Max-Planck-Institute for Physiological and Clinical Research (H.H.M.), Department of Molecular Cell Biology, D-61231 Bad Nauheim, Germany; Institute of Physiology (D.M.K., K.F.W., B.S., R.H.W.) and Clinic of Anaesthesiology (K.F.W.), Medical University of Lübeck, D-23538 Lübeck, Germany; and Department of Obstetrics and Gynaecology (L.S.), University Hospital Zürich, CH-8091 Zürich, Switzerland
Address all correspondence and requests for reprints to: Roland H. Wenger, Ph.D., Carl-Ludwig-Institut für Physiologie, Universität Leipzig, Liebigstrasse 27, D-04103 Leipzig, Germany. E-mail: wenr{at}medizin.uni-leipzig.de
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ABSTRACT |
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INTRODUCTION |
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During nuclear elongation of haploid spermatids, the rate of transcription declines and becomes undetectable in elongated spermatids. Nevertheless, ongoing translation of a number of sperm cell-specific structural proteins and isoforms of metabolic enzymes is required for the production of spermatozoa. Therefore, specific mRNA isoforms containing long poly(A) tails are stored as translationally inactive ribonucleoprotein particles. In transcriptionally inactive states, these mRNA isoforms are recruited into translationally highly active polysomes to ensure ongoing protein synthesis (reviewed in Refs. 2, 3, 4). Prominent examples include the nuclear transition proteins (5, 6) and later in spermiogenesis the protamines (7, 8, 9), which replace the histones and lead to compaction of the chromatin.
There are also various testis-specific isoforms of glycolytic enzymes, which are expressed in the haploid stages of spermatogenesis and which are still active in mature spermatozoa, including phosphoglycerate kinase 2 (10, 11), glyceraldehyde 3-phosphate dehydrogenase-2 (12), and lactate dehydrogenase C (13). Because of the greater oxygen diffusion distance, the luminal regions of the seminiferous tubuli as well as the epididymis are likely to be hypoxic when compared with the basal regions of spermatogonial self-renewal. In addition, the high proliferative capacity of the germinal epithelium suggests a pronounced oxygen consumption, thereby further decreasing the oxygen concentration. Thus, testis-specific glycolytic enzyme isoform expression might be related to hypoxic adaptation by altered anaerobic energy metabolism. Indeed, sperm capacitation, motility changes, acrosome reaction, and fertilization is exclusively dependent on anaerobic glycolysis and can occur under strictly anaerobic conditions (14). However, the influence of oxygen concentration, consumption, and metabolism on the molecular events during spermatogenesis, spermatozoa release, in ejaculated sperm, and during fertilization is largely unknown.
The hypoxia-inducible factor 1 (HIF-1) is an ubiquitously expressed
transcriptional master regulator of many genes involved in mammalian
oxygen homeostasis, including the hormones and related compounds
erythropoietin, vascular endothelial growth factor, inducible nitric
oxide synthase, and heme oxygenase-1 (reviewed in Refs. 15, 16). HIF-1 is also a major regulator of the glycolytic
capacity (17). HIF-1 is a
1ß1 heterodimer
specifically recognizing the HIF-binding site within
cis-regulatory hypoxia response elements. Under normoxic
conditions, the von Hippel-Lindau tumor suppressor protein targets the
HIF-1
subunit for rapid ubiquitination and proteasomal degradation
(18). The von Hippel-Lindau tumor suppressor protein
binding requires oxygen-dependent prolyl hydroxylation of HIF-1
(19, 20). We previously cloned the mouse HIF-1
gene
(Hif1a) and found that its expression is driven by two
different promoters located 5' to two alternative first exons
designated exon I.1 and exon I.2 (21, 22). Whereas the
upstream exon I.1 promoter exhibits tissue-specific features, the
downstream exon I.2 promoter is a typical housekeeping-type promoter
driving ubiquitous transcription (22). The mouse
mHIF-1
I.1 mRNA isoform encodes for a predicted protein product that
is 12 amino acids shorter than the predicted mHIF-1
I.2 protein.
Despite its vicinity to the basic-helix-loop-helix DNA binding domain,
this N-terminal deletion does not affect DNA binding efficiency
(23). To define the cell types expressing the
tissue-specific mHIF-1
I.1 mRNA, in situ hybridization of
the two HIF-1
mRNA isoforms was performed in mouse tissues.
