Mol. Hum. Reprod. Advance Access originally published online on December 10, 2004
Molecular Human Reproduction 2005 11(1):53-64; doi:10.1093/molehr/gah132
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Novel testis-expressed profilin IV associated with acrosome biogenesis and spermatid elongation
Department of Andrology, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany
1 To whom correspondence should be addressed at: IHF Falkenried 88 (CiM), D-20251 Hamburg, Germany. Email: kirchhoff{at}ihf.de
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Abstract |
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A novel profilin, named profilin IV, was cloned and characterized as a testicular isoform, distinct from the previously described testis-specific profilin III. Profilin IV showed only 30% amino acid identity with the other mammalian profilins; nevertheless, database searches produced significant alignments with the conserved profilin domain. Northern blot analysis and in situ transcript hybridization suggested that profilin IV, like profilin III, is transcribed in the germ cells. However, the timing of their expression during post-natal development of rat testis and in the rat spermatogenetic cycle was distinct. In the human testis, profilin IV mRNA expression correlates with the presence of germ cells suggesting that it may be a suitable molecular diagnostic parameter to supplement conventional histopathological diagnostics in the assessment of testicular biopsies. The predicted profilin IV protein was verified employing an anti-oligopeptide antibody. Western blot analysis detected an immunorelated testicular protein of approximately 14 kDa. Immunohistochemistry revealed an intracellular protein of the rat, the mouse and the human testis accumulating asymmetrically in the cytoplasm of round and elongating spermatids with its perinuclear location coinciding with the position of the developing acrosome–acroplaxome and the manchette. Profilin IV thus may regulate testicular actin cytoskeleton dynamics and play a role in acrosome generation and spermatid nuclear shaping.
Key words: acrosome/human testicular biopsy/manchette/puberty/rat/spermatid
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Introduction |
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Actin filaments are concentrated at specific sites within the testis and appear to be central to testicular function and sperm development (reviewed in Vogl, 1989
). In the Sertoli cells, they are abundant in the ectoplasmic specializations (reviewed in Lee and Cheng, 2004) and in regions adjacent to the tubulobulbar complexes (Guttman et al., 2004). In the germ cells, besides being concentrated in the intercellular bridges, F-actin is present in the subacrosomal space of early spermatids before depolymerizing to G-actin in late spermatids and spermatozoa (reviewed in Vogl, 1989; Fouquet and Kann, 1994). It is contained in the acroplaxome which anchors the acrosome to the sperm nucleus (Kierszenbaum et al., 2003a; Kierszenbaum et al., 2004) and presumably also in the transient manchette (Kierszenbaum et al., 2003b), suggesting an important role for controlled actin polymerization during acrosome biogenesis and spermatid nuclear shaping. However, how the correct polarity of the acrosome, manchette and axoneme becomes established during spermiogenesis still remains undefined.
The non-muscle actin cytoskeleton of the testis, like that of other tissues, is highly dynamic and may be regulated by various proteins that either promote or inhibit actin polymerization. The existence of a number of actin-interacting proteins in the testis (for example actin-capping proteins, Hurst et al., 1998; calicin, Lécuyer et al., 2000; and Arc, Maier et al., 2003) underscores the important role of actin dynamics within the testis. Among the proteins that bind actin are the profilins, small cytosolic proteins (
15 kDa) which are able to bind actin monomers in a stable 1:1 complex (Schutt et al., 1993). Profilins are actively involved in the regulation of the actin cytoskeleton and are crucial for the creation and maintenance of a polarized cytoskeleton in each cell type investigated thus far. Loss-of-function mutations of profilins disrupt multiple actin-dependent processes, often causing lethal phenotypes (Drosophila, Verheyen and Cooley, 1994) or severe growth reduction (Saccharomyces, Haarer et al., 1990). Profilin was the first actin-monomer-binding protein to be discovered (Carlsson et al., 1977). Since then, profilins have been found in each species where they have been searched for and have been implicated in ligand-induced actin reorganization (for a review, see Pollard and Borisy, 2003). Their amino acid sequences are only moderately conserved across many phyla; despite this, their structure and function are remarkably well conserved (Schutt et al., 1993; Rothkegel et al., 1996).
