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Endocrinology Vol. 142, No. 4 1567-1577
Copyright © 2001 by The Endocrine Society

ARTICLES

Irradiation Selectively Inhibits Expression from the Androgen-Dependent Pem Homeobox Gene Promoter in Sertoli Cells1

Sourindra Maiti, Marvin L. Meistrich, Gene Wilson, Gunapala Shetty, Marco Marcelli, Michael J. McPhaul, Patricia L. Morris and Miles F. Wilkinson

Departments of Immunology (S.M., M.F.W.) and Experimental Radiation Oncology (M.L.M., G.W., G.S.), University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030; Department of Medicine, Baylor College of Medicine (M.M.), Houston, Texas 77030; Department of Internal Medicine, University of Texas Southwestern Medical School (M.J.M.), Dallas, Texas 77235; The Population Council, and The Rockefeller University (P.L.M.), New York, New York 10021

Address all correspondence and requests for reprints to: Miles F. Wilkinson, Departments of Immunology, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030. E-mail: mwilkins{at}mail.mdanderson.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
How radiation blocks spermatogenesis in certain strains of rats, such as LBNF1, is not known. Because the block depends on androgen, we propose that androgen affects Sertoli cell function in irradiated LBNF1 rats, resulting in the failure of spermatogonial differentiation. To begin to identify genes that may participate in this irradiation-induced blockade of spermatogenesis, we investigated the expression of several Sertoli genes in response to irradiation. The expression of the Pem homeobox gene from its androgen-dependent Sertoli-specific proximal promoter (Pp) was dramatically reduced more than 100-fold in response to irradiation. In contrast, most other genes and gene products reported to be localized to the Sertoli cell, including FSH receptor (FSHR), androgen receptor (AR), SGP1, and the transcription factor CREB, did not exhibit significant changes in expression, whereas transferrin messenger RNA (mRNA) expression dramatically increased in response to irradiation. Irradiation also decreased Pp-driven Pem mRNA levels in mouse testes (approximately 10-fold), although higher doses of irradiation than in rats were required to inhibit Pem gene expression in testes of mice, consistent with their greater radioresistance. The decrease in Pem gene expression in mouse testis was also selective, as the expression of CREB, GATA-1, and SGP1 were little affected by irradiation. We conclude that the dramatic irradiation-triggered reduction of Pem expression in Sertoli cells is a conserved response that may be a marker for functional changes in response to irradiation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ALTHOUGH irradiation of certain strains of rat, including the LBNF1 hybrid, fails to kill most type A spermatogonia, the surviving spermatogonia do not undergo differentiation, resulting in the failure of spermatogenic recovery (1). This failure, which is at the level of maturation of the undifferentiated A spermatogonia or the survival of their progeny, is reversed by GnRH antagonists and other treatments that suppress intratesticular testosterone levels (2). The success of these hormone treatments implies that a testosterone-responsive cell is responsible for this block of spermatogonial differentiation. Because spermatogonia do not have androgen receptors (AR), testosterone must be acting on another cell, which then alters its signals (directly or indirectly) to the spermatogonia such that it promotes their survival and/or differentiation. The most likely candidate cell is the Sertoli cell, as it is in intimate physical contact with spermatogonia.

Although Sertoli cells are not killed by irradiation, there are few data as to whether irradiation affects these cells in some other manner, thereby altering their ability to support germ cell differentiation by paracrine interactions. A decline in Sertoli cell function in response to irradiation has been suggested by indirect measurements, including a rise in serum FSH (a measure of reduced inhibin production by Sertoli cells) and a decline in serum levels of androgen-binding protein (a Sertoli cell product) (3). Another study showed a loss of interleukin-1{alpha} mRNA following radiation or cytotoxic drug treatment, which was attributed to altered Sertoli cell function as a result of germ-cell depletion (4). To our knowledge, there is only one example of direct irradiation-induced alterations of Sertoli gene expression. In this case, irradiation of Sertoli cells in vitro increased transferrin secretion and interleukin-6 activity (5).

In the present study, we screened several rat Sertoli genes for alterations in gene expression after in vivo irradiation. We identified the Pem gene as a major target of irradiation. Pem is a homeobox gene that we originally cloned by subtraction hybridization from a T-lymphoma cell clone (6). Pem is not expressed in normal T cells, but rather it is aberrantly expressed in tumor cells from many different organs. Pem expression in normal tissues is primarily restricted to specific cell types in reproductive organs (7, 8). Given that Pem is a homeobox protein, it is likely to be a transcription factor that regulates downstream genes important in these male reproductive organs. In fact, Pem is the founding member of a new homeobox transcription factor subfamily whose members are all expressed in reproductive tissues. This PEPP homeobox subfamily, which also includes the Esx-1 (Spx), Psx-1, and Psx2 (Gpbox) genes, encodes proteins containing related homeodomains (DNA-binding domains) that are most similar to (but nevertheless distinct from) those encoded by the Prd homeobox subfamily (9, 10). All known PEPP homeobox genes are present on the mouse X chromosome and all appear to be derived from a common precursor gene (10).

The Pem gene is transcribed from two promoters to produce the same protein (11). The proximal promoter (Pp) is androgen dependent and is expressed in Sertoli cells and somatic cells of the epididymis, but not in any other of several adult mouse and rat tissues that have been examined (7, 8, 12, 13). In contrast, the distal promoter (Pd) is more ubiquitously expressed; it is not only transcribed in testes but also in the female reproductive tissues, such as placenta and mural granulosa cells in the ovary (11, 12).

