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Testis |
Department of Pathology and Laboratory Medicine,3
Howard Hughes Medical Institute,4
Department of Neuroscience,5 University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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acrosome reaction, circadian rhythm, spermatid, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although the core oscillator is highly studied, little is known about how the central clock in the SCN regulates the functions of peripheral organs. One proposal holds that peripheral tissues have their own innate clocks, so-called peripheral oscillators. Experimental evidence supports this hypothesis. In mice, all the circadian genes are cyclically expressed in peripheral tissues [4, 6, 7, 10–14]. Moreover, cyclic expression of clock genes in peripheral organs can be experimentally uncoupled from cyclic expression in the SCN [15–17]. The subset of clock genes expressed by a particular tissue varies, suggesting that clock proteins have tissue-specific activities. These proteins may interact with tissue-specific proteins to control circadian functions such as the release of hormones from glands. Alternatively, clock proteins may control noncircadian biological timing in the periphery, such as the timing of cell division and differentiation during development of an organism.
Reproduction is a peripheral activity that is strongly influenced by a circadian clock in most organisms [18–20]. Insights concerning the circadian control of reproduction come from elegant studies with insects [21–23]. Insect reproductive tissue displays cyclic production of core circadian clock proteins, and the clock controls sperm release. Moreover, strains of Drosophila with mutations in the core circadian genes display decreased fertility [24]. In mammals, experiments concerning the influence of circadian rhythms on reproduction are not as complete as with insects. However, it is clear that many mammals exhibit a seasonal reproductive capacity that is under the influence of the photoperiod in a light:dark cycle. For example, in hamsters, short photoperiods (similar to wintertime) cause decreased sperm production and testicular regression [25]. How the circadian clock controls these aspects of reproduction is unknown. Hormonal activity may be involved, as there is circadian control of reproductive hormone levels in all organisms [26–30]. Of course, how this hormonal rhythm is established is also unknown. In contrast, some data support the existence of a circadian component to meiosis [31].
The murine testis is an attractive model for studying circadian clock protein activities in peripheral organs. It contains a defined number of cell types with well-described functions including germ cells, Sertoli cells (supportive cells), and Leydig cells (testosterone-producing cells). However, spermatogenesis is the primary function of the testis, and the germ cells are by far the predominant cell type. Cells in various stages of spermatogenesis, for example, spermatogonia (mitosis), primary spermatocytes (meiosis I), secondary spermatocytes (meiosis II), and spermatids (postmeiotic), can be identified by light microscopy. The entire developmental cycle displays tight temporal regulation. Murine spermatogenesis takes 35 days, and each developmental stage has a defined length of existence. For example, the primary spermatocyte stage lasts 12.5 days. The mechanisms that impart this temporal regulation are unknown.
If a circadian clock is functioning in the testis, cyclic expression of clock genes should be observed in this tissue. However, results concerning such cycling are inconsistent. To resolve this issue, we examined expression of multiple circadian clock genes in the murine testis. Furthermore, we localized expression of mPER1 and CLOCK to specific spermatogenic cell types. We found that circadian gene expression is constant in the testis and that the proteins are expressed in a stage-specific manner. In addition, we found that cyclic expression of clock genes is attenuated in the thymus, suggesting that this is a general feature of differentiating tissues.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Six- to 8-wk-old C57BL/6 male mice (Jackson Laboratories, Bar Harbor, ME) were housed individually on a 12L:12D (0700 h, lights-on; 1900 h, lights-off) for at least 2 wk after arrival at the animal facility. Female mice (bred within the animal facility) were housed together on a 12L:12D cycle. Care of mice was in accordance with Institutional Animal Care and Use Committee guidelines at the University of Pennsylvania.