In mammals, to date, three profilin genes have been described encoding at least four protein isoforms, which exhibit highly divergent expression patterns, with profilin I being ubiquitously expressed, profilin II being most abundant in the brain (Witke et al., 2001) and profilin III representing a testis-specific isoform (Braun et al., 2002). The lethal phenotype of profilin I gene knockout mice, which is not compensated for by profilin II (Witke et al., 1998), emphasizes its crucial importance for cell survival and cell division in early development. In comparison, very little is known about profilin expression in the mammalian testis and the involvement of testis-specific isoforms in the development and function of the male germ cell. However, a detailed analysis of profilin expression in the testis might contribute greatly to our understanding of mammalian spermiogenesis and spermatid nuclear shaping.
A central role for profilins in spermatogenesis and sperm function has already been postulated earlier. In the acrosomal process of evertebrate spermatozoa, a profilin protein governs unidirectional addition of monomers to actin bundles (Tilney et al., 1983). In Drosophila, male meiosis is severely disturbed in mutants of the chickadee gene that encodes the Drosophila profilin (Verheyen and Cooley, 1994). In this study, a novel mammalian profilin, named profilin IV, is described, which may be centrally involved in spermiogenesis and sperm function. It represents another testicular isoform which is distinct from profilin III (Braun et al., 2002) based upon its primary structure and expression pattern.
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Materials and methods |
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Animals
Male Wistar rats and NMRI mice were obtained from the Hamburg University Hospital animal house. ‘The Guiding Principles in the Care und Use of Animals’ (DHEW Publication, NIH, 80-23) was observed in all cases. The mice were 5 (n=90), 15 (n=24), 30 (n=5), and 60 (n=10) days old and the rats were 10, 15, 20, 22, 26, 28, 30, 45, and 60 days old (n=10 animals per age group). Animals were killed by decapitation (5- and 10-day-old) or CO2 asphyxiation (all the others), the testes removed and snap-frozen in liquid nitrogen for RNA and protein extraction (see below). Various control tissues were taken from the 60-day-old animals. Testes of the three remaining animals per age group were Bouin-fixed for use during in situ hybridization and immunohistochemistry (see below).
Human testis tissues
Tissue specimens were taken from 16 azoospermic patients who underwent a testicular sperm extraction (TESE) according to a protocol previously described (Jezek et al., 1998; Schulze et al., 1999). This protocol comprises the mode of removal and processing of at least four fragments per testis for histological analysis, a post-surgical trial-TESE, and cryopreservation. An additional small tissue fragment of approximately half rice grain size was used for the purpose of the present study. Informed consent and ethics committee approval were obtained and studies were conducted in accordance with the guidelines of the ‘Helsinki Declaration’. The morning baseline serum concentrations of FSH, LH and testosterone were measured prior to testicular biopsy. The average age of patients undergoing testicular biopsy was 38.6±6 years (mean±standard deviation). Individual serum hormone concentrations, age and status of spermatogenesis (modified Johnsen score according to De Kretser and Holstein) are given in Table I. One tissue sample (No. 10) showed RNA degradation upon extraction (see below) and was excluded from this study.
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RNA preparation and Northern blot analysis
Total RNA was isolated from frozen tissues by the RNAclean method (AGS, Heidelberg, Germany). Poly(A) + RNA was isolated using standard procedures employing oligo(dT) cellulose type 7 (Amersham Pharmacia Biotech, Freiburg, Germany). Ten micrograms of total RNA or 3 µg of poly(A) + RNA, per lane, were separated on denaturing agarose gels and transferred to Hybond N membrane (Amersham). Profilin IV cDNA-fragments (for primer sequences see Table II), a mouse profilin III cDNA fragment (see Table II) and an actin cDNA fragment (accession no. NM_001101 [GenBank] ) were 32P-labelled employing the Prime-It II random primer labelling Kit (Stratagene, Amsterdam, the Netherlands), denatured and employed as hybridization probes under standard conditions. Autoradiograms were exposed to Kodak BioMax MS autoradiography film (Amersham) and developed after 2 h to 5 days of exposure, depending on the experiment.
