This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Merico, V. Articles by de Talamoni, N. T. Search for Related Content PubMed PubMed Citation Articles by Merico, V. Articles by de Talamoni, N. T.

A mitochondrial mechanism is involved in apoptosis of Robertsonian mouse male germ cells

Dipartimento di Biologia Animale, Università degli Studi di Pavia, Piazza Botta, 9-10, I-27100 Pavia, Italy¹ Bioquimica y Biologia Molecular, Facultad de Ciencias Medicas and² Quimica y Fisica Biologicas, Facultad de Odontologia, Universidad Nacional de Cordoba, 5000 Cordoba, Argentina

Correspondence should be addressed to S Garagna; Email: silvia.garagna{at}unipv.it

	Abstract

Top Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
The aim of this study was to determine whether the intrinsic mechanism of apoptosis is involved in the death of germ cells in Robertsonian (Rb) heterozygous adult male mice. Testes from 5-month-old Rb heterozygous CD1xMilano II mice were obtained and compared with those from homozygous CD1 (2n=40) and Milano II (2n=24) mice. For histological evaluation of apoptosis, TUNEL labelling and immunohistochemistry were used to localise Bax and cytochrome c. Expression of calbindin D_28k (CB), an anti-apoptotic molecule, was also analysed by immunohistochemistry and immunoblotting. Testicular ultrastructure was visualised by electron microscopy. Morphology and cell associations were abnormal in the Rb heterozygous seminiferous epithelium. An intense apoptotic process was observed in tubules at stage XII, mainly in metaphase spermatocytes. Metaphase spermatocytes also showed Bax and cytochrome c redistributions. Mitochondria relocated close to the paranuclear region of spermatocytes. CB was mainly expressed in metaphase spermatocytes, but also in pachytene spermatocytes, spermatids and Sertoli cells at stage XII. The co-localisation of CB and TUNEL labelling was very limited. Sixty per cent of metaphase spermatocytes were apoptotic and calbindin negative, while 40% were calbindin positive without signs of apoptosis. Ten per cent of the Bax- and cytochrome c-positive cells were also calbindin positive. These data suggest that apoptosis of the germ cells in heterozygous mice occurs, at least in part, through a mitochondrial-dependent mechanism. Calbindin overexpression might prevent or reduce the apoptosis of germ cells caused by Rb heterozygosity, which could partially explain the subfertility of these mice.

	Introduction

Top Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
Spermatogenesis is a complex process by which male germ cells mature from spermatogonia via spermatocytes and spermatids to spermatozoa and involves a controlled balance between cell proliferation and death (Lizama et al. 2007). Infertility or testicular disorders may appear when this balance is deregulated (Francavilla et al. 2000, 2002, Eguchi et al. 2002).

There are many different, poorly understood causes of deregulation of cell proliferation/cell death during spermatogenesis. However, a normal karyotype is an essential prerequisite for proper spermatogenesis as shown by the detrimental effects of structural rearrangements of the karyotype on this process (Redi et al. 1985, Wallace et al. 2002). The extent of the impairment of spermatogenesis depends on various factors such as the type and number of fusions, the configuration of diakinesis during meiosis and genic incompatibilities. In Robertsonian (Rb) heterozygous mice, Merico et al. (2003) found a high percentage (up to 59%) of defective seminiferous tubules in 5-month-old animals in which the typical cell-to-cell associations were almost unrecognisable. In these animals, the spermatid/spermatocyte ratio was as low as 1.4 when compared with the expected 4.0. Massive germ cell depletion was detected at each step of germ cell differentiation in both meiotic and post-meiotic germ cells indicating cell death. The impairment of spermatogenesis exerted by Rb heterozygosity was found to be partially relieved with time because the percentage of defective tubules was lower, and germ cell survival and sperm morphology better in 7-month-old mice than in 3- and 5-month-old animals. Eaker et al. (2001) demonstrated a high frequency of misaligned, condensed chromosomes in their Rb heterozygous mice, which could account for a significant proportion of the apoptotic spermatocytes suggesting a checkpoint process that identifies aberrant meiosis, although this mechanism would not be efficient enough to avoid the high frequency of gametic aneuploidy.

