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- Original Article -
Localization of AKAP4 and tubulin proteins in sperm with
reduced motility
Elena Moretti, Giacomo Scapigliati, Nicola Antonio Pascarelli, Baccio Baccetti, Giulia Collodel
Department of General Surgery, Biology Section, University of Siena, Regional Referral Center for Male Infertility,
Siena 53100, Italy
Abstract
Aim: To perform screening, related to A-kinase anchoring proteins 4 (AKAP4) and tubulin proteins, in spermatozoa
with absent or severely reduced motility in order to detect the status of the fibrous sheath and the axonemal structure.
Methods: An immunocytochemical study of tubulin, used as a positive control, and AKAP4 was carried out to detect
the presence and the distribution of these proteins in different sperm samples. The morphological characteristics of
sperm were studied by transmission electron microscope (TEM) and the results were elaborated using a formula
reported in previous studies. PCR was carried out on DNA extracted from peripheral blood lymphocytes to analyse
partial sequences of the Akap4 and Akap3 genes.
Results: Immunolabelling of tubulin and AKAP4 showed different
patterns, which led us to divide the patients into groups. In group I, the absence of AKAP4 and tubulin was revealed,
although these patients did not show alterations in the Akap4/Akap3 binding site. TEM evaluation highlighted that a
high presence of necrosis was associated with total sperm immotility. In group II, a regular AKAP4 and tubulin signal
was present, although motility was reduced and TEM analysis revealed the presence of immaturity. In group III, in
which a weak AKAP4 label associated with normal tubulin staining and reduced motility was observed, a severe
disorganization of the fibrous sheath was highlighted by TEM.
Conclusion: While the role of AKAP4 in sperm
motility is unclear, absent or weak AKAP4-labelling seems to be associated with absent or weak sperm
motility. (Asian J Androl 2007 Sep; 9: 641_649)
Keywords: AKAP4; immunocytochemistry; motility, sperm; transmission electron microscope
Correspondence to: Dr Giulia Collodel, Department of General Surgery, Biology Section, University of Siena, Policlinico S. Maria alle
Scotte, Siena 53100, Italy.
Tel: +39-0577-2335-39 Fax: +39-0577-2335-27
E-mail: collodel@unisi.it
Received 2006-06-07 Accepted 2007-01-22
DOI: 10.1111/j.1745-7262.2007.00267.x
1 Introduction
Male infertility is a significant problem in humans and may be caused by different pathologies, such as anatomical
problems, infections, hormonal imbalances, chromosomal alterations and gene anomalies. However, 30% of infertile
men are affected by idiopathic oligoasthenoteratozoospermia and the c
ause of infertility is still unknown as reported
by Cavallini [1]. The analysis of sperm motility plays a central role in the evaluation of male
fertility because it is known that a high percentage of poorly motile or immotile sperm will not be able to fertilize. The clinical relevance of
sperm motility is evident, but the molecular mechanisms involved in this process have not been fully understood yet.
Flagellar sperm structure has been extensively described in the published literature, and correct organization of
the axonemal pattern and of periaxonemal structures is pivotal for ensuring normal motility. During the past few
years, attention has been paid on the fibrous sheath, a cytoskeletal structure surrounding the axoneme and outer dense
fibers that defines the extent of the region of the
principal piece of sperm flagellum. It consists of two
longitudinal columns connected by closely arrayed
semicircular ribs that assemble from the distal to the proximal part
of the tail throughout the spermiogenetic process.
It is generally accepted that the fibrous sheath plays
a role as a mechanical support of sperm flagellum.
Fibrous sheath is able to modulate flagellar bending and to
define the shape of flagellar beats as described by Fawcett
[2].
Recently, extensive studies have been carried out to
identify proteins that constitute the fibrous sheath and
that are in some way actively involved in sperm motility.
Eddy et al. [3] found that in the human fibrous
sheath, A-kinase anchoring proteins 3 and 4 (AKAP3,
AKAP4) are the most abundant structural proteins,
anchoring cyclic-AMP (cAMP)-dependent protein kinase
A to the fibrous sheath through the regulatory subunit of
kinase. cAMP-dependent phosphorylation of flagellar
proteins is involved in the beginning and maintenance of
sperm motility. The second messenger cAMP mediates
its intracellular effects in spermatozoa through
cAMP-dependent kinase (PKA). The intracellular organization
of PKA is controlled by its association with AKAPs.
