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Testis |
Division of Reproductive Biology,3 Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, Maryland 21205
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gene regulation, Sertoli cell, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As mammalian spermatogenic cells divide and differentiate, they interact extensively with the adjacent Sertoli cells (reviewed in [8]). Distinct populations of spermatogonia, spermatocytes, and spermatids mature synchronously, progressing through the stages of the cycle of the seminiferous epithelium [9]. During this progression, the male germ cells engage Sertoli cells in specific and cyclic sets of cellular interactions. In the mouse and rat testes, the progression of the male germ cells through the stages of the cycle has differential effects on the expression of specific Sertoli cell mRNAs. Transcripts of several genes that encode cell adhesion molecules, growth factors, transport proteins, hormone receptors, metabolites, proteases, and protease inhibitors are expressed at specific stages of the cycle of the seminiferous epithelium [10–12]. It has been proposed that the product of these genes expressed in a stage-specific manner may regulate the development of the male germ cells present in those stages [13–15]. However, contrary to the progress achieved in identifying promoter regions that control gene expression in the developing male germ cells, no promoter has been identified that regulates stage-specific expression in Sertoli cells in a manner comparable to the endogenous gene.
In order to gain insights into this fundamental issue, we have been analyzing the transcriptional regulation of the cathepsin L gene in Sertoli cells. Cathepsin L is a cysteine protease synthesized in a proenzyme form that requires proteolytic processing in order to be active (reviewed in [16]). Levels of cathepsin L mRNA in rat Sertoli cells are highly regulated with respect to the stages of the cycle of the seminiferous epithelium. During the end of puberty in the rat (from 30 to 60 days of age), levels of cathepsin L mRNA per Sertoli cell increase 6-fold [17]. Simultaneously, germ cells at specific stages of the cycle of the seminiferous epithelium begin to stimulate or to repress expression of cathepsin L mRNA in Sertoli cells. At 30 days of age, low levels of this transcript in rat Sertoli cells are detected at all stages of the cycle, as determined by in situ hybridization. However, by 40 days of age, this transcript is only detected in Sertoli cells of tubules at stages VI–VIII. Upon completion of the first round of spermatogenesis (about 45 days of age), expression of this transcript is markedly increased in Sertoli cells of tubules at stages VI–VIII [13, 18]. This stage-specific expression of the cathepsin L gene in Sertoli cells of mature animals is regulated by sequential inhibitory and stimulatory signals from germ cells [18]. Thus the cathepsin L gene represents an excellent candidate for identification of a promoter region that mediates the response of Sertoli cells to stage-specific interactions with male germ cells.
In vivo, to ensure the timely division and differentiation of the male germ cells, complex interactions take place between the germ cells and the somatic Sertoli cells. Since these interactions cannot be fully replicated in vitro, we have used a transgenic mouse model to identify the promoter that regulates in vivo the stage-specific transcription of the cathepsin L gene in Sertoli cells. The present study establishes that regulatory elements that are sufficient to recapitulate the expression pattern of the endogenous cathepsin L gene in Sertoli cells are located within the 3-kilobase (kb) region located immediately upstream of the translation start site of this gene. To our knowledge, this is the first identification of a promoter region that conveys stage-specific expression in Sertoli cells in a manner comparable to that of the endogenous gene.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The 9.5-kb transgenic construct cath L (-2065/ +977)-LacZ was derived from the construct cath L (-2065/+977)-Luc that contains a 3042-bp genomic fragment upstream of the translation start site of the rat cathepsin L gene [19], fused to the firefly luciferase coding sequence. The 3042-bp fragment (GenBank accession AF025476) includes 2065 bp of genomic sequence upstream of the rat cathepsin L transcription start site, the first exon (74 bp), the first intron (891 bp), and the first 11 bp of exon 2 (see Fig. 1, top). PCR was performed with the Expand High Fidelity PCR System (Roche, Indianapolis, IN) according to the manufacturer's instructions. The forward primer 5'-GATCT
GAGCTCACCTCATTTTTGT-3' and the reverse primer 5'-GACGT
GGTTCAAACACCTGGGGAA-3' were used to amplify a DNA fragment spanning -2065 to +977. After digestion with KpnI and HindIII (underlined sequences), the amplified DNA fragment was subcloned into pGL2-Basic (Promega Corp., Madison, WI) digested with KpnI and HindIII, yielding the construct cath L (-2065/+977)-Luc. The nucleotide sequence of the cathepsin L fragment was verified by DNA sequencing.
