Molecular Human Reproduction, Vol.16, No.11 pp. 811–817, 2010
Advanced Access publication on May 27, 2010 doi:10.1093/molehr/gaq044
ORIGINAL RESEARCH
Comparative analysis of the germ cell
markers c-KIT, SSEA-1 and VASA in
testicular biopsies from secretory and
obstructive azoospermias
J.V. Medrano 1,2, A.I. Marqués-Marı́ 1, C.E. Aguilar 1, M. Riboldi 1,2,
N. Garrido 3,4, A. Martı́nez-Romero 5, E. O’Connor 5, M. Gil-Salom 3,
and C. Simón 1,2,3,4,*
1
Valencia Stem Cell Bank, Centro de Investigación Prı́ncipe Felipe (CIPF), Valencia 46012, Spain 2Fundación Instituto Valenciano de
Infertilidad (FIVI), Valencia University, C/Guadassuar, 1, Valencia 46015, Spain 3Instituto Universitario IVI (IUIVI), Valencia University,
Valencia 46015, Spain 4Instituto de Investigación Sanitaria del Hospital Clinico de Valencia (INCLIVA), Valencia University, Valencia 46010,
Spain 5Cytomics Laboratory, CIPF-UVEG Mixed Unit, Centro de Investigación Prı́ncipe Felipe (CIPF), Valencia 46012, Spain
*Correspondence address. E-mail: [email protected]
Submitted on October 20, 2009; resubmitted on May 13, 2010; accepted on May 25, 2010
abstract: Testicular biopsy is needed to confirm diagnosis in azoospermic patients and to recover spermatozoa, if possible. This report
aims to quantitatively analyse the germline markers stage-specific embryonic antigen (SSEA-1), c-KIT and VASA in testicular biopsies with
distinct azoospermic aetiologies. Twenty-three testicular biopsies were analysed by flow cytometry and RT-qPCR for c-KIT, SSEA-1 and
VASA. In all the Sertoli cell-only (SCO) samples, significantly lower VASA mRNA expression and fewer VASA+ cells were found compared
with obstructive controls. Maturation arrest (MA) cases showed significant differences only with the non-mosaic SCO samples when compared for VASA mRNA expression and percentage of VASA+ cells, but not with the mosaics. However, the normalized VASA– KIT parameter obtained by subtracting the percentage of c-KIT+ cells from the percentage of VASA+ cells showed significant differences between
the MA and all the SCO samples. RT-qPCR consistently found differences for the VASA expression between SCO mosaic and non-mosaic
samples. However, by flow cytometry, only VASA –KIT showed significant differences between them. Conversely, the percentage of SSEA1+ cells revealed no inter-group differences. In conclusion, testicular biopsies display different expression profiles for c-KIT and VASA
depending on the azoospermic aetiology. These results can be used as a complementary tool to create new molecular categories for diagnoses in azoospermic patients, particularly useful to discriminate between mosaic and non-mosaic SCO patients.
Key words: c-KIT / male infertility / SSEA-1 / testicular biopsy / VASA
Introduction
Spermatogenesis is a unique process in which diploid spermatogonia
become haploid mature spermatozoa. A disorder in this process may
provoke azoospermia with different aetiologies, resulting in male fertility
problems. Of these disorders, obstructive azoospermia (OA) represents
a problem in the ejaculation ducts that impedes the presence of sperm
within the seminal fluid; maturation arrest (MA) is a disorder that stops
sperm maturation in different possible stages of spermatogenesis; and
the Sertoli cell-only (SCO) syndrome is the diagnosis with the poorest fertility outcome given the total absence of germline (Anniballo et al., 2000).
However, in some mosaic SCO patients the recovery of sperm is possible.
It is estimated that 2000 genes are implicated in the regulation of
spermatogenesis (Hargreave, 2000). Within this pool of genes, VASA
has become a specific germ cell marker which is present from when
germ cells arrive at the gonadal ridge in fetal development until
mature functional gametes in adults are produced (Castrillon et al.,
2000; Toyooka et al., 2000; Noce et al., 2001). The VASA protein
is localized in the cytoplasm of germline cells (Castrillon et al.,
2000). VASA is functionally relevant for germline establishment. Disruption of exons 9 and 10 of the Mvh (Mouse VASA homologue)
gene generates Mvh-deficient male mice with a phenotype without
the presence of germline (Tanaka et al., 2000). Given the specific
expression of VASA, it is considered a robust quantitative marker of
total germ cell contribution in gonads (Guo et al., 2007).
