| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Testis |
a Department of Biochemistry
b Department of Urology, Yamagata University School of Medicine, Yamagata 990-9585, Japan
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
gene regulation, spermatid, spermatogenesis, stress
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Many genes are differentially expressed in germinal cells, especially in primary spermatocytes. For example, several cell- and stage-specific histone variants play a role in modulating chromatin changes that mediate the replacement of histone-rich nucleoproteins of spermatocytes to the protamine-rich nucleoproteins of spermatids [9]. The nuclear compaction of the sperm nucleus during spermatogenesis involves the sequential replacement of histones by transition proteins and, finally, by protamine. YB2, a rat Y-box binding protein, and RYB-a, an alternatively spliced product of the YB2 gene, are highly expressed in spermatogenic cells [10]. The Y-box proteins contain a cold-shock domain (CSD), the sequences of which are conserved over 40% in small bacterial cold-shock proteins [11, 12] and bind single-stranded nucleic acids [13]. In addition to CSD, all vertebrate Y-box proteins contain basic/aromatic islands that bind RNA [14, 15] in their C-terminal region. In previous work, we reported the localization of the YB2/RYB-a protein in the rat and mouse testis and the induction of gene expression around the prepubertal stage [10]. Both the YB2/RYB-a mRNA and the protein appeared in prepubertal testes prior to the expression of mouse protamine 2 (mP2) mRNA. The protein was present at high levels in spermatocytes, decreased in round to elongated spermatids, and was absent in spermatozoa. Because mP2 mRNA was present at high levels in round and elongating spermatids but was not translated until the elongated spermatid stage had been reached, the proposed function of the YB2/RYB-a protein appears to be a translational repressor of the mRNA in mouse.
In this study, we investigated the effects of heat stress caused by artificial cryptorchidism or the local heating of the lower abdomen on the expression of the YB2/RYB-a and the mP2 mRNA in adult mouse testes. The data further support the view that YB2/RYB-a interacts with the mP2 mRNA, thus protecting it from degradation during spermiogenesis.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Eight- to 12-wk-old BDF1 mice were purchased from Japan SLC (Shizuoka, Japan) and maintained under conventional conditions at the Laboratory Animal Center, Yamagata University School of Medicine, at least 2 wk before use. This study was conducted in accordance with the Guiding Principles in the Care and Use of Animals (DHEW Publication, NIH, 80-23). Experiments using animals were performed in accordance with the Declaration of Helsinki under the protocol approved by the Animal Research Committee of this institution. Mice for immunohistochemical studies were anesthetized with diethyl ether and killed by perfusion with Bouin fixative through the heart. The resulting tissue samples were used for immunohistochemical analysis and in situ hybridization. Fresh tissue samples were dissected from the mice under anesthesia, frozen in liquid nitrogen, and preserved at -80°C until used for protein and mRNA assays. All reagents were of the highest grade available.
Culture of Testicular Germ Cells
Testicular cells were isolated by the method of Nagao [16] with modifications [17]. Briefly, testes were removed and decapsulated mechanically. The individual seminiferous tubules were gently teased apart and incubated in PBS containing 0.25% collagenase (Wako Pure Chemicals, Osaka, Japan) for 15 min at 32.5°C with occasional shaking. The seminiferous tubules were then washed and incubated again in PBS containing 0.25% trypsin (Difco, Detroit, MI) for 15 min at 32.5°C with gentle shaking. After incubation, the trypsin treatment was terminated by adding fetal bovine serum (FBS) to 10% (v/v). The resultant cell suspension was filtered through a metal mesh to remove cell aggregates and tissue debris, after which the cells were collected by centrifugation. The recovered cells were resuspended in F12-L15 medium supplemented with 1 mg/ml of sodium bicarbonate, 100 U/ml of penicillin-G, 100 µg/ml of streptomycin sulfate, 15 mM Hepes, and 10% FBS. The final concentration of testicular cells in the medium was adjusted to approximately 5 x 105/ml. The cell suspension was plated in a 6-cm plate (Sumitomo Bakelite, Akita, Japan). The cells were incubated in a humidified atmosphere of 5% CO2 in air at 32.5°C. At 1 day after isolation, the cultured cells were incubated at either 32.5 or 37°C for up to 5 days. Some cells were also exposed to 43°C for 30 min, followed by incubation up to 5 more days at 32.5°C.
