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Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University Medical Center, Stanford, California 94305
Address all correspondence and requests for reprints to: Marco Conti, M.D., Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University Medical Center, Stanford, California 94305-5317. E-mail: marco.conti{at}forsythe.stanford.edu
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Abstract |
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Introduction |
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In spite of these data indicating that cAMP-regulated gene expression is essential for spermatogenesis, the signals regulating cAMP levels and the exact pathway linking cAMP and transcription regulation are at present largely unknown. Previous data from several laboratories, including our own, have shown that all the components of the cAMP-dependent signal transduction are present in developing germ cells (9). Spermatids express adenylyl cyclase (AC) activity that is both soluble and particulate, indicating that multiple AC isoforms are expressed at this stage of gamete development (10, 11, 12, 13). Cyclic nucleotide phosphodiesterases are expressed during spermatogenesis, and a complex pattern of expression of the genes encoding enzymes with unique properties accompanies sperm differentiation (14, 15). Unique cAMP-dependent protein kinase isoforms are expressed during spermiogenesis, and the expression of the different regulatory and catalytic subunits is associated with sperm differentiation (16, 17).
In the attempt to define the structure and function of the ACs involved in spermatogenic cell differentiation, we have characterized the AC genes expressed in germ cells. The data reported here demonstrate that type III AC (ACIII) messenger RNA (mRNA) and protein are expressed in rat round spermatids. This form has been previously characterized as the major cyclase expressed in the olfactory system (18). Since olfactory receptors have already been localized on spermatids and spermatozoa (19, 20, 21), our results add strength to the hypothesis that common mechanisms may exist in the olfactory and reproductive systems to detect and modulate chemical signals.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-[32P]uridine triphosphate (400–800 Ci/mmol) and
-[32P]deoxycytidine triphosphate (3000 ci/mmol) were
from DuPont NEN (Boston, MA), and 125I-labeled Protein A
(2–10 µCi/µg) was from Amersham Corp. (Arlington Heights, IL).
pBluescript KS was from Stratagene (La Jolla, CA). Except as otherwise
indicated, all chemicals were the purest grade available from Sigma
Chemical Co. (St. Louis, MO).
Cell isolation and cultures
Male Sprague-Dawley rats of different ages were used in all
experiments. Animal housing and procedures were performed following
approved protocols at Stanford University. Sertoli cell-enriched
primary cultures were prepared from explants of seminiferous
epithelium, according to established procedures (22) from rat testes of
15-day-old animals and cultured at 32 C in MEM without serum. The
supernatant of the collagenase digestion obtained during Sertoli cell
procedure preparation was used to obtain a heterogeneous population of
peritubular cells. After dilution, the collagenase-dispersed cell
suspension was centrifuged at 1200 rpm for 10 min at room temperature
and, after several washings, the cells thus obtained were cultured in
DMEM in the presence of 5% FBS. Cells were cultured for 3 days and
contaminating germ cells were removed by a short hypotonic treatment
(23). Because of this treatment, less than 1% of the contaminating
cells were germ cells, as determined by phase contrast microscopy.
Cells were harvested at least 24 h after hypotonic shock to
extract total RNA or proteins. Using the alkaline phosphatase staining
(24), the contamination of Sertoli cell cultures by peritubular myoid
cells was evaluated to be less than 5%. Sertoli cells were recognized
by phase contrast microscopy in peritubular cell cultures, and the
contamination was evaluated to be 5%.