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cDNA synthesis and PCR amplification
For standard RT–PCR, oligo(dT)-primed cDNA was synthesized from 5 µg of total RNA in a 20 µl reaction using 200 U of Superscript III reverse transcriptase (Invitrogen, Karlsruhe, Germany), 1 mM dNTP, and 0.5 µg oligo(dT)12–18. Incubation was at 50°C for 50 min. PCR amplification was performed in a 50 µl volume with 0.5 U Biotherm Taq-Polymerase (Genecraft, Münster, Germany), 1 µl single-stranded cDNA, 200 nM dNTP and 20 pmol each of oligonucleotide primers in the PCR buffer provided (Gene-craft). Generation of the 3'-ends was achieved by RACE (rapid amplification of 3'-ends) technique employing 0.5 µg of the anchor primer; the following PCR was performed in the presence of 20 pmol of RACE primers (see Table II). The sequences of primer pairs and PCR conditions were as shown in Table II. Except for primers profilin IV r3, all primers spanned at least one intron to control DNA contamination. PCR products were isolated and ligated into the TA-cloning vector pGEM-T Easy (Promega, Mannheim, Germany). Plasmid DNA was sequenced from both strands (MWG, Ebersberg, Germany) and sequence analysis performed using the Lasergene software package (DNASTAR Inc., Madison, WI, USA).
In situ transcript hybridization
Bouin-fixed and paraffin-embedded rat testes were sectioned (7 µm) and processed as described (Pusch et al., 2000). Briefly, sense and antisense profilin III and IV cRNA comprising the open reading frames were transcribed in vitro in the presence of digoxigenin-11-dUTP (Roche, Indianapolis, USA), following the supplier's instructions and were applied to the sections for an overnight incubation at 40°C. After stringent washing and blocking with 20% normal sheep serum, sections were incubated with sheep anti-digoxygenin (DIG) alkaline phosphatase conjugate (Roche) at a dilution of 1:5000. Colour development of the S-bromo-
-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate proceeded in the dark. Tissue sections were investigated using normal brightfield microscopy (Nikon, Japan) and images captured with a Leica DC 300 digital camera (Leitz, Bensheim, Germany).
Generation of anti-peptide polyclonal antibodies
A chemosynthetic oligopeptide was obtained (Pineda-Antikörper Service, Berlin, Germany) according to the amino acid sequence deduced from the mouse profilin IV cDNA. The sequence of the 26-mer oligopeptide employed in the immunization was: NH2-CRREGLYFKEKDYKCVRADDYSLYAK-COOH (see Figure 2); an N-terminal cysteine residue was added for convenient coupling. The peptide was conjugated to keyhole limpet haemocyanin as a carrier using the terminal cysteine, and the conjugate employed to immunize female rabbits. Preimmune sera were obtained on the day of immunization, while immune sera were obtained after 90 and 120 days.
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Western blot analysis
Proteins extracted from the mouse testis and brain were separated on 15% standard Laemmli sodium dodecyl sulphate-polacrylamide gels (50 µg per lane) and transferred to polyvinylidene difluoride membranes (Amersham, Braunschweig, Germany) in a discontinuous buffer system using a semi-dry blotter (Phase, Lübeck, Germany). Immunodetection was carried out by blocking for 1 h in 1% blocking solution (Boehringer-Mannheim), followed by incubation with the polyclonal profilin IV antiserum (dilution 1:500). Antibody binding was recognized by a peroxidase-coupled goat anti-rabbit antibody (Sigma). For detection, the chemoluminescent horse-radish peroxidase (CL-HRP) substrate system (Pierce Chemical Company; Rockford, IL) was used at a dilution of 1:10 and the blots exposed to X-ray film (Fuji Photo film, Tokyo, Japan). The specificity of antibody binding was shown by competition with the chemosynthetic oligopeptide (see Figure 2) pre-incubating the antiserum with the peptide (20 µg/ml of diluted antibody; 100 µg of total profilin IV peptide).