During spermatogenesis, spermatogonia undergo several cell divisions and spermatocytes divide twice to build the germ cell population of the testis (Russell et al. 2002). Some authors have found that immature spermatogonia show features of apoptosis, while more mature spermatocytes do not (Allan et al. 1987, Russell 1999); however, others have found that in the first wave of spermatogenesis primarily mid-pachytene spermatocytes die by apoptosis (Jahnukainen et al. 2004). Cell death is, therefore, a normal process during spermatogenesis, limiting the number of germ cells to ensure that Sertoli cells can provide enough nutrients to nurture the continuous generation of germ cells (Lee et al. 2006). The Fas system has been shown to be involved in the regulation of germ cell apoptosis in the rat testis. Through binding to its ligand, FasL, Fas promotes activation of caspase-8, which proteolytically activates executive caspases that ultimately kill the cells. Another set of caspases, involved in the intrinsic apoptotic pathway, has recently been reported to be involved in spermatocyte apoptosis from the first round of spermatogenesis in the rat (Lizama et al. 2007). When apoptosis in pachytene spermatocytes occurs as a result of stimuli such as heat or hormone deprivation, it appears that only the intrinsic pathway is stimulated (Sinha Hikim et al. 2003). A caspase-independent mechanism triggered by calpain activation is another mechanism that has been suggested to be involved in germ cell apoptosis (Coureuil et al. 2006). Some members of the p53 family and high [Ca²⁺]_i also appear to be important regulators of apoptotic death in spermatogenic cells (Mishra et al. 2006, Petre-Lazar et al. 2007). However, the pathways leading to apoptosis of germ cells in the testes from the Rb heterozygous mice remain elusive.

Data from studies employing extrinsic modulators of Ca²⁺ homeostasis have suggested that the increment in [Ca²⁺]_i is a signal sufficient to produce apoptosis. Calpains are frequently activated in apoptosis models involving enhanced [Ca²⁺]_i. Furthermore, the modulated expression of calcium-binding proteins, such as calbindin D_28k (CB) and others, can confer protein protection against apoptotic stimuli (Mathiasen et al. 2002). CB is a protein that was originally discovered in chick intestine (Wasserman & Taylor 1966), where its expression is vitamin D dependent. CB expression is low in both mice and chick testes and is vitamin D independent and dependent respectively (Walters et al. 1991, Inpanbutr et al. 1996). Christakos & Liu (2004) showed that CB has a major role in different cell types in protecting against cell death by preventing calcium-mediated mitochondrial damage and the subsequent release of cytochrome c.

Using a mouse model with reduced fertility, we studied whether the intrinsic mechanism of apoptosis is involved in the partial arrest of spermatogenesis caused by the heterozygous Rb translocations. We also analysed the expression of CB as a protective mechanism against apoptosis.

	Results

Top Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
Rb heterozygous mice exhibit high apoptosis of germ cells
We used 5-month-old mice in our study since Merico et al. (2003) had previously shown that the spermatogenic process is more impaired in Rb heterozygous mice of that age than in 3- or 7-month-old animals. The testicular histology of CD1 and Mil II mice had a normal appearance while heterozygous CD1xMil II (hereafter also named Rb heterozygous) mice showed a large proportion of defective seminiferous tubules with atypical cellular associations and severe germ cell loss, confirming previous findings (Merico et al. 2003). In order to estimate apoptosis we employed the TUNEL assay, which is a marker of DNA fragmentation. Figure 1 shows that TUNEL-positive cells were scarce in the seminiferous tubules from CD1 or Mil II mice, while TUNEL staining was strong in seminiferous tubules from Rb heterozygous mice. The percentage of TUNEL-positive tubule cross sections was significantly higher in the testes of Rb heterozygous mice than in those of either CD1 or Mil II animals (Table 1). Most of the tubules showing apoptotic cells were at stage XII, involving germ cells in both metaphases I and II. The mean number of TUNEL-positive cells/tubule section was seven- to nine-fold higher in Rb heterozygous mice than in the Mil II or CD1 mice, indicating that massive cell death of germ cells occurred by apoptosis in the Rb heterozygotes. No significant differences were found in either the percentage of TUNEL-positive tubule cross sections or in the mean number of TUNEL-positive cells/tubule section between CD1 and Mil II mice, but these latter exhibited higher levels of TUNEL-positive tubules (Table 1).

