* Department of Molecular Genetics and Cell Biology, Department of Biochemistry and Molecular Biology, The Howard
Hughes Medical Institute, The University of Chicago, Chicago, Illinois 60637
Basonuclin is a zinc finger protein that was thought to be restricted to keratinocytes of stratified squamous epithelia. In epidermis, basonuclin is associated with the nuclei of mitotically active basal cells but not in terminally differentiating keratinocytes. We report here the isolation of a novel form of basonuclin, which we show is also expressed in stratified epithelia. Most unexpectedly, we find both forms in testis, where a surprising localization pattern was uncovered. While basonuclin RNA expression occurs in mitotically active germ cells, protein was not detected until the meiotic stage, where basonuclin localized to the appendage of the distal centriole of spermatocytes and spermatids. Near the end of spermiogenesis, basonuclin also accumulated in the acrosome and mitochondrial sheath surrounding the flagellum. Intriguingly, a perfect six- amino acid residue mitochondrial targeting sequence (Komiya, T., N. Hachiya, M. Sakaguchi, T. Omura, and K. Mihara. 1994. J. Biol. Chem. 269:30893-30897; Shore, G.C., H.M. McBride, D.G. Millar, N.A. Steenaart, and M. Nguyen. 1995. Eur. J. Biochem. 227: 9-18; McBride, H.M., I.S. Goping, and G.C. Shore. 1996. J. Cell. Biol. 134:307-313) is present in basonuclin 1a but not in the 1b form. Moreover, three distinct affinity-purified peptide antibodies gave this unusual pattern of basonuclin antibody staining, which was confirmed by cell fractionation studies. Our findings suggest a unique role for basonuclin in centrosomes within the developing spermatid, and a role for one of the protein forms in germ cell mitochondrial function. Its localization with the acrosome suggests that it may also perform a special function during or shortly after fertilization.
Mammalian testes consist of numerous seminiferous tubules, which converge toward common
ducts, i.e., the epididymis, through which mature
sperm travel to exit the male animal (Fig. 1 A). Within
each seminiferous tubule, the epithelial cells, referred to
as Sertoli cells, anchor and provide nourishment for the
developing spermatozoa (Fig. 1 A; Browder et al., 1991
As spermatogonia differentiate, they leave the basement membrane and transit towards the lumen of the tubule. During the early stages, the cells continue mitoses,
but when they differentiate first into primary and then secondary spermatocytes, they undergo two sequential meiotic divisions resulting in the production of haploid spermatids (Fig. 1 A). In the later stages of differentiation, referred to as spermiogenesis, spermatids mature into fully
motile sperm. An acrosomal cap forms at the anterior tip
of the nucleus and continues to spread over an entire half
(Fig. 1 B). The acrosome appears to be a storage vessel for
factors needed in the fertilization process. As this cap develops, the spermatid nucleus becomes elongated and flattened, and nuclear chromatin condenses and moves towards the periphery of the caudal hemisphere. A cylinder
of microtubules, referred to as the manchette, assembles
downward from the posterior margin of the acrosomal
cap. The manchette has been implicated in sperm head
elongation and in organizing condensed chromatin on the
opposing side of the nuclear envelope. The pair of centrioles migrates and attaches to the base of the nucleus,
where the distal centriole, distinguished at the ultrastructural level by its association with pericentriolar "satellite"
appendages, serves as the organizing center for the 9 + 2 axoneme of the sperm flagellum (Fig. 1 B; for review see
Browder et al., 1991 The process of spermatogenesis and spermiogenesis involves three distinct and unusual cytoskeletal networks of
microtubules, which are likely to assemble from specialized organizing centers. During meiosis 2, the spindle must
form in the absence of a preceding round of DNA synthesis. While the mechanism underlying this process in spermatogenesis is still not well understood, genetic differences between these stages have been identified, and morphological differences between meiotic and mitotic centrosomes have been reported (Gonzalez et al., 1988 Very few of the genes involved in sperm development
have been characterized at a molecular level. We report
here the cloning and characterization of murine and human
cDNAs that encode a novel form of a previously identified
zinc finger protein called basonuclin. We demonstrate that
basonuclin mRNA is expressed in the differentiating germ
cells of seminiferous tubules, and the protein is made later during spermatogenesis and spermiogenesis. Using three
affinity-purified, monospecific antibodies that we have made
to different peptide sequences within the basonuclin protein, we show that the protein localizes to several interesting places during sperm morphogenesis. We first detect
basonuclin in the centrosomes of meiotic spermatocytes. As differentiation proceeds, it maintains its centriolar location, but in addition, it accumulates in the acrosome. Basonuclin antibody also labels the mitochondrial sheath encompassing the midpiece of the flagellum, and intriguingly
one of the two basonuclin forms has a perfect mitochondrial localizing signal. Our findings, supported by cell fractionation studies, are entirely unexpected and have important implications for our understanding of the specialized centrosomes, microtubule arrays, and mitochondria of late
stage spermatogenesis and spermiogenesis.
Preparation of a Keratinocyte cDNA Library
Human epidermal keratinocytes were cultured (Rheinwald and Green,
1977 Isolation of Genomic Clones
A 688-bp EcoRI/EcoRD fragment encoding a 5 Preparation and Characterization of
Basonuclin Antibodies
Three peptides corresponding to segments of the published basonuclin
protein sequence (Tseng and Green, 1992 Immunoblot, Northern and In Situ Hybridizations
Immunoblot analyses were performed as described (Yang et al., 1996 Immunofluorescence Microscopy on Frozen
Tissue Sections
Frozen tissue sections (10 µm) were cut onto Superfrost plus slides. Sections were briefly fixed with methanol ( Ultrastructural Analysis
For regular EM, tissues were fixed at room temperature for at least 1 h
with 2.5% glutaraldehyde and 4% paraformaldehyde in 0.2 M sodium cacodylate buffer, pH 7.4. Samples were trimmed and washed three times
with the same buffer and then postfixed with 1% aqueous osmium tetroxide for 1 h at room temperature. These samples were further washed with
sodium cacodylate buffer, followed by maleate buffer, pH 5.1, and stained
en bloc with 1% uranyl acetate for 1 h at room temperature. The samples
were then washed several times with maleate buffer, dehydrated with cold
ascending grades of ethanol and propylene oxide, embedded with LX-112
medium, and polymerized at 70°C for 48 h before sectioning.
