Simultaneous Accumulation of Hyaluronan Binding Protein 1 (HABP1 ...

Journal of Andrology, Vol. 33, No. 1, January/February 2012 Copyright E American Society of Andrology

Simultaneous Accumulation of Hyaluronan Binding Protein 1 (HABP1/p32/gC1qR) and Apoptotic Induction of Germ Cells in Cryptorchid Testis VINOD KUMAR,* MAN MOHAN MISRO,{ AND KASTURI DATTA* From the *Biochemistry Laboratory, School of Environmental Sciences, Jawaharlal Nehru University, New Delhi, India; and the ÀDepartment of Reproductive Biomedicine, National Institute of Health and Family Welfare, Baba Gang Nath Marg, New Delhi, India

ABSTRACT: In the experimentally cryptorchid rat, spermatogenic arrest is associated with the formation of multinuclear giant cells, leading to large-scale apoptosis and elimination of germ cells from the seminiferous epithelium. Using this model, the role of Hyaluronan Binding Protein 1 (HABP1), which expresses a stage specifically in post-meiotic cells during spermatogenesis, was examined. Cryptorchidism induced complete arrest of spermatogenesis by 2 days, and by 3–5 days many large and small multinucleated giant cells populated the affected tubules. Ultrastructure of the giant cells revealed both single and multiple chromatin aggregation, with some less compact and distorted, and others broken down into tiny

fragments. These cells along with other germ cells were stained terminal deoxynucleotidyl transferase dUTP nick-end labeling positive, demonstrating strong expression of Bax and Heat Shock Protein 70. Simultaneously, there was an up-regulation of the proprotein form of HABP1 in these cells and a decrease in the mature form of protein. The above findings indicate a possible role for HABP1 proprotein in apoptosis induction of germ cells in the cryptorchid testes. Key words: Cryptorchidism, spermatogenic arrest, giant cells, germ cell apoptosis, HABP 1. J Androl 2012;33:114–121

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recently reported multifunctional protein, annexin A5 (Yao et al, 2009). On the other hand, a large number of inducible heat shock factors have been reported to counter the apoptotic affect of cryptorchidism. Expression of Heat Shock Proteins (hsp) 60, 70, and 90 have been reported during different developmental and degenerative changes in rat testis (Ogi et al, 1999). However, there may be other factors associated with germ cell apoptosis that are yet to be identified. For the last few years, our laboratory has been working on a novel protein, Hyaluronan Binding Protein 1 (HABP1), a member of the hyaladherin family, which has been shown to have specific affinity toward hyaluronan (Gupta et al, 1991). Sequence analysis of cDNA encoding HABP1 (Deb and Datta, 1996) suggests its multifunctional nature, because it is identical to p32, a protein copurified with splicing factor SF2 (Krainer et al, 1991) and receptor for globular head component factor C1q (Ghebrehiwet et al, 1994). Its localization is confirmed on human chromosome 17p12–13 (Majumdar and Datta, 1998), with a synonym HABP1/p32/gC1qR. HABP1 is synthesized as a 272amino acid–long, highly labile proprotein, which was purified to homogeneity as confirmed in two-dimensional gel analysis (Deb and Datta, 1996). It is posttranslationally and prototypically cleaved of 73 Nterminal amino acids to give its mature form of 34 kd.

uring fetal development, mammalian testes descend down into the extra-abdominal scrotal sac, where temperature is conducive for spermatogenesis. Failure of the process leads to cryptorchidism, a congenital abnormality. Higher abdominal temperature during cryptorchidism affects the normal functioning of the testis with the formation of multinucleated giant cells, which facilitates large-scale removal of germ cells from the seminiferous epithelium. It also induces spermatogenic arrest associated with an increase in germ cell apoptosis (Chaki et al, 2005). Germ cell apoptosis is induced by multiple factors, such as oxidative stress (Peltola et al, 1995; Makker et al, 2009), heat stress (Yin et al, 2002), differential response to gonadotropins (Iizuka et al, 1996), and mechanisms that include activation of nuclear factor kappa B (Mizuno et al, 2009) and testicular expression of a Supported by the Indian Council of Medical Research, Government of India. Financial support was given to V.K. in the form of fellowship from the University Grants Commission, Government of India, New Delhi. Correspondence to: Kasturi Datta, 103 Biochemistry Laboratory, School of Environmental Sciences, Jawaharlal Nehru University, New Delhi 110067, India (e-mail: [email protected] or datta_k@ hotmail.com). Received for publication July 20, 2010; accepted for publication February 4, 2011. DOI: 10.2164/jandrol.110.011320