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RESULTS |
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Evidence that Induction of the Testis-Specific mHIF-1I.1 mRNA
Isoform Starts in Late Post-Meiotic Germ Cells
Four different cell lines corresponding to Leydig cells (TM3),
Sertoli cells (TM4), spermatogonia (GC-1 spg), and spermatocytes (GC-2
spd(ts)) were chosen for expression studies. These cells were exposed
to hypoxic conditions (1% O2) for 4 h and
analyzed by immunoblotting. For comparison, three non-testis-
derived cell lines, HeLa cervic carcinoma, MCF-7 adenocarcinoma,
and Jurkat T cell leukemia, were treated accordingly. As shown in Fig. 2A, HIF-1
protein was strongly induced
by hypoxia in nuclear extracts isolated from all cell lines. When
cultured under hypoxic conditions, the levels of HIF-1
in the
testis-derived cell lines were clearly higher than the levels in the
non-testis-derived cell lines. Compared with HIF-1
, the differences
in the levels of HIF-1ß/ARNT under hypoxic conditions were less
pronounced among the cell lines analyzed. Under normoxic conditions,
the levels of ARNT were decreased in nuclear extracts of some of the
cell lines, despite the fact that ARNT is normally expressed in a
constitutive manner. We previously showed that, when HIF-1
is
absent, ARNT is decreased in normoxic nuclear extracts due to leakage
out of the nucleus during preparation rather than to a true
oxygen-dependent regulation (25). The signals obtained
with an antibody derived against an unrelated transcription factor
(Sp-1) confirmed that similar protein amounts were loaded in each
lane.
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Initially, the GC-2 spd(ts) cell line has been shown to consist of both diploid spermatocyte and haploid early spermatid-like cells (26), but haploid cells could not be detected in later studies (27). Nevertheless, our in vitro results indicate that a change in transcriptional activity does not occur during the premeiotic stages of spermatogenesis and hence cannot explain the strong expression of the I.1 mRNA isoform and the complete lack of the I.2 mRNA isoform in the haploid mature spermatids as observed by in situ hybridization. Thus, the I.1 mRNA isoform accumulation might be due to a change in transcriptional activity at the postmeiotic stages of spermiogenesis that, however, cannot be investigated in cell culture models in vitro.
Hypoxia Induces HIF-1 Protein Expression in
Spermatocytes
To investigate oxygen-regulated HIF-1 protein expression in
testis, adult male mice were exposed to hypobaric hypoxia for 6 h,
and the testes were analyzed by indirect immunohistochemistry. As shown
in Fig. 3A
, under normoxic conditions we
were able to detect moderate HIF-1
expression that is induced under
hypoxic conditions in a region overlapping with Sertoli cells and
spermatocytes. HIF-1
is mainly expressed in the nuclei of
pachytene spermatocytes (as characterized by their large nuclei) and to
a lesser extent in the adjacent round spermatids (Fig. 3B
). In
contrast, HIF-1
was undetectable in the nuclei of the basal
spermatogonial layer and the innermost layer of mature elongated
spermatids (Fig. 3B
). Control experiments using the secondary antibody
alone indicated that the weak staining of the matrix surrounding the
seminiferous tubuli but not the cellular staining was due to unspecific
antibody binding (Fig. 3
, AC). Surprisingly, there was also a
positive HIF-1
protein staining in the lumen of the seminiferous
tubuli where the spermatozoal tails are located (Fig. 3
, B and C).
Under higher magnification, it became apparent that hypoxia-inducible
HIF-1
protein is located to part of the flagellum adjacent to the
head rather than to the typically shaped end-stage spermatid nuclei
(Fig. 3D
). Thus, the spermatid-specific mHIF-1
I.I mRNA isoform might
give rise to a protein isoform that occupies a distinct cellular
compartment of mature spermatozoa.
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Discussion |
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In marked contrast, the detection of the mHIF-1I.1 mRNA isoform in
the head of mature haploid spermatids and of the HIF-1
protein in
the midpiece of the flagellum of spermatozoa represents to our
knowledge the first example of testis-specific transcription factor
expression beyond the release of spermatozoa from the seminiferous
tubuli. To date, the function of HIF-1
at this specific site is
unclear. Intriguingly, many HIF-1 target genes, especially the
glycolytic enzymes, are also expressed in the testis as specific
isoforms (10, 11, 12, 13). However, because the spermatozoal
nuclei are thought to be transcriptionally inactive, it seems unlikely
that HIF-1 functions as a transcription factor in sperm. Rather, the
high levels of HIF-1
might be required in oocytes after
fertilization that occurs in a hypoxic environment. Because mice
containing a null mutation in the second exon of the Hif1a
gene are nonviable (33), an exonI.1-specific mouse
knockout model will be required to elucidate the functional
significance of specific HIF-1
isoform expression during mouse
spermiogenesis.
