1 Department of Physiology and Pharmacology, State University of New York,
Health Science Center at Brooklyn, Brooklyn, New York 11203, USA
2 Department of Neurology, State University of New York, Health Science Center
at Brooklyn, Brooklyn, New York 11203, USA
3 Institute of Experimental Pathology/Molecular Neurobiology, University of
Münster, D-48149 Münster, Germany
4 Department of Biology, Queens College and Graduate Center of CUNY, Flushing,
New York 11367, USA
* Author for correspondence (e-mail: tiedge{at}hscbklyn.edu )
Accepted 13 December 2001
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Summary |
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Key words: Neuronal, Testicular RNA, Repetitive elements, Retroposition, Spermatogonia, Spermatogenic development
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Introduction |
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The original hypothesis of ID elements as markers for brain-specific gene
expression (Sutcliffe et al.,
1984) could not be substantiated (reviewed by
Chikaraishi, 1986
). Thus,
subsequently it has been proposed that BC1 RNA is the actual functional entity
(DeChiara and Brosius, 1987
),
and that its single gene is in fact a master gene from which ID elements were
derived, either directly or through intermediates, by way of retroposition
(Kim et al., 1994
;
Shen et al., 1997
). In this
mechanism, an RNA is first reverse transcribed into cDNA, which is then
inserted into genomic DNA. A fundamental question that remained to be
addressed in this scenario was whether BC1 RNA is in fact expressed in germ
cells: only retroposition in such cells would result in germ-line transmission
and amplification in the genome (Kim et
al., 1994
; Shen et al.,
1997
).
It was important, for this reason, to examine expression of BC1 RNA in germ cells. We report here that BC1 RNA is expressed at significant levels during early phases of male germ cell development. These results validate the retroposition model for the origin of repetitive ID elements. At the same time, they indicate that aspects of protein synthesis in male germ cells, such as RNA localization and regulation of local translation, may be subject to BC1-mediated control during spermatogenic development.
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Materials and Methods |
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Separation of mouse testis cells was performed as described earlier
(Wolgemuth et al., 1985). This
preparation yields enriched populations of spermatogenic cells in meiotic
prophase (mostly pachytene), of early (round) spermatids, and of cytoplasmic
fragments of elongating spermatids and residual bodies
(Ponzetto and Wolgemuth, 1985
;
Wolgemuth et al., 1985
;
Zakeri et al., 1988
).
Isolation and analysis of RNA
Total and poly(A)+ RNA were isolated as described before
(Zakeri et al., 1988;
Chen et al., 1997a
;
Chen et al., 1997b
). RNA was
fractionated on agarose-formaldehyde gels, transferred to GeneScreen Plus
membranes (New England Nuclear, Boston, MA), and immobilized by
UV-illumination (Church and Gilbert,
1984
). Integrity as well as equal loading and transfer of RNA was
verified by ethidium bromide staining or by UV illumination of the membrane
subsequent to transfer (Sambrook and
Russell, 2001
). Membranes were used for hybridization only if 28S
and 18S rRNA bands were of equal intensities in all loaded lanes. Membranes
were then hybridized to 32P-endlabeled oligodeoxynucleotide probe
HT005 (Chen et al., 1997b
).
This probe is complementary to the 60 3'-most nucleotides of BC1 RNA
(DeChiara and Brosius, 1987
).
It was hybridized at 42°C in 1 M NaCl, 0.5 M Tris-HCl (pH 7.5), 5x
Denhardt's reagent (Sambrook and Russell,
2001
), 1% sodium dodecyl sulfate (SDS), 0.1 mg/ml yeast tRNA. The
membranes were washed three times at 55°C in 0.5x SSC (1x SSC
is 0.15 M NaCl, 0.015 M sodium citrate) and 0.1% SDS for 30 minutes.
In situ hybridization
Freshly removed testis tissues were quick-frozen in liquid nitrogen and
embedded in TissueTek OCT embedding medium (Sakura Finetek, Torrance, CA).
Tissue sections, prepared at 10 µm thickness on a Bright Microtome Cryostat
(Hacker, Fairfield, NJ), were collected on gelatin and poly-L-lysine coated
microscope slides (Tiedge,
1991).
Plasmid pMK1 (Tiedge et al.,
1991) was used to generate RNA probes for the detection of BC1 RNA
in tissue sections. 35S-labelled probes were transcribed from
linearized templates, using T3 (for sense strand) or T7 (for antisense strand)
RNA polymerase. In situ hybridization experiments were performed as described
previously (Tiedge, 1991
;
Tiedge et al., 1991
). Prior to
pre-hybridization, tissue sections were fixed with 4% formaldehyde (made
freshly from paraformaldehyde) in 0.1 M sodium phosphate buffer pH 8.0.