The germline marker c-KIT is a transmembrane protein receptor
associated with the maturation of several cell types (Lammie et al.,
1994; Izquierdo et al., 1995), including germ cells (Matsui et al.,
& The Author 2010. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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812
1990; Rossi et al., 2000; Sette et al., 2000). Mutations in the c-KIT
gene can cause deficiencies in germ cell development, as well as haematopoiesis and melanogenesis disorders (Loveland and Schlatt,
1997). In male germ cells, c-KIT is implicated in the transduction of
extracellular signals that control cell proliferation, survival and differentiation (Mauduit et al., 1999; Prabhu et al., 2006). In mice, c-KIT may
play a role in the regulation of spermatogonial proliferation and in the
maturation of round spermatids (Sorrentino et al., 1991; Albanesi
et al., 1996). In the adult human testes, it is expressed along the basement membrane of seminiferous tubules with intense protein localization in spermatogonia stages I – III during the proliferative phase in
which Adark (Ad) spermatogonia produce many Apale and B spermatogonia. However, a low expression is observed at stages IV –VI when
the B spermatogonia differentiate to early spermatocytes and enter
meiosis (Clermont, 1963; de Rooij and Russell, 2000; Amann,
2008). This pattern suggests a stage-specific expression, so c-KIT
could be established as a marker of pre-meiotic human spermatogenesis stages. However, the Leydig cells present in the testicular biopsies
also express c-KIT (Motro et al., 1991) and are able to increase the
percentage of the c-KIT+ cell population within testicular biopsies.
This fact reflects the need for a marker at both the mRNA and
protein levels (Ostermeier et al., 2002), as well as the combined analysis of several markers to improve data analysis accuracy (Park et al.,
2009).
Stage-specific embryonic antigen (SSEA-1) is another germline marker
that is also expressed in granulocytes. In the germline, SSEA-1 is
expressed on the surface of gonocytes and is considered an early germ
cell development marker (Kerr et al., 2008; Park et al., 2009). Little is
known about the expression pattern of SSEA-1 in adult human testes.
It has been studied at the protein level by different groups as a marker
of early human germline development in differentiation studies with
human embryonic stem cells (Bucay et al., 2008; Tilgner et al., 2008;
Park et al., 2009). These reports suggest that SSEA-1 could be a
marker of the very early stages of spermatogenesis, specifically immature
gonocytes and germ stem cells within adult human testes.
Despite current knowledge about these stage-specific molecular
markers of spermatogenesis, the gold standard diagnostic tool
employed to determine the aetiology behind a spermatogenesis
problem is the pathological study of testicular biopsy samples.
However, mistakes in the diagnoses of some samples are possible,
specially in mosaic SCO samples in which the analysed section
might not be representative of the whole sample. The aim of this
work is to quantitatively compare the expression of c-KIT, SSEA-1
and VASA at the mRNA and protein levels in testicular biopsies
obtained from patients with obstructive and secretory azoospermia.
We propose this methodology as a complementary diagnostic molecular tool for azoospermia diagnosis. This could be specially useful
to discriminate between mosaic and non-mosaic SCO patients.
Materials and Methods
Samples and processing
Twenty-three testicular biopsies consisting of small pieces of testicular
tissue composed of seminiferous tubules and interstitial tissue were
obtained from azoospermic patients as part of their diagnosis and
work-up at the Instituto Valenciano de Infertilidad. One part of the
Medrano et al.
samples was designated for histological study and other part was used in
this study after obtaining signed informed consent, which had been previously approved by the institution’s Ethics Committee in accordance
with Spanish legislation. Samples were classified into four groups depending on their histological diagnosis as follows: (i) obstructive controls (OC)
(n ¼ 11); (ii) non-OA with sperm MA (n ¼ 5); (iii) putative mosaic
secretory azoospermia due to the SCO syndrome (SCO SPZ+) (n ¼ 3)
and (iv) putative non-mosaic secretory azoospermia due to the SCO syndrome (SCO SPZ2) (n ¼ 4). SCO samples were classified as putative
mosaics (SCO SPZ+) when motile sperm was found and as non-mosaics
(SCO SPZ2) when sperm was not present within the biopsies.