Total germ cells were isolated from adult testis by two subsequent collagenase digestions (0.33% Collagenase type I, 220 U/mg, Worthington Biochemical Corp., Freehold, NJ). After the first digestion, collagenase was diluted with PBS, and the supernatant containing interstitial cells was discarded. After a second digestion of the cell clumps, the collagenase was removed by several washings with PBS, and the tubules were disrupted mechanically. After 5 min of sedimentation, a pellet containing Sertoli cells and peritubular cells was discarded, and the supernatant was centrifuged at 1,200 rpm in a tabletop centrifuge. After several washings of the pellet in PBS, the total germ cell suspension was processed for Western blot analysis or immunohistochistry as described below. The purity of total germ cell suspension from adult testis was determined by phase contrast microscopy and staining with alkaline phosphatase. A contamination of less than 1% by somatic cells was determined by these methods. Germ cell fractions, enriched in round spermatid (F3) or pachytene spermatocyte (F5), were obtained by using sedimentation at unit gravity in albumin gradient (STAPUT) (25, 26). The two germ cell fractions used (F3 and F5) had the following composition: round spermatids (80–86%), intermediate and elongated spermatids (5–10%), early meiotic cells (1%), and unidentified cells (1–5%); middle late pachytene spermatocytes (85–90%), spermatid symplasts (10%), aggregates of elongated spermatids (0–5%), unidentified cells (> than 1%). Using cell preplating, it was estimated that less than 5% of the contaminating cells, presumably somatic cells, attached to the culture dish.
RNA preparation
Using a Quick prep micro mRNA purification kit (Pharmacia,
Piscataway, NJ), mRNA were extracted from rat and mouse testis
following the manufacturer’s protocol and were precipitated by
potassium acetate and ethanol. Total RNA was extracted from rat testis,
round spermatids, peritubular cells, or Sertoli cells cultured using
TriReagent (Molecular Research Center Inc., Cincinnati, OH) following
the manufacturer’s protocol and were precipitated with cold ethanol.
The dried pellets for each preparation were dissolved in
ribonuclease-free H2O and stored at -80 C for further
analysis.
Design of degenerated and nested primers to amplify AC from
mammalian germ cells
Design of primers was based on the comparison of the catalytic
domain C2a (amino acids 859-1122; highly conserved domain) of various
ACs. In Fig. 1
, the alignment of the
amino acid sequences of the conserved C2a domain of seven ACs is
reported, including the AC from Dictyostelium discoideum
expressed during its sporulation (DDIADCYG), the AC from
Drosophila melanogaster rutabaga (DRORAC), the bovine AC
type I (BOVADC), the rat AC type III (RATADCY3), AC type V (MUSADCYC),
AC type VI (MMU12919), and AC type IX (MMU30602) from mouse. The four
last ACs were the only ones cloned in rodents at the time this study
was initiated. Based on the comparison of both the amino acid sequence
and the nucleotide sequence, two sets of degenerate primers were
designed: an external set A (5'-G/A A/C G/C/A A A G/A A T C/T A A G/A A
C C A/G T T/C/A G G-3') and B (5'-A T C/T T/C C/T C/A C C T/C T T N C C
C/T T T C/G A C-3') and an internal set C (5'-C C N G T T/G/C G/A T
A/G/T G/T C T/G/A G G A/C/T G T-3') and D (5'-G G C/A C/G/A A C/T A/G T
T G/C A C N G T G/A T-3').
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gt11 cDNA library for mouse round
spermatids (1 µg). The oligonucleotides employed for these
experiments are reported above. Incubation mix was composed of 50
mM KCl, 10 mM Tris HCl, pH 8.3, 1.5
mM MgCl2, 200 µM of
deoxynucleoside triphosphate and 0.7 µM of each external
primer (A+B) or 2.5 µM of each internal primer (C+D). PCR
was carried out with low stringency for 50 cycles of denaturation (45
sec, 94 C), annealing (1 min, 37 C), and extension (3 min, 55 C) with
2.5 U of Taq DNA polymerase (GIBCO-BRL). At the end of PCR
amplification, products were analyzed on 1.6% or 3% agarose gel
stained with ethidium bromide and visualized with UV light. The
amplified fragments of the correct size were excised from the gel and
purified by a gel extraction kit (Quiagen, Chatsworth, CA) for
subcloning and sequencing. To monitor DNA contamination in the reagents
or samples, RT was omitted from some reactions to exclude genomic or
cDNA contamination. Reaction without RNA was used to monitor
contamination of RT or the primers. All of these controls gave negative
results.