View larger version (47K): [in this window] [in a new window]   Figure 1 TUNEL labelling in seminiferous tubules from (A) CD1, (B) Mil II and (C) CD1xMil II mice. (B) shows a section of Mil II seminiferous tubule in which the highest number of TUNEL-positive metaphase spermatocytes was detected. The arrows indicate metaphase spermatocytes from tubules at stage XII. Bar=50 µm.

View larger version (83K): [in this window] [in a new window]   Figure 2 Electron microscopy pictures of male germ cells from (A) CD1 and (B) CD1xMil II mice. The arrow in (A) indicates sparse mitochondria within a spermatocyte and the arrow in (B) indicates a conglomeration of mitochondria close to the nucleus of a spermatocyte. The asterisk indicates a defective acrosome in a spermatid. Bar=5 µm.

View larger version (137K): [in this window] [in a new window]   Figure 3 Seminiferous tubules at stage XII from CD1xMil II mice showing (A) Bax, (B) cytochrome c, (C) calbindin D28k and (D) TUNEL staining. Most positive cells are metaphase spermatocytes (arrows) as shown in the insets. The asterisk indicates co-localisation of calbindin and TUNEL labelling of a metaphase spermatocyte. Bar=50 µm.

View larger version (58K): [in this window] [in a new window]   Figure 4 Representative Western blots obtained using anti-Bax, anti-cytochrome c and anti-calbindin D28k antibodies on whole homogenates (10 µg protein) of testis from CD1, Mil II and CD1xMil II mice. Anti-actin antibody was used to detect the housekeeping protein. The molecular weights were calculated by comparison with molecular weight markers run simultaneously with the protein samples. Calbindin D28k presents two bands, one of 28 kDa (monomer) and the other of 56 kDa (dimer).

	Discussion

Top Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
The purpose of this study was to determine whether the intrinsic mechanism of apoptosis is involved in the death of germ cells in the testis of Rb heterozygous mice. Our findings clearly show that testis from Rb heterozygous mice exhibits intense apoptosis of germ cells, which is accompanied by the redistributions of Bax and cytochrome c. The expression of these proteins, as revealed by Western blotting, was similar to that in CD1 or Mil II mouse testis, indicating that the synthesis of these two pro-apoptotic molecules in Rb heterozygous mice is not greater than that in the other two groups of animals. Therefore, Bax redistribution might be responsible for the release of cytochrome c from the mitochondria, which, through several steps, would finally promote DNA fragmentation. It has been shown that the insertion of Bax into mitochondrial membranes is involved in the release of cytochrome c from the mitochondrial intermembrane space into the cytosol in heat-induced testicular germ cell death (Vera et al. 2004) and in the apoptotic process of the first round of rat spermatogenesis (Lizama et al. 2007). Moreover, the relocation of mitochondria to the paranuclear area in spermatocytes from Rb heterozygous mice is another indication that these organelles are involved in the apoptosis of germ cells, which has been also demonstrated in heat-induced germ cell death (Vera et al. 2004). Thus, our study shows that the intrinsic apoptotic pathway is involved in the death of testicular germ cells triggered by karyotype rearrangements. Other apoptotic mechanisms were not explored, but cannot be excluded. It has been reported that Fas/FasL is involved in spermatogenic cell death during development, adulthood and after toxic exposure (Mishra et al. 2006). Calpain and the p53 subfamily have also been reported to be involved in male germ cell death (Coureuil et al. 2006, Petre-Lazar et al. 2007).

Most of the DNA fragmentation in Rb heterozygous testis was observed in tubules at stage XII, during which spermatocytes undergo meiotic divisions, in agreement with the previous data showing that the frequency of cell death is higher in this stage. Although apoptosis of male germ cells was significantly higher in Rb heterozygous mice than in CD1 or Mil II mice, most DNA fragmentation occurred mainly in metaphase spermatocytes in all three groups of animals. Eaker et al. (2001) have also reported increased apoptosis in metaphase spermatocytes with misaligned chromosomes in other Rb heterozygous mice (2n=36), suggesting that a spindle checkpoint mechanism identifies aberrant meiosis. Apoptotic metaphase spermatocytes at stage XII were also found in the testis of Mlh1^–/– mice (Eaker et al. 2002). Our study further supports the idea that in metaphase spermatocytes at stage XII a checkpoint mechanism is active, which might be activated in response to meiotic defects.