Induction of Artificial Cryptorchidism
Artificial cryptorchidism was induced in mice as described previously [18]. A group of mature male mice was made cryptorchid surgically under sodium barbital anesthesia (4 mg/kg body weight). Both testes were translocated and sutured to the lateral abdominal wall via the fat pad. Care was taken not to injure blood vessels or the epididymis. The animals were killed at appropriate times after cryptorchidism. Mice were used for analyses only when both testes were located abdominally at postmortem and were atrophic. Testes from successfully operated males were always atrophic.
Local Heating of Lower Abdomen
Local heating of the lower abdomen was carried out as described previously [8]. The mice were anesthetized with an i.p. injection of sodium pentobarbital and placed in a specially constructed holder. The lower abdomen, including the scrotum, were then immersed in hot water at 43°C for 15 min. After drying, they were maintained under conventional conditions.
Digoxygenin Labeling of Complementary RNA Probes for YB2/RYB-a and mP2 mRNA Detection
The cRNA probes used were the same as described previously [10]. Full-length RYB-a cDNA [19] was excised by digestion with HaeIII and SmaI, and the resultant 396-base pair DNA fragment, nucleotides 757–1152, was ligated into the EcoRV site of the pBluescript KS+ vector. The vector that contained the RYB-a cDNA, was linealized by HindIII and EcoRI digestion to produce sense and antisense cRNA, respectively. The vector that contained the mP2 insert was linealized by XhoI and EcoRI to produce sense and antisense cRNA, respectively. In vitro transcription to produce digoxygenin (DIG)-labeled cRNAs was performed using the RNA-DIG labeling mix (Roche, Mannheim, Germany) and T3 and T7 RNA polymerases. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antisense cRNA probe was also prepared in the same way.
Northern Blot Analysis
RNA samples were electrophoresed in 1% agarose-formaldehyde gels, transferred to nylon membranes (MSI, Westborough, MA), and hybridized at 60°C overnight in hybridization buffer (50% formamide, 0.5% SDS, 5% Irish Cream Liquor [R & A Bailey & Co., Dublin, Ireland], 0.75 M NaCl, 43 mM Na2PO4, and 6.25 mM EDTA) containing DIG-labeled cRNA probes. The nylon membranes were washed sequentially, finally with 0.1x standard saline citrate containing 0.1% SDS at 65°C. The membranes were then incubated for 30 min at room temperature with an anti-DIG Fab-antibody conjugated with alkaline phosphatase (Roche). The membranes were reacted with disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclodecan}-4-yl)phenyl phosphate (Roche) and exposed to x-ray film to detect the signals from the enzymatic reaction. After stripping the probe, the same blot was rehybridized with the GAPDH antisense cRNA probe and then with the mP2 antisense cRNA probe.
Western Blot Analysis
Tissues dissected from mice were pulverized under liquid nitrogen and suspended in a buffer containing 25 mM Tris-HCl, 50 mM NaCl, 0.5% Na-deoxycholate, 100 mM NaF, 2% NP-40, 0.2% SDS, and 200 mM NaVO3 supplemented with protease inhibitors (10 µg/ml aprotinin, 10 µg/ml leupeptin, 0.57 mM PMSF, and 10 µg/ml pepstatin). Cultured cells were collected in 1.5-ml tubes, washed twice with PBS, and lysed in the same buffer. The lysate was centrifuged at 12 000 rpm for 5 min in a microcentrifuge. Protein concentrations of the supernatant were determined using a BCA kit (Pierce, Rockford, IL). Total proteins, 10–20 µg, were separated on 10% SDS-polyacrylamide gels and electroblotted onto PVDF membranes (Amersham Biosciences, Piscataway, NJ). The blots were blocked with 10% nonfat dry milk in PBS and then incubated with the polyclonal, anti-YB2/RYB-a antiserum [10] diluted in PBS overnight at 4°C. After washing twice in PBS containing 0.1% Tween 20 and twice in PBS for 30 min, the blots were incubated with goat anti-rabbit IgG antibody conjugated with horseradish peroxidase. After washing as above, the presence of the enzyme was determined by chemiluminescence with an ECL plus detection reagent (Amersham Biosciences) and exposed to X-ray films. The antibody was stripped off and reacted with the anti-SOD1 antibody. For the control experiment, nonimmunized serum was used with the same dilution.