cDNA subcloning and sequencing
The 400-bp fragment amplified with the external primers (A+B)
from rat round spermatids was subcloned into pUC18 using the SureClone
ligation kit (Pharmacia) at a SmaI site. Plasmid DNA
extraction and purification were performed using column chromatography
(Promega, Madison, WI). The nucleotide sequences of the inserted
fragments were determined by DNA sequencing using the Sequenase
(version 2) kit (Amersham Corp.), which is based on the dideoxy chain
termination method. Some of the sequencing was carried out by the
automatic sequencing in the DNA sequencing facility of the Beckman
Center located at Stanford University.
Northern blot analysis
Rat ACIII cDNA (gift from R. Reed) was digested by
PstI and StuI, and a 1120-bp fragment was
recovered (3034 bp to 4153 bp) which contained 699 bp/792 bp of C2a
domain, the complete C2b domain, and 357 bp of the 3'- untranslated
region. This fragment was labeled to a specific activity of
109 cpm/µg of DNA using
[-32P]deoxycytidine triphosphate and Random Primers
DNA labeling System (GIBCO-BRL). A rat multiple tissue blot from
Clontech (Palo Alto, CA) containing 2 µg of poly A+ RNA from eight
different tissues was hybridized with this probe using Express Hyb TM
hybridization solution from Clontech following manufacturer’s
procedure. Membranes were washed four times for 10 min in SCC 2x + SDS
0.05% and two times for 10 min in SCC 0.1x + SDS 0.1%.
Autoradiographs were obtained after 48 h of exposure at -70
C.
Ribonuclease (RNase) protection assay
The 400-bp PCR product was excised from pUC18 by SalI
and EcoRI and was subcloned in pBluescript KS-
(Stratagene). In this new construct the PCR product was under the
control of T7 polymerase promoter. After a linearization with
SalI and purification, this plasmid was ready to use as a
template to generate an RNA probe. A glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) construct from Ambion Inc. (Austin, TX) was
linearized by StyI to generate a probe of 190 bp and a
protected probe of 135 bp, if the transcription was performed using
also T7 RNA polymerase. The RNA Century Marker Template Set (Ambion
Inc.) includes five linearized plasmids for use as templates in an
in vitro transcription reaction for synthesis of RNA size
standards.
In vitro transcription was performed on each template (1
µg) using Riboprobe System II (Promega) following the manufacturer’s
instructions and using T7 polymerase (20 U/µg of probe) and 50 µCi
of -[32P]uridine triphosphate. At the end of the
reaction, digestion with 20 U of RNAse-free DNAse I per reaction was
performed at 37 C for 15 min. Probes were then precipitated to remove
unincorporated hot nucleotides with ethanol and ammonium acetate. The
pellets were dissolved in a loading buffer and loaded on a 6%
urea/acrylamide gel that was run to isolate only the full-length
probe.
RNAse protection assays were then performed using the RPA II kit (Ambion) following the manufacturer’s instructions. Forty micrograms of total RNA were hybridized with 106 cpm of 32P-labeled probe overnight at 45 C. Free probe was further digested with RNAse, and protected fragments were run on a 6% urea/acrylamide gel.
Transient transfection of 293 cells
ACIII cDNA was ligated in pCMV5 in EcoRI site in
sense or antisense orientation. Human embryonic kidney 293 cells grown
in DMEM-F12 (10% FBS) at 70% of confluence were transfected with 10
µg of pCMV5-ACIII by CaCl2 precipitation (125
mM) in 2-[bis(2-hydroxyethyl)-amino]ethane sulfonic
acid. After 18 h cells were incubated in fresh medium for 24
h. Cells were then harvested for Western blot analysis. For
immunohistochemistry experiments, cells were seeded on Lab-Teck wells
(Nunc, Naperville, IL).
