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research-article |
Shanghai Key Laboratory for Molecular Andrology,3 State Key Laboratory of Molecular Biology,
Key Laboratory of Proteomics,4 Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, People's Republic of China
Laboratories for Reproductive Biology,5 University of North Carolina, Chapel Hill, North Carolina 27599-7500
Shanghai Institute of Planned Parenthood Research,6 Shanghai 200032, People's Republic of China
Graduate University of Chinese Acedemy of Sciences,7 Beijing 100049, People's Republic of China
ABSTRACT
Members of the RNase superfamily participate in a diverse array of biological processes, including RNA degradation, antipathogen activities, angiogenesis, and digestion. In the present study, we cloned the rat RNase9 gene by in silico methods and genome walking based on homology to the Macaca mulatta (rhesus monkey) epididymal RNase9. The gene is located on chromosome 15p14, spanning two exons, and is clustered with other members of the RNase A superfamily. It contains 1279 bp and encodes 182 amino acids, including a 24-amino acid signal peptide, and it has unique features known from other RNases. Unlike those other members, the rat RNase9 mRNA was specifically expressed in the epididymis, especially in the caput and corpus, and exhibited an androgen-dependent expression pattern but was downregulated in an epididymitis animal model. The RNASE9 was expressed in a principal cell-specific pattern. Interestingly, most of the principal cells in the caput expressed the RNASE9; however, in the distal caput, the principal cells showed a checkerboard-like pattern of immunoreactivity. We also observed that the RNASE9 was bound on the acrosomal domain of sperm. Its potential roles in sperm maturation are discussed.
epididymis, gene regulation, male reproductive tract, principal cells, sperm maturation, testosterone
The epididymis is an essential male reproductive organ that is composed of four major distinctive regions: the initial segment and the caput, corpus, and cauda. The epididymis is responsible for sperm maturation, storage, and protection [1]. Mammalian spermatozoa are transported from the testis into this organ, where they undergo a series of physical, chemical, and morphological alterations to acquire maturity. The luminal fluid microenvironment provided by the epididymis, which is certainly the most complex found in any exocrine gland [2], plays an important role in sperm maturation. At least 200 proteins are present in the epididymal lumen [3], but the functions of most of them in male fertility are unknown. A few have been identified that seem to be directly involved in sperm maturation in the epididymis.
In recent years, we constructed and analyzed a rhesus monkey epididymis-specific cDNA library. From this library, 36 nonoverlapping novel cDNAs were isolated and sequenced. A total of 11 full-length cDNAs have been cloned [4]. These opened new opportunities for further investigating the roles of the epididymis in sperm maturation. Among them, ESC461 showed homology to RNases of diverse physiological functions, prompting the search for orthologs in the human [5] mouse [1], and rat [6]. They are members of the secreted pancreatic RNase A superfamily and are known as RNase A family member 9 (RNASE9).
The RNase A superfamily is a well-documented protein family. To date, there are 13 known human RNASEs, including RNASE1 (known as the pancreatic RNase), RNASE2 (known as EDN, eosinophil-derived neurotoxin), RNASE3 (known as ECP, eosinphil-cationic protein), RNASE4, ANG (RNASE5), RNASE6 (previously known as RNase k6), RNASE7, and RNASE8. RNASE9 and RNASE10 were described only recently [5–8], and RNASE11–RNASE13 were identified only from whole-genome databases based on TBLASTN [6]. With the exception of RNASE2 and RNASE3 and RNASE7 and RNASE8, all human RNASE genes are represented (in one or multiple copies) in the mouse and rat [6]. They have some shared features. The genes are closely linked on the same chromosome. In each, a single exon contains the entire open reading frame (ORF). The encoded proteins contain a signal peptide at the N-terminus and six to eight conserved cysteines. Most RNASEs have the H-K-H catalytic triad and several other conserved motifs, although the level of conservation varies. Most RNASE genes are expressed in a wide range of tissues, except for RNASE8, which is expressed prominently in the placenta [9]. In contrast, RNASE9–RNASE11 are all specifically or highly expressed in male reproductive organs. Members of the RNase A superfamily participate in a diverse array of biological processes, including RNA degradation, antipathogen activities, angiogenesis, and digestion [10, 11]. The RNASE7 expressed in epithelial tissues, especially in human skin, exhibits very high enzymatic activity and broad-spectrum antimicrobial activity [12, 13]; the RNASE3 and RNASE2 found in the secretory granules of human eosinophilic leukocytes have antibacterial or antiviral activities. It has been shown that the angiogenesis activity is unique to the angiogenins [14]. But recently, the angiogenins were described as a new class of microbicidal proteins involved in innate immunity [15]. The activities of RNASE9–RNASE13 have not been well examined. The epididymis-specific mouse RNase9 and RNase10 [1] and the human RNASE9 [5] were described only from the mRNA level. The RNASE10 [6, 7] was purified and identified only from the porcine epididymis. To our knowledge, the literature contains no description of the orthologs of these two epididymis-specific RNases in the rat. In this article, we describe the cloning of the RNASE9 ortholog in the rat and the characterization of this gene at both the mRNA and protein levels.
