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Endocrinology Vol. 144, No. 3 975-988
Copyright © 2003 by The Endocrine Society

ARTICLE

Gene Expression Is Differentially Regulated in the Epididymis after Orchidectomy

Nadine Ezer and Bernard Robaire

Departments of Pharmacology and Therapeutics and Obstetrics and Gynecology, McGill University, Montréal, Québec H3G 1Y6, Canada

Address all correspondence and requests for reprints to: B. Robaire, Department of Pharmacology and Therapeutics, McGill University, 3655 Promenade Sir-William-Osler, Montréal, Québec H3G 1Y6, Canada. E-mail: brobaire{at}pharma.mcgill.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The epididymis is the site for the transport, maturation, and storage of spermatozoa. Regulation of epididymal structure and function is highly dependent on the ipsilateral testis. At the molecular level, however, few studies have been undertaken to determine which genes are expressed in the epididymis under testicular regulation. The goal of this study was to identify genes for which expression is regulated after orchidectomy, both throughout the epididymis and in a segment-specific manner. Microarrays spotted with 474 rat cDNAs were used to examine gene expression changes over the first 7 d post orchidectomy in the initial segment, caput, corpus, and cauda epididymidis of the adult Brown Norway rat. Using k-means cluster analysis, we show that four patterns of gene expression are activated in each epididymal segment over the first week following orchidectomy. Transient up-regulation of gene expression in the epididymis after orchidectomy is described for the first time. Potential androgen-repressed genes, including Gpx-1, show increased expression in the epididymis after orchidectomy. Several glutathione-S-transferases and calcium-binding proteins decline throughout the epididymis after orchidectomy, indicating that these may be novel androgen-regulated epididymal genes. Other genes coding for metabolism-associated proteins, transporters, and {alpha}-1 acid glycoprotein show segment-specific regulation in the epididymis after orchidectomy. Finally, we describe the expression of the previously uncharacterized heat shock proteins, and apoptosis-associated genes in the epididymis after orchidectomy. Thus, gene expression in the epididymis is differentially affected over time after orchidectomy. These results provide novel insight into androgen-dependent and segment-specific epididymal function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE EPIDIDYMIS, A HIGHLY convoluted tubule that links the efferent ducts to the vas deferens, functions in the transport, maturation, and storage of spermatozoa (1). As early as 1926, it was established that maintenance of epididymal structure and function was dependent on an unknown testicular factor (2), purified 5 yr later as testosterone (3). Subsequent studies showed clearly that epididymal functions are highly dependent on the circulating androgen, testosterone; in the absence of androgens, spermatozoa become immotile, lose the ability to fertilize, and die (4, 5).

Orchidectomy removes hormonal androgenic support arriving to the epididymis via the circulation as well as testicular factors entering the epididymal lumen directly via the efferent ducts. Following orchidectomy, epididymal weight is decreased to 25% of control over a 2-wk period. Testosterone replacement, even at supraphysiological levels, can restore epididymal weight but only to 50% of control; this is due to the large proportion (nearly half) of epididymal weight that is attributable to spermatozoa and luminal fluid (6, 7).

Of the four major epididymal epithelial cell types (principal cells, basal cells, clear cells, and narrow cells), principal cells are particularly sensitive to removal of circulating androgens, whereas other cell types appear unaffected (8, 9). Following orchidectomy, altered principal cell morphology, protein secretion, and apoptotic cell death in the caput, corpus, and cauda epididymidis can be reversed or prevented by androgen replacement (6, 10, 11, 12). However, testosterone replacement is not sufficient to reverse these regressive changes in the initial segment of the epididymis (6, 10, 13). Efferent duct ligation studies, in which testicular contributions to the epididymis are removed but circulating androgen concentrations are maintained, have established that testicular factors arriving through the efferent ducts are essential for maintenance of proper initial segment structure (10), secretion (13, 14), enzyme activity (15, 16), and prevention of principal cell apoptosis (12).

At the mRNA level, several epididymal genes have been described as androgen dependent; such genes respond by a decline in expression after androgen withdrawal [DE/AEG/CRISP-1 (17), glutathione peroxidase (Gpx)-5 (18), Gpx-3 (14), carbonic anhydrase (19), cyclooxygenase 2 (20), angiotensinogen (21)]. In addition, testicular factors play a role in maintaining normal expression of proximal epididymal genes in the initial segment and caput epididymidis [proenkephalin (22), cystatin-related epididymal specific (23), 5-{alpha} reductase 1 (24), {gamma}-glutamyl transpeptidase (GGT) (25), and EP17 (26)]. Candidate testicular factors include high intraluminal androgens, basic fibroblast growth factor (16), and androgen-binding protein (15). The possibility that some genes are repressed by androgens and therefore induced, at least in some epididymal segments, in the absence of androgens has been established [Clusterin/Trm-2/Apo-J (27), TGFß (28)]. These two patterns of gene expression in the epididymis after orchidectomy suggest that regulation of epididymal gene expression is complex.