Antibody
The antibody used for this study is a commercially available
anti-ACIII antibody (C-20; Santa Cruz Biotechnology, Santa Cruz, CA).
The antiserum was raised in rabbits by injecting a peptide
corresponding to amino acids 1125–1144 mapping at the carboxy terminus
of the rat ACIII sequence. The same peptide has been successfully used
to generate a ACIII-specific antibody (18). The rabbit antiserum was
affinity purified on a column with immobilized peptide. According to
the manufacturer, it is specific for ACIII since it does not
cross-react with AC types I, II, IV, V, and VI. As described below, its
specificity was further assessed in Western blot analysis and
immunohistochemistry using 293 cells transfected with a ACIII cDNA
inserted in the sense or antisense orientation.
Western blot analysis
Organs or cells were homogenized in 1% SDS (vol/vol) + 1%
ß-mercaptoethanol (vol/vol) and centrifuged for 20 min at 16,000
x g to remove cellular debris. The protein concentration of
the samples was measured according to the method of Bradford (27).
Samples were diluted in 2x sample buffer [62.5 mM Tris
HCl (pH 6.8), 10% glycerol, 2% (wt/vol) SDS, 0.7 M
ß-mercaptoethanol, 0.0025% (wt/vol) blue bromophenol], the samples
were boiled 3 min, and proteins were transferred to an Immobilon
membrane (Millipore Corp., Bedford, MA). Blocking of nonspecific
binding sites was performed by incubating the membrane overnight at 4 C
in 5% BSA (wt/vol) dissolved in TBS-T solution (0.1% Tween-20, 20
mM Tris HCl, and 14 mM NaCl, pH 7.6).
Incubation with ACIII antiserum diluted 1:100 (vol/vol) in TBS-T
containing 0.1% BSA and 1% normal goat serum (Vector Laboratories,
Burlingame, CA) was carried out for 1 h, and after extensive
washing, the membrane was incubated for 1 h with
peroxidase-conjugated secondary antibody (Amersham Corp.) diluted
1:5000 in TBS-T. After several washings with TBS-T, bound antibodies
were detected using a luminescence method (ECL from Amersham Corp.) and
recorded after exposure to x-ray films (usually 5–30 sec).
Alternatively, proteins were transferred to a nitrocellulose membrane,
and the incubation with ACIII antibody was performed at 1:1000 dilution
or ACIII antibody saturated with a synthetic peptide (1 µg/ml). After
washings, the membrane was incubated for 1 h with
125I-labeled protein A (0.5 µCi/ml) in a blocking buffer
(5% nonfat dry milk in PBS), rinsed extensively with TBS-T, and
autoradiographed.
Immunofluorescence
Adult Sprague-Dawley rat testes were cut into small pieces and
quickly embedded in Tissue-Teck (Miles, Inc., Diagnostic Division,
Elkhart, IN) and frozen in liquid nitrogen. Approximately 5-µm
sections were obtained from the embedded specimens and mounted on
poly-L-lysine covered slides. Total germ cells were
isolated from adult testis as described above and loaded at a
concentration of 0.5 x 106 cells/ml on
poly-L-lysine-coated slides. After an incubation of 10 min
at room temperature in a humidified chamber, unattached cells were
removed from the slides by washing with PBS. Germ cell preparations on
slides were stored at -20 C. Sections and germ cell preparations were
dried at room temperature and fixed in ethanol/acetone (vol/vol) at
-20 C (10 min). After washing in cold PBS (10 mM, 150
mM NaCl, pH 7.4), the preparations were incubated in 1%
normal goat serum (Vector Laboratories) for 30 min to reduce
nonspecific staining. This was followed by overnight incubation in a
humidified chamber at 4 C with ACIII antibody at a dilution of 1:1000
(0.1 µg/ml). After washing in PBS, the preparations were incubated
with a goat anti-rabbit IgG coupled to fluorescein (7.5 µg/ml)
(Vector Laboratories) for 45 min in the dark. Slides were washed
several times with PBS and mounted in Vectorshield mounting medium
(Vector Laboratories). Specificity for ACIII staining was monitored
using several controls. The antibody was preadsorbed with 1 µg/ml of
the peptide used for immunization (Santa Cruz Biotechnology), or with 2
µg/ml of membrane preparations from 293 cells transfected with ACIII
cDNA in sense and antisense orientation. Transfected cells were
harvested in an isotonic buffer (250 mM sucrose, 20
mM Tris HCl, pH 7.8, 1 mM EDTA, 10
mM ß-mercaptoethanol) and a mixture of protease
inhibitors (50 mM benzamidine, 0.5 µg/ml leupeptine, 0.7
µg/ml pepstatin, 4 µg/ml aprotinin, 10 µg/ml soybean trypsin
inhibitor, 2 mM phenylmethylsulfonylfluoride) and
centrifuged 30 min at 20,000 x g. The particulate
fraction was resuspended in 10 mM PBS (pH 7.4) and
incubated for 30 min at room temperature with 1% normal goat serum.