Immunohistochemistry
Bouin-fixed rat, mouse, hamster and human testes specimens were embedded in paraffin wax and 4–5 µm sections prepared. Sections were de-waxed, and submerged in phosphate-buffered saline. Antiprofilin IV antiserum was employed at dilutions from 1:500 to 1:2000; the corresponding preimmune serum served as a negative control. A conventional double-PAP–ABC procedure was adopted with the modifications as described by Balvers et al., 1998. Briefly, biotinylated anti-rabbit IgG was used as the second antibody at a dilution of 1:500. In the following steps peroxidase–antiperoxidase complexes from rabbits (1:500), avidin–biotin complexes, and diaminobenzidin reagent were used for detection. Tissue sections were investigated using normal brightfield microscopy (Nikon, Japan) and images captured with a Leica DC 300 digital camera (Leitz, Bensheim, Germany).
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Results |
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Profilin IV is a novel member of the mammalian profilin family
A human cDNA sequence had recently been annotated during the National Institutes of Health Mammalian Gene Collection Program showing moderate homology with the profilins (Strausberg et al., 2002
, Genbank accession no. BC029523
[GenBank]
). It was tentatively named profilin IV, but was not further characterized. The predicted protein showed only approximately 30% amino acid identity with the previously known human profilins, as revealed by an alignment on the peptide level. Nevertheless, database searches and structural prediction programmes produced significant alignment scores with the conserved profilin domain, the highest scores being reached with the profilins of yeasts, protozoa and Drosophila. Aided by homology searches, closely related sequences were identified in mammals; and rat, mouse and human profilin IV partial cDNAs were cloned (Figure 1). Sequence comparisons revealed that the open reading frame (ORF) was highly conserved (approximately 90% identity on the nucleic acid level). A ‘full-length’ rat profilin IV cDNA (Genbank accession no. AY682392 [GenBank] ) was obtained by an RT–PCR and 3'-RACE cloning strategy based on the cloned partial rat cDNA and the genomic and expressed sequence tag sequences found in the databases. An unusually long transcript leader region of 884 nucleotides was identified upstream of the ORF (Figure 1), which was highly similar to that found in the mouse. In this species, cDNA sequences which had been deposited in the databases (accession nos. AY495949 [GenBank] and AK013595 [GenBank] ) suggested alternative splicing within the leader involving intron splicing or retention while the protein-coding region remained unaffected. By 3'-RACE, two alternative 3'-ends were identified in the rat containing two non-canonical ATTAAA polyadenylation signals (Figure 1). These sequences were also found in the homologous mouse sequences. Indeed, rat and mouse profilin IV cDNAs were highly conserved (>90% identity) not only in their ORFs but also in their non-coding sequences. In comparison, in the human profilin IV cDNA only the ORF was conserved while the leader, 3'-untranslated region (UTR) and the exon/intron structure (Figure 1) appeared to be largely divergent. On comparing with the genomic databases, the human gene was tentatively localized to chromosome 2 (http://www.ncbi.nlm.nih.gov/genome/guide/human), and the homologous mouse gene on chromosome 12 in a region showing synteny to the human chromosome 2. Chromosomal assignment in the rat is still uncertain. The proposed exon/intron structure of the human and the rodent profilin IV genes which may give rise to different mRNA variants in each species is shown in Figure 1.