It is not readily feasible to simultaneously measure changes in expression of a large number of genes in the epididymis by using conventional techniques such as differential display, Northern blot analysis, RNase protection assays, or Q-RT-PCR. Therefore, a more comprehensive understanding of testis-dependent regulation of epididymal gene expression remains to be attained. DNA microarrays provide a powerful tool to analyze differential gene expression (29). We anticipated that several patterns of gene expression are activated in the epididymis after orchidectomy and specific gene families respond differentially to the removal of androgens and/or testicular factors. To investigate this hypothesis, we used gene expression profiling to examine the segment-specific response of 474 stress-related genes in the rat epididymis over the first 7 d post orchidectomy. Using k-means cluster analysis, we found four clearly distinctive expression profiles in the epididymis after orchidectomy. Novel genes that respond to orchidectomy along the epididymis and in a segment-specific manner were identified. Finally, we have described the expression of previously uncharacterized gene families in the epididymis after orchidectomy. By providing insight into regulation of epididymal gene expression after orchidectomy, these results contribute to our understanding of testis-dependent epididymal functions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male Brown Norway rats (3 months old) were purchased from the National Institute on Aging (Bethesda, MD) and supplied by Harlan Sprague Dawley, Inc. (Indianapolis, IN). Rats were housed at the McIntyre Animal Resources Centre, McGill University, under controlled light (14-h light, 10-h dark) and temperature (22 C); animals had free access to food and water. All animal studies were conducted in accordance with the principles and procedure outlined in the Guide to the Care and Use of Experimental Animals prepared by the Canadian Council on Animal Care (Animal Use Protocol no. 206).

Bilateral orchidectomy was done through the abdominal route. Efferent ducts were ligated on both sides, and testes were removed above the ligation. Animals were killed at 0.5, 1, 2, 3, or 7 d post orchidectomy by decapitation. Control group animals (C) were sham operated and killed at various time points thereafter in parallel to animals that underwent orchidectomy. At the time of death, epididymides were collected; sectioned into initial segment, caput, corpus, and cauda segments; and immediately frozen in liquid nitrogen. Sections were stored at -80 C until used for RNA extraction. Blood was collected at time of death for hormone analysis.

Serum testosterone analysis
We used a commercially available testosterone ELISA kit to establish the time course of the decline in total serum testosterone after bilateral orchidectomy. At the time of death, serum was obtained by centrifuging blood for 20 min (2700 x g, 4 C); collecting the supernatant; and centrifuging again. Supernatants were collected and frozen at -20 C. Total serum testosterone was measured using ELISA (Research Diagnostics, Flanders, NJ) according to the manufacturer’s instructions. Sensitivity of the assay was 0.1 ng/ml, and the intraassay coefficient of variation was less than 11%.

RNA extraction
Total RNA was extracted from each sample using guanidine thiocyanate as described previously (30). Following isolation, RNA samples were DNase-treated (Atlas pure isolation kit user manual, section IV, CLONTECH Laboratories, Inc., Palo Alto, CA), and RNA concentration was assessed by OD determination at 260 nm (DU7 spectrophotometer, Beckman, Montréal, Québec, Canada). To verify the quality of each sample, 5 µg RNA was run on a denaturing gel containing 1% agarose-formaldehyde. Each sample consisted of single epididymal segment obtained from an individual rat; no tissues were pooled.

cDNA arrays and hybridization
RNA samples were used to probe cDNA arrays (Atlas Rat Stress Toxicology II array, CLONTECH Laboratories, Inc.) according to the manufacturer’s instructions. Five arrays per epididymal segment per time point post orchidectomy (n = 5/time/segment) were probed and referred to as replicates, with the following exceptions: four replicates per group were used (n = 4 group) at 0.5 d post orchidectomy for all segments, at 3 d post orchidectomy for the initial segment and 2 d post orchidectomy for the corpus epididymidis; three arrays (n = 3) per group were used at 7 d post orchidectomy for the corpus epididymidis. A total number of 112 samples were probed for this experiment. Arrays were exposed to PhosphorImager plates (Molecular Dynamics, Inc., Sunnyvale, CA) 24 h before scanning with a PhosphorImager (Storm, Molecular Dynamics, Inc.). Analysis of array images with Atlas Image (version 2.0, CLONTECH Laboratories, Inc.) was done to quantify the intensity of each cDNA spot, which reflects the relative abundance of RNA in the sample. The raw data for each gene (intensity minus the background) were imported into GeneSpring 4.0.7 (Silicon Genetics, Redwood, CA) for further analysis. For each replicate array, a gene was considered detected if its intensity was above threshold, with threshold defined as two times the average background of that array. A gene was considered expressed at any given time post orchidectomy if it was detected in at least three replicates in that group.

To minimize experimental variation and to allow for comparison of different time groups post orchidectomy, data were normalized with one of two different methods (GeneSpring 4.0.7). For the standard experiment-to-experiment normalization, the median level of expression on each array was defined as 1 and expression of each gene was normalized relative to 1; this value was averaged for all replicates in a group to generate what is referred to as the relative intensity for a given gene. For cluster analysis only, gene-to-gene normalization was done in addition to experiment-to-experiment normalization. Using this method, the signal strength of each gene was normalized relative to the median of all measurements taken for that gene in each experiment, defined as 1. This normalization allows for clearer visualization of expression profiles with all genes on the same vertical scale. All individual gene profiles are shown using the standard experiment-to-experiment normalization.