Briefly, 1–2 mg of the residual tissue derived from the clinical processing
of testicular biopsies was placed on ice and transported to the lab for its
processing within 90 min after surgery. Then mechanical dissection was performed to obtain small pieces of 1 mm3. Samples were washed twice in
phosphate buffered saline (PBS) (Invitrogen, San Diego, CA, USA) and incubated for enzymatic digestion with Collagenase IA (1000 UI/ml) (Sigma, St
Louis, MO, USA) for 20 min at 378C, followed by a second incubation with
Tryple Select (Invitrogen) for 10 min at 378C. The resulting single cell suspension was filtered through nylon membranes with a pore diameter of
30 mm (Partec GmbH, Görlitz, Germany), counted in a haemocytometer
and split to be used for flow cytometry and RT-qPCR analysis.
RNA extraction and RT-qPCR
The total RNA of the samples was extracted with Tripure reagent (Invitrogen) according to the manufacturer’s protocol, and was analysed in a
NanoDrop Spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA) for RNA quantification and purity. cDNA was synthesized
in RT– PCR with an MMLV enzyme (Clontech, BD, Palo Alto, CA, USA),
whereas 750 – 1000 ng of RNA per sample were used as a template.
RT– PCR was initiated with a denaturation at 948C for 5 min and cycled
35 times at 948C for 30 s, 578C for 30 s and 728C for 1 min. A final extension at 728C for 10 min was performed after cycling. PCR primers were
designed using freely available web-based software (Primer3, Genefisher).
Water was included as a negative control.
PCR products were resolved on a 1.5% agarose gel, stained with ethidium bromide, and visualized in a transilluminator (BioRad).
qPCR was carried out in a DNA thermal cycler LightCycler 2.0 (Roche
Diagnostics, GmbH Mannheim, Germany). The SYBRw Green I doublestranded DNA binding dye (Roche Diagnostics, GmbH Mannheim,
Germany) was chosen as the fluorescent marker for these assays.
Between 25 and 50 ng of cDNA was used as a template per PCR or
qPCR reaction in duplicate. After the first denaturalization step at 958C
for 10 min, the samples for qPCR were subjected to 45 cycles, each consisting of a denaturalization step at 958C for 10 s, an annealing step at
598C for 6 s and an extension at 728C for 10 s. The negative controls
were the same RT reaction samples without addition of enzyme. The
primers used for amplification were commercially synthesized (Sigma)
with both forward and reverse primers in different gene exons to
ensure that amplicons were cDNA-specific without genomic crosscontamination. RPL19 was used as an internal housekeeping control
(Al-Bader and Al-Sarraf, 2005). The expression profile of each sample
was calculated by the DCt method and normalized to the SCO sample
with lowest expression levels for all markers as reference. The primer
sequences for PCR were as follows: VASA F 5′ -ATGGATGATGG
ACCTTCTCG-3′ and VASA R 5′ -CCTCTGTTCCGTGTTGGATT-3′
(GenBank accession no. NM_024415.2, positions 228 and 456, respectively), c-KIT F 5′ -GTCTCCACCATCCATCCATC-3′ and c-KIT R 5′ -TTT
CCGACAGCACTGACTTG-3′ (GenBank accession no. NM_000222.2,
positions 188 and 662, respectively). The primer sequences for
qPCR were as follows: VASA F 5′ -ATGGATGATGGACCTTCTCG-3′
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Analysis of c-KIT, SSEA-1 and VASA in testicular biopsies
and VASA R 5′ -CCTCTGTTCCGTGTTGGATT-3′ (GenBank accession no.
NM_024415.2, positions 228 and 456, respectively), c-KIT F 5′ -GCAAAT
ACACGTGCACCAAC-3′ and c-KIT R 5′ -GCACCCCTTGAGGGAAT
AAT-3′ (GenBank accession no. NM_000222.2, positions 364 and 520,
respectively). The RPL19 primers were the same in both the PCR
and qPCR assays: RPL19 F 5′ -CGAATGCCAGAGAAGGTCAC-3′ and
RPL19 R 5′ -CCATGAGAATCCGCTTGTTT-3′ (GenBank accession no.
NM_000981.3, positions 323 and 460, respectively). PCR products were
validated by sequencing in all cases (See supplementary data).
