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Agricultural Research Service, United States Department of Agriculture, Grand Forks Human Nutrition Research Center, Grand Forks, ND 58202-9034
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGMENTS
LITERATURE CITED
ABSTRACT 
Zinc deficiency results in reduced testicular angiotensin-converting enzyme (ACE) activity and reduced amounts of ACE protein in the testes of young rats. In the present study, we examined the effect of zinc deficiency on the relative abundance of testicular ACE mRNA and its relationship to ACE activity over time. Forty-five male rats at 7 wk of age were placed on one of three feeding regimens: 1) a diet adequate in zinc, 2) a diet deficient in zinc and 3) a diet adequate in zinc that was fed in an amount equal to that consumed by a paired mate fed the zinc-deficient diet. Rats were killed after 3, 5 and 7 wk. Rats fed the zinc-deficient diet had significantly lower (P < 0.01) body weight gain and testis weight at each week sampled than the other groups. They also showed compromised zinc status as evidenced by significantly lower (P < 0.01) serum and testis zinc concentrations. At each period, rats fed the zinc-deficient diet had significantly lower (P < 0.01) testicular ACE activity than rats fed either of the zinc-adequate diets. Coinciding with low ACE activity, there was a lower (P < 0.01) relative abundance of ACE mRNA in the group for the zinc-deficient diet than in either of the zinc-adequate groups. The results suggest that much of the low ACE activity in the testis of rats in the latter stages of zinc deficiency is attributable to a reduction in ACE gene transcription. However, an effect of the deficiency on ACE mRNA turnover is not ruled out.
Key words: zinc, angiotensin-converting enzyme, testes, mRNA, rats.INTRODUCTION
Zinc deficiency in males of many species, including humans, causes an impairment of sexual development (Millar et al. 1958
and 1960, Sandstead et al. 1967). Previously, this impairment was shown to manifest itself primarily in young animals and consisted of hypogonadism that resulted in arrested development of secondary sex characteristics (Mason et al. 1982). More recent investigations found that adult animals are affected similarly to young animals in that germ cell maturation is suppressed and the testes begin to atrophy when the animals are fed zinc-deficient diets for up to 5 wk (Reeves and Stallard 1995).
The exact role that zinc plays in male sexual maturation and fertility is unknown, but it may be related partly to the enzymes that require zinc for full activity. A zinc-metalloenzyme that is closely related to maturation of the testes, and to fertility in general, is angiotensin-converting enzyme (ACE,5 EC 3.4.15.1) (Cushman and Cheung 1971, Strittmatter and Snyder 1984, Strittmatter et al. 1985). Angiotensin-converting enzyme is not expressed in the testes until puberty and then a large increase in activity occurs, which reaches a plateau as the organs mature (Strittmatter et al. 1985). It was shown recently that a mouse model carrying an insertion mutation that inactivates testicular ACE has low rates of fertility (Krege et al. 1995). Zinc deficiency in prepubertal and adult rats was shown to severely reduce ACE activity (Reeves 1990, Reeves and O'Dell 1988, Reeves and Rossow 1993, Reeves and Stallard 1995). Although these studies suggest that testicular ACE is important in sexual development and fertility and that its activity is dependent upon an adequate supply of dietary zinc, its function remains unresolved.
In an ongoing effort to determine the association between zinc status and ACE activity, we showed that a reduced amount of ACE protein in zinc-deficient rat testes may be the cause of low ACE activity (Reeves and Rossow 1993, Rossow and Reeves 1993). To further examine this question, we designed the following experiment to determine whether the reduction in ACE protein in the testes of zinc-deficient rats is related to the relative abundance of ACE mRNA.
MATERIALS AND METHODS
) but with the following changes: egg white solids were substituted for casein, extra biotin (2 mg/kg diet) was added, the mineral mix did not contain a source of zinc, and the amount of phosphorus was changed so that the finished diet provided 3 g phosphorus/kg diet when the mineral mix was used at 35 g/kg diet. This diet was designated -Zn. Another group was fed the basal diet supplemented with 35 mg zinc/kg diet (+Zn). Each rat in the third group was fed daily an amount of the +Zn diet equal to that consumed the previous day by its mate in the -Zn group; this diet was designated +ZnPF.
