1 Institute of Reproductive and Developmental Biology, Imperial College Faculty of Medicine, Hammersmith Campus, Du Cane Road, London W12 ONN, 2 Institute of Human Genetics, The International Centre for Life, Central Parkway University of Newcastle-Upon-Tyne, NE1 3BZ, UK and 3 The Biological Imaging Center, Beckman Institute 139-74, California Institute of Technology, Pasadena, CA 91125, USA
4 To whom correspondence should be addressed: Email: readhead{at}.caltech.edu
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Abstract |
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Key words: GATA-1/gonocytes/mouse spermatogenesis/proliferating cell nuclear antigen/RNA binding motif
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Introduction |
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The ensuing multi-step differentiation of spermatogonia depends on specific cellular interactions within the seminiferous tubules as well as on local environmental factors, and leads to the production of spermatozoa (Berruti, 1998). Spermatogonia exist as a heterogeneous group of cells of different subtypes. They are derived from primitive spermatogonial stem cells (As), which undergo an asymmetric mitosis to form the subsequent type A14, intermediate, and type B spermatogonia. The As are renewed, thus maintaining their own numbers throughout the life of the animal (de Rooij and Russell, 2000
). In the mouse, meiosis is initiated at postnatal day 10 (P10) and leads to the differentiation of the type B spermatogonia into primary spermatocytes (Bellve et al., 1977
, 1998). The spermatocytes proceed through meiosis and differentiate into haploid spermatids. The main visible changes during spermatogenesis occur via a dramatic cytoplasmic remodelling of the spermatid, i.e. the process of spermiogenesis. This phase culminates in the formation of mature spermatozoa (Johnson and Everitt, 2000
). The number of spermatogonial stem cells in the mouse testicle is
35 000 (de Rooij and Grootegoed, 1998
) and this population is responsible for the initiation and maintenance of spermatogenesis (Russell and Griswold, 2000
). Isolation and characterization of these cells has been carried out predominantly though antibody staining and more recently through flow cytometric analysis (Kubota et al., 2003
). Although spermatogonial stem cells have proven difficult to isolate, they have important implications for several areas of research. These include: the generation of transgenic animals (Brinster and Zimmermann, 1994
), infertility treatments (Mahato et al., 2000
), and preservation of fertility in male cancer patients (Aslam et al., 2000
). For all these reasons, molecular analysis of germ cells has important applications.
Several molecular markers are associated with testicular cells. The proliferating cell nuclear antigen (PCNA) is a 36 kDa auxiliary protein to DNA polymerase- that is required during DNA replication (Jaskulski et al., 1988
) and nucleotide excision repair (Nichols and Sancar, 1992
). It also interacts with cyclin dependant kinases, which are associated with cell cycle control (Xiong et al., 1992
). PCNA displays a multifunctional role through interactions with other cell cycle proteins (Fukuda et al., 1995
) and is expressed variably at different stages of the cell cycle. Elevated levels appear in the nucleus during late G1 phase, peak at S phase, and decline after the G2 phase (Miyachi et al., 1978
). PCNA has been used extensively in the identification of proliferating spermatogonia and spermatocytes in a number of species, notably human (Steger et al., 1998
), rhesus and cynomolgus monkeys (Schlatt and Weinbauer, 1994
) and rat (Godlewski et al., 1999
).
RNA binding motif (RBM) is a highly conserved nuclear protein encoded by the azoospermia factor (AZFb and AZFc) regions of the human Y chromosome (Elliott et al., 1997). Six subfamilies (RBMIVI) of the RBM family of genes have been described; however analyses of cDNA libraries indicate that only the members of the RBMI subfamily encode functional proteins (Chai et al., 1997
). Various studies suggest a possible role for RBM in the storage, metabolic stability, or transport, of mRNA from the nucleus during spermatogenesis. RBM may also be important for mRNA translation (Hecht, 1998
). It has been used as a marker for germ cells in testicular biopsies from infertile men and those with possible germ-cell neoplasia (Lifschitz-Mercer et al., 2000). These studies show that RBM displays germ-cell specific expression in fetal, pubertal and adult human testicular tissue. RBM has also been detected using semi-quantitative RTPCR within the mouse testis during development. Its expression peaks at E15.5 post coitum, and P4 and P14 post partum (Elliott et al., 1996
).