To maintain testicular homeostasis, pro-survival and pro-apoptotic molecules work together, regulating the extent of apoptosis to produce a supply of high-quality gametes (Mishra et al. 2006). As reported, the quality of male gametes is poor in Rb heterozygous mice, and these mice are subfertile (Redi et al. 1984, Hauffe & Searle 1998, Wallace et al. 2002). Therefore, some anti-apoptotic mechanisms in germ cells must be ‘turned on’ to limit apoptosis. Among the various apoptotic molecules, CB has been suggested to protect different cell types against apoptotic cell death induced by both calcium-independent and calcium-dependent pathways (Christakos & Liu 2004). In our study, we have demonstrated that CB is overexpressed in Rb heterozygous testis, with maximal expression in metaphase spermatocytes. CB overexpression occurs at stage XII, during which TUNEL labelling is also maximal. However, co-localisation of TUNEL labelling and CB is very limited. Sixty per cent of metaphase spermatocytes die by apoptosis and 40% of them exhibit high expression of CB, without signs of DNA apoptosis. Only 3% of metaphase spermatocytes show simultaneous apoptosis and CB expression. These data suggest that CB overexpression might protect against spermatogenic apoptosis. The concomitant presence of CB- or TUNEL-positive cells at stage XII in the same tubule section suggests a potential, immediate surveillance mechanism for preventing cell death, which has rapidly triggered the expression of CB.

Whether CB arrests mitochondrial apoptosis before the onset or during the apoptotic process remains unknown. Co-localisation of CB with molecules involved in the early steps of apoptosis, such as Bax or cytochrome c, could indicate that CB expression either might not be sufficient to block the already initiated apoptosis or might block apoptosis downstream of cytochrome c release. In fact, it has been found that CB is capable of directly inhibiting caspase-3 in MC3T3-E1 osteoblastic cells after treatment with tumour necrosis factor- (Bellido et al. 2000). In pancreatic β-cell lines, CB protects against cytokine-induced β-cell death by buffering calcium, preventing mitochondrial damage and inhibiting the generation of oxygen free radicals (Rabinovitch et al. 2001).

The CB overexpression not only occurs in metaphase spermatocytes but also in other germ cells and Sertoli cells. It is well known that Sertoli cells provide structural support, create an immunological barrier and nourish cells through secretory products (Yuan et al. 2006). Germ cell–Sertoli cell interactions during spermatogenesis have been described, but the molecules regulating these interactions have yet to be identified (Saunders 2003). Further studies are required to determine whether CB expressed in Sertoli cells acts as an anti-apoptotic molecule for these cells themselves or it is a secretory product to protect germ cells from apoptosis.

In summary, Rb heterozygous mice present intense apoptosis of germ cells mediated, at least in part, by a mitochondrial apoptotic mechanism. The redistributions of Bax and cytochrome c suggest that Bax moves to the mitochondria where it causes the release of cytochrome c, which might produce a cascade of events leading to DNA fragmentation. Apoptosis occurs at stage XII, mainly in metaphase spermatocytes. CB, an anti-apoptotic molecule, is also highly expressed at the same stage and in the same type of cell. We suggest that CB overexpression might be a mechanism to prevent or reduce spermatogenic apoptosis caused by Rb rearrangements. It is quite possible that the ability of CB to prevent and maybe rescue cells from apoptosis could explain the subfertility in these mice. The knowledge of specific mechanisms controlling germ cell apoptosis could have particular importance for understanding some aspects of male infertility.

	Materials and Methods

Top Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
Animals
We used adult male laboratory mice (Mus musculus domesticus, CD1 strain; weight: 40 g) carrying the standard chromosome complement of 2n=40 acrocentric chromosomes, wild-derived mice carrying 2n=24 chromosomes (M. m. domesticus, Milano II population, Mil II; Gropp et al. 1982, weight: 30 g) and heterozygous animals (2n=32; weight: 40 g) obtained by crossing CD1 female mice with Mil II males. The mice were maintained according to the Guide for Care and Use of Laboratory Animals. Three 5-month-old male mice of each karyotype group were killed by cervical dislocation. Every effort was made to minimise the number of animals used and their suffering.

