help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2007-0348
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Puglisi, R.
Right arrow Articles by Boitani, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Puglisi, R.
Right arrow Articles by Boitani, C.
Endocrinology Vol. 148, No. 9 4302-4309
Copyright © 2007 by The Endocrine Society

Mice Overexpressing the Mitochondrial Phospholipid Hydroperoxide Glutathione Peroxidase in Male Germ Cells Show Abnormal Spermatogenesis and Reduced Fertility

Rossella Puglisi, Arturo Bevilacqua, Gianfranco Carlomagno, Andrea Lenzi, Loredana Gandini, Mario Stefanini, Franco Mangia and Carla Boitani

Department of Histology and Medical Embryology (R.P., G.C., M.S., C.B.); Department of Psychology, Section of Neuroscience (A.B., F.M.), Department of Medical Pathophysiology (A.L., L.G.), Istituto Pasteur-Fondazione Cenci Bolognetti (F.M.), Sapienza University of Rome, 00161 Rome, Italy

Address all correspondence and requests for reprints to: Dr. Carla Boitani, Department of Histology and Medical Embryology, Via A. Scarpa 14, 00161 Roma, Italy. E-mail: carla.boitani{at}uniroma1.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To investigate the physiological effects of mitochondrial phospholipid hydroperoxide glutathione peroxidase (mPHGPx) overexpression during early male germ cell differentiation, we have generated transgenic mice bearing the rat mPhgpx coding sequence driven by the mouse synaptonemal complex protein 1 promoter, allowing the transgene to be specifically activated in the testis from the zygotene to diplotene stages of the first meiotic division. Northern/Western blotting and immunocytochemical analyses of endogenous mPHGPx expression during spermatogenesis showed a low enzyme level in middle-late pachytene spermatocytes, but not in earlier meiotic stages, and a significant increase in mPHGPx content in round spermatids. The histological and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling analysis of transgenic testes revealed a number of spermatogenetic defects, including primary spermatocyte apoptosis, haploid cell loss, and seminiferous epithelium disorganization. In line with these features, adult transgenic male mice also displayed a reduction in fertility. Results obtained in this study suggest that mPHGPx expression is tightly regulated in pachytene spermatocytes, with any spatial-temporal increase in mPHGPx expression resulting in damage to spermatogenesis and eventual loss of haploid cells. Present findings in the mouse may be of interest to human male fertility.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SELENIUM PLAYS A CRITICAL role in normal spermatogenesis of mammals. Animals fed on a selenium-deficient diet develop a number of reproductive disorders, including impaired sperm motility, abnormal tail morphology, and reduced fertility (1, 2). In addition, offspring of extremely selenium-deficient dams exhibit severe testicular atrophy that can be reversed by a selenium-adequate diet (3). Almost the entire selenium content of the testis is associated with the enzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx), a selenoprotein belonging to the family of glutathione peroxidases. This enzyme has unique structural and functional properties, among which is the ability to reduce peroxidated membrane phospholipids by oxidizing not only the thiol groups of glutathione but also those of proteins, resulting in polypeptide chain cross-linking (4, 5). To date, three PHGPx isoforms have been identified, having specific subcellular localization in mitochondria, cytosol, and nuclei, respectively, and differing in their N-terminal amino acid sequence. The N termini of mitochondrial (mPHGPx) and cytosolic variants derive from different translation initiation sites of the exon 1a. In contrast, the N terminus of the nuclear isoform (nPHGPx) is generated by the alternative exon 1b, under the direction of a promoter located in the first intron of the gpx-4 gene (6). We recently showed that nPHGPx and mPHGPx have different patterns of expression during male germ cell maturation in rat testis, with nPHGPx expressed only in the haploid phase of spermatogenesis, whereas mPHGPx is also expressed in late primary spermatocytes (7).

Together with other lines of evidence, these data support the view that PHGPx behaves like a moonlighting protein having an array of functions during sperm maturation. The mitochondrial variant is certainly involved in the enzymatic defense against oxidative damage to mitochondria in mPHGPx-overexpressing RBL-2H3 cells (8, 9, 10). Likely, this is also true for mammalian spermatogenesis, in light of the high levels of PHGPx catalytic activity found in rat spermatocytes and round spermatids (7). In epididymal sperm, however, PHGPx catalytic activity is apparently lost or strongly reduced (5), despite the high levels of immunoreactivity found in the midpiece of human and rat spermatozoa (4), where PHGPx constitutes more than half of the capsule material embedding mitochondrial helix (5). Indeed, such an inactive enzyme is apparently essential for the integrity and fertilizing ability of male gametes, as also suggested by the finding that spermatozoa of infertile men with asthenozoospermia show a significant, albeit controversial, decrease of rescued PHGPx-specific activity and expression (11, 12, 13). As for the nuclear variant, it was proposed to be involved in the stabilization of condensed chromatin during sperm maturation (14) according to a number of findings, including that 1) the sperm of selenium-deficient mice display abnormal heads (15, 16), 2) the expression of nPHGPx is concomitant with the chromatin remodeling process in haploid cells (7), and 3) the thiol-oxidase activity of PHGPx is preferentially addressed to protamines, the nuclear proteins required for sperm head condensation (4).