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Results |
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We used in situ hybridization in our initial approach to identify and localize BC1 RNA in male gonads. BC1 expression patterns were highly distinctive in mouse testis (Fig. 1). Labeling signal indicating the presence of BC1 RNA was seen concentrated in the basal (peripheral) layers of seminiferous tubules. Highest relative BC1 levels were detected in the outermost periphery. These layers are rich in spermatogonia but also contain other cell types such as non-spermatogenic Sertoli cells and, during epithelial stages VI to VIII (Fig. 1A,B), significant numbers of preleptotene spermatocytes. BC1 expression levels were seen decreasing in a peripheral-to-central gradient in seminiferous tubules. Labeling in layers adjacent to basal layers (rich in spermatocytes) was thus weaker than in basal layers. Little if any signal was detectable in central adluminal regions that are rich in elongating spermatids (Fig. 1A-D). Equivalent results were obtained with rat testis (data not shown). No specific BC1 labeling signal was detectable in either rat or mouse epididymis (not shown).
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The distribution of the labeling signal in seminiferous tubules is
consistent with the notion that BC1 RNA is expressed at highest relative
levels in spermatogonia and early spermatocytes, but disappears from germ
cells as these progress through subsequent spermatogenic development. At
times, particularly during epithelial stages VI and VII, remarkably strong
labeling was observed concentrated in distinct patches, typically overlying
clusters of several neighboring cells along the extreme tubular periphery
(Fig. 1C-F). Location, light
nuclear counterstain, and a relatively large size would provisionally identify
such cells as Aal spermatogonia (aligned type A spermatogonia).
These are undifferentiated spermatogonia (proliferating predominantly through
stages VII to XII of the spermatogenic cycle) that are aligned in a chain-like
fashion and are connected by intercellular cytoplasmic bridges (reviewed by
De Rooij, 1998;
De Rooij and Grootegoed,
1998
). Such syncytial arrangements are considered important for
the synchronization of subsequent spermatogonial differentiation
(De Rooij, 1998
). While the
intensity of the labeling signal over such cellular clusters typically gives
them a striking appearance, it should be emphasized that these cells
constitute a clear minority among all labeled cells. Thus, the bulk of
BC1-expressing cells is likely to consist of other types of spermatogonia
and/or spermatocytes (e.g. preleptotene, see above), even though relative
expression levels per cell may not be quite as high.
These data are potentially directly relevant to the function of BC1 RNA in germ cells. However, the results described above required substantiation because light microscopic in situ hybridization cannot provide the resolution necessary to ascribe the labeling signal unambiguously to a particular cell type. Thus, in the above experiments, no definitive distinction could be made between spermatogonia and spermatocytes on one hand, or between spermatogenic and non-spermatogenic cell types (e.g. Sertoli cells) on the other. It was therefore necessary to ascertain the presence of BC1 RNA in spermatogonia, spermatocytes, and/or other testicular cells by using additional experimental approaches.
Expression of BC1 RNA in testis is developmentally regulated
In developing prepubertal testis, different spermatogenic cell types make
their initial appearances in sequential order. In murine testis, meiosis does
not usually begin until after postnatal day 8, prior to which date most
seminiferous tubules contain spermatogonia (and Sertoli cells) but not
subsequent germ cell types (Nebel et al.,
1961; Bellvé et al.,
1977
). However, it should be cautioned that because of strain
differences, the precise timing may be variable
(Kluin et al., 1982
). By
postnatal day 17, seminiferous tubules contain (in addition to spermatogonia)
spermatocytes at various phases of meiotic development. Spermatids typically
first appear after postnatal day 17. We took advantage of this well-defined
temporal progression to establish the regulation of BC1 expression in
developing testis, and to identify BC1-expressing cell types at various
developmental stages.