K-means cluster analysis partitions genes into groups based on similar expression patterns. The data are divided into k different clusters of greatest possible distinction by starting with k random clusters and moving the genes between clusters to minimize variability within clusters and maximize variability between clusters. Before clustering by k means, the data were normalized (gene to gene), k was defined as 4, and the smooth correlation was selected. Clustering was done independently on the data sets (total number of genes) for each epididymal segment. The total number of genes represents the number of genes detected in control groups as well as those that are detected post orchidectomy.

Changes in gene expression were considered relative to control and when the difference in expression level was at least 2-fold in either direction, i.e. 100% increase or 50% decrease. Genes showing transiently increased expression were described by expression at the peak, in which peak value was expressed relative to control. Because of the normalization procedures, standard statistical comparisons were not done. However, SEM is shown for expression of individual genes to show variability among replicates in a given group.

We examined genes on our array that have known expression profiles post orchidectomy; this is similar to the method we previously used to validate gene expression profiling (30). Clusterin mRNA was detected most abundantly in the proximal epididymis, with highest expression in the control caput epididymidis, followed by the initial segment, cauda, and corpus epididymidis (31); consistent with published observations, clusterin mRNA expression showed the greatest increase in the cauda epididymidis (~10-fold), followed by the corpus epididymidis (~2.3-fold) but was unchanged in the initial segment and caput segments of the epididymis after orchidectomy (27). GGT mRNA was expressed most abundantly in the initial segment followed by the caput epididymidis and was undetected at all time points in the corpus and cauda epididymides. As expected, GGT mRNA declined most dramatically in the initial segment, followed by the caput epididymidis at 7 d post orchidectomy (25). Finally, metallothionein-3 mRNA expression was highest in the caput epididymidis, followed by the initial segment, and its expression declined only in the caput epididymidis at 7 d post orchidectomy (32).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Serum testosterone
The average total serum testosterone in the control (sham-operated) group was 4.53 ± 2.26 ng/ml (mean ± SEM), with values ranging from 2.3 ng/ml to 6.0 ng/ml. By 0.5 d post orchidectomy average serum-free testosterone fell to less than 0.3 ng/ml and remained below this level at all subsequent times studied post orchidectomy.

Genes detected in the epididymis post orchidectomy
Of the 474 genes examined, transcripts for 39% of genes (184) were detected in the control initial segment, 43% (203) in the control caput epididymidis, 39% (185) in the control corpus epididymidis, and 42% (201) in the control cauda epididymidis. In addition, a comparison of genes expressed in the control and 7 d postorchidectomy time point for each epididymal segment is presented in the supplemental data for this article (see The Endocrine Society’s Journals Online Web site at http://endo.endojournals.org). The number of genes used for cluster analysis was 243 in the initial segment, 239 in the caput, 235 in the corpus, and 244 in the cauda epididymidis. This number includes all genes detected in the control group plus any additional genes that became expressed over the 7-d time course post orchidectomy.

K-means cluster analysis of gene expression in each epididymal segment after orchidectomy
We used k-means clustering to visualize trends in gene expression that occurred over time post orchidectomy (Fig. 1Go). Four distinct gene expression profiles were generated in each epididymal segment. Decreased gene expression after orchidectomy was classified into one of two profiles. Genes that partitioned into the early-declining profiles (profile 1) showed a large decline in expression before 2 d post orchidectomy, followed by smaller changes in expression between 2 d and 7 d post orchidectomy. For example, thiopurine methyltransferase was associated with this profile in each epididymal segment. GGT was associated with this profile in the initial segment and caput epididymis only; this gene was not detected in the corpus and cauda epididymidis. The second profile (profile 2) grouped genes with expression that declined more progressively; these profiles also included genes that showed minimal changes in expression in response to orchidectomy.



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  		Figure 1. Profiles of gene expression obtained by k-means cluster analysis in the epididymis after orchidectomy. 1, Early-declining gene expression. 2, Progressively declining gene expression. 3, Transiently increased gene expression. 4, Progressively increasing gene expression. The level of gene expression is expressed as relative intensity. Relative intensity was obtained by normalizing the signal for any given gene relative to the median intensity of all the measurements for that gene, defined as 1. Time post orchidectomy is indicated on the horizontal axes. Inset, A schematic of the profile of gene expression. IS, Initial segment; CA, caput; CO, corpus; CD, cauda.

 
A subset of genes in each epididymal segment showed transiently increased expression in response to orchidectomy (profile 3). This profile was particularly evident in the caput epididymidis. In the initial segment, this trend was less apparent; however, upon examination of these genes individually using the standard experiment-to-experiment normalization, transient up-regulation of these genes was clearly observable. Interestingly, c-jun was assigned to this profile in each epididymal segment (Fig. 2Go).



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  		Figure 2. Expression of c-jun and gpx-1 in the epididymis over the first week post orchidectomy. A, C-jun. B, Gpx-1. Gene expression is presented as relative intensity, mean ± SEM for three to five replicates per group. Time post orchidectomy is indicated on the horizontal axis. IS, Initial segment; CA, caput; CO, corpus; CD, cauda.

