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Keywords:

  • ionizing radiation;
  • rat strains;
  • spermatogenesis;
  • spermatogonia

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. Author contributions
  10. References

Recently, we reported large differences between rat strains in spermatogenesis recovery at 10 weeks after 5-Gy irradiation suggesting that there are interstrain as well as interspecies differences in testicular radiation response. To determine whether these interstrain differences in sensitivity might be a result of the particular dose and time-point chosen, we performed dose–response and time–course studies on sensitive Brown-Norway (BN) and more resistant spontaneously hypertensive rats (SHR) and Sprague–Dawley (SD) rats. Type A spermatogonia were observed in atrophic tubules at 10 weeks after irradiation in all strains indicating that tubular atrophy was caused by a block in their differentiation, but the doses to produce the block ranged from 4.0 Gy in BN to 10 Gy in SD rats. Although the numbers of type A spermatogonial were unaffected at doses below 6 Gy, higher doses reduced their number, indicating that stem cell killing also contributed to the failure of recovery. After 10 weeks, there was no further recovery and even a decline in spermatogonial differentiation in BN rats, but in SHR rats, sperm production returned to control levels by 20 weeks after 5.0 Gy and, after 7.5 Gy, differentiation resumed in 60% of tubules by 30 weeks. Suppression of testosterone and gonadotropins after irradiation restored production of differentiated cells in nearly all tubules in BN rats and in all tubules in SHR rats. Thus, the differences in recovery of spermatogenesis between strains were a result of both quantitative differences in their sensitivities to a radiation-induced, hormone-dependent block of spermatogonial differentiation and qualitative interstrain differences in the progression of post-irradiation recovery. The progression of recovery in SHR rats was similar to the prolonged delays in recovery of human spermatogenesis after cytotoxic agent exposure and thus may be a system for investigating a phenomenon also observed in men.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. Author contributions
  10. References

The mammalian testis is sensitive to ionizing radiation: low doses can temporarily reduce sperm production, moderate doses can cause prolonged reductions in sperm count, and high doses can result in permanent azoospermia. In humans, the testis appears more sensitive and exhibits longer delays before spermatogenesis recovers than in most rodent models. Single doses as low as 0.15 Gy cause temporary reductions in spermatogonial numbers and sperm count that can last as long as 6 months (Paulsen, 1973; Rowley et al., 1974; Clifton & Bremner, 1983). Higher doses can produce azoospermia that lasts from about 8 months after 0.5 Gy to about 2 years after 6 Gy, and it then takes several years for sperm production to return to normal. The delays indicate that there are surviving spermatogonial stem cells that are blocked at some point in their differentiation, but the mechanisms of the block and subsequent recovery of spermatogenesis in human are not known. The fractionated radiation therapy used in cancer treatment is more toxic to the testis than the single doses (Meistrich & Beek, 1990) and can result in no recovery of spermatogenesis occurs even 5 years after treatment (Sandeman, 1966; Speiser et al., 1973; Hahn et al., 1982), suggesting that all stem cells may have been killed.

An animal model that simulates the response of the human testis to radiation is needed to improve our understanding of this process. Non-human primates (macaques) show the most similarities to human including the histological types of spermatogonia (Ehmcke & Schlatt, 2006) and drastic declines in spermatogonial numbers and sperm count after 2 or 4 Gy lasting 6 months before recovery begins, and incomplete recovery even after 18 months (van Alphen et al., 1988; Foppiani et al., 1999; Kamischke et al., 2003). However, studies on primates are limited by theirs cost and lack of genetic tools. Rodent models are inexpensive, have more detailed literature, inbred lines and genetic tools, and are most amenable to laboratory studies, but they have not so far shown the delayed recovery phenomenon.