To examine the mechanism and specificity of the radiation-induced reduction in Pem expression in the rat, we determined if androgen levels or receptors were involved. In addition, we compared the effects of irradiation on Sertoli gene expression in rat testis to that in mouse testis. In contrast with the irradiation sensitivity of LBNF1 rat testes, mouse testes are capable of supporting the differentiation of spermatogonia even after irradiation doses of 10 Gy or more (14). Our results demonstrate that Pem’s androgen-regulated promoter is selectively radiation sensitive in rodents.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult LBNF1 male rats were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, IN). LBNF1 rats were irradiated at about 8 weeks of age. C3H/Kam mice, bred at the University of Texas M. D. Anderson Cancer Center, were irradiated at 8–10 weeks of age. Animals were housed in standard lighting (12 h of light, 12 h of darkness) and allowed food and water ad libitum. They were maintained in facilities approved by the American Association for the Accreditation of Laboratory Animal Care.

Irradiation
The lower part of the body of anesthetized rats, extending anteriorly about 6 cm from the base of the scrotum, was irradiated with {gamma} rays (1.25 MeV) from a 60Co {gamma} ray unit (1) at a dose rate of 0.96 Gy/min. Rats were placed on their backs and 5 mm of tissue-equivalent bolus material was placed over the scrotum to provide a build-up layer.

Mice were given localized radiation {gamma} rays (0.66 MeV) using a pair of 137Cs sources with a 3-cm diameter field (14). They were awake at the time of irradiation and were restrained in Lucite boxes and positioned so that only the testes and parts of surrounding organs received the irradiation. Although different sources were used for mouse and rat irradiations, there is no significant difference in the biological effectiveness of {gamma} rays of these slightly different energies.

Preparation of purified populations of testicular cells
Sertoli cells were freshly isolated from the testes of twelve 65-day old Sprague Dawley rats (15). The testes were saline-perfused in situ. Each tunica was removed, the tubules were gently separated and treated with collagenase, hyaluronidase (Worthington, Lakewood, NJ), DNase (Roche Molecular Biochemicals, Germany) in the presence of soybean trypsin inhibitor (STI, Sigma, St. Louis, MO). Following a 25-min incubation at 34 C with moderate shaking, fresh F12/DMEM was added, and the tubules were sedimented by low-speed centrifugation. Enzymatic treatments and additions of medium to the tissue were repeated twice. The loosely associated tubule elements were then treated with collagenase/dispase (Roche Molecular Biochemicals, Mannheim, Germany), hyaluronidase, DNase, and STI for 30 min as above. Tubules were then dispersed through repeated pipettings. The suspensions were repetitively diluted, sedimented at unit gravity, and resuspended in medium. The large number of free germ cells and peritubular myoid cells were aspirated each time. The suspensions were monitored to be certain loose germ cells and single cells were removed. The resulting cell aggregate pellet was resuspended in 2% BSA and filtered with stirring through several 53-µm nylon mesh filters (TETKO, Inc., Briarcliff Manor, NY).

The resulting highly enriched populations were repeatedly rinsed with fresh culture medium, resedimented at unit gravity, and the supernatants were discarded. The pellet contained Sertoli cell aggregates at a purity of approximately 85–90%. Most contaminating cells were spermatozoa and sperm tails that adhered to the larger Sertoli cell aggregates in the final suspension.

Interstitial cells were separated from tubular cells by two methods. In the first procedure, tubules and interstitial tissue were separated by microdissection. In the second procedure, the blood was first flushed from the testis by passing saline through the testicular artery. Then the decapsulated tubules were incubated with trypsin and DNase for 15 min as described previously, except that the tubules were not first chopped (16). The solution was filtered through an 80-µm mesh. Of the cells that passed through, about 50% were interstitial cells. The tubules that remained on the mesh were incubated with trypsin and DNase for another 15 min and filtered again to obtain a highly enriched tubular preparation. In one experiment interstitial cells were further purified by Percoll density-gradient centrifugation (17).

Pachytene spermatocytes, round spermatids, and late spermatids were separated by centrifugal elutriation, and in one experiment, followed by equilibrium density centrifugation (17). The purity of the fractions is given in Table 1Go.


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  		Table 1. Percentage cellular composition of fractions of cells obtained by elutriation followed by Percoll density gradient centrifugation (values in parentheses are with elutriation only)

 
Immunohistochemistry
Testes from control and irradiated rats (killed at 14 weeks after 6 Gy of irradiation) were fixed in formalin and embedded in paraffin. Paraffin sections of 6 µm were mounted onto poly-L-lysine coated slides, deparaffinized in xylene, rehydrated through graded ethanol, and treated in a microwave oven in 0.01 M sodium citrate buffer for 10 min. One percent hydrogen peroxide in buffered saline was used to block endogenous peroxidase activity and 3% normal goat serum was used to block nonspecific antigens. Sections were incubated for 90 min with primary polyclonal AR antibody (U402) (18) diluted 1 to 250 in buffered saline. Specimens were stained using the avidin-biotin peroxidase complex method (ABC Kit, Vector Laboratories, Inc., Burlingame, CA). The specificity of the reaction using the AR peptide from which this antibody is derived has already been reported (18). To determine reaction specificity in the experiments presented in this paper, some sections were incubated with buffered saline substituting for the primary antibody. Specific AR immunostaining was defined as brown staining present in sections incubated with AR antibody but absent in sections without the primary antibody. All specimens of the various studies were stained simultaneously. Counterstaining, which obscures low intensity immunostaining, was not done.

