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Male Reproductive Tract |
School of Life Sciences,4 Arizona State University, Tempe, Arizona 85287
Molecular and Cellular Biology Graduate Program,5 Arizona State University, Tempe, Arizona 85287
Center for Evolutionary Functional Genomics,6 Arizona BioDesign Institute, Arizona State University, Tempe, Arizona 85287-4501
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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male reproductive tract, male sexual function, sperm motility and transport, vas deferens
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The vas deferens is a thick-walled muscular duct that connects the distal cauda of the epididymis to the urethra at the level of the prostate. The duct is lined with a pseudostratified epithelium with columnar cells bearing stereocilia projecting into the lumen [3]. The epithelium is supported by a thin lamina propria, which is characterized by an intricate network of elastic fibers [2]. The epithelial lining and lamina propria in the distal vas deferens rise into longitudinal folds that permit expansion of the duct during ejaculation. The distal end of the vas deferens forms the ampulla, a region possessing a wider lumen with more epithelial folds than the main portion. The ampulla and the seminal vesicle form the ejaculatory duct that empty into the urethra at the level of the prostate.
ATF4 is member of the large activating transcription factor/cyclic AMP-responsive element-binding protein (ATF/CREB) transcription factor family of basic region-leucine zipper (bZip) containing proteins that are able to bind to the consensus cyclic AMP-responsive element (CRE) site (TGACGTCA) [4]. ATF4 is able to form heterodimers through the bZip domain with members of the AP-1 and C/EBP families, including Fos [5], Jun [5, 6], JunD [7], and several C/EBP proteins, C/EBPa, C/EBPb/CRP2, C/EBPg (IgEBP or GPE-BP), and C/EBP1/CRP1 [8–11]. The choice of heterodimer partner can alter DNA-binding specificity and transcriptional activity, thus increasing the complexity by which these factors regulate gene expression. In addition, ATF4 can interact with components of the general transcription machinery, including TFIIB, TFIIF, and TATA-binding protein, as well as the CREB-binding protein (CBP), a factor associated with chromatin remodeling [12].
ATF4 (CREB-2) can act as a transcriptional activator or repressor, dependent on the cell and promoter system being tested [4]. Unlike other members of the CREB/ATF family that promote cell proliferation, ATF4 expression is associated with growth arrest. Overexpression of ATF4 in NIH3T3 fibroblasts countered the transforming activity of ectopically expressed ras(V12/G12) oncogene [13]. Further, ATF4 appears to repress the ras promoter and prevent ras-mediated cell transformation. Another proposed mechanism by which ATF4 regulates cell proliferation is through the transcriptional activation of CHOP in response to a broad spectrum of cell stresses [14]. CHOP is a member of the C/EBP transcription factor family that has been implicated in inducing either growth arrest at the G1/S boundary or cell death [15].
Here we present evidence that male mice deficient for ATF4 are infertile due to obstructions in the vas deferens. Histologic analysis revealed the presence of hyperplastic foci in the lamina propria layer where cells overproliferated. Further, they failed to adopt a differentiated morphology, as they did not produce elastic fibers and lacked interstitial cells of Cajal (ICC). These studies demonstrate that ATF4 is an important regulator of the lamina propria layer of the vas deferens.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A targeted null ATF4 allele was generated by replacing the second exon of ATF4 with the neomycin-resistance gene [16]. This mutation removes the basic leucine zipper domain that is essential for the function of the protein. Mice carrying the ATF4- allele were identified using a PCR genotyping strategy [16]. Genomic DNA was isolated from tail clippings (
2 mm) by incubating overnight at 55°C in PCR buffer with nonionic detergents (50 mM KCL; 10 mM Tris [pH 8.3]; 2.5 mM MgCl2; 0.1 mg/ml gelatin, 0.45% v/v NP40, 0.45% v/v Tween-20, and proteinase K 0.15 mg/µl) [17]. The tissue was vigorously vortexed to break up cellular debris and 1–2 µl was used for PCR. The mutant ATF4 allele was amplified using an upstream primer specific to ATF4 (5'-AAGCAAAGCTAAGCCTCCAT CTTGTGC-3') and a downstream primer specific for the neomycinR gene (5'-ATCG CCTTCTATCGCCTTCTTGACGA-3'). The wild-type allele was amplified using the upstream ATF4 primer and a downstream primer specific for the ATF4 gene (5'-AAAG GAATGCTCTGGAGTGGAAGACAG-3'). The amplified product for both primer sets was approximately 1000 base pairs. The experiments were carried out according to the principles and procedures outlined in the Guide for Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at Arizona State University.