We examined mouse testes from animals between postnatal days 7 and 8 (p7/8; spermatogenic cell types represented: mostly spermatogonia), from animals at postnatal day 17 (p17; spermatogenic cell types represented: mostly spermatogonia and spermatocytes), and from adult animals (all spermatogenic cell types represented). Northern hybridization with a probe specific for BC1 RNA revealed that relative BC1 expression levels were highest at p7/8, and showed a substantial decrease between then and adulthood (Fig. 2). The strong BC1 hybridization signal obtained with testes from p7/8 animals indicates that developing spermatogonia express significant amounts of BC1 RNA, although we cannot rule out contributions from early spermatocytes that may have been present at this time. The sharp decrease in BC1 signal intensities between postnatal day 17 and adult age is most probably due to dilution of BC1 expression in testis as a result of increasing development of non-expressing cell types. However, a simultaneous decrease of BC1 RNA levels in expressing cell types is also possible.
|
These data were confirmed and extended by in situ hybridization (Fig. 3). The earliest time point analyzed was postnatal day 7 (p7) (i.e. slightly earlier than in the above northern experiments). A robust BC1 hybridization signal was seen in seminiferous tubules of this developmental stage (Fig. 3A,B). Care was taken to ascertain cell types present in the tubules screened. We detected Sertoli cells and spermatogonia, but not subsequent types of spermatogenic cells, at this developmental stage. The results therefore indicate that BC1-expressing spermatogenic cells at this developmental stage are spermatogonia. The BC1 labeling pattern in p7 seminiferous tubules was heterogeneous as a significant percentage of cells in any given tubule remained unlabeled. These unlabeled cells are presumably non-spermatogenic Sertoli cells that do not express BC1 RNA (see below). Nonetheless, both northern hybridization and in situ hybridization confirmed that, compared with adult testis, average signal intensities were substantially higher at the end of the first postnatal week, thus suggesting rather high BC1 copy numbers per cell in expressing spermatogonia. At p17, labeling remained strong, but the more centrally located cells (most likely spermatocytes) exhibited a much lower BC1 hybridization signal (Fig. 3C). In adult testis, overall BC1 labeling signals were lower than at postnatal day 17: signal intensities remained low in adluminal tubular regions, but they also decreased in peripheral regions (Fig. 3D; please note that autoradiographic exposure time was three times longer for Fig. 3D than for Fig. 3A-C).
|
In summary, the results obtained with developing testes suggest that BC1 RNA is initially expressed in spermatogonia at rather robust levels. In subsequently differentiating cell types, levels of BC1 RNA appear to decrease gradually until little or no labeling remains detectable in elongating spermatids. In addition, expression levels in peripherally located BC1-positive cell types were observed to decrease during the late phase of postnatal development, indicating that BC1 expression in testis is not only cell-type specific, but also developmentally regulated.
BC1-expressing cells in testis are identified as spermatogonia and
spermatocytes
The morphological and developmental data point to spermatogonia and
spermatocytes as the main BC1-expressing cell types in testis. However, for
lack of resolution, they do not allow us to discriminate these cell types from
non-germinal ones. To establish whether BC1 RNA is in fact expressed in germ
cells and/or in non-spermatogenic cells in testis, we used germ-cell deficient
testes obtained from homozygous progeny (WV/W) of W series mutant
mice (Zakeri and Wolgemuth,
1987). Gonads from such homozygous animals are virtually devoid of
germ cells (Coulumbre and Russell,
1954
; Mintz and Russell,
1957
). We analyzed WV/W mutant testes for the presence
of BC1 RNA by both northern and in situ hybridization.
Northern hybridization revealed a robust signal in testis from wildtype (+/+) mice (Fig. 4E). In germ-line deficient WV/W animals, in contrast, a hybridization signal was barely detectable. These results were confirmed by in situ hybridization. With germ cells absent, non-germinal cells are located peripherally in mutant seminiferous tubules. Low-level labeling was observed over such cells, but labeling intensities here were not significantly higher than over central lumina that are devoid of cells (Fig. 4A-D). We conclude that little or no BC1 RNA is expressed in non-germinal cells of WV/W mutant testis, and that BC1 expression in wildtype testis is therefore attributable to germ cells.
|
These results, together with the developmental data and the fact that the
highest relative BC1 expression levels were observed in the extreme periphery
of wildtype seminiferous tubules, support the notion that spermatogonia and
spermatocytes are the most prominent BC1-expressing cell types in testis. To
test this hypothesis, and to evaluate the respective relative contributions to
BC1 expression by different types of germ cells, we analyzed enriched
populations of testicular cells at specific stages of spermatogenesis. Methods
for separation of such cells have previously been described and are routinely
used by us (Ponzetto and Wolgemuth,
1985; Wolgemuth et al.,
1986
; Zakeri et al.,
1988
). Using this approach, cells were separated into enriched
populations of spermatocytes in the prophase stage of meiosis (mainly
pachytene), of early (i.e. round) spermatids, and of cytoplasmic fragments of
elongating spermatids and residual bodies. RNA isolated from these fractions
was then analyzed by northern hybridization.
