| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Departments of Urology3 and Cell Biology,4 University of Virginia Health Science System, Charlottesville, Virginia 22908
Discovery Translational Medicine,5 Wyeth Research, Collegeville, Pennsylvania 19426
Biological Technologies,6 Molecular Profiling and Biomarker Discovery, Wyeth Research, Cambridge, Massachusetts 02140
ABSTRACT
The epididymis has traditionally been divided into the caput, corpus, and cauda regions, which are further organized into intraregional segments. In the rat and mouse, these segments have high degrees of transcriptional differentiation, and what has traditionally been called the initial segment of the rat epididymis actually consists of three transcriptionally different intraregional segments. These segments are regulated by endocrine, lumicrine, and paracrine factors, whose relative importance remains a topic of investigation. In the present study, 15-day unilateral efferent duct ligation (EDL) was used to deprive ipsilateral rat epididymides of lumicrine regulation. Segments 1–4 of EDL epididymides and contralateral, sham-operated tissues were collected individually. Microarray analysis of gene expression was used to determine the effect of lumicrine factor deprivation on the transcriptome-wide gene expression of each segment studied. More than 11 000 genes were detected as being expressed in each of the four segments examined. More than 2000 genes responded significantly to EDL in segment 1, although this number of genes declined in each succeeding segment. Segments 1 and 2 of control tissues were the most different transcriptionally and the most affected by EDL. In the absence of lumicrine factors, the four segments regressed to a transcriptionally undifferentiated state, which was consistent with the less-differentiated histology seen after EDL. Interestingly, for an individual gene, lumicrine factor deprivation could stimulate expression in some segments and suppress expression in other segments. These results reveal a higher complexity to the regulation of rat epididymal segments than heretofore appreciated.
epididymis, gene regulation, male reproductive tract
Luminal factors from the testis, in addition to androgens, are important for both epididymal development [1, 2] and the maintenance of adult tissues [3–6]. Efferent duct ligation (EDL) obstructs testicular outflow, causes the loss of lumicrine factors, such as androgen-binding protein and basic FGF, from all points distal to the ligation, and induces a number of alterations in the epididymal tubule [7, 8].
Many studies of the epididymis have divided the organ into the traditional regions of caput, corpus, and cauda, with the initial segment of the caput sometimes being separated for further definition. It has been known for some time that each epididymal region of the common rodent models is made up of intraregional segments or lobules of coiled tubule that are separated by septae [9, 10]. Recently, microdissection of the mouse [11, 12] and rat [13] epididymides has demonstrated that these epididymides are divided into 10 and 19 intraregional segments, respectively, and it has been shown that the connective tissue septae (CTS) that separate the segments can establish borders for epididymal gene expression, protein presence, and epithelial responses to lumicrine factors [11, 12, 14]. Furthermore, based on their unique gene expression profiles, some segments appear to be separate functional units within the epididymis [12, 13]. This type of segmentation has been noted in species from rats and mice [11] to dogs [15]. In addition, the marmoset, which is a primate, shows histological evidence of epididymal segments separated by CTS (T.T. Turner and D. Bomgardner, unpublished results), and preliminary evidence suggests that the adult cynomolgous monkey (Macaca fasicularis) epididymis microdissects into approximately 50 segments (T.T. Turner and D. S. Johnston, unpublished results).
EDL has often been reported to have effects on specific epididymal gene or protein expression [7, 8], although these reports have focused on relatively few genes or have made reference to only the standard epididymal regions or the initial segment. Microarray analysis of gene expression allows quantitative detection of thousands of gene transcripts, and when performed on individual segments rather than entire regions it increases the sensitivity of transcript detection. This increase in sensitivity is in the range of 17% to 25% for the mouse and rat epididymides [12, 13].
Since EDL and the deprivation of testicular factors have been shown to alter dramatically epithelial structure, especially in the proximal segments of the epididymis [3, 6] as well as secreted protein composition [4], gene expression [5], and epithelial cell apoptosis [16–18], and since microarray analysis of individual segments allows a broadly based yet highly specific evaluation of epididymal gene expression, we have used this approach to examine the effects of lumicrine signaling on both general and specific gene expression in the most proximal four segments of the rat epididymis.