In Vivo Fertility
ATF4+/+ female mice (6–8 wk of age) were introduced into the cages of male ATF4-/- and ATF4+/+ mice of the same age. Copulation was determined by the presence of a vaginal plug on subsequent mornings. Females that copulated were isolated for 21 days to allow the pregnancy to come to term. The success of mating was scored based on the ability of the male to successfully copulate over a 14-day period, the ability to impregnate, and the number of pups per litter.
Isolation of Sperm and In Vitro Fertilization
The caudae epididymides of 8-wk-old ATF4+/+ and ATF4-/- mice were quickly excised into human tubal fluid (HTF; Specialty Media, Phillipsburg, NJ). Each epididymis was diced, and the sperm were allowed to disperse into the medium for 10 min at 37°C. Aliquots of the epididymal suspension were counted on a hemocytometer, accompanied by a further dilution to 1 x 106 sperm/ml. Sperm were capacitated in HTF for 1 h in an incubator at 37°C and 5% CO2 as described by Jeffs et al. [18]. Sperm morphology was analyzed by spreading epididymal sperm suspended in HTF on microscope slides and visualized by phase-contrast microscopy.
CD-1 females were superovulated by injection with 5 international units (IU) of eCG (Sigma, St. Louis, MO) followed, after 48 h, by 5 IU of hCG (Sigma). Females were killed 14 h after hCG injection and cumulus-oocyte complexes were isolated from the oviducts and placed in simplex optimized medium with elevated potassium (KSOM; Specialty Media, Phillipsburg, NJ) supplemented with 7.5% calf serum. One hundred microliters of sperm from wild-type and ATF4-/- mice at concentration of 1 x 106 sperm/ml were added to 15 to 20 eggs in an equal volume of KSOM. Eggs were exposed to sperm for 2 h, after which they were washed twice in KSOM, then transferred to fresh KSOM medium where they were cultured at 37°C and 5% CO2. Twenty-four and 48 h after the addition of sperm, the eggs were scored for progression to the two- and four-cell stages. Eggs that had undergone apoptosis as judged by cytofragmentation were scored. Experiments were repeated three times with sperm from three different mice of each genotype. A statistical comparison of the ability of the sperm from the ATF4+/+ and ATF4-/- mice to fertilize eggs was carried out using a two-way ANOVA. Significant difference was defined as P < 0.05.
Immunohistochemistry
The vas deferens, epididymis, and duodenum were dissected from 5- and 10-wk-old C57Bl/6 male mice and fixed in 10% neutral buffered formalin. The tissue was dehydrated through a graded series of alcohol and xylene incubations and embedded in paraffin. Transverse 10-µm histologic sections were generated from the distal and proximal vas deferens, cauda of the epididymis, and proximal region of the duodenum. Immunohistochemistry was performed as described previously [19]. Briefly, thin sections were deparaffinized in xylene and rehydrated. The tissue was treated with boiling citrate buffer (pH 6.0) for 10 min in order to improve antigenicity. Immunodetection was performed using the HistoStain SP Kit (Zymed Laboratories Inc., San Francisco, CA) using the protocol recommended by the supplier. For detection of ATF4, a rabbit anti-CREB-2 polyclonal IgG (Santa Cruz Biotechnologies, Santa Cruz, CA) [20–22] was used at a 1:200 dilution. Goat anti-rabbit IgG conjugated to peroxidase (Zymed Laboratories Inc.) at a 1:500 dilution was used as a secondary antibody. Neuron-specific enolase was detected with an anti-neuron-specific enolase (Polysciences, Inc., Warrington, PA) [23–25] and a goat anti-rabbit secondary antibody at a dilution of 1:1000 (Zymed Laboratories Inc.). Micrographs were taken on a Nikon Eclipse TE300 with a Nikon Coolpix 4500 camera (Nikon Corporation, Tokyo, Japan).