Flow cytometry
The single cell suspensions obtained by processing samples were fixed in
paraformaldehyde (PFA) 1% for 20 min at 48C and washed with 1% bovine
serum albumin (BSA) (Sigma) in PBS (Invitrogen). For the surface markers,
cells were incubated in 100 ml of 1% BSA in PBS containing conjugated
antibodies for c-KIT-PE 1:50 (R&D Systems, Minneapolis, MN, USA)
and for SSEA-1-FITC 1:50 (BD Bioscience, San Diego, CA, USA) on ice
for 30 min. For the intracellular staining of VASA protein, cells were permeabilized in 250 ml Perm/WashTM buffer (BD Bioscience). Cells were
labelled with goat anti-human VASA antibody 1:100 (R&D Systems) in
1× BD Perm/WashTM buffer on ice for 45 min. Cy5-conjugated rabbit
anti-goat IgG 1:500 (Jackson ImmunoResearch Laboratory, West Grove,
PA, USA) was used as a secondary antibody. The analysis was performed
using a Cytomics FC500 (Beckman-Coulter, Fullerton, CA, USA) flow cytometer and FlowJo software (Tree Star Inc., Ashland, OR, USA). For validation of antibody reactivity, the following cell lines were used as controls:
for c-KIT, positive control was MALME-3M cell line and negative control
human foreskin and the same MALME-3M cells without antibody labelling;
for SSEA-1, positive control were human granulocytes from peripheral
blood and negative control human foreskin and the same granulocytes
without antibody labelling; for VASA, positive control were testicular
germ cells within a testicular biopsy from an obstructive azoospermic
patient and negative control was human foreskin and the same germ
cells without antibody labelling (Supplementary Fig. 1– R).
Immunohistochemistry
For paraffin embedding, samples were fixed in 4% PFA overnight at 48C.
The next day, samples where washed in PBS (Invitrogen) for 15 min at
48C and then dehydrated at 48C for 15 min in 50% ethanol followed by
two washes in 70% ethanol and embedded using standard procedures.
Sections were cut 5 mm thick and mounted on slides (ProbeOn Plus;
Fisher Scientific). For staining, sections were deparaffinized and rehydrated
through a series of graded alcohols at room temperature. For antigen
retrieval, tissue sections were incubated in 0.01 M citrate buffer (pH
6.0) for 15 min and then cooled for 3 min. Sections were incubated in
10% BSA and 10% normal donkey serum (Sigma) in PBS for 1 h at
room
temperature
to
block
non-specific
binding.
For
immunohistochemical staining, sections were incubated overnight at 48C
in a sealed, humidified chamber with the same primary antibodies used
in flow cytometry and examined with an Olympus IX81 microscope
(Olympus, GmbH Mannheim, Germany). Negative controls were performed by excluding primary antibodies.
Statistic analysis
Non-parametric Kruskal – Wallis test was performed to compare the
samples among the groups for all the markers investigated. When the
Kruskal – Wallis test showed significant inter-group differences, multiple
pair-wise comparisons were performed using the Mann– Whitney test
to find the particular inter-group differences. Statistical analysis was
done with the Statistical Package for Social Sciences, v. 17.0 (SPSS Inc.,
Chicago, IL, USA). Significance was defined as P , 0.05 in all cases.
Results
Germline markers c-KIT and VASA were quantitatively analysed by
RT –PCR and RT-qPCR and these same markers plus SSEA-1 were
also analysed by flow cytometry in obstructive azoospermic controls
in whom spermatogenesis is conserved (OC) (n ¼ 11), secretory
azoospermia with sperm MA (n ¼ 5), putative mosaic SCO syndrome
(SCO SPZ+) (n ¼ 3) and putative non-mosaic SCO syndrome (SCO
SPZ2) (n ¼ 4). The epidemiological data of patients involved in this
study are presented in Table I.