BC1 RNA was detectable at significant levels in total RNA and in
poly(A)+ RNA from entire testis, but not in poly(A)- RNA
(Fig. 4F). BC1 RNA is known to
contain a central A-rich region, and previously it has been identified in the
poly(A)+ RNA fraction from rat neural tissues
(DeChiara and Brosius, 1987).
Total RNA from spermatogenic cells in meiotic prophase (predominantly
pachytene spermatocytes) showed a significantly lower BC1 RNA hybridization
signal than total RNA from entire testis
(Fig. 4F). Little or no BC1
signal was observed with total RNA from early spermatids and total RNA from
elongating spermatids/residual bodies. In these latter cases, the
hybridization signal was barely above background
(Fig. 4F), confirming that
spermatids contain little if any BC1 RNA.
In summary, the combined results of these and previous experiments indicate that BC1 expression in seminiferous tubules is to a significant part attributable to spermatogonia, with additional contributions from spermatocytes. The identification of spermatogonia as a prominent BC1-expressing cell type in testis also supports our original notion (see above) that the clustered, most strongly labeled cells observed at the extreme periphery of seminiferous tubules are in fact syncytial ensembles of spermatogonia. These results are of direct relevance for the functional role of BC1 RNA in male germ cells, as will be discussed below.
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Discussion |
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Germ-line retroposition
By showing that BC1 RNA is expressed in spermatogenic germ cells, we
established what has until now been a missing link between BC1 RNA and
repetitive ID elements. ID elements, which show a high degree of sequence
similarity to the 5' domain of BC1 RNA, form a subtype of short
interspersed repetitive elements (SINEs) that are distributed at high but
divergent copy numbers throughout rodent genomes
(Sapienza and St-Jacques,
1986; Kass et al.,
1996
). Our results thus provide prerequisite support for the
theory that ID elements can be generated by germ-line retroposition of BC1 RNA
(DeChiara and Brosius, 1987
;
Deininger et al., 1992
;
Kim et al., 1994
;
Shen et al., 1997
;
Aleman et al., 2000
). Following
self-priming through association of the 3' terminal series of U-residues
with the central A-rich region (Kim et
al., 1994
; Shen et al.,
1997
), BC1 RNA becomes a substrate for cellular or viral reverse
transcriptase (Brosius and Tiedge,
1996
). Reverse transcription of the BC1 5' domain and
subsequent integration into germ-line DNA can thus account for the generation
of high numbers of ID elements in the genome. The fact that BC1 RNA is
expressed in male germ cells, as reported here, indicates that germ-line
retroposition and transmission of the 5' domain are potentially ongoing
processes in modern rodents, thus continuing to contribute to genomic
diversification (Brosius,
1999
). Furthermore, this process is likely to be efficient given
the rather robust steady-state levels of BC1 RNA observed in spermatogonia.
Although germ-line retroposition will often prove neutral or even deleterious,
depending on the site of genomic integration, insertion of 5' BC1
domains into target gene sites corresponding to untranslated mRNA regions may
in certain cases add functionality to such transcripts. `Retroposed
functionality' may encompass altered RNA stability, translatability or
localization competence, hypotheses that we plan to address in future
research.
Cell-type specific expression of BC1 RNA in the course of
spermatogenic development
Spatiotemporal BC1 expression patterns in seminiferous tubules are highly
distinctive. Results with germ-cell deficient animals indicate that BC1 RNA is
not expressed in Sertoli or other non-spermatogenic cells in testis. Early
onset of BC1 expression in p7 testes documents substantial amounts of the RNA
in spermatogonia. Developmental analysis, cell separation experiments and in
situ hybridization further identify BC1 RNA in spermatocytes, albeit at lower
average levels. In round and elongating spermatids, the RNA is not detectable
at significant levels. Taken together, these results therefore suggest that
BC1 RNA is expressed at high levels in spermatogonia (as well as in cells
directly derived from them, i.e. preleptotene spermatocytes). By the time
spermatogenic development has reached the pachytene spermatocyte stage, BC1
expression levels have significantly decreased. BC1 RNA disappears from male
germ cells by the time development has proceeded to the early spermatid
stage.
Since spermatocytes derive from spermatogonia, the origin of BC1 RNA in the
former may be explained by `carry-over' from the latter. Alternatively, BC1
RNA may still be transcribed in spermatocytes; if so, transcription would have
to be assumed to proceed at gradually reduced levels (and/or be offset by an
increasingly higher rate of degradation), particularly in pachytene
spermatocytes. At any rate, BC1 transcription ceases, and pre-existing BC1 RNA
is eliminated, by the end of spermatocyte development at the latest. BC1 RNA
levels remain negligible during spermiogenesis (the developmental interval
spanning differentiation from early spermatids to spermatozoa), even though
RNA synthesis is known to continue about halfway through this period
(Kierszenbaum and Tres, 1978).