In particular, AKAP3 is synthesized in round spermatids, incorporated into the fibrous sheath
concurrently with the formation of rib precursors and is
reported to be involved in organizing the basic structure of
the fibrous sheath [4]. AKAP4 is synthesized and
incorporated into a nascent fibrous sheath late in spermatid
development and it plays a major role in completing
fibrous sheath assembly as reported by Brown et
al. [4]. A targeted disruption of the Akap4 gene causing defects
in mice sperm flagellum and motility has also been
demonstrated by Miki et al. [5]. Recently, Baccetti
et al. [6] used transmission electron microscope (TEM) to
determine the genetic defect "dysplasia of the fibrous
sheath" (DFS) in sperm from a group of infertile men.
In these cases, immunolabelling of tubulin confirmed the
presence of typical short and thick tails whereas AKAP4
protein staining showed a weak signal, revealing a
disorganized and incompletely assembled fibrous sheath.
Moreover, polymerase chain reaction (PCR) for
detecting the presence of a partial sequence of
Akap4/Akap3 binding regions produced positive results according to
Turner et al. [7].
Based on our data showing alteration of the fibrous
sheath in immotile sperm containing genetic defects, the
present study was undertaken to determine if similar
alterations were present in sperm with reduced motility
but without genetic defects.
As previously described, AKAP4 labeling was performed to detect the typical spatial organization of
fibrous sheath components and tubulin staining was
carried out in order to check axomene assembly.
Morphological characteristics were studied by TEM, a valuable
tool for a more detailed evaluation of sperm ultrastructure,
and the results were elaborated using the formula by
Baccetti et al. [8]. TEM analysis was performed to try
to explain the structural causes of absent or reduced
motility in this group of patients. PCR was carried out on
DNA extracted from peripheral blood lymphocytes to
analyse partial sequences of the Akap4 and Akap3 genes.
2 Materials and methods
2.1 Patients
Semen samples were obtained from 16 patients (aged
24 to 33 years old) with idiopathic infertility referred to
the Regional Referral Center for Male Infertility for
semen analysis after 3 years of sexual intercourse without
conception. The lymphocyte karyotypes were normal
in all cases. Written consent was obtained from all
patients donating samples, both infertile men and controls.
2.2 Semen analysis
2.2.1 Light and electron microscopy
Semen samples were collected by masturbation after 3_4 days of sexual abstinence and examined after
liquefaction for 30 min at 37ºC. Any delay between
ejaculation and sample processing was recorded. Volume,
pH, concentration and motility were evaluated according
to WHO guidelines [9]. Supravital eosin staining was
used for evaluating sperm viability.
For TEM, semen was fixed in cold Karnovsky fixative and maintained at 4ºC for 2 h. Fixed semen was
washed in 0.1 mol/L cacodylate buffer (pH 7.2) for 12
h, postfixed in 1% buffered osmium tetroxide for 1 h at
4ºC and dehydrated and embedded in Epon Araldite.
Ultra-thin sections were cut with a Supernova
ultramicrotome (Reickert Jung, Vienna, Austria), mounted on
copper grids, stained with uranyl acetate and lead citrate and
then observed and photographed with a Philips CM10
transmission electron microscope (TEM; Philips Scientifics, Eindhoven, The Netherlands). Three
hundred ultra-thin sperm sections (approximately 50% heads
and 50% tails) were analyzed for each patient. Major
submicroscopic characteristics were recorded, applying
the same evaluation criteria, by highly trained examiners
who were blind to the experiment. TEM data were
elaborated using the mathematical formula by Baccetti
et al. [8], based on Bayesan's technique. This formula
considers 16 selected submicroscopic characteristics of
sperm organelles to define sperm function and
calculates the number of spermatozoa free of structural
defects ("healthy") and the percentages of three main
phenotypic sperm pathologies: immaturity, necrosis and
apoptosis as demonstrated by Baccetti et
al. [10]. Moreover Baccetti et al. [8] observed that the lowest number
of spermatozoa free of defects and assuring normal
fertility was slightly over two million.
The controls were five men with normal karyotype
who had fathered a child during the previous 1 to 2 years.
2.2.2 Immunofluorescence
Semen samples were washed twice in phosphate buffered saline (PBS), smeared on glass slides, air dried,
rinsed in PBS and fixed for 15 min in methanol at _20ºC.