RNA Preparation
At the indicated times, mice were killed by asphyxiation with CO2 gas. Organs were collected in light. For most experiments, decapsulated testes were immediately homogenized in RNAWiz reagent (Ambion, Austin, TX), and RNA was isolated according to the manufacturer's instructions. For samples shown in Figure 1A, germ cells were isolated as follows. Decapsulated testes were incubated in 30 ml of KREBs solution (0.1 mM KH2PO4, 12 mM NaCl, 0.1 mM MgSO4·7H2O, 0.2% dextrose, 0.1 mM CaCl2·2H2O, 0.5 mM KCl, 2.5 mM NaHCO3) supplemented with collagenase (0.75 mg/ml, Sigma, St. Louis, MO) on a shaking (80–90 rpm) water bath at 33°C for 20 min to isolate seminiferous tubules. Tubules were washed with cold KREBs solution three times and then incubated with KREBs supplemented with trypsin (0.5 mg/ml, Sigma) and DNase (1 µg/ml, Sigma) in a shaking water bath at 33°C for 20 min. The solution was mixed by pipetting at the end of the incubation, filtered through 80-µm mesh, and centrifuged at 1500 rpm for 5 min. Cells were resuspended in RNAWiz, and RNA was isolated. RNA from the lower pole of the right kidney and from the thymus was isolated using RNAWiz.
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RNase Protection Assays
RNase protection probe constructs were provided by P. McNamara [32] (University of Pennsylvania) and E. Meyer-Bernstein (University of Pennsylvania Medical School). Constructs were carried in the PCRII vector (Invitrogen, Carlsbad, CA), and probes were homologous to the following nucleotides of the respective genes: mNpas2 ( 627–922 of GenBank accession no. U77969), mPer1 (380–886 of no. AF022992), mPer2 (146–429 of no. AF035830), mCry1 (1374–1656 of no. AF156986), Bmal1 (522–793 of no. NM007489), and Clock (693–969 of no. AF000998). Probe constructs were linearized by digestion with EcoRV. Control probe was the pTRI-Actin (Ambion). Radiolabeled probes were produced with uridine 5'-[
-32P]-triphosphate (3000 Ci/mmol, 10 mCi/ml; Amersham Corp., Piscataway, NJ) using the Riboprobe System-SP6 (Promega, Madison, WI) according to the manufacturer's instructions. RNase protection assays were carried out using 10 µg of the appropriate RNA sample and the RPA III kit (Ambion) according to the manufacturer's instructions. Protected fragments were resolved on 5% denaturing polyacrylamide gels (National Diagnostics, Atlanta, GA). Gels were examined using a Storm PhosphorImager (Molecular Dynamics, Sunnyvale, CA), and transcript levels were quantified using ImageQuant software (Molecular Dynamics).
Immunohistochemistry
At the indicated times, mice were killed by asphyxiation with CO2 gas. Testis were dissected and fixed in Bouin's fixative (Sigma) for between 12 and 24 h followed by 10% neutral buffered formalin (Sigma) for at least 12 h. Following fixation, testes were dehydrated through graded increases in ethanol (50%, 70%, 85%, 90%, 95%, 100%) each for 30 min followed by xylene (2 x 30 min), embedded in paraffin, sectioned on a microtome at a thickness of 6 µm, and then mounted on Plus slides (Fisher Scientific, Pittsburgh, PA). Sections were rehydrated by incubation in xylene (3 x 5 min) followed by gradually decreasing concentrations of ethanol (100%, 100%, 95%, 95%, 80%, 70%, 50%, each for 5 min) and ddH2O for 5 min. Following rehydration, sections were incubated in 0.5% NaBH4 and rinsed in ddH2O, and endogenous peroxidase activity was quenched with 1.5% H2O2 for 30 min. Slides were rinsed in PBS, blocked with 20% normal goat serum (NGS) in PBS/0.5% Triton-X-100 (PBST) for 45 min at room temperature, washed with PBST, and then incubated with primary antibody diluted in PBST (1:500 for anti-mPER1, 1:1000 for anti-CLOCK) overnight at 4°C in a humidified chamber. Both antibodies were bought from Affinity Bioreagents (Golden, CO). Slides were washed three times with PBST for 5 min and incubated with biotin-conjugated goat anti-rabbit IgG (Jackson Immunochemicals, West Grove, PA) diluted 1:500 in PBST for 1 h at room temperature in a humidified chamber. Slides were washed three times with PBST for 5 min, followed by incubation with ABC reagent (Vector Laboratories, Burlingame, CA) diluted 1:100 in PBS for 1 h at room temperature in a humidified chamber. Slides were rinsed in PBS and incubated with stable DAB (Research Genetics, Huntsville, AL) for 10 min (anti-mPER1) or 5 min (anti-CLOCK). Sections were counterstained as follows: Gills Hematoxylin (Sigma) for 30 sec, rinsed in dH2O, 4% acetic acid for 20 sec, rinsed in dH2O, saturated Li2CO3 for 20 sec, and rinsed in dH2O. Sections were dehydrated with gradually increasing concentrations of ethanol followed by incubation with xylene. Coverslips were applied, and the slides were viewed using a light microscope.