VASA and c-KIT RT –PCR products presented inter-group differences in the intensity of the bands for these markers (Fig. 1A). Moreover, the SCO SPZ2 group showed no VASA expression due to the
absence of germ cells. Subsequent RT-qPCR led to a significantly
decreased VASA mRNA level in the SCO SPZ+ (3874.78 +
1752.63) and the SCO SPZ2 (5.89 + 1.61) groups compared with
the OC (58 324.84 + 8937.86) group (P ¼ 0.016 and P ¼ 0.004,
respectively) (Fig. 1B). Interestingly, the MA samples only displayed
significantly higher values for VASA (18 851.40 + 3346.92) when compared with the SCO SPZ2 samples (P ¼ 0.014), but not when compared with the SCO SPZ+ samples. Furthermore, the SCO SPZ+
group showed significantly higher values of VASA than the SCO
SPZ2 group (P ¼ 0.034). Non-significant differences were found
between the OC and MA groups for VASA expression. On the
other hand, the RT-qPCR analysis for c-KIT expression revealed nonsignificant inter-group differences (Fig. 1C).
The immunohistochemistry of the representative testicular biopsy
samples from the OC, MA and non-mosaic SCO patients confirmed
the stage-specific expression (Fig. 2A) and the cellular localization
(Fig. 2B) of the three germ cell markers under study. Flow cytometry
Table I Epidemiological data of etiological groups included in this study.
Group
Age
FSH
Testosterone
Prolactin
.............................................................................................................................................................................................
Reference values
35–44
4 –13 ng/ml
3– 10.6 ng/ml
1.6 –18.7 ng/ml
Obstructive controls (n ¼ 11)
42.63 + 1.56
4.58 + 0.46
4.57 + 0.14
10.27 + 2.34
MA (n ¼ 5)
38.20 + 1.81
14.45 + 0.48
5.75 + 0.88
10.4 + 0.55
Mosaic SCO (n ¼ 3)
38.00 + 2.25
30.50 + 2.43
3.36 + 0.35
15.20 + 1.25
Non-mosaic SCO (n ¼ 4)
35.67 + 0.44
28.23 + 3.55
3.25 + 0.45
8.57 + 0.08
Normal values for FSH are 4–13 ng/ml; for testosterone are 3– 10.6 ng/ml and for Prolactin are 1.6– 18.7 ng/ml. Data are represented as mean + SEM.
814
Medrano et al.
Figure 1 RT– PCR analysis of samples for the germline markers VASA and c-KIT. (A): Representation of amplification bands by PCR for VASA and
c-KIT in each group of samples: OC (obstructive control), MA (maturation arrest), SCO+ (Sertoli cells only with sperm), SCO2 (Sertoli cells only
without sperm). (B): Quantitative mRNA expression among the four groups of patients for VASA. (C): Quantitative mRNA expression among the
four groups for c-KIT. Significant differences in the comparison with the Obstructive, MA and SCO SPZ+ groups are indicated by the symbols *, + and ^,
respectively. The expression profile of each sample was calculated by DCt method and normalized to the housekeeping RPL19. Data are represented as
mean + SEM.
results (Fig. 2C) also revealed significant differences for VASA among
groups. Using the Mann– Whitney test, we concluded that the percentage of VASA+ cells was statistically lower in the SCO SPZ+ (5.41 +
1.44) and in the SCO SPZ2 groups (1.29 + 0.43) when compared
with OC (21.96 + 2.14) (P ¼ 0.039 and P ¼ 0.004, respectively).
Reinforcing our results obtained by RT-qPCR, the percentage of
VASA+ cells within the MA group (23.62 + 2.60) was significantly
higher than in the SCO SPZ2 group (1.29 + 0.43) (P ¼ 0.014), but
non-significant differences were found when compared with the
SCO SPZ+ group. Unlike the results obtained by RT-qPCR, nonsignificant differences were found between both SCO SPZ+ and
SCO SPZ2 groups. The percentages of the SSEA-1+, c-KIT+ cells
and SSEA-1+/c-KIT+ double positive cells were similar among the
groups (data not shown).
We analysed the meiotic and post-meiotic germ cell population
within biopsies by subtracting the percentage of c-KIT+ cells from
the percentage of VASA+ cells. This new parameter was called
VASA–KIT. The Mann–Whitney test found significantly higher
values for VASA –KIT in the OC group (10.46 + 1.85) when compared with both SCO SPZ+ (22.00 + 1.01) and SCO SPZ2
(27.88 + 0.66) groups (P ¼ 0.035 and P ¼ 0.019, respectively). Significantly higher values for VASA –KIT were also found between the
MA group (16.37 + 1.79) and both SCO SPZ+ and SCO SPZ2
groups (P ¼ 0.034 and P ¼ 0.021, respectively) (Fig. 2B). Moreover,
the SCO SPZ+ group presented significantly higher values for
VASA–KIT compared with the SCO SPZ2 group (P ¼ 0.034).