 
The fourth profile of gene expression identified by k-means clustering showed a progressive increase in expression over the 7-d period after orchidectomy in each epididymal segment (profile 4). An example of one such gene is Gpx-1 (Fig. 2Go).

Gene expression changes in the epididymis post orchidectomy
The number of genes demonstrating a minimum 2-fold change in expression (increased or decreased) at each time post orchidectomy relative to control was obtained for each epididymal segment (Fig. 3Go). The number of genes that showed increased expression varied little over the first week post orchidectomy. At 7 d post orchidectomy, the proportion of genes that remained increased represented 5%, 8%, 9%, and 9%, respectively, of the genes expressed in the initial segment, caput, corpus, and cauda epididymidis. In contrast, the population of genes that showed a decline in expression increased with time post orchidectomy. At 1 d post orchidectomy, several genes showed decreased expression in each epididymal segment, similar to the pattern of early-declining gene expression identified by k-means cluster analysis (Table 1Go). By 7 d post orchidectomy, genes that showed decreased expression represented 30%, 42%, 51%, and 29% of genes expressed in the initial segment, caput, corpus, and cauda epididymidis, respectively. Expression of a large proportion of genes remained unchanged along the epididymis at 7 d post orchidectomy: 65%, 50%, 40%, and 62% of genes expressed in the initial segment, caput, corpus, and cauda epididymidis, respectively.



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  		Figure 3. Number of genes showing changes in gene expression at each time post orchidectomy in each epididymal segment. The vertical scale represents the total number of genes that showed at least a 2-fold change in expression in either direction (100% increase or 50% decrease). This number was obtained independently at each time post orchidectomy relative to control. The numbers above and below the horizontal zero-line represent genes that show increased or decreased expression respectively. IS, Initial segment; CA, caput; CO, corpus; CD, cauda.

 

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  		Table 1. Genes with decreased expression at 1 d post orchidectomy in each epididymal segment

 
To identify orchidectomy-responsive gene families in the epididymis, we compared gene expression changes 7 d post orchidectomy across segments. Expression of five genes was increased in all four segments of the epididymis 7 d post orchidectomy. The segment-specific expression and increase in expression for these genes at 7 d post orchidectomy is presented (Table 2Go). Similar to Gpx-1 (Fig. 2Go), each of these genes showed a progressive increase in expression with time post orchidectomy. The expression of 27 genes was decreased throughout the epididymis at 7 days post orchidectomy; the decline in expression for these genes at 7 d post orchidectomy is presented (Table 3Go). Among these were several genes belonging to the family of glutathione-S-transferases (GSTs) (Fig. 4Go). Expression of GST Yf, which was most abundant in the initial segment, declined in the initial segment and caput epididymidis by 79% and 59%, respectively, but was unchanged in the corpus and cauda epididymal segments (Fig. 4Go). Expression of calcium-binding proteins (CaBPs) also declined along the epididymis after orchidectomy (Fig. 5Go); expression of CaBP-2 declined by 73%, 85%, and 66% in the caput, corpus, and cauda epididymidis, respectively, at 7 d post orchidectomy, whereas CaBP-2 expression in the initial segment remained unchanged.


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  		Table 2. Genes with increased expression throughout the epididymis at 7 d post orchidectomy

 

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  		Table 3. Genes showing decreased expression throughout the epididymis at 7 d post orchidectomy

 


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  		Figure 4. Expression of GSTs in the epididymis over the first week post orchidectomy. A. GST Yb2 and GST Yc1. C. GST Yf. Gene expression is presented as relative intensity, mean ± SEM for three to five replicates per group. Time post orchidectomy is indicated on the horizontal axis. IS, Initial segment; CA, caput; CO, corpus; CD, cauda.

 


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  		Figure 5. Expression of CaBPs in the epididymis over the first week post orchidectomy. A, CaBP-2 and CaBP-3/calreticulin. B, CaBP-1 and calnexin. Gene expression is presented as relative intensity, mean ± SEM for three to five replicates per group. Time post orchidectomy is indicated on the horizontal axis. IS, Initial segment; CA, caput; CO, corpus; CD, cauda.

 
Segment-specific changes in gene expression were observed in the epididymis after orchidectomy. At 7 d post orchidectomy, several genes expressed along the epididymis showed increased expression in various segments. The segment-specific increase in expression for these genes at 7 d post orchidectomy is presented (Table 4Go). Others, such as vascular endothelial growth factor D, were newly detected at 7 d post orchidectomy in the caput, corpus, and cauda segments of the epididymis.