Chemical reagents
All reagents were purchased from Sigma Chemical Co. unless otherwise stated.

Histology
The right testis of each mouse was fixed in Bouin's fluid and embedded in paraffin wax. Five micrometre serial transverse cross sections were made for the subsequent co-localisation analyses. At least four serial sections of each testis were mounted on each glass slide. One slide was stained by the periodic acid–Schiff reaction and counterstained with haematoxylin to identify the stages of seminiferous epithelium according to Oakberg (1956).

TUNEL assay
For histological evaluation of apoptosis, the sections were processed with the TUNEL method, using an ApopTag Plus Peroxidase In Situ Apoptosis Kit (Chemicon-Millipore, Billerica, MA, USA), according to the manufacturer's instructions. Sections were permeabilised by incubation with proteinase K (20 µg/ml) for 10 min, and then exposed for 5 min to 3% (v/v) H₂O₂ for endogenous peroxidase inhibition, incubated in the terminal deoxynucleotidyl transferase (TdT) labelling reaction mix for 1 h at 37 °C, washed in PBS, incubated with the anti-digoxigenin and finally developed with 3,3'-diaminobenzidine (DAB). Positive and negative controls were also set up. The positive controls were established using the slides contained in the same kit and following the manufacturer's instructions. For the negative controls, sections were processed without TdT enzyme in the labelling reaction mix. The sections were counterstained with 0.5% (w/v) methyl green for 10 min at room temperature.

Immunohistochemical analysis
Serial sections were processed according to the avidin–biotin–peroxidase complex method. Following deparaffinisation, sections were hydrated and incubated for 10 min in 0.5% (v/v) H₂O₂ diluted in methanol to reduce endogenous peroxidase activity. After having been rinsed in PBS, the slides were incubated with normal bovine serum at 10% (v/v) in PBS for 10 min to prevent non-specific binding of the primary antibody. Thereafter, the primary antibodies mouse anti-bovine CB monoclonal antibody (clone CB-955; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-mouse Bax (P-19, polyclonal antibody, Santa Cruz Biotechnology) and purified mouse anti-mouse cytochrome c monoclonal antibody (BD Pharmingen, Franklin Lakes, NJ, USA) were applied at a dilution of 1:1000 in PBS at 37 °C for 1 h. Later, the sections were washed thrice in PBS and then incubated with appropriate secondary biotinylated antibodies diluted in PBS. After 30 min, the sections were incubated with peroxidase-conjugated streptavidin (Molecular Probes, Carlsbad, CA, USA) and developed with DAB (Dako, Glostrup, Denmark). The control sections were incubated using the same procedure but only with the secondary antibodies. Cytochrome c and Bax immunoreacted sections were counterstained with haematoxylin. Sections were visualised using a Leica DM LB microscope and images were obtained with a Leica DC 180 Camera.

Immunoblot analysis
Western blot analysis was performed to detect the expression of CB, cytochrome c and Bax proteins. The left testis of each mouse, freed from the albuginea membrane, was homogenised in PBS. The total protein suspension of each testis was centrifuged for 10 min at 13 000 g. The protein concentration of supernatants was measured using Bradford's method. Supernatants (10 µg/sample) were mixed with loading buffer (2% w/v SDS in Tris–HCl (pH 8) containing 25% v/v glycerol, 1% v/v β-mercaptoethanol and 0.1% v/v bromophenol blue). The mixture was denatured for 5 min at 95 °C and aliquots of 15 µl homogenate were separated in 12.5% w/v SDS-polyacrylamide minigels. Gels containing the separated proteins were immersed in the transfer buffer (25 mM Tris–HCl, 192 mM glycine, 0.05% w/v SDS and 20% v/v methanol). Nitrocellulose membranes (0.2 µm) were blocked for 2 h with 3% w/v skimmed milk in PBT (1xPBS plus 0.05% v/v Tween-20) and incubated for 1 h at 37 °C with the primary antibodies at 1:1000 dilution (CB, cytochrome c), 1:5000 dilution (purified goat anti-human actin polyclonal antibody, Santa Cruz Biotechnology) or 1:500 dilution (Bax) in PBT with 1% w/v BSA. After extensive washing with PBT, the membranes were incubated with anti-rabbit (Bax), anti-goat (actin) or anti-mouse (CB and cytochrome c) biotinylated antibodies for 1 h at 37 °C using a 1:2000 dilution in PBT with 1% w/v BSA and then washed thoroughly. The appropriate Western Blot Amplification Module (Bio-Rad) and the Opti-4CN Substrate Kit (Bio-Rad) were used, according to the manufacturers' instructions, for detection. Actin was used as a marker to normalise the relative expression of the other proteins. The band intensity was quantified by the Image J software (Wayne Rasband, NIH, Bethesda, MD, USA) in order to obtain the relative expression of proteins.