Semithin sections (0.75 µm) were stained with toluidine blue, and these
were visualized by light microscopy. Ultrathin sections (~80 nm) were cut
with a regular diamond knife, collected on 200-mesh, uncoated copper
grids, and double stained with 50% saturated uranyl acetate and 0.2%
lead citrate. These sections were then examined with a transmission electron microscope (model CX100; JEOL U.S.A., Inc., Peabody, MA) operated at 60 kV.
For postembedding immunolabeling, samples were placed in warm fixative containing 2% fresh paraformaldehyde, 0.05% glutaraldehyde in
0.1 M PBS, pH 7.4, for 10 min. The samples were trimmed, washed several
times with the same PBS at 4°C, then dehydrated at Ultrathin sections (~90 nm) were cut with a diamond knife, collected
on Nickel grids coated with Formvar and thin carbon films, and labeled
with specific antibodies according to the tested dilution, followed by incubation with 10-nm colloid gold-conjugated secondary antibodies. The sections
were briefly stained with uranyl acetate and lead citrate and examined
with an electron microscope (model CX100; JEOL U.S.A., Inc.) operated
at 60 kV.
Isolation of a Novel cDNA Encoding a Known
Zinc Finger Protein, and Genomic Mapping of Its
Encoded Exons
In the course of our studies on epidermal differentiation,
we hybridized a human keratinocyte cDNA library with a
517-bp PCR fragment corresponding to the zinc finger
domain of human basonuclin (nucleotides 2657-3174 of
hBSN1a), a protein known to be expressed in mitotically
active basal epidermal cells (Tseng and Green, 1992 To assess whether both 5
Interestingly, both mouse and human forms of BSN1a
but not BSN1b contain the sequence RRPEPG, which has
been shown to be a mitochondrial targeting sequence for
proteins (Komiya et al., 1994 Basonuclin mRNAs Are Abundantly Expressed
in the Testis
Previously, basonuclin was thought to be restricted in its expression to mitotically active cells of stratified squamous
epithelia (Tseng and Green, 1994
To assess whether our new form of basonuclin was expressed in both stratified epithelia and testis, we used reverse transcriptase and PCR on mouse skin and testis
mRNAs in the presence of a 5 To examine BSN expression in testis in more detail, we
first conducted Northern blot analysis. As shown in Fig.
4 A, a single RNA band of ~4,600 nucleotides was obtained from mouse testis. This band was comparable in
size to that seen in human keratinocyte mRNA preparations
(Tseng and Green, 1994
To verify that the hybridizing band(s) detected in the
testis corresponded to bona fide BSN mRNA and to examine the testis-specific expression of BSN mRNA during
sexual maturation, we conducted RNase protection assays.
In mouse germ cell development, meiosis in the seminiferous tubules begins at about 2 wk postnatally, and production of mature sperm occurs by 5 wk of age. As shown in
Fig. 4 B, a single band of the expected size was protected when mRNAs were used from mouse testes taken at 2 and
5 wk after birth and adult. The overall levels of BSN
RNAs appeared to be comparable. These data demonstrated unequivocally that BSN mRNAs are expressed in
testis and that their expression exists before sexual maturity.
Basonuclin mRNA Is Detected Early in
Spermatogenesis
To determine where BSN mRNAs are expressed within
the testis, we conducted in situ hybridizations on frozen
sections of mouse testes isolated at various stages of postnatal development. A digoxygenin-labeled antisense BSN
cRNA hybridized strongly in the seminiferous tubules of
all testis samples examined (Fig. 5). Hybridization was detected at the periphery of the tubules and appeared to be
present even at birth, before spermatogenesis (Fig. 5, A).
Hybridization remained high throughout most of spermatogenesis. At 2 wk of postnatal development, hybridization was strongest in the centers of the tubules, where
the primary spermatocytes are located, and weaker at the
periphery, where the spermatogonia reside (Fig. 5, B). By
4-5 wk of age, spermatid formation, i.e., spermiogenesis,
had begun (Rugh, 1990
Basonuclin mRNAs Are Translated in the Testis,
Where They Produce a 120-kD Protein
Both BSN1a and BSN1b cDNAs are predicted to encode
120-kD polypeptides; however, this has never been confirmed by immunoblot analysis. We therefore raised monospecific rabbit antibodies to three different peptide sequences present in the shared regions of these proteins
(see Materials and Methods). As judged by immunoblot analysis, each of the three different affinity-purified peptide antibodies (UC56, 372, and 176) detected a single crossreacting band of 120 kD in protein extracts from mouse
testis (Fig. 6 A). A band of this size was also detected in
protein extracts from mouse skin and from other epithelial
tissues known to contain keratinocytes (Fig. 6, A and B).
Collectively, these findings: (a) establish the size of basonuclin in testis, skin, and other stratified epithelia; (b) verify the
specificity of our antisera; and (c) suggest that, if other major
forms of basonuclins exist, they either must be 120 kD in size
or alternatively must have a most peculiar splice pattern,
missing three different domains of the basonuclin protein.
Basonuclin Localizes to the Centrosomes of
Spermatocytes and Developing Spermatids
To assess the location of basonuclin within differentiating
male germ cells, we conducted indirect immunofluorescence
on frozen sections of developing mouse testes. Basonuclin
protein was first detected in testis at 2 wk postnatally (Fig.
7 A). In contrast to BSN RNAs, which are expressed in
mitotic spermatogonia, protein was not detected until the
cells had differentiated into primary spermatocytes located at the midregion of the 2-wk seminiferous tubules.