In any case, the reason that PHGPx is so relevant to spermatogenesis and male fertility still remains to be unraveled. The inactivation of all exons of the gpx-4 gene in knockout mice caused embryonic lethality between 7.5 and 8.5 d postcoitum (17, 18), making that animal model inappropriate to provide new insights into the role of this gene in the testis. In contrast, knockout mice specifically lacking nPHGPx displayed defective chromatin condensation in caput epididymis sperm. This defect, however, was apparently overcome during subsequent epididymal sperm maturation (14), suggesting that nPHGPx is dispensable for the maintenance of male fertility in the mouse. As for mPHGPx, information presently available on the alteration of reproductive potential of male mice fed diets with increased/decreased selenium levels (19, 20, 21) cannot give a clear-cut insight on this issue, owing to the very large number of selenoprotein-coding genes and the multiple body regions where these genes are expressed (22). In this study, we have overcome these difficulties by producing a transgenic mouse line overexpressing mPHGPx in the testis during the prophase of the first meiotic division, when the endogenous mPHGPx level is markedly lower than in the postmeiotic phase. Here we demonstrate that such mPHGPx overexpression is associated with male germ cell apoptosis, seminiferous tubule disorganization, and reduced fertility.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Animals used were mice from the C57BL/6 and B6D2F1 strains as well as transgenic mice. Animals were housed in accordance with the guidelines for animal care of the Sapienza University of Rome and were killed by CO2 asphyxia.

Germ cell preparation
Highly purified pachytene spermatocytes and round spermatids (steps 1–8) were obtained from 28- and 30-d-old mouse testes, as previously described (23). Briefly, the cell suspension obtained by enzymatic digestion of testicular tissue was fractionated by velocity sedimentation at unit gravity on 0.5–3% albumin gradient. Purity of cell fractions was verified by flow cytometry and morphology of cytospun and stained cell preparations (7).

Northern blot analysis
Total RNA was extracted from testes of mice at different ages (8, 14, 30, and 60 d after birth) or from isolated germ cell populations, using the guanidinium thiocyanate-cesium chloride ultracentrifugation method (24). Northern blotting was performed as previously described (7). Blots were hybridized overnight at 42 C with a 32P-labeled probe encompassing the region from nucleotide (nt) 3–146 of the mitochondrial PHGPx cDNA sequence (accession no. AF045768). For the standardization of different lanes, blots were rehybridized with an 18S rRNA cDNA probe.

Protein extraction and immunoblotting
After fractionation, meiotic and postmeiotic cells were lysed in RIPA buffer [150 mM NaCl, 1% deoxycholate, 0,1% SDS, 50 mM Tris-HCl (pH 8.00), and a protease and phosphatase inhibitor cocktail (Sigma, St. Louis, MO)], sonicated 12 times for 15 sec each time, and centrifuged at 20000 x g for 40 min at 4 C. Protein concentration of each sample was determined using the bicinchoninic acid method (Pierce Chemical, Rockford, IL) with albumin as standard. Equal amounts of proteins were fractionated by 15% SDS-PAGE (25), and then transferred to a nitrocellulose membrane (Hybond-C extra; Amersham Pharmacia, Piscataway, NJ). Blotted membranes were probed with 1:4000 rabbit polyclonal anti-PHGPx antibody (a generous gift of Prof. E. Panfili, University of Trieste, Italy), followed by 1:4000 goat peroxidase-labeled antirabbit IgG (Zymed Laboratories Inc., South San Francisco, CA). Detection was eventually performed using a chemiluminescence system (ECL; Amersham Pharmacia).

Histology and immunohistochemistry
Transgenic testis histology was routinely analyzed on 10–40 animals at each age examined, taking wild-type mice belonging to the same litter as control. Testes were fixed for at least 24 h in Bouin’s fixative at room temperature, dehydrated, and then paraffin embedded. Mounted sections (5 µm) were deparaffinized, rehydrated, and then stained with carmalum or used for immunohistochemistry/terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. For immunohistochemistry, endogenous peroxidase activity was quenched by incubating sections with 3% H2O2. Sections were saturated and permeabilized in PBS containing 1% BSA and 0.1% Triton X-100 at room temperature and then incubated overnight at 4 C with the rabbit polyclonal anti-PHGPx antibody mentioned above, diluted 1:40. Sections were incubated with a goat peroxidase-labeled antirabbit IgG, developed using diaminobenzidine as substrate (Roche/Roche Molecular Biochemicals, Indianapolis, IN) and counterstained with carmalum.

TUNEL assay
Evaluation of apoptotic cells was performed by the in situ cell death detection kit (Roche). Histological sections were treated with 15 µg/ml proteinase K in Tris-HCl (pH 7.5) at room temperature, rinsed with PBS, and incubated in 0.3% H2O2 in methanol for 30 min. After permeabilization in 0.1% Triton X-100 in sodium citrate buffer, sections were incubated with the TUNEL reaction mixture and then with anti-fluorescein-peroxidase-conjugated antibody, according to the manufacturer’s instructions. Positive controls were prepared by adding 1 U/µl DNase for 10 min, and negative controls were performed by omitting terminal deoxynucleotidyl transferase. The number of apoptotic cells per seminiferous tubule was determined by counting TUNEL-positive cells in at least 300 tubule cross-sections, selected at random within 4- and 26-wk-old transgenic and wild-type animals.