The transcription factor GATA-1 is a member of a family of transcription factors, GATA-1 to 6, which all have a consensus zinc finger DNA binding motif, WGATAR. It is a fundamental regulator of genes in haematopoietic cell lineages (Ito et al., 1993). GATA-1, originally identified in erythroid, megakaryocytic, and mast cell types (Evans and Felsenfeld, 1989
), has since been found within the murine seminiferous tubules, expressed specifically in the Sertoli cell lineage. Furthermore GATA-1 expression in Sertoli cells displays both a developmental stage and spermatogenic cycle specific expression. Yomigida et al. (1994)
have demonstrated that GATA-1 expression is initiated during the first wave of spermatogenesis in the prepubertal mouse and is then synthesized synchronously during the spermatogenic cycle in the mature adult testis. GATA-1 expression is first seen at approximately day P7 (Yomigida et al., 1994
), and later in development it is expressed only in adult Sertoli cells of seminiferous tubules at stages VIIIX. Recent in vitro studies have shown that GATA-1 has a role in the transactivation of the inhibin-
and the inhibin/activin-
-B-subunit genes, important for regulating testicular function (Feng et al., 1998
; Feng et al., 2000
).
This study was designed to investigate the dynamics of neonatal development in the mouse testis using these specific markers. The three markers togetherGATA-1, PCNA and RBM were used to identify the mitotic and migration stages that germ and Sertoli cells undergo within the seminiferous tubules. Immunohistochemistry, semi-quantitative and quantitative RTPCR (QRTPCR) were used to follow the expression patterns of these genes.
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Materials and methods |
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The experiments were approved according to the Animals (Scientific Procedures) Act 1986 (UK) and the Guiding Principles in the Care and Use of Animals was adhered to (DHEW Publication, NIH, 80-23). Tissue was obtained and fixed in neutral buffered formalin, processed and paraffin embedded using standard protocols.
RBM
Five-micrometer sections were dewaxed and rehydrated for 2 min in Histoclear (National Diagnostics, UK), 100% and 70% ethanol. The standard antigen retrieval technique was carried out by microwaving the tissue for 20 min in 0.01 M citrate buffer, pH 6.0. After cooling, the sections were blocked with 0.3% H2O2 in methanol to quench the endogenous peroxidase activity. After washing twice with phosphate-buffered saline (PBS), the sections were preincubated with 0.01% blocking solution of goat serum for 1 h at room temperature. Sections were then incubated with diluted primary antibody (rabbit anti-mouse RBM; 1:100) at 4°C overnight in a humidified chamber. The following day, sections were washed three times in PBS and incubated with prediluted secondary antibody (goat anti-rabbit immunoglobulin G, Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature. After three washes in PBS, the ABC (Streptavidinhorseradish peroxidase, Santa Cruz Biotechnology) reagents were added for 45 min followed by three washes in PBS. Staining was detected using the diaminobenzidine (DAB) chromogen system (Santa Cruz Biotechnology) following the manufacturer's instructions. The sections were counterstained with haematoxylin (Zymed, San Francisco, CA) and dehydrated and mounted using DePeX medium (BDH, Gurr).
GATA-1 staining
Antigen retrieval was carried out using a 55 mM glycine solution for 10 min under standard conditions. After blocking peroxidase activity with 0.1% H2O2 in methanol, sections were preincubated with 0.01% goat serum for 1 h at room temperature. Sections were incubated overnight with diluted primary antibody (rat anti-mouse GATA-1; 1:100) and for 1 h with prediluted secondary antibody, goat anti-rat antibody (both from Sigma). The standard development and mounting system was used as before.
PCNA
Sections were treated as described above as per GATA-1 staining but antibody staining was carried out with an anti-PCNA kit (Zymed) following the manufacturer's protocol.
Immunohistochemical controls
To ensure that staining was specific, the primary antibody was omitted on control sections which were incubated in PBS alone. The remainder of the protocol was followed as described for each antibody above.
Semi-quantitative RTPCR
Total RNA was extracted from testis tissue of mice aged between days 0 and 14, and weeks 3, 6, 8, 16 and 36 using Trizol reagent (Gibco) according to the manufacturer's instructions. cDNA templates were made using oligo-dT and reverse transcriptase (Promega, Southampton, UK). The standard buffer, enzyme and RNase inhibitor were used as supplied by Promega and the tubes were incubated at 37°C for 1 h before heating to 95°C for 5 min. One microlitre of the cDNA templates was used in each PCR.
The primer sets used to detect PCNA expression were: 5'-GATGTGGAGCAACTTGGAAT and 5'-AGCTCTCCACTTGCAGAAAA and for RBM expression: 5'-GTGGTCCTTCATGTGAAGGG and 5'-CTCGCCCTCTTAGTCCAGTA. Annealing temperatures of 53 and 59°C were used respectively and the final products were 160 bp and 179 bp on an agarose gel. DNA controls were carried out to ensure the results of the product were specific to RNA, with a reverse transcriptase negative control. Sets of primers for -actin were used as a positive internal control.
