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Male Reproductive Tract |
a Division of Research, Department of Ob, Gyn, and Women's Health, University of Louisville Health Sciences Center, Louisville, Kentucky 40292
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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levels. Twenty-one-day testosterone replacement therapy in 30-day-old knockout animals reversed some, but not all, morphological and biochemical phenotypes. Those that did not reverse include luminal diameters of proximal and distal caput and cauda epididymis, the percentage of ciliated principal cells in caput epididymis, and nuclear AR localization. In summary, while our results reaffirm that androgens are important for normal epididymal morphology and function, they indicate that LH could be required for certain facets of epididymal morphology and/or function.
androgen receptor, epididymis, estrogen receptor, gene regulation, luteinizing hormone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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LH, a glycoprotein hormone and a member of the cystine knot growth factor family [25, 26], binds to the same receptor as its structural and functional homolog, hCG [27, 28]. Studies have demonstrated the presence of these receptors not only in testis but also in epididymis and other accessory sex organs [18–21, 29–36]. Functional studies have demonstrated that LH can regulate epididymal functions that are important for sperm maturation [16, 21].
LH-receptor knockout mice have recently been made by homologous DNA recombination [37, 38]. The knockout males have markedly decreased serum testosterone levels, markedly elevated LH levels, and moderately increased estradiol and FSH levels [37, 38]. The phenotype of these animals includes micropenis, small abdominal testes, spermatogenic arrest at the round spermatid's stage, the absence of adult-type Leydig cells, the presence of a few fetal-type Leydig cells in adult testes, an increase in Sertoli cell number, and a rudimentary epididymis and other accessory sex organs. These animals presented us with an opportunity to test the hypothesis that testosterone replacement therapy alone may not fully restore epididymal phenotype, which implies that direct LH actions could be required.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mice were maintained and all the procedures were performed as described in the NIH guide for the Care and Use of Laboratory Animals. Adult male and female heterozygous mice were mated to obtain wild-type (+/+), heterozygous (±), and homozygous (-/-) animals. The zygosity was determined by Southern blotting with genomic tail DNA digested with Stu I and hybridized to [32P]-labeled DNA probe for the LH-receptor gene as previously described [37]. The probe was designed to detect an 8-kilobase (kb) DNA fragment of the 5'-flanking region of a wild-type LH-receptor gene and 10-kb mutant fragments that include the neomycin gene used in the disruption strategy. Thus, a single Southern blot performed on each sample allowed unambiguous identification of all three genotypes. All animals were maintained on a 12L:12D cycle with regular, unrestricted diet. For each experiment, three to six 7–8-week-old mice of each genotype were used.
Histology and Computerized Quantitative Morphometry
Caput and cauda epididymis were chosen for morphological analysis because they represent two extreme ends of the organ with distinct morphology and function. The tissues were fixed in 10% formalin and embedded in paraffin. Five-micrometer-thick sections were cut and then stained with hematoxylin and eosin or periodic acid-Schiff reagent (PAS). The tissue sections were examined under bright-field microscopy for subsequent quantitative morphometry and photography.
Morphological changes were determined by a computerized Bioquant IV System (R & M Biometrics, Nashville, TN). The diameters of the ductal lumen were measured in several cross-sections covering a total area of 10 000 µm2. Principal epithelial cell heights were measured from the basement to the apical membrane in cross-sections of 10 tubules. The number of nonciliated and ciliated cells was counted in several cross-sections covering a total area of 10 000 µm2. The cilia lengths were determined by linear measurements from the apical membrane to the cilia tip in 10 tubule cross-sections.
Semiquantitative Reverse Transcription-Polymerase Chain Reaction
Total RNA was isolated from epididymis using a single-step acid guanidine thiocyanate-chloroform extraction method [37]. Two micrograms of total RNA were reverse transcribed into cDNA with oligo dT primers and AMV reverse transcriptase (Promega, Madison, WI). The cDNA was then coamplified with ß-actin primers, [32P]-dCTP (3000 Ci/mmol from New England Nuclear-Dupont Biotechnology Systems, Boston, MA), dNTP mix (Promega), and one of the following primer sets (top strand is 5'-primer and bottom strand is 3'-primer). These are designed from published mouse sequences using a designer polymerase chain reaction (PCR) computer program (Research Genetics, Huntsville, AL) and synthesized by Operon Technologies (Alameda, CA).