Healthy male Sprague-Dawley (SD) rats and male New Zealand white rabbits (body weight ~2.5 kg) were purchased from the Animal Center of the Chinese Academy of Sciences (Shanghai, China). They were housed for an additional 7–10 days before manipulation in the animal house of the institute. Food and water were freely available throughout the experiments. Experiments were conducted according to a protocol approved by the Institute Animal Care Committee. The protocol conforms to internationally accepted guidelines for the humane care and use of laboratory animals.
DNA and Protein Sequence Analysis
The mouse ortholog of monkey RNASE9 was obtained by homology, searching the mouse expressed sequence tag (EST) database at http://www.ncbi.nlm.nih.gov/BLAST. The mouse EST was used to search the rat genome to obtain the rat ortholog. The signal peptide cleavage sites were predicted by use of the Website http://www.cbs.dtu.dk/services/SignalP. N-glycosylation sites and phosphorylation sites were predicted by use of the ProfileScan Website http://myhits.isb-sib.ch/cgi-bin/PFSCAN. The cDNA fragments of rat RNase9 were amplified by RT-PCR. Total RNA isolated from the rat epididymis was reverse transcribed by SuperScript reverse transcriptase (Gibco/BRL, Grand Island, NY) according to the manufacturer's recommendations. The rat RNase9 cDNA fragments were amplified by PCR with the forward primer F1, 5'-ACCTCCTTTTCCTGTGCC-3', and the reverse primer R, 5'-TGGCATCTGTCTGCTGGA-3', at denaturing, annealing, and extension temperatures of 95°C for 30 sec, 55°C for 40 sec, and 72°C for 1 min, respectively, with Ex-Taq (Takaro). This cDNA fragment was used as a probe to screen the rat epididymis cDNA library to obtain the full-length cDNA.
RNA Isolation and Northern Blot Analysis
Tissue samples were obtained from male rats after they were killed, and the samples were frozen immediately in liquid nitrogen. Total RNA was extracted with Trizol (Invitrogen, Carlsbad, CA) following the manufacturer's recommendations. Northern blot analysis was performed according to the procedure described previously [16]. Twelve micrograms of total RNA from each sample was loaded in each lane. The probe was a 32P-labeled 898-bp cDNA fragment of rat RNase9. An 18S rRNA hybridization signal was used as a loading control. Autoradiographs with pronounced differences in expression were analyzed by densitometry.
Castration and Androgen Replacement
One hundred twenty-day-old normal male SD rats were castrated bilaterally by sodium pentobarbital anesthesia. Animals were divided into nine groups (4–7 rats per group) and killed on Days 0, 1, 3, 5, and 7 after castration as well as 1, 3, 5, and 7 days after the initial testosterone propionate injection. The epididymides were excised and used for RNA extraction. Androgen supplementation began on the seventh day after castration, and rats were injected with testosterone propionate (3 mg/kg body weight) every 2 days. Pooled serum samples for each group were sent to the Shanghai Zhong-Shan Hospital for the measurement of testosterone concentration by RIA.