). Tissue (0.2 g) was removed from a decapsulated frozen testis and immediately homogenized in a glass/glass tissue grinder containing 1.0 mL of buffer (6 mol guanidinium thiocyanate, 40 mmol sodium citrate and 90 mmol 2-mercaptoethanol/L). The homogenate was mixed with 100 µL of 2 mol sodium acetate/L and 1.2 mL of phenol-chloroform (5:1 ratio), pH 4.7. The mixture was placed on ice for 15 min and then centrifuged at 10,000 × g for 20 min at 4°C. The upper aqueous phase was mixed with an equal volume of cold isopropyl alcohol and kept at -20°C for at least 1 h. After centrifugation at 10,000 × g for 30 min, the pellet was dried briefly and then suspended in 300 µL of TE buffer (10 mmol Tris-HCl and 1 mmol EDTA/L, pH 8). Then 300 µL of phenol-chloroform (5:1), pH 4.7, was added and the mixture centrifuged at 10,000 × g for 20 min. The upper aqueous phase was transferred to a fresh tube and 45 µL of 2 mol sodium acetate/L was added followed by 700 µL of cold 100% ethanol. The tube was set aside at -20°C overnight before centrifugation at 13,000 × g for 30 min. The supernatant was decanted and the pellet washed with 80% ethanol and then dried. The pellet was suspended in TE buffer and the RNA quantified spectrophotometrically. Both quantity and quality of the RNA sample were verified by gel electrophoresis and staining with ethidium bromide. The RNA samples were stored at -80°C.
Northern analysis of RNA.
The isolated testes RNA (10 µg) was denatured and separated on a 1% agarose gel containing 2.2 mol formaldehyde/L and transferred to a nylon membrane (Quantum Yield, Promega, Madison, WI) by capillary blotting (Sambrook et al. 1989). Three gels were run, one for each week sampled. Every gel contained lanes representing each dietary treatment as well as a size marker. Blots were prehybridized for 4 h at 42°C in 10 mL of hybridization buffer (5× Denhardt's reagent, 40% formamide, 10% dextran sulfate, 1 mol NaCl and 50 mmol Tris-HCl/L, pH 8, 0.1% SDS, and 100 mg denatured salmon sperm DNA/L) (Bernstein et al. 1988).
). The ACE.5 cDNA was cloned into phagemid Bluescript (Stratagene, La Jolla, CA) and used to transform bacterial cells (Epicurian Coli XL1-Blue competent cells; Stratagene). The cDNA was separated from the vector by agarose gel electrophoresis after digestion with EcoRI. A control cDNA probe, human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (CLONTECH, Palo Alto, CA), was used to verify equal RNA loading per lane and to determine the specificity of dietary treatment effects. Both probes were labeled with [
-32P]dCTP (Amersham, Arlington Heights, IL) by random hexamer primer labeling (RadPrime Labeling System, Gibco BRL, Gaithersburg, MD). Blots were hybridized sequentially, first with GAPDH cDNA at 48°C for 16 h followed by washing at 65°C with the following conditions: 1) 2× SSC, 15 min; 2) 2× SSC, 0.1% SDS, 30 min; 3) 0.1× SSC, 10 min. Then blots were hybridized with ACE.5 cDNA at 42°C for 16 h and washed as follows: 1) 6× SSC, 0.1% SDS at room temperature for 15 min; 2) 0.2× SSC at 62°C for 15 min. Blots were then exposed to autoradiography film (Kodak X-OMAT AR) at -80°C with intensifying screens.
. Tissue was homogenized in a mixture containing 1 mol NH4OH and 20 g Triton X-100/L. One milliliter of homogenate was incubated with 100 ng of bisbenzimidazole (Hoechst 33258) and the fluorescence read at 350 nm excitation and 455 nm emmission.
Other analyses.
Angiotensin-converting enzyme activity was measured fluorometrically by detecting His-Leu released from hippuryl-histidyl-leucine by the method of Santos et al. (1985) as modified by our laboratory (Reeves and Stallard 1995). Activity was expressed in the katal unit (kat), which corresponds to moles per second. Protein concentrations in testes preparations were determined by using the bicinchoninic acid method (Sigma Chemical).
, Ryan 1960, Welsch 1977). For two variables, testes ACE activity and ACE mRNA relative density, we could not assume that the data were sampled from a Gaussian population; therefore, we use the Kruskal-Wallis nonparametric ANOVA, and the t statistic for pairwise comparisons between means (Kruskal and Wallis 1952). Data are expressed as means ± SEM.
RESULTS
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Table 1. Zinc deficiency affects the body weight gain, testis weight and testis weight:body weight ratio of rats1,2 |
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Table 2. Zinc deficiency affects the concentration of zinc in serum, liver, and testis of rats without affecting the amount of testicular DNA1,2 |
-32P]dCTP-labeled probes, ACE.5 and glutaraldehyde-3-phosphate dehydrogenase. Only examples from wk 5 are shown in this autoradiograph. Testicular ACE mRNA gave a signal at 2.7 kb and the control probe at 1.4 kb. Although all lanes gave near equal intensity for the control probe, the intensity of the ACE.5 probe in lanes containing RNA from -Zn rats was barely detectable.