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The assembly of the transgenic construct required three steps: 1) removal of the firefly luciferase coding sequence plus an intron and the polyadenylation signal from the construct cath L (-2065/+977)-Luc. The luciferase-based construct was cut with HindIII and SalI, giving rise to two DNA fragments: a 5907-bp DNA fragment that contains the cathepsin L 5'-upstream region and a 2697-bp fragment that included the firefly luciferase coding sequence plus the SV40 intron and polyadenylation sequence. After fractionating the cut plasmid DNA on an agarose gel, the 5907-bp DNA fragment was isolated and purified using the GENECLEAN II system (BIO 101, Vista, CA). 2) Assembly of the construct cath L (-2065/+977)-SH in which a single SalI site is located upstream of a single HindIII site. The 5907-bp DNA fragment was ligated with a 34-bp synthetic double-stranded oligomer, yielding the construct cath L (-2065/ +977)-SH. The oligomer contained a 5'-mutated HindIII overhang, a SalI restriction site located upstream of a HindIII restriction site, and a 3'-mutated SalI overhang (the overlapping 5' and 3' ends of the double-stranded oligomer were designed to ensure that only the internal HindIII and SalI sites found within the double-stranded oligomer would remain functional). The location of these two restriction sites directed the insertion of the LacZ sequence in the sense orientation. 3) Insertion of the LacZ coding sequence into the construct cath L (-2065/ +977)-SH. The DNA fragment that contains the Escherichia coli LacZ coding sequence plus an intron and polyadenylation sequence from the mouse metallothionein II gene was excised from the plasmid pLacI [20] following digestion with SalI and HindIII and purified as described above. The DNA fragment was then subcloned into the construct (-2065/+977)-SH cut with SalI and HindIII, giving rise to the transgenic construct cath L (-2065/ +977)-LacZ. In this construct, the ATG initiation codon of the LacZ gene is located 44-bp downstream of the nucleotide at position +977 in the cathepsin L sequence. The structure of the transgenic construct was verified by analysis with restriction enzymes, and the 3-kb upstream region derived from the rat cathepsin L gene present in the transgenic construct was completely sequenced.
Production and Identification of Transgenic Mice
The 6.6-kb DNA insert was purified from the construct cath L (-2065/+977)-LacZ as previously described [21] and injected into single cell B6SJL embryos. The embryos were transferred to eight pseudopregnant ICR female recipients and 55 offspring were born. Tail samples were collected from the offspring at 3 wk, and genomic DNA was isolated as previously described [21]. The identification of the transgenic founders was performed by Southern blot hybridization with a LacZ DNA. Each founder was bred to obtain heterozygous F1, F2, and F3 male transgenic offspring. The identification of transgenic F1, F2, and F3 offspring was carried out by polymerase chain reaction as previously described [22], using a forward primer corresponding to a 21-bp sequence in the cathepsin L upstream region (5'-TTGAGAGATGGAGGGGCATTG-3') and a reverse primer corresponding to a 21-bp sequence in the LacZ gene (5'-CATCGTAACCGTGCATCTGCC-3'). The use of mice for all studies described herein was approved by the Institutional Animal Care and Use Committee of Johns Hopkins University.