Adult male Sprague Dawley rats (400–500 g) were obtained from vivarium sources and maintained on ad libitum food and water in a constant temperature (22°C) room on a 12L:12D cycle. All experiments were conducted according to protocols approved by the IACUC of the University of Virginia.
Animals were anesthetized with sodium pentobarbitol (50 mg/kg body weight). Unilateral EDL and contralateral sham operations were performed as described previously [18, 19]. Fifteen days later, the testes and epididymides were exposed, and the epididymides were removed after ligation of the spermatic and vasal arteries. Epididymides were immediately placed in ice-cold saline in a Petri dish and carefully defatted. These and all subsequent manipulations were performed with the use of a dissecting microscope while maintaining the dissection medium on ice at all times. Each defatted epididymis was transferred to fresh, ice-cold saline and the epididymal tunica albuginea was removed from the entire caput epididymidis using sharp microdissection. With removal of the tunica and blunt dissection along the plane of the segment-dividing CTS, the first four epididymal segments were isolated. As described previously for the mouse [12], the exact track of the CTS borders varied somewhat from animal to animal, although the general appearances of the segments and their CTS (Fig. 1) were relatively consistent. As each segment was isolated, it was placed immediately in RNALater (>10x tissue volume; Ambion, Austin, TX) in a 1.5-ml Eppendorf tube on ice. All four segments of each epididymis were place in separate tubes of RNALater within 15 min of epididymal extirpation. The samples were immediately stored at –80°C. This procedure was repeated until each epididymal segment was represented by five or six separate samples, each from a different animal, for RNA extraction.
|
Five additional animals were subjected to unilateral EDL and 15 days later both the contralateral control epididymides and the ipsilateral EDL epididymides were extirpated, paraffin embedded, and stained with hematoxylin-eosin for subsequent measurements of tubule diameter and epithelial height.
Immediately prior to processing, the samples were thawed at room temperature and the RNALater was removed. The samples were washed twice with 1 ml of ice-cold TRIzol (GIBCO BRL, Gaithersburg, MD). The tissues were homogenized in 600 µl of TRIzol using a PowerGen 700 automatic homogenizer (Fischer Scientific, Hampton, NH). RNA was extracted according to the manufacturer's instructions and further purified through RNAeasy columns (Qiagen, Valencia, CA). All samples were treated with DNase on the column and eluted with water. RNA was quantified by the absorbance at 260 nm (NanoDrop, Willmington, DE) and RNA quality was assessed using the Agilent Bioanalyzer (Palo Alto, CA).
Five micrograms of total RNA were used to generate biotin-labeled cRNA using an oligo(T7) primer in a reverse transcription reaction, followed by in vitro transcription with biotin-labeled UTP and CTP. Ten micrograms of cRNA were fragmented and hybridized to the RAE230 2.0 array (Affymetrix, Santa Clara, CA), which monitors over 30 000 transcripts. The hybridized arrays were stained in the Fluidics Station 450 and scanned on the Affymetrix Scanner 3000. All of the array images were visually inspected for defects and quality. Arrays with excessive background, low signal intensity or major defects within the array were eliminated from further analysis. The final numbers of arrays used for analysis were three for all the control segments and six, five, six, and six for EDL segments 1 to 4, respectively.
Signal values were determined using Gene Chip Operating System 1.0 (GCOS; Affymetrix). For each array, the transcript expression values were normalized to a mean signal intensity value of 100. The default GCOS statistical values were used for all analyses. Signal values and absolute detection calls were imported into Expressionist Analyst 3.0 (Genedata, Basle, Switzerland) for analysis.
The transcripts of a gene were considered to be detectable if their mean expression level in any segment was greater than 50 signal units and the percentage of samples with a Present call, as determined by the GCOS default settings, was equal to or greater than 67% for the samples within a group. Such transcripts are hereinafter referred to as qualifiers. Normalized signal values were transformed to log base 10, and pair-wise comparative analysis of the qualifiers in each segment was performed. A qualifier was considered to be regulated if the following conditions were met: 1) the qualifier was detected in at least 67% of the samples of at least one of the segments analyzed; and 2) a significant difference at the level of P 
0.01 (Welch test) existed between the control and EDL values. Qualifiers that met these conditions were used for further analysis. For some computations, the expression values for each qualifier were normalized to a mean of zero and a standard deviation of 1 (z-score normalization). This allowed direct comparison of patterns within the data without regard to absolute expression levels.