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Optimal conditions and a PCR-cycle number for each primer set were predetermined to ensure that coamplification was within the linear range. PCR products were resolved by electrophoresis in polyacrylamide gels and bands were identified by autoradiography. The band intensities were quantified by a Z-gel scanning system (Zaxis, Hudson, OH) and expressed as ratios with ß-actin.
Western Blotting
This procedure was performed by homogenizing epididymides at 4°C in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 1% phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin using a tissue tearor [39]. Then protein concentrations were determined and 20-µg aliquots were separated by discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and transferred to Immobilon P membranes (Millipore, Bedford, MA). After blocking nonspecific binding sites with 5% nonfat powdered milk, the membranes were incubated with 1:1000 dilutions of polyclonal antibodies to LH receptor (a gift from Dr. Patrick Roche, Mayo Clinic, Rochester, MN), AR, ER, and ERß (Santa Cruz Biotechnology, Santa Cruz, CA). The antibody bound receptors were detected by using a 1:1000 dilution of horseradish peroxidase-labeled second antibody and enhanced chemiluminescence detection system (Amersham-Pharmacia Biotech, Piscataway, NJ). The molecular sizes of the proteins were determined by running standard molecular-weight marker proteins in an adjacent lane. The densities of bands were measured by the Z-gel scanning system and expressed as ratios with ß-actin.
Immunocytochemistry
This procedure was performed with an avidin-biotin immunoperoxidase kit (Vector Laboratories, Burlingame, CA) as previously described [40]. Briefly, deparaffinized epididymal sections were treated for 10 min with 0.75% H2O2 in methanol to block endogenous peroxidase. For antigen retrieval, the sections were boiled for 10 min in a microwave oven in a buffer containing 10 mM citric acid (pH 6.0). After washing, the sections were exposed to normal horse serum; a 1:500 dilution of LH receptor; proliferating cell nuclear antigen (PCNA); progesterone receptor; AR, ER, and ERß antibodies; and were incubated overnight at 4°C. Substitution of primary antibodies with nonspecific IgG served as procedural controls.
Testosterone Replacement Therapy
We chose 30-day-old animals for testosterone replacement therapy because this age corresponds to puberty, when circulating testosterone levels normally begin to raise. Twenty-one-day time-release pellets containing 5 mg testosterone were subcutaneously implanted (Innovative Research America, Sarasota, FL). Null mice for controls were implanted with placebo pellets. The animal's age at the end of therapy corresponds to the approximate age of sexual maturity.
Measurement of Testosterone Levels
The levels were measured by a Coat-A-Count total testosterone radioimmunoassay kit (Diagnostic Products, Los Angeles, CA). The kit instructions were followed in the measurement. Serum levels were measured without any extraction. Tissue levels were measured in supernatants obtained from centrifuging the testicular homogenates for 10 min at 10 000 x g. The inter- and intrassay coefficients of variation were less than 10% in the range of testosterone levels found in the samples. The cross-reactivity was less than 1% for most of the steroid hormones.
Statistical Analysis
The data presented are the means and their standard errors. Analysis of variance and Duncan multiple range tests were used for determining significant differences [41].
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The presence of LH receptors in wild-type epididymis and their absence in null epididymis were verified by Western blotting and immunocytochemistry. Testes from the same animals were used for comparison in Western blotting. Figure 1A shows that epididymis, like testis from wild-type animals, contained an 80-kDa receptor protein. This protein was not detected either in epididymis or in testis from null animals. Immunocytochemistry revealed the presence of LH-receptor immunostaining primarily in the principal cells of epididymal tubules of wild-type animals (Fig. 1B). Blood vessels in intertubular stroma were also receptor positive. The punctate staining reflects some principal cells staining more than others. Null epididymis showed no receptor immunostaining (Fig. 1C), as did the wild-type epididymis used in a procedural control (Fig. 1D).
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Epididymal Morphology
The epididymal tubules were much smaller and lumens were completely devoid of spermatozoa in null animals as compared with wild-type and heterozygous littermates, which are indistinguishable (Fig. 2C versus Figure 2, A and B). Although it may not be apparent from low-magnification photographs, clear or halo cells, which represent minor cell populations in the epithelial cell lining, were not seen. Also, the number of principal cells decreased and they became cubic with centrally located nuclei. Only a few of the principal cells contained cilia, and their length decreased. These changes were quantified by computerized morphometry and are presented in Figure 3 and Table 1.