ß-Galactosidase Assay
ß-Galactosidase enzymatic activity in different organs was assayed as previously described [22]. Reactions were carried out for 10 min at room temperature, and light emissions were measured for 10 sec using a Monolight 3010 luminometer (Analytical Luminescence Laboratory, Sparks, MD). Activity was expressed as (relative light) units per minute per milligram of protein or as (relative light) units per minute per testis.
Histology
Testes were fixed and incubated in 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal) for 48 h in a 30°C water bath as previously described [22]. Prior to being processed for histology, testes were photographed using a Wild M400 photomacroscope with a zoom objective. Testes were then embedded in paraffin, cut into serial sections (5–6 µm), mounted on glass slides, and deparaffinized. Testis sections were counterstained either with nuclear fast read or with periodic acid Schiff and photographed using a Nikon Eclipse E800 microscope equipped with a digital camera (Princeton Instruments, Trenton, NJ).
Staging of the Seminiferous Tubules
The seminiferous tubules that had a discernible lumen and contained at least two Sertoli cells that expressed the reporter gene were grouped into one of three sets of stages (I–V, VI–VIII, or IX–XII). Stages were identified either by the acrosomal morphology (as revealed by periodic acid Schiff reaction) or by the nuclear morphology of the spermatids, their position within the seminiferous epithelium, and the distribution of their cytoplasm. Based on these criteria, two different analyses were performed to evaluate the stage-specific expression of the transgene. In an initial evaluation, testis sections from three different animals were examined, and for each animal the stages of the cycle of 15–20 tubules that expressed the transgene were identified. For a more precise analysis of stage-specific expression, serial sections of two transgenic testes were examined, and the stage of the cycle of each tubule that expressed the transgene was determined. In this analysis, a tubule that was recovered on more than one histological section was only counted once. A total of 83 separate tubules expressing the transgene were identified and the percentage of positive tubules in each of the three sets of stages noted above was calculated. The same sections were then re-evaluated for the stages of the cycle of all tubules regardless of whether or not they expressed the transgene. Stage-specific expression of the transgene was tested by comparing the percentage of tubules at stages I–V, VI–VIII, or IX–XII that expressed the transgene with the percentage of all tubules at these stages, regardless of whether or not they expressed the transgene.
Statistical Analyses
Statistical comparison of ß-galactosidase enzymatic activity in tissue homogenates was conducted by ANOVA using StatView 5.0.1 (SAS Institute, Inc., Cary, NC). Chi-square analysis was used to compare the percentage of tubules at stages I–V, VI–VIII, or IX–XII that expressed the transgene with the percentage of all tubules at those stages, regardless of whether or not they expressed the transgene. Statistically significant differences were defined as P 
0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The primary goal of the experiments presented below was to test if the 5' region located immediately upstream of the translation start site of the rat cathepsin L gene could drive expression of a reporter gene in murine Sertoli cells in a manner comparable to that of the endogenous gene. To validate our choice of testing the promoter activity of a DNA fragment derived from a rat gene in mice, it is important to stress the similarities between mice and rats as far as spermatogenesis and expression of the cathepsin L gene are concerned: 1) both murine and rat spermatogenesis take place in distinct stages, 14 (I–XIV) in the rat [9] and 12 (I–XII) in the mouse [23]. In both species, spermiation takes place during stage VIII. 2) The proenzyme form of cathepsin L is secreted by both mouse and rat mature Sertoli cells [24]. 3) In vivo, mature mouse Sertoli cells express cathepsin L mRNA in the same stage-specific manner as mature rat Sertoli cells [24].