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Profilin IV mRNA variants are expressed in the mammalian testis
Tissue distribution and lengths of profilin IV-encoding mRNAs were studied by Northern blot analysis. Total RNAs extracted from rat, mouse and human tissues were hybridized with the respective homologous cDNA probes. Within the limits of Northern blot detection, profilin IV mRNA was only found in the testis in each of the species investigated (Figure 2). In the rat, multiple-hybridizing mRNAs were seen under stringent conditions, indicative of the presence of multiple profilin IV transcripts. A prominent band doublet was observed representing the two major rat transcripts of approximately 1.6 and 1.4 kb. This was considerably shorter than predicted from our cloned cDNA sequence (Figure 2A), suggesting that the first AUUAAA signal caused termination in the majority of transcripts. However, minor transcripts of increased and decreased lengths were also observed. A probe comprising the prolonged 3'-UTR sequence of rat profilin IV (after the first AUUAAA signal sequence, see Figure 1) hybridized only to the longest, at least 1.8 kb-long mRNA species, suggesting that our cDNA sequence information was indeed ‘full-length’. Transcripts of decreased length may be explained by alternative splicing, which by analogy with the mouse may involve the long 5'-leader (Figure 2A). In comparison, rehybridization of the same blot with a rat profilin III cDNA probe revealed a single band of approximately 1 kb in the testis. The weak 4 kb-signal seen in the rat kidney with this probe may result from a different transcript (possibly the sodium-phosphate transport gene 2) which had previously been shown in the mouse to include the antisense profilin III sequence in its 3'-UTR (Braun et al., 2002
).
Two broad profilin IV-hybridizing bands of approximately 1.6 and 1.4 kb were detected in testicular RNA extracts of the mouse, starting from day 15 of post-natal development to the adult. However, testes from 5-day-old prepubertal mice, where only spermatogonia are present (Malkow et al., 1998), showed no hybridization signal (Figure 2). Mouse profilin IV-encoding sequences (GenBank accession nos. AY495949
[GenBank]
and AK013595
[GenBank]
) suggested alternative splicing of exon 1, encoding the 5'-UTR, which might account for the two different lengths. In the human testis, a highly heterogeneous hybridization signal was observed with three different testicular RNA extracts, the lengths of the profilin IV-hybridizing mRNAs ranging from approximately 1.2 to 1.6 kb (Figure 2). Although partial degradation of human RNA cannot be excluded, it appeared that in the human testis, similar to the testes of rats and mice, the occurrence of multiple mRNA species is characteristic of testicular profilin IV expression.
Profilin III and IV mRNAs show distinct stage-specific expression in the adult rat testis
To confirm and extend the Northern blot results and to examine the cellular and temporal expression of profilin III and IV mRNAs within the testis, in situ transcript hybridization was performed in the rat. Employing DIG-labelled antisense cRNA probes (see Materials and methods) interstitial cells did not exhibit any hybridization signal above the background. Rather, different stage-specific hybridization patterns were observed within the seminiferous tubules. Profilin III antisense signals were confined to round spermatids during a relatively short period ranging from stages IV–V to VIII (Leblond and Clermont, 1952) of the rat spermatogenetic cycle (Figure 3). Profilin IV hybridization signals, in comparison, were weaker and occurred in germ cells during a longer period ranging from stages X to stages IV–V of the next cycle (Figure 3). This pattern was consistent with mRNA expression starting from spermatocytes of the late pachytene stages and proceeding throughout meiosis to the round spermatids. No reactivity with either probe was ever observed in spermatogonia, and no signals above the background were obtained with the corresponding sense cRNAs (Figure 3).
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Profilin III and IV mRNAs are differentially regulated during post-natal testicular development
To exploit the synchronous nature of the onset of spermatogenesis and to monitor how mRNA expression proceeds during the first spermatogenic wave, we prepared Northern blots of total testicular RNA from rats of different post-natal developmental stages and compared their profilin III and IV mRNA levels with those of adult rat testes. Radioactively labelled cDNA probes comprising the ORFs of profilin III and IV, respectively, were hybridized subsequently on the same blot (Figure 4). By employing the profilin IV probe, low levels of hybridizing mRNAs were first detected in testes of 20–22-day-old animals. Between days 22 and 26 of post-natal development, hybridization signals increased dramatically and remained high after that, increasing gradually towards adulthood. The increase in profilin IV mRNA levels overlaps with the onset of meiosis and the FSH surge in prepubertal male rats (Killian et al., 2003
). At this time, germ cells start to increase greatly in number, finally dominating the testis and diluting any signals from somatic cells in whole testicular tissue extracts.