Preparation for Tissue Sections
After perfusion with Bouin fixative, the testes were removed from mice, cut into pieces, and immersed in Bouin fixative for 3 h to overnight. They were then embedded in paraffin, sectioned at 4 µm thickness, and used for both in situ hybridization and immunohistochemical detection.
In Situ Hybridization
In situ hybridization was performed using a previously reported methods [20] with minor modifications [10]. Briefly, sections on the slides were deparaffinized, digested with proteinase K (20 µg/ml) for 5 min at room temperature, and postfixed in 4% paraformaldehyde, 0.2% glutaraldehyde for 20 min, followed by incubation in a prehybridization buffer containing 50% formamide, 0.5% SDS, 5% Irish Cream Liquor, 0.75 M NaCl, 43 mM Na2PO4, and 6.25 mM EDTA. They were hybridized with the DIG-labeled sense or antisense mP2 RNA (2 µg/ml) in a hybridization buffer containing 50% formamide, 1% SDS, 0.75 M NaCl, 10 mM PIPES, 0.05% heparin, and 100 µg/ml yeast tRNA (Sigma, Tokyo, Japan) at 50°C overnight. After washing with PBS, the tissue samples were incubated for 30 min at room temperature with an anti-DIG Fab-antibody conjugated with alkaline phosphatase (Roche). Positive signals were visualized by the reaction of the alkaline phosphatase with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Roche) under protection from light in a humid chamber. After dehydration by passing the slides through a series of graded ethanol solutions, they were mounted. For each test, control experiments using the DIG-labeled sense cRNA probe were performed.
Immunohistochemical Staining
The paraffin sections were deparaffinized in xylene and hydrated in a series of graded ethanol solutions. After hydration, endogenous peroxidase was inactivated in 3% hydrogen peroxide. Prior to immunostaining, the nonspecific binding of the antibody was blocked with 2% swine serum in PBS for 10 min. The slides were immersed in 50 µl of a solution containing the anti-YB2/RYB-a antiserum [10] at dilution of 1:250 in PBS, with the tissue face down, and incubated at room temperature in a humid chamber overnight. The control experiment was carried out using nonimmunized rabbit serum with the same dilution. Following three consecutive washes in PBS for 5 min each, the sections were incubated at room temperature for 30 min with horseradish peroxidase-conjugated goat anti-rabbit IgG polymer (DAKO, Carpinteria, CA). To visualize the signals, the reaction was completed by incubating the sections in diaminobenzidine tetrahydrochloride (DAB) reaction reagent (DAKO) for several seconds. The resulting slides were then washed with water, dehydrated by passing through a series of graded ethanol, and mounted. Photographs were obtained using a digital camera under light microscopy BX50 (Olympus, Tokyo, Japan).