Verhoeff Stain
Tissue was stained for elastic fibers using a standard Verhoeff stain for elastic tissue. Histologic sections were deparaffinized in xylene and rehydrated in a graded series of ethanol solutions. Tissue was rinsed in tap water for 3 min followed by 15 min in the Verhoeff solution (2.8% alcoholic hematoxylin, 2% aqueous ferric chloride, 20% Lugol iodine). Tissue was then washed in distilled water and differentiated by briefly dipping in 2% ferric chloride. The excess ferric chloride was removed by rinsing in tap water. Tissue was treated with 5% sodium thiosulfate for 1 min, followed by washing in tap water and counterstaining with Von Gieson stain. Micrographs were taken on a Nikon Eclipse TE300 with a Nikon Coolpix 4500 camera.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The fertility of ATF4-/- male mice of reproductive age was examined by introducing age-matched ATF4+/+ females into the cage. We scored for both the ability to impregnate females over a 2-wk period of time and the size of the resulting litters. Of the eight ATF4-/- males tested, only one was able to impregnate a wild-type female, which resulted in a litter of four pups. In contrast, all of the ATF4+/+ males were able to successfully impregnate a female within 6 days, with an average litter size of 8.5 pups.
The failure of the ATF4-/- males to sire offspring indicates that the mice are subfertile. The most common cause of infertility is disruption of the production of viable sperm. To examine the role of ATF4 in spermatogenesis, sperm production, and the ability to fertilize an egg, in vitro fertilization was assessed in ATF4-/- and ATF4+/+ littermates. Sperm were isolated from the epididymis and vas deferens of 10-wk-old mice, capacitated in HTF for 1 h and counted and examined for morphological defects (Table 1). In the eight ATF4-/- males examined, there was no significant difference (P < 0.28) in the production of sperm when compared with ATF4+/+ males. Sperm were scored for morphological abnormalities, including bent or kinked tails, no tails, or no heads. Over 90% of the sperm from the ATF4-/- were judged normal (Table 1), which was comparable with 88% normal in the ATF4+/+ mice. Seven percent of the ATF4-/- and 10% of the ATF4+/+ mice had bent or kinked tails.
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The capacity of the mutant and wild-type sperm to fertilize an egg was examined by in vitro fertilization. Sperm were diluted to 1 x 106 sperm/ml in KSOM supplemented with 7.5% calf serum and incubated with eggs isolated from CD-1 mice. Fertilization was determined at 48 h based on successful cleavage and progression to a four-cell embryo (Table 2). From three independent experiments, there was no significant difference (P < 0.7778) between the percentage of eggs fertilized by ATF4-/- and ATF4+/+ sperm.
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Morphological Changes in the Vas Deferens of the ATF4-/- Mice
Male infertility may also be due to obstructions of the seminal ducts and prostate. The epididymis, vas deferens, and prostate isolated from 10-wk-old ATF4-/- andATF4+/+ mice were fixed in 10% neutral buffered formalin and cleared in xylene. This method allows for the visualization of the lumen of these ducts in the context of the entire tissue. In Figure 1, representative ATF4-/- and ATF4+/+ proximal vas deferens images revealed distinct differences in the thickness of the tissue layers. In the ATF4+/+ vas deferens, the smooth muscle layer, the pseudostratified epithelium (epithelial layer and the associated lamina propria), and sperm-filled lumen were easily distinguishable. The smooth muscle and pseudostratified epithelium were also present in the ATF4-/- mice, though the thickness of the pseudostratified epithelium appeared thicker and the lumen was reduced in diameter. Interestingly, there were focal regions where the epithelial layer appeared dramatically thicker (Fig. 1, asterisk). Focal regions were found in all of the ATF4-/- vas deferens, though the position was not consistent. There was no difference in the organization or lumenal diameter of the ducts of the epididymis or prostate in the ATF4-/- mice (data not shown), suggesting that these lesions were specific to the vas deferens.