Plasmid construction and validation
The full-length rat mPhgpx cDNA sequence (RP1) (26) was kindly provided by Dr. D. Driscoll, The Cleveland Clinic Foundation, Cleveland, OH. This sequence encodes for a product with 98% amino acid identity with the mouse mPhgpx, having an identical catalytic site and an N-terminal 27-amino-acid residue acting as mitochondrial targeting signal (27). The internal EcoRI restriction site (nt 189) was modified by introducing a sense mutation (GAA->GAG) by recombinative PCR. Next, the mutated mPhgpx-coding sequence was cloned into the pNASSß vector (Clontech, Mountain View, CA) containing the mouse synaptonemal complex protein 1 (Sycp1) promoter fragment from nt –722 to +102 (kindly provided by Dr. F. Cuzin, University of Nice, France). Construct expression was determined by transfecting GC-1 spg cells, an SV40 large T antigen-immortalized germ cell line that corresponds to a spermatogenetic stage between spermatogonia type B and primary spermatocyte (28), with 2 µg DNA/dish of either the full-size or control constructs (the empty vector or a construct containing the antisense sequence), using Fugene (Roche, Boehringer/Mannheim). Transfection efficiency was monitored by cotransfection with 0.1 µg/dish of a pCMV-LacZ DNA construct (Clontech). ß-Galactosidase activity was assayed in cell extracts using 4-methylumbelliferyl-ß-D-galactoside as substrate, as described (29). The expression of mPHGPx was analyzed by Western blotting as described above. For standardization of different lanes, blots were probed again with anti-{alpha}-tubulin monoclonal antibody diluted 1:2000 (Sigma) and with a rabbit peroxidase-labeled antimouse IgG diluted 1:1000 (Dako A/S, Glostrup, Denmark).

Generation of mPHGPx transgenic mice
The transgene construct described above (see Fig. 3AGo) was isolated by digestion with EcoRI and XbaI and microinjected (~2 pg/nucleus) into the male pronucleus of fertilized eggs obtained from B6D2F1 x B6D2F1 crosses, according to standard techniques (30). Transgenic founders were genotyped by Southern blotting of genomic DNA extracted from tail biopsies and digested with BglII. A fragment specific to Phgpx exon 7 was used as a probe. The F1–F4 founder progeny were genotyped by Southern blot analysis and/or PCR of genomic DNA, using the following primers: sense, 5'-CCATGCACGAGTTCTCAGCC-3', and antisense, 5'-CAATGTATCTTATCATGTCTGG-3'.


Figure 3
View larger version (14K):
[in this window]
[in a new window]

  		FIG. 3. Transgenic mice overexpressing mPHGPx during meiosis. A, A schematic representation of the Sycp1-mPhgpx transgene. A nt –722/+102 fragment of mouse Sycp1 promoter was fused to a full-length rat mPhgpx cDNA, as described in Materials and Methods. Representation is in approximate scale. The horizontal white bar indicates the length and position of the probe used for Southern blot analysis. Arrows indicate PCR primers (s, sense; as, antisense; not in scale) used for genotyping the founder’s progeny. B, Western blot analysis of Sycp1-mPhgpx transgene expression in the GC1-spg cell line. Cells were transfected with the following: lane 1, the empty DNA vector; lane 2, the transgene construct; lane 3, an antisense Sycp1-mPhgpx construct. The amounts of proteins loaded on the gel were monitored by probing the membrane with anti-{alpha}-tubulin antibody. C, Southern blot analysis of genomic DNA extracted from wild-type and founder transgenic mice. Filters were hybridized with the probe indicated in A. BglII-digested DNA revealed the presence of endogenous genes/pseudogenes in all samples (arrowheads). Lane 1, DNA of a wild-type mouse; lane 2, wild-type DNA supplemented with five transgene copies per genome (small arrow); lane 3, DNA of transgenic founder SCP9, used in this study. Note the presence of two hybridization bands, suggestive of two transgene copies integrated into different sites (white arrows); lane 4, DNA of transgenic mouse SCP10, showing integration of a single transgene copy (white arrow).

 
Genomic DNA (100 ng) was amplified in a 50-µl reaction volume, with 40 cycles (94 C for 1 min, 62 C for 1.30 min, and 72 C for 2 min).

Semiquantitative RT-PCR analysis
Total RNA extracted from testes of transgenic and wild-type mice of different ages was treated with DNase I (Life Technologies, Inc., Invitrogen, Milan, Italy) and reverse-transcribed with oligo dT, using the Superscript II RT kit (Life Technologies, Inc.) according to manufacturer’s recommendations. Amounts of exogenous mPHGPx mRNA were determined using the same pair of primers described for genotyping but with 35 cycles. The internal control was the amplification of S16 ribosomal protein mRNA, performed as previously described (31), with 25 cycles. This cycle number was in the linear range of amplification. Negative controls were performed by omitting the cDNA template from PCR and by performing a RT without the reverse transcriptase enzyme.

Fertility assessment
Transgenic and wild-type male mice belonging to the same litter were mated continuously with C57BL/6 females (one male with two females) for a period of 2 months. Fertility was assessed by recording the number and the size of litters produced by each pairing. In a second test, transgenic and wild-type male mice were sequentially mated with C57BL/6 wild-type females (one male with two females) for a period of 2 months. Females were checked for vaginal plugs each morning. Plug-positive females were separated from the male and substituted with new ones. Reproductive organs were harvested for weight determination and sperm counts. Epididymal spermatozoa were collected from adult mice by squeezing the cauda epididymides in PBS. Sperm morphology was determined by light microscopy, and quantity was measured by hematocytometry.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mitochondrial PHGPx expression in differentiating male germ cells
To determine the pattern of mPHGPx expression during mouse testis development, we performed a Northern blot analysis of total testicular RNA isolated from mice at different postnatal ages (8–60 d) (Fig. 1AGo). Little signal was observed during the first 2 wk of animal age. In contrast, an intense mRNA band became apparent from 30 d and onward, coinciding with the appearance of a large number of haploid cells in the testis. To better characterize the germ-cell stage of spermatogenesis at which mPHGPx was expressed, RNA was prepared from highly enriched fractions of middle-late pachytene spermatocytes and round spermatids at steps 1–8. Northern hybridization analysis of RNA preparations obtained from the two germ cell types showed that the mPHGPx transcript was expressed in both meiotic and postmeiotic cells, being markedly more abundant in the haploid cells (Fig. 1BGo).