These cells, in the first meiotic phase of differentiating male germ cells, displayed a dotlike pattern of staining with the UC56 anti-BSN (
The dotlike staining pattern suggested that basonuclin
might be localizing to centrosomes. To explore this possibility in greater detail, we used double immunofluorescence labeling with the H1 human autoimmune serum
( Basonuclin Also Localizes to Acrosomes and to the
Middle Piece of Developing Spermatids
In sexually mature adult testes, anti-BSN antibodies
strongly stained the spermatid heads (Fig. 8). Costaining
with propidium iodide, which labels chromatin, indicated
that this labeling was not nuclear. The crescent-shaped
staining pattern, coupled with the appearance of this strong
staining in the spermatid region of the seminiferous tubules, was reflective of that seen for acrosomal proteins in
spermatids (Lepage and Roberts, 1995
Finally, we observed Again, as was the case for the centrosomal staining,
all three affinity-purified antibodies against basonuclin labeled the acrosomes and the middle piece. This said, the
UC56 and 176 antibodies showed significantly stronger
staining in the acrosome than did the 372 antibody. Since
the three antibodies detected a single major 120-kD band
by immunoblot analysis, we posit that these differences reflect variation in masking of the basonuclin epitopes in
centrosomes and acrosomes.
Cell Fractionation Supports the Complex Localization
Pattern of Basonuclin
The pattern of BSN antibody staining was unexpected and
diverse. To verify that the staining patterns reflected multiple locations for basonuclin protein, we conducted cell
fractionation studies. Although procedures for isolation of
centrosomes from testis tissue have not yet been developed, it is possible to dissociate isolated sperm into tail,
acrosome, and headpiece by sonication and to subsequently resolve these fractions by sucrose gradient ultracentrifugation (Walensky and Snyder, 1995
Sperm fractions were examined by phase contrast and
immunofluorescence microscopy to verify that the separation procedure was successful (not shown). Proteins from
each fraction were then resolved by SDS-PAGE, and the
gel was stained with Coomassie blue to visualize the proteins (Fig. 9 B). All three fractions contained different sets
of proteins. As judged by immunoblot analysis, basonuclin
was present in the sperm tail and acrosome fractions, but it
was not present in appreciable amounts in the nuclear
fraction (Fig. 9 C). The purity of fractions was confirmed by
immunoblot analysis using an antibody against the established acrosomal marker PLC Immunoelectron Microscopy of Spermatids Reveals
Basonuclin Protein in the Centriolar Appendages, in the
Acrosomal Membrane, and in the Mitochondria of the
Tail Middle Piece
To further examine basonuclin expression during spermiogenesis, we conducted electron and immunoelectron microscopy (Fig. 10). Fig. 10 A provides an example of the
typical pair of centrioles that associates with the nuclear
envelope during the acrosomal cap phase of spermiogenesis (also see diagram in Fig. 1 B). At this stage of differentiation, centrioles migrate to the nuclear pole opposing the
acrosomal cap. The 9 + 2 axoneme assembly of the flagellum always initiates from the end of the distal centriole (Fig. 10, A and C, dis), which contains a prominent electron-dense satellite appendage often in close proximity to
the nuclear envelope (Fig. 10 A and B, arrowheads). The
proximal centriole (Fig. 10, A and C, px), laterally aligned
with the nuclear envelope, is not directly involved in
flagellar assembly, and its fate is unknown. Concomitant
with the attachment of centrioles to the nuclear envelope, the cylinder of manchette microtubules forms around the
lower half of the nucleus as it elongates (Fig. 10 B, ma).
Antibodies against basonuclin specifically labeled the
satellite appendages of distal centrioles (Fig. 10, C and D).
Perfect cross-sections of these appendages revealed a hollow ringlike structure (Fig. 10 C, inset). The labeling of these
structures with For years, it has been known that zinc plays an important
role in testis development, and a number of zinc finger
proteins are expressed in male germ cells (Burke and
Wolgemuth, 1992 Given the prior studies of Tseng and Green (1992, 1994),
we were surprised to find basonuclin expressed in testis at
all, since it had been thought to be restricted to stratified
squamous epithelia. However, BSN RNA expression was
as high or higher in testis than in any other organ examined. BSN RNAs were detected early in the differentiative
pathway of mouse germ cells, i.e., long before the animals
reached sexual maturity. Despite basonuclin RNA expression in mitotically active spermatogonia, basonuclin protein was not detected until later, where antibody labeling was first seen in meiotic spermatocytes. While antibody
masking is always a formal possibility, three different affinity-purified peptide antibodies failed to reveal labeling
in spermatogonia. Thus, we conclude that if basonuclin
protein is expressed earlier in development, it is present at
reduced levels or in a very different complex than its location in spermiogenesis.
In meiotic spermatocytes, basonuclin appeared to be
concentrated in centrosomes, a location that it then maintained throughout spermiogenesis. At least at later stages
of spermiogenesis, basonuclin seemed to be localized to
hollow ringlike appendages that were largely if not fully
confined to the centriole forming the sperm flagellum. This
was reminiscent of mitotic cells, where satellite structures
are generally unique to the mature centriole and are not
found on newly synthesized (immature) centrioles (for review see Lange and Gull, 1996 Little is known about the functions or molecular complexity of satellite structures associated with centrioles. In
fact, the first molecular marker for centriole maturation,
cenexin (96 kD), was only recently discovered in mitotically active cells (Lange and Gull, 1995 It is puzzling that BSN antibodies and cell fractionation
studies also detect this protein in the acrosome and mitochondrial sheath of the mature sperm. Since the acrosome
is a storage vessel for proteins used in fertilization, we surmise that basonuclin might perform a specialized role in
this process. This notion is particularly interesting in light
of the facts that: (a) Basonuclin appears to associate with
the acrosome late in spermiogenesis; and (b) centrioles are
absent in the oocytes of many species, including mouse
(Schatten, 1994 While the localization of basonuclin in sperm acrosomes
is consistent with the hypothesis that basonuclin performs
a function in centrosomes and/or microtubule organization, we are at a loss to explain why basonuclin was also
detected in the mitochondria of the flagellar midpiece.