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Figure 3 shows that luminal diameters of tubules and height of principal cells in proximal and distal caput (Fig. 3, upper panels) and cauda (Fig. 3, lower panels) epididymis dramatically decreased in homozygosity as compared with wild-type and heterozygous littermates. Table 1 shows that the percentage of ciliated epithelial cells and their cilia height decreased in homozygous animals.
PAS staining, reflecting the secretion of glycogen and glycoproteins, was very weak in the epithelial cells and in lumen of epididymal tubules of homozygous animals as compared with wild-type and heterozygous littermates (data not shown).
We previously reported that epididymides were barely recognizable in null animals, with a dramatic decrease in their weights [37]. This gross epididymal underdevelopment could either be due to an increase in apoptosis and/or a decrease in cell proliferation. The apoptosis, as tested by the TUNEL method, was found to be minimal in both wild-type and null animals (data not shown). In contrast, testicular apoptosis, especially in spermatocytes, dramatically increased in null as compared with wild-type testes [42].
Cell proliferation as determined by PCNA immunostaining showed a marked reduction in null epididymis as compared with wild-type and heterozygous littermates (data not shown). Quantitation revealed that, while less than 10% of principal cell nuclei were PCNA positive in null epididymis, approximately 98% of them were positive in wild-type and heterozygous littermates (data not shown).
Epididymal Steroid Hormone Receptor Levels
The epididymal steroid hormone receptor levels were determined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Semiquantitative RT-PCR revealed that, while progesterone receptor was undetectable (data not shown), AR, ER, and ERß transcripts were present in epididymis of all three genotypes (Fig. 4A). While the AR and ERß levels decreased, ER levels increased in null as compared with wild-type epididymis.
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Western blot analysis revealed the presence of a 110-kDa AR protein, a 67-kDa ER protein, and a 60-kDa ERß protein in epididymis of all three genotypes (Fig. 4B). All three receptor proteins changed in parallel with their cognate mRNAs.
Immunocytochemistry was used not only to determine cellular localization but also to independently verify AR, ER, and ERß protein changes in null epididymis. It demonstrated that principal cell nuclei primarily contained AR (Fig. 5A), ER (Fig. 5D), and ERß (Fig. 5G) immunostainings, and they changed in parallel with their mRNAs and proteins by Western blotting in null epididymis.
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Effect of Testosterone Replacement Therapy
If the epididymal changes in homozygous animals were completely due to a decrease in serum testosterone levels, then restoring them by replacement therapy should reverse the changes. This possibility was tested by placing 30-day-old homozygous animals on 21-day replacement therapy with 3-mm pellets representing a matrix-driven delivery system integrating the principles of diffusion, erosion, and concentration gradient resulting in a biodegradable matrix that effectively and continuously releases testosterone. The therapy resulted in similar intratesticular testosterone levels (1.3 ± 0.2 ng/µg protein), but serum levels were elevated by 6.6-fold (8 ± 0.1 ng/ml) as compared with wild-type littermates (1.2 ± 0.03 ng/ml). The control homozygous animals, placed on empty pellets, showed very low serum testosterone levels, as did untreated homozygous animals (
0.1 ng/ml).