Transmission electron microscopy
Small pieces of seminiferous tubules were fixed by immersion in a mixture of 1.5% (v/v) glutaraldehyde and 4% (v/v) formaldehyde in 0.1 M cacodylate buffer, and then treated with 1% w/v osmium tetroxide, dehydrated and embedded in Araldite at 60 °C. For ultrastructural studies, thin sections were cut with a diamond knife on a Porter-Blum MT2 and JEOL JUM-7 ultramicrotome, counterstained with lead citrate and uranyl acetate, and examined in a Zeiss LEO 906E electron microscope.

Statistical methods
Four sections were selected at random from each testis for both the immunohistochemical studies and the TUNEL assay. At least nine transverse sections of seminiferous tubules for each epitope, TUNEL labelling or their combinations were randomly chosen and pictures were taken at 400x magnitude by two independent researchers in a blinded fashion. A cross section of a tubule was considered apoptotic when three or more TUNEL-positive cells were present within the seminiferous epithelium (Eaker et al. 2001). For each animal testis, 100 cross-sectioned tubules were scored to evaluate the frequency of the immunopositive or apoptotic sections. The percentage of positive cells for each epitope or TUNEL labelling was calculated taking into account the total number of cells per tubule section at the same stage of differentiation. Abercrombie's (1946) correction was applied to all cell counts. Two serial sections were processed with two different antibodies (CB and Bax, CB and cytochrome c) or with an antibody and TUNEL assay (CB and TUNEL) in order to compare and analyse the co-localisation of the different markers. Statistical significance between means of the different groups was assessed by the one-way ANOVA, followed by Bonferroni's post hoc test. A P value <0.05 was considered statistically significant.

	Acknowledgements

Top Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
This work was supported by a Bilateral Grant 2005–2007 from MAE-SECYT (Italia–Argentina), funds from the Olympus Europa Foundation, Millipore France and Ministero dell'Università e della Ricerca FIRB 2005 (Dr Silvia Garagna) and from FONCYT (PICT 2005), CONICET (PIP (2005–2006) and SECYT (UNC 2006–2007) (Dr Nori Tolosa de Talamoni). Dr Nori Tolosa de Talamoni is a Member of Career from CONICET (Argentina). Valeria Rodriguez is a recipient of a CONICET fellowship. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

Received 18 October 2007
First decision 8 February 2008
Revised manuscript received 2 January 2008
Accepted 25 February 2008

	References

Top Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 

Abercrombie M 1946 Estimation of nuclear population from microtome sections. Anatamoical Record 94 239–247.[CrossRef]

Allan DJ, Harmon BV & Kerr JFR 1987 Cell death in spermatogenesisCS PottenIn Perspectives on Mammalian Cell Death Oxford: Oxford University Press: 229–258.

Bellido T, Huening M, Raval-Pandya M, Manolagas SC & Christakos S 2000 Calbindin-D_28k is expressed in osteoblastic cells and suppresses their apoptosis by inhibiting caspase-3 activity. Journal of Biological Chemistry 275 26328–26332.[Abstract/Free Full Text]

Christakos S & Liu Y 2004 Biological actions and mechanism of action of calbindin in the process of apoptosis. Journal of Steroid Biochemistry and Molecular Biology 89–90 401–404.