In preparation for the production of transgenic mice and to avoid possible cloning artifacts, the construct cath L (-2065/+977)-LacZ was tested for reporter gene activity in transient transfection assays using the murine Sertoli cell line TM4 [25]. ß-Galactosidase enzymatic activity in cells transfected with the construct cath L (-2065/+977)-LacZ was 125-fold higher than that measured in cells transfected with the plasmid lacking the cathepsin L promoter region (data not shown). A 6.6-kb DNA fragment derived from the construct cath L (-2065/+977)-LacZ was then used to generate transgenic mice. The DNA fragment used for injection included a 3-kb DNA fragment located immediately upstream of the rat cathepsin L translation start site, fused to the LacZ coding sequence (Fig. 1, bottom). Four independent founders were identified: two males (founders 904 and 908) and two females (founders 934 and 943). Slot-blot analysis established that founders 904, 908, 934, and 943 had eight, four, one, and three copies of the transgene, respectively (data not shown). Each founder was bred with wild-type male or female B6SJL-F1 mice to produce heterozygous F1, F2, and F3 progeny for analysis. Transmission of the transgene proceeded according to Mendelian laws in the transgenic lines studied.
The Expression of the Reporter Gene Is Restricted to the Testes of Transgenic Mice
To determine which organs of the transgenic mice express the reporter gene, ß-galactosidase enzymatic activity was measured in extracts from various tissues of mice from the four founder lines. High levels of ß-galactosidase enzymatic activity were detected in the testis of male mice from lines 904 and 908, compared with the testis of nontransgenic litter mates (Fig. 2). Mean ß-galactosidase enzymatic activity measured in testis extracts from line 904 was 
87 000 U min-1 mg-1 of protein, compared to 50 000 U min-1 mg-1 of protein in testis extracts from line 908. Since mice from line 904 have twice the number of copies of the transgene as mice from line 908, there is a correlation between transgene copy number and ß-galactosidase enzymatic activity. We found that levels of ß-galactosidase enzymatic activity in all of the seven other organs tested were comparable with background levels exhibited in nontransgenic control mice (Fig. 2). Finally, all organs of mice from lines 934 and 943, including testes, exhibited the same background level of ß-galactosidase enzymatic activity as those from nontransgenic controls (data not shown). This was presumably due to the insertion of the transgene at a chromosomal location that suppressed transcription.
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The Expression of the Transgene Is Heterogeneous Within Individual Seminiferous Tubules
Following immersion in buffer containing the substrate X-gal, the testes of male mice from line 904 stained blue, indicative of expression of the LacZ reporter gene (Fig. 3A). Moreover, expression of the reporter gene was confined to the seminiferous tubules. The stained tubules were randomly distributed over the surface of the testis. X-gal reaction product is clearly visible in some seminiferous tubules, yet it is undetectable in the immediate adjacent tubules. An identical result was also obtained with testes of mice from line 908 (data not shown). The testes from nontransgenic control mice show a faint blue staining (Fig. 3B) due to endogenous ß-galactosidase activity observed in Leydig cells, which reside in the interstitial compartment between the seminiferous tubules. Close inspection of the testes of mice from lines 904 and 908 revealed heterogeneity in the expression of the reporter gene within the individual seminiferous tubules. As shown in Figure 3C, X-gal reaction product is visible only in a portion of the tubule. A higher magnification of the area of the tubules that express the transgene revealed a punctate blue staining pattern (Fig. 3D).
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Somatic Sertoli Cells Are the Only Cells Within the Seminiferous Epithelium that Express the Transgene
To establish the identity of the X-gal positive cells within the seminiferous tubules, the testes of mice from line 904 were embedded in paraffin, sectioned, and counterstained with nuclear fast red. Examination of these sections by light microscopy revealed that positive blue staining was clearly evident in the somatic Sertoli cells (Fig. 4). The staining was detected throughout the major axis of the Sertoli cells, which give rise to the punctate blue staining pattern observed in the intact seminiferous tubules (Fig. 3D). No staining was detected in the spermatogenic cells or the peritubular myoid cells (Fig. 4). X-gal positive staining was also observed only in Sertoli cells of testes of mice from line 908 (data not shown). No staining was observed in any cell populations of the seminiferous epithelium of nontransgenic litter mates (data not shown).