Principal Component Analysis (PCA) was performed on the log-transformed data and visualized using Spotfire 7.2 (Somerville, MA). The outcome this analysis of datasets is a set of variables that is visible in a two- or three-dimensional space. The process allows analysis of very large datasets in a way that is useful for functional and biological interpretation of complex data [20].
The Database for the Annotation, Visualization, and Integrated Discovery (DAVID) was used for gene annotation and characterization. Using the Functional Annotation tool, each gene was annotated based on a number of sources including but not limited to GO ontology, Kegg pathways, and Biocarta pathway. The Fisher exact test was used to determine if a particular category of genes was overrepresented in the dataset. Related genes are usually annotated within the same functional categories, which aids in the identification of biologically relevant processes.
The primer sequences were chosen from the published sequences for prostaglandin D2 synthase (Ptgds), CD52 antigen (Cd52), glutathione peroxidase 3 (Gpx3), cystatin 8, (Cst8; also known as CRES for cysteine-related, epididymal-specific protein), defensin ß1 (Defb1), 5
-reductase I (Srd5a1), phosphatidylethanolamine-binding protein (Pebp1), lipocalin 5 (Lcn5; also known as ERABP or epididymal retinoic acid-binding protein), and superoxide dismutase 1 (Sod1). The GenBank accession numbers and the forward, reverse, and probe sequences, respectively, for each gene were: for Ptgds (NM_013015), 5'-CGGCCTCAACCTCACCTCTA-3', 5'-CCGGCTGCAGTACCATCAC-3', and 5'-CTTCCTAAGGAAAAACCAGTGTGAGACC-3'; for Cd52 (NM_053983), 5'-TGGGAAGGGTTGATACCAGAGA-3', 5'-CCAAGGAGGTTCAAGTTGACAGCCCA-3', and 5'-CCCAGCACCTCGACGTTCT-3'; for Gpx3 (NM_022525), 5'-TTGAACTGAATGCACTACAAGAAGAA-3', 5'-TTGGCCCATTCGGCCTGGTC-3', and 5'-TGCAAGGGAAGCCCAGAA-3'; for Cst8 (NM_019258), 5'-ACAAGACACTCCATGCCACACT-3', 5'-TGCGGGAGATCTGAACATCA-3', and 5'-CAGATCACAGACCGCATGGAATACCACA-3'; for Defb1 (NM_031810), 5'-TCTTGGACGCAGAACAGATCA-3', 5'-CAGCTGGAGCGGAGACAGA-3', and 5'-TACCGATGCCTCCAAAATGGAGG-3'; for Srd5a1 (NM_017070), 5'-CTTGACCCAGTTTGCGGTTT-3', 5'-TGCTGAAGACTGGGTGACCCATCCC-3', and 5'-TGCTGAAGACTGGGTGACCCATCCC-3'; for Pebp1 (NM_017236), 5'-GGTTACAGCTCTAGGATGTCTTCCA-3', 5'-TGATAAGCCCACCCCAAAAG-3', and 5'-TTTGTCCAGGACCAGGCCCAGTAACA-3'; for Lcn5 (NM_024136), 5'-GAGATTGCCTTTGCCTCCAA-3', 5'-CACCATGGCTCCCATCTTCT-3', and 5'-ACACCTGGCTTGGCACACAAGGA-3'; and for Sod1 (NM_017050), 5'-CGGATGAAGAGAGGCATGTTG-3', 5'-TTGGCCACACCGTCCTTT-3', and 5'-AGACCTGGGCAATGTGGCTGCTG-3'.
Briefly, mRNA from each epididymal segment was analyzed by RT-PCR using 2.5 ng of total RNA in a final volume of 25 µl that contained 300 nM of the target-specific PCR primers (Invitrogen, Carlsbad, CA), 100 nM fluorescently-labeled oligonucleotide probe (Eurogentec, San Diego, CA), and 1x Quantitect Probe RT-PCR Mix (Qiagen). Reverse transcription was performed for 30 min at 48°C, followed by 40 thermal cycles of 30 sec at 94°C and 1 min at 60°C using the 7900HT Fast Real-Time PCR System (Applied Biosystems). Target mRNA was normalized to 18S ribosomal RNA, as determined using TAQMAN Ribosomal RNA Control Reagents (Applied Biosystems).