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Using the profilin III probe on the same blot, a very faint hybridization signal was observed in rat testes of day 26. A clear signal became obvious only on day 28, paralleling the increasing numbers of round spermatids (Figure 4). Testes of 30-day-old rats revealed a stronger signal, while in the testes of 45-day-old animals, i.e. during the termination of the first spermatogenic cycle, hybridization signals appeared to decrease slightly. In the adult testis, differing from the continuously increasing profilin IV mRNA levels, profilin III mRNA levels experienced a strong, sudden increase. Rehybridization with a ß-actin probe as a control revealed a larger mRNA encoding the cytoplasmic ß- and
-actin isoforms, and the smaller 1.5 kb mRNA presumably encoding the testicular smooth muscle -actin (Kim et al., 1989). However, while the 2.1 kb mRNA was present at comparable levels throughout rat spermatogenesis, the 1.5 kb actin mRNA species was not consistently observed in the peripubertal rat testis (see Figure 2).
Profilin IV mRNA in the human testis is correlated with the presence of germ cells
To determine whether profilin IV mRNA could be detected in biopsy specimens from azoospermic patients undergoing biopsy in preparation for TESE (see Materials and methods), an RT–PCR analysis was performed based on 15 individual human testicular cDNA samples prepared from biopsy specimens, including patients showing the testicular histology of Sertoli Cell Only syndrome (SCOS) or maturation arrest (Figure 5). Histopathological examination revealed SCOS in five cases. In four others, biopsy findings showed partial tubular atrophy with maturation arrest (MA). The remaining specimens presented a normal feature of the parenchyma with intact spermatogenesis. PCR products indicative of profilin IV mRNA expression were identified in tissues showing normal histology and MA, however, profilin IV mRNA expression was largely reduced or absent in cDNA samples of patients with SCOS.
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Profilin IV protein expression parallels acrosome biogenesis and spermatid elongation
To verify the predicted profilin IV protein, monospecific antibodies were raised against a chemosynthetic oligopeptide. The amino acid sequence was deduced from a conserved stretch of the mouse profilin IV ORF (see Figure 1B). Specificity of the antiserum obtained was tested by Western blot analysis of whole testis protein extracts. The analysis revealed an immunoreactive band representing a testicular protein of approximately 14 kDa which did not react with the corresponding preimmune serum. Also, the reactivity of this band could be blocked by pre-absorption of the profilin IV antiserum with the antigenic oligopeptide (Figure 6), suggesting that the reactivity of the antiserum was specific.
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Profilin IV-related immunostaining was then monitored in paraffin sections through the pre- and peripubertal rat testis, tissues taken successively at 15–30 days after birth (Figure 7). Staining was first observed on day 26 when a weak signal became visibly associated with the cytoplasm of pachytene spermatocytes. Perinuclear staining of round spermatids was first observed 28 days after birth. The profilin IV-related immunoreactivity appeared to be distributed asymmetrically in the spermatids' cytoplasm, coincident with the position of the developing acrosome system. Perinuclear staining significantly increased in the testes of 30-day-old animals, the perimeter of staining expanding to cover approximately one-third of the nucleus of round spermatids. Immunostaining of these structures did not occur when preimmune serum was employed. Thus, the protein seemed to be localized mainly in the developing acrosome of haploid germ cells, closely following the time course of profilin IV mRNA expression during rat puberty (see above).