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HABP1 Expression and Germ Cell Apoptosis

The mature form of HABP1 was shown to have a significant role in sperm (rat, mouse, and human) function (Ranganathan et al, 1994) and water buffalo sperm oocyte interaction (Ghosh and Datta, 2003). The proprotein form of HABP1 is found to be present only in testis but not in any other somatic tissue, like spleen or liver. Further, it is expressed specifically in pachytene and round spermatids, compared with the mature form present in epididymal and ejaculated sperm. The above data suggest a stage-specific expression of the proprotein form of HABP1 in testicular germ cells (Bharadwaj et al, 2002). During normal spermatogenesis only one transcript of HABP1 mRNA was detected. The testisspecific proprotein form was found to be immunologically identical with the proprotein form of HABP1 using antibody raised against a decapeptide present in the proprotein region of HABP1. The HABP1 expression is also developmentally regulated and absent in immature testis (Thakur et al, 2006). The reduced expression of mature HABP1 in sperm from infertile patients suggests its possible role in the regulation of sperm function (Ghosh et al, 2001). To identify its functional characterization, constitutive expression of HABP1 in fibroblasts demonstrates growth inhibition and apoptosis induction (Meenakshi et al, 2003). When overexpressed, it accumulates in mitochondria, leading to reactive oxygen species generation, formation of apoptosome, and caspase activation (Chowdhury et al, 2008). A significant increase in the expression level of HABP1 is also detected in HeLa cells treated with cisplatin, a well-established apoptosis inducer. Apoptosis among cisplatin-treated HeLa cells is significantly decreased if expression of HABP1 is knocked down using short interfering RNA specific to HABP1 (Kamal and Datta, 2006), suggesting its possible role in apoptosis induction. Keeping in view the involvement of HABP1 in a stage-specific manner in spermatogenesis, the present study was initiated to find out the role of HABP1 in apoptosis induction of germ cells in the cryptorchid rat testis.

Materials and Methods Chemicals All chemicals were obtained from Sigma Chemical Co (St Louis, Missouri). Resource-Q column and molecular weight markers were obtained from Pharmacia Biotech Inc (Uppsala, Sweden). Chemicals for reverse transcription–polymerase chain reaction (RT-PCR) were purchased from PerkinElmer (Waltham, Massachusetts). M-MuLV-RT was purchased from Promega Corp (Madison, Wisconsin). Fast Red substrate system was purchased from Dako (Glostrup, Denmark).

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Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay kit was purchased from R&D Systems (Minneapolis, Minnesota). All antibodies used were purchased from Santa Cruz Biotechnology (Santa Cruz, California), with the exception of anti-rabbit and anti-mouse alkaline phosphatase conjugate, procured from Sigma.

Animals and Treatment Wistar rats and New Zealand white rabbits were maintained in the animal house of Jawaharlal Nehru University, New Delhi. The animals were fed ad libitum standard feed purchased from Hindustan Lever Ltd (New Delhi, India). To induce experimental cryptorchidism, adult Wistar rats weighing 200–220 g were anesthetized, and a small incision was made in the left lower quadrant of the abdomen. Processus vaginalis was freed from the surrounding scrotum, and testis was pushed to abdomen. Processus vaginalis was tucked back into itself and sutured at the level of the internal inguinal ring (Watts et al, 2000). Abdominal cut was ligated, and Neosporin powder was sprayed over the cut to avoid infection. Sham-operated animals were used as control. Animals were kept in separate cages for recovery. Animal experiments were carried out under strict compliance with the guidelines for laboratory animal facilities prepared by the Committee for the Purpose of Control and Supervision on Experiments on Animals, India, and the Institutional Guidelines for Animal Care.