BC1 expression is thus a hallmark of early but not late stages of
spermatogenic development.
Functional relevance of BC1 expression in spermatogonia and
spermatocytes
The characteristic and specific expression of BC1 RNA in male germ cells,
in particular in spermatogonia and early spermatocytes, indicates a functional
role in spermatogenic development. In neurons, BC1 RNA has been suggested to
participate in the regulation of translation-related processes in postsynaptic
microdomains (reviewed by Brosius and
Tiedge, 2001). Translation regulation also plays an important role
in the modulation of gene expression in male germ cell development. A large
number of mRNAs have been reported to be subject to translational control in
murine testis (reviewed by Schäfer et
al., 1995
). In seminiferous tubules, overall protein synthetic
activities are highest in basal layers, decreasing in a peripheral-to-central
gradient (Monesi, 1965
;
Dadoune et al., 1981
) in a
fashion similar to the pattern of BC1 expression.
Since general transcription ceases about midway through spermiogenesis,
protein synthesis in the late phase of spermiogenesis can be achieved only by
translation from stored mRNAs that have been transcribed previously
(Schäfer et al., 1995).
However, in view of our results that BC1 RNA is virtually undetectable in
spermatids, a direct role in translation regulation during late spermiogenesis
must appear unlikely. Conversely, high BC1 expression levels in spermatogonia
and early spermatocytes clearly point to a more prominent role of the RNA in
these cell types. It is notable in this context that at times strikingly high
BC1 expression levels were observed in clusters of neighboring spermatogonia.
Such paired (Apr) or aligned (Aal) A spermatogonia are
undifferentiated spermatogonia, derived from single A (As)
spermatogonia through several rounds of mitosis (reviewed by
De Rooij, 1998
;
De Rooij and Grootegoed,
1998
). Forming pairs/chains, Apr/Aal
spermatogonia of the same clonal origin remain connected through intercellular
cytoplasmic bridges, allowing for the intercellular transport of cytoplasmic
components. It is assumed that such interconnectivity between Aal
spermatogonia is required for the exquisite synchronization of subsequent
differentiation into B spermatogonia and spermatocytes
(De Rooij, 1998
). However, the
mechanisms underlying synchronous spermatogonial differentiation, and the
possible role of cytoplasmic interconnectivity in such mechanisms remain
unclear.
In nuerons, BC1 RNA is specifically transported to dendritic microdomains
(Muslimov et al., 1997) where
it has been suggested to operate in the modulation of local protein synthesis
(Brosius and Tiedge, 2001
). We
suggest that the synchronization of spermatogonial differentiation is a
translation-controlled process that requires intercellular molecular
communication between spermatogonia. We further propose that BC1 RNA may be a
mediator of such coordinated intercellular modulation at the translational
level, in an activity that may necessitate movement of this RNA (and possibly
other RNAs and/or proteins) through spermatogonial cytoplasmic bridges. Since
BC1 RNA remains detectable, albeit at gradually decreasing levels, in
spermatocytes up to the pachytene stage, it is certainly possible that the RNA
continues to be operational in early spermatocytes. Our suggestions are
conjectural at this time but establish a testable hypothesis that can be
addressed in future research. We find it noteworthy at this point that more
than 100 years ago, Wilhelm His was one of the first to comment on
similarities between developing male germ cells and neurons
(His, 1890
), a prescient
observation that would be extended to the molecular level by the
identification of equivalent RNA transport in both cell types.
Intercellular transport of RNAs and proteins between spermatogenic cells
has previously been suggested to subserve an important role in male germ cell
development (Willison and Ashworth,
1987). Movement of RNA has also been reported to occur between
post-meiotic spermatids, where it is assumed to serve a different purpose,
namely, to facilitate the sharing of gene products among genotypically
different haploid cells (Morales et al.,
1998
). It should also be noted that other small RNAs
(lin-4 and let-7, no apparent relationship with BC1 RNA)
have been implicated in the regulation, through translational modulation, of
developmental timing in C. elegans
(Lee et al., 1993
;
Pasquinelli et al., 2000
;
Reinhart et al., 2000
). There
is thus increasing evidence for roles of, and interplay between, small RNAs,
RNA transport and translation regulation in various developing eukaryotic
systems.
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Acknowledgments |
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