This said, BSN1a has a perfect mitochondrial targeting sequence at its amino terminus, and this sequence is evolutionarily conserved. Although mBSN1a seems to utilize a
downstream ATG, the two putative forms may perform
unique functions in separate compartments of the differentiating male germ cell.
To make matters more intriguing, basonuclin has what
appears to be a reasonably bona fide nuclear localization
signal. While computer analysis of known proteins indicates that basonuclin has only a 40% chance of being localized to the nucleus, the protein does associate with the
nucleus in epidermal keratinocytes (Tseng and Green,
1994 One possibility is that basonuclin might be transiently
associated with chromatin after nuclear envelope breakdown of meiotic germ cells. Since the cell cycle of meiotic
mouse germ cells is so long, meiotic germ cells in the act of
nuclear envelope breakdown and spindle formation are
rare, making such analysis difficult. However, in this regard, it may be relevant that CP190, a recently described
zinc finger protein in mitotic cells of Drosophila, is associated with centrosomes during mitosis and with chromatin during interphase (Oegema et al., 1995).
The germ stem cells within each tubule reside at the tubule
periphery and give rise to proliferating spermatogonial
cells. Only the most primitive spermatogonia, i.e., those
that contribute to the stem cell population, complete their
cytoplasmic divisions. All other daughter cells are linked
through their cytoplasms and undergo a series of synchronous differentiation steps that culminate in the production
of mature sperm.
Fig. 1.
Schematic of male
germ cell development. (A)
In the seminiferous tubules,
stem cells and mitotically active spermatogonia are located at the periphery. As
the germ cells differentiate, they move inward toward the
lumen of the tubule. Spermatocytes undergo two rounds
of meiotic divisions before
they embark upon spermiogenesis, a process culminating in the production of mature spermatids. Spermatids
are released into the lumen
as sperm, leaving their cytoplasm behind. The large Sertoli cells provide nutrients and support for the developing germ cells; seminiferous
tubules are surrounded by interstitial space containing
blood vessels and also Leydig
cells, the hormone-producing
cells of the testis. (B) A diagram of a mature sperm, revealing in more detail its unique structures. The tail flagellum is a 9 + 2 axoneme
of microtubules, which at its upper end is surrounded by a sheath of specialized mitochondria (Middle Piece). The head of the sperm is
composed of an elongated nucleus, which in its upper hemisphere is surrounded by a membrane-encased acrosome. The acrosome is
thought to be a storage vessel used during fertilization. At the base of the crescent-shaped nucleus is the pair of sperm centrioles. The
proximal centriole attaches to the nuclear envelope but does not appear to have microtubule organizing activity. It is surrounded at its base by pericentriolar material but is otherwise naked. The distal centriole is decorated by an appendage or satellite structure and is the
only centriole capable of assembling the 9 + 2 axoneme of the flagellum. We have only visualized one appendage at any one time on ultrathin sections of mouse spermatid centrioles (artwork by Christa Wellman; adapted from Larsen, 1993).
[View Larger Version of this Image (52K GIF file)]
; Lange and Gull, 1995
). As the sperm
tail matures, the upper portion (middle piece) becomes ensheathed by mitochondria, the manchette disappears,
and the sperms become motile and are released into the
lumen, leaving the cytoplasm of the spermatids behind
(Fig. 1 B).
, 1990;
Staiger and Cande, 1990
; Messinger and Albertini, 1991
; Kubiak et al., 1992
; Wickramasinghe and Albertini, 1992
; Fuge, 1994
; Matthies et al., 1996
; de Vant'ery et al., 1996). Another unusual feature of the microtubule architecture
in sperm is the development of a tail flagellum. The ability
of spermatid centrioles to assemble these 9 + 2 axonemes
implies that the distal centriole acquires some component(s) that enables it to orchestrate this unique microtubule assembly process. Finally, the formation of the
manchette is perhaps the least understood of the microtubule assembly processes that occur during spermatogenesis. The manchette does not seem to emanate from a centrosome, but rather assembles from a membranous ring
that circumvents the equator of the spermatid nucleus.
Materials and Methods
), and poly(A)+ mRNAs were isolated using the procedure of Chomzcynski and Sacchi (1987). The RNA preparation was translated in a reticulocyte lysate system in the presence of [35S]methionine, and proteins
were resolved by SDS-PAGE and autoradiography. Proteins >200 kD
were translated from the mRNAs, verifying the quality of the preparation.
These mRNAs were then used to engineer a
-zap phage library (Stratagene, La Jolla, CA), made from a mixture of cDNAs that were synthesized using oligo dT and random hexamer oligonucleotide primers.
segment of the human
basonuclin 1b mRNA was used to screen a 129/sv mouse genomic library
(Stratagene). Three hybridizing clones were identified and subsequently
purified. Two clones, mBSN-1 and mBSN-2, were subcloned as ~17 kb
NotI restriction fragments into Bluescript KS+. These clones were then
subjected to restriction map analyses and partial sequencing.
) were synthesized, coupled to
keyhole limpet hemacyanin, and used for generating polyclonal antisera
in rabbits (Zymed Labs, Inc., S. San Francisco, CA). The three basonuclin
peptides are: UC56, CRPPPSYPGSGEDSK (human sequence, corresponding to the published amino acid residues 449-463; Tseng and Green,
1992
); Ab176, ESCGHRSASLPTPVD (mouse sequence, equivalent to
human residues 208-222); and Ab372, ASPNPRLHAMNRNNR (mouse
sequence, equivalent to human residues 404-419). All antisera were purified by affinity chromatography using the appropriate peptide-conjugated
Sepharose columns. Antibodies were tested by immunoblot analyses on
proteins extracted from the skin and testes of adult mice.
).
RNA blots used for Northern analysis were purchased from Clontech
(Palo Alto, CA), and hybridizations were performed as described by the
manufacturer. A 576-bp radiolabeled human cDNA corresponding to sequences within exons 2-4 of basonuclin was used for hybridization. In situ
hybridizations of frozen sections of mouse testes were performed using
digoxygenin-labeled antisense and sense riboprobes as described (Chiang
and Flanagan, 1996
).