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The Expression of the LacZ Gene under the Control of the Rat Cathepsin L Promoter Is Predominantly Detected in Sertoli Cells Within Stage VI to VIII Seminiferous Tubules
In order to establish if the reporter gene was expressed at the same stages as the endogenous cathepsin L gene, the stages of the cycle of the seminiferous epithelium that contained X-gal reaction product were determined according to morphological criteria [26]. In an initial analysis of three different transgenic mice, 70%–100% of the X-gal positive tubules in each testis were at stages VI–VIII (data not shown). A detailed analysis of the stage-specific expression of the transgene was then performed. The stage of the cycle of 83 individual X-gal positive tubules in serial sections of testes from transgenic mice was determined (group A). The same sections were re-examined and the stage of all the tubules (X-gal negative and positive tubules) was determined (group B). The seminiferous tubules in both groups A and B were divided into three sets: stages I–V, VI–VIII, and IX–XII. If the transgene is expressed in a stage-specific manner, the stage-specific distribution in group A should be significantly different than in group B. When the stage of the cycle of the X-gal positive tubules was established, 62 of the 83 tubules (75%) were at stages VI–VIII (Fig. 5). Only eight tubules (10%) were at stages I–V, and most were at stage V. Finally, 13 tubules (15%) were at stages IX–XII, with most at stage IX. When the stage of all of the tubules (X-gal negative and positive tubules) in the same sections was established, 31% of these tubules were at stages I–V, whereas 37% were at stages VI–VIII and 32% were at stages IX–XII. It is important to note that this distribution is consistent with that reported in previous studies [26]. The difference in the stage-specific distribution of tubules in groups A and B was statistically significant and clearly established that the transgene was expressed in a stage-specific manner. Expression of the transgene in stage VI–VIII Sertoli cells was also observed in mice from line 908 (data not shown). These results clearly demonstrate that the transgene was predominantly expressed in stage VI–VIII Sertoli cells, mimicking the expression pattern of the endogenous cathepsin L gene in Sertoli cells.
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In addition to the stage-specific expression of the cathepsin L gene in mature Sertoli cells, levels of rat cathepsin L transcript per Sertoli cell increase with the completion of testis maturation [17]. Testes from mature 64-day-old rats (producing adult numbers of spermatozoa) express 6-fold higher levels of cathepsin L mRNA than testes from immature 40-day-old rats (containing no spermatozoa, but up to step 10 spermatids). In order to test if expression of the transgene increased in a similar fashion, ß-galactosidase enzymatic activity was measured in immature testes collected from 25-day-old transgenic mice and in mature testes from adult transgenic mice (90 to 130 days old). Sertoli cell proliferation stops by Postnatal Day 12 in mice [27], and the number of Sertoli cells does not change thereafter. Testes from 25-day-old mice contain up to step 9 spermatids, whereas the testes of 90- to 130-day-old mice have undergone several rounds of spermatogenesis. If the transgene behaved like the endogenous cathepsin L gene, we predicted that ß-galactosidase enzymatic activity in testis extracts of 90- to 130-day-old mice should be significantly higher than that in testis extracts of 25-day-old mice.
The data in Figure 6 are expressed as units per minute per testis (instead of units per minute per milligram of protein) to account for the fixed number of Sertoli cells versus the increased number of male germ cells in the testis extracts of mice at different ages. These results reveal that activity of the transgene in testis extracts of adult mice was 4-fold higher than that in testis extracts of 25-day-old mice. These results suggest that the maturation-dependent increase in the expression of the transgene parallels the maturation-dependent increase in transcript levels observed for the endogenous rat cathepsin L gene at the end of puberty.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As mentioned above, the 3-kb promoter region drives the expression of the reporter gene only in the testes of mice from two independent transgenic lines (Fig. 2). However, the expression of the endogenous rat cathepsin L gene is not restricted to the testis. High levels of rat cathepsin L transcripts are also detected in kidney and liver, whereas low mRNA levels are measured in brain, heart, lung, and spleen [13]. It is of interest that, except for the testis, the genomic fragment used in this study did not drive transcription of the reporter gene in organs in which the endogenous cathepsin L gene is normally highly expressed (Fig. 2). This lack of expression indicates that the regulatory elements that drive high levels of cathepsin L mRNA in kidney and liver are missing from the 3-kb promoter region used in this study. The use of different regulatory elements to drive transcription in different somatic tissues has been previously reported. For example, in the case of the apolipoprotein B gene, the regulatory elements required for expression in the liver are located within a 5-kb region upstream of the transcription start site, whereas the intestinal control region is located 57-kb 5' of this gene [28].