Tissue and Lysate Preparation Rats were killed with CO2 asphyxiation. For immunohistochemical studies, testes were fixed by immersion in Bouin fixative for 16–18 hours and then washed immediately in 70% alcohol to get rid of excess fixative. After thorough washing, the tissues were processed further by dehydration through graded alcohol, as well as xylene, and were finally embedded in paraffin wax. Sections (5 mm) of testis were cut using a microtome. For sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, the testis lysate was prepared in Laemmli buffer containing b-mercaptoethanol (Laemmli, 1970). The lysate was centrifuged at 12 000 6 g for 15 minutes at room temperature, and the supernatant containing the lysate proteins was stored at 220uC until use. Protein was estimated by Bradford assay.

Purification of HABP1 and Generation of Antibodies HABP1 was purified to homogeneity using a Resource Q (6 mL) column (Amersham Bioscience, Uppsala, Sweden), interfaced with AKTA Basic10 system (Amersham Bioscience; Jha et al, 2002), followed by hyaluronan-Sepharose affinity column chromatography as reported previously (Deb and Datta 1996). Polyclonal anti-rHABPl antibodies were raised against the purified recombinant HABP1 (rHABPl) in rabbit as previously described (Deb and Datta, 1996).

Tissue Preparation for Electron Microscopic Study Seminiferous tubules from normal and cryptorchid testes were collected after removal of tunica albuginea, and individual tubules were separated. For fixation, some tubules were kept in

116 phosphate-buffered saline (PBS) containing 5% glutaraldehyde for 6 hours. Vacuum suctioning was done to ensure penetration of fixative into the tubules. The tissue was then embedded in epoxy resin, and 60-nm sections were cut with the help of an ultramicrotome; they were subsequently stained with uranyl acetate and lead citrate. Sections were examined under Morgagni 268D Transmission Electron Microscope (FEI, Eindhoven, The Netherlands).

SDS-PAGE and Western Blot Analysis Linear slab gel electrophoresis under a denaturing condition (in the presence of 0.1% SDS) was performed according to the method of Laemmli. Protein (60 mg per well) was loaded in each well and electrophoresed along with the standard molecular weight marker. b-Actin was used as an internal control. The proteins in the gel were transferred onto the nitrocellulose membrane by applying a 0.8 mA/h current in a wet transfer unit (Bio-Rad, Hercules, California) and were immunodetected with specific antibodies of anti-rHABP1 (1:1000, raised in the laboratory), anti-hsp70 (0.2 mg/mL), and anti-Bax (0.2 mg/mL), and visualized with nitro blue tetrazolium 5-bromo-4-chloro-3-indolyl phosphate using antirabbit or anti-mouse secondary antibody (0.02 mg/mL) conjugated to alkaline phosphatase.

Immunohistochemistry Testis sections were deparaffinized and rehydrated, and the nonspecific antibody binding was blocked with 3% (wt/vol) bovine serum albumin in 10 mM PBS for 1 hour at room temperature. After 5 washes of 5 minutes each in PBS, the slides were incubated with primary antibodies, 1:100 dilution of anti-rHABP1 (which was raised in the laboratory) and antiBax, or anti-hsp70 at 2 mg/mL each for 1 hour in a humid chamber. Sections were washed (5 times for 5 minutes each in PBS) and incubated with secondary antibody (goat anti-rabbit immunoglobulin G [IgG]; 0.2 mg/mL) conjugated to alkaline phosphatase (AP) or goat anti-mouse IgG conjugated to AP for 1 hour at room temperature. Fast Red substrate was used to develop color. After the desired color intensity, the sections were washed in PBS, counterstained in hematoxylin, and mounted in glycerol. At no point during the whole procedure were the sections allowed to remain dry.

RNA Isolation and Semiquantitative RT-PCR Analysis Total RNA was isolated from the testes using TRIZOL reagent (Invitrogen Life Technologies, Carlsbad, California) according to the manufacturer’s instructions. Single-step RTPCR was performed on 2 mg of total RNA from normal and cryptorchid testes with the help of rTth enzyme (PE Applied Biosystems, Branchburg, New Jersey). The forward HABP1 primer 59-GAAGGCCCTTGTGTTGGACTGTCA-39 and the reverse HABP1 primer 59-GTATCAAAGTACCGTCCGAAACC-39 were used. The RT-PCR was performed for 25 cycles to ensure linear amplification. b-Actin was simultaneously amplified as an internal control using forward primer 59-CGAGCACGGCATCGTCACCAAC-39 and reverse primer 59-GTGGATGCCACAGGACTCCATGCC-39.