Brain Perfusion
Mice were killed at the indicated times and perfused with cold PBS followed by 4% paraformaldehyde. Brains were collected and postfixed in 4% paraformaldehyde, followed by equilibration in 30% sucrose. Coronal sections were cut to a thickness of 30 µm on a cryostat. Staining was performed essentially as described previously except sections were free floating in buffer for each step. Additionally, anti-mPER1 antibody was used at a concentration of 1:750, anti-CLOCK antibody was used at a concentration of 1:1500, and secondary antibody was used at a concentration of 1:1000.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To determine which circadian clock gene transcripts are expressed in a cyclic manner in the testis, we performed RNase protection assays. Eight- to 12-wk-old C57BL/6 male mice were individually housed and kept on a 12L:12D cycle. Germ cells were purified from testes at various zeitgeber times (ZTs). By definition, the time of lights-on is ZT0, and the time of lights-off is ZT12. Germ cell RNA was purified from the germ cells, and transcript levels of mPer1, mPer2, Clock, Bmal1, mCry1, and Npas2 were determined. Npas2 is a circadianly transcribed gene that is not considered a component of the core clock, but its expression cycles in some peripheral oscillators [32, 33]. Surprisingly, the transcript levels of all these genes were constant over the course of the day (Fig. 1A).
To ensure that the RNase protection assay could detect cycling of these genes, we collected RNA from kidneys and from testes at six different times of day. In the kidney, robust cycling of mPer1, mCry1, and Bmal1 transcript levels was observed. However, in the same animal, there is clearly a lack of cyclic expression in the testis (Fig. 1B). Clock expression showed minimal cycling in the kidney, which is consistent with its reported expression in both the SCN and peripheral tissues [32, 34–36]. Clock expression did not cycle in the testis (Fig. 1B).
In the kidney, the time of peak and trough levels of the gene transcripts corresponded to previously published reports [34–36]. Comparison of the levels of the various transcripts in the kidney and in the testis revealed strong differences (Fig. 1C). In the testis, mPer1 and mCry1 displayed high levels of expression, but in the kidney, expression of these genes was quite low, even at peak expression times. Clock transcript levels were about half as abundant in the kidney as in the testis. In contrast, the levels of Npas2 and Bmal1 transcripts were lower in the testis than in the kidney.
Expression of mPER1 Is Constant in the Testis and Is Restricted to Specific Developmental Stages
The finding that clock transcript levels are constant in the testis may indicate that transcript cycling does not occur at all in the testis. Alternatively, expression may cycle only in specific cell types and be constant in others. If this were the case, cell-specific cyclic expression might be obscured by such constant expression elsewhere. Use of the RNase protection assay does not allow determination of the particular cell types that express the genes of interest. Additionally, these studies only characterize the RNA levels of the genes. Indeed, it is clear that RNA cycling is not always necessary for protein cycling [36, 37]. To determine whether circadian clock proteins are cyclically expressed in the testis, immunohistochemistry was utilized. Immunohistochemistry also allowed localization of protein expression to specific cell types. Because mPer1 is highly expressed in testes, we decided to localize this protein product. Testes were collected from animals at six different times of day, and sections were stained with a commercially available anti-mPER1 antibody. Two distinct subpopulations of cells stained positively for mPER1 expression: Type B spermatogonia and condensing spermatids (Fig. 2). Staining of both these cell types was observed at all time points, indicating that cyclic expression of mPER1 does not occur within specific cells.
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Seminiferous tubules in mice can be histologically classified into 12 different categories, each composed of spermatogenic cells in different substages of development. The tubules that contain the positively staining Type B spermatogonia represent stages V–VII of the seminiferous cycle (Fig. 2A). In contrast, the tubules that contain positively staining condensing spermatids represent stages IX–XI of the seminiferous cycle (Fig. 2B). Notably, the two mPER1 expressing subpopulations were never found in the same seminiferous tubules, indicating that expression of mPER1 is restricted to specific substages of development.