Quantification of the Protein and mRNA Bands and Statistical Analysis
The amounts of YB2/RYB-a protein and mP2 mRNA were quantified by densitometric scanning using Densitography (Atto, Tokyo, Japan) of the x-ray films and were normalized by the amount of SOD1 protein and GAPDH mRNA, respectively. Statistical analyses of the data were carried out using the Mann-Whitney U-test.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
To investigate the effects of heat stress on YB2/RYB-a protein levels in the spermatogenic cells in vitro, we carried out a primary culture of the testicular cells at the scrotal temperature, 32.5°C. At 24 h after isolation, the cultured cells were exposed to higher temperature, 37 or 42°C, for 30 min. We actually counted the number of cells at each day point and found the decreased number of cells during the culture period. The density of living cells decreased to 40% and 20% at 32 and 43°C, respectively, while cell viability decreased to 70% and 54% of that at the starting time at 32 and 43°C, respectively. It appears that dead cells immediately fragmented to small pieces and were uncountable. Thus, contribution of the dead cells to the protein or mRNA would be small. Immunoblot analysis using the anti-YB2/RYB-a antiserum showed that levels of the YB2/RYB-a proteins in the cultured testicular cells decreased gradually during the incubation period, even in the case of untreated cells (Fig. 1). Exposure of the cells to the elevated temperature led to a more rapid decrease in YB2/RYB-a protein levels than for those at the scrotal temperature.
|
Effects of Artificial Cryptorchidism on the YB2/RYB-a Protein and the mP2 Expression
To examine the effects of heat stress on YB2/RYB-a expression in the testis in vivo, we employed cryptorchidism. Immunoblot analysis of proteins prepared from whole testes revealed that the YB2/RYB-a protein disappeared abruptly in the cryptorchid testis at Day 10 after surgery, while YB2/RYB-a mRNA and mP2 mRNA declined after 14 days (Fig. 2). It is noteworthy, however, that both YB2/RYB-a and mP2 mRNA slightly increased at early phases of the cryptorchidism.
|
Altered Distribution of the YB2/RYB-a Protein and the mP2 mRNA in the Cryptorchid Testes
Distribution and alteration of the YB2/RYB-a protein and the mP2 mRNA were examined by immunohistochemistry and in situ hybridization, respectively, at various times after the induction of artificial cryptorchidism. Pictures at selected times are shown in Figure 3. The anti-YB2/RYB-a antiserum specifically detected the YB2/RYB-a protein in the serial sections. No specific staining was obtained by a control experiment using nonimmunized rabbit serum (data not shown). Immunoreactivity was seen in spermatocytes and, to a lesser extent, in round to elongated spermatids but not in spermatozoa as previously reported [10]. The number of immunoreactive cells decreased after the induction of cryptorchidism, and only trace levels were seen at Day 14. A control incubation with the DIG-labeled mP2 cRNA sense probe was completely negative (data not shown). The mP2 mRNA was detected in round and elongating spermatids and, to a lesser extent, in early elongated spermatids of some seminiferous tubules. The levels of both the YB2/RYB-a protein and mP2 mRNA were gradually diminished and were almost extinguished by Day 14 after surgery.
|
Effects of Local Heating of the Lower Abdomen
We next investigated the alteration in the levels of the YB2/RYB-a protein and the mP2 mRNA after the local heating of the lower abdomen by bathing in water at 43°C for 15 min. In the heat-stressed testes, the levels of the YB2/RYB-a protein were decreased after Day 3 and then began to recover between Days 14 and 21 after the heat treatment (Fig. 4). The mP2 mRNA decreased at Day 6 and recovered after Day 28, when YB2/RYB-a recovered. Immunohistochemistry and in situ hybridization revealed that testicular damage was already evident at Day 1, although the damaged cells still contained both the YB2/RYB-a protein and the mP2 mRNA (Fig. 5). At Day 9, some seminiferous tubules showed the presence of the mP2 mRNA but at much lower extent than that of the YB2/RYB-a protein. At Day 28, numerous seminiferous tubules had recovered from the damage and possessed spermatogenic ability. The YB2/RYB-a protein was present in all seminiferous tubules, while the mP2 mRNA was found only in some. The time lag could be due to the extent of differentiation of the spermatogenic cells because the mP2 mRNA is expressed in more differentiated cells than the YB2/RYB-a protein [10].