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TUNEL Assay In situ apoptosis detection kit (R&D Systems) was used to detect DNA fragmentation of apoptotic germ cells in deparaffinized testis sections. To make the DNA accessible to the labeling enzyme, the cell membranes were permeabilized with proteinase K. Endogenous peroxidase activity was quenched using hydrogen peroxide, and biotinylated nucleotides were incorporated into the 3-OH ends of the DNA fragments by terminal deoxynucleotidyl transferase. The biotinylated nucleotides were detected by using streptavidin–horseradish peroxidase conjugate followed by the substrate diaminobenzidine (DAB). The enzyme reaction generates an insoluble colored precipitate where DNA fragmentation has occurred. DAB-stained samples were counterstained with methylene green, mounted with DPX, and examined using a Nikon Eclipse E600 microscope image analyzer (Nikon, Tokyo, Japan) and photographed.

Statistical Analysis Each figure (for Western blotting and RT-PCR) is a representative of 3 independent experiments with similar results. Statistical analysis was performed where appropriate using Student’s t test. Results were considered statistically significant at P , .05.

Results Arrest of Spermatogenesis and Formation of Giant Cells Arrest of spermatogenesis, shrinkage of seminiferous tubule diameter, and widening of intertubular space was observed starting from day 2 of cryptorchidism (Figure 1b) compared with untreated control testis (Figure 1a). Number of germ cells per tubule declined drastically by day 3 (Figure 1c) with the appearance of multinuclear giant cells, which grew in number and attained a maximum by day 5 (Figure 1d). Higherresolution studies of giant cells (Figure 1e) showed a mass of aggregated nuclear chromatin with intact or disintegrated nuclear envelope.

Increased Expression of hsp70 Immunopositive staining of hsp70 was intense and found to be restricted to the germ cells of day 3 (Figure 2if) or day 5 (Figure 2ih) cryptorchid seminiferous epithelium. No germ cell was found to be immunopositive for the protein, either in the contralateral control testis (Figure 2ib) or in the testis after 2 days (Figure 2id) of cryptorchidism. Figure 2ia, c, e, and g represent preimmune serum-treated controls for normal and cryptorchid testes of 2, 3, and 5 days, respectively. Expression of hsp70 could not be probed by Western blotting in normal or day 2 cryptorchid testis. However, it was up-regulated by day 3 (Figure 2ii, lane 4) and day 5 (Figure 2ii, lane 5) of cryptorchidism, which was

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Figure 1. (a, b, c, d) Representative histologic sections of rat testes stained with hematoxylin and eosin. Normal spermatogenesis is seen in the control testis (a). Arrest of spermatogenesis in day 2 (b) and formation of giant (gnt) cells in days 3 (c) and 5 (d) cryptorchid testes are seen. Original magnification 6400. (e) Ultrastructure of a gnt cell reveals aggregation of germ cells with intact (im) or disintegrated (dm) chromatin content and nuclear envelope. Original magnification 63000. Color figure available online at www.andrologyjournal.org.

identical to the immunolocalization of the protein in testicular sections.

Evidence of the Apoptotic Nature of Giant Cells When testicular sections were immunostained using antibody to Bax, an apoptotic marker protein, positive and intense staining was observed in most of the germ cells in the day 5 cryptorchid testis (Figure 3if), which also included the multinucleated giant cells. Although no staining was visible in germ cells of control testis (Figure 3ib), moderate staining of Bax was resolved in 3-day cryptorchid testis (Figure 3id). Figure 3ia, c, and e represent the corresponding negative controls for normal and cryptorchid testis for 3 days and 5 days, respectively. In the Western blots, expression of Bax could not be resolved in testicular lysates of 2-day cryptorchid (Figure 3ii, lane 1) or normal (Figure 3ii, lane 2) testis. However, the expression was very much up-regulated in the lysates of day 3 (Figure 3ii, lane 3) and day 5 (Figure 3ii, lane 5) cryptorchid testis compared with contralateral testis of respective (Figure 3ii, lanes 4 and 6, respectively) groups. TUNEL-positive cells were found to be prevalent on day 2 (Figure 3iiib) and day 3 (Figure 3iiic) of cryptorchidism compared with control testis (Figure 3iiia). Multinuclear giant cells were also observed to be TUNEL positive (Figure 3iiid).

