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Male Reproductive Tract |
a Station de Physiologie de la Reproduction et des Comportements, UMR INRA-CNRS 6073, 37380 Nouzilly, France
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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epididymis, male reproductive tract, puberty
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The establishment of regionalized epididymal protein secretion takes place progressively during postnatal development and before puberty, following testis development. Other researchers have described the changes in epididymal secretory activity in rodents [4, 5] and swine [6] during postnatal development. The molecular mechanisms resulting in the establishment of regionalized gene expression in the epididymis are under testicular control and involve numerous and complex regulatory elements that have not yet been fully elucidated. The biochemical changes in the epididymis during postnatal development coincide with the development of testicular activity, in particular increases in blood and luminal androgen levels, differentiation of germ cells, and secretion of Sertoli-derived factors in the epididymal tubule. For example, the mRNA level of the proenkephalin gene in the rat increases when the first spermatozoa reach the epididymal tubule, suggesting direct regulation by sperm-related factors [7]. Another example is the expression of the murine epididymal retinoic acid-binding protein gene, which is correlated with the increase in androgens and androgen receptor content during postnatal development, suggesting direct regulation via blood androgens [8].
Most of the previous studies concerning the developmental establishment of the epididymis were performed in rodents. However, the domestic animal model provides larger organs from which tissues from different regions can be easily obtained, even in the early stages of postnatal development. The larger epididymis allows the investigation of protein secretion in the lumen, as previously performed in our laboratory [6], whereas these areas are less accessible in rodents.
We have previously reported that one of the major sheep epididymis proteins that presents a high level of spatial specificity is lipocalin prostaglandin D2 synthase (PGDS). In the adult sheep, both PGDS mRNA and protein expression are restricted to the proximal caput of the epididymis. The expression and synthesis of PGDS is not dependent on endocrine or exocrine testicular factors because efferent duct ligation or castration does not abolish mRNA expression or protein synthesis in the caput epididymis [9].
In the present study, we investigated PGDS expression in terms of mRNA and protein in the ovine epididymis during postnatal development. The results obtained showed three mechanisms of regulation contributing to the definitive pattern of PGDS expression, all occurring during the prepuberty period: 1) regulation at the mRNA level, as revealed by the progressive restriction of the presence of mRNA in the proximal part of the epididymis, 2) translation regulation, as revealed by spatial and temporal differences between the presence of mRNA and protein synthesis, and 3) posttranslation regulation, as revealed by different molecular characteristics on two-dimensional electrophoresis of the protein in the developing versus mature epididymis. The results obtained contribute to a better understanding of the spatial establishment of gene expression in the epididymis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dulbecco modified Eagle medium without methionine and cysteine (DMEM;ms), x-ray films (Kodak X-OMAT-XAR5), and goat anti-rabbit IgG coupled to horseradish peroxidase were purchased from Sigma Chemical Co. (St. Louis, MO). [35S]Methionine and [35S]cysteine ([35S] Protein Labeling Mix, EXPRE35S35S) were purchased from NEN (Les Ulis, France), [
32P]dCTP was from Amersham (Les Ulis, France), acrylamide (30% acrylamide, 0.8% N,N-methylenebisacrylamide) was from Millipore (St. Quentin, France), ampholytes pH 2–11 (Servalytes) were from Serva (Heidelberg, Germany), and ampholytes pH 3–10 (Ampholytes) and the electrophoresis calibration kit (standard proteins) were from Pharmacia (Saclay, France). All other chemicals were of molecular biology grade (Sigma).
Animals and Organ Sampling
To investigate the expression of PGDS in the ovine epididymis during postnatal development, we characterized PGDS expression in Ile de France sheep at the age of 120 days (4 mo), 135 days (4.5 mo), 165 days (5.5 mo), and 195 days (6.5 mo: adult). Expression was also investigated in the fetal testis and epididymis at 50 days of gestation.