The surgical procedure was performed as described previously, which induced inflammatory changes in the epididymis [16]. Adult male SD rats (age = 120 days) were divided into two groups. After an i.p. injection of sodium pentobarbital, the left vas deferens was tied, avoiding damage to the regional blood vessels. The right side served as the control. The rats (n = 4 per interval) were killed 2 and 6 wk after surgery.
Anti-Rat RNASE9 Polyclonal Antisera
The cDNA fragment for the rat RNASE9 mature peptide of 158 amino acids was amplified by PCR with the following primers. The forward primer 5'-CATATGAACTACTGGGATGAA-3' has an NdeI site on its 5' end, and the reverse primer 5'-AAGCTTCTACTCAGGCAGTGA-3' adds a HindIII site to the 3' end of the amplicon. The fragment was inserted into a pET28 (a) vector (Novagen). The expression vector was constructed according to the standard protocol in the pET expression manual. The recombinant protein in the inclusion bodies was induced by isopropylthiogalactoside from the strain Escherichia coli BL21 Codon Plus RP (Novagen). The purification of the recombinant protein from inclusion bodies was performed as described previously [17]. The antisera were obtained according to our modified immunization methods [18]. Six hundred micrograms of antigen was injected into rabbits on Days 1, 3, and 28. On the 35th day, the antisera were harvested from the arteriae carotis. The polyclonal antisera against the N-terminal part of the rat RNASE9 have been raised in the same way.
Protein Extracts and Western Blot Analysis
Total protein extracts of the rat epididymis were prepared as described previously [19] with a minor modification, i.e., the addition of a protease inhibitor cocktail (PIERCE) in the homogenizing buffer instead of individual protease inhibitors. Protein extracts for two-dimensional gel were prepared as described [20].
Total protein extracts for each sample (20 µg) were separated on 15% SDS-PAGE gels and semidry blotted to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech). The polyclonal antiserum against rat RNASE9 recombinant protein was used as the primary antibody (dilution 1:8000). The second antibody was a goat horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin G (IgG) (dilution 1:16 000; CalBiochem). The peroxidase activity was demonstrated with a chemiluminescent substrate (Western blot chemiluminescence Reagent Plus; Amersham Pharmacia Biotech). The Western blot of two-dimensional gel electrophoresis was performed as described previously [20], except that the primary antibody was diluted 1:5000, and the second antibody was diluted 1:10 000.
Peptide N-Glycosidase F Treatment
The total tissue protein extracts (30 µg) in 1x Glycoprotein Denaturing Buffer were denatured by boiling at 100°C for 10 min. To the sample solution were added 1:10 volumes of 10x G7 Reaction Buffer and 10% Nonidet P-40. Peptide N-Glycosidase F (PNGase-F, 500 U/µl; BioLabs) was added to the test samples, and the digestion reaction was allowed to proceed at 37°C overnight. In the control sample, no enzyme was added. The N-glycosylation modification of rat RNASE9 was then evaluated by Western blot analysis.
Immunohistochemical and Immunofluorescence Staining of Tissues
Immunohistochemical staining was performed as described previously [19], except that the tissues were fixed in Bouin fluid. Primary and secondary antibodies were diluted in PBS containing 10% normal goat serum. The 1:300-diluted anti-rat RNASE9 antiserum was applied to the tissues overnight at 4°C, and the 1:200-diluted HRP-conjugated goat anti-rabbit IgG was incubated for 1 h at room temperature. As a negative control, serial sections were subjected to the same procedure, with normal rabbit serum replacing the primary antibody.