Discussion
Our results are consistent with previous studies, suggesting that
VASA is a robust germline marker during spermatogenesis (Fujiwara
et al., 1994; Castrillon et al., 2000; Tanaka et al., 2000; Toyooka
et al., 2000; Noce et al., 2001), and that it may even be considered
a diagnostic tool for spermatogenic disorders (Guo et al., 2007).
The stage-specific expression of several markers has been reported
in rodent and human testes (Yoshinaga et al., 1991; Rossi et al.,
1992; Vincent et al., 1998; Hakovirta et al., 1999; Schrans-stassen
et al., 1999; Zhang et al., 2004; Shah et al., 2005). Two studies
have reported a lower c-KIT expression in the testes of infertile
men compared with those of fertile men (Sandlow et al., 1996;
Feng et al., 1999), and Unni et al. (2009) have reported that c-KIT-positive cells were found in the spermatogonia stages I –III in
human testes.
In this study, the RT-qPCR data show that the VASA mRNA
expression in both mosaic and non-mosaic SCO samples is statistically
decreased compared with the OC samples, thus suggesting a lower
contribution of germline cells within the total biopsy since the VASA
expression is germline-specific and is expressed throughout
Analysis of c-KIT, SSEA-1 and VASA in testicular biopsies
815
Figure 2 Results of flow cytometry and fluorescence microscopy analysis of samples for the germline markers VASA, c-KIT and SSEA-1.
(A): Staining for VASA, c-KIT and SSEA-1 in testicular biopsies from OC, MA and SCO SPZ2 patients. The scale bar represents a distance of
100 mm. (B): Staining for the three germline markers at higher magnification in obstructive controls. VASA is localized at the cytoplasm of all
germ cells. c-KIT is localized on the surface of spermatogonia stages I – III, but also on Leydig cells surrounding seminiferous tubes. SSEA-1 is a
surface marker of most immature germ cells within the seminal epithelium. The scale bar represents a distance of 50 mm. All pictures were taken
with an Olympus IX81 fluorescence microscope. (C): Percentages of positive cells for all three markers within testicular biopsies obtained by flow
cytometry analysis. Significant differences in the comparison with the Obstructive, MA and SCO SPZ+ groups are indicated by the symbols
*, + and ^, respectively. Data are represented as mean + SEM.
spermatogenesis. However, the MA samples only showed significantly
higher values when compared with the SCO SPZ2 group. One explanation for this could be the presence of complete spermatogenic areas
in the putative mosaic SCO SPZ+ biopsies which were not detected
in the pathologic analysis, and which increase the levels of VASA
mRNA to levels comparable to the MA samples (Fig. 1B). We also
found higher values for VASA expression in putative mosaic SCO
SPZ+ samples when compared with the SCO SPZ2 samples.
These results indicate that VASA could be a robust marker to discriminate between mosaic and non-mosaic SCO samples. On the other
hand, the c-KIT mRNA expression did not prove to be a useful parameter to discriminate among pathologies (Fig. 1C).
We also detected at the protein level the stage-specific expression
of VASA, c-KIT and SSEA-1 (Fig. 2A and B). Subsequent flow cytometry analysis of VASA corroborates the RT-qPCR results obtained at
the protein level, except that no significant differences were found
between mosaic and non-mosaic SCO. Given that the percentage of
VASA+ cells represents the total amount of germ cells implicated in
spermatogenesis, and that the percentage of c-KIT+ cells corresponds to the spermatogonia stages I –III (Unni et al., 2009), we
created the VASA–KIT parameter by subtracting the percentage of
c-KIT+ cells from the percentage of VASA+ cells to analyse the
meiotic and post-meiotic germ cell population within samples. This
parameter represents the germ cell population that has passed the
spermatogonia stage. VASA –KIT data showed significant differences
between mosaic and non-mosaic SCO samples. Significantly lower
values of this parameter in both mosaic and non-mosaic SCO
groups, when compared to the other two groups, are due to the
decrease of meiotic and post-meiotic cells, which is indicative of
early spermatogenesis detention (Fig. 2C).
816
Medrano et al.