Another intriguing question is the exact mechanism of the mHIF-1I.1
mRNA accumulation in the differentiated spermatids. Our results with
in vitro cultured spermatogonia and spermatocyte cell lines
indicate that the exon I.1 promoter does not become activated during
spermatogenesis in these premeiotic stages. Interestingly, the proximal
exon I.1 promoter contains an open-reading frame for a so-called
premordial peptide (21). These repetitive elements have
been reported to be transcribed in the sex-determining region of the Y
chromosome (34). While the mouse Hif1a gene is
not located on the Y chromosome, this sequence might be responsible for
activating the exon I.1 promoter during male postmeiotic
spermiogenesis. Clearly, in vitro transcription assays and
transgenic mouse models are needed to clarify this point.
While exposure to hypoxia induced mHIF-1I.2 protein levels in the
testis, it did not further induce mHIF-1
I.1 protein in epididymal
spermatozoa or after in vitro exposure of isolated
spermatozoa. The most obvious explanation for this effect might be the
lack of proteasomal activity in mature spermatozoa, rendering
mHIF-1
I.1 protein stability already under normoxic conditions.
Although we cannot formally exclude that the mHIF-1
I.2 protein
corresponds to the isoform detected in spermatozoa, the switch from
mHIF-1
I.2 to mHIF-1
I.1 mRNA isoform expression during
spermatogenesis and the complete lack of detectable mHIF-1
I.2 mRNA
in spermatids strongly suggest that the mHIF-1
I.1 mRNA is translated
in postmeiotic spermatids. The definitive proof would require
antibodies specific for the two different predicted N-termini (the
actual N-termini are unknown for both isoforms and might differ from
the prediction due to posttranslational processing). However, as we did
not find any functional differences of the two isoforms in
vitro (23), the biological significance of our
findings probably lies in the fact that an alternative promoter is
active to ensure the expression of HIF-1
at unusually late stages of
spermiogenesis, rather than the expression of a structurally or
functionally different protein isoform.
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MATERIALS AND METHODS |
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In Situ Hybridization
The technique used for in situ hybridization
was essentially as described by Breier et al.
(35). A cDNA clone containing mouse Hif1a exon
I.1 was constructed by inserting a 134-bp
SspI-HincII fragment, derived from the
Hif1a genomic clone H13 (21), into the
SmaI site of the pBluescriptSKII vector
(Stratagene, La Jolla, CA). The plasmid for
Hif1a exon I.2 was constructed by inserting a 218-bp
NcoI-StuI fragment, derived from a
ZAP mouse
Hif1a genomic clone, into the SmaI site of the
pBluescriptSKII vector. Single-stranded antisense or sense cRNA probes
were generated by in vitro transcription of these plasmids
using 100 µCi 35S-UTP and T7 or SP6 RNA
polymerases as described by the manufacturer (Stratagene).
Adult male C57BL/6 mice from an in-house breeding facility were killed
by decapitation, both testes were removed, embedded in Tissue Tek
O.C.T. (Miles Scientific, Naperville, IL) and transferred into a
mixture of methyl butane and dry ice until frozen. The blocks were
stored at -70 C. Ten-micrometer sections were cut with a cryostat and
melted on silane-coated glass slides. Sections were incubated in 2x
SSC (20x SSC = 3 M NaCl, 0.3 M
Na3-citrate) at 70 C, digested with Pronase (40 µg/ml),
fixed in 4% paraformaldehyde, and acetylated with acetic anhydride
diluted 1:400 in 0.1 M triethanolamine.
Hybridization was performed in buffer containing 50% formamide, 10%
dextran sulfate, 10 mM Tris-HCl (pH 7.5), 10
mM sodium phosphate (pH 6.8), 2x SSC, 5
mM EDTA, 150 µg/ml yeast tRNA, 0.1
mM UTP, 1 mM adenosine
5'-O-(2-thiodiphosphate), 1 mM
adenosine 5'-O-(3-thiotriphosphate), 10
mM dithiothreitol, 10 mM
2-mercaptoethanol, and 2.5 x 104 cpm/ml
35S-labeled RNA probe overnight at either 48 C
(exon I.1) or 60 C (exon I.2). Slides were washed in 2x SSC/50%
formamide at 37 C (exon I.1) or 60 C (exon I.2) for 4 h, digested
with RNase (20 µg/ml) for 15 min, washed again with 2x SSC/50%
formamide overnight, and dehydrated in graded ethanol. Slides were
coated with Kodak NTB-2 emulsion (Eastman Kodak Co., Rochester, NY) diluted 1:1 in water, and exposed for
21 d (exon I.1) or 50 d (exon I.2), respectively. Slides were
developed and counterstained with 0.02% Toluidin Blue, air dried, and
mounted.