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Expression of ATF4 in the Vas Deferens
Cell-specific expression of ATF4 in components of the vas deferens was examined by indirect immunohistochemistry. In transverse histologic sections, ATF4 immunoreactivity was detected in all of the cells of the lamina propria layer that lie between the basal layer of the epithelium and the smooth muscle (Fig. 2A). Staining in the lamina propria was consistent along the length of the vas deferens. A weak signal was detectable in the smooth muscle layers, but no ATF4 was present in the epithelium. In the epididymis, which forms a contiguous duct with the vas deferens and is derived from the same embryonic tissue [26], ATF4 was detected in both the lamina propria and the epithelium (Fig. 2C). The expression of ATF4 is differentially regulated between these two tissues. ATF4 was also found in the epithelium and lamina propria of the villi of the duodenum (Fig. 2D), suggesting that regulation of this gene is conserved in mucosal membranes.
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Disruption of the Lamina Propria Layer of the Vas Deferens in the ATF4-/- Mice
A histologic analysis was performed on the vas deferens of 5- and 10-wk-old ATF4+/+ and ATF4-/- mice. Transverse histologic sections along the length of the vas deferens of mutant and wild-type mice were stained with hematoxylin and eosin (Fig. 3). In sexually mature 10-wk-old mice, the lamina propria normally consists of a thin layer of elongated cells located between the basal surface of the epithelial and the inner layer of the smooth muscle (Fig. 3, A through C). In the distal vas deferens, the lamina propria is also present as structural support for the longitudinal folds in the epithelium (Fig. 3C). The morphology of the ATF4-/- lamina propria ranged from subtle to a dramatic increase in thickness (Fig. 3F). The thickening of the lamina propria was associated with a constriction of the lumen of the vas deferens that could reduce the efficiency of sperm transport along the duct (Fig. 3, A and F). At a higher magnification, the cells in the ATF4-/- lamina propria were rounder and appeared to infiltrate the smooth muscle layer (Fig. 3, G and H). A morphometric analysis was performed to determine if these differences in the width and number of cells in the lamina propria of the ATF4+/+ and ATF4-/- vas deferens were significant (Table 3). The mean width of the lamina propria from randomly selected histologic section was significantly larger (P < 0.017) in the ATF4-/- (27.2 µm) vas deferens when compared with the ATF4+/+ (18.5 µm) deferens. In the most severe regions, the lamina propria was as wide as 180 µm. The increase in width of the lamina propria was associated with a significant increase in the number of cells (P < 0.0001). Almost three times as many cells were present within a 60-µm circumferential length of the lamina propria in randomly selected histologic sections (Table 3). These data indicate that the expression of ATF4 is required to regulate the shape and number of cells in the lamina propria. The changes in the morphology of the lamina propria were not observed in the epididymis despite the fact that ATF4 is expressed in these cells (Fig. 3, E and J).
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To determine whether ATF4 plays a role in regulating the maturity of the lamina propria, the morphology of the lamina propria of 5-wk-old ATF4-/- and ATF4+/+ vas deferens were compared (Fig. 3, D and I). In the juvenile vas deferens, the cells of the lamina propria in both the ATF4-/- and ATF4+/+ mice were similar in shape and organization. The cells were rounded instead of flat as seen in the adult wild-type lamina. The width of the lamina propria was not significantly different (P < 0.19) between ATF4-/- and ATF4+/+ mice (Table 3). Similarly, there was little difference in the number of cells (P < 0.04) in the lamina propria. This demonstrates that the influence of ATF4 on the morphology of the lamina propria occurs at the time of sexual maturity.