The segmentation of the proximal caput epididymidis of the rat was evident under epi-illumination (Fig. 1A). EDL reduced the size of the organ and made the appearance of the segment surfaces more indistinct (Fig. 1B). Segment histology showed significant reductions in lumen diameter and epithelial height (Fig. 1, C and D, Table 1), with the most marked changes in both features occurring in segment 1 (Table 1). The reductions in lumen diameter became progressively less severe as the segments become more distal, even in this very proximal part of the organ. Epithelial height reduction was also most pronounced in segment 1 (Table 1). Clearance of sperm from the epididymis had typically reached segment 17 (mid-cauda) by Day 15 post-EDL (see [13] for schematic of distal segments).
|
Overall Changes in Gene Transcription Induced by EDL
In all, 2255 qualifiers were regulated in response to EDL in segment 1 (Table 2). The numbers of qualifiers that were either upregulated or downregulated after EDL declined sequentially in each more-distal segment, with segment 4 showing only 420 genes (
4% of total) that were regulated in response to EDL (Table 2). The caliber of these changes varied from gene to gene and from segment to segment, with EDL again having its largest effects in segment 1, in which 1595 qualifiers were induced to at least a 2-fold change, 150 to at least a 10-fold change, and 11 to at least a 100-fold change (Table 3). The number of qualifiers induced to change expression at each level (2-, 5-, 10- or 100-fold) decreased as the segments became more distal to the site of EDL (Table 3), although the proportions of upregulated versus downregulated qualifiers were relatively constant, i.e., approximately 50%, 30%, and 15% of the altered genes were upregulated at 2-, 5-, and 10-fold levels, regardless of the segment studied (not shown). All changes at the 100-fold level were downregulatory.
|
|
Among the qualifiers that responded to EDL, there were many genes with relatively low expression in control segment 1 that gradually increased in expression in the control segments thereafter (Fig. 2). At the same time, there were many qualifiers with high expression in control segment 1 that decreased in subsequent segments (Fig. 2). EDL eliminated these differences by sharply decreasing the expression of highly expressed qualifiers, especially in segments 1 and 2, while allowing moderately increased expression of qualifiers that had been poorly expressed (Fig. 2). Thus, the net effect of EDL was to eliminate segmental differences, not only making segments 1–4 resemble each other in terms of gene expression, but also resemble segment 4 of the control tissue (Fig. 2) and segments 5 and 6 of the control epididymis [13].
|
PCA grouped samples with similar expression patterns, which when displayed in two-dimensional space, illustrated rat segment 1 as a unique gene expression unit, as was segment 2 (Fig. 3). Control segments 3 and 4 spanned a common space and, thus, were not different. EDL caused all four segments to become transcriptionally different from their control counterparts and eliminated segmental differences (Fig. 3).
|
Individual Gene Expression Levels After EDL: Verification of Microarray Results
Gene expression was assayed by qRT-PCR, to verify the results of the microarray analysis. The selected genes were those previously known to be expressed in the epididymis. Ptgds, Cd52, and Gpx3 are examples of genes whose expression levels increased in at least one segment after EDL according to the microarray results. Cst8, Defb1, and Srd5a1 are examples of genes that were generally downregulated after EDL according to the microarray analysis, and the gene expression levels of Pebp1, Lcn5, and Sod1 were unaltered by EDL. These microarray results were verified by qRT-PCR (Fig. 4). In both methods of analysis, Ptgds expression increased from segments 1 to 4 in the control tissues, whereas EDL upregulated Ptgds expression in segment 1 and downregulated Ptgds expression in segment 4 (Fig. 4A). Both methods of analysis demonstrated that Cyst8 decreased sharply after segment 2 in the rat epididymis and virtually vanished in all studied segments after EDL (Fig. 4B). The expression levels of Pebp1, Lcn5, and Sod1 were generally not significantly altered by EDL (data not shown).
|
DAVID analysis revealed changes induced by EDL in functional categories of qualifiers that were not necessarily detectable by evaluation of individual expression patterns (Table 4). The DAVID annotations include over 500 different categories. Table 4 illustrates 18 selected categories from those overrepresented among the qualifiers that changed after EDL in the four segments studied. Consistent with the data in Tables 1 and 2, segment 1 typically had the largest number of affected qualifiers in a given category, and the number of these qualifiers typically declined sequentially in the more distal segments (Table 4).
|
The individual gene data generated in the present study will be made available at the Mammalian Reproductive Genetics website (http://mrg.genetics.washington.edu).