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  		Table 4. Genes that showed a segment-specific increase in expression at 7 d post orchidectomy in the four epididymal segments

 
A segment-specific decline in the expression of metabolism-associated genes was observed in the epididymis after orchidectomy (Fig. 6Go). Mitochondrial aspartate aminotransferase (mAAT) declined in the caput and corpus epididymal segments by 63% and 81%, respectively, but was unchanged after orchidectomy in the initial segment and cauda epididymidis. In contrast, the cytoplasmic aspartate aminotransferase was not affected in the epididymis after orchidectomy. Expression of ornithine decarboxylase (ODC), which increased from the proximal to distal epididymis, declined by 63%, 74%, and 50%, respectively, in the caput, corpus, and cauda epididymidis but was unchanged in the initial segment (not shown). Expression of D-amino acid oxidase (DAAO), which was detected only in the caput, corpus, and cauda epididymidis, declined to undetectable levels in these segments by 7 d post orchidectomy. Expression of two genes coding for transporters also declined in a segment-specific manner after orchidectomy (Fig. 7Go). The transcript for organic cation transporter N2 (OCTN2), which was most abundant in the corpus epididymidis but not expressed in the initial segment, declined to undetectable levels in the caput, corpus, and cauda epididymidis by 7 d post orchidectomy. Sodium-dependent neutral amino acid transporter (ASCT2) was most abundantly expressed in the caput epididymidis, in which it declined by 60% at 7 d post orchidectomy. Lastly, the gene coding for {alpha}-1-acid-glycoprotein (AGP), detected only in the initial segment of the epididymis, declined by 84% at 7 d post orchidectomy in this segment (Fig. 8Go).



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  		Figure 6. Expression of metabolism-associated genes in the epididymis over the first week post orchidectomy. A, mAAT. B, DAAO. Gene expression is presented as relative intensity, mean ± SEM for three to five replicates per group. Time post orchidectomy is indicated on the horizontal axis. IS, Initial segment; CA, caput; CO, corpus; CD, cauda.

 


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  		Figure 7. Expression of transporters in the epididymis over the first week post orchidectomy. A, OCTN2. B, ASCT2. Gene expression is presented as relative intensity, mean ± SEM for three to five replicates per group. Time post orchidectomy is indicated on the horizontal axis. IS, Initial segment; CA, caput; CO, corpus; CD, cauda.

 


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  		Figure 8. Expression of AGP in the initial segment of the epididymis over the first week post orchidectomy. Gene expression is presented as relative intensity, mean ± SEM for three to five replicates per group. Time post orchidectomy is indicated on the horizontal axis.

 
Expression by gene family
Heat shock proteins.
Members of the heat shock protein (Hsp) family showed several patterns of gene expression in the epididymis after orchidectomy (Fig. 9Go). The glucose-regulated proteins (grp)94 and grp78 (not shown) showed a steady decline in expression in all segments of the epididymis over the first week post orchidectomy. By 7 d post orchidectomy, grp94 expression declined by 62%, 86%, 71%, and 78%, respectively, in the initial segment, caput, corpus, and cauda epididymidis; expression of grp78 declined by 69%, 72%, 77%, and 82%, respectively, in the initial segment, caput, corpus, and cauda epididymidis. Other Hsps, such as Hsp27 and Hsp47, demonstrated a transient increase in expression post orchidectomy. At the peak of transiently increased expression, Hsp27 expression was increased by 70%, 489%, 226%, and 140%, respectively, in the initial segment, caput, corpus, and cauda epididymidis; Hsp47 expression was increased by 207%, 270%, 254%, and 81%, respectively, in the initial segment, caput, corpus, and cauda epididymidis. Hsp90ß, the most highly expressed Hsp along the epididymis, was unaffected by orchidectomy. The small Hsp crystallin-ß B1 (CRYßB1), which was expressed only in the initial segment and caput epididymidis, showed a steady increase in expression in the initial segment post orchidectomy; at 7 d post orchidectomy, CRYßB1 expression was increased by 475% in this segment.



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  		Figure 9. Expression of Hsp in the epididymis over the first week post orchidectomy. A, Grp94. B, Hsp47 and Hsp27. Gene expression is presented as relative intensity, mean ± SEM for three to five replicates per group. IS, Initial segment; CA, caput; CO, corpus; CD, cauda.

 
Apoptosis.
Genes coding for several apoptosis-associated proteins responded to orchidectomy throughout the epididymis (Fig. 10Go). Defender against cell death protein 1 (Dad1) was highly expressed in the proximal epididymis, particularly in the initial segment. Dad1 mRNA expression declined after orchidectomy by 88%, 88%, 91%, and 76%, respectively, in the initial segment, caput, corpus, and cauda epididymidis, respectively. Of the five bcl-2 family members [myeloid cell differentiation protein1 (Mcl1), bcl2-associated death promoter, bcl-x, Bax-{alpha}, and bcl2] that were expressed in the four epididymal segments, Mcl1 was the most highly expressed along the epididymis and, following orchidectomy, showed transiently increased expression. At the peak of transiently increased expression, Mcl1 expression was increased by 17%, 160%, 85%, and 76% in the initial segment, caput, corpus, and cauda epididymidis, respectively. TNF receptor 1 (TNFR1) was expressed at similar levels along the epididymis; TNFR1 showed a steady increase in expression before 7 d post orchidectomy when TNFR1 expression levels were increased by 87%, 166%, 128%, and 82% in the initial segment, caput, corpus, and cauda epididymidis, respectively.