Figure 1
View larger version (29K):
[in this window]
[in a new window]

  		FIG. 1. Northern blot analysis of mPHGPx expression during postnatal testis development and in middle-late pachytene spermatocytes and round spermatids. Total RNA was extracted and hybridized with a probe specific to the mitochondrial isoform, taking 18S rRNA as internal control, as described in Materials and Methods. A, The gel was loaded with similar amounts of RNA preparations from testes of increasing age. B, The gel was loaded with RNA preparations from purified middle-late pachytene spermatocytes (lanes 1 and 3) and round spermatids (lanes 2 and 4). The mPHGPx mRNA abundance in total RNA preparations from spermatocytes/round spermatids was compared with each other by layering on the gel similar amounts of RNA preparations (lanes 1 and 2) and amounts of RNA extracted from similar numbers of spermatocyte/round spermatids cells (lanes 3 and 4).

 
The expression profile of mPHGPx protein was determined by Western blotting of proteins extracted from germ cells at definite stages of differentiation (Fig. 2AGo), giving results consistent with those of Northern blotting. A band corresponding to the 20-kDa isoform of PHGPx was detected in both pachytene spermatocytes and round spermatids (steps 1–8), being expressed at a higher level in the haploid cells. In addition, we examined the localization of mPHGPx in the adult testis by immunohistochemistry. Positive immunostaining was observed in the inner cell layer of seminiferous tubules, where middle-late pachytene spermatocytes, round spermatids, and elongated spermatids were positive (Fig. 2Go, B–D). No appreciable staining was detected in the basal layer of seminiferous tubules, indicating that the mPHGPx isoform was not expressed in spermatogonia and early primary spermatocytes. Sertoli cells were also negative.


Figure 2
View larger version (110K):
[in this window]
[in a new window]

  		FIG. 2. Western blot and immunohistochemical analyses of mPHGPx expression in mouse testis. A, Protein preparations from purified populations of middle-late pachytene spermatocytes (spc) and round spermatids (spt) were fractionated by SDS-PAGE and probed with an anti-PHGPx antibody as described in Materials and Methods. B–D, Histological sections from adult testis were immunolabeled using the anti-PHGPx antibody used in A. Insets on the left of the figures show a x2 enlarged detail of the tubule. The seminiferous tubule stage is indicated by Roman numerals at the center of the figure. PHGPx-positive cells were middle-late pachytene spermatocytes and round/elongated spermatids. E, The section was stained by omitting the primary antibody. Scale bars, 50 µm.

 
Generation of mPHGPx-overexpressing transgenic mice
The demonstration that mPHGPx expression depended on male germ cell maturation prompted us to investigate the in vivo function of this selenoprotein by generating transgenic mice that overexpressed mPHGPx in a stage-specific manner, when the endogenous protein level is very low, namely in early primary spermatocytes. In fact, the mPHGPx transgene (Fig. 3AGo) was driven by the promoter of synaptonemal complex protein 1 (Sycp-1), a testis-specific gene that is activated at the zygotene-diplotene stages of meiosis (32). Proper construct transcription was preliminarily determined by transfecting GC-1 spg cells with Sycp1-Phgpx as described in Materials and Methods and monitoring transfection efficiency by a cotransfected CMV-LacZ vector. The Sycp1 promoter was efficiently activated in GC-1 spg cells. In fact, Western blot analysis of transfected cells showed that the level of mPHGPx expression was markedly increased with respect to mock cells (Fig. 3BGo), validating the use of the Sycp1-mPhgpx construct for generating transgenic mice. Subsequent microinjection experiments led to the production of two transgenic founders, named SCP9 and SCP10, respectively (Fig. 3CGo). Southern blot analysis of SCP9 detected the presence of two copies of the transgene, having distinct integration sites. These transgene copies did not segregate from each other in founder’s F1–F4 progeny, suggesting linkage to the same chromosome. Founder SCP10 also bore a transgene copy, which had Mendelian inheritance but was not expressed in the progeny (not shown). Therefore, this line was not used for further study.

Transgene expression and histological analysis of SCP9 transgenic testes
Testes of transgenic and wild-type mice belonging to the same litter were subjected to RT-PCR evaluation of transgene expression at increasing ages from early prepubertal to adulthood (Fig. 4Go). The transgene was consistently expressed at all animal ages examined, validating the use of the SCP9 progeny for further study. Testes of sibling transgenic and wild-type animals were also analyzed by conventional histology (Fig. 5AGo). Spermatogenesis in 2-wk-old transgenic mice appeared normal (data not shown). In contrast, testes of 3-, 4-, 6-, and 26-wk-old transgenic mice revealed various abnormalities, which were absent in wild-type animals: 1) the number of spermatocytes and spermatids was markedly reduced; 2) several apoptotic cells were present in the seminiferous tubules, together with multinucleated cells (giant cells) and epithelium vacuolization; and 3) the lumen of altered tubules was filled with a concentric nest of cells composed of amorphous cellular debris and degenerating cells. Even though spermatogenesis did not arrest at a specific epithelial stage, stages I–VIII of the seminiferous epithelium were the most affected, displaying a marked reduction in round and elongated spermatids, which resulted in lack of spermatozoa in the lumen of the most compromised tubules. On the contrary, spermatogonia and the interstitium were apparently normal.


Figure 4
View larger version (13K):
[in this window]
[in a new window]

  		FIG. 4. Expression of the Sycp1-mPhgpx transgene during postnatal mouse development. Total testicular RNA preparations from wild-type (WT) and two representative transgenic (Tg) mice analyzed at various ages were subjected to RT-PCR amplification using the primers indicated in Fig. 3AGo. Primer specificity for the transgene is shown by lack of amplification in RNA preparations from wild-type mice.