Figure 3 Stage-specific expression of the germ line markers VASA, c-KIT and SSEA-1 during human spermatogenesis. Arrows indicate stage-specific
expression of each marker during the spermatogenesis process. (A): In obstructive controls, VASA is expressed throughout all the spermatogenesis
stages until mature sperm is formed. c-KIT expression is limited to the first stages of spermatogonia development until initiation of meiosis when
spermatogonia B differentiate to spermatocytes I. SSEA-1 expression is specific for the Adark (Ad) spermatogonia, which are the earliest cells that
initiate the spermatogenesis process. Pathology reports confirmed that spermatogenesis in obstructive azoospermic patients and controls were
similar. (B): MA patients have an arrest in spermatogenesis that usually affects the transition from Spermatocyte I (2n) to Spermatocyte II (n), so
the VASA expression is truncated at this stage. On the other hand, expressions of c-KIT and SSEA-1 are maintained. (C): SCO patients have a complete depletion of the spermatogenesis process, so expressions of VASA, c-KIT and SSEA-1 are reduced until it almost disappears in the case of VASA.
However, mosaic SCO patients may have germ epithelium areas with conserved and complete spermatogenesis.
SSEA-1 has been suggested to be an early germline marker for
gonocytes (Bucay et al., 2008; Tilgner et al., 2008; Park et al., 2009).
In this study, this marker has also been analysed. However, our data
suggest that SSEA-1 has no discriminatory capacity among the different
aetiologies investigated.
We present a model of the stage-specific expression of the
three spermatogenesis markers: SSEA-1, c-KIT and VASA. This
model could be helpful to understand the spermatogenesis status in
patients with OA (Fig. 3A), MA (Fig. 3B) and SCO syndrome (Fig. 3C).
At the mRNA level, VASA is a robust quantitative marker of the
germline contribution to the whole sample and its analysis makes it
possible to discriminate among pathologies, specially between mosaic
SCO SPZ+ and non-mosaic SCO SPZ2 samples. However, the flow
cytometry study of VASA itself does not have the same strength to discriminate among pathologies. A focused analysis of meiotic and postmeiotic germ cell population by the VASA –KIT parameter might help
to discriminate between the early or post-meiotic arrest in azoospermic
patients and also between mosaic and non-mosaic SCO patients. We
propose that the combined quantitative analysis by flow cytometry
and RT-qPCR of these germline markers could be used as a complementary diagnostic tool to study and diagnose azoospermia from testicular biopsies, thus improving the qualitative information of the
pathology report. The analysis of germline markers that we propose
is a definitive and quantitative method that allows us to detect mosaic
SCO cases. This molecular tool will detect mosaicism in histologically
diagnosed SCO azoospermic patients and will offer the possibility of
recovering the sperm when the first biopsy has not been successful.
Our study must be considered as a first pilot to quantify stagespecific germline markers in testicular biopsies from azoospermia.
However, further combined studies with a more complete collection
of samples from different pathologies and other germline markers,
such as meiotic-specific markers, would be beneficial to improve the
clinical translational opportunities of this study.
Authors’ roles
J.V.M. designed, carried out and analysed most of the experiments,
and wrote the manuscript. A.I.M.-M. carried out an important part
of the experiments and revised the manuscript. C.E.A. helped in discussion of the results and statistical analysis. M.R. helped in recruiting
samples and contributed in some experiments. N.G. helped in the discussion of the results and statistical analysis. A.M.-R. helped in flow
cytometry analysis. E.Ó. helped in flow cytometry analysis. M.G.-S.
practised the surgery to the patients and helped in the discussion of
the results. C.S. designed experiments, helped in the discussion of
the results and revised the manuscript.
Supplementary data
Supplementary data are available at http://molehr.oxfordjournals.
org/.
Acknowledgements
We thank all the clinical technicians at the Instituto Valenciano de
Infertilidad, and specially Rafael Salinas for his help in recruiting the
samples used in this study and Dr Nuria Bosch for the pathologic
analysis of samples.
Analysis of c-KIT, SSEA-1 and VASA in testicular biopsies
Funding
This work has been supported by a grant from the Instituto de Salud
Carlos III from the Spanish Ministry of Science and (FI07/00 011) and
by a Santiago Grisolı́a grant from the Generalitat Valenciana (Regional
Valencian Government).
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