For immunofluorescence staining, the method was as described by Hu et al. [21]. The first antibodies that were applied to the tissues were rabbit anti-rat RNASE9 antibody (dilution 1:300), chicken anti-ATP6E immunoglobulin Y (IgY) antibody (dilution 1:200; Genway Biotech), rabbit anti-GSTP1 antibody (dilution 1:200; CalBiochem), and goat anti-CLU antibody (dilution 1:100; Santa Cruz). The second antibodies were fluorescein isothiocyanate (FITC)-labeled anti-rabbit IgG (dilution 1:500; Sigma), Rhodamine-conjugated bovine anti-chicken IgY (dilution 1:200; Santa Cruz) and tetramethylrhodamine isothiocyanate (TRITC)-labeled goat anti-rabbit IgG (H + L) (dilution 1:200; Jackson ImmunoResearch Laboratories), and Rhodamine-conjugated anti-goat IgG. The sections were mounted in 80% glycerol and examined with a confocal microscope (Leica TCS SP2 AOBS) or an Olympus BX-52 microscope.
Indirect Immunofluorescence Detection of Proteins Associated with Spermatozoa
The epididymis was separated into three segments: the caput, corpus, and cauda. Spermatozoa were collected from the different segments by puncturing the ductus, and the first drop was placed in tissue culture wells and incubated at 37°C for 30 min with 5% CO2 in human tubule fluid culture medium. The spermatozoa were collected and washed with PBS and then placed on polylysine-coated slides and air dried. The slides that contained spermatozoa were blocked for 1 h at room temperature with 10% goat serum in PBS. They were then incubated in polyclonal anti-rat RNASE9 serum (diluted 1:200 in PBS containing 10% goat serum) overnight at 4°C, with preimmune rabbit serum as the control. After three washes with PBST (PBS containing 0.1% Tween-20), the corresponding second antibody was applied (FITC-conjugated anti-rabbit IgG, 1:500 diluted in PBS containing 10% goat serum). The slides were washed three times with PBST and mounted in 80% glycerol. Slides were examined with an Olympus BX-52 microscope.
Cloning of the Rat RNase9 cDNA
The rat RNase9 ortholog to monkey ESC461 was obtained by the monkey cDNA sequence for a BLASTN query of the mouse EST database to obtain the mouse RNase9 EST (AY303816) that was then used to query the rat genome. On the basis of the rat genomic sequence, two primers (F1 and R) were designed and used to amplify a rat cDNA fragment by RT-PCR with rat epididymal total RNA. The correct 898-bp fragment was amplified and verified by automated sequencing. The cDNA fragment was also used as a probe to screen the rat epididymal cDNA library to obtain the full-length cDNA of rat RNase9 (1279 bp). The sequence was further confirmed by sequence analysis from four healthy male SD rats.
The gene of rat RNase9 is located on rat chromosome 15p14, which is closely linked to other RNases. It spans 3578 bp and includes one intron (2299 bp) and two exons (104 and 1175 bp). The entire ORF of 549 bp from the start codon to the stop codon is located in the second exon (Fig. 1, A and B). The RNase9 cDNA sequence corresponds to the predicted rat UniGene Rn.128555, under which no tissue or developmental expression data are yet available. The predicted protein contains 182 amino acids with an estimated size of 21.3 kDa. The N-terminal 24 amino acids probably form a signal peptide, as predicted with the SignalP 3.0 server. Cleavage of this peptide would lead to a mature protein of 158 amino acids and a calculated isoelectric point (pI) of 5.12. One potential N-glycosylation site was predicted at N177. Six other sites of potential posttranslational modification are present in this sequence: three casein kinase II phosphorylation sites at amino acid positions T64VGE, S101YEE, and S179LPE; one N-myristoylation site at amino acid position G119VKFCR; and two tyrosine kinase phosphorylation sites at amino acid positions R68PLQDYDY and R142MVDCMY (Fig. 1).
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Compared with the sequences of the other members of the RNase A family, rat RNASE9 has eight conserved cysteine residues characteristic of the RNase A superfamily (Figs. 2 and 3) but lacks the catalytic triad (H12-K41-H119, numbers according to human RNASE1). Additional amino acids are inserted at the N-terminus following the signal peptide. These features are present in the RNASE9 either from primates or rodents (Fig. 3). Interestingly, the male organ-abundant rat RNASE10 and RNASE11 showed similar characteristics, but they also had a longer additional N-terminal peptide in front of the eight conserved cysteine residues than did the rat RNASE9 (Fig. 2). The rat RNASE9 amino acid sequence is 35% similar to the human RNASE9, 38% similar to the monkey RNASE9, and 69% similar to the mouse RNASE9.