Up-Regulation of HABP1 Expression Cell-specific distribution of HABP1 was found mostly localized in the germ cells and multinucleated giant

cells in the adluminal area of the seminiferous epithelium after 3 days (Figure 4id) and 5 days (Figure 4if) of cryptorchidism. On the other hand, in control testis expression of HABP1 was extended to all of the cells in the periluminal and adluminal area of the seminiferous epithelium, including pachytene spermatocytes, elongating spermatids, and sperm (Figure 4ib). The immunostaining, however, was mostly concentrated among the tubular cells surrounding the periluminal area (Figure 4ib). Sham-operated animals depict immunoexpression of testicular HABP1 identical to that of controls (data not shown). Negative controls for normal (Figure 4ia) and cryptorchid testis 2 days (Figure 4ic) and 5 days (Figure 4ie) are shown. When testicular lysates were immunoprobed with antirHABP1 antibody, the mature form (34 kd) and the precursor form (55 kd) of HABP1 were detected in the control testis (Figure 4ii, lane 2). Following 2 days of cryptorchidism, the mature form of the protein declined (Figure 4ii, lane 3), and in subsequent days (3 and 5) it failed to be probed in the gel (Figure 4ii, lanes 5 and 4, respectively). During the same period, the precursor form of the protein demonstrated an upregulated expression, more in day 5 (2.9-fold vs control) than in day 3 (1.4-fold vs control) cryptorchidism. This increase in expression was further confirmed by the RT-PCR data, which showed 1.5-fold to 3.2-fold increases (densitometry) in transcript levels of HABP1 in 3 days and 5 days of cryptorchidism (Figure 4iii), respectively.

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Figure 2. (i) Representative sections showing immunoexpression of heat shock protein (hsp) 70 in normal and cryptorchid rat testes. Giant (gnt) cells and germ (gc) cells in 3-day (f) and 5-day (h) cryptorchid testis demonstrated strong immunoexpression. No immunopositivity of cells is seen in normal (a) and 2-day cryptorchid (d) testis. Negative controls (a, c, e, g) represent normal, and 2-day, 3-day, and 5-day cryptorchid testis, respectively. Immunohistochemical stain of normal testis (b) wherein no stain is observed for hsp70. Original magnification 6400. (ii) Up-regulated expression of hsp70 in day 5 (lane 5) and day 3 (lane 4) in Western blots coincides well with the immunopositivity of cells in testicular sections. The protein fails to be probed in normal (lane 2) and 2-day cryptorchid (lane 3) testis. Color figure available online at www. andrologyjournal.org.

Discussion The present findings establish that the proprotein form of HABP1 is overexpressed, replacing the mature form of protein specifically in the apoptotic and multinucleated giant cells, which are in fact aggregations of germ cells under elimination in the cryptorchid rat testis. The apoptotic transformation in these cells is confirmed by the identical rise in the expression of Bax and hsp70 following 3 and 5 days of cryptorchidism. Although loss of germ cells may occur during various hormonal (reviewed in Sofikitis et al, 2008) or pathologic (reviewed in Tung, 1995) conditions, elevated temperature in undescended testis is one of the main causes of germ cell

removal by apoptosis (Chaki et al, 2005). Heat-induced apoptosis is reported to involve both the mitochondriadependent and endoplasmic reticulum–dependent pathways of apoptosis. Initiation of apoptosis is preceded by a redistribution of Bax from a cytoplasmic to a paranuclear localization in heat-susceptible germ cells. Such relocation of Bax is further accompanied by sequestration of mitochondria and endoplasmic reticulum into paranuclear areas, cytosolic translocation of cytochrome c leading to activation of the initiator caspase 9 and the executioner caspases 3, 6, and 7, and cleavage of PARP (Hikim et al, 2003). The redistribution of Bax triggers apoptotic induction in germ cells (Damavandi et al, 2002; Du¨ndar et al, 2005). Bax immunopositivity of germ cells and giant

