contribute to the development of the
quiescent gonocytes to the As, Apr, and Aal
spermatogonia. Further studies on the role of PP2A during germ cell
development are currently being performed.
The remaining 4 clones that have been isolated aligned to sequences that have been submitted to GenBank but were of unknown nature. It would be interesting to investigate the nature and function of these products in general, and especially in the gonocytes, because they may be important for the development of gonocytes toward the As, Apr, and Aal spermatogonia.
Taken together, the gonocyte and the spermatogonia cDNA libraries offer exciting possibilities for studying the gene expression of the germ cells during spermatogenic stem cell development. In this study, differential screening of the gonocyte library has shown to be a powerful method for identifying candidate genes involved in gonocyte development. The role of the identified differentially expressed genes during germ cell development and the detection of additional differentially expressed genes in gonocytes and As, Apr, and Aal spermatogonia will be a subject for future studies.
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References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K. Current Protocols in Molecular Biology. New York: Wiley Interscience; 1993.
Bellve AR, Cavicchia JC, Millette CF, O'Brien DA, Bhatnagar YM, Dym
M. Spermatogenic cells of the prepuberal mouse. Isolation and morphological
characterization. J Cell Biol.
1977; 74:
68
-85.
Braissant O, Wahli W. A simplified in situ hybridization protocol using non-radioactively labeled probes to detect abundant and rare mRNAs on tissue sections. Biochemica. 1998; 1: 10 -16.
Clermont Y, Perey B. Quantitative study of the cell population of the seminiferous tubules of immature rats. Am J Anat. 1957; 100: 241 -268.[Medline]
Cohen P. The structure and regulation of protein phosphatases. Annu Rev Biochem. 1989; 58: 453 -508.[Medline]
De Rooij DG. Stem cells in the testis. Int J Exp Pathol. 1998;79: 67 -80.[Medline]
Dieckmann KP, Skakkebaek NE. Carcinoma in situ of the testis: review of biological and clinical features. Int J Cancer. 1999;83: 815 -822.[Medline]
Franchi LL, Mandl AM. The ultrastructure of germ cells in foetal and neonatal male rats. J Embryol Exp Morphol. 1964; 12: 289 -308.[Medline]
Hilscher B, Hilscher W, Bulthoff-Ohnolz B, Kramer U, Birke A, Pelzer H, Gauss G. Kinetics of gametogenesis. I. Comparative histological and autoradiographic studies of oocytes and transitional prospermatogonia during oogenesis and prespermatogenesis. Cell Tissue Res. 1974; 154: 443 -470.[Medline]
Huckins C, Clermont Y. Evolution of gonocytes in the rat testis during late embryonic and early post-natal life. Arch Anat Histol Embryol. 1968;51: 341 -354.[Medline]
Kemper CH, Peters PW. Migration and proliferation of primordial germ cells in the rat. Teratology. 1987; 36: 117 -124.[Medline]
Kremmer E, Ohst K, Kiefer J, Brewis N, Walter G. Separation of PP2A core enzyme and holoenzyme with monoclonal antibodies against the regulatory A subunit: abundant expression of both forms in cells. Mol Cell Biol. 1997;17: 1692 -1701.[Abstract]
McCright B, Rivers AM, Audlin S, Virshup DM. The B56 family of
protein phosphatase 2A (PP2A) regulatory subunits encodes
differentiation-induced phosphoproteins that target PP2A to both nucleus and
cytoplasm. J Biol Chem.
1996; 271:
22081
-22089.
Morita K, Nishikawa M, Kobayashi K, et al. Augmentation of retinoic acid-induced granulocytic differentiation in HL-60 leukemia cells by serine/threonine protein phosphatase inhibitors. FEBS Lett. 1992;314: 340 -344.[Medline]
Novi AM, Saba P. An electron microscopic study of the development of rat testis in the first 10 postnatal days. Z Zellforsch Mikrosk Anat. 1968;86: 313 -326.[Medline]
Prinetti A, Bassi R, Riboni L, Tettamanti G. Involvement of a ceramide activated protein phosphatase in the differentiation of neuroblastoma Neuro2a cells. FEBS Lett. 1997; 414: 475 -479.[Medline]
Sapsford CS. Changes in the cells of the sex cords and the seminiferous tubules during development of the testis of the rat and mouse. Austr J Zool. 1962; 10: 178 -192.
Sasaki K, Ikeda K, Nagai M, Shima H, Nagao M, Takahara J, Irino S. Protein phosphatase 2A alpha involvement in granulocytic differentiation of HL-60 cells. Hematol Oncol. 1993; 11: 1 -5.
Skakkebaek NE, Rajpert-De Meyts E, Jorgensen N, et al. Germ cell cancer and disorders of spermatogenesis: an environmental connection? APMIS. 1998;106: 3 -11.[Medline]
Van den Ham R, van Pelt AM, de Miguel MP, van Kooten PJ, Walther N, van Dissel-Emiliani FM. Immunomagnetic isolation of fetal rat gonocytes. Am J Reprod Immunol. 1997; 38: 39 -45.
Van Dissel-Emiliani FM, de Rooij DG, Meistrich ML. Isolation of rat gonocytes by velocity sedimentation at unit gravity. J Reprod Fertil. 1989;86: 759 -766.
Van Dissel-Emiliani FM, van Kooten PJ, Boer-Brouwer M, de Rooij DG, van der Donk JA. A monoclonal antibody recognizing a differentiation marker on rat gonocytes. J Reprod Immunol. 1993; 23: 93 -108.[Medline]
Van Haaster LH, de Rooij DG. Spermatogenesis is accelerated in the immature Djungarian and Chinese hamster and rat. Biol Reprod. 1993;49: 1229 -1235.[Abstract]
Van Pelt AM, de Rooij DG. Synchronization of the seminiferous epithelium after vitamin A replacement in vitamin A-deficient mice. Biol Reprod. 1990a; 43: 363 -367.[Abstract]
Van Pelt AM, de Rooij DG. The origin of the synchronization of the seminiferous epithelium in vitamin A-deficient rats after vitamin A replacement. Biol Reprod. 1990b; 42: 677 -682.[Abstract]
Van Pelt AM, de Rooij DG, van der Burg B, van der Saag PT,
Gustafsson JA, Kuiper GG. Ontogeny of estrogen receptor-beta expression in rat
testis. Endocrinology.
1999; 140:
478
-483.
Van Pelt AM, van Dissel-Emiliani FM, Gaemers IC, van der Burg MJ, Tanke HJ, de Rooij DG. Characteristics of A spermatogonia and preleptotene spermatocytes in the vitamin A-deficient rat testis. Biol Reprod. 1995;53: 570 -578.[Abstract]
Van Pelt AM, Morena AR, Dissel-Emiliani FM, Boitani C, Gaemers IC, de Rooij DG, Stefanini M. Isolation of the synchronized A spermatogonia from adult vitamin A-deficient rat testes. Biol Reprod. 1996; 55: 439 -444.[Abstract]
Wilkinson DG, Green J. In situ hybridization and the three-dimensional reconstruction of serial sections. In: Copp AJ, Cockroft DL, eds. Postimplantation Mammalian Embryos: A Practical Approach. Oxford, United Kingdom: Oxford University Press; 1990 : 155-171.
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