The replacement therapy stimulated epididymal growth to a size comparable with that in wild-type littermates. This increased growth appears to be due to an increase in cell proliferation as reflected by PCNA staining (data not shown). The principal cells became columnar, with nuclei returned to the basal part of the cells (Fig. 2D). The number of ciliated cells and cilia lengths were restored (Table 1). The clear and halo cells seem to have been recovered, but their numbers have not been quantified because they represent only minor cell populations in the epithelial lining. The lumen contained polynucleated epithelial cells mixed with spermatozoa (Fig. 2D). As determined by PAS staining, the secretory activity of epididymis appears to have been restored (data not shown). The AR, ER, and ERß changes were reversed, as determined by semiquantitative RT-PCR, Western blot, and immunocytochemical analyses (Figs. 4 and 5). The therapy, however, failed to completely restore the luminal diameters in proximal and distal caput and cauda epididymis (Fig. 3, lower left panel) and principal cell heights in caput epididymis (Fig. 3, upper right panel). In addition, AR immunostaining remained diffuse cytoplasmic rather than nuclear, as in the case of wild-type littermates (Fig. 5C versus Fig. 5A).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-reductase present in the epididymal tissue [1, 2]. The actions of dihydrotestosterone are mediated by AR, which are abundant in epididymal tissue [1, 2]. Although androgens are very important [1, 2, 7], other molecules can also regulate epididymal functions [1, 2, 8–24]. The evidence for potential LH regulation of epididymal functions comes from the following data. Epididymis contains LH-receptor transcripts and/or receptor protein [18, 19, 21], hCG treatment stimulates epididymal growth in infant monkeys [43], increases in chloride ion effluxes, and the activity of tissue-type and urokinase-type plasminogen activators, both of which are associated with sperm maturation [16, 21]. These findings have led us to test whether the epididymal phenotype in LH-receptor knockout animals could be completely reversed by testosterone replacement therapy. If it did, androgen deficiency alone would be entirely responsible; otherwise, lack of LH action could also be responsible for epididymal phenotype. The results with testosterone replacement therapy demonstrate that much of the wild-type morphologic and biochemical epididymal phenotype was restored, except for luminal diameters in proximal and distal caput and cauda epididymis, height of principal cells in cauda epididymis, and nuclear AR localization. It is possible that luminal diameter is determined by the presence of sperm and fluid, both of which may not have completely recovered after testosterone replacement therapy. We do not know whether varying the testosterone dose or length of replacement would restore these other features to wild-type epididymis. This is probably unlikely considering that the present replacement regimen reversed most of the epididymal changes. Nevertheless, variations in the testosterone replacement therapy, including starting the therapy shortly after birth and intraepididymal lumen delivery of testosterone, which is technically demanding considering the small size of null epididymis, need to be tested. In the meantime, however, because LH has a direct role in epididymal functions, the lack of reversal of a subset of epididymal features suggests that LH actions could be required.
It is well known that not only androgens but also estrogens can regulate epididymal functions through their cognate receptors [1–3]. Thus, it would be of interest to determine the effect of LH-receptor knockout on epididymal AR, ER, and ERß levels. Our studies revealed differential effects on epididymal and testicular steroid hormone-receptor levels. For example, while epididymal AR decreased, testicular AR levels were unaffected [37]. While ER increased and ERß decreased in epididymis, ER decreased and ERß increased in testes [37]. Testosterone replacement therapy reversed ER and ERß changes in epididymis but not in testes [44]. These differences suggest that androgens mediate the LH actions in regulating epididymal but not testicular ER and ERß levels. Thus, androgens up-regulate ERß along with AR and down-regulate ER in epididymis, and in their absence, ERß and AR decrease and ER increases. It is of interest to the note that, while testosterone replacement therapy reversed AR, it remained cytoplasmic rather than nuclear. This may indicate that direct LH actions may be required for nuclear import of AR.
Whether AR, ER, and ERß changes were related to the lack of epididymal growth in knockout animals is not known. However, their reversal, concomitant with growth stimulation after testosterone replacement therapy, suggests that they may somehow be related. ER may also participate in fluid reabsorption in caput epididymis because its disruption results in its impairment and infertility [45]. The role of ERß in epididymal functions is unknown. However, its presence and reciprocal changes from ER in LH-receptor knockout animals suggest that both ERs may have distinct roles in the regulation of epididymal functions.
As previously described, sperm appeared in epididymal lumen, due to resumption of spermatogenesis in testes, after testosterone replacement therapy [44]. However, their concentrations appeared to be lower than in wild-type animals, which could in part be due to impaired fluid reabsorption mechanisms. Nevertheless, sperm in cauda epididymis have normal morphology and are able to fertilize wild-type oocytes in vitro at a lower rate than wild-type sperm [44]. This lower rate may also reflect a lack of adequate sperm maturation.
In summary, our results document in detail the epididymal phenotype in LH-receptor knockout animals. This phenotype was mostly, but not entirely, due to a decrease in testosterone levels, and direct LH actions could be required for a subset of epididymal features.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Z.M. Lei or Ch.V. Rao, Department of Ob, Gyn, and Women's Health, 438 MDR Building, University of Louisville, Health Sciences Center, Louisville, KY 40292. FAX: 502 852 0881; e-mail: zhenmin.lei{at}louisville.edu or e-mail: cvrao001{at}louisville.edu
Received: 25 July 2002.
First decision: 20 August 2002.
Accepted: 19 September 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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