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Heat-induced stress results in the degradation of many mRNAs in the testis. The effects of the heat stress on the translation of mRNAs for the histone H1t variant and the transition proteins TP1 and TP2 have been reported in rat primary spermatocytes and elongated spermatids, respectively [21]. The decreased resistance of the primary spermatocytes to the abdominal temperature can be attributed to the reduction in the proportion and the size of polysomes translating H1t mRNAs. The ribosome spacing, which affects the relative rates of the initiation and the elongation of the nascent polypeptide from the translationally active mRNA, is a cause for translational regulation. Messenger RNA species, which are translated in meiotic and early haploid spermatogenic cells, exhibit a wider ribosome spacing, 100–150 bases, than those in the somatic cells [23]. The ribosome spacing on the mRNA species that are translatable in elongated spermatids are quite variable, ranging from 40–50 bases for protamine 1 and 2 mRNA to 143 bases for the cysteine-rich protein mRNA of sperm mitochondria. The closer ribosome spacing in elongated spermatids implies that qualitative or quantitative differences in the translational apparatus are responsible for the difference in the temperature sensitivity of protein synthesis in pachytene spermatocytes and elongated spermatids [23]. Although we do not yet have direct evidence for the involvement of the Y-box protein in the translational apparatus, free mRNP fractions in pachytene spermatocytes to round spermatids contain correspondingly high levels of the Y-box protein [24].
Stress response is generally thought to be activated by denaturation of proteins [25, 26]. Sarge [22] has proposed that some proteins that are synthesized specifically in meiotic and haploid spermatogenic cells may be denatured at abdominal temperatures. The heat-resistance of elongated spermatids suggests either that the expression of these putative thermolabile proteins is restricted to primary spermatocytes and round spermatids or that their denaturation in elongated spermatids does not lead to the inhibition of translational initiation [21]. We previously provided supporting evidence to show that the YB2/RYB-a could interact with mP2 mRNA and prevent them from being translated in normal spermatogenesis [10]. If heat stress affects mRNA-protecting proteins, such as YB2/RYB-a, by altering their conformation or their levels in pachytene spermatocytes and round spermatids or both, the mP2 mRNA protected by them should be easily degraded in the elongated spermatids. Our data, which show concerted changes in the YB2/RYB-a protein and the mP2 mRNA under heat-stressed conditions, provide further support for this hypothesis.
The translational regulation in spermatogenic cells raises important questions. The mechanisms that inhibit the translational initiation of various mRNA species in a mRNA-specific manner have not been elucidated, although several candidates for sequence-specific translational repressors have been identified [10, 24, 27–29]. Moreover, the reason for why nearly all the mRNA species in meiotic and haploid spermatogenic cells are translationally repressed is not fully understood. Protamine mRNAs represent well-known members of a large group of mRNAs that are translationally repressed in round spermatids and are actively translated in elongated spermatids after the cessation of transcription [30].
In conclusion, the concomitant decline in YB2/RYB-a protein levels and mP2 mRNA under heat-stressed conditions supports the notion that the YB2/RYB-a protein is involved in the protection of mP2 mRNA. It is possible that other proteins also exert similar protective effects. The interaction of Y-box proteins and protamine mRNAs represent an excellent model for studying not only translational regulation during spermatogenesis but also mechanisms of damage caused by heat stress.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence: Junichi Fujii, Department of Biochemistry, Yamagata University School of Medicine, 2-2-2 Iidanishi, Yamagata City, Yamagata 990-9585, Japan. FAX: 81 23 628 5230; {at}med.id.yamagata-u.ac.jp
Received: 1 March 2002.
First decision: 20 March 2002.
Accepted: 5 August 2002.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  R. Oliva Protamines and male infertility Hum. Reprod. Update, July 1, 2006; 12(4): 417 - 435. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Takano, H. Matusi, and T. Takahashi Granzyme N, a Novel Granzyme, Is Expressed in Spermatocytes and Spermatids of the Mouse Testis Biol Reprod, December 1, 2004; 71(6): 1785 - 1795. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