The suspension was then recentrifuged for 30 min at 20,000 x
g. The resuspended pellet was incubated with ACIII antibody
for 2 h at room temperature and recentrifuged for 30 min at
20,000 x g. Recovered supernatant was used for the
immunohistochemistry.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, RT-PCR was performed on mRNA prepared from total
testis and enriched spermatid fractions. Amplification of first-strand
cDNAs from rat testis and rat round spermatids with the external pair
of primers (A+B) yielded a PCR product that had the expected size (400
bp) (Fig. 2). No signal was detected in
the controls in which mRNA from rat testis or RT was omitted. These PCR
products were subjected to a second amplification with an internal pair
of primers (C and D of Fig. 1). PCR products of the expected size were
obtained from rat testis, as well as from rat and mouse round
spermatids. In the latter case, an amplified fragment was observed in
spite of the fact that the first amplification did not show a
detectable product. An amplification of products of the correct size
was also obtained with mRNA from mouse testis (data not shown).
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). One of the clones showed 100% of sequence identity
with the corresponding region of type I AC (Fig. 1). Seven clones
contained fragments missing one or both of the primers and did not
correspond to any known sequences when compared with known deposited
sequences using the BLAST (NCBI, Bethesda, MD) search routine. This
semiquantitative analysis provided a first indication that ACIII mRNA
is present in significant amounts in germ cells.
Northern blot analysis of ACIII in the testis and other tissues
To confirm whether the ACIII mRNA is indeed expressed in the
testis, Northern blot analysis was performed on mRNAs from different
rat tissues using a cDNA probe corresponding to 1120 bp sequence unique
to rat ACIII (Fig. 3). A 4.7-kb
transcript corresponding to rat ACIII was detected in all rat organs
surveyed with variable intensity dependent on the tissue used. The
strongest signal was detected in brain, spleen, and lung; a signal of
medium intensity was detected in kidney and of low intensity in
skeletal muscle and testis (Fig. 3). In the testis, two additional
transcripts were detected: an abundant 7.5-kb transcript and one of low
intensity at 5.6 kb. The former transcript was also detected at a low
level in spleen (Fig. 3). When the blot was stripped and hybridized
with an actin probe, similar hybridization was observed in all lanes
(data not shown). Although the signals detected are lower than that
reported for the olfactory system, these data are consistent with the
hypothesis that ACIII is expressed in organs and tissues other than the
olfactory epithelium.
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). When a 520-bp
ACIII probe was hybridized with total RNA from different cell
populations of the testis, protected fragments were clearly detectable
in round spermatids and, at a lower level, in pachytene spermatocytes
(Fig. 4). With RNA from somatic cells of the seminiferous epithelium,
protected fragments were observed in peritubular cells, but not in
Sertoli cells (Fig. 4) or Leydig cells (data not shown). The signal
with the total testis was approximately 50% of that in round
spermatids, confirming that not all testicular cells express this
message. Similar results were obtained with a shorter 212-bp probe
(data not shown). The GAPDH signal was reduced in germ cells because a
probe corresponding to the somatic GAPDH rather than germ cell GAPDH
was used in these experiments (28).