Real-time QRTPCR
Total RNA was isolated and cDNAs made as previously decribed. Reactions of cDNA were set in duplicate. The amplification reaction contained 1 µl of the cDNA for each time point, 35 µl of TaqMan universal PCR mastermix (PE Applied Biosystems, Foster City, CA), 0.38 µl of each 0.5 pmoles primer and 1 µl of TaqMan probe. Reactions were performed with an ABI PRISM 7700 sequence detection system (PE Applied Biosystems, Foster City, CA). RBM was compared against the internal control ribosomal gene L-19 throughout the study to determine RNA quality and cDNA quantity. RBM cDNA levels, measured in triplicate, were normalized to the mean value of the internal control.
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Results |
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RBM and GATA-1 are expressed in germ cells and Sertoli cells, respectively, during later postnatal development and adulthood (Days P8, 14 and adult)
Similar to earlier stages of development, the cells from P8 onwards of the seminiferous tubules showed a dark brown, punctate appearance on staining with anti-RBM. Type A spermatogonia were dark brown, and their large spherical nuclei became more obvious; they were consistently located close to the basement membrane. From P8, both type B spermatogonia and primary spermatocytes were deeply stained with anti-RBM antibodies (Figure 2A). The type B spermatogonia and spermatocytes were located more distant from the basement membrane than the type A spermatogonia (de Rooij and Russell, 2000). These cells also stained deeply with PCNA indicating their actively dividing state at this time (Figure 2A). Surrounding Sertoli cells, identified by irregular triangular shaped nuclei projecting toward the lumen, were only weakly stained with PCNA and not with RBM. The position and morphology of the RBM-positive stained cells on P14 showed that RBM was expressed in all pre-meiotic germ cells. There was no difference in expression in these cells or in primary spermatocytes. The same cells also stained positively for PCNA. Sertoli cells of adult mice over 4 months old did not show PCNA staining (data not shown). These results confirm that proliferative activity of Sertoli cells is high at birth, gradually decreasing up to day 17 post partum (Vergouwen et al., 1993
). Type A, type B spermatogonia, and primary spermatocytes displayed intensely stained nuclei with PCNA (Figures 2A and B). The spermatogonia and the early spermatocytes were stained with anti-RBM antibodies, however round and elongated spermatids did not appear to stain with either RBM or PCNA antibodies (Mahadevaiah et al., 1998
; Steger et al., 1998
; Lifschitz-Mercer et al., 2000). It was difficult to distinguish between Sertoli cells and germ cells in sections from older mice (P8+), because of increasing numbers of cell types during later development. However, this distinction was made easier since in those sections stained with RBM antisera, only germ cells were stained and somatic cells were negative (including Sertoli cells). These were compared to the sections stained with PCNA antisera where most cells (both Sertoli and germ cells) were stained positive. In order to test our ability to discriminate between the somatic and germ cells, serial sections from P8 onwards were stained with GATA-1 and were analysed in parallel with PCNA stained sections. These confirmed that these cells were correctly identified as Sertoli cells (Figures 2A and B). Again, consistent with the lack of RBM gene expression in Sertoli cells, no RBM staining was seen in sections of adult Sxra mice, which are Sertoli cell only.
Semi-quantitative RTPCR of PCNA and RBM
Semi-quantitative RTPCR analysis was made on total RNA from the testis in the neonatal period, during later development, and in adults of various ages. RBM expression was seen throughout development. This was low in the early neonate, increasing gradually throughout postnatal development. The highest levels were seen during the prepubertal period (36 weeks of age) (Figure 3). RBM expression levels appeared to be reduced between 68 weeks of age. It was completely absent in very old adults (>18 months) (data not shown). PCNA showed similar patterns of expression indicating that the highest level of testicular cell proliferation and expansion occurs just before puberty, around day 14
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Discussion |
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In our study, actively dividing cells were identified through staining with anti-PCNA antibodies. PCNA staining seen in Sertoli cells suggests that these cells proliferate most actively at birth. Proliferation and growth steadily decrease thereafter until division ceases around day P17 postpartum (Pelliniemi et al., 1993; Orth et al., 1998
). This is consistent with the changes in the ratios of cell types within the mouse testis. For instance, the proportion of Sertoli cells in the total population decreases from 84% to 3% from day 6 to day 84 post partum (Bellve et al., 1977
).