In Ile de France sheep, spermatogenesis begins at around 100–110 days (first spermatids), spermatozoa appear in seminiferous tubules between 120 and 130 days, and plasma LH, FSH, and testosterone levels increased regularly between the 1st and 12th week of age [10].
The epididymis was subdivided into 9 or 10 regions (0 to 8/9) as previously described in the ram [9, 11, 12]. One epididymis for each animal was used for in vitro biosynthesis to analyze total epididymal protein secretion and for Western blotting to investigate the putative presence of PGDS in tissue extracts. The second epididymis was used for mRNA and epididymal fluid analysis. For mRNA analysis, epididymal and testicular tissues were collected, frozen in liquid nitrogen, and kept at -80°C until extraction. For protein analysis, the epididymal luminal fluids were obtained by perfusion with PBS as previously described [11]. Samples were centrifuged (1500 x g for 15 min) to remove sperm. The supernatants were then centrifuged (15 000 x g for 10 min) and kept at -20°C until used.
In Vitro Secretion of [35S]Methionine-Cysteine-Labeled Proteins from Tissue Samples and Isolated Tubules
In vitro secretion of [35S]methionine-cysteine-labeled proteins was estimated from all epididymal regions (isolated tubules) according to previously described methods [2, 3, 11]. Closed-end tubules were incubated in 0.5 ml DMEM;ms in the presence of 100 µCi [35S] Protein Labeling Mix and under 95% O2-5% CO2 at 32°C. After 5 h of incubation, the luminal fluid of each tubule was collected by perfusion with DMEM solution. The fluids were then centrifuged (16 000 x g for 10 min), and the supernatants were stored at -20°C until used. After perfusion of the lumen, the tissues were kept at -20°C until processed for protein extraction.
Protein Extraction
Proteins were extracted from epididymal tissues after incubation and perfusion by homogenization in 50 mM Tris-HCl pH 7.4, 0.5 mM EDTA pH 8, and a cocktail of protease inhibitors (10 µg/ml antipain, leupeptine, bestatine, and pepstatine A, 0.5 mM PMSF). The tissue homogenates were centrifuged twice at 15 000 x g for 30 min at 4°C, and the concentration of total proteins in the supernatants was estimated using a Bradford assay (Bio-Rad, Hercules, CA). Supernatants were kept at -20°C until used.
Gel Electrophoresis
SDS-PAGE for protein separation was carried out for all samples according to the method of Laemmli [13] on gels (14 x 16 cm or 6 x 8 cm) formed with 6%–16% linear concentrations of polyacrylamide. The quantities of protein were 30 µg for one-dimensional electrophoresis. Isoelectric focusing was performed using the O'Farrell technique [14], modified as previously described [15], with 150 µg of protein. The pH gradient was obtained with 1% Ampholytes pH 3–10 and 1% Servalytes pH 2–11. Isoelectric focusing was performed at 20 mA, 0.1 W/tube, 700 V for a total of 10 000 V/h followed by 20 mA, 0.1 W/tube, 3000 V for a total of 2000 V/h. The second-dimension separation was performed on a 6%–16% acrylamide gel (1.5 mm thick) running at 40 mA.
Fluorography
For detection of radioactive proteins the gels were impregnated with a fluorography enhancer (Amplify; Amersham) after silver staining [16] and were then dried and exposed on preflashed x-ray film for several days at -80°C or on a phosphoimaging screen (Storm; Molecular Dynamics, Paris, France).
Western Blot
The specific antibody against PGDS was characterized in our previous study [9]. This antibody was used to detect the protein in the epididymal fluids and tissues after in vitro biosynthesis. For immunodetection, the proteins separated by electrophoresis were electrotransferred (0.8 mA/cm2 for 2 h) by a semidry technique to a 0.2-µm nitrocellulose membrane (Schleicher et Schuell, Dassel, Germany). After transfer, the proteins were stained with Ponceau red S and incubated overnight in 10% goat serum in 1x Tris-buffered saline (TBS).