Cell Culture
The following cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA): human
HeLa cervic carcinoma (ATCC no. CCL-2); mouse TM3 Leydig cells
(CRL-1714); TM4 Sertoli cells (CRL-1715); GC-1 spg spermatogonia
(CR-2053); and GC-2 spd(ts) spermatocyte (CRL-2196). The human MCF-7
adenocarcinoma cell line was kindly provided by J. Lisztwan and W. Krek
(Basel, Switzerland). The human Jurkat T cell leukemia line was kindly
provided by M. Balzer (Zürich, Switzerland). All cell lines were
cultured in DMEM (high glucose, Life Technologies, Inc.,
Gaithersburg, MD) supplemented with 10% heat-inactivated FCS
(Roche Molecular Biochemicals, Mannheim, Germany), 100
U/ml penicillin, 100 µg/ml streptomycin, 1x nonessential amino
acids, and 1 mM Na-pyruvate (all purchased from Life Technologies, Inc.) in a humidified atmosphere containing 5%
CO2 at 37 C. Oxygen partial pressures in the
hypoxic workstation (InVivoO2-400, Ruskinn
Technology, Leeds, UK) or in the incubator (Forma Scientific,
model 3319, Marietta, OH) were either 140 mm Hg (20%
O2 vol/vol, normoxia) or 7 mm Hg (1%
O2 vol/vol, hypoxia).
Immunoblot Analysis
Nuclear extracts were prepared as described previously
(25). Protein concentrations were determined by the
Bradford protein assay (Bio-Rad Laboratories, Inc.,
Hercules, CA) or the BCA assay (Pierce Chemical Co.,
Rockford, IL) using BSA as a standard. Nuclear extracts (50
µg) were electrophoresed through SDS-polyacrylamide gels and
elctrotransferred to nitrocellulose membranes (Schleicher & Schuell, Inc., Dassel, Germany) using standard procedures.
Membranes were stained with Ponceau S (Sigma, St. Louis,
MO) to confirm equal protein loading and transfer. HIF-1 was
detected using an affinity-purified chicken polyclonal IgY antibody
raised against a bacterially expressed GST-HIF-1
530825 fusion
protein (28) followed by a rabbit antichicken secondary
antibody (Promega Corp., Madison, WI). ARNT was
detected using the mouse monoclonal antibody MA1515 (Affinity BioReagents, Inc., Golden, CO) followed by goat antimouse
secondary antibody (Pierce Chemical Co.). Sp1 was detected
using the rabbit polyclonal antibody sc59 (Santa Cruz Biotechnology, Inc., Glaser AG, Basel, Switzerland) followed by
a goat antirabbit secondary antibody (Sigma). The
horseradish peroxidase-coupled secondary antibodies were detected by
using luminol (Sigma) chemiluminescent substrate, exposure
to x-ray films (Fuji Photo Film Co., Ltd., Tokyo, Japan)
and laser densitometric scanning (Molecular Dynamics Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire,
UK).
Isoform-Specific RT-PCR Analysis
Total RNA was isolated from normoxic and hypoxic cell cultures
according to the method described by Chomczynski and Sacchi
(36). For cDNA synthesis, 6 µg RNA was heat denatured (3
min at 70 C) and reverse transcribed in 100 µl 50 mM
Tris/Cl pH 8.3, 60 mM KCl, 3 mM
MgCl2, 10 mM dithiothreitol, 0.5
mM deoxy-NTPs, and 1 U/µl RNAsin (Promega Corp.), using 5 µg
(deoxythymidine)12-18 primers (Amersham Pharmacia Biotech) and 250 U Stratascript reverse transcriptase
(RT) (Stratagene). After incubation for 30 min at 37 C and
for 30 min at 42 C, the reaction was stopped by heating to 95 C for 5
min. An aliquot (2 µl) of each cDNA reaction was subjected to PCR
amplification using each 50 pmol of the forward primers mHIFexI.1
(5'-TTTCTGGGCAAACTGTTA-3') and mHIFexI.2 (5'-CGCCTCTGGACTTGTCT-3'), and
the reverse primer mHIFexIII (5'-TAACCCCATGTATTTGTTC-3') in 50 µl 1x
PCR buffer (Stehelin, Basel, Switzerland), 0.2 mM
deoxy-NTPs, and 0.2 U SuperTaq DNA polymerase (Stehelin). After 35
cycles of 94 C for 30 sec, 48 C for 30 sec, and 72 C for 2 min, the PCR
products were analyzed by agarose gel electrophoresis and ethidium
bromide staining.