Identification of a Promoter Region that Conveys Stage-Specific Expression in Sertoli Cells in a Manner Comparable to that of the Endogenous Gene
Prior to the analysis of the promoter region of the cathepsin L gene, three other promoter regions had been reported to drive stage-specific gene expression in Sertoli cells of transgenic mice. They include the promoter regions of the murine Müllerian inhibiting substance (MIS) gene, the bovine oxytocin gene, and the human androgen-binding protein/sex hormone-binding globulin (ABP/SHBG) gene.
In the case of the murine MIS promoter [29], high expression of the transgene was detected in mature Sertoli cells of tubules at stages VII–VIII. However, since expression of the endogenous MIS gene is barely detectable in mature Sertoli cells [30], it appears likely that the insertion of the transgene at a chromosomal location that activates transcription in mature Sertoli cells was responsible for the aberrant stage-specific expression of the reporter gene. Sertoli cells of transgenic mice that carry the bovine oxytocin gene under the control of its own promoter expressed the oxytocin protein in tubules at stages I–V and X–XII [31]. However, the endogenous oxytocin gene is not expressed in murine testes, nor has it been established that the endogenous bovine oxytocin gene is expressed in Sertoli cells in a stage-specific manner [32]. Finally, Sertoli cells of transgenic mice that carry the human ABP/SHBG gene under the control of its own promoter expressed high levels of ABP/SHBG transcripts in tubules at stages VI–X [33]. However, it is not known if the endogenous human or mouse ABP/SHBG genes are expressed in a stage-specific fashion.
In contrast, the results from the present study clearly demonstrate that the transgene was predominantly expressed in seminiferous tubules at stages VI–VIII, mimicking the expression pattern of the endogenous mouse and rat cathepsin L genes in Sertoli cells. In addition, ß-galactosidase enzymatic activity in testis extracts of adult transgenic mice was shown to be 4-fold higher than that in testis extracts of 25-day-old transgenic mice. These results suggest that the maturation-dependent increase in the expression of the transgene parallels the maturation-dependent increase in transcript levels observed for the endogenous rat cathepsin L gene at the end of puberty [17]. To our knowledge, the promoter region of the rat cathepsin L gene is the first promoter region to convey stage-specific expression in Sertoli cells in a manner comparable to that of the endogenous gene.
The Regulatory Elements Responsible for Stage-Specific Expression in Sertoli Cells Are Located Within a 3-kb DNA Fragment
The results from the present study have established that the regulatory elements required for stage-specific expression of the cathepsin L gene in Sertoli cells are located proximal to the transcription start site. It is interesting to consider whether or not regulatory elements required for stage-specific gene expression in Sertoli cells are usually located proximal to the transcriptional start site.