20°C) for 10 min and then
washed 2× with PBS. Sections were preblocked with a solution containing
1% BSA, 0.1% Triton X-100, and 1% gelatin in PBS. Primary antibodies
were then added to fresh solution and incubated with sections at room
temperature for 1 h. Antibody concentrations used were: anti-BSN antibodies (1:20 to 1:500); anti-
tubulin antibodies (1:200 dilution; gift of Dr.
Harish C. Joshi, Emory University, Atlanta, GA; gift of Dr. Bruce Alberts, University of California at San Fransisco, San Fransisco, CA); H1
human autoantisera against centrosomes (1:250 dilution; gift of Dr. Thomas Medsgar, University of Pittsburgh, Pittsburgh, PA); and anti-PLC
1
(purchased from Santa Cruz Biotechnology, Santa Cruz, CA). After
washing the slides 3× with PBS for 10 min each, sections were incubated
with fresh solution containing secondary Texas red or FITC-conjugated
antibodies (1:100 dilution) for 30 min before washing as before and mounting. Nuclei were stained either with propidium iodide or with 4,6-diamidino-2-phenylindole (DAPI)1. Sections were examined using either a confocal microscope (model LSM 410; Carl Zeiss, Inc., Thornwood, NY) or
an immunofluorescence microscope (model Axiophot; Carl Zeiss, Inc.).
25°C with ascending
grades of ethanol, infiltrated with different concentrations of Lowicryl
K4M medium, embedded in gelatin capsules with fresh 100% Lowicryl
K4M medium, and polymerized at
25°C with UV light for 5-7 d.
Results
). One
clone contained a 4,606-bp insert, which upon sequencing was shown to harbor a complete open reading frame, followed by 1,547 bp of 3
sequence containing a polyadenylation signal at nucleotide 4583. This cDNA encoded a
protein that was nearly identical to the published sequence
from amino acid residues 33-993 (Tseng and Green, 1992
).
However, it differed in its 5
segment by the absence of a
32-amino acid residue sequence and the replacement of a
novel 166-nucleotide residue sequence.
basonuclin sequences were
bona fide, we screened a genomic library and mapped the
positions of the two 5
upstream sequences relative to the
remainder of the basonuclin gene. Each sequence was contained within individual exons that were found within a
single genomic clone (data not shown). The new sequence
was located in an exon 3
to the one present in the previously published sequence. These data confirmed the existence of the two sequences within the basonuclin gene. We
refer to the two predicted forms as BSN1a (Tseng and
Green, 1992
) and BSN1b (this report), based upon the positioning of their respective exons within the basonuclin
gene. The two basonuclin sequences were characterized
from both mouse and human and are provided in Fig. 2.
The BSN1b exon is highly conserved and is nearly identical between mouse and human. The BSN1a exon found in the
originally reported sequence (Tseng and Green, 1992
) is
less conserved between the two species, and different
translation start sites are predicted.
Fig. 2.
Sequence relation between the two forms of basonuclin. Shown is a schematic of the mouse gene structure (top) and
the two basonuclin forms (bottom). Hatched bars denote regions
not fully sequenced; intron sizes are unknown. Human BSN1a sequence shown is from Tseng and Green (1992); the mBSN1a
RNA is likely to use a downstream ATG for translation (underlined). The hBSN1b sequence, determined from a full-length
cDNA, differs only in its 5 sequence from hBSN1a. It is possible
that an ATG shared by 1a and 1b is used for translation, since in
vitro transcription/translation yields a >110-kD protein. The sequences encoding the unique segments of BSN1a and BSN1b are
located on individual exons. Arrows denote splice sites; small
case nucleotides represent intron sequences; putative mitochondrial localization sequence is boxed.
[View Larger Version of this Image (45K GIF file)]
; Shore et al., 1995
; McBride
et al., 1996
). While not previously noted, a computer survey of all protein sequences known indicates a 60% chance
of the BSN1a form localizing to the mitochondria, a prediction that we address experimentally later in the text.
). In the course of examining basonuclin RNA expression in different mouse tissues, we were surprised to discover that PCR primers corresponding to the shared sequences of BSN1a and BSN1b
detected a band in testis mRNA in addition to RNAs isolated from tissues known to contain keratinocytes (Fig. 3 A).
Fig. 3.
Expression of both
basonuclin RNA forms in testis as well as in stratified squamous epithelia. (A) Reversetranscriptase-PCR (RT-PCR)
Analysis I. RNAs were isolated from the various mouse tissues indicated and subjected to RT-PCR as described above. Primer sets to a
shared portion of BSN1a and BSN1b were used, along with an actin control. HT, heart; LN, lung; MS, muscle; SL, spleen; KD, kidney;
FS, forestomach; ES, esophagus; SK, skin; OV, ovary; TS, testis. (B) RT-PCR Analysis II. RNAs were isolated from mouse skin and testis. These RNAs were subjected to RT-PCR analysis using primer sets specific for each of the two unique exons and for a shared segment
of mouse BSN1a and BSN1b RNAs, respectively. Appropriate primer sets for -actin were used as controls. DNA fragments generated
were resolved by electrophoresis through 1% agarose gels. Lanes 1-4, skin RNAs; lanes 5-8, testes RNAs. Fragments shown were generated using primers specific for: lanes 1 and 5, BSN1a; lanes 2 and 6, BSN1b; lanes 3 and 7, BSN1a/1b; lanes 4 and 8, actin.
[View Larger Version of this Image (22K GIF file)]
oligonucleotide primer to
one or the other of the 1a and 1b sequences. In each case,
the 3
primer corresponded to sequence shared by both
cDNAs. As shown in Fig. 3 B, both primer sets produced a band of the expected size, indicating that both sequences
are expressed by skin and testis. In our initial study, we
have focused on using cRNA probes and antibodies that
are shared by the two forms, and we refer generally to the
properties of basonuclin (BSN).