Furthermore, immunohistochemical staining of testes from neonatal mice revealed mPER1 expression in primitive Type A spermatogonia at 7 days of age (Fig. 3). Interestingly, at 14 days of age, there is less staining of primitive Type A spermatogonia. In contrast, primary spermatocytes are the predominant cell population that expresses mPER1. Currently, it is not clear why there is variability in terms of the spermatogenic stages that express mPER1 at different stages of murine development. One possibility is that the positively staining primary spermatocytes observed at Day 14 represent daughter cells of the positively staining spermatogonia observed at Day 7. Primary spermatocytes are not positive for mPER1 in adults, perhaps because those that express mPER1 at Day 14 are eliminated through apoptosis. Therefore, the adult pattern of mPER1 expression may not be established until sexual maturity.
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The immunohistochemical stain for mPER1 was specific, as no staining was seen with rabbit immunoglobulin, and staining was blocked by preincubation of the antibody with immunogenic peptide (data not shown). Also, staining with another commercially available anti-mPER1 antibody revealed the same results (data not shown). Specificity of the antibody was also demonstrated by staining SCN sections (see the following discussion).
Expression of CLOCK Is Found in the Acrosome and Does Not Colocalize with mPER1
In the pacemaker cells of the SCN, mPER1 works with CLOCK to set up a transcription translation feedback loop that underlies the circadian clock. Therefore, if mPER1 and CLOCK function in the testis as they do in the SCN, expression of these proteins should be found in the same cells. Strikingly, immunohistochemical localization of CLOCK in the testis revealed that expression was localized to the acrosome, which is an extranuclear structure involved in fertilization (Fig. 4). This staining is seen beginning at the round spermatid stage. To verify that mPER1 and CLOCK were not expressed in the same cells, serial sections of the same testes were stained. As seen in Figure 5, these proteins are expressed at completely different developmental stages. Indeed, the proteins are expressed in separate seminiferous tubules, indicating that they are expressed at different stages of spermatogenesis. As with mPER1, CLOCK staining was specific. No staining was seen with rabbit immunoglobulin or when the antibody was preincubated with immunogenic peptide (data not shown).
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Both Anti-mPER1 and Anti-CLOCK Antibodies Are Specific for These Proteins
The finding of noncyclic, stage-specific expression of both mPER1 and CLOCK was unexpected. To rule out the possibility that these antibodies were reacting with proteins other than mPER1 and CLOCK, their ability to stain the SCN was tested. Immunohistochemistry was performed on brains isolated from mice at different times of day. Abundant expression of mPER1 was found in the SCN at ZT12, but very little expression was found at ZT0 (Fig. 6). This result is consistent with the known cyclic nature of mPER1 expression in the SCN [38]. Additionally, a light pulse delivered in the middle of the night will induce mPER1 expression in the SCN [39]. The anti-mPER1 antibody used in our experiments also detected an increase in mPER1 staining in the SCN 1 h after a light pulse (data not shown). Overall, the staining pattern detected in the SCN with the anti-mPER1 antibody is consistent with previous reports.
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Strong immunohistochemical staining for CLOCK was found in the SCN of mice at all times of day (Fig. 6). Such constant expression is consistent with previously reported expression patterns of CLOCK [34]. Notably, the anti-mPER1 and anti-CLOCK antibodies detected bands of the correct size on Western blots of protein extracts from testis (data not shown).
Circadian Clock Genes Do Not Cycle in the Thymus
It is not clear why noncyclic expression of circadian clock genes occurs in the testis but, to our knowledge, nowhere else in the body. One difference of the testis compared to other organs in the body is that cellular differentiation is still occurring. In contrast, cells in organs such as the kidney are postmitotic and fully differentiated. If a connection exists between cellular differentiation and a lack of cycling, presumably another organ with differentiating cells would also lack cycling. The thymus is an example of such an organ. Thymocytes develop into mature T-cells within the thymus through distinct developmental stages. Analysis of expression of various clock genes in the thymus by RNase protection analysis revealed that expression of mCry1 is constant at all times of day (Fig. 7). Expression of Bmal1 and Npas2 were quite low in the thymus, but there was no variation in transcript level (data not shown). Expression of mPer1 showed minimal cycling but was clearly dampened in amplitude (Fig. 7). Expression of mPer2 was low but did show minimal, dampened cycling (data not shown). The complete lack of cycling of some genes and the attenuated cycling of others in the thymus supports the hypothesis that circadian expression is suspended in differentiating cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Whether there are any endogenous circadian functions in the testis is unclear. Of course, the type of animal under study has to be considered. Circadian rhythms regulate spermatogenesis in insects, but a function in murine spermatogenesis is not known yet. However, in photoperiodic mammals, such as the hamster, the length of day affects sperm production [25]. Whether the testes of such animals also show constant expression of circadian genes as is seen in the mouse would be interesting to determine. Additionally, there are well-described diurnal outputs from the murine testis. For example, testosterone levels are at a peak in the morning and decrease as the day progresses [42]. Interestingly, neither mPER1 nor CLOCK expression was observed in Leydig cells. It is possible that other circadian proteins in these cells regulate testosterone production. Alternatively, Leydig cells may respond solely to extracellular signals that are generated in a circadian manner from other organs.