DISCUSSION 
Fig. 3.
Zinc deficiency affects the relative abundance of angiotensin-converting enzyme (ACE) mRNA in the testes of adult rats. Rats were placed on one of three feeding regimens: 1) a diet adequate in zinc (+Zn), 2) a diet deficient in zinc (-Zn), and 3) a diet adequate in zinc that was paired-fed in an amount equal to that consumed by a mate fed the -Zn diet (+ZnPF). At 3, 5 and 7 wk, 10 µg of testicular RNA was isolated and separated on denaturing gels (one for each week sampled). RNA was transferred to nylon membranes and hybridized with two [-32P]dCTP-labeled probes, ACE.5 and glutaraldehyde-3-phosphate dehydrogenase (GAPDH). Following autoradiography, differences in band intensities were determined by densitometric analysis. Peak heights of each probe were compared within lanes. Bars represent the mean ± SEM of four or five rats. Different letters within weeks denote significant differences by the Kruskal-Wallis rank means test.
[View Larger Version of this Image (32K GIF file)]
). It also cleaves the vasodilator bradykinin. This isozyme has a molecular mass of approximately 160 kDa and contains 2 mol of zinc per mole of enzyme protein. The other isozyme is found only in the testes, has a molecular mass of approximately 95 kDa, and contains only 1 mol of zinc per mole of enzyme (Ehlers and Riordan 1991). The testicular ACE proteins in mice (Bernstein et al. 1989), humans (Ehlers et al. 1989) and (presumably) rats are unique in that they are produced from the exons identical to those used to assemble the carboxyl terminal half of somatic ACE.
). Although we also demonstrated that zinc deficiency reduced the activity of somatic ACE in serum (Reeves and O'Dell 1985), it seemed to depend on the type of substrate used in the assay, and it required a sufficient supply of zinc in the assay medium. In addition, we could not find a negative effect of the deficiency on lung ACE (Reeves and O'Dell 1988), which seemed to suggest that rat testicular ACE was unique in its response to zinc deficiency. However, we have since shown that ACE activity in the intestinal mucosa of zinc-deficient young male rats is about 40% less than that in zinc-adequate rats (not published), suggesting that indeed somatic ACE can be affected by zinc deficiency.
, Strittmatter et al. 1985), we initially thought that the absence of a sufficient supply of dietary zinc during this period was inhibiting the general development of the testes. However, more recently we showed that zinc deficiency also causes a reduction in testicular ACE activity in adult rats (Reeves and Stallard 1995). In addition, we demonstrated that the reduction in activity was apparently caused in part by a reduction in ACE protein (Reeves and Rossow 1993, Rossow and Reeves 1993). This observation led us to the present study in which the effect of zinc deficiency on the relative abundance of testes-specific ACE mRNA was examined. The results clearly show that zinc deficiency in adult male rats will eventually lead to a reduction in ACE mRNA. In turn, this apparently leads to lower ACE protein, which then causes the reduced activity of ACE as observed in zinc-deficient rats. We are interpreting our results to suggest that ACE mRNA transcription is reduced by zinc deficiency. Because we did not measure mRNA turnover, however, the possibility of increased ACE mRNA degradation by zinc deficiency is not ruled out.
). Sibony et al. (1994) determined the step-wise expression of germinal ACE mRNA and ACE protein in testes of normal rats and mice as the spermatids matured through various stages. By using in situ hybridization with a testis-specific ACE cDNA probe, they found that ACE mRNA was first expressed in stages four to seven, with maximal expression at stages eight to 12. Expression declined through the remaining stages. Immunolocalization of ACE protein showed a similar progression; however, ACE protein remained elevated throughout the latter stages of maturation. Sibony et al. (1994) concluded that ACE is produced exclusively in haploid germ cells of these species. Because zinc is involved in so many aspects of metabolism, it is possible that spermatid development is arrested in the early stages of zinc deficiency and the spermatids never reach the stage where ACE mRNA is normally expressed.
FOOTNOTES
Manuscript received 6 May 1996. Initial reviews completed 24 June 1996. Revision accepted 10 September 1996.
ACKNOWLEDGMENTS
The authors thank Brenda Skinner for technical assistance, Jim Lindlauf and Karin Tweton for mixing the animal diets, and Denice Schafer and her staff for care of the animals. We also thank Barry Milavetz, Department of Biochemistry and Molecular Biology, University of North Dakota, Grand Forks, ND, for advice concerning RNA isolation and Northern blotting.
LITERATURE CITED
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