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  		Figure 10. Expression of apoptosis-associated genes in the epididymis over the first week post orchidectomy. A, Dad1. B, Mcl1. C, TNFR1. Gene expression is presented as relative intensity, mean ± SEM for three to five replicates per group. Time post orchidectomy is indicated on the horizontal axis. IS, Initial segment; CA, caput; CO, corpus; CD, cauda.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we examined the expression of a large number of genes simultaneously at several time points post orchidectomy in the epididymis in a segment-specific manner. This design permitted us to visualize global trends in gene expression in the epididymis post orchidectomy, identify novel orchidectomy-responsive genes, and follow the expression of previously uncharacterized gene families in the epididymis after orchidectomy.

Patterns of gene expression in the epididymis after orchidectomy identified by k-means cluster analysis
K-means cluster analysis of gene expression is a powerful analytical tool that can provide important information on the responses of genes. Genes following the early-declining gene expression profile are likely regulated by circulating factors derived from the testis, such as androgens, because their decline in expression parallels the decrease circulating androgenic support to the epididymis observed by 12 h post orchidectomy. In support of this, a known androgen-dependent gene, metallothionein-3, showed an early-declining expression profile in the epididymis after orchidectomy. In the present study, we identified some potentially novel androgen-regulated genes such as thiopurine methyltransferase, DAAO, and OCTN2 (Table 1Go).

A major strength of cluster analysis is that it allowed us to detect transiently increased gene expression in each epididymal segment within the first week after orchidectomy. Transient up-regulation of gene expression in the epididymis after orchidectomy has not been reported previously. Transiently increased gene expression may reflect transcriptional changes occurring in the population of principal cells that undergoes apoptotic cell death in the epididymis after orchidectomy (12). Apoptosis is an active process that requires protein and RNA synthesis (33). Up-regulation of the transcription factor c-jun has been characterized in numerous models of apoptosis (34) as well as in apoptotic cells of the regressing rat ventral prostate after cadmium treatment (35). Alternatively, during regression of hormone-dependent tissues, such as the epididymis, expression of a number of mRNAs that are not involved in the apoptotic process may be induced. These genes may be induced as part of a futile stress response in cells that die after hormone ablation, or, alternatively, these genes may be induced in cells that resist apoptotic cell death as part of a survival mechanism. Transiently up-regulated gene expression may, at least partially, explain why the majority of epididymal epithelial principal cells survive the insult of androgen withdrawal after orchidectomy, whereas more than 95% of epithelial cells in the rat ventral prostate undergo apoptosis after orchidectomy (36).

The pattern of gene expression associated with a progressive increase in expression in the epididymis over the first week post orchidectomy included the genes that were up-regulated by at least 2-fold throughout the epididymis. At 7 d post orchidectomy, up-regulated gene expression most likely reflects transcriptional changes in surviving epididymal cells because apoptotic cell death is no longer detected in the rat epididymis at 7 d post orchidectomy (12). We propose that some of these genes, such as Gpx-1, are novel androgen-repressed genes in the epididymis. Only clusterin and TGFß have been previously identified as potential testosterone-repressed genes in this tissue (27, 28). As a family, Gpx enzymes catalyze the reduction of organic hydroperoxides and hydrogen peroxide, using glutathione as a reducing agent, thereby protecting cells from oxidative damage caused by normal oxidative metabolism (37). Gpx-1 is the cellular cytosolic Gpx enzyme, whereas Gpx-3 and Gpx-5 are secretory in mammals. The latter two have been previously localized in the epididymis and their expression is androgen dependent (38). Consistent with our hypothesis that Gpx-1 is androgen repressed in the epididymis, a previous report (39) has shown that Gpx-1 mRNA expression was unchanged after efferent duct ligation.

Gene expression changes in the epididymis after orchidectomy
Our analysis of 2-fold changes in gene expression in each epididymal segment revealed that there is a simultaneous increase and decrease in the expression of several genes at various time points over the first week after orchidectomy. Increased expression of several genes has been noted during regression of other androgen-dependent organs such as the prostate (36). By comparison, decreased gene expression was clearly the dominant response observed in the epididymis at 7 d post orchidectomy. The fact that expression of several genes was unchanged at this time suggests that the observed decrease in gene expression in each epididymal segment is a consequence of testis-dependent regulation rather than an overall decline in RNA synthesis (40).

Analysis of orchidectomy studies in the epididymis is somewhat complex because orchidectomy removes testis-derived circulating androgen support as well as direct testicular input to the epididymis. Presumably genes that respond by a decline in expression in all segments of the epididymis by 1 wk after orchidectomy are regulated by circulating androgens and not testicular factors because the latter have been implicated in the regulation of gene expression in the proximal epididymis but not in the distal epididymis (41). Among others, these include several glutathione GSTs and CaBPs. Interestingly, the pattern of decreased expression for the GSTs and CaBPs is similar to that of early-declining gene expression identified by k-means cluster analysis, which further supports androgen-dependent regulation of these genes.