Slides were then treated with blocking solution (PBS,
1% bovine serum albumin, 5% normal goat serum) for
20 min at room temperature (RT) and incubated overnight at 4ºC with mouse monoclonal anti-tubulin (Sigma
Chemical, St. Louis, MO, USA) and mouse monoclonal
anti-AKAP82 (BD Biosciences, Erembodegem, Belgium)
specific for human AKAP4 protein, diluted at 1:100 and
1:50 respectively in PBS, 0.1% BSA, 1% NGS. After
three washes in PBS, the samples were treated with goat
anti-mouse IgG-Texas Red conjugated antibody (Southern
Biotechnology, Birmingham, AL, USA). Finally, the
samples were washed three times in PBS and mounted
with Vectashield (Vector Labs, Burlingame, CA, USA).
Incubation in primary antibodies was omitted in control
samples. Observations and photographs were made with
a Leitz Aristoplan light microscope (Leica, Wetzlar,
Germany) equipped with a fluorescence apparatus. A
total of 200 spermatozoa from each sample were counted
and scored as either labelled or not labelled with the
respective antibody. For the AKAP4 experiment, only those
spermatozoa stained throughout the length of the
principal piece were counted. The same five samples from
healthy men of proven fertility were examined and used
as controls.
2.3 PCR analysis
DNA was extracted from peripheral blood lymphocytes using the QIAamp DNA Blood Kit (QIAGEN,
Valencia, CA, USA).
PCR products corresponding to a region of hAkap4
involved in binding to Akap3 (site 1) and to a region of
Akap3 involved in binding to hAkap4 (site 2) were
amplified according to Turner et al. [7]. Oligonucleotide
primers flanking the respective binding sites were used:
site 1: sense primer 5'-TCAGTGCCCTTATAGGTGAG-3', antisense primer
5'-GCAGAGCTTCATCACAGATTC-3';
site 2: sense primer 5'-TTGAGGAATCTCCACAGCG-3'; antisense primer
5'-CCAACGGTCTTTCACACAACTTC-3').
Control DNA was extracted from the blood of five
fertile men.
3 Results
Sixteen semen samples from men with idiopathic
infertility were examined by light and electron microscopy
to determine sperm concentration, motility and
morphology. In all samples, immunocytochemistry for the
localization of AKAP4 and tubulin was performed.
Among the group of infertile patients, sperm
concentration was normal in eight out of 16 patients
according to WHO guidelines [9]. Rapid (a) and slow (b)
progressive motility was absent or severely reduced in all
analysed samples. These parameters are reported in
Tables 1, 2 and 3.
Immunolabeling of AKAP4 protein and tubulin, allowed us to separate the patients into three groups (Tables
1_3):
Group I: patients 1_5 in whom the label was
negative for both antibodies and motility was totally absent
(Table 1);
Group II: patients 6_10 in whom the AKAP4 signal
(Figure 1A, 1B) and the tubulin label (Figure 1C, 1D)
were 100%, except in patient 9 in whom AKAP4 labelling was 95%. Sperm motility was reduced in all
patients (Table 2);
Group III: patients 11_16 in whom the AKAP4 signal
(Figure 1E, 1F) was weaker (at least 20%), the tubulin
label ranged from 80% to 100% (Figure 1H) and motility
was strongly reduced.
The five patients with proven fertility, who were used
as controls (Table 4, A_E), showed normal
immunofluorescent staining for tubulin (Figure 1H) and AKAP4
(Figure 1G) in 95%_100% of tails (Table 4).
All sperm samples from infertile and fertile men were
analysed by TEM and the data obtained were processed
using the mathematical formula by Baccetti et
al. [8].
Mathematically elaborated TEM analysis (Tables
1_4) confirmed that 16 examined patients were infertile
(Tables 1_3), showing a number of healthy sperm of
< 2 000 000. Necrosis (Figure 2A) was extremely high
(Table 1) in group I (89.045± 10.936) versus the other
groups (Tables 2 and 3) and controls (Table 4). This
sperm pathology is characterised by absent or reacted
acrosomes, misshapen nuclei with disrupted chromatin,
swollen and disassembled mitochondria and altered
axonemal and periaxonemal structures. In these cases, eosin
Y staining confirmed the presence of dead sperm at >
80%.
Group I also showed a high percentage of sperm with apoptotic characteristics (12.059
± 6.596) such as misshapen acrosomes, nuclei with marginated
chromatin and swollen mitochondria irregularly organized into
large cytoplasmic residues with translucent vacuoles
(Figure 2B).
Group II showed a mean of percentage of sperm necrosis (Table 2) very similar to that of controls (Table
4), which was considered normal. However, a very high
presence of apoptosis and immaturity (Figure 2C)
compared to controls (Table 4) was observed. Immature
spermatozoa generally showed altered acrosomes and
round or elliptical nuclei with uncondensed chromatin.