The primary antibody was diluted to 1:2000 in 5% goat serum in TBS and incubated for 1 h at 37°C. Blots were then washed with the same buffer and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG diluted to 1:10 000 in 5% goat serum in 1x TBS for 30 min at 37°C. After several washings, immunoreactive proteins were detected by chemiluminescent substrate (Western Blot Chemiluminescence Reagent Plus; NEN, Boston, MA) according to the manufacturer's instructions.
RNA Extraction
Total RNA was prepared from 200 mg frozen tissue according to the isothiocyanate guanidinium technique described by Chomczynski and Sacchi [17]. RNA was extracted from the testes, efferent ducts, and various epididymal regions. For each total RNA sample, 10 µg was separated by electrophoresis in a 1% agarose formaldehyde gel in the presence of RNA markers (Promega, Charbonnières, France). RNA was then transferred to a nylon membrane (Hybond N+; Amersham) by capillary blotting in 10x saline-sodium citrate (SSC; 1.5 M NaCl, 0.15 M Na3 citrate, pH 7) and fixed for 2 h at 80°C. The membrane was stored at -20°C until prehybridization.
Northern Blot Hybridization
The production of the cDNA probe for PGDS was described previously [9]. The DNA was labeled with [32P]dCTP using the Prime-a-Gene labeling system (Promega). Hybridization was performed using 32P-labeled probes as described by Thomas [18]. The membrane was prehybridized in 6x SSC, 5x Denhardt solution, 0.5% SDS, 100 µg/ml herring sperm DNA, and 50% formamide for 2 h at 42°C. Overnight hybridization with the labeled probe was performed in the same solution at 42°C but without Denhardt solution. The membrane was washed once in 2x SSC, 0.1% SDS and twice in 0.1x SSC, 0.1% SDS. The PGDS transcript was visualized by exposure on preflashed x-ray film.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PGDS mRNA and Protein in the Testis and Epididymis of the Ovine Fetus and 4-Mo-Old Lamb
A single 0.75- to 0.8-kilobase mRNA band was detected in the fetal testis and epididymis at 50 days of gestation; the mRNA signal was stronger in the testis than in the epididymis (Fig. 1). Four months after birth, the mRNA signal was also more intense in the testis than in the epididymis. Messenger RNA was detected with similar intensity in the epididymis from the caput (region E2) to the cauda (E7, Fig. 1). Using in vitro biosynthesis analysis, most of the epididymal regions appeared to be active in protein secretion. The major proteins detected were secreted in a region-specific manner. At least two major proteins, of 25 kDa (glutathione peroxidaxe [GPX5]) and 45–35 kDa (clusterin), were secreted in the anterior region. However, PGDS was not secreted in any epididymal region (Fig. 2A). Moreover, no PGDS was detected by Western blotting in the tissue extracts of the corresponding incubated tubules (data not shown). These results suggest that PGDS was not synthesized by the epididymal epithelium under our experimental conditions. However, PGDS was immunodetected in the epididymal fluid of the proximal caput (E0 and E1, Fig. 2B). The isoforms of the protein characterized by two-dimensional electrophoresis (Fig. 2C) had the same molecular characteristics (30 kDa, isolelectric point [pI] 4.2–5.5) as the testicular form of PGDS previously described in the ram and were highly resorbed in the first part of the epididymis [9].
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PGDS mRNA and Protein in the Testis and Epididymis of 4.5-Mo-Old Lambs
At 4.5 mo of age, as in the 4-mo-old lambs, PGDS mRNA was detected in the testis and in all parts of the epididymis; the testis signal was still stronger than that in any epididymal region (Fig. 1). In the epididymis, the mRNA signal was more intense in the caput than in the cauda.
In contrast to the 4-mo-old lambs, PGDS in the 4.5-mo-old lambs was secreted in the lumen (Fig. 3A). However, despite the presence of mRNA throughout the epididymis, the protein was secreted only in the proximal caput regions (E0, E1, and E2) but not in more distal regions.