Spermatozoa produce reactive oxygen species that are essential for capacitation and chromatin condensation (42). The GST family of enzymes function in cellular detoxification by acting as molecular scavengers that conjugate harmful electrophiles with reduced glutathione (43). In the epididymis, GSTs are expressed at high levels in principal and basal cells throughout the epididymal epithelium (44, 45) and are thought to form part of the epididymal antioxidant system that protects luminal spermatozoa and the epididymal epithelium from oxidative damage (46). The decline in epididymal expression of several GST transcripts after orchidectomy suggests that androgens regulate epididymal antioxidant functions. In support of this concept, our observation that the mRNA for GST Yf declined after orchidectomy in the proximal epididymis is in accordance with histochemical observations that GST Yf protein declines in these regions after androgen withdrawal by orchidectomy, but not after efferent duct ligation (47). The lack of an effect of orchidectomy on the expression of GST Yf in the corpus and cauda epididymidis is consistent with the observation that this subunit is selectively located in basal cells in these segments (46) and these cells seem to be minimally affected by orchidectomy (8). Moreover, the GST Yf promoter in the mouse contains several androgen-response elements (48), indicating that GST Yf expression in the epididymis may be regulated by androgens at the transcriptional level. Androgens have also been implicated in regulating epididymal GST enzyme activity (49).

In contrast, expression and regulation of CaBPs has not been previously described for the epididymis. CaBPs, originally isolated from rabbit endoplasmic reticulum (50), play an important role in maintaining cellular Ca2+ homeostasis. Our observations that several CaBPs are highly expressed throughout the epididymis and CaBP expression declines throughout the epididymis after orchidectomy suggest that circulating androgens are involved in the regulation of intracellular Ca2+ in the epididymal epithelium. In the prostate, expression of CaBP-3/calreticulin mRNA is androgen dependent (51); down-regulation of this gene increases sensitivity to apoptosis induced by the calcium ionophore A23187 in the androgen-sensitive prostate cell line LNCaP (52). This evidence suggests that the decreased expression of CaBP-3/calreticulin after androgen withdrawal by orchidectomy may be linked to androgen-dependent apoptosis of principal cells in the epididymis after orchidectomy (12). Alternatively, a recent study (53) has shown that differing expression patterns of high molecular weight CaBPs are observed in spermatozoa collected from the caput and cauda epididymal segments, indicating that CaBPs may be secreted from the epididymal epithelium during sperm maturation. The precise function of CaBPs in the epididymis and the significance of their regulation after orchidectomy remain to be elucidated.

Segment-specific up-regulation of genes in the epididymis has not been previously described. In contrast, segment-specific down-regulation of gene expression after the withdrawal of androgens or testicular factors is a hallmark characteristic of the epididymis (54). ODC is involved in the biosynthesis of polyamines, and ODC enzyme activity in the epididymis is regulated by androgens (55). Similarly, expression of mAAT, which we describe for the first time in the epididymis, is likely to be regulated by androgens. In the prostate, citrate production is an androgen-dependent process that requires mAAT (56); mAAT expression and stability in that tissue are regulated by androgens (57), likely through an androgen response element in its promoter (58). DAAO is a ubiquitous flavoenzyme that catalyzes the oxidative deamination of D-amino acids in many species including mammals (59). Although the existence of in vivo substrates for DAAO was not known for many years, recent evidence has demonstrated modulation of N-methyl-D-aspartate neurotransmission in the brain by D-ser, with a role for DAAO in brain function by regulating the level of these compounds (60). The significance of DAAO expression and regulation in the epididymis remains to be resolved.

Our observation that several transporters are expressed and differentially regulated along the epididymis is of interest in light of the changing microenvironment bathing spermatozoa in the epididymal lumen (61). OCTN2 has recently been characterized in the epididymis (62); OCTN2 is thought to function to transport carnitine into the epididymal lumen. ASCT2 has been proposed to be involved in transport of amino acids across the brush-border membrane of the intestine and kidney (63). Transport of amino acids has been characterized in the epididymis, particularly in the caput region (64), although specific transporters have yet to be identified. Our observation that ASCT2 is expressed and regulated in the epididymis after orchidectomy suggests that this particular transporter may be involved in androgen-dependent transport of amino acids across the epididymal epithelium.

Finally, we describe for the first time the expression and regulation of AGP mRNA specifically in the initial segment of the epididymidis. AGP belongs to the family of acute phase proteins synthesized by the liver and released into the circulation in response to tissue injury, inflammation, or infection. Drug binding to AGP, including retinoic acid and the endogenous steroid cortisol, has also been reported (65). It is possible that AGP is involved in transport of androgens across the epididymal epithelium in a manner similar to the well-characterized epididymal androgen-binding protein (66).

Expression by gene family
Hsps.
The expression of Hsps has not been described previously in the epididymis. Hsps are highly conserved proteins that act as molecular chaperones within the cell. In addition to their housekeeping functions, which include stabilization against protein aggregation, protein folding, intraorganellar transport, and refolding of denatured proteins (67), the expression of several family members can be modulated by cellular stress. Inducible Hsps, including Hsp27 and Hsp70, can accumulate in cells under a variety of stressful stimuli such as heat or oxidative stress (68). The family of grp, which include grp94 and grp78, can be regulated by perturbations in function such as Ca2+ depletion (69). Hsp47 is a collagen-specific molecular chaperone essential for collagen synthesis in the endoplasmic reticulum (70). We have observed that throughout the epididymis, progressively decreased expression of grp94 coincides with transient up-regulation of Hsp47 and Hsp27 over the first week post orchidectomy. Interestingly, transiently increased expression of TGFß1 has been noted also in the epididymis after orchidectomy (28), and TGFß1 has been implicated in the transcription of Hsp47 in mouse osteoblast cells (71). Transient up-regulation of Hsp47 after orchidectomy suggests that epididymal collagen production is altered in the epididymis after androgen withdrawal.