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Figure 3. (i) Representative sections showing immunoexpression of Bax in normal and cryptorchid rat testes. No expression in normal (b) but moderate expression at 3 days (d) to strong expression at 5 days (f) is observed among giant (gnt) cells and germ (gc) cells of the cryptorchid testis. Testis sections (a, c, e) represent negative controls for normal, and 3-day and 5-day cryptorchid testis, respectively. Original magnification 6400. (ii) Up-regulated expression of Bax is observed in day 2 (lane 1), day 3 (lane 3), and day 5 (lane 5) cryptorchid testis. Lanes 2, 4, and 6 represent the protein expression in control testis. (iii) Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay for germ cell apoptosis. Both gnt and gc cells are found to be TUNEL positive. Normal testis (a) and cryptorchid testis at 2 (b), 3 (c), and 5 (d) days are shown. Original magnification 6400. Color figure available online at www.andrologyjournal.org.

cells in the cryptorchid testis (Figure 3if), as seen in the present study, supports the above contention. Hsps are a large family of evolutionarily conserved proteins that function as molecular chaperones (Castelli et al, 2004). In multiple cell types, these specialized proteins play important roles in cellular defense mechanisms in cells under stress. They are also involved in proper folding of the proteins that are functionally active (Guzhova et al, 1997; Garrido et al, 2003). Cryptorchidism-induced stress results in overexpression of hsp70 in the giant cells as well as other germ cells of 5day cryptorchid testis (Figure 2ih), as observed in the present study. The TUNEL positivity in multinucleated giant cells (Figure 3iiid) indicates that under adverse conditions, it is one of the natural processes of quick removal of apoptotic cells from the seminiferous epithelium (Chaki et al, 2005).

The role of HABP1 in apoptosis has been suggested for a long time on the basis of the crystal structure, but there are very few reports for this hypothesis to suffice. Meenakshi et al (2003) have already reported that overexpression of HABP1 in fibroblast cells causes apoptosis induction in normal fibroblast cells. Also, we have recently reported the up-regulation of HABP1 in cisplatin-induced HeLa cells. The number of apoptotic cells can be reduced by using short interfering RNA specific for HABP1 (Kamal and Datta, 2005). It may be important here to mention that the overexpression of HABP1 resulted in the phenotypic changes of multinucleation and abnormal cell septum formation in Schizosaccharomyces pombe (Mallick and Datta, 2005). Testes behave differently than somatic tissues (Bharadwaj et al, 2002) because of their unique gene expression during meiosis, and a higher level of HABP1

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Figure 4. (i) Representative sections showing immunoexpression of Hyaluronan Binding Protein 1 (HABP1) in normal and cryptorchid rat testes. Strong expression in germ (gc) cells around the periluminal area in normal testis (b) is later found to be restricted to gc cells and giant (gnt) cells of 3-day (d) and 5-day (f) cryptorchid testis. Sections (a, c, e) represent negative controls for normal, and 3-day and 5-day cryptorchid testes, respectively. Original magnification 6400. (ii) Up-regulated expression of HABP1 proprotein (upper lane) is observed in normal (lane 2) as well as 2-day (lane 3), 3-day (lane 5), and 5-day cryptorchid rat testis. In contrast, the mature form of HABP1 (lower lane), which is found to be present in normal (lane 2) and 2-day (lane 3) cryptorchid testis, cannot be probed in subsequent 3-day (lane 5) and 5-day (lane 3) cryptorchidism. (iii) Reverse transcription–polymerase chain reaction of HABP1 (lower lane) showing up-regulated transcript levels in day 3 (lane 5) and day 5 (lane 9) cryptorchid testis compared with normal testis in the corresponding adjacent lanes. Upper lane shows equal loading with b-actin. Color figure available online at www.andrologyjournal.org.

transcript in cryptorchid rat testis (Figure 4iii) could be due to mRNA storage or up-regulated transcriptional activity or a delayed posttranslational cleavage during meiosis. The HABP1/gC1qR-associated phosphorylated proteins were observed recently in germ cell lines. Because gC1qR is localized predominantly in the mitochondrial matrix, the gC1qR in germ cells may be requisite for transducing mitochondrial stress or apoptotic signals to apoptosis (Kitazawa et al, 2006). However, future studies will determine the exact mechanism as to how HABP1 is

associated with apoptotic induction of germ cells in the cryptorchid testis.

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