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Epididymis-Specific and Androgen-Upregulated Expression of Rat RNase9 mRNA
To determine whether RNase9 is predominantly expressed in the epididymis, total RNAs from three regions (caput, corpus, and cauda) of the epididymis and other tissues of adult male rats were analyzed by Northern blot hybridization (Fig. 4A). A strong hybridizing signal (~1.3 kb) was detected only in the rat epididymis, especially in the caput and corpus regions. A weak signal in the cauda also could be seen. No signal was observed in the testis, seminal vesicle, brain, spleen, lung, or kidney. Expression only in the epididymis implies that it must have some special roles in this organ, such as sperm maturation.
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Since sperm maturation is an androgen-dependent process, we analyzed RNase9 mRNA expression under conditions of androgen manipulation. Total RNAs were obtained from the epididymides of adult rats that were sham operated, castrated, or castrated but given an injection of 3 mg/kg (per body weight) of testosterone propionate every 2 days from the seventh day after castration. In the castrated animals, the serum testosterone level declined rapidly and was almost undetectable from the first day (Fig. 4C). In parallel, an obvious decrease was found in the RNase9 mRNA level on the first day of postcastration and was nearly undetectable by the third to seventh day after surgery (Fig. 4B). Testosterone replacement for the animals 7 days after castration resulted in a rapid increase of the serum testosterone concentration. At the same time, the RNase9 mRNA in the epididymis followed the same trend as the serum testosterone level (Fig. 4, B and C), suggesting that RNase9 mRNA expression was regulated by testosterone in vivo.
RNase9 mRNA Expression in the Inflamed Caput after Vas Deferens Ligation
Reports concerning RNases in inflammation [15, 22] prompted us to examine the expression of RNase9 mRNA in the rat model of chronic inflammation induced by unilateral vas deferens ligation [16]. In all epididymides from the operated sides, spermatic granulomas were found only in the cauda region at both 2 and 6 wk after vas deferens ligation (data not shown). The untreated epididymis was the same as in the normal controls. Northern blot hybridization analysis showed that the RNase9 mRNA was slightly reduced (15%) by the surgical obstruction after 2 wk and was further reduced (23%) after 6 wk (Fig. 5). These results showed the responsive decrease of rat RNase9 mRNA after vas deferens ligation.
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Evidence for Posttranslational Modification of Rat RNASE9
Two polyclonal antisera showed the same specificity, and the following data were generated by use of the antisera against the whole mature rat RNASE9. Two molecular mass species of purified recombinant RNASE9 were detected, even in the lowest loading quantity (0.5 ng) (Fig. 6A). The size of the main band of approximately 19.7 kDa matches the value that was predicted by the antigen amino acid sequence. The higher molecular mass of the less intense extra band was 38.3 kDa, which was double that of the major band and thus may have represented the dimerized form of the protein (Fig. 6A, left). To confirm this hypothesis, different amounts of 2-mercaptoethanol were added to the antigen sample before electrophoresis to ensure the complete reduction of any disulfide bridges. The results (Fig. 6A, right) showed that the 38.3-kDa band disappeared, which indicates that the extra band represents dimerization by intermolecular disulfide linkage.
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A single band of approximately 26.3 kDa was detected in rat epididymal protein extracts but not in the testis or other tissues tested (Fig. 6B), which is consistent with the tissue distribution pattern of the RNase9 mRNA (Fig. 4A). Two-dimensional gel Western blot analysis (Fig. 6C) showed proteins with a molecular mass of 26–26.7 kDa and a pI of 4.5–5.0 exclusively in the extracts from the caput region. Compared with the data for the deduced mature peptide (molecular mass = 18.6 kDa; pI = 5.12), the molecular mass is about 8 kDa larger, and the pI is slightly more acidic. To determine if glycosylation accounted for these differences, N-glyconase was used to remove the carbohydrate. After the deglycosylation of the native protein, a decreased molecular mass of rat RNASE9 (Fig. 6D) was detected, suggesting that a posttranslational carbohydrate addition accounts for these differences.