A transient increase in the expression of Hsp27 has been reported in epithelial cells during involution of the rat ventral prostate after castration/orchidectomy (72); Hsp27 has been shown recently to have antiapoptotic functions (73), suggesting that it may be directly involved in the survival of epididymal principal cells after orchidectomy. It is interesting to note that increased expression of the transcription factor c-jun coincided with the rise in Hsp27 and Hsp47 expression along the epididymis, with the greatest increase for all three genes observed in the caput epididymidis. This suggests that c-jun may be involved in the transcription of these genes in the epididymis after orchidectomy.

Expression of Hsp90ß, which regulates androgen receptor activation (74), was unchanged in the epididymis after orchidectomy. This observation was surprising in light of reports that have shown a decline in androgen receptor expression in the epididymis after androgen withdrawal (75, 76). Similar to clusterin, the small Hsp CRYßB1 showed increased expression in the initial segment of the epididymis after orchidectomy. CRYßB1 may be an androgen-repressed gene in the initial segment of the epididymis in a manner analogous to the androgen-repressed status of clusterin in the corpus and cauda epididymidis (27).

Apoptosis-associated proteins.
Mechanisms underlying apoptotic cell death in the epididymis after androgen withdrawal by orchidectomy (12) or efferent duct ligation (12, 77) are largely unknown. The involvement of the FAS pathway in epididymal apoptotic cell death after orchidectomy is controversial; an initial study indicated that FAS signaling was involved in apoptosis of male reproductive organs after orchidectomy (78), although a subsequent report on the FASR and FAS ligand null mutants failed to show prevention of apoptotic cell death in male reproductive organs (79), indicating that the FAS-FAS ligand pathway is not essential in mediating apoptosis after orchidectomy in these tissues.

In the present study, we show differential regulation of three apoptosis-associated transcripts after orchidectomy in the epididymis, Dad1, the bcl-2 family member Mcl1, and TNFR1. Dad1 has largely been implicated in N-linked glycosylation (80). In cell culture, inhibition of N-linked protein glycosylation in cell lines that undergo apoptosis after loss of Dad1 function suggests that loss of N-linked glycoproteins is associated with onset of apoptotic cell death (81). In the mouse, the null mutation for Dad1 is lethal and causes abnormal N-linked glycoproteins and increased embryonic apoptosis (82). High levels of Dad1 expression, particularly in the initial segment of the epididymis, indicate that there is a differential requirement for N-linked protein glycosylation along the epididymis. This decline in the expression of Dad1 along the epididymis suggests that androgens modulate Dad1 expression and protein glycosylation, although the link with apoptotic cell death in the epididymis after orchidectomy remains unclear. Interestingly, Dad1 has been shown to bind Mcl1 (83), which we have shown to be transiently increased along the epididymis after orchidectomy. Mcl1 is an antiapoptotic member of the bcl-2 family of proteins (84), which form a key group of intracellular factors that regulate apoptosis by binding to each other to form heterodimers (85). Transient up-regulation of Mcl1 expression may reflect an antiapoptotic mechanism by which the majority of principal cells are able to survive the apoptotic insult of orchidectomy. Studies in cell lines have shown that Mcl1 is capable of suppressing cell death induced by various stimuli (86).

TNFR1 is a transmembrane glycoprotein receptor that binds intracellular proteins to control signaling within the target cell. TNFR1-dependent signaling can be either pro- or antiapoptotic (87). At present, the significance of increased TNFR1 expression in the epididymis after orchidectomy is unclear, although studies on caspase-8, a downstream target of TNFR1 signaling, should help clarify this observation.

In summary, our study of gene expression in the epididymis after orchidectomy has provided a clearer understanding of patterns of mRNA expression underlying epididymal regression after orchidectomy. The identification of novel genes that are regulated in the epididymis after orchidectomy, both throughout the epididymis and in a segment-specific manner, enhances understanding of androgen-dependent and segment-specific epididymal functions.


   Footnotes
 
This work was supported by a program project grant from the National Institute on Aging, NIH (Grant AG08321).

Abbreviations: AGP, {alpha}-1-Acid-glycoprotein; ASCT2, sodium-dependent neutral amino acid transporter; CaBP, calcium-binding protein; CRYßB1, crystallin-ß B1; DAAO, D-amino acid oxidase; Dad1, defender against cell death protein 1; GGT, {gamma}-glutamyl transpeptidase; Gpx, glutathione peroxidase; grp, glucose-regulated protein; GST, glutathioneS-transferase; Hsp, heat shock protein; mAAT, mitochondrial aspartate aminotransferase; Mcl1, myeloid cell differentiation protein 1; OCTN2, organic cation transporter N2; ODC, ornithine decarboxylase; TNFR1, TNF receptor 1.

Received July 10, 2002.

Accepted for publication November 14, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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