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In the adult rat testis, the profilin IV antibody strongly stained nearly all stages of spermiogenesis (Figure 8). Counterstaining of nuclei with haemalaun revealed close association with the nuclear membrane of round and elongating spermatids. Round spermatids after step 4 of spermiogenesis were intensely stained (Figure 8), the intensity still increasing during the acrosome phase (steps 8–12), and the maturation phase (steps 13–16). In the round spermatids of steps 4–5, the staining sometimes appeared associated with the nucleus as a little ring, in which there was a small hole, the size of the acrosomal granule, that was devoid of staining (Figures 8 and 9). Immunostaining then seemed to spread in the acrosome–acroplaxome region following the extending form of the future acrosomal head cap until it extended across approximately 30–40% of the spermatid nuclei. After the release of step 19 spermatozoa, staining of the successive step 9 spermatids was characterized by a typical asymmetry, following compression of the elongating nuclei, indicative of a staining of the transient manchette and possibly the Sertoli cell ectoplasmic region. During progressive elongation, the staining appeared as a V-shaped region in sagittal sections covering the narrow lateral width of the condensing spermatid head (Figure 8). Immunohistochemical staining patterns comparable to the rat were obtained in sections from mouse and human testes (Figure 9).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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A novel mammalian profilin was cloned and characterized, named profilin IV, representing a testicular isoform. Closely related species counterparts were identified in rat, mouse and human. Although highly conserved in its protein-coding region, the novel profilin showed only moderate amino acid sequence identity with the previously described profilins I (Kwiatkowski and Bruns, 1988
), II (Honore et al., 1993), and III (Hu et al., 2001; Braun et al., 2002). Nevertheless, database searches produced significant alignments with the profilin domain. Even profilins of highly distant evolutionary origin and of only 20% sequence identity can functionally substitute for each other (Rothkegel et al., 1996). Thus, it is most likely that profilin IV is indeed a functional profilin. Similar to profilin III (Braun et al., 2002), it might be involved in the regulation of testicular actin dynamics. The identification of a second testicular profilin which is specifically associated with acrosome biogenesis and spermatid elongation emphasizes the complexity of these processes. Their disturbance may underlie many of the abnormalities in sperm head morphology and acrosome function observed in infertile men.
Combining Northern blot analysis, in situ transcript hybridization and immunohistochemistry, our results suggested germ cell expression of profilin IV. During post-natal development, the first appearance of the mRNA coincided with the appearance of pachytene spermatocytes. Thus, profilin IV mRNA expression started earlier than profilin III, which was observed post-meiotically. Profilin III mRNA had previously been assumed to be expressed in ‘cells in the late stages of spermatogenesis’ (Braun et al., 2002). Our in situ hybridization results showed germ cell-specific transcription during stages V–VI to VIII of the rat spermatogenic cycle for profilin III, and during stages IX to IV–V for profilin IV mRNA, emphasizing that both testicular isoforms represented distinct gene products which may be required at subsequent stages of spermatogenesis and which might as well exert different specific functions.
Multiple mRNAs were characteristic of testicular profilin IV expression in each species investigated. In the case of profilin II, the variant mRNAs generate structurally and functionally distinct protein isoforms, named profilin 2a and 2b (Di Nardo et al., 2000; Lambrechts et al., 2000). We have as yet no indication that alternative splicing may give rise to different profilin IV protein isoforms. Rather, the untranslated regions seem to contribute to the observed length heterogeneity. The long 5'-leader of profilin IV mRNA seemed to be alternatively spliced in the mouse and could indicate that protein translation is tightly controlled. Testis-specific mRNAs that contain extended 5'-leaders are often translationally less active (Yiu et al., 1994; Gu et al., 1995). Translational repression, however, can also be an efficient way to localize and concentrate proteins in certain subcellular domains of germ cells (Kleene, 2003).
Aspects of 3'-end formation appear to be unique to male germ cells as well (reviewed in MacDonald and Redondo, 2002), and a large number of mRNAs from male germ cells lack the canonical AAUAAA polyadenylation signal but are nevertheless polyadenylated. Our cDNA sequence analysis showed that the canonical polyadenylation signal was likewise absent from the profilin IV mRNAs in each species investigated. Instead, two AUUAAA sequences were found in the cDNA sequences of rats and mice at homologous positions giving rise to two alternative transcript lengths. In the human counterpart, these signal sequences were lacking at homologous positions, suggesting that still other potential polyadenylation signals may be used in this species.