 

Figure 5
View larger version (58K):
[in this window]
[in a new window]

  		FIG. 5. Histological analysis of testes from wild-type (WT) and transgenic (Tg) mice. A, Sections representative of most severe histopathological features observed in transgenic mice were prepared from animals of different ages and stained with carmalum. Note pyknotic cells (brackets), seminiferous tubule disorganization (asterisks), and nests of cells in the lumen of tubules (arrows). Germ cells were sloughing off from Sertoli cell cytoplasm, spermatocytes were degenerating, and haploid cells were markedly reduced. Scale bars, 50 µm. B, Frequency of most severely damaged transgenic phenotypes as a function of animal age. Values on the ordinate represent pooled frequencies of medium- and high-severity classes.

 
Approximately 40% of transgenic mice examined displayed testicular deficiencies with large interindividual variation in the type and severity. The remaining 60% of transgenic mice had an apparently normal phenotype. We grouped the transgenic phenotypes into four histopathological classes according to their severity (normal, low, medium, and high). The occurrence of testicular damage consistently decreased through animal age in terms of both severity and distribution in the testis, the medium/high phenotypes being observed in approximately only 20% of adult transgenic mice (Fig. 5BGo).

Germ cell apoptosis was also analyzed by TUNEL labeling of testicular sections from transgenic and wild-type mice of increasing ages (4 and 26 wk) (Fig. 6AGo). In transgenic mice, apoptotic cells were significantly more abundant than in wild-type animals and predominantly localized in germ cell layers containing primary spermatocytes (Fig. 6BGo).


Figure 6
View larger version (70K):
[in this window]
[in a new window]

  		FIG. 6. Germ cell apoptosis in wild-type and mPHGPx-overexpressing transgenic testes. A, Apoptotic cells in representative histological sections of wild-type (WT) and transgenic (Tg) testes were revealed by TUNEL assay, as described in Materials and Methods. Apoptotic spermatocytes were very abundant in transgenic testes (brackets) compared with wild-type testes (arrows). Scale bars, 50 µm. B, A comparison of TUNEL-positive cell numbers in wild-type (open bars) and transgenic (solid bars) testes. Difference between transgenic and wild-type testes was calculated by Student’s t test: 4 wk, P < 0.001; 26 wk, P < 0.001.

 
Transgenic males are subfertile
The marked germ cell loss observed in the haploid cell compartment of transgenic testes suggested that transgenic mice would undergo a reduction in fertility. This issue was assessed by maintaining transgenic/wild-type mice in the continuous presence of C57BL6 females (one male with two females) and determining the number and size of the litters produced in a period of 2 months (Table 1Go). A limited number of transgenic males produced fewer litters than wild-type males, even though no difference was observed in the size of the litter. Parallel experiments recording the plugs produced by transgenic males mated with C57BL6 females demonstrated that transgenic and wild-type males had similar plug frequencies (not shown), indicating that the transgene did not negatively affect the male copulatory activity per se. In addition, transgenic and wild-type animals did not differ in body and testis weight (not shown). Histological analyses of the testes of subfertile transgenic mice showed the presence of medium/high-severity defects. These mice had also a decreased number of epididymal sperm and a higher frequency of sperm abnormalities (Table 1Go), among which microcephalic spermatozoa were the most abundant.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Fertility of mPHGPx-overexpressing male mice with medium/high testicular defects

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The need to produce novel in vivo models for studying mPHGPx functions during spermatogenesis is underscored by previous observations that knockout mice lacking PHGPx die in utero by midgestation (17, 18). In this study, we have used the promoter of the Sycp1 gene to specifically overexpress the mPHGPx isoform in the testis and during the prophase of the first meiotic division, from zygotene to diplotene stages (32). Present biochemical/morphological findings have shown that endogenous mPHGPx transcripts first appear at the middle-late pachytene stage. Therefore, the expression of the mPHGPX transgene was not only increased in amount but also developmentally anticipated. Transgenic mice were fully viable and developed normally, indicating that mPHGPx overexpression did not affect embryogenesis and postnatal life and ruling out the possibility of nonspecific effects due to the sites of transgene insertion. According to the testis- and stage-specific expression of the transgene, primary spermatocytes specifically underwent an increase in the frequency of apoptosis, which eventually resulted in a severe loss of haploid germ cells and in tubular epithelium disorganization. It is also interesting to note that the degree of spermatogenesis impairment was variable between transgenic mice, a large fraction of them having a histologically normal phenotype, further arguing against the possibility that spermatogenesis damage was due to the transgene genomic insertion per se. Moreover, the inverse relationship between the extent of testicular damage and the age of the animal suggests that a compensatory mechanism(s) takes place with testis maturation. Regional damage is frequently observed in injured adult testis of mammals (33).

mPHGPx is a key enzyme in the protection of biomembranes exposed to oxidative stress (10). Indeed, mPHGPx overexpression in RBL-2H3 cells increases the resistance to oxidative injury and suppresses mitochondrial death pathway-mediated apoptosis (8, 9). In addition, the forced expression of a human Phgpx transgene in mouse tissues increased the liver cell resistance to apoptosis after animal treatment with a potent prooxidant (34). Therefore, the present findings of defective spermatogenesis in mice overexpressing this selenoprotein were unexpected. It should be kept in mind, however, that the antioxidant/antiapoptotic role of mPHGPx in somatic tissues/cell lines does not necessarily reflect the role this enzyme actually plays in the testis, with particular reference to primary spermatocytes. Indeed, increasing evidence pinpoints PHGPx as a moonlighting protein, with a plethora of functions, which may vary according to the expression, the intracellular localization, and different interacting partners in a given cell type (35). Another source of mPHGPx multifunctionality is the presence of two alternative mRNA translation initiation sites, resulting in the potential production of either mitochondrial or cytosolic PHGPx isoforms. Cytosolic PHGPx is abundant in somatic tissues (26, 36) and was also described in sperm nuclei (37), although its role in the testis is still obscure. Unfortunately, due to the molecular weight similarity of cytosolic and mitochondrial isoforms, the issue of whether the expression of cPHGPX was activated/increased in transgenic testes could not be addressed in this work.