To address this question, we summarize below the information available on the promoter of two other genes that have been shown to be expressed in a stage-specific fashion in rat Sertoli cells. They are the promoter of the follicle-stimulating hormone receptor (FSHR) gene and of the inhibin-
gene. Levels of FSHR transcripts are highest in rat Sertoli cells from tubules at stages XIII–II and decrease to a minimum during stages VII and VIII [34], whereas levels of inhibin- mRNA are highest in rat Sertoli cells from tubules at stages XIV–IV [35]. A 5-kb DNA fragment that spans the transcription start site of the rat FSHR gene (spanning -5000 to +123) was used to drive expression of the Cre recombinase reporter gene in transgenic mice. Although testes from eight independent transgenic lines were shown to express the reporter gene, the expression of the Cre recombinase gene was not detected in Sertoli cells, but in male germ cells [36]. To test the activity of the inhibin- promoter in Sertoli cells, a 6-kb DNA fragment that spans the transcription start site of the murine gene (spanning -6000 to +71), fused to the coding sequence of the human Bcl-2 gene was used to generate transgenic mice [37]. In this case, the expression of the reporter gene was observed in Sertoli cells, but the 6-kb promoter region failed to direct expression of the reporter gene in a stage-dependent manner. The negative outcome from these two studies indicates that regulatory elements located upstream or downstream of the DNA regions used are required for stage-specific expression in Sertoli cells. In contrast to the compact nature of the male germ cell-specific promoters, it appears that Sertoli cell promoters span several kilobases.
One structural feature that distinguishes the cathepsin L gene from the FSHR and inhibin- genes is the presence of an intron upstream of the exon that contains the translation start site (Fig. 1). As discussed below, the first intron of the rat cathepsin L gene contains regulatory elements that enhance transcription (unpublished results). Therefore, it appears that the organization of the rat cathepsin L promoter may be unique because of the proximal location of the regulatory elements around the transcription start site and the presence of regulatory elements within an intron located upstream of the translation start site. Clearly, the identification of additional stage-specific promoters that activate transcription in Sertoli cells will be needed to validate this observation.
Three Functional Domains Within the Rat Cathepsin L Promoter
The next step in the characterization of the cathepsin L promoter will be to identify the regulatory elements that are required for the stage-specific expression of this gene in Sertoli cells. We have started to delineate such elements by performing transient transfection analyses of reporter gene constructs that contain different portions of this 3-kb promoter region in primary Sertoli cell cultures. The results from these experiments support a working model in which the cathepsin L promoter can be divided into three functional domains.
The first domain is a 200-bp region that flanks the transcription start site of the rat cathepsin L gene. Transfection analyses using primary rat Sertoli cell cultures have established that this 200-bp region of the rat cathepsin L gene is critical for promoter activity in mature Sertoli cells [19] (unpublished results). The second domain corresponds to the first intron of the cathepsin L gene (spanning +75 to +966). Transfection analyses in primary rat Sertoli cell cultures indicate that the first intron of the rat cathepsin L gene contains regulatory elements that activate transcription in both immature and mature rat Sertoli cells (unpublished results). Finally, a third domain appears to be responsible for the repressive effects mediated by male germ cells. Transfection analysis has demonstrated that two constructs that contain either 2065-bp or 244-bp upstream of the transcription start site of the rat cathepsin L gene had similar activities in mature Sertoli cells. However, addition of pooled male germ cells to the transfected Sertoli cells only affected the activity of the longer construct, which was reduced by 30% [19]. This result, coupled with the observation that transcription of the cathepsin L gene in mature Sertoli cells is repressed at most stages of the cycle of the seminiferous epithelium, are consistent with the hypothesis that male germ cells modulate transcription of the cathepsin L gene in Sertoli cells [18].
The biological functions of these three domains have now been validated by the results presented in this report demonstrating that the transcriptional activators and repressors that regulate the stage-specific expression of the rat cathepsin L gene in Sertoli cells in vivo are contained within a 3-kb promoter region. Future experiments using a combination of both in vitro and in vivo experimental approaches should enable us to define the specific regulatory elements and the transcription factors that confer stage-specific expression of the cathepsin L gene in Sertoli cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: William W. Wright, Johns Hopkins University Bloomberg School of Public Health, Department of Biochemistry and Molecular Biology, Room 3508, 615 North Wolfe St., Baltimore, MD 21205. FAX: 410 614 2356; wwright1{at}jhem.jhmi.edu
Received: 24 September 2002.
First decision: 16 October 2002.
Accepted: 22 November 2002.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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