Our results show that on P0, gonocytes are located centrally in the sex cord, when only a small number express PCNA and so are proliferating. Immediately subsequent to this, high PCNA levels in germ cells occur at day P5, at which time spermatogonia start to undergo many mitotic divisions before the initiation of meiosis, thereby giving rise to primary spermatocytes (Cooke et al., 1998). Studies in the rat have shown that
7% of gonocytes resume mitotic activity while the rest make contact with the basement membrane before they start proliferating. Previous studies also imply that the first gonocytes to undergo proliferation do so at day P3 at the earliest (McGuiness and Orth, 1992
). Our results indicate that a small subset of gonocytes proliferate as early as day P0 prior to migration to the periphery of the tubule, whilst most gonocytes make contact with the basement membrane before resuming proliferative activity. We show that this occurs between days P2 and P5, rather than between P3 and P5 as suggested by the earliest work using gravitational cell separation carried out by Bellve et al., 1977
). Semi-quantitative RTPCR showed an overall increase in PCNA expression during the early postnatal period and a decrease during the prepubertal period and adulthood.
This extensive developmental study of gene expression confirms the findings of Elliott et al., 1997, 1998, 2000
) which showed that mouse RBM expression is confined to male germ cells and is not seen in the Sertoli cells. Similar results have been obtained for rat RBM (D.J.Elliott, unpublished data). While Elliott et al. and other investigators (Turner et al., 2002
; Fernandez-Capetillo et al., 2003
; Saunders et al., 2003
; Szot et al., 2003
; Lee et al., 2004
) have found RBM to be expressed only in germ cells in mice and humans, Osterlund et al., 2001
reported that an antibody specific to RBM1a produced staining within the nuclei of Sertoli cells in the human, but this effect was not as strong as it was in germ cell nuclei. At stages such as P04 during early neonatal life, the germ cells occupy only a small proportion of the seminiferous tubules and this is reflected by a reduced number of positive staining cells with the anti-RBM. At these stages of development, the Sertoli cells make up the bulk of the seminiferous tubules and if RBM were also expressed in Sertoli cells positive staining of these cells may be expected at these stages, which is not seen in our work (Figure 1). Secondly, our work on the Sxra mouse testis shows that there is no RBM staining in these Sertoli cell only testes. Thirdly, the RBM antibody used in this study was raised against a 25kDa peptide encoded by the SRGY box of the RBM gene, (Elliott et al., 1997
). Further evidence for the germ cell specific nature of RBM comes from compelling studies by Mahadevaiah et al. (1998)
where no RBM transcripts were detected by RTPCR in XXSxrb mouse testis, which lacks germ cells.
In our study, immunostaining showed RBM positive gonocytes, spermatogonia and primary spermatocytes in the developing mouse, however, in the 4-month old adult, RBM was confined to the type A spermatogonia. This finding in mice differs with that in humans where adult testicular biopsies from normal and azoospemia patients showed RBM positive spermatogonia, spermatocytes and round spermatids (Lifschitz-Mercer et al., 2000; Maymon et al., 2001). Chandley et al. (1994) reported high levels of RBM RNA in situ in pachytene spermatocytes in man, whilst low levels were detected in mice (Chandley et al., 1994). RBM expression may be switched off after spermatocyte formation in mice because of formation of the XY body, since during the formation of the XY body the Y chromosome is condensed (Richler et al., 1994
).
RBM expression is seen in the gonocytes on the day of birth of the mouse and expression levels increase throughout postnatal development, reaching peak levels in the juvenile between 17 and 21 days. These findings are in agreement with Elliott et al. (1996). Mahadevaiah et al. (1998)
also reported a steady increase in transcript levels as spermatogonia proliferate from birth to P14.5. They observed a marked drop as large numbers of non-transcribing pachytene spermatocytes appeared. We found that RBM levels dropped dramatically in the 56-day-old (8 week) mouse. When the ratio of RBM levels and RBM positive cells was calculated it showed that on average the level of RBM in germ cells was highest at P21 but that by P56 when RBM was confined to spermatogonial stem cells it had dropped to very low levels. As mentioned before, RBM is a marker of pre-meiotic cells and so this decrease in expression could be explained by the altered ratios of pre-meiotic and meiotic cells as the composition of the testis changes. However, our data suggest that the decrease in RBM expression seen is not solely due to a decrease in the number of cells expressing the protein, but rather due to a decrease in expression of RBM per cell, indicating that it may be down-regulated. These findings have not been shown before and pose an interesting question of why this should happen. This may indicate an altered nature of the stem cell in adults compared to juvenile mice.
It would be of interest to know whether there is a functional difference between: spermatozoa derived from spermatogonia with high levels of RBM and to those from spermatogonia with low RBM levels. Older animals (>56 days) have spermatogonia with very low levels of RBM. Recent studies have shown that paternal age may be associated with altered fertility due, in part, to age related changes in the methylation patterns of male germ cells (Oakes et al., 2003). Molecular changes, such as RBM levels, in the spermatogonial cells may be associated with altered fertility during aging.
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Acknowledgements |
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Submitted on May 15, 2003; resubmitted on July 22, 2004; accepted on September 20, 2004.
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