), and it corresponded to the size
expected for BSN1a and BSN1b mRNAs, which have a
long 3
untranslated sequence. For human testis, two bands
were detected in approximately comparable levels: one
was an ~4,600-nucleotide band, as expected, and the other
was an ~3,200-nucleotide band. This smaller band is large
enough to encode full-length basonuclin, although our focus for the remainder of the study was on mouse, and we
have not pursued the identity of this smaller band in human testis.
Fig. 4.
Northern blot and
RNase protection analyses.
Northern blots of mouse and
human RNAs were purchased from Clontech. Blots
contained 2 µg polyA+
mRNAs, which were hybridized with a radiolabeled
probe corresponding to a
576-bp fragment shared by
BSN1a and BSN1b. As control, a radiolabeled probe
corresponding to a 1-kb fragment of glyceraldehyde dehydrogenase was used. Note
the presence of a 4,600-nucleotide band, predicted for the
bona fide BSN1a and BSN1b RNAs, in testes samples (TS) from both mouse (m) and human (h); note the presence of an additional
3,200-nucleotide band in human testis. PB, peripheral blood; CO, colon; SI, small intestine; OV, ovary; TS, testis; PS, prostate; TH, thymus; SL, spleen. (B) RNAs were isolated from the testes of mice at 2 wk, 5 wk, and adult postnatally. RNAs were hybridized with radiolabeled mouse cRNas generated to either (a) a 576-nucleotide sequence common to BSN1a and BSN1b or (b) a 359-nucleotide actin sequence. After hybridization, RNase treatment was performed as described by Faus et al. (1994). Duplexes were resolved by PAGE, and
the gel was exposed to x-ray film overnight. Note the presence of 487- and 250-nucleotide bands expected for BSN1a/BSN1b and actin
RNAs, respectively, after hybridization with the probes and RNase digestion.
[View Larger Version of this Image (38K GIF file)]
), and BSN RNAs were still detected throughout the tubules (Fig. 5, C-E). The persistence of BSN mRNA in late-stage spermiogenesis suggests
that BSN RNAs are stable, as is the case with many mRNAs
that are translated at this time (for review see Browder et
al., 1991
). Basonuclin cRNA hybridization was largely specific for derivatives of the germ cell population within the
testis and was not detected in the interstitial Leydig cells.
If present at all in Sertoli cells, the signal was reduced over
that seen in germ cells. No hybridization was seen with the
sense control cRNA (Fig. 5 F).
Fig. 5.
In situ hybridization of BSN RNAs in
mouse testis. Testes were isolated from mice at various times after birth, and frozen sections (10 µm)
were hybridized with a 576-nucleotide digoxygeninlabeled cRNA (A-E) or sense (F) control corresponding to the shared region of BSN1a and BSN1b
RNAs. After hybridization, sections were washed extensively and developed for equal times (Yang et al.,
1996). Shown are samples from: A, postnatal day zero
(p0); B, p14; C-F, p35. Bar: (A and B) ~175 µm; (C)
420 µm; (D) 100 µm; (E and F) 40 µm.
[View Larger Version of this Image (81K GIF file)]
Fig. 6.
Immunoblot analysis with affinity-purified antiBSN UC56, 372, and 176 antibodies. (A) Total proteins
were isolated from mouse testis and skin, and samples
were resolved by electrophoresis through 8.5% SDS-
polyacrylamide gels. Proteins
were transferred by electroblotting to Immobilon-P
membranes (Millipore Corp., Bedford, MA), and blots were
subjected to immunoblot
analysis as described by the
manufacturer. Each of the three anti-BSN peptide antibodies were affinity column purified before use. In both testis and skin samples, a
single band of 120 kD was detected; this band was not detected by secondary antibody alone or by preimmune sera. (B) Total proteins
were isolated from a variety of adult mouse tissues and subjected to immunoblot analysis as outlined in A. Abbreviations are as indicated in the legend to Fig. 3, except: hEP, human epidermal keratinocytes; BR, brain.
[View Larger Version of this Image (38K GIF file)]
BSN) antibody. Double immunofluorescence with DAPI to stain chromatin indicated that the
labeling was located near the nucleus (Fig. 7 A). Staining
was more prevalent by 4 wk (Fig. 7, B and C), when primary and secondary spermatocytes exist (Rugh, 1990
).
While the majority of these spermatocytes contained single dots, a few seemingly contained double dots positioned at opposing sides of the nucleus (Fig. 7 B, inset). Similar
staining patterns were observed with all three affinitypurified BSN antibodies, although antibodies UC56 (shown)
and 372 gave the strongest staining. The pattern was not
seen with secondary antibody alone.
Fig. 7.
Detection of antibasonuclin immunofluorescence in the centrosomes of meiotic spermatocytes. Frozen sections (~10 µm) of
mouse testis (2, 4, or 8 wk old, as indicated in the panels) were subjected to double or triple immunofluorescence, and sections were
viewed with a confocal microscope (Carl Zeiss, Inc.). Sections stained with anti-BSN antibodies (shown here are UC56 BSN profiles)
were visualized by costaining with an FITC-conjugated anti-rabbit IgG secondary antibody (green). Sections stained with human H1 autoantisera (
H1) were visualized by costaining with a Texas red-conjugated anti-human IgG secondary antibody (red). Nuclei were labeled with DAPI (blue). (A) Seminiferous tubule at 2 wk, containing a mixture of mitotically active spermatogonia at the periphery and
primary spermatocytes in the midsection (Rugh, 1990
);
BSN stained only midsection cells; (B and C) Seminiferous tubule at 4 wk, containing spermatogonia, primary spermatocytes, and secondary spermatocytes, located from the periphery to the centers of the tubules,
respectively; view is of midsection, double stained with:
BSN (B and C) and DAPI (C). Arrows denote typical single dot pattern. Inset
in B shows what is probably a secondary spermatocyte in late G2 or prophase, after duplication of the centrosomes; double labeling of
dots is with
BSN and
H1 antiserum. (D-F) Region of spermatocytes, triple labeled with
BSN (D and F),
H1 (E and F), and DAPI
(F). Yellow reveals costaining with
BSN and
H1. Portion of F to the left of the double arrows is the region shown in D and E. (G) 8-wk
sexually mature seminiferous tubule costained with
BSN,
H1, and DAPI. View is of spermatogonia, primary spermatocytes, and secondary spermatocytes. Note that spermatogonial centrosomes at the periphery only labeled with
H1 (arrowheads), while spermatocytes colabeled with
BSN and
H1 (arrows). Additional note:
BSN antibodies 372 and 176 gave similar immunofluorescence staining
patterns to those shown here. Dotted lines denote nuclear borders. Bars, 10 µm.