The contribution of the lamina propria to the proper functioning of the vas deferens has not been well studied. However, in other tissues that undergo peristaltic contractions including the small intestines, colon, and ureter, the lamina propria has been shown to play a critical role in coordinating contraction of smooth muscle. This occurs through the action of ICC and the flexibility provided by the network of elastic fibers [27–29]. In characterizing the lamina propria of the vas deferens in the ATF4-/- mice, we examined the presence of elastic fibers and ICC in this tissue.
The lamina propria layer of the vas deferens is associated with elastic fibers that are organized into an inner layer of circumferentially oriented fibers and an outer meshwork [30]. The elastic fibers are believed to participate in peristaltic movement of sperm and provide passive recoil of the vas deferens following contraction and dilatation at ejaculation [31]. Elastin, the amorphous protein that multimerizes to form elastic fibers, can be visualized in histologic sections with Verhoeff stain (purple). It has been reported in humans that multimerization of elastin occurs during sexual maturation [30]. Consistent with that report, elastin was diffusely distributed over the region of the lamina propria of pubescent 5-wk-old ATF4+/+ mice (Fig. 4A). Elastic fibers were not present in any of the sections of ATF4+/+ 5-wk-old mice. A similar distribution of elastin in the absence of elastic fibers was observed in the lamina propria of 5-wk-old ATF4-/- mice (Fig. 4D). In contrast, elastic fibers were clearly visible in a circumferential pattern and in the longitudinal folds of the epithelium of sexually mature ATF4+/+ mice (Fig. 4, B and C). The elastin staining in the 10-wk-old ATF4-/- mice was dependent on the morphology of the lamina propria. In regions where the cells were hyperplastic, the elastin staining was diffuse with a few elastic fibers present at the base of the epithelium (Fig. 4, E and F, yellow arrowhead). In other regions of the lamina propria, where the thickness of the layer and cell shape are similar to wild type, elastic fibers were prevalent. These data demonstrate that elastic fiber formation in the lamina propria of the mouse vas deferens is regulated by signals associated with sexual maturation. Further, a failure of elastin to polymerize into elastic fibers of the ATF4-/- mice is associated with focal disruptions in cell morphology.
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In tubular tissues that undergo peristaltic contractions, the lamina propria layer contains ICC. These cells have been best studied in the enteric plexi, where they coordinate the contractions of gastrointestinal smooth muscle fibers via a reticular network of processes [31, 32]. Coordinating the rhythmic smooth muscle contractions is necessary for unidirectional movement of the lumenal contents. ICC have been identified in the lamina propria of the vas deferens, where they are proposed to regulate the pulsatile transport of semen from storage in the epididymis [33–35]. To further characterize the lamina propria of the ATF4-/- mice, we looked for the presence of ICC using neuron-specific enolase (NSE) as a marker [35]. Transverse histologic sections from the distal region of the vas deferens of three ATF4-/- and ATF4+/+ mice were used to examine the expression of NSE by immunohistochemistry. Staining was punctate in the cells, consistent with the sublocalization of the enolase within the cells. NSE was present in ICC within the lamina propria as well as the neuromuscular junctions in the smooth muscle layer (Fig. 5A) [36]. In the ATF4-/- mice, NSE-positive cells were detectable in the smooth muscle layer and in regions of the lamina propria that were minimally disrupted (Fig. 5B). However, NSE-positive cells were reduced in the more severely affected regions.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Elastic fibers play a critical role in the repeated cycles of extension and recoil associated with connective tissues such as the lamina propria of the vas deferens [30, 31]. The lamina propria undergoes a number of morphological changes associated with sexual maturation. The thickness of the cell layer is reduced, due in part to a change in cell shape from round to flat, and elastin polymerizes into elastic fibers. Elastic fibers arise from the deposition of cross-linked elastin chains on a preformed template of fibrillin-rich microfibrils [37]. Initially, proelastin molecules polymerize to form elastin chains, which are then cross-linked through oxidative deamination of certain lysine residues by lysyl oxidases [38]. Microfibrils derived from fibrillin monomers are cross-linked by the actions of transglutaminases. It is clear from the current studies that polymerization of elastin is linked to the change in cell morphology during the sexual maturation process. This raises the possibility that elastic fiber formation is dependent on androgens. It is unlikely that ATF4 directly regulates this process because the disruption of elastic fibers was not observed throughout the vas deferens.