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) (18) was observed with
cells transfected with a plasmid containing the ACIII cDNA in the sense
orientation. Conversely, no signal was detected when cells were
transfected with ACIII inserted in antisense orientation (Fig. 5A).
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The intensity of the signal increased with the age. A signal was also
detected in total germ cell preparations from adult rat testis while a
very faint signal was present with peritubular cell extracts. The
immunoreactivity of the 170-kDa band was specific, since it was blocked
when the ACIII antibody was preincubated with the synthetic peptide
used as immunogen (Fig. 5B). It should be noted that, in the olfactory
epithelium, the ACIII protein migrates as a glycosylated form with a
molecular size between 180 kDa and 200 kDa (18, 29).
Immunolocalization of ACIII in the rat testis
To test the specificity of the ACIII antibody in
immunofluorescence, staining was performed on 293 cells transfected
with pCMV5-rat ACIII expression vector (Fig. 6a). Approximately 10% of the 293 cells
exhibited a positive immunoreactivity for ACIII (Fig. 6, a and b). No
signal was detected if the ACIII antibody was preincubated with the
synthetic peptide used to generate this antibody (Fig. 6c) or if the
cells were transfected with ACIII cDNA inserted in pCMV5 expression
vector in antisense orientation (data not shown).
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) in round spermatids,
elongated spermatids, and maturing spermatids. On the most
differentiated spermatids (Fig. 7b) the intensity of the signal
decreased. An experiment performed on epididymal spermatozoa showed a
very faint staining of these more mature germ cells (data not shown).
No staining was detected in Sertoli, myoid, or interstitial cells of
the testis (Fig. 7, a and b). Blood vessels (Fig. 7, a and b) and
endothelial cells (Fig. 7b, arrows) presented a positive
immunoreactivity for ACIII, although the signal was lower than that
localized on spermatids.
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) or with membrane preparations from 293
cells previously transfected with ACIII cDNA inserted in sense
orientation in pCMV5 expression vector (Fig. 7e). Conversely, signal
was still present if the antibody was preadsorbed with the membrane of
293 cells transfected with ACIII cDNA inserted in pCMV5 expression
vector in antisense orientation (Fig. 7d). All these controls confirmed
the specificity of the signal observed with sections of the rat testis.
When the immunofluorescence experiment was performed on 15-day-old rat
testis, only vessels and endothelial cells showed a positive
immunoreactivity for ACIII (data not shown).
Subcellular localization of the ACIII staining in rat
spermatids
In Fig. 8, ACIII staining of the
spermatids at a high magnification (Fig. 8, a, e, and g) with their
corresponding phase contrast photomicrographs (Fig. 8, b, f, and h) are
presented. At this higher magnification and by comparing the staining
in section and in isolated germ cells (Fig. 8, c and d), the staining
was localized on the acrosomal membrane of round spermatids. Figure 8
documents a similar localization of the staining for elongated
spermatids (step 13 of spermiogenesis) and maturing spermatids (step 16
of spermiogenesis), respectively. Immunofluorescence performed on adult
mouse testis showed the same localization of ACIII in the acrosomal
membrane of spermatids from round spermatids to maturing spermatids
(data not shown), demonstrating that this localization is observed in
more than one species.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To date, eight or nine different AC genes have been identified in
mammals (32). This large multigene family has a complex transmembrane
topology that consists of a short cytoplasmic amino terminus followed
by six transmembrane spans termed M1 and a large 40-kDa cytoplasmic
domain C1. This motif is then repeated once (32). The overall amino
acid sequence similarity among the different ACs is roughly 50%;
however, two regions within the catalytic domain, the cytoplasmic
domains designated as C1a and C2a, are highly conserved. Analysis of a
series of truncations and alanine scanning mutants of mammalian AC
indicated that both C1a and C2a, but not C1b and C2b, are necessary for
catalytic activity (33). Recently, the crystal structure of the C2
region of ACIII has been determined in a complex with the activating
dipterpene forskolin (34). The active site of AC is formed by
dimerization of both catalytic monomers, and the binding sites of ATP,
forskolin, and Gs protein are in close association in this C2
domain.