[View Larger Version of this Image (64K GIF file)]
H1), known to cross-react with centrosomal proteins
(Shu and Joshi, 1995
). As shown in Fig. 7, D-F, the two
antibodies displayed staining that was superimposable at
the confocal microscopy level. This was further verified by staining serial cross-sections with same-species antibodies
against
-tubulin (not shown). Interestingly, only the centrosomes near the midregion of the seminiferous tubules
costained with
BSN and
H1; centrosomes at the periphery stained with
H1, but not the UC56 sera (Fig. 7 G).
Based upon these data, basonuclin appeared to be a specific component of the centrosomes of postmitotic, differentiating male germ cells.
; Walensky and Snyder, 1995
; Yoshiki et al., 1995
). Interestingly, despite
the fact that spermiogenesis in mouse is initiated by 4 wk,
and that acrosomal caps are seen throughout the centers
of the 4-wk seminiferous tubules, these caps did not stain
with
BSN (not shown). The relatively late acquisition of
anti-BSN staining in the acrosome suggested that basonuclin is a component of late-stage sperm acrosomes.
Fig. 8.
Anti-BSN antibodies label the acrosomes and middle piece of developing spermatids. Frozen sections of adult mouse testis
were processed as described in the legend to Fig. 7, except that propidium iodide (red) was used instead of DAPI to label chromatin. (A) Low magnification to show strong staining of spermatids (Sp) near the lumen (Lu) of all seminiferous tubules. Sg, spermatogonia; 1° and
2°, spermatocytes at the first and second meiotic stage, respectively. (B) Higher magnification of spermatids showing BSN staining of
both the head region and also the middle piece of the tail (arrows). (C) Lower magnification to show that spermatids in all seminiferous
tubules of the adult testis labeled strongly with anti-BSN antibodies; note dotlike staining of spermatocyte centrosomes as well. (D)
Double label of spermatids with propidium iodide and
BSN to show that the majority of
BSN labeling is in the acrosome and not the
nucleus. Bar: (A) 20 µm; (B) 9 µm; (C) 30 µm; (D) 7 µm.
[View Larger Version of this Image (106K GIF file)]
BSN staining within the middle
piece of the tail of maturing spermatids (Fig. 8 B, arrows).
This structure contains the mitochondrial sheath at the upper portion of the 9 + 2 axoneme (Fig. 1 B). This observation was surprising, given that mBSN1a was not predicted
to contain mitochondrial localization signal seen at the
amino end of hBSN1a.
). We applied
this procedure to mature sperm that we removed from the
epididymis of adult mice. First, we verified that mature
sperm, similar to spermatids, display
BSN UC56 immunofluorescence staining in the acrosome, middle piece of the
tail, and centrosome. (Fig. 9 A; sperm centrosomal staining was more readily visible with the 372 antibody, which did not stain acrosomes so brightly.)
Fig. 9.
Fractionation of sperm proteins confirms presence of the majority of basonuclin in the acrosome and sperm tail. Sperm were isolated from the epididymis of adult mice. They were first stained with BSN UC56 antibodies to confirm the presence of acrosomal (ac) and middle piece (mp) staining in mature sperm (two sperm shown in A; bottom middle piece has a 90° kink). Sperm were then fractionated by sonication and sucrose gradient centrifugation as described by Walensky and Snyder (1995)
. Proteins were solubilized in
10 mM DTT, 2% SDS, and samples were resolved by electrophoresis through 8.5% SDS-polyacrylamide gels. Gels were analyzed by either staining with Coomassie blue (B) to visualize total proteins or immunoblot analysis (C) with antibodies against basonuclin or PLC
1. E, total protein extract from epidermal keratinocytes; S, total sperm proteins; A, acrosomal fraction; T, tail fraction; N, nuclear
fraction. Note: Sperm centrioles were lost in the fractionation procedure, as judged by immunofluorescent staining of each fraction.
[View Larger Version of this Image (37K GIF file)]
1 (Walensky and Snyder,
1995
; Fig. 9 C) and a protamine nuclear antibody (not
shown). These data were consistent with our immunofluorescence analysis. The absence of BSN immunoblot reaction in the nuclear fraction indicated that our failure to
observe
BSN staining in sperm nuclei was due to the absence of protein, rather than the masking of BSN epitopes.
We do not yet know whether basonuclin is present in nuclei of germ cells at earlier stages of spermatogenesis.
Fig. 10.
Electron and immunoelectron microscopy of mouse spermatids. Mouse testes from adult animals were fixed and processed
for either regular or immunoelectron microscopy as described in the Materials and Methods. For immunoelectron microscopy, we used
either BSN (UC56) or anti-
-tubulin (Shu and Joshi, 1995
) antibodies. (A and B) Negatively stained sections of spermatid centrioles.
The tip of the proximal centriole (px) is always associated with the nuclear envelope through implantation foci (if). Its free tip is always
encased by a cloud of pericentriolar material; the distal centriole (dis) is always the site of 9 + 2 axoneme assembly and most sections revealed an attached appendage(s) or satellite structure(s) (arrowhead) located near the junction of the two centrioles and often in close
proximity to the nuclear envelope. The manchette (ma) of microtubules that surrounds the lower hemisphere of the nucleus is formed at
the acrosomal cap phase and disappears shortly after nuclear elongation and flagellar assembly (B). (C and D) Immunogold labeling of
centrioles with
BSN. Note that the appendages of the distal centrioles (arrowheads) labeled specifically with the antibody. Inset shows
a cross section of an appendage, revealing a hollow center to the structure. (E) Immunogold labeling of centrioles with anti-
-tubulin.