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The protein was immunodetected in the fluids by one-dimensional (Fig. 3B) and two-dimensional electrophoresis (Fig. 3, C and D) throughout the epididymis, with more protein in the caput than in the cauda. The compound in the cauda had a molecular mass (30 kDa) and a pI (4.2–5.5) similar to those of the compound in the caput and testicular fluid.
PGDS mRNA and Protein in the Testis and Epididymis in 5.5-Mo-Old Lambs and Adult Sheep
In 5.5-mo-old animals, PGDS mRNA was detected in the testis, efferent ducts, and proximal caput epididymis (E0, E1, and E2) but not in the cauda. In contrast to the above findings, the testis signal was less intense than that in the efferent ducts or epididymis. Within the proximal region, the PGDS mRNA signal was stronger in E0 than in E1 and E2 (Fig. 4A). The protein was secreted in the lumen only in the E0 region (Fig. 4B) but was present in the lumen throughout the tubule, with a smaller quantity in the cauda than in the caput (Fig. 4C). The PGDS isoforms secreted and present in the caput had molecular characteristics (27 kDa and pI 4.5–6.5) similar to those of the PGDS secreted by the epididymis in the adult (Fig. 5) [9]. However, at 5.5 mo there were no differences in molecular mass and pI between the caput and cauda PGDS, in contrast to reports in the adult (Fig. 5) [9].
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Stage 1: Expression of PGDS Gene Without mRNA Regionalization and Without the Presence of the Protein in the Epididymis
Transcription of the PGDS gene in the male genital tract appeared early during ontogeny; PGDS mRNA was detected at 50 days of gestation in both the testis and the epididymis. At 4 and 4.5 mo of age, PGDS mRNA was present throughout the epididymal tubule from the caput to the cauda, and regionalization was similar (i.e., restricted to the proximal region) to that in the adult at 5.5 mo of age. Because androgen production in the lamb increases progressively through the prepuberty period [19], mRNA expression probably is not directly regulated by androgen.
The presence of the PGDS messenger in the epididymal tissues was not associated with the presence of the corresponding protein, either in the tissue or in the luminal secretion. In 4-mo-old animals, no PGDS secretion was evident in any epididymal region, as investigated using in vitro neosynthesis and immunodetection of PGDS in the tissues. However, the presence of PGDS of testicular origin in the lumen of the anterior caput suggests that PGDS mRNA translation is already effective in the gonad, the protein probably secreted by the Sertoli cells as previously suggested [20]. The lack of translation of PGDS mRNA in the epididymis is not likely to be related to an immature stage of the translation machinery for epididymal cells; several other proteins are already synthesized and secreted in the lumen. Thus, the absence of mRNA translation seems to be specific at least to PGDS gene expression, indicative of differential regulation between the testis and epididymis.
Stage 2: Regionalization of the Secretion of PGDS Protein with No Regionalization of mRNA
Synthesis and secretion of the PGDS protein occurred in 4.5-mo-old animals and was restricted to the anterior caput, although PGDS mRNA was still present in all the epididymal regions. The protein concentration decreased toward the caudal region, as previously shown in adult sheep [9]. The localization of the secretion was similar to that in the adult epididymis, but the protein secreted was not the same isoform characteristic of the mature epididymis, suggesting that glycosylation of the protein was incomplete in 4.5-mo-old animals.
According to the developmental characteristics of the Ile de France breed [21], 4.5 mo of age was not present under a notable period for changes in androgen status of the gonad but corresponded to the entrance of the first waves of spermatozoa carried by the rete testis fluid into the epididymis. In our study, spermatozoa were found in the fluid collected in the anterior part of the epididymis at this age. Thus, the translation trigger of PGDS mRNA might be related to sperm-related factors or to a specific secretion(s) of Sertoli cells. Further studies are needed to identify the trigger factors involved in the PGDS mRNA translation.