RNA preparation
Total cellular RNA from tissues was prepared as described previously by either guanidinium isothiocyanate lysis and centrifugation over a CsCl cushion (19) or by a single-step acid guanidinium thiocyanate/phenol/chloroform extraction (20). In the case of Sertoli cells, total RNA was extracted using the TRIzol reagent (Life Technologies, Inc., Grand Island, NY).

The recoveries of RNA from the different control and irradiated mouse testes were very reproducible with an average yield of 1640 ± 300 (SD) µg/g tissue (range, 1160 to 2410 µg/g tissue). The recovery from the different control and irradiated rat testes were somewhat more variable, with an average yield of 350 ± 190 (SD) µg/g tissue (range 150 to 800 µg/g tissue). Most of the variability came from the three 4-week samples, which had approximately twice the RNA yield (average of ~500 µg RNA/g tissue) as the other samples.

Northern blot analysis
RNA was electrophoresed in 1%-agarose formaldehyde gels, and the separated RNA was transferred to Nytran membranes by capillary action (21). After transfer, the membrane was cross-linked by UV irradiation (Stratalinker, Stratagene Inc.) and stained with methylene blue to evaluate the transfer and loading of RNA in each lane (22). Blots were prehybridized in buffer (50% formamide, 5x Denhardt’s solution, 5x SSPE, 0.5% SDS, and 100 µg/ml sheared salmon sperm DNA) for 1 to 4 h at 42 C. Blots were then hybridized overnight at 42 C with random oligomer-primed 32P-labeled complementary DNA (cDNA) in prehybridization buffer plus 10% dextran sulfate, as described (8).

The rat SGP1 cDNA probe was isolated from the pSGP1–1A plasmid (G-107) that was kindly provided by Dr. Michael Collard (University of Illinois, Carbondale, IL) (23). The rat transferrin cDNA probe is a 1.0-kb EcoRI/HindIII 3' fragment released from the pSP65 vector (G-182) (24) that contains sufficient sequence identity with rat hemiferrin to also hybridize with hemiferrin mRNA (25). The mouse CREB cDNA probe is a 341-bp HindIII/XbaI fragment derived from the RC-RSV vector CREB 341 vector (G-187) provided by Dr. Richard Goodman (Oregon Health Sciences University, Portland, OR) (26). This probe hybridizes with a transcript migrating between 18S and 28S ribosomal RNA (rRNA) that is enriched in Sertoli cells and a band below 18S that is enriched in germ cells (27, 28). The rat FSHR genomic probe is a 729-bp AccI/BamHI fragment within exon 10 (29). The mouse GATA1 probe is a 1.9-kb EcoRI fragment from the pXM vector (G-186) kindly provided by Celeste Simon (University of Chicago). The RXRß probe is a 1.5-kb EcoRI/AccI cDNA fragment derived from a RSV-H2RIIBP (G-258) vector kindly provided by Dr. Ronald Evans (Salk Institute, San Diego, CA) (30). The rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe is a 0.35-kb HindIII/EcoRI fragment encompassing part of the GAPDH coding region (obtained from Ambion, Inc.). In addition to the somatic form of GAPDH, this probe might also recognize the testis-specific isozyme, Gapd-s (31) (J. E. Welch, personal communication), as there is 61% sequence identity in this region.

Ribonuclease protection analysis (RPA)
Three probes were used for RPA: rat Pem, mouse Pem, and rat GAPDH. The mouse and rat Pem probes are the same as Pem probes A and B, respectively, described previously (12). GAPDH probes were generated from the pTR1 vector (G-159) (Ambion, Inc.) cleaved with either or DdeI or StyI; they contain either 66 or 134 nt, respectively, of rat 3' GAPDH cDNA sequences (the shorter and longer probes were used for mouse and rat RNA annealing, respectively). These GAPDH probes protect only somatic GAPDH mRNA, not mRNA from the divergent germ-cell isoform, Gapd-s (31) (Dr. Jeffrey Welch, U.S. Environmental Protection Agency, Research Triangle Park, NC; personal communication).

[32P]UTP-labeled RNA probes were generated, and RPA was performed on total cellular RNA as described (8). The riboprobes were purified in 8 M urea, 6% polyacrylamide denaturing gels. After exposure to film, the appropriately sized bands were excised from the gel, mashed in 100 µl of diethylpyrocarbonate-treated water, and the probe was eluted by two incubations in 600 µl of 1x proteinase K (PK) buffer [0.3 M NaCl, 0.5% SDS, 10 mM Tris (pH 7.5), 200 µg/ml PK, and 20 µg/ml transfer RNA] for 15 min at 37 C. The suspended probe from both incubations was filtered through a 0.45-µm Acrodisc filter, extracted with 200 µl of phenol/chloroform, and ethanol precipitated. The appropriate gel-purified [32P]UTP-labeled probe was then coprecipitated with the sample RNA or transfer RNA (negative control). The pellet was resuspended in 30 µl of annealing buffer (40 mM PIPES [pH 6.4], 0.4 M NaCl, 1 mM EDTA, 80% formamide) and allowed to hybridize overnight at 44 C. Unhybridized RNA was digested with RNase A and RNase T1 for 20 min at 37 C at concentrations of 25 µg/ml and 5 µg/ml, respectively. The samples were then treated with proteinase K and extracted with phenol/chloroform/isoamyl alcohol (25:24:1 by volume). After ethanol precipitation, the RNA pellet was resuspended in 90% formamide loading buffer, denatured at 85 C, and electrophoresed in an 8 M urea, 6% polyacrylamide gel. A set of RNA size markers generated from the Century ladder template (Ambion, Inc.) was included in all gels.