In the present report, we have used both molecular and immunological approaches to identify and elucidate the properties of the AC expressed in mammalian spermatids. All our findings indicate that an AC form thought to be expressed predominantly in the olfactory system (18) is expressed in developing germ cells. RT-PCR initially designed to detect transcripts for all known AC RNAs, including the enzyme present in D. discoideum and D. melanogaster, consistently yielded an amplification product corresponding to ACIII. Furthermore, Northern blot analysis confirmed that a transcript derived from ACIII is expressed in the testis. ACIII was originally cloned by Bakalyar and Reed (18) and described as being mainly expressed in the olfactory system as a 4.7-kb transcript. Conversely, a 7.5-kb transcript is the predominant mRNA species present in the testis, opening the possibility that a unique splicing variant of ACIII may be expressed during spermatogenesis. RNAse protection analysis confirmed and extended the above findings, demonstrating that an mRNA coding, at least for the catalytic domain of ACIII, is expressed in germ cells at meiotic and postmeiotic stages of differentiation. Finally, an immunoreactive polypeptide with the properties of ACIII was identified in testis and germ cell extracts. The commercial antibody used is directed toward the carboxyl terminus end of ACIII, a region that does not share any homology with other ACs. This domain is absent in ACIV and ACII, and when it is present as in ACI, it has no detectable homology to the corresponding ACIII region (35). The same antiserum (36) or antibodies against the same epitope (18) have been used to study the properties and localization of ACIII in the olfactory system and in transfected HEK-293 cells. Thus, the properties of this antibody exclude the possibility of cross-reactivity with other ACs.
In agreement with the above conclusions, the immunocytochemical localization of ACIII in the seminiferous tubule showed a strong signal on spermatids at different steps of their differentiation, including round spermatids in cap phase and mature elongated spermatids. The specificity of the staining was confirmed by several controls including preadsorption of the antibody with synthetic peptide or with the protein expressed on 293 cells transfected with ACIII. The intensity of the staining decreases for the last step of the maturation just before spermiation, and only very faint staining was detected on epididymal spermatozoa. Further experiments are required to clarify whether ACIII is still present in significant amounts in mature spermatozoa.
Transcripts corresponding to ACIII were detected not only in germ cells but also in peritubular cell preparations. Although little signal was obtained with Western blot analysis, some staining above the background was observed in the endothelial cells by immunocytochemistry of testis sections. Thus, low levels of ACIII may be expressed in cells other than germ cells of the rat and mouse testis. The possibility that the signal detected in germ cells is due to contamination from this minor cell population is unlikely for the following reasons. RNAse protection was performed on enriched germ cell populations where somatic cell contamination was estimated to be less than 5%. Since the expression detected in somatic cells is low, a minor cell contaminant could not account for the intensity of the signal observed in germ cells. In addition, an increase in the ACIII signal was observed when enriched germ cell preparations were compared with total testis by Western blot analysis, by RNAse protection or PCR, suggesting that germ cells are a major site of expression of ACIII. Finally, the immunocytochemical localization is again consistent with the conclusion that a major site of expression of ACIII is in postmeiotic germ cells.