Temporal, Regional, and Cell-Specific Expression of Rat RNASE9 in the Epididymis
Rat RNASE9 was detected in the caput and corpus regions of the mature rat epididymis (Fig. 7, b–e) but not in the initial segment or cauda regions (Fig. 7, a and f), consistent with the mRNA distribution (Fig. 4A). The RNASE9 also exhibited a cell-specific localization pattern. It is noteworthy that the 69-day-old rat epididymis (Fig. 7A) showed that in the caput region (Fig. 7 Bb), nearly all the principal cells demonstrated strong RNASE9 immunoreactivity. However, in the distal caput and distal corpus (Fig. 7, Bc and Be), the protein showed a checkerboard pattern. No expression at all was detected in the cauda region (Fig. 7Bf).
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To identify the different cell types that express RNASE9, we used the following antibodies: anti-ATP6E IgY for clear cells [23], anti-GSTP1 antibody for basal cells [24], and anti-CLU antibody for the principal cells [25]. The epithelial cells in the caput region were used as the reference cells. The results of the immunofluorescent staining of the same or sequential sections (Fig. 8, A–C) showed that RNASE9 was secreted by the principal cells but not by the clear cells or the basal cells (Fig. 8, A and B). Figure 8C shows that in the corpus region, only some of the principal cells expressed RNASE9.
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To determine if RNASE9 could be involved in developmental as well as mature functions, expression of the RNase9 mRNA and protein was analyzed in the tissues from animals of different ages (Fig. 9). The RNase9 mRNA began to be detected at the age of 30 days or so; after that, expression increased gradually and remained at a high level (Fig. 9, A and B). The result of immunohistochemistry was consistent with Northern blot data but revealed much more information (Fig. 9C). At Postnatal Day 21, the epithelium of the entire epididymis was not reactive to anti-rat RNASE9 sera, but by Day 28, the epithelium began to show RNASE9 immunoreactivity. Positive signals could be seen in some of the epithelial cells, but no regional differences were apparent. By Day 69, the signals increased considerably and established the region-specific expression as described above. From Days 90 to 120, strong immunoreactivity was maintained in the epithelial cells of the caput region but faded in the corpus. Interestingly, positive signals appeared in the luminal fluid of the 90-day-old corpus that remained in the aged animal, while there was little rat RNASE9 expressed in the caput principal cells (Fig. 9C). Similar results were obtained with antisera against the N-terminal fragment (data not shown).
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Immunolocalization of Rat RNASE9 in Sperm
We noted in Figure 9C that the immunopositive signals were more intense in the lumen of the 90-day-old rat epididymis than in the 69-day-old animal, where sperm first appeared. Western blot analysis confirmed that there was less RNASE9 in the 69-day-old epididymis lumen protein extracts than in the 90- or 120-day-old animals (data not shown). Nevertheless, the presence of rat RNASE9 in the epididymal lumen indicated that it is available to interact with spermatozoa. Indirect immunofluorescence staining analysis showed that RNASE9 was concentrated over the acrosomal region of spermatozoa (Fig. 10B). The testis is not a likely origin of the protein associated with the spermatozoa, since the RNase9 mRNA was not detected in the testis (Fig. 4A). In the caput, most spermatozoa can bind rat RNASE9, whereas only a small percentage of spermatozoa showed a weak fluorescence in the corpus. Furthermore, the cauda spermatozoa did not show fluorescent staining (Fig. 10), which suggests the loss of rat RNASE9 during epididymal transit. The negative control (normal rabbit serum) had no signal at all (data not shown).
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In the present study, we identified and characterized the RNase9 gene in the rat epididymis and found that it is the first androgen-upregulated member among the presently known RNases. Although the rat RNASE9 sequence does possess some similarities to other RNASEs in addition to the eight highly conserved cysteine residues, it lacks the H-K-H catalytic triad and the CKXXNTF that are signature motifs of this superfamily and are required for the RNase activity of the canonical RNASEs (RNASE1–RNASE8). The pI of RNASE9 is lower than that of any other mammalian RNASEs. A basic pI is believed to be important for RNase activity, conferring the affinity of the enzyme to its negatively charged RNA substrate [26–29]. Therefore, RNase9 may not function as a typical RNase. Our preliminary studies showed that the renatured recombinant protein lacks both RNase activity and antibacterial activity (data not shown). In addition, RNase9 mRNA expression was not inducible in the rat epididymitis model, which is consistent with an unimportant role in the promotion or resolution of inflammation. Rat RNASE9 has an even lower sequence similarity to its orthologs in the human and monkey than other RNASEs [8]. Androgen dependence is another unique feature of the RNase9 gene. In contrast, human RNASE10 (mouse RNase10 and porcine RNASE10), which is also an epididymis-specific gene, seems to be more sensitive to testicular factors. Its mRNA expression and protein secretion decreased following castration but did not reappear by testosterone replacement [1, 7]. Together, these unique features of the rat RNase9 imply that it has unique functions in the epididymis, perhaps playing a role in some aspects of sperm maturation, a major function of this organ.
In the epididymis, the principal cells outnumber all the other cell types combined by at least 3:1 [30]. The principal cells in all regions exhibit the morphological features of cells actively involved in absorption and secretion. Rat RNASE9 expression showed a principal cell-specific pattern, but not all of the principal cells expressed RNASE9. Most principal cells in the caput expressed RNASE9, but only a subset expressed it in the corpus. This type of expression pattern for rat RNASE9 may define a previously unrecognized subtype of principal cells for which RNASE9 might serve as a marker. Determination of the functional differences in these RNASE9-expressing principal cells is an attractive aspect for further study that could lead to a better understanding of the roles of the epididymis in sperm maturation.
That rat RNASE9 expression is mainly restricted to the principal cells and is secreted into the lumen fluid throughout sexual maturity suggest its potential role in sperm maturation. It is well established that the sperm plasma membrane undergoes substantial remodeling to induce sperm maturation during epididymal transit. Many epididymal proteins take part in this process. In the present study, we observed that the fluorescence was not seen after the spermatozoa were fixed with 4% paraformaldehyde. So we deduced that paraformaldehyde might affect the binding sites of rat RNASE9 and its antibody to spermatozoa. The nature of the association between rat RNASE9 and the maturing spermatozoa is unknown. Whether carbohydrate or protein receptor interacts to mediate this association with spermatozoa is not clear. The reason that secreted rat RNASE9 was not detected on sperm from the cauda region is also not known but may be due to masking. Further functional studies will analyze the mechanism of rat RNASE9 binding to caput sperm and its apparent disappearance during corpus transit and cauda storage.
Another interesting phenomenon was that the level of rat RNASE9 was even lower in the lumen fluid but relatively more abundant in the principal cells of the 69-day-old rat than in the 90-day-old animal. Whether the rat RNASE9 presence mainly in principal cells suggests a specific role in these cells should be addressed in future experiments. Additional approaches are under way, including studies to obtain activated rat RNASE9 to determine its function and to analyze the reproductive biology of the RNase9 knockout mouse.
FOOTNOTES
1Supported by the Fogarty International Center Research Collaboration Award (1R03TW01490), the National Natural Sciences Foundation of China (30230190 and 30570684), Shanghai Science and Technology funding (03JC14080 and 05DZ22103), State 863 High Technology R & D project of China (2004AA221120), and 973 program (2006CB504002). 
Correspondence: 2 Yong-Lian Zhang, 320 Yue-Yang Rd., Shanghai, P. R. China 200031. FAX: 86 21 54921011; e-mail: ylzhang{at}sibs.ac.cn
Received: 13 June 2006.
First decision: 6 July 2006.
Accepted: 22 September 2006.
REFERENCES
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 |
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