A more likely possibility, not excluding the one discussed above, is that transgenic spermatocytes were directly damaged by the excessive production of mPHGPx per se, perturbing the redox status of these cells. A series of oxidative events takes place during spermatogenesis, leading to a finely regulated redox status, which is needed to stabilize sperm nucleus and tail. Any modification of this status may damage germ cells and impair fertility (38, 39). The relevance of redox status to cell viability was also suggested by a number of observations performed on MCF-7 cells overexpressing mPHGPx, whose G1 phase of the cell cycle was significantly delayed (40). Moreover, it was thoroughly demonstrated in lens cells and retinal pigment epithelial cells that the cellular redox status regulates the activity of thiol enzymes of the ubiquitin system (41, 42, 43). It is therefore possible that in primary spermatocytes of our transgenic mice, mPHGPx overexpression caused an increase in oxidized thiols, which eventually resulted in cell cycle defects and apoptosis. Supporting this hypothesis, knockout mice in which the ubiquitin-conjugating DNA-repair enzyme HR6B was inactivated displayed an increase in primary spermatocyte apoptosis and altered spermatogenesis (44), a testicular phenotype similar to that described in the present transgenic animals.

A deficiency or an excess of selenium in the diet results in increased oxidative stress negatively affecting spermatogenesis, and this effect is modulated by redox-sensitive transcription factors nuclear factor-{kappa}B and activator protein-1, controlling cell proliferation (45, 46, 47). These findings, as well as present observations, suggest that high levels of antioxidants are not beneficial to the progression of normal germ cell differentiation. Consistent with this idea, dietary administration of a mixture of the antioxidant agents vitamins C and E caused increased sperm head abnormalities and reduced sperm production in the mouse (48).

Another effect of mPHGPx overexpression was the subfertility of transgenic mice displaying a damaged testis phenotype. Combining this finding with the notion that in normal testis, haploid germ cells represent the testicular compartment bearing the highest PHGPx content (4, 7, 49) (present observations), it can be concluded that subfertility was likely due to spermatid loss and/or production of spermatozoa compromised in morphology and fertilization potential. It is worth noting that the modest subfertility of transgenic mice was likely related to focal testicular damage in adults, i.e. the animals in which fertility tests were performed. Although a correlation between a deficiency in PHGPx catalytic activity/protein content and human male infertility is still debatable (11, 12, 13), the present findings demonstrating that the up-regulation of mPHGPx expression leads to alteration of mouse spermatogenesis further suggest that PHGPx is relevant to human male reproduction.


   Acknowledgments
 
We are grateful to Prof. Donna M. Driscoll for the gift of recombinant PHGPx cDNA, Prof. Francois Cuzin for the gift of Sycp1 promoter, Prof. Enrico Panfili for the gift of PHGPx antibody, and Ms. Tiziana Menna for her technical assistance.


   Footnotes
 
This work was supported by grants from The European Union (STREP PIONEER FOOD-2005-513991) to C.B.; Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza University of Rome, to F.M.; and PRIN 2005-055188 to A.B.

Disclosure Statement: The authors of this manuscript have nothing to disclose.

First Published Online May 31, 2007

Abbreviations: mPHGPx, Mitochondrial phospholipid hydroperoxide glutathione peroxidase; nPHGPx, nuclear isoform of PHGPx; nt, nucleotide; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.

Received March 13, 2007.