Coureuil M, Fouchet P, Prat M, Letallec B, Barroca V, Dos Santos C, Racine C & Allemand I 2006 Caspase-independent death of meiotic and postmeiotic cells overexpressing p53: calpain involvement. Cell Death and Differentiation 13 1927–1937.[CrossRef][Web of Science][Medline]

Eaker S, Pyle A, Cobb J & Handel MA 2001 Evidence for meiotic spindle checkpoint from analysis of spermatocytes from Robertsonian-chromosome heterozygous mice. Journal of Cell Science 114 2953–2965.[Web of Science][Medline]

Eaker S, Cobb J, Pyle A & Handel MA 2002 Meiotic prophase abnormalities and metaphase cell death in MLH1-deficient mouse spermatocytes: insights into regulation of spermatogenic progress. Developmental Biology 249 85–95.[CrossRef][Web of Science][Medline]

Eguchi J, Koji T, Nomata K, Yoshii A, Shin M & Kanetake H 2002 Fas–Fas ligand system as a possible mediator of spermatogenic cell apoptosis in human maturation-arrested testes. Human Cell 15 61–68.[Medline]

Francavilla S, D'Abrizio P, Rucci N, Silvano G, Properzi G, Straface E, Cordeschi G, Necozione S, Gnessi L, Arizzi M et al. 2000 Fas and Fas ligand expression in fetal and adult human testis with normal or deranged spermatogenesis. Journal of Clinical Endocrinology and Metabolism 85 2692–2700.[Abstract/Free Full Text]

Francavilla S, D'Abrizio P, Cordeschi G, Pelliccione F, Necozione S, Ulisse S, Properzi G & Francavilla F 2002 Fas expression correlates with human germ cell degeneration in meiotic and post-meiotic arrest of spermatogenesis. Molecular Human Reproduction 8 213–220.[Abstract/Free Full Text]

Gropp A, Winking H, Redi C, Capanna E, Britton-Davidian J & Noack G 1982 Robertsonian karyotype variation in wild house mice from Rhaeto-Lombardia. Cytogenetics and Cell Genetics 34 67–77.[Web of Science][Medline]

Hauffe HC & Searle JB 1998 Chromosomal heterozygosity and fertility in house mice (Mus musculus domesticus) from Northern Italy. Genetics 150 1143–1154.[Abstract/Free Full Text]

Inpanbutr N, Reiswig JD, Bacon WL, Slemons RD & Iacopino AM 1996 Effect of vitamin D on testicular CaBP28K expression and serum testosterone in chickens. Biology of Reproduction 54 242–248.[Abstract]

Jahnukainen K, Crysis D, Hou M, Parvinen M, Eksborg S & Soder O 2004 Increased apoptosis occurring during the first wave of spermatogenesis is stage-specific and primarily affects midpachytene spermatocytes in the rat testis. Biology of Reproduction 70 290–296.[Abstract/Free Full Text]

Lee NPY, Leung KW, Wo JY, Tam PC, Yeung WSB & Luk JM 2006 Blockage of testicular connexins induced apoptosis in rat seminiferous epithelium. Apoptosis 11 1215–1229.[CrossRef][Web of Science][Medline]

Lizama C, Alfaro I, Reyes JG & Moreno RD 2007 Up-regulation of CD95 (Apo-1/Fas) is associated with spermatocyte apoptosis during the first round of spermatogenesis in the rat. Apoptosis 12 499–512.[CrossRef][Web of Science][Medline]

Luu KC, Nie GY, Hampton A, Fu GQ, Liu YX & Salamonsen LA 2004 Endometrial expression of calbindin (CaBP)-d28k but not CaBP-d9k in primates implies evolutionary changes and functional redundancy of calbindins at implantation. Reproduction 128 433–441.[Abstract/Free Full Text]

Mathiasen IS, Sergeev IN, Bastholm L, Elling F, Norman AW & Jäätelä M 2002 Calcium and calpain as key mediators of apoptosis-like death induced by vitamin D compounds in breast cancer cells. Journal of Biological Chemistry 277 30738–30745.[Abstract/Free Full Text]

Merico V, Pigozzi MI, Esposito A, Merani MS & Garagna S 2003 Meiotic recombination and spermatogenic impairment in Mus musculus domesticus carrying multiple simple Robertsonian translocations. Cytogenetic and Genome Research 103 321–329.[CrossRef][Web of Science][Medline]