Our results also show a striking difference in expression levels of the various genes between two different organs: testis and kidney. For example, strong expression of mPer1 was observed in the testis, but little expression was seen in the kidney. Notably, recent microarray experiments show that, within peripheral tissues, hundreds of genes cycle over the course of the day [43–47]. Surprisingly, there is little overlap between the specific subsets of genes that cycle in the various tissues examined. Differences in the subsets of cycling genes in peripheral organs may result from differential expression of core clock genes in these organs.
The fact that there is no cycling of circadian genes in the testis naturally raises the question as to their function. It is tempting to speculate that these genes participate in spermatogenesis. Although gene-targeted animals of the circadian genes are reported to be fertile, such findings do not rule out a function for these genes in spermatogenesis [48–53]. Multiple isoforms and family members of these proteins exist; therefore, it is possible that these act redundantly in the testis. A test of this possibility awaits the production of multiple knockout animals. Another possibility is that deletion of these genes has a subtle effect on spermatogenesis that has not been detected yet. To our knowledge, a careful study of fertility and spermatogenesis has not been performed on any of the circadian gene knockout animals. There may be effects on fertility, such as on litter size. It is also possible that the timing of the specific stages of development is disrupted. That is, if circadian gene expression is necessary to properly time the transition from one stage to the next, sperm may still be produced, but the histological architecture of the seminiferous tubules may be disrupted. This could affect the quantity and/or quality of sperm produced.
In support of a developmental function for circadian clock proteins, we found CLOCK and mPER1 expression restricted to separate and specific developmental stages of spermatogenesis. Protein expression at these stages was found at all times of day, consistent with the lack of transcript cycling. CLOCK was expressed in the developing acrosome of round spermatids, and mPER1 was expressed in a subpopulation of spermatogonia and in condensing spermatids. The distinct expression patterns of CLOCK and mPER1 indicate that these two proteins do not set up a transcription translation feedback loop as they do in cells of the SCN. Given the constant expression of circadian clock genes, such a result is not completely surprising; however, the acrosomal expression of CLOCK is unexpected. This extranuclear structure is necessary for sperm to penetrate the zona pellucida, the extracellular matrix that surrounds the egg, during fertilization. To date, the only known function of CLOCK is to promote transcription; therefore, its function in a structure such as the acrosome is difficult to explain. However, this is not the only example of a transcription factor being expressed in the acrosome. Basonuclin is a zinc finger transcription factor found in both basal keratinocytes and the acrosome, although the acrosomal function is unknown [54, 55]. Possibly CLOCK has a completely new function in the acrosome other than regulating transcription.
In a larger sense, a lack of cycling may not be restricted solely to spermatogenesis and reproductive biology. We hypothesize that the circadian cycle is suspended while cells are undergoing differentiation. In support of this hypothesis, we found that cyclic expression of many of the clock genes does not occur in the thymus. The thymus is another organ where stagewise cellular differentiation occurs in the adult, much like the testis. It is possible that the circadian clock must be "shut off" to allow another clock, such as a developmental clock, to take precedence. Although a circadian clock clearly is at work in the fetus, it is not clear at what point during development it becomes active. It will be interesting to determine if other tissues that are undergoing differentiation, such as intestinal epithelium or epidermis, also show a suspension of clock gene cycling.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Amita Sehgal, Department of Neuroscience, 232 Stemmler Hall, University of Pennsylvania School of Medicine, 35th Street and Hamilton Walk, Philadelphia, PA 19104. FAX: 215 573 2015; amita{at}mail.med.upenn.edu
Received: 27 September 2002.
First decision: 1 November 2002.
Accepted: 6 February 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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