Reporter Gene Assays
Two fragments of 1.0 kb and 1.4 kb, corresponding to the exon
I.1-specific (21) and the exon I.2-specific promoters
(22), respectively, were inserted in front of a luciferase
reporter gene. For transient transfections, 0.5 x
106 cells in 350 µl medium without FCS were
mixed with 50 µg DNA in 50 µl 10 mM Tris/Cl (pH 7.4), 1
mM EDTA and electroporated at 250 V and 960 microfarads
(GenePulser, Bio-Rad Laboratories, Inc.). Luciferase
reporter genes were coelectroporated into cells together with the
ß-galactosidase reference vector pCMVlacZ (37). The
cells were split and incubated for 3872 h under normoxic or hypoxic
conditions. After stimulation, transiently transfected cells were lysed
in reporter lysis buffer (Promega Corp.) and luciferase
and ß-galactosidase activities were determined according to the
manufacturers instructions (Promega Corp.) using a
Lumat LB9501 luminometer (EG&G Berthold, Bad Wildbad, Germany)
and a DigiScan 96-well plate photometer (ASYS), respectively.
Differences in the transfection efficiency and extract preparation were
corrected by normalization to the corresponding ß-galactosidase
activities, and the results were displayed as fold induction above the
activity of a promoterless vector.
Immunohistochemistry
For hypobaric exposure, male C57Bl/6C3F1 mice (57 months old)
were housed in an airtight chamber in which the air pressure was slowly
(1 h) lowered to a final pressure of 228 mm Hg (corresponds to a
normobaric oxygen concentration of 6%), and kept under these
conditions for 6 h. Immediately thereafter, the mice were killed
by cervical dislocation, and testis and epididymis were excised and
frozen in liquid nitrogen/isopentane. Frozen testes and epididymes of
normoxic and hypoxic mice were cut into serial sections of 6 and 2
µm, respectively, dried on a 50 C hot plate for 2 min, and fixed in
4% formaldehyde in PBS for 10 min. All antibodies were diluted in 50
mM Tris/Cl, pH 7.4, 154 mM NaCl, 0.1% Tween
20, 10% FCS. Sections were incubated with the chicken anti-HIF-1
polyclonal antibody described above or 10% FCS alone overnight at 4 C.
A peroxidase-conjugated rabbit antichicken IgY (Pierce Chemical Co.) was incubated for 45 min at RT, followed by a peroxidase
conjugated goat antirabbit Ig (DAKO Diagnostika GmbH,
Hamburg, Germany) for 45 min. The slices were developed with
diaminobenzidine (Sigma) for 15 min and mounted in
distrene/dibutyl phthalate/xylene medium.
Immunofluorescence
Sperm cells were obtained from mouse epididymis and vas
deferens, washed in PBS and resuspended in DMEM. After incubation under
20% or 1% oxygen for 4 h at 37 C, the sperm cells were fixed
with 3.5% formaldehyde, pelleted, resuspended in 80% ethanol, spread
on glass slides, and air- dried. After rehydration, unspecific binding
sites were blocked with PBS containing 10% FCS. Subsequently, the
sperm cells were incubated with the polyclonal anti-HIF-1 chicken
IgY antibody described above or a monoclonal
IgG2b antibody (clone H1
67, Novus Biologicals,
Littleton, CO) or 3% BSA in PBS alone. FITC-labeled rabbit
antichicken IgY (Promega Corp.) or rabbit antimouse IgG
(DAKO Corp.) secondary antibodies were used for the
detection by fluorescence microscopy (Axioplan 2000, Carl Zeiss Vision GmbH, Mannheim, Germany).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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H.H.M. and D.M.K. contributed equally to this work.
1 Present address: Institute of Physiology, University of
Zürich-Irchel, CH-8057 Zürich, Switzerland.
Abbreviations: HIF, Hypoxia-inducible factor; TBP, TATA-binding protein.
Received for publication July 10, 2001. Accepted for publication November 1, 2001.
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REFERENCES |
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