In each species investigated, profilin IV protein expression seemed to closely accompany acrosome biogenesis and spermatid elongation. In comparison, mRNA expression, at least in the rat, seemed to have already started in spermatocytes of the late pachytene. The pattern of profilin IV expression thus resembles that of acrosin and other acrosomal proteins whose transcription also starts already during meiosis (Kashiwabara et al., 1990; Kremling et al., 1991; Raab et al., 1994). These earlier studies provided evidence that, in rodents, the process of acrosome biogenesis begins during the diploid stage and proceeds post-meiotically into haploid cells, apparently different from the haploid-restricted expression of proacrosin described for the bull and the boar (Adham et al., 1989).
Our studies revealed that the pattern of profilin IV mRNA and protein expression in the human testis is similar to that observed in rodents. Moreover, RT–PCR analysis of human testicular biopsy specimens suggested that profilin IV expression is a molecular diagnostic parameter suitable for supplementing conventional histopathological diagnostics in the assessment of testicular biopsies. TESE with subsequent intracytoplasmic injection of haploid germ cells is the common therapy for patients with non-obstructive azoospermia. One problem with this approach is the lack of prognostic parameters for the presence of haploid testicular germ cells. The molecular-biological detection of germ cell-specific gene expression of profilin IV in these specimens (Patrizio et al., 2000; Schrader et al., 2002) may provide a possibility to improve the diagnostic value of testicular biopsies.
Considering the close association with the actin cytoskeleton of all profilins analysed thus far (for review, see Pollard and Borisy, 2003), our findings support the idea that the testis-expressed mammalian profilins III and IV may interact with the specific actin cytoskeleton of developing male germ cells at distinct time points and subcellular sites. Specifically, our immunostaining results showed that profilin IV is located in the right place at the right time to play a distinct role in the assembly–disassembly of F-actin within the acrosome–acroplaxome complex, in the transient manchette and presumably also the tubulobulbar extensions of Sertoli cells. It is thus tempting to speculate that profilin IV might play a role during acrosome formation, spermatid nuclear shaping and possibly spermiation. Male germ cells are endowed with a unique cytoskeleton of both ubiquitous and testis-specific actin isoforms, some of which arise from haploid-specific gene expression (Kim et al., 1989; Tanaka et al., 2003). It is conceivable that these testis-specific actin isoforms require specific profilins.
In addition to these proposed functions during spermiogenesis, the testis-expressed profilins may play a more direct role in the acrosome reaction of mammalian spermatozoa. In evertebrates, profilins have long been known to be centrally involved in the acrosome reaction, causing actin filament elongation in the acrosomal process (Tilney et al., 1983). It may thus be speculated that the novel profilin IV described here and/or the previously cloned profilin III (Braun et al., 2002) likewise function as regulators of actin dynamics during functional maturation of spermatozoa during epididymal transit (Howes et al., 2001; Scarlett et al., 2001; Lin et al., 2002) as well as during sperm capacitation and zona pellucida-induced acrosome reaction (Brener et al., 2003). Exposure of actin during in vitro capacitation in the equatorial region of human spermatozoa correlates with zona binding capacity (Liu et al., 1999, 2002).
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Acknowledgements |
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The authors thank Ms Annemarie Samalecos for her skilled technical assistance. We also thank Drs Laura Tres, Abraham Kierszenbaum, New York, and Richard Ivell, Adelaide, for an enlightening and stimulating introduction into mammalian spermatogenesis and for many helpful discussions. We are indebted to Professor Dr Freimut Leidenberger, IHF Hamburg, for his support and provision of excellent work facilities. Serono GmbH Deutschland kindly supported parts of this study.
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Notes |
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Supported by the German Research Association (DFG contract Ki 317/9 and GRK336).
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Submitted on September 7, 2004; resubmitted on October 11, 2004; accepted on October 27, 2004.
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