Accepted for publication May 24, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Wu SH, Oldfield JE, Whanger PD, Weswig PH 1973 Effect of selenium, vitamin E, and antioxidants on testicular function in rats. Biol Reprod 8:625–629[Abstract]
  2. Wu AS, Oldfield JE, Shull LR, Cheeke PR 1979 Specific effect of selenium deficiency on rat sperm. Biol Reprod 20:793–798[Abstract]
  3. Behne D, Weiler H, Kyriakopoulos A 1996 Effects of selenium deficiency on testicular morphology and function in rats. J Reprod Fertil 106:291–297[Abstract/Free Full Text]
  4. Godeas C, Tramer F, Micali F, Soranzo M, Sandri G, Panfili E 1997 Distribution and possible novel role of phospholipid hydroperoxide glutathione peroxidase in rat epididymal spermatozoa. Biol Reprod 57:1502–1508[Abstract]
  5. Ursini F, Heim S, Kiess M, Maiorino M, Roveri A, Wissing J, Flohe L 1999 Dual function of the selenoprotein PHGPx during sperm maturation. Science 285:1393–1396[Abstract/Free Full Text]
  6. Maiorino M, Scapin M, Ursini F, Biasolo M, Bosello V, Flohe L 2003 Distinct promoters determine alternative transcription of gpx-4 into phospholipid-hydroperoxide glutathione peroxidase variants. J Biol Chem 278:34286–34290[Abstract/Free Full Text]
  7. Puglisi R, Tramer F, Panfili E, Micali F, Sandri G, Boitani C 2003 Differential splicing of the phospholipid hydroperoxide glutathione peroxidase gene in diploid and haploid male germ cells in the rat. Biol Reprod 68:405–411[Abstract/Free Full Text]
  8. Arai M, Imai H, Koumura T, Yoshida M, Emoto K, Umeda M, Chiba N, Nakagawa Y 1999 Mitochondrial phospholipid hydroperoxide glutathione peroxidase plays a major role in preventing oxidative injury to cells. J Biol Chem 274:4924–4933[Abstract/Free Full Text]
  9. Nomura K, Imai H, Koumura T, Arai M, Nakagawa Y 1999 Mitochondrial phospholipid hydroperoxide glutathione peroxidase suppresses apoptosis mediated by a mitochondrial death pathway. J Biol Chem 274:29294–29302[Abstract/Free Full Text]
  10. Imai H, Nakagawa Y 2003 Biological significance of phospholipid hydroperoxide glutathione peroxidase (PHGPx, GPx4) in mammalian cells. Free Radic Biol Med 34:145–169[CrossRef][Medline]
  11. Imai H, Suzuki K, Ishizaka K, Ichinose S, Oshima H, Okayasu I, Emoto K, Umeda M, Nakagawa Y 2001 Failure of the expression of phospholipid hydroperoxide glutathione peroxidase in the spermatozoa of human infertile males. Biol Reprod 64:674–683[Abstract/Free Full Text]
  12. Foresta C, Flohe L, Garolla A, Roveri A, Ursini F, Maiorino M 2002 Male fertility is linked to the selenoprotein phospholipid hydroperoxide glutathione peroxidase. Biol Reprod 67:967–971[Abstract/Free Full Text]
  13. Tramer F, Caponecchia L, Sgro P, Martinelli M, Sandri G, Panfili E, Lenzi A, Gandini L 2004 Native specific activity of glutathione peroxidase (GPx-1), phospholipid hydroperoxide glutathione peroxidase (PHGPx) and glutathione reductase (GR) does not differ between normo- and hypomotile human sperm samples. Int J Androl 27:88–93[CrossRef][Medline]
  14. Conrad M, Moreno SG, Sinowatz F, Ursini F, Kolle S, Roveri A, Brielmeier M, Wurst W, Maiorino M, Bornkamm GW 2005 The nuclear form of phospholipid hydroperoxide glutathione peroxidase is a protein thiol peroxidase contributing to sperm chromatin stability. Mol Cell Biol 25:7637–7644[Abstract/Free Full Text]
  15. Watanabe T, Endo A 1991 Effects of selenium deficiency on sperm morphology and spermatocyte chromosomes in mice. Mutat Res 262:93–99[CrossRef][Medline]
  16. Pfeifer H, Conrad M, Roethlein D, Kyriakopoulos A, Brielmeier M, Bornkamm GW, Behne D 2001 Identification of a specific sperm nuclei selenoenzyme necessary for protamine thiol cross-linking during sperm maturation. FASEB J 15:1236–1238[Free Full Text]
  17. Imai H, Hirao F, Sakamoto T, Sekine K, Mizukura Y, Saito M, Kitamoto T, Hayasaka M, Hanaoka K, Nakagawa Y 2003 Early embryonic lethality caused by targeted disruption of the mouse PHGPx gene. Biochem Biophys Res Commun 305:278–286[CrossRef][Medline]
  18. Yant LJ, Ran Q, Rao L, Van Remmen H, Shibatani T, Belter JG, Motta L, Richardson A, Prolla TA 2003 The selenoprotein GPX4 is essential for mouse development and protects from radiation and oxidative damage insults. Free Radic Biol Med 34:496–502[CrossRef][Medline]
  19. Kaur P, Bansal MP 2004 Influence of selenium induced oxidative stress on spermatogenesis and lactate dehydrogenase-X in mice testis. Asian J Androl 6:227–232[Medline]
  20. Kaur P, Bansal MP 2004 Effect of selenium-induced oxidative stress on the oxidation reduction system and reproductive ability of male mice. Biol Trace Elem Res 97:83–93[CrossRef][Medline]
  21. Kaur P, Bansal MP 2005 Effect of selenium-induced oxidative stress on the cell kinetics in testis and reproductive ability of male mice. Nutrition 21:351–357[CrossRef][Medline]
  22. Kohrle J, Jakob F, Contempre B, Dumont JE 2005 Selenium, the thyroid, and the endocrine system. Endocr Rev 26:944–984[Abstract/Free Full Text]
  23. Boitani C, Geremia R, Rossi R, Monesi V 1980 Electrophoretic pattern of polypeptide synthesis in spermatocytes and spermatids of the mouse. Cell Differ 9:41–49[CrossRef][Medline]
  24. Chirgwin JM, Przybyla AEMRJ, Rutter WJ 1979 Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294–5299[CrossRef][Medline]
  25. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685[CrossRef][Medline]
  26. Pushpa-Rekha TR, Burdsall AL, Oleksa LM, Chisolm GM, Driscoll DM 1995 Rat phospholipid-hydroperoxide glutathione peroxidase. cDNA cloning and identification of multiple transcription and translation start sites. J Biol Chem 270:26993–26999[Abstract/Free Full Text]