The profound effect of EDL on the epididymis was apparent by direct observation (Fig. 1A and B), and tissue histology revealed marked changes (Fig. 1C and 1D) in the measured features, verifying the effects (Table 1) reported previously for the rat and mouse [3, 19, 21, 22], i.e., EDL causes the loss of testicular contribution from the epididymal lumen, as well as reductions in lumen diameter and epithelial height. Furthermore, the present results demonstrate that histological changes in the proximal rat epididymis are most profound in segment 1, with lower effects in progressively more distal segments (Table 1).
The present study differs from previous studies in that the tissue morphology and transcriptome-wide gene expression were examined in both sham-operated control tissues and tissues after 15 days of EDL in a segment-by-segment manner through the first four segments of the rat epididymis. Preliminary assurance that conventional histology confirmed the effects of EDL was fundamental to undertaking the gene expression analysis.
It is well known that interruption of the luminal contribution from the testis alters epididymal gene and protein expression [7, 8]. However, the extents of these changes have been difficult to assess from studies of specific genes or proteins after EDL. Microarray analysis of gene expression has previously been used in rats [23] and mice [24, 25], to evaluate more broadly the changes in epididymal gene expression after orchidectomy in the presence or absence of androgen replacement. These studies have documented widespread changes in gene expression throughout the epididymis when androgen and other testicular factors are removed. Typically, these studies have shown that non-androgen testicular factors, as well as the androgens themselves play regulatory roles in the epididymis. Unfortunately, direct comparisons of these studies are difficult owing to the use of different species, different epididymal dissections, e.g., some studies divide the caput epididymis into the initial segment and the remainder of the caput, whereas other studies use the intact caput, and different microarray platforms. Although direct comparison with the present study is difficult, some broad correlations can be made.
In the present study, we used unilateral EDL, which preserved the circulating androgens while eliminating lumicrine factors from the ipsilateral epididymis. The results make it clear that the expression patterns of thousands of gene are altered by loss of lumicrine factors, which can be suppressive (more than 1200 qualifiers were upregulated after loss of lumicrine factors) or stimulatory (more than 1000 qualifiers were downregulated after loss of lumicrine factors; Table 2). It has been demonstrated previously that loss of testicular factors can upregulate or downregulate the expression of individual genes in the rat [22] and mouse [24, 25] epididymis. The present study focused specifically on the first four segments of the rat epididymis, as these segments receive intraluminal factors directly from the testis and are probably the portions of epididymal tubule that are most sensitive to lumicrine factors [8].
The first three segments of the rat epididymis make up what has historically been referred to in the rat as the initial segment, and originally referred to by Reid and Cleland [9] as Zones 1a, 1b, and 1c, respectively. The initial segment nomenclature presents difficulties that have been described elsewhere [13], not the least of which is that under this single heading all three or even four segments have been assayed together as if they are a single unit, which is not the case. The segments are transcriptionally different (Figs. 2 and 3), and the more proximal the segment the more profoundly its gene transcription is affected by EDL (Fig. 2, Table 3).
Nearly 20% of the segment 1 transcriptome was altered by 15-day EDL, and this proportion declined to approximately 4% by segment 4 (Table 3). This is probably due to the fact that segments that were highly differentiated in control tissues became undifferentiated after EDL (Fig 2), a point that was validated by PCA (Fig. 3). This establishes that the transcriptome of control segment 1 is transcriptionally distinct from control segment 2, which is distinct from segments 3 and 4, which are identical to each other (Fig. 3). After 15-day EDL, none of the segments were transcriptionally different from each other, but they were all different from their respective control tissues (Fig. 3). In other words, EDL induces dedifferentiation of the proximal segments of the rat epididymis.