Note that this antibody heavily labeled the pericentriolar cloud at the tip of the proximal centriole and, to a lesser extent, showed some
labeling near the distal appendages. (F)
BSN immunogold labeling of the acrosomal cap (ac) of a late-stage spermatid. Note that most
of the labeling is concentrated near the outer membrane (om) of the cap rather than the inner membrane (im) or acrosomal space. Note
the elongated nucleus, characteristic of nearly mature sperm. (G) Immunogold labeling of the sheath of mitochondria (mi) in the middle
piece surrounding the axoneme (Ax). Additional abbreviations: Nu, nucleus; A, annulus. Bar: (A-D) 0.2 µm; (E and F) 0.3 µm; (G) 0.4 µm.
[View Larger Version of this Image (139K GIF file)]
BSN was largely distinct from anti-
-
tubulin, which specifically labeled the pericentriolar material surrounding the end of the proximal centriole (Fig.
10 E).
-Tubulin labeling was not detected at the end of
the distal centriole, i.e., at the site of assembly of the 9 + 2 axoneme. Given that the fate of the proximal tubule seems
to be variable dependent upon species, this might explain why in Xenopus sperm,
-tubulin has not been found associated with the pericentriolar material of flagellar centrioles (Stearns et al., 1991
; Felix et al., 1994
; Stearns and
Kirschner, 1994
), whereas in mouse sperm, it has (Palacios
et al., 1993
).
BSN also labeled acrosomes of late-stage spermatids
that had undergone nuclear elongation (Fig. 10 F). By immunoelectron microscopy, the labeling was most dense at
the inner surface of the outer acrosomal membrane. Finally, as predicted from our immunofluorescence data, the
mitochondria within the middle piece of the sperm tail were specifically and uniformly labeled with anti-BSN antibodies (Fig. 10 G). Based upon the human sequence,
we would have presumed that this labeling represented
BSN1a rather than BSN1b. Further studies will be necessary to determine whether there are multiple forms of basonuclin that are differentially localized in germ cells.
Discussion
; Noce et al., 1992
, 1993
; Hosseini et al.,
1994
; Zambrowicz et al., 1994
; Kundu and Rao, 1995
; Passananti et al., 1995
; Stassen et al., 1995
; Mello et al., 1996
;
Supp et al., 1996
). Where tested, these proteins have been
found to be strictly nuclear. Our discovery that basonuclin
is a testis protein adds another zinc finger protein to this
growing list, but its location sets it apart from the others.
). The restriction of appendages to the axonemal centriole of male germ cells has been
described before (Browder et al., 1991
; Lange and Gull,
1996
).
), and as yet cenexin has not been cloned. In contrast to cenexin, which is
regulated with the cell cycle of mitotic cells, basonuclin
may represent the first example of a centriolar appendage
marker that, in male germ cells, is largely specific for spermatocytes and spermatids. The primarily distal centriole location in spermatids is particularly intriguing because:
(a) This is the only centriole that nucleates microtubule assembly in the spermatid, and pericentriolar material surrounding centrioles has been implicated in orchestrating
microtubule organizing activity (Gould and Borisy, 1977
;
Telzer and Rosenbaum, 1979
; Calarco-Gilliam et al., 1983;
Doxsey et al., 1994
; Lange and Gull, 1995
); and (b) so little
is known about how microtubules organize into their unique
and diverse arrays during spermiogenesis. Through its association with the distal centrioles of developing spermatids, basonuclin becomes a candidate for a protein that
could be involved in tailoring the organization of the microtubules during male germ cell meiosis and spermatid
differentiation. Moreover, by identifying one protein involved in these centrioles, basonuclin becomes a powerful
tool for identifying additional proteins involved in centriole
maturation during postmeiotic spermatogenesis. This should
allow us to probe deeper into the differences that exist between the centrosomes of meiotic versus mitotic germ cells.
; Lange and Gull, 1996
). In the future, it
will be important to examine basonuclin expression during
oogenesis and to track the fate of sperm basonuclin during fertilization.
), and we have confirmed this with our antibodies (unpublished results). We did not detect basonuclin in isolated sperm nuclei, nor did we detect
BSN labeling in
germ cell nuclei. Thus, it seems unlikely that basonuclin is
nuclear in germ cells, although we cannot rule out the possibility that in the early stages of spermatogenesis, its antigenic determinants are masked by association with other
nuclear proteins. Additionally, while evidence that basonuclin is a DNA-binding protein is lacking, its structural
features predict that it has this potential.
; Whitfield et al.,
1995
). Despite the lack of sequence similarity between CP190
and basonuclin, these findings suggest collectively that: (a)
Zinc finger proteins may play important roles in the formation, structure, positioning, or function of centrosomes;
and (b) the requirements for these proteins may differ in
meiosis and mitosis. As future studies are conducted, the
mysteries underlying the dynamic roles of basonuclin during spermiogenesis should become increasingly apparent.
Received for publication 27 January 1997 and in revised form 21 February 1997.
1. Abbreviations used in this paper: BSN, basonuclin; DAPI, 4,6-diamidino-2-phenylindole.We thank Christa Wellman for her artistic talents exerted in the design of
Fig. 1 and Chuck Welleck for his artwork in preparation of the color figures. We thank Dr. Thomas Medsgar for his autoimmune sera against centriolar proteins, Dr. Bruce Alberts, and Dr. Harish Joshi for antibodies
against -tubulin. We thank Dr. Gary Smith for his valuable discussions
on spermiogenesis, Dr. Xiaoming Wang for her advice on RNase protection assays, and Dr. Satrajit Sinha for his computer analysis and identification of the mitochondrial localization signal.