Contrary to the somatic ACs, the soluble AC described in germ cells is activated poorly by forskolin and does not interact with a G protein. The germ cell enzyme is insensitive to GTP, analogs and the enzyme activity requires Mn++ rather than Mg++. On the basis of these properties, it is unlikely that this soluble AC form expressed in spermatids corresponds to ACIII, since this enzyme is activated by forskolin and is coupled to G proteins. Since it has been shown that both C1a and C2a domains are necessary for catalysis, it is also difficult to envisage that the soluble germ cell cyclase is generated by proteolysis of ACIII. To reconcile these differences, we propose the hypothesis that multiple ACs are expressed in spermatids. This hypothesis is supported by our previous observation that both soluble and particulate AC are detected in isolated pachytene spermatocytes and round spermatids (12). A soluble, or easily solubilized, enzyme originating from a unique gene is expressed predominantly in spermatids, perhaps with a structure similar to the D. discoideum ACG (30). Conversely, one or more particulate forms with multiple transmembrane domains including ACIII are expressed in haploid germ cells and may be retained in spermatozoa.
A detailed study at high magnification of the immunohistochemistry data
together with the results obtained on germ cell preparations from adult
testis indicate that ACIII is localized in the acrosome membrane of
spermatids rather than on the plasma membrane. There are precedents to
localization of the cyclase in membranes other than plasma membrane, as
a cyclase appears to be confined to T tubules of cardiac myocytes (37).
The acrosome is an exocytotic vesicle containing a variety of
hydrolytic enzymes (38) and lying over the anterior portion of the
sperm head. This organelle is essential for fertilization since it
allows the penetration of the cumulus mass and the zona pellucida
surrounding the oocyte (39, 40). Morphological features of acrosome
formation and development during spermiogenesis have been described in
detail for several species (41, 42, 43, 44). However, the biochemical events
accompanying these structural changes are not well defined.
Interestingly, a study done in the mouse indicated the localization of
Gi protein in the proacrosomal granules and in the developing
acrosome of spermatids (45), suggesting a role of G proteins in the
formation of this organelle. Immunoreactive Gi protein has been
detected in the sperm acrosome of mouse and Guinea pig (45, 46), a
localization consistent with a role of this G protein in acrosomal
exocytosis (46, 47, 48). Finally, a GRK4 kinase has been found in the
acrosomal membrane of human and rat spermatozoa (49). Our finding that
ACIII is expressed on the acrosomal membrane adds another component of
membrane signaling expressed in this organelle during spermiogenesis,
reinforcing the possibility of the involvement of this transduction
machinery in the acrosome biogenesis or regulation.
A subset of the olfactory receptor gene family (seven-transmembrane domain receptors coupled to G protein) is specifically expressed in male germ cell lines of dog, human (19, 20), and rat (21) with little or no expression in olfactory mucosa. Selective localization of putative odorant receptors and associated desensitizing proteins, G protein-coupled receptor kinase 2 (GRK2), and ß-arrestin 2 have been shown in elongated spermatids and the midpiece of the sperm tail of the rat (21). The midpiece is characterized by the presence of tightly packed mitochondria contributing to the production of energy required for motility (50). It has been proposed that these olfactory receptors may be implicated in sperm chemotaxis during fertilization. Although we have not been able to demonstrate an overlap in the localization of ACIII and the olfactory receptor, further experiments are required to test the possibility that this cyclase may be an effector for these receptors as it is in the olfactory organ. Interestingly, a type 4A phosphodiesterase mainly expressed in odorant receptor neurons of the olfactory neuroepithelium (51) is also expressed at a high level in round spermatids (15) and possibly in spermatozoa. All these findings are then consistent with the hypothesis that a signal transduction system used in olfaction may also be used during gamete development and/or in the function of the mature spermatozoon. Although the signals activating this pathway in germ cells remain to be identified, understanding the nature of these signals may uncover an important regulatory mechanism in gamete production and fertilization.
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Acknowledgments |
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Footnotes |
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2 Recipient of a fellowship from L’ Association Française pour
la Recherche Thérapeutique, Paris, France.
3 Supported by a fellowship from Fondazione Cenci Bolognetti, Rome,
Italy, and from the Dean’s Fellowship at Stanford University.
Received September 22, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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i is associated with the
developing acrosome. Dev Biol 152:393–402[CrossRef][Medline]
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