Stage 3: Similarity in the Spatial Location of PGDS mRNA and Protein Expression
The third stage in the establishment of postnatal PGDS gene expression was the restriction of both PGDS mRNA and protein expression to the same epididymal region, i.e., the proximal caput. Definitive regionalization of mRNA expression was established between 4.5 and 5.5 mo. At 5.5 mo, the mRNA and protein secretion were localized to the anterior region, as in the adult epididymis. No PGDS messengers were detected by Northern blotting in corpus or cauda epididymal tissues. Expression of other developmentally regulated genes has previously been described in the epididymis. Bone morphogenetic protein 7 (Bmp7) transcripts have been detected in mice in all epithelial cells throughout the epididymis during early postnatal development. As the mice aged, Bmp7 expression was gradually restricted to the initial segment and was undetectable after 4 wk of age in the rest of the epididymis [22].
During the postnatal differentiation of the epididymis, the regionalization of protein secretion may be different from that in adult animals [6]. Syntin et al. [6] showed in the boar that the earlier the protein was secreted during postnatal development the greater the difference in localization from the adult. Secretion generally began in a more posterior region than in the adult. For proteins secreted later, the secretion site was the same as the adult site. This situation applied to PGDS secretion, which appeared late in the epididymal development (2 mo before puberty) and was detected in the same region as in the adult.
This is the first time to our knowledge that temporal and spatial differences between mRNA expression and translation have been reported for an epididymal gene during postnatal development. Other similar studies on the epididymis have shown that mRNA and protein secretion appear at the same time and in the same location (e.g., CRISP-1 [23, 24] and GPX5 [25] proteins in mice).
For PGDS mRNA our results suggest a complete blockage of mRNA translation in corpus and cauda regions and a transitory blockage of mRNA in the caput before the age of 4.5 mo. Several factors such as the rate of initiation of translation, mostly regulated by sequences in the 5' untranslated region, the presence of specific RNA-binding proteins, and the length of the poly(A) tail are involved in the modulation of mRNA translation. Inefficiency of mRNA translation has previously been described in the male genital tract, e.g., beta 2-adrenergic receptor mRNA in the prostate after castration [26] and SVS IV mRNA in seminal vesicles during the first stage of postnatal development [27]. A CD52 mRNA present in all parts of the epididymis in the rat is not efficiently translated in the cauda region, where the mRNA molecules possess a longer poly(A) tail [28, 29]. No modifications in the length of PGDS mRNA were observed by Northern blotting at 4.5 mo compared with 4 mo of age, suggesting that modifications of poly(A) tail lengths may not be involved in this regulation of translation (Fig. 1). Thus, the mechanisms of regulation of PGDS mRNA translation in the epididymis remain to be elucidated
Testicular factors such as testicle-derived growth factors [30] and spermatozoa-associated factors [7] are involved in the developmental regulation of gene transcription in the proximal regions of the epididymis [6, 31]. The results of our study strongly suggest that testicular factors also may be involved in translation factors that control PGDS mRNA translation and secretion of the corresponding protein. These factors seem to be necessary only during the first stage of the establishment of secretion, because in adult animals the suppression of these factors either by efferent duct ligation or castration does not abolish mRNA expression or protein synthesis of PGDS in the epididymal caput [9].
The results of the present study, using PGDS expression as a model to study the differentiation of the epididymis, confirm that developmental regulation of epididymal secretory activity is a complex process involving gene regulation at transcription, translation, and posttranslation levels. The translation and posttranslational processes are fairly new concepts and offer new perspectives for a better understanding of gene expression regulation in the epididymis. Further investigations are necessary to characterize the processes involved in the spatial and temporal differences in PGDS mRNA and protein expression in the epididymis.
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FOOTNOTES |
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2 Correspondence. FAX: 33 02 47 42 77 43; jdacheux{at}tours.inra.fr
Received: 22 April 2002.
First decision: 13 May 2002.
Accepted: 17 July 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This article has been cited by other articles:
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  H. Zhu, H. Ma, H. Ni, X.-H. Ma, N. Mills, and Z.-M. Yang Expression and Regulation of Lipocalin-Type Prostaglandin D Synthase in Rat Testis and Epididymis Biol Reprod, April 1, 2004; 70(4): 1088 - 1095. [Abstract] [Full Text] [PDF]
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