Calculation of relative RNA levels
The relative levels of mRNA per testis were calculated by first determining the relative intensities of the bands by either phosphorimage or densitometric analysis of autoradiographic films. The band intensities were then multiplied by the volume in which the recovered RNA was resuspended and divided by the volume loaded on the gel. The levels of mRNA per Sertoli cell were determined by normalizing against the Sertoli-specific transcript SGP1. In the case of Northern blots, the intensities of the bands were normalized by dividing by the intensity of the SGP1 band for each lane. In the case of RPA, the intensities of the bands were normalized per Sertoli cell using GAPDH to relate the data on the northern blots to that on the RPAs. To do this, the intensities of the bands were divided by the intensities of the GAPDH band in the same lane. These values were then multiplied by the ratio of GAPDH to SGP1 for the same sample on the Northern blot; i.e. band-RPA/SGP1-Northern = (band-RPA/GAPDH-RPA) x (GAPDH-Northern/SGP1-Northern). Lastly, the normalized values for irradiated rat samples obtained from both the RPAs and northern blots were corrected by dividing by the value obtained from control unirradiated rats.

Hormone measurements
Blood was collected by cardiac puncture at the time the rats were killed. Serum FSH concentrations were measured by a double-antibody RIA, using NIDDK assay kits and standards (32). Intratesticular testosterone (ITT) concentrations were measured in testicular homogenates using coated-tube RIA kits (DSL 4000, Diagnostics Systems Laboratories, Inc., Webster, TX) (33).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of irradiation on the LBNF1 rat testis
We have shown previously that 6 Gy irradiation depletes most germ cells from seminiferous tubules of LBNF1 rats, leaving only spermatogonia and Sertoli cells (1). Here we present the time course of this depletion process (Table 2Go). Damage was apparent after 2 weeks by the loss of spermatogonia. At 4 and 6 weeks, there was some recovery of A spermatogonia but the spermatocytes and spermatids were lost by maturation depletion. At 10 and 20 weeks, there was a more complete failure of spermatogenesis. There was a reduction in the numbers of tubules containing A spermatogonia and those that were present had apparently lost the ability to differentiate.


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  		Table 2. Effect of 6 Gy irradiation on testis weight, histology, and hormone levels of LBNF1 rats

 
To determine whether irradiation causes Sertoli cell gene expression to be altered by loss of gonadotropic or steroid hormone-dependent stimulation, we measured testosterone and FSH levels. We found that neither intratesticular testosterone (ITT) nor serum FSH were depleted in response to irradiation. In fact, both serum FSH and ITT levels increased after irradiation (Table 2Go).

Irradiation extinguishes expression from the Pem proximal promoter (Pp)
To examine the effect of irradiation of Pem gene expression, RNase protection analysis (RPA) was performed with total cellular RNA from testes obtained from irradiated and nonirradiated (control) LBNF1 rats. We used a probe that distinguishes between transcripts from the Pem proximal (Pp) and distal (Pd) promoters (Fig. 1AGo). RPA with this probe showed that control (nonirradiated) LBNF1 rats expressed approximately equal levels of Pp and Pd transcripts (Fig. 1BGo). This is consistent with the expression pattern we had previously established for Sprague Dawley rats (12).



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  		Figure 1. Pem Pp transcripts are selectively eliminated in response to irradiation. A, Genomic organization of the rat Pem gene, including its distal (Pd) and proximal (Pp) promoters. The location of the probe used for RPA is indicated. B, Time course of the decline of Pem mRNA between 2 and 10 weeks after 6 Gy of irradiation assessed by RPA. Forty micrograms of RNA was annealed per lane. A GAPDH control probe was included in the annealing reactions that also contained the Pem probe. Two different exposures of the same gel are shown to permit visualization of both Pem mRNA (modestly expressed) and GAPDH mRNA (highly expressed).

 
Figure 1BGo shows an RPA autoradiogram of equal amounts of total testis RNA obtained 0, 2, 4, 6, and 10 weeks after 6 Gy irradiation. We found that 10 weeks after 6 Gy irradiation, Pem Pp mRNA was reduced to undetectable levels in all three rats tested (Fig. 1BGo). A decrease in Pem Pp mRNA levels was observable as early as 2 weeks after irradiation; the decline in Pp levels was progressively more dramatic at later times. The absence of detectable Pem Pp mRNA was maintained for at least 20 weeks after irradiation (data not shown). In contrast to Pp transcripts, Pd transcripts persisted after irradiation, although their levels modestly declined. Thus, we conclude that the transcripts from the two Pem promoters were independently regulated in response to irradiation.

In the same RPA annealing reactions that were used to determine Pem mRNA levels, we also analyzed the expression of transcripts from the GAPDH housekeeping gene. The relative abundance of GAPDH mRNA increased after irradiation (Fig. 1BGo). This increase probably results from the fact that the somatic form of GAPDH is expressed primarily by nongerminal cells, which represent a much greater proportion of the testis after irradiation-induced germ cell depletion.

To determine the relative levels of Pem Pp, Pem Pd, and GAPDH transcripts at different times after irradiation, the band intensities were measured by densitometry and then normalized to reflect RNA levels per testis (Fig. 2AGo). We normalized per testis because the numbers of Sertoli cells (and other nongerminal cells) per testis were unaffected by irradiation. Thus, for Sertoli-specific genes, this value should be unaffected by the dramatic decrease in testes weight and RNA yield per testis that results from the irradiation-induced depletion of germ cells. Although this analysis assumes that the recovery of RNA is equal in all samples, we found that the variation in recovery, as measured by µg RNA/g testis, was much smaller than the effect of irradiation on Pem Pp mRNA expression. This small variation in recovery was further confirmed by northern-blot analysis of the expression of other Sertoli cell genes (see below).



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  		Figure 2. Effect of irradiation on expression of different genes in the rat testis. A and C, Relative mRNA levels based on values generated from RPA. B and D, Relative mRNA levels based on values generated by Northern blot analysis. Values in panels A and B were corrected to reflect expression per testis (see Materials and Methods). Values in panels C and D were corrected to reflect levels per Sertoli cell by normalizing against SGP1 mRNA, which is a Sertoli-specific transcript whose levels per testis remained nearly constant after irradiation (see B).

 
This analysis demonstrated that Pem Pp transcript levels per testis decreased by greater than 5-fold after 2 weeks, by approximately 10-fold after 4 weeks, and by greater than 50-fold after 6 weeks (note the log scale in Fig. 2AGo). Pem Pd transcripts also decreased in levels per testis, but this decrease (~3-fold) was much more modest than the down-regulation observed for Pp transcripts.

Regulation of SGP1, CREB, FSHR, and transferrin gene expression in response to rat irradiation
We wanted to determine whether irradiation specifically inhibits Pem gene expression or whether instead it has a general inhibitory effect on the expression of Sertoli genes. Toward this end, we examined the effect of irradiation on the expression of SGP1, CREB, FSHR, and transferrin by Northern-blot analyses. In the case of CREB, we only quantified the expression of the approximately 3-kb transcript, which appears to be Sertoli specific; see Materials and Methods. Figure 3Go shows the autoradiogram of the Northern-blot data and Fig. 2BGo provides the quantitative analysis. We found that the levels of SGP1, 3-kb CREB, and FSHR transcripts were only modestly affected by irradiation. Unlike Pem Pp mRNA, none of these other mRNAs decreased in level within the first 6 weeks after irradiation (we attribute the slight increase of all transcripts at 4 weeks to the higher yield of RNA per testis at this time point; see Materials and Methods). The behavior of transferrin was different from that of the other transcripts, as its mRNA levels were dramatically increased in response to irradiation (Fig. 2BGo).



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  		Figure 3. Effect of irradiation on the levels of the SGP1, CREB, transferrin, hemiferrin, and GAPDH transcripts. Northern blot analysis of the time course of the changes in the expression of the transcripts between 2 and 10 weeks after 6 Gy of irradiation. Ten micrograms of RNA were loaded per lane.

 
GAPDH, a ubiquitously expressed gene, also exhibited little change in expression after irradiation. RPA analysis showed there was less than a 2-fold decline in mRNA levels per testis (Fig. 2BGo). Note that the probe used for RPA protects only somatic GAPDH mRNA, not the Gapd-s germ cell-specific isoform (see Materials and Methods). The result obtained by RPA was confirmed by northern-blot analysis, which also indicated little or no change in GAPDH mRNA levels (Fig. 3Go). In contrast to RPA, Northern analysis can detect cross-hybridizing signals and thus the GAPDH probe might detect 1.5-kb Gapd-s mRNA by northern (31). However, we found that the size of Gapd-s mRNA was too similar to that of 1.3-kb somatic GAPDH mRNA to distinguish between the two on agarose gels. Because we found that the level of the GAPDH/Gapd-s band was not significantly affected by irradiation, even at time point when there was massive germ-cell depletion, this suggests that the probe only weakly cross-hybridized with Gapd-s mRNA (under the conditions used) or that Gapd-s mRNA levels were significantly lower than GAPDH mRNA levels.

In contrast to these Sertoli cell genes, the levels of hemiferrin mRNA per testis, which is germ-cell specific (25), and the lower band of CREB decreased as a result of the germ cell depletion caused by irradiation.

Because we found that Sertoli-specific SGP1 mRNA (23) exhibited little change in level in response to irradiation, we also used it as a baseline to estimate the expression of the other genes per Sertoli cell. These normalized values are shown in Fig. 2CGo (RPA) and Fig. 2DGo (Northern-blot analysis). Pem Pp transcripts precipitously decreased per Sertoli cell, declining by more than 100-fold 6 or 10 weeks after irradiation. In contrast, the CREB and FSHR transcripts remained at high levels, and transferrin mRNA levels were dramatically elevated after irradiation. Hemiferrin germ-cell specific transcripts declined rapidly as a result of the massive germ-cell depletion after irradiation. Pem Pd transcripts, which unlike Pp transcripts are expressed primarily by interstitial and germ cells (see later), also decreased.

Androgen receptor (AR) expression in irradiated rat testes
We also examined the expression of AR in Sertoli cells after irradiation. We reasoned that if irradiation abolished AR expression, this would explain the dramatic reduction in the levels of the Pem Pp transcripts, as the expression of this transcript is testosterone dependent in both rats and mice and fails to be expressed in mice deficient in AR (7, 8, 11, 12).

We analyzed AR protein levels by immunohistochemistry. Control testes showed a stage-specific pattern of Sertoli nuclear staining, which was maximum at about stage VII, declined rapidly at stage VIII, and then increased gradually in stages I to VI (Fig. 4Go). The signal obtained with the anti-AR antibody was specific, as the Sertoli cells that stained positive with this antibody were at the same stages that have previously been reported to be AR+ (34, 35). Furthermore, testes sections incubated with only the secondary antibody showed no staining (data not shown). Staining of Leydig and peritubular cell nuclei was also observed with the AR antibody. Staining of the acrosome of spermatids and the residual bodies was deduced to be nonspecific because it was not observed uniformly across the slide and on all slides. We did not detect any specific staining of elongating spermatid nuclei and the cytoplasm of elongated spermatids, as observed by some workers (35) but not by others (34).



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  		Figure 4. Immunohistochemical analysis of AR in testes from control and irradiated rats. A, Normal, control LBNF1 rat. Approximate stage of tubules is indicated by the Roman numerals. B, Irradiated LBNF1 rat, 14 weeks after 6 Gy irradiation. S, Moderately and strongly stained Sertoli cells; L, Leydig cells; ImL, immature Leydig cell in peritubular region; P, peritubular myoid cell.

 
In irradiated rats, all of the Sertoli cells showed a uniform level of staining, intermediate between the maximal staining observed at stage VII in control rats and the undetectable levels at stages IX-XIV. Leydig cells, especially the immature ones developing around the tubules (Russell, L. D., and M. L. Meistrich, unpublished observations), displayed increased levels of anti-AR immunoreactivity after irradiation. Peritubular cells, as well as blood vessel cells, were strongly stained in both control and irradiated rats. In irradiated rats, it was difficult to identify all of the undifferentiated A spermatogonia in these formalin-fixed, paraffin-embedded tissues, but of the ones that were identified, none showed the presence of AR.

We conclude that Sertoli cells in irradiated rats possess AR and hence are most likely capable of responding to stimulation by the high levels of androgen present in the irradiated testes (Table 2Go). Thus, the dramatic decline in Pem Pp mRNA levels in response to irradiation does not result from a lack of androgen receptors in Sertoli cells.

Pem is predominantly expressed in Sertoli cells in rat testes
The observation that Pem Pp transcripts are dramatically down-regulated in response to irradiation suggests that the Pp promoter is tightly regulated in rat Sertoli cells by one or more irradiation-sensitive factors. However, this interpretation is tempered by the fact that our previous studies had only shown that Pem protein and Pp transcripts are restricted to Sertoli cells in the mouse (8, 12); this had not been determined in the rat. Thus, it was critical to determine whether Pp transcripts are also Sertoli specific in the rat.

We determined the cellular source of Pem by RPA of fractionated testicular cell RNA. Pem mRNA, rather than Pem protein, was assessed for several reasons: 1) Pem protein could be translated from either Pp or Pd transcripts, and thus only RNA analysis permitted us to distinguish between expression from the two promoters. 2) Pem is expressed at lower levels (at least 10-fold lower) in the rat compared with the mouse testis (12) and thus its expression in rat testis may be insufficient to permit detection of rat Pem protein. 3) The only anti-Pem antibody available is against mouse Pem (12, 36), which we find only weakly binds to rat Pem.

Initially we analyzed preparations of tubular and interstitial cells, isolated either mechanically or enzymatically. The results of both methods (data not shown) demonstrated that Pem Pp expression was approximately 5-fold higher (relative to GAPDH) in the tubular preparations than in the interstitial cell preparations, which, based on the purities of the preparations, is consistent with Pem Pp transcription occurring exclusively in the tubular compartment. In contrast, there were 2.5 times more Pem Pd transcripts per µg RNA in the interstitial than in the tubular cell fraction (data not shown).

To determine the cell types that express Pem Pp and Pd transcripts within the seminiferous tubules, purified rat Sertoli cells and rat germ-cell fractions were analyzed by RPA. We found that purified Sertoli cells had about 50-fold higher levels of Pp transcripts than total testis (Table 3Go). In contrast, the enriched round and elongated spermatid fractions had less Pp transcripts than total testis. Enriched pachytene spermatocytes had levels of Pp transcripts similar to total testis, but this is probably a result of contamination of this fraction with Sertoli cells (Table 1Go). Pem Pd transcripts exhibited a pattern of expression reciprocal to that of Pp transcripts. Pd transcripts were present at similar levels in all fractions except for the Sertoli fraction, where its expression was undetectable.


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  		Table 3. Relative levels of mRNA species in different cell types per µg of RNA loaded and normalized to 1.0 for total testis

 
For comparison, we examined the levels of SGP1, transferrin, and hemiferrin mRNA. Similar to Pem, the Sertoli-specific gene SGP1 was expressed at high levels in the Sertoli fraction, at modest levels in the pachytene spermatocyte fraction contaminated with Sertoli cells, and at trace levels in the spermatid fractions (Table 3Go). Transferrin mRNA, which is known to be expressed by both Sertoli and Leydig cells (37), was indeed expressed at high levels in both of these fractions. Hemiferrin mRNA, which is known to be maximally expressed by round spermatids (25), was indeed expressed at the highest level in this fraction and was almost undetectable in the Sertoli fraction. These results provide molecular evidence supporting the morphological characterization of the fractions and enable us to conclude that Sertoli cells transcribe the Pem Pp, whereas both germ cells and interstitial cells transcribe the Pem Pd.

Decrease of Pem Pp gene expression in mouse testis in response to irradiation
To determine whether the irradiation-induced decrease in Pem Pp expression in LBNF1 rat testis is a response specific to this rat model system, we examined the effect of irradiation on testicular Pem mRNA levels in mice (C3H strain). Mouse testes are more radiation resistant than rat testes (38). Irradiation of mice with doses of 6 Gy (Table 4Go) or 10 Gy (14) only transiently inhibits spermatogenesis, whereas such doses cause spermatogenesis to irreversibly fail in the rat, despite the survival of the spermatogonial stem cells (1). Higher doses of irradiation (over 20 Gy) permanently block spermatogenesis, even in mice, as a result of stem-cell death (39).


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  		Table 4. Testis weight and germ cell loss after irradiation of mice; comparison with rat

 
The levels of Pem mRNA in mouse testes after different doses of irradiation were analyzed by RPA (Fig. 5Go). In control (nonirradiated) mice, we could detect only Pp transcripts and not Pd transcripts, consistent with our past studies (12). Pem Pp mRNA levels per testis decreased about 10-fold after high doses of irradiation (14 and 24 Gy), but exhibited only modest reductions (less than 2-fold) after lower doses (6 and 10 Gy) (Fig. 6AGo). It should be noted that the maintenance of Pem Pp levels per testis at 8 weeks after 10 Gy, when germ cell number is drastically reduced, supports our previous observation that Pem is strictly localized to a nongerminal cell type, namely the Sertoli cell (12).



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  		Figure 5. Expression of Pem mRNA in normal and irradiated mouse testes. Pem mRNA levels in nonirradiated mouse testes and testes from mice at 8 and 20 weeks after 6 Gy and at 8 weeks after 10, 14, and 24 Gy of irradiation as determined by RPA. GAPDH was also measured for comparison. Forty micrograms of RNA were annealed per lane.

 


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  		Figure 6. Pem Pp mRNA levels are selectively decreased in mice testes in response to high doses of irradiation. All data shown are from mice killed at 8 weeks after irradiation. Values for mice irradiated with 6 and 22 Gy and killed at 20 weeks after irradiation did not differ significantly from the values at 8 weeks. A, Relative mRNA levels based on values generated from RPA. B, Relative mRNA levels based on values generated by Northern blot analysis.

 
This reduction in Pem Pp mRNA levels was selective, as RPA analysis showed that the somatic form of GAPDH decreased only 2-fold, even after 24 Gy. Furthermore, Northern blot analysis showed that SGP1, CREB, and GATA-1 transcripts exhibited little or no decline at doses up to 14 Gy irradiation (Figs. 6BGo and 7Go). Only the highest dose of irradiation (24 Gy) modestly reduced the levels of these four transcripts (by less than 2-fold), but the decline was much less marked than that observed for Pem Pp. In contrast, we found that the transcript encoding the nuclear receptor transcription factor RXRß was reduced, even at low doses of irradiation. Although one report indicates that RXRß mRNA is expressed exclusively by Sertoli cells in mice testes (40), another indicates that it is present in germ and Leydig cells, in addition to Sertoli cells (41). Thus, we do not know whether the decline in RXRß mRNA levels after irradiation results from a bona fide decrease in its expression in Sertoli cells or from a loss of germ cells.



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  		Figure 7. Levels of expression of RXRß, SGP1, CREB, and GATA-1 transcripts in response to irradiation of mice. Northern blot analysis of control and irradiated mouse testes was performed with 10 µg of total cellular RNA.

 
We conclude that irradiation selectively decreases the expression of the Pem gene in mouse testis, whereas SGP1, CREB, GATA-1, and GAPDH gene expression were maintained at all but the highest dose of irradiation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this paper, we report the expression of genes in somatic testicular cells after irradiation in vivo. We identified one gene, Pem, that is specifically down-regulated from its male organ-specific proximal promoter (Pp) in response to irradiation. The expression of all other Sertoli transcripts and gene products that we tested, including SGP1, CREB, AR, GATA-1, and FSHR, were, at most, only slightly affected by irradiation, and transferrin mRNA levels were dramatically increased after irradiation.

In many respects, the expression pattern of Pem from its male organ-specific promoter is conserved between mice and rats. In both species of rodents, the Pp is exclusively expressed in Sertoli cells in the testis and somatic cells in the epididymis. Its expression in both of these organs is exquisitely androgen dependent, as demonstrated in hypophysectomized mice and rats, as well as mice mutant for AR or GnRH (7, 8, 11, 12). Here, we show that the Pem Pp was the most irradiation sensitive of all of the Sertoli gene promoters that we tested in both mice and rats.

However, in other respects, Pem displays differences in expression between mice and rats. The basal level of Pp mRNA expression is much higher in mouse than in rat testis. Furthermore, its sensitivity to radiation is much greater in the rat than in the mouse. This latter observation correlates with the greater sensitivity of the rat testis to radiation-induced inhibition of spermatogonial differentiation, but as will be discussed below, the loss of Pem Pp expression is not the primary cause of failure of spermatogonial differentiation, although it is possible that it plays a contributory role.

The down-regulation of Pem Pp transcription in Sertoli cells could either be a direct effect of radiation or an indirect effect of hormonal changes or the subsequent loss of germ cells. We believe that it is unlikely that Pem expression is eliminated because of insufficient hormonal stimulation of Sertoli cells. First, both FSH and intratesticular testosterone levels are high after rat irradiation (Table 2Go). Second, irradiation does not abolish AR expression in Sertoli cells (Fig. 4Go). Third, the levels of FSHR mRNA per Sertoli cell were not affected by irradiation (Fig. 2Go) and thus FSHR protein is probably also present. Although we have no proof as to whether the transcriptional coactivators necessary for expression of androgen-responsive genes are present or whether the signal transduction pathway downstream of FSHR are intact, the presence of the ligand and receptors indicate that at least these components of the hormone response machinery of the Sertoli cell are intact. Because AR is positively regulated by androgen (42), the persistence of AR in the face of irradiation suggests that the AR pathway is active in Sertoli cells from irradiated rats.

The time course of Pem Pp mRNA decline in Sertoli cells after rat irradiation is consistent with the possibility that germ-cell loss is responsible for the decline. We found that the level of Pp transcripts declined 2 weeks after 6 Gy of irradiation and was undetectable at 6 weeks, paralleling the loss of testis weight (Table 2Go). There is indeed precedent for positive regulation by germ cells of Sertoli cell gene products, such as inhibin-{alpha} (43), interleukins (44), and transferrin (45). In contrast, in mice there was no marked decline in Pem Pp transcripts at 8 weeks after 6 or 10 Gy irradiation, despite the absence of most of the germ cells at this time (Table 4Go). Such findings support the notion of direct dose-dependent radiation effect on Pem gene expression in mouse Sertoli cells. This germ cell-independent regulation of the Pp in irradiated mice is consistent with Pem’s normal expression in WVWV germ cell-deficient mice (8).

Irrespective of whether irradiation directly or indirectly regulates Pem Pp mRNA levels, it is likely to result from either the down-regulation of a factor required for Pp transcription or the induction of a repressor that inhibits Pp transcription. To identify the cis elements responsible for Pp regulation, we have recently generated transgenic mice containing the Pp transcription initiation site and upstream sequences. This has revealed that 0.6 kb sequences upstream from the Pp transcriptional start site are sufficient for Sertoli-specific expression in the mouse testis. We do not yet know if these sequences are sufficient to confer the down-regulation observed with the endogenous gene in response to androgen, radiation, or both.

In contrast to genes such as SGP1, FSHR, CREB, GATA-1, and GAPDH, whose levels were relatively constant in irradiated testes, transferrin exhibited a dramatic increase in levels in response to irradiation and/or germ cell depletion (Fig. 2Go, B and D). This might be a compensatory response to provide iron to irradiation-damaged testes. Our results are in agreement with several other studies that have examined the effect of toxic agents on transferrin gene expression, although the level of induction that we observed was much more marked than that observed in other studies. One study (37) showed a modest approximately 50% increase in transferrin mRNA levels in rats after partial depletion of germ cells with methoxyacetic acid. The reason transferrin mRNA levels were increased more dramatically in our study is not known. There were several differences between the two studies, including the fact that germ-cell depletion was more complete in our study (in response to irradiation) than in response to methoxyacetic acid. An issue of future interest is to determine whether the increase in transferrin mRNA levels is in Sertoli cells, Leydig cells, or both. Recently, it was shown that transferrin protein levels are up-regulated by irradiation in rat Sertoli cells in vitro (5), suggesting that at least one target of irradiation is the Sertoli cell.

Because Pem has evolved a unique promoter regulated in a stage-specific manner in particular somatic cell types in the testis and epididymis from both mouse and rat, it may well function in both of these male reproductive organs. However, the observation that mice deficient for the Pem gene are capable of normal spermatogenesis indicates that Pem is not essential for this process, at least in the mouse (13), and that other genes may substitute for Pem’s function in Pem null mutant mice. If so, elucidating the function of Pem requires generating compound-knockout mice defective both in Pem and genes that participate with Pem. Another possibility is that Pem plays a context-specific role in male reproductive organs, which is not evident in mice under typical laboratory conditions. For example, Pem may play a role in responses to temperature shock, hypoxia, or oxidative stress. In this regard, it is of interest that Pem has a conserved redox domain (CPAC) known to function as the catalytic center of several redox enzymes (46, 47). Recently, Pem was shown to be a potent regulator of endodermal differentiation (48). Thus, by analogy, Pem may regulate the differentiation of Sertoli cells in either a redundant or context-specific manner.

Because Pem is not essential for spermatogenesis in normal mice, its depletion in irradiated rats and mice is probably not responsible for the defect in sperm production in irradiated animals. Instead the irradiation-imposed block of Pem expression in Sertoli cells may be part of a cascade of events caused by irradiation that is downstream from the critical event inhibiting spermatogonial differentiation. Mice and LBNF1 rats differ considerably in their sensitivity to irradiation-induced blockade in spermatogenesis. Although mice initially experience a dramatic decline in spermatogenesis after irradiation, spermatogenesis is eventually restored in most mice strains, even at doses (10 Gy) that exceed those required to irreversibly block LBNF1 rat spermatogenesis (49). Even doses as high as 16 Gy do not block spermatogonial differentiation, although the efficiency of differentiated cell production from surviving stem cells is compromised (38). In C3H mice, 12 Gy irradiation permitted only 10% recovery of sperm production, as compared with 80% recovery after 6 Gy irradiation (39). This is the same dose range in which the decline of Pem Pp mRNA levels is observed in the mouse (Fig. 6Go). Although the functional consequences of this decline in Pem expression is not known, these observations suggests that Pem expression may be a marker of irradiation-altered Sertoli cell function.

In conclusion, Pem mRNA transcribed from the male organ-specific Pp promoter is selectively down-regulated after irradiation. The levels per testis of other Sertoli cell-specific or enriched transcripts, such as SGP1, FSHR, GATA-1, and CREB remained nearly constant after irradiation. However, we believe that other Sertoli genes, including ones essential for spermatogonial differentiation in the LBNF1 rat, will be affected by radiation in the same manner as is Pem. Once these genes and their products are identified, it will be possible to discern how they interact to dictate spermatogenesis arrest in response to irradiation in an androgen-dependent manner.


   Acknowledgments
 
We thank Dr. Ilpo Huhtaniemi for performing the FSH measurements, Dr. Ming Zhao for assistance with the densitometry, Yun Zhang for performing germ and interstitial cell separations, and Drs. Michael D. Griswold and Walter Tribley and Jeong S. Kim for performing the northern blotting of FSHR. We thank Walter Pagel for editorial assistance.


   Footnotes
 
1 This work was supported by NIH Grants ES-08075 (to M.L.M.), CA-78023 (to M.W.), DK-03892 (to M.M.), HD-29428 (to P.L.M.), and Core Grant CA-16672. Back

Received September 27, 2000.


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Top
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 Introduction
 Materials and Methods
 Results
 Discussion
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