The microarray results, which documented the segmentation of gene expression in control tissues, upregulation of many genes after EDL, downregulation of other genes, and the lack of change in still other genes, were validated by qRT-PCR. The results show general fidelity between the two techniques (Fig 4). Each expression profile illustrated in Figure 4 has been reported previously for the epididymis at either the gene or protein level [7, 12, 14, 26–28], although typically not on a segment-by-segment basis. Ptgs expression was upregulated in segment 1 after EDL but downregulated in segment 4 according to both the microarray analysis and qRT-PCR (Fig. 4A). Cd52 and Gpx3 were upregulated in segments 1 and 2 after EDL according to both techniques. The expression levels of Cst8, Defb1, and Srd5a1 were all heavily dependent upon testicular factors in virtually all the segments according to both analyses (Fig. 4B), and the expression levels of Lcn5, Pebp, and Sod1 (data not shown) were typically unaltered by the presence or absence of lumicrine factors. The results from the two different assay systems generally corroborate each other and confirm the patterns of gene expression in both control epididymal segments and in segments after EDL.
Interestingly, the data illustrate that removal of lumicrine factors from the epididymis causes more upregulation than downregulation of gene expression, which supports the results obtained in the mouse by Sipila et al. [25], while adding the complexity of removal of luminal factors, which can suppress the expression of a particular gene in one segment and stimulate expression in another segment, e.g., Ptgs (Fig. 4A). This emphasizes the complexity implied by epididymal segmentation and draws attention to the fact that gene regulation needs to be addressed on a segment-by-segment basis.
Changes in gene expressions across a category of related genes after a specific treatment imply broad functional changes associated with that category. This implication is even stronger when related categories change simultaneously. In the present case, categories, such as extracellular matrix, collagen, cell differentiation, blood vessel development, apoptosis, and cell-matrix junction, were overrepresented among the qualifiers that showed changed expression after EDL (Table 4). Another interpretation is that the same sets of genes are represented in each of these categories, since each gene can be represented in multiple annotation categories. This may indicate continuous remodeling of the tubule epithelium and potentially, of the peritubular/interstitial tissue 15 days after EDL. An overrepresentation of qualifiers from certain categories, such as lipid metabolism, carboxylic acid metabolism, ATPase activity, and mitochondrion, suggest a profound change in basic metabolism within the tissue, which is not surprising given the tissue remodeling. Interestingly, the Development category is most overrepresented among the genes that show changes in expression after EDL (e.g., 260 qualifiers in segment 1; Table 4). Development genes are found in the categories of cell differentiation (113 qualifiers with expression altered by EDL), cellular morphogenesis (55 qualifiers), embryonic development (30 qualifiers), tube morphogenesis (12 qualifiers), urogenital development (6 qualifiers), and Wnt signaling pathway (5 qualifiers) with expression altered by EDL (data not shown). Future investigations will determine if these changes in gene expression represent true dedifferentiation of the proximal epididymal segments in the absence of luminal factors from the testis.
In summary, the first four segments of the rat epididymis show three different transcriptome-wide expression patterns. Segments 1 and 2 are different from each other and from segments 3 and 4, which are not different from each other. Testicular lumicrine factors are critical for the maintenance of these differential expression patterns and their removal induces a loss of differentiation of the segments both histologically and transcriptionally. These findings imply that even in the patent tract, normal testicular secretions in addition to testosterone are required for normal epididymal function in a system that is much more complex than was previously appreciated. A more complete determination of important lumicrine molecules is necessary, as well as the determination of the specific roles of these molecules in both direct signaling to the tubule epithelium and indirect signaling through cells of the epididymal interstitium.
ACKNOWLEDGMENTS
The technical assistance of Sonja Usanovic is gratefully acknowledged, as is the histology preparation carried out by the Cell Science Core Facility of the University of Virginia School of Medicine.
FOOTNOTES
1Supported by NIH grant DK45179 (T.T.T.) and Wyeth Research. 
Correspondence: 2Terry T. Turner, Department of Urology, University of Virginia School of Medicine, P.O. Box 800422, Charlottesville, VA 22908. FAX: 434-924-8311; e-mail: ttt{at}virginia.edu
Received: 13 December 2006.
First decision: 20 January 2007.
Accepted: 8 March 2007.
REFERENCES
-glutamyl transpeptidase messenger ribonucleic acid transcripts in the adult epididymis is differentially regulated by androgens and testicular factors. Endocrinology 1994; 135:1225–1233This article has been cited by other articles:
|
|
 |
|
  T. T. Turner De Graaf's Thread: The Human Epididymis J Androl, May 1, 2008; 29(3): 237 - 250. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