  27. Nam S, Nakamuta N, Kurohmaru M, Hayashi Y 1997 Cloning and sequencing of the mouse cDNA encoding a phospholipid hydroperoxide glutathione peroxidase. Gene 198:245–249[CrossRef][Medline]
  28. Hofmann MC, Narisawa S, Hess RA, Millan JL 1992 Immortalization of germ cells and somatic testicular cells using the SV40 large T antigen. Exp Cell Res 201:417–435[CrossRef][Medline]
  29. Bevilacqua A, Fiorenza MT, Mangia F 1997 Developmental activation of an episomic hsp70 gene promoter in two-cell mouse embryos by transcription factor Sp1. Nucleic Acids Res 25:1333–1338[Abstract/Free Full Text]
  30. Nagy AGM, Vintersten KBR 2003 Manipulating the mouse embryo: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press
  31. Fragale A, Puglisi R, Morena AR, Stefanini M, Boitani C 2001 Age-dependent activin receptor expression pinpoints activin as a physiological regulator of rat Sertoli cell proliferation. Mol Human Reprod 7:1107–1114[Abstract/Free Full Text]
  32. Sage J, Martin L, Meuwissen R, Heyting C, Cuzin F, Rassoulzadegan M 1999 Temporal and spatial control of the Sycp1 gene transcription in the mouse meiosis: regulatory elements active in the male are not sufficient for expression in the female gonad. Mech Dev 80:29–39[CrossRef][Medline]
  33. Russell LD, Ettlin RA, Sinha Hikim AP, Clegg ED 1990 Histological and histopathological evaluation of the testis. Clearwater, FL: Cache River Press
  34. Ran Q, Liang H, Gu M, Qi W, Walter CA, Roberts LJ, Herman B, Richardson A, Van Remmen H 2004 Transgenic mice overexpressing glutathione peroxidase 4 are protected against oxidative stress-induced apoptosis. J Biol Chem 279:55137–55146[Abstract/Free Full Text]
  35. Puglisi R, Tramer F, Carlomagno G, Gandini L, Panfili E, Stefanini M, Lenzi A, Mangia F, Boitani C 2005 PHGPx in spermatogenesis: how many functions? Contraception 72:291–293[CrossRef][Medline]
  36. Roveri A, Casasco A, Maiorino M, Dalan P, Calligaro A, Ursini F 1992 Phospholipid hydroperoxide glutathione peroxidase of rat testis. Gonadotropin dependence and immunocytochemical identification. J Biol Chem 267:6142–6146[Abstract/Free Full Text]
  37. Maiorino M, Mauri P, Roveri A, Benazzi L, Toppo S, Bosello V, Ursini F 2005 Primary structure of the nuclear forms of phospholipid hydroperoxide glutathione peroxidase (PHGPx) in rat spermatozoa. FEBS Lett 579:667–670[CrossRef][Medline]
  38. Lenzi A, Gandini L, Picardo M, Tramer F, Sandri G, Panfili E 2000 Lipoperoxidation damage of spermatozoa polyunsaturated fatty acids (PUFA): scavenger mechanisms and possible scavenger therapies. Front Biosci 5:E1–E15
  39. Aitken RJ, Baker MA 2006 Oxidative stress, sperm survival and fertility control. Mol Cell Endocrinol 250:66–69[CrossRef][Medline]
  40. Wang HP, Schafer FQ, Goswami PC, Oberley LW, Buettner GR 2003 Phospholipid hydroperoxide glutathione peroxidase induces a delay in G1 of the cell cycle. Free Radic Res 37:621–630[CrossRef][Medline]
  41. Jahngen-Hodge J, Obin MS, Gong X, Shang F, Nowell Jr TR, Gong J, Abasi H, Blumberg J, Taylor A 1997 Regulation of ubiquitin-conjugating enzymes by glutathione following oxidative stress. J Biol Chem 272:28218–28226[Abstract/Free Full Text]
  42. Obin M, Shang F, Gong X, Handelman G, Blumberg J, Taylor A 1998 Redox regulation of ubiquitin-conjugating enzymes: mechanistic insights using the thiol-specific oxidant diamide. FASEB J 12:561–569[Abstract/Free Full Text]
  43. Shang F, Gong X, Taylor A 1997 Activity of ubiquitin-dependent pathway in response to oxidative stress. Ubiquitin-activating enzyme is transiently up-regulated. J Biol Chem 272:23086–23093[Abstract/Free Full Text]
  44. Roest HP, van Klaveren J, de Wit J, van Gurp CG, Koken MH, Vermey M, van Roijen JH, Hoogerbrugge JW, Vreeburg JT, Baarends WM, Bootsma D, Grootegoed JA, Hoeijmakers JH 1996 Inactivation of the HR6B ubiquitin-conjugating DNA repair enzyme in mice causes male sterility associated with chromatin modification. Cell 86:799–810[CrossRef][Medline]
  45. Brigelius-Flohe R 2006 Glutathione peroxidases and redox-regulated transcription factors. Biol Chem 387:1329–1335[CrossRef][Medline]
  46. Shalini S, Bansal MP 2005 Role of selenium in regulation of spermatogenesis: involvement of activator protein 1. Biofactors 23:151–162[Medline]
  47. Shalini S, Bansal MP 2007 Alterations in selenium status influences reproductive potential of male mice by modulation of transcription factor NF{kappa}B. Biometals 20:49–59[CrossRef][Medline]
  48. Ten J, Vendrell FJ, Cano A, Tarin JJ 1997 Dietary antioxidant supplementation did not affect declining sperm function with age in the mouse but did increase head abnormalities and reduced sperm production. Reprod Nutr Dev 37:481–492[Medline]
  49. Godeas C, Tramer F, Micali F, Roveri A, Maiorino M, Nisii C, Sandri G, Panfili E 1996 Phospholipid hydroperoxide glutathione peroxidase (PHGPx) in rat testis nuclei is bound to chromatin. Biochem Mol Med 59:118–124[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Hum Reprod UpdateHome page
J. Ramalho-Santos, S. Varum, S. Amaral, P. C. Mota, A. P. Sousa, and A. Amaral
Mitochondrial functionality in reproduction: from gonads and gametes to embryos and embryonic stem cells
Hum. Reprod. Update, September 1, 2009; 15(5): 553 - 572.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Puglisi, R.
Right arrow Articles by Boitani, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Puglisi, R.
Right arrow Articles by Boitani, C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals