| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Testis |
a Center for Reproductive Biology, School of Molecular Biosciences, Washington State University,> Pullman, Washington 99164-4660
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
epididymis, Sertoli cell, spermatogenesis, testis
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Sertoli cells produce specific products that are necessary for germ cell survival and development, and those products combine to form a unique and essential environment in the adluminal compartment. A number of proteins synthesized and secreted by Sertoli cells have been described (see [3–5] for a review), including proteinase inhibitors, which are believed to be important in tissue remodeling processes during spermiation and movement of preleptotene spermatocytes into the adluminal compartment.
The cystatins are a superfamily of cysteine proteinase inhibitors that consist of three evolutionarily related families [6]. In vitro studies have established that cystatins are specific inhibitors against papain-like cysteine proteinases such as the mammalian cathepsins B, H, L, and S [7]. However, the in vivo function of cystatins is not well understood. Some researchers have suggested that they may play an important regulatory role in normal body processes such as bone resorption [8] and prehormone processing [9], and in numerous pathological conditions such as cancer progression and metastasis [10], and inflammatory reactions such as rheumatoid arthritis [11], purulent bronchiectasis [12], and periodontitis [13].
In this paper we report the identification of two novel cystatin members from Sertoli cells, termed cystatin SC (cystatin-related gene expressed in Sertoli cells) and cystatin TE-1 (cystatin-related gene highly expressed in testis and epididymis). These genes were identified with the use of differential display-reverse transcriptase-polymerase chain reaction (DD-RT-PCR) by comparing expression profiles of Sertoli cells with those of brain and liver tissues. Comparison of the gene sequences and structural motifs suggests that cystatins SC and TE-1 may be new members of the male reproductive subgroup of the cystatin type 2 family, whereas gene expression studies suggest that cystatins SC and TE-1 may play a specialized role in the testis and epididymis.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
All animal procedures were approved by the Institutional Animal Care and Use Committee of Washington State University and by the National Institutes of Health. Sertoli cells were isolated from 20-day-old C57/B6 mice or 20-day-old Sprague-Dawley rats as previously described [14]. Mouse Sertoli cells were cultured in tissue culture dishes that were precoated with 1 mg/ml of gelatin and maintained in serum-free media with 50% (v/v) Ham F12 media (Gibco BRL Life Sciences, Grand Island, NY) and 50% (v/v) Dulbecco modified Eagle medium (DMEM; Sigma-Aldrich, St. Louis, MO) for 4 days at 37°C in an atmosphere of 5% CO2. Rat Sertoli cells were maintained in serum-free Ham F12 media for 4 days at 32°C.
Isolation of RNA and Messenger RNA
Total RNA was isolated using the Trizol method (Gibco) following the manufacturer's recommendations. The polyadenylated (poly[A]+) RNA was prepared using the standard oligo-(deoxythymidine) affinity chromatography method (Sigma-Aldrich). Brain and liver samples for differential display analysis were taken from 20-day-old C57/B6 mice.
Differential Display-Reverse Transcriptase-Polymerase Chain Reaction
The mRNA DD-RT-PCR technique was performed using the Hieroglyph mRNA Profile kit (Genomyx Corp., Foster City, CA) according to the manufacturer's instructions. Briefly, total RNA samples were digested with RNase-free DNase I to remove contaminating genomic DNA [15]. Reverse transcriptase reactions were carried out in duplicate using oligo(dT)-anchored primers with each of the RNA samples isolated from cultured mouse Sertoli cells, liver, or brain. Differential display-PCRs were also performed in duplicate using arbitrary primers and anchored primers with dNTP mix and [
-33P]dATP. Amplified products were resolved on denaturing polyacrylamide gels (4.5% LR-Optimized, HR-1000; Genomyx) using a Genomyx LR GX100 DNA sequencer. Autoradiographs of dried gels were prepared by overnight exposure to BioMAX MR film (Eastman Kodak, Rochester, NY). Bands corresponding to differentially expressed mRNAs in Sertoli cells were excised from the acrylamide gel and reamplified by PCR using T7 (5'-ACGACTCACTATAGGGC-3') and M13 reverse (5'-ACAATTTCACACAGGA-3') primers, and then cloned into pGEM-T Easy vector (Promega, Madison, WI). The plasmids containing the candidate inserts were extracted with the GenElute PlasmidMiniprep Kit (Sigma-Aldrich) for sequencing.
Oligonucleotide Synthesis and DNA Sequencing
Both the primer synthesis and DNA sequencing analysis were performed at the Bioanalysis and Biotechnology Laboratory at Washington State University (Pullman, WA). T7 END (5'-TTGGACCCGACGTCGCA-3') and SP6 END (5'-AGCTATGCATCGAACGCGTT-3') primers were used for sequencing the DD-RT-PCR clones. The sequences of cloned DD-RT-PCR fragments were analyzed with the GenBank and European Molecular Biology Laboratory databases using the basic local alignment search tool (BLAST) for homology identification and assignment of potential functions. Entire cDNAs were submitted to GenBank using the Bankit internet program at the National Center for Biotechnology Information Web site (http://www.ncbi.nlm.nih.gov/).
Northern Blot Analysis
Ten micrograms of total RNA extracted from testes of 0- to 62-day-old mice or 2 µg of mRNA isolated from organs of adult mice such as brain, heart, kidney, liver, lung, skeletal muscle, spleen, pancreas, ovary, uterus, mammary gland, prostate, epididymis, testis, and cultured Sertoli cells were fractionated on a 1.0% agarose/formaldehyde gel. The RNA was transferred to Hybond N membranes (Amersham Pharmacia Biotech, Piscataway, NJ) and cross-linked to the membranes by exposure to 120 mJ of UV energy in a Stratalinker 1800 (Stratagene, La Jolla, CA).
The cDNA fragments obtained from DD-RT-PCR, about 375 base pairs (bp) of mouse cystatin SC (mCystatin SC) and 385 bp of mouse cystatin TE-1 (mCystatin TE-1), were radiolabeled with [-32P]dATP using the Rad Prime DNA Labeling Kit (Gibco BRL). The membranes were hybridized overnight at 65°C in a hybridization solution consisting of 1% BSA, 7% SDS, 1 mM EDTA, 0.5 M sodium phosphate pH 7.2, and a corresponding cDNA probe. After hybridization, the blots were washed with 2x SSC/0.1% SDS for 5 min at room temperature, 2x SSC/0.1% SDS for 15 min at 65°C, and 0.2% SSC/0.1% SDS for 15 min at 65°C. The blots were then exposed to a phosphor screen (Molecular Dynamics, Sunnyvale, CA) for 16–48 h. The signals were detected and analyzed using a Molecular Dynamics PhosphorImager 445 SI and ImageQuant (Molecular Dynamics) and Microsoft Excel (Microsoft, Seattle, WA) software. Ribosomal protein S2 (S2) cDNA probe was used for normalizing the amount of loaded RNA [16].
Rapid Amplification of cDNA Ends
Full-length, RNA ligase-mediated (RLM) rapid amplification of 5' and 3' cDNA ends (RACE) was performed with a GeneRacer TM kit (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. Briefly, 5 µg of Sertoli cell total RNA was treated with calf intestinal phosphatase at 50°C for 1 h to remove the 5' phosphates of truncated mRNA and non-mRNA, then treated with tobacco acid pyrophosphatase at 37°C for 1 h, and the decapped RNA was ligated with the GeneRacer RNA oligo (5'-CGACUGGAGCACGAGGACACUGACAUGGACUGAAGGAGUAGAAA-3') at 37°C for 1 h. The RT reaction was performed using avian myeloblastosis virus RT and the GeneRacer oligo(dT) primer (5'-GCTGTCAACGATACGCTACGTAACGGCATGACAGTG[dT]18-3'). The 5' and 3' end cDNAs of cystatin SC and cystatin TE-1 were amplified by initial PCR followed by nested amplification. The specific primers used for RACE reactions were as follows:
The initial PCR was carried out under the following cycling conditions: 2 min of denaturation at 94°C followed by 5 cycles at 94°C for 30 sec and 72°C for 1 min; 5 cycles at 94°C for 30 sec, 67°C for 30 sec, and 72°C for 1 min; and 20 cycles at 94°C for 30 sec, 64°C for 30 sec, and 72°C for 2 min. The nested RACE reaction was done using the following program: 1 cycle at 94°C for 2 min; 30 cycles at 94°C for 30 sec, 67°C for 30 sec, and 72°C for 2 min; and a final extension at 72°C for 10 min. PCR products were gel-purified, cloned into pGEM-T Easy Vector (Promega), and sequenced using T7 END and SP6 END primers. Three clones were sequenced from each nested RACE reaction. The entire cDNA sequences of cystatins SC and TE-1 were amplified from Sertoli cell RNA in RT-PCRs using primers targeting the 5' untranslated region (UTR; p5 primer) and 3' UTR (p1 primer) portions of the cDNA. Potential identity of the proteins encoded by cystatins SC and TE-1 were determined by a homology search with the protein-protein BLAST method (http://www.ncbi.nlm.nih.gov/blast). Multiple protein sequences were aligned by the CLUSTAL W method [17]. Signal peptide and its putative cleavage site were predicted by Nielsen [18] using SignalP v2 (http://www.cbs.dtu.dk/services/SignalP). The prediction of the N-glycosylation site was made using PROSITE established by the Swiss Institute of Bioinformatics (http://ca.expasy.org/cgi-bin/scanprosite/scanprosite). The putative phylogenetic tree was constructed using GeneBee-NET (http://www.genebee.msn.su) established by A.N. Belozersky at the Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia [19, 20]. The phylogenic distances were obtained using the method described by Chumakov and Yushmanov [21, 22].
Reverse Transcriptase-Polymerase Chain Reaction
Five micrograms of DNase-treated total RNA from rat Sertoli cells was annealed with oligo(dT)12–18 at 42°C for 90 min in a solution with SuperScript RT (Gibco BRL) and RNasin (Promega). Primers of mCystatin SC-p1 and -p3 and mCystatin TE-1-p1 and -p3 were used to amplify rat cystatin SC (rCystatin SC) and rat cystatin TE-1 (rCystatin TE-1) from rat Sertoli cell cDNA. The PCR reactions were performed using the following thermal cycle profile: 2 min of denaturation at 95°C followed by 30 cycles at 95°C for 30 sec, 52°C for 30 sec, and 72°C for 1 min; and a final extension step of 72°C for 10 min.
In Situ Hybridization Analysis
In situ hybridization analysis was performed as previously described [23, 24] with slight modifications. Mouse full-length cystatin SC cDNA and cystatin TE-1 cDNA were cloned into pBluescript SK+ (Stratagene). The plasmids were linearized with either NcoI (antisense probe) or PstI (sense probe) to produce a template for in vitro transcription. Radiolabeled antisense or sense probes were generated from a reaction consisting of 10 µl of [-33P]UTP (NEG-607H; NEN, Boston, MA), 20 units of RNase inhibitor (Promega), and 50 units of either T3 or T7 RNA polymerase (Gibco BRL). The cRNA probes were cleaved to about 200 bp by alkaline hydrolysis so that they had similar access to cells in each tissue section. Testes and epididymides were removed from adult mice. Tissues were fixed in freshly prepared 4% (v/v) paraformaldehyde/0.25% glutaraldehyde for 12 h at 4°C. Sections (6 µm thick) were deparaffinized with xylene and treated with 10 µg/ml of proteinase K (Gibco BRL) for 30 min at 37°C, then acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine pH 8.0. Slides were hybridized overnight at 50°C in a hybridization mixture containing 1 x 106 cpm probe/30 µl hybridization solution, stringently washed the next day, dipped in Kodak emulsion NTB-2 (Kodak, Rochester, NY), and exposed for 4–30 days at room temperature. The sections were lightly counterstained with hematoxylin and eosin-Y and mounted with GVA mounting solution (Zymed, South San Francisco, CA).
Photomicroscopy
Sections were observed with a Nikon Microphot-FX microscope (Meridian Instrument Company, Kent, WA), and photomicrographs were taken under brightfield and darkfield illumination with an Olympus OLY-200 digital camera (Olympus America Inc., Melville, NY) using MagnaFire software version 1.0 (Olympus America Inc.). Digital images were captured and assembled using Adobe Photoshop 5.0 (Adobe Systems, Seattle, WA) and Microsoft PowerPoint (Microsoft, Redmond, WA) software.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The purpose of this study was to identify genes specifically expressed in mouse Sertoli cells. DD-RT-PCR was used to compare patterns of mRNA expression from cultured Sertoli cells, liver, and brain taken from 20-day-old mice. The PCR bands present in Sertoli cells but not in brain or liver were isolated, cloned, and sequenced. Two cDNA fragments with no homology to known gene sequences in GenBank but with similarity to expressed sequence tags (ESTs) from mouse and rat testis cDNA libraries were selected for further study.
To determine the entire sequence of these two cDNAs, 5' and 3' RLM-RACE reactions were performed. Single products were obtained after the RACE reaction (data not shown), which suggested that full-length cDNAs had been amplified. One open reading frame (ORF) was identified for each of the two genes, potentially encoding a protein of 130 amino acids and 128 amino acids, respectively. The ORF of each gene starts from the first methionine codon, and multiple stop codons were present after the coding sequence. A nonconventional polyadenylation signal, ATTAAA, was located 14–16 bases upstream of the poly(A) stretch on both genes. The gene encoding the putative 130 amino acid protein was named mCystatin SC (GenBank accession number AF440735), whereas the gene encoding the putative 128 amino acid protein was named cystatin TE-1 (GenBank accession number AF440737).
According to the deduced amino acid sequences, mCystatin SC and mCystatin TE-1 show similarities to family 2 cystatins. Both genes have four conserved cysteine residues at the C-terminal end in precise alignment with those of other members of the family (Fig. 1A). These transcripts also contain secretory signal sequences with putative cleavage sites between position 20 (T) and 21 (I) of mCystatin SC, and between 21 (F) and 22 (K) of mCystatin TE-1. These cleavage sites are at similar positions to those of other cystatin family members (Fig. 1A). Mouse cystatin TE-1 contains a predicted C-terminal N-glycosylation site NST at N122, whereas no N-glycosylation site was found in mCystatin SC. A phylogenetic tree (Fig. 1B) shows the evolutionary relationship between mCystatin SC, mCystatin TE-1, and selected known cystatins. There is a close phylogenetic relationship between mCystatin SC and mCystatin TE-1, and between these two genes and mTestatin. The level of sequence homology between mCystatin SC and mCystatin TE-1 is 45%. The homology between these two genes and other closely related cystatins it is 26%–34%.
|
Although cystatins SC and TE-1 have characteristics of the family of cystatins, they lack some of the three short conserved amino acid regions believed to be important for cysteine proteinase inhibitory function. Cystatin SC protein possesses the Pro103-Trp104 motif, and the Trp104 motif is conserved in cystatin TE-1 (Fig. 1A), whereas other regions commonly present in cystatins are absent.
Molecular Cloning of Rat Cystatin SC and Cystatin TE-1
In order to obtain similar sequences from a rat, RT-PCR was performed using rat Sertoli cell total RNA and primers of mouse cystatins SC and TE-1 under conditions of low stringency. RLM-RACE was used to obtain the full-length cDNA sequences. Two cDNA sequences with long ORFs, named rat (r) cystatin SC (GenBank accession number AF442205) and rCystatin TE-1 (GenBank accession number AF440736), were obtained. The cDNAs for rCystatin SC and rCystatin TE-1 encode 130 and 128 amino acid putative proteins, respectively. Potential signal cleavage sites are between 20 (T) and 21 (I) of rCystatin SC and between 21 (F) and 22 (K) of rCystatin TE-1. These two cDNAs also contain a nonclassical polyadenylation signal, ATTAAA, located 14–16 bases upstream of the poly(A) stretch on both of the genes. The identity between mCystatin SC and rCystatin SC is 96%, and the similarity between mCystatin TE-1 and rCystatin TE-1 is approximately 90%. Rat cystatin TE-1 contains a predicted C-terminal N-glycosylation site, NST at N122, whereas a potential N-terminal N-glycosylation site, NNS at N49, was found in rCystatin SC.
Tissue-Specific Expression of Cystatin SC >and Cystatin TE-1 mRNA
To determine the tissue expression pattern of mCystatins SC and TE-1, we performed Northern blot analyses using mRNA isolated from cultured mouse Sertoli cells and other tissues from adult male and female animals. Figure 2, A and B show that mCystatin SC mRNA transcript was detected only in testis and cultured Sertoli cells, and that the cystatin TE-1 gene was present in epididymis, cultured Sertoli cells, and testis. The cystatin TE-1 transcript was also observed in ovary and prostate, but the signal was very weak. Expression of these two genes was not detected in the other organs we examined.
|
Developmental Expression of mCystatin SC> and mCystatin TE-1 mRNA in Testis
We used Northern blot analysis to examine total testis RNA (10 µg) from mice of different ages (
2 mice/age). The results indicated that mRNA of mCystatins SC and TE-1 was present from Day 0 through 62 days of age, but the signal varied during testis development (Fig. 3, A and B). Both genes displayed similar patterns of developmental expression in testis (Fig. 3C). Expression of mCystatins SC and TE-1 was detected at Day 0. Levels of both transcripts increased sharply at Day 15, remained high between 20 and 30 days of age, and decreased at Day 35. However, cystatin TE-1 mRNA expression decreased between 0 and 10 days of age, whereas that of cystatin SC did not show a significant change. Expression of cystatins SC and TE-1 at Day 62 was consistent with that expression at Day 10, when the lowest expression level occurred.
|
Localization of Cystatin SC and Cystatin TE-1 mRNA> in Testis and Epididymis
We performed in situ hybridization of mouse cystatins SC and TE-1 in order to localize their expression to specific cells in the testis and epididymis. Tissue sections of mouse adult testis and epididymis were incubated with antisense and sense -33P-labeled UTP cRNA probes generated from mCystatin SC and mCystatin TE-1 cDNA. Figure 4, A and B, show that mCystatin SC mRNA was detected only in seminiferous tubules, and there was no expression in testis interstitial tissues. The signal varied among different seminiferous tubules, with low expression during stages VI to VIII. The mCystatin SC transcript was present predominantly in the regions that overlay Sertoli cells, and we detected no apparent expression in spermatogenic cells (Fig. 4, C and D), nor did we observe any mRNA signal in any epididymal region (data not shown). These results suggest that mCystatin SC has a Sertoli cell-specific and stage-dependent expression pattern.
|
Cystatin TE-1 mRNA was localized primarily to Sertoli cells with no apparent signal detected in germ cells or Leydig cells, and no stage-specific expression (Fig. 5, A–D). Hybridization of mCystatin TE-1 antisense probe to a longitudinal section of epididymis is shown in Figure 6, A and B, and indicates that mCystatin TE-1 mRNA was localized to only the very proximal caput epididymis, with no signal in the midcaput, distal caput, corpus, or cauda epididymis. Higher magnification (Fig. 6, C and D) revealed that the mCystatin TE-1 transcript was expressed at different levels in different segments of the proximal caput epididymis. Mouse cystatin TE-1 expression in the boundary between the proximal caput and midcaput is shown in Figure 6, E and F. Messenger RNA expression is high only in the epithelial cells of proximal caput, and we detected no signal in sperm, conjunctive tissue, or in the epithelial cells of midcaput epididymis. We also detected no signal when the sense probe was used (Fig. 4, E and F; Fig. 5, E and F; Fig. 6, G and H). Thus, expression of mCystatin TE-1 mRNA is highly restricted to testis Sertoli cells and to epithelial cells of the proximal region of mouse epididymis, whereas mCystatin SC transcript is expressed only in Sertoli cells with a stage-dependent expression pattern among seminiferous tubules.
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Cystatins SC and TE-1 have several features similar to those of family 2 cystatins, including four characteristic cysteines located in the C-terminus, which suggests they have a common ancestry [27]. However, cystatins SC and TE-1 lack some of the conserved regions believed to be critical for binding to a cysteine proteinase of the papain family [28], such as the N-terminal glycine (Gly-9 in chicken egg white cystatin) and Gln53-X-Val55-X-Gly57 in the first hair loop. Likewise, cystatin SC has the Pro103-Trp104 motif in the second hair loop, whereas only the Trp104 motif is conserved in cystatin TE-1 (Fig. 1A). Three other cystatin-related genes, CRES [29], testatin [30], and cystatin T [31], share this feature with mCystatins SC and TE-1. All these genes contain an ORF that has four conserved cysteine residues, and they share homology with cystatins but they lack the N-terminal and QXVXQ region (Fig. 1A). It is interesting that three cystatins, mTestatin, and mCystatins SC and TE-1, which are expressed in Sertoli cells, have the closest evolutionary relationship (Fig. 1B). This suggests that cystatins SC and TE-1 are new members of the male reproductive subgroup within the family 2 cystatins [30].
The diversity in the inhibitory regions in the male reproductive cystatins may meet the inhibitory requirements of a range of proteinases involved in spermatogenesis and sperm maturation. The effect of some of the conserved amino acid substitutions has been studied by site-directed mutagenesis. When the G11 of hCystatin C was mutated to R, which naturally occurs in hCRES and testatin, the dissociation constant (Ki) for the mutant cystatin C complexes with the endopeptidases papain and cathepsin B were increased by 2000-fold, whereas the Ki value for the cysteine exopeptidase cathepsin C interaction was affected less than 10-fold [32]. These results indicate that substitution of conserved amino acid influences the binding affinity and the efficient inhibition of target cysteine proteinases and affects interactions with different enzymes [32].
Members of the cystatin 2 family have different tissue and cell distributions. Cystatin C is found mainly in cerebrospinal fluid, seminal fluid, and milk; cystatins S, SA, and SN are found in saliva, tears, urine, and seminal fluid [26]; and cystatins E/M and F are expressed in most tissues [33, 34]. In contrast to this wide expression, cystatins SC and TE-1 show tissue- and cell-specific expression patterns. Mouse cystatin SC transcript is expressed only in Sertoli cells in a stage-dependent pattern (Figs. 2A and 4), whereas mCystatin TE-1 mRNA expression is highly restricted to testis Sertoli cells (Figs. 2B and 5) and to epithelial cells in the proximal region of mouse epididymis (Fig. 6). Three other cystatins have been reported expressed predominantly in the male reproductive tract. CRES is highly restricted to the proximal region of the epididymis [29] and shows little expression in testis [29] and anterior pituitary gonadotropes [35], whereas testatin is expressed only in the pre-Sertoli cells of fetal testis and in Sertoli cells of adult testis [30], and spermatogonia [36]. Cystatin T is expressed exclusively in the testis [31] and is predominant in spermatogenic cells (unpublished data). Although the CRES expression pattern in epididymis is similar to that of mCystatin TE-1, differences in mCystatin TE-1 mRNA levels have been found among epididymal tubules. Moreover, cystatin TE-1 gene expression in the testis is localized to Sertoli cells in no stage-dependent pattern, whereas CRES mRNA is restricted to spermatogenic cells and is stage-specific [37]. The complicated expression pattern of these cystatins suggests that they might interact with different proteinases in male reproductive tract function.
Several cystatins have been studied in testis and epididymis. For example, cystatin C is expressed in Sertoli cells in a stage-dependent manner and its mRNA levels show a peak expression between stages XIII and II and a nadir between stages VI and VIII [38]. Cathepsin L activity can be inhibited by cystatin C in vitro [6] and has been reported to have an opposite pattern of mRNA expression in seminiferous epithelia. Some researchers have proposed that cathepsin L may facilitate the movement of condensing spermatids toward the lumen at stages VI through VIII [39, 40], and that cystatin C may inhibit cathepsin L activity in testis seminiferous tubules [41]. CRES protein, which is localized in sperm acrosomes and is released during the acrosome reaction, is believed to regulate the intra-acrosomal protein processing or it may be involved in the fertilization process [42]. Because cystatins SC and TE-1 mRNAs are highly restricted to testis Sertoli cells, these two genes, like other proteinase inhibitors expressed by Sertoli cells and their corresponding proteases, might work synergistically in germ cell adherence to Sertoli cells and the subsequent formation of intercellular junctions during germ cell migration [43]. Cystatin TE-1, which shares a similar expression pattern to that of CRES mRNA in the epididymis, may also regulate the release and activity of enzymes that are critical for sperm maturation and fertilization [42].
The unique expression patterns of mCystatin SC and TE-1 during testis development, which are different from that of cystatin C [41], indicate that cystatins SC and TE-1 play specific roles in testis. In mice and rats, during the first weeks after birth, the seminiferous cords undergo morphological changes that are extremely important to spermatogenesis; these include relocation of gonocytes to the basement membrane of the cord, development of Sertoli-Sertoli junctional complexes leading to epithelial compartmentalization, and the beginning of meiosis by germ cells. During this period, gonocytes and Sertoli cells interact in potentially important ways [44]. The decrease of mCystatin TE-1 mRNA expression from Day 0 to Day 10 after birth (Fig. 3) raises the possibility that unknown corresponding proteinases may have an opposite expression pattern and may participate in the process of migration of postnatal gonocytes. The process of Sertoli-Sertoli cell junction formation is essentially completed between 10 and 16 days in mice [45, 46]. Likewise, the steep increase in mRNAs of mCystatin SC and TE-1 in 15-day-old testis that remains high until 30 days after birth indicates that these two genes may play a role in tight junction formation. The initiation of meiosis by germ cells also occurs in most mammals at about the same time as the appearance of Sertoli-Sertoli junctional complexes [44]. These two new cystatin-like proteins may take part in germ cell-Sertoli cell interaction and may play a role in germ cell meiosis during testis development.
In summary, we identified and characterized two new members of type 2 cystatins: cystatins SC and TE-1. Their deduced amino acid sequences lack some of the three short conserved regions believed to be important for cysteine proteinase inhibitory activity. Both genes show specific expression in the male reproductive system. The mCystatin SC transcript is expressed in Sertoli cells with a stage-dependent expression pattern, whereas mCystatin TE-1 mRNA is localized predominantly to testis Sertoli cells and to epithelial cells of proximal region of mouse epididymis. The results indicate that these two genes are new members of the male reproductive subgroup of family 2 cystatins. Further study will be necessary to determine which enzymes can be inhibited by the male reproductive cystatins and to determine the roles of cystatins SC and TE-1 in spermatogenesis.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence. FAX: 509 335 9688; griswold{at}mail.wsu.edu
Received: 16 January 2002.
First decision: 8 February 2002.
Accepted: 27 June 2002.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  P J O'Shaughnessy, L Hu, and P J Baker Effect of germ cell depletion on levels of specific mRNA transcripts in mouse Sertoli cells and Leydig cells Reproduction, June 1, 2008; 135(6): 839 - 850. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. A. Jelinsky, T. T. Turner, H. J. Bang, J. N. Finger, M. K. Solarz, E. Wilson, E. L. Brown, G. S. Kopf, and D. S. Johnston The Rat Epididymal Transcriptome: Comparison of Segmental Gene Expression in the Rat and Mouse Epididymides Biol Reprod, April 1, 2007; 76(4): 561 - 570. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R.-S. Wang, S. Yeh, L.-M. Chen, H.-Y. Lin, C. Zhang, J. Ni, C.-C. Wu, P. A. di Sant'Agnese, K. L. deMesy-Bentley, C.-R. Tzeng, et al. Androgen Receptor in Sertoli Cell Is Essential for Germ Cell Nursery and Junctional Complex Formation in Mouse Testes Endocrinology, December 1, 2006; 147(12): 5624 - 5633. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. J. Asirvatham, M. Schmidt, B. Gao, and J. Chaudhary Androgens Regulate the Immune/Inflammatory Response and Cell Survival Pathways in Rat Ventral Prostate Epithelial Cells Endocrinology, January 1, 2006; 147(1): 257 - 271. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Li, C. A. Putnam-Lawson, H. Knapp-Hoch, P. J. Friel, D. Mitchell, R. Hively, and M. D. Griswold Immunolocalization and Regulation of Cystatin 12 in Mouse Testis and Epididymis Biol Reprod, November 1, 2005; 73(5): 872 - 880. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Tohonen, J. Frygelius, M. Mohammadieh, U. Kvist, L. J. Pelliniemi, K. O'Brien, K. Nordqvist, and A. Wedell Normal Sexual Development and Fertility in testatin Knockout Mice Mol. Cell. Biol., June 15, 2005; 25(12): 4892 - 4902. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. A. L. Tan, K. De Gendt, N. Atanassova, M. Walker, R. M. Sharpe, P. T. K. Saunders, E. Denolet, and G. Verhoeven The Role of Androgens in Sertoli Cell Proliferation and Functional Maturation: Studies in Mice with Total or Sertoli Cell-Selective Ablation of the Androgen Receptor Endocrinology, June 1, 2005; 146(6): 2674 - 2683. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Johnston, P. J. Baker, M. Abel, H. M. Charlton, G. Jackson, L. Fleming, T. R. Kumar, and P. J. O'Shaughnessy Regulation of Sertoli Cell Number and Activity by Follicle-Stimulating Hormone and Androgen during Postnatal Development in the Mouse Endocrinology, January 1, 2004; 145(1): 318 - 329. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. G. Cooper, A. Wagenfeld, G. A. Cornwall, N. Hsia, S. T. Chu, M.-C. Orgebin-Crist, J. Drevet, P. Vernet, C. Avram, E. Nieschlag, et al. Gene and Protein Expression in the Epididymis of Infertile c-ros Receptor Tyrosine Kinase-Deficient Mice Biol Reprod, November 1, 2003; 69(5): 1750 - 1762. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P.J. O'Shaughnessy, L. Fleming, P.J. Baker, G. Jackson, and H. Johnston Identification of Developmentally Regulated Genes in the Somatic Cells of the Mouse Testis Using Serial Analysis of Gene Expression Biol Reprod, September 1, 2003; 69(3): 797 - 808. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Li, P. J. Friel, D. J. McLean, and M. D. Griswold Cystatin E1 and E2, New Members of Male Reproductive Tract Subgroup Within Cystatin Type 2 Family Biol Reprod, August 1, 2003; 69(2): 489 - 500. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