Mishra DP, Pal R & Shaha C 2006 Changes in cytosolic Ca²⁺ levels regulate Bcl2-xS and Bcl2-xL expression in spermatogenetic cells during apoptotic death. Journal of Biological Chemistry 281 2133–2143.[Abstract/Free Full Text]

Oakberg EF 1956 A description of spermatogenesis in the mouse and its use in analysis of the cycle of the seminiferous epithelium and germ cell renewal. American Journal of Anatomy 99 391–413.[CrossRef][Web of Science][Medline]

Petre-Lazar B, Livera G, Moreno SG, Trautmann E, Duquenne C, Hanoux V, Habert R & Coffigny H 2007 The role of p63 in germ cell apoptosis in the developing testis. Journal of Cell Physiology 210 87–98.[CrossRef][Web of Science][Medline]

Pochet R, Blachier F, Malaisse W, Parmentier M, Pasteels B, Pohl V, Resibois A, Rogers J & Roman A 1989 Calbindin-D28 in mammalian brain, retina, and endocrine pancreas: immunohistochemical comparison with calretinin. Advances in Experimental Medicine and Biology 255 435–443.[Medline]

Rabinovitch A, Suarez-Pinzon WL, Sooy K, Strynadka K & Christakos S 2001 Expression of calbindin-D(28k) in a pancreatic islet beta-cell line protects against cytokine-induced apoptosis and necrosis. Endocrinology 142 3649–3655.[Abstract/Free Full Text]

Redi CA, Garagna S, Pellicciari C, Manfredi Romanini MG, Capanna E, Winking H & Gropp A 1984 Spermatozoa of chromosomally heterozygous mice and their fate in male and female genital tracts. Gamete Research 9 273–286.[CrossRef][Web of Science]

Redi CA, Garagna S, Hilscher B & Winking H 1985 The effects of some Robertsonian chromosome combinations on the seminiferous epithelium of the mouse. Journal of Embryology and Experimental Morphology 85 1–19.[Web of Science][Medline]

Russell LD 1999 Cell loss during spermatogenesisS Hammamah, R Mieusset, F Olivennes & R FrydmanIn Male Sterility and Motility Disorders New York: Springer: 103–124.

Russell LD, Chiarini-Garcia H, Korsmeyer SJ & Knudson CM 2002 Bax-dependent spermatogonia apoptosis is required for testicular development and spermatogenesis. Biology of Reproduction 66 950–958.[Abstract/Free Full Text]

Saunders PTK 2003 Germ cell–somatic cell interactions during spermatogenesis. Reproduction S61 91–101.[Medline]

Sinha Hikim AP, Lue Y, Diaz-Romero M, Yen PH, Wang C & Swerdloff RS 2003 Deciphering the pathways of germ cell apoptosis in the testis. Journal of Steroid Biochemistry and Molecular Biology 85 175–182.[CrossRef][Web of Science][Medline]

Vera Y, Diaz-Romero M, Rodriguez S, Lue Y, Wang C, Swerdloff R & Sinha Hikim AP 2004 Mitochondria-dependent pathway is involved in heat-induced male germ cell death: lessons from mutant mice. Biology of Reproduction 70 1534–1540.[Abstract/Free Full Text]

Wallace BM, Searle JB & Everett CA 2002 The effect of multiple simple Robertsonian heterozygosity on chromosome pairing and fertility of wild-stock house mice (Mus musculus domesticus). Cytogenetic and Genome Research 96 276–286.[CrossRef][Web of Science][Medline]

Walters MR, Bruns ME, Carter RM & Riggle PC 1991 Vitamin D independence of small calcium-binding proteins in nonclassical target tissues. American Journal of Physiology 260 794–800.

Wasserman RH & Taylor AN 1966 Vitamin D₃-induced calcium-binding protein in chick intestinal mucosa. Science 152 791–793.[Abstract/Free Full Text]

Yuan W, Leisner TM, McFadden AW, Clark S, Hiller S, Maeda N, O'Brien DA & Parise LV 2006 CIB1 is essential for mouse spermatogenesis. Molecular and Cellular Biology 26 8507–8514.[Abstract/Free Full Text]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Merico, V. Articles by de Talamoni, N. T. Search for Related Content PubMed PubMed Citation Articles by Merico, V. Articles by de Talamoni, N. T.