In particular, a high percentage of spermatozoa with large
cytoplasmic residues, often embedding coiled axonemes
(Figure 2C, 2D) was highlighted although the axonemal
and periaxonemal structures, including the fibrous sheath,
were generally normal (Figure 2D), as also revealed by
immunocytochemical analyses.
Group III showed a mean percentage of sperm pathologies (Table 3) similar to the previous group (Table
2). Moreover, despite reduced motility, TEM analysis
showed "9 + 2" pattern axonemes with structural defects,
particularly involving the fibrous sheath, that appeared
poor and badly assembled (Figure 2E).
PCR products corresponding to a region of hAkap4
involved in binding to Akap3 (site 1) and to a region of
Akap3 involved in binding to hAkap4 (site 2) were present
in all examined patients and controls.
4 Discussion
Although sperm motility is one of the most
important predictors of fertilizing ability, the mechanisms
underlying motility abnormalities are still poorly understood.
Recent research performed by Li et al. [11] has
furnished new clues regarding the key molecular mediators
of sperm motility. However, it is well known that sperm
motility is regulated by the cAMP-dependent protein
kinase (protein kinase A)-mediated phosphorylation of
groups of flagellar proteins. In mouse, human and bull
spermatozoa, two major fibrous sheath proteins, AKAP4
(also called AKAP82 or fibrous sheath component 1) and
its precursor proAKAP4, have been identified as
members of the A-kinase anchor protein (AKAP) by Carrera
et al. [12, 13]. The hypothesis is that, by anchoring the
activity of PKA in the fibrous sheath, AKAPs play central
roles in the regulation of normal sperm motility. Turner
et al. [14] did not find evidence of an association
between the degree of processing of pro-hAKAP4 and
increases or decreases in motility in spermatozoa from
normal men.
Recently, Brown et al. [4] reported that AKAP4
anchors AKAP3 and two novel spermatogenetic cell
specific proteins, Fibrous sheath interacting proteins 1 and
2 (FSIP1; FSIP2).
Miki et al. [5] demonstrated that targeted disruption
of the Akap4 gene causes the absence of sperm motility
together with a total lack of fibrous sheath on the
principal piece of mature mice sperm.
Baccetti et al. [15] described a rare sperm tail defect
characterized by absence of the fibrous sheath in humans.
AKAP4 labelling was present at the testicular level in
cytoplasmic residues and residual bodies, yet it was
totally absent in ejaculate spermatozoa. Moreover, in a
case of disorganized and incompletely assembled fibrous
sheath, such as fibrous sheath sperm, Baccetti et
al. [6] found moderate and diffused immunofluorescent
staining of AKAP4.
The aim of this study was to assess the status of the
fibrous sheath and the axonemal structure by
performing screening, related to AKAP4 and tubulin proteins, in
spermatozoa with absent or severely reduced motility.
Immunolabelling of tubulin and AKAP4 in sperm
flagella showed different patterns, leading us to divide the
patients into groups. In group I, in which sperm motility
was 0%, no AKAP4 or tubulin labelling was detected.
When sperm motility was greater than 0%, a variable
pattern of AKAP4 and tubulin staining was observed
(groups II and III).
In group I, TEM evaluation highlighted that a high
presence of necrosis was associated with cases of total
immotility and the absence of AKAP4 and tubulin,
indicating a loss of antigenicity caused by post-necrotic
protein degradation. In order to exclude a genetic origin of
the absence of AKAP4, PCR analysis was performed to
detect the presence of a partial sequence of
Akap4/Akap3 binding regions and it produced normal results.
In group II, despite reduced motility, regular AKAP4
and tubulin signals were observed. This apparent
inconsistency was justified by TEM analysis that revealed the
presence of sperm immaturity. We observed numerous
cytoplasmic residues, typical markers of this pathology
and responsible for the decrement of motility. The
axonemal and periaxonemal structures embedded in these
cytoplasmic residues, including the fibrous sheath, were
generally normal as also revealed by
immunocytochemical analyses.
In group III in which a weak AKAP4 label was observed, associated with good tubulin staining, TEM
analysis showed a severe disorganization of the fibrous
sheath and a normal "9 + 2" axonemal pattern. This
pattern was quite similar to that observed in cases of
dysplasia of the fibrous sheath (DFS) as already described
by Baccetti et al. [6]. However, none of these patients
were affected by this genetic sperm defect, characterized
by a typical ultrastructural feature as highlighted by Chemes
et al. [16].
In conclusion, while the role of AKAP4 in sperm
motility is unclear, absent or weak AKAP4 labeling seems
to be associated with absent or weak sperm motility.
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