ICC is able to coordinate contractions by acting as pacemaker cells for the basal electrical rhythm [31, 39]. The presence of ICC in the lamina propria of the vas deferens raised the possibility that the pulsatile transport of semen is controlled by a similar mechanism of coordinating the rhythmic contraction of smooth muscle [35]. This is supported by the observation that a null mutation in the P2X2 receptor, an ICC marker gene, led to male sterility by reducing the sperm count in the ejaculate [40]. We observed an overall reduction in the number of ICC detected in the lamina propria of the ATF4-/- vas deferens with a complete absence in focal regions of severe disorganization. Thus, it is possible that the subfertile phenotype of the ATF4-/- mice is due in part to a lack of coordinated muscle contraction.
The differential expression of ATF4 in the vas deferens and epididymis may reflect a fundamental difference in the function of the protein in these two tissues. Despite the fact that these tissues are both derived from the Wolffian duct and are responsive to androgens, they perform distinct functions associated with sperm storage, maturation, and transport [41]. Several genes have been identified that are transcribed in the lamina propria of the vas deferens and the lamina propria and epithelium of the epididymis, including the androgen receptor [42], the estrogen receptor alpha [43], bone morphogenetic factor 7 [44], and the Hoxa-11 cofactor, Meis 1 [45]. All of these genes play a role in regulating differentiation and proliferation in several tissues in the body. This suggests that the differential transcription of these genes and ATF4 may reflect fundamental differences in the function of the epithelial layer between the two ducts.
The presence of hyperplasia in the lamina propria in the ATF4-/- predicts that ATF4 functions to arrest growth of these cells during the development of the vas deferens. This is consistent with reports that ATF4 regulates the arrest of cell proliferation in cell culture. Ectopic expression of ATF4 is able to block proliferation of NIH3T3 fibroblasts cells transformed by ras(V12/G12) [13]. Repression of cell proliferation is occurring in part by suppressing the transcription of cyclin A, an essential regulator of G1/S transition during the cell cycle [7]. Further, ATF4 has been shown to regulate cell proliferation in response to a broad spectrum of cell stresses through the transcriptional activation of CHOP [14]. CHOP is a member of the C/EBP transcription factor family that has been implicated in the commitment to, or implementation of, growth arrest at the G1/S boundary or cell death [15].
The present study predicts that ATF4 is required for changes in cell morphology and elastic fiber formation in the lamina propria associated with sexual maturation. It is tempting to predict that ATF4 is required for the responsiveness of the cells to androgens.
Androgen-dependent transcription of the mouse vas deferens protein gene, a member of the aldo-keto reductase superfamily, is modulated by the AP1 complex [46]. Proteins of the jun, fos, and CREB/ATF families that are components of the AP1 complex are known to play an important role in the control of the balance between proliferation, differentiation, and apoptosis [47]. This raises the possibility that ATF4 may act by modifying the activity of the androgen receptor in the differentiation of the lamina propria. Further experiments will be required to determine if ATF4 directly interacts with the androgen receptor.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Alan Rawls, School of Life Sciences, Life Sciences C Bldg., Rm. 544, Arizona State University, Tempe, AZ 85287-4501. FAX: 480 965 2519; jrawls{at}imap4.asu.edu
3 Current address: Vincent Center for Reproductive Biology, Department of Obstetrics and Gynecology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114
Received: 26 July 2003.
First decision: 14 August 2003.
Accepted: 23 September 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(but not estrogen receptor ß) during postnatal development of the epididymis and vas deferens of the rat and disruption of this pattern by neonatal treatment with diethylstilbestrol. Endocrinology 2001 142:874-886This article has been cited by other articles:
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