The recovery from toxic effects of radiation in mice is much more rapid and robust than in humans. The stem spermatogonia surviving irradiation begin to differentiate almost immediately after doses even as high as 6 Gy and restore spermatogonial numbers to control levels after only 2 weeks (Erickson & Hall, 1983). Sperm production begins to increase within 7 weeks after 2 Gy and within 11 weeks after 6–12 Gy and reaches 60% of control values within 23 weeks after 6 Gy (Searle & Beechey, 1974; Meistrich et al., 1978; Meistrich & Samuels, 1985). Killing of stem spermatogonia first becomes significant at a dose of 4 Gy (de Ruiter-Bootsma et al., 1976; Erickson, 1981), and their numbers are further reduced with higher doses, with 9 Gy resulting in only 25% of tubules recovering production of differentiated cells within 5 weeks (Withers et al., 1974; Lu et al., 1980). As a negligible number of the atrophic tubules contain spermatogonia (Kangasniemi et al., 1996a), these atrophic tubules are primarily owing to stem cell killing and not a block in spermatogonial differentiation, although there is some reduction in the yield of later differentiated cells after high doses (van den Aardweg et al., 1983).

The rat testis appears somewhat more sensitive to damage produced by irradiation than the mouse and shows less recovery. However in Sprague–Dawley (SD) rats, the most widely studied and most resistant strain, spermatogonial numbers recovered after 3 Gy to control levels within 5 weeks (Dym & Clermont, 1970), and epididymal sperm counts to 40% of control after 19 weeks (Jégou et al., 1991). However, after 6 Gy, recovery was far from complete at 16 weeks, as testis weights were only 52% of control and 44% of the tubules had incomplete spermatogenesis (Erickson & Hall, 1983). Following 9 Gy of radiation, less than 10% of tubules showed differentiating cells at 8 weeks (Delic et al., 1986), and not until 26 weeks did sperm production reach 10% of control (Pinon-Lataillade et al., 1991). Other strains of rats, such as LBNF1 [F1 hybrids of Lewis and Brown-Norway (BN)], were much more sensitive and, despite the survival and maintenance of stem spermatogonia, the testis showed progressive failure of recovery (Kangasniemi et al., 1996b; Shetty et al., 2000). Although some recovery of differentiated cells was transiently observed at 6 weeks after irradiation, this declined progressively to zero at 60 weeks after 3.5 Gy and by 10 weeks after 5 Gy, thus indicating a permanent failure of spermatogenic recovery. The only other rats previously studied, various Wistar substrains, did show some recovery after 5 Gy, but to levels below that of SD (Delic et al., 1986, 1987).

To systematically characterize these strain differences, we directly compared the recovery of spermatogenesis at 10 weeks after 5-Gy irradiation in seven rat strains and observed dramatic differences (Abuelhija et al., 2012). There was no recovery of differentiating germ cells in the Lewis and BN stains despite the presence of type A spermatogonia in many tubules. Thus, they showed the complete block in spermatogonial differentiation as had been previously observed in the LBNF1 hybrids (Kangasniemi et al., 1996b). In contrast, in two Wistar-derived inbred strains, Wistar–Kyoto and spontaneously hypertensive rats (SHR), recovery of spermatogenesis was observed in 55 and 94% of the tubules respectively. Sperm production was still markedly reduced, as it was only 3% of control levels in both Wistar strains. SD rats showed the best recovery of spermatogenesis, as 98% of tubules showed recovery and sperm production was 6% of controls. Nevertheless, the atrophic tubules in all strains contained type A spermatogonia, indicating that the tubular atrophy observed after 5 Gy was due primarily to a block in spermatogonial differentiation and not stem cell killing.

However, questions remain regarding the differences in sensitivity between strains. For example, is not known whether the different strains would show the same qualitative patterns of recovery, but differ in quantitative doses to produce blocks in recovery, and could we find a strain and dose that results in recovery after a delay. Furthermore, the role of testosterone in blocking spermatogenic recovery in different strains needs to be investigated to determine whether the recovery in the resistant strains is a result of their insensitivity to the action of testosterone, which we have previously shown is responsible for the block in spermatogonial differentiation in LBNF1 rats (Shetty et al., 2000). In addition, we must determine whether the presence of tubules at different stages of differentiation at 10 weeks after irradiation represented a block at a later stage of differentiation or just a delay in initiation of differentiation. Finally, we wanted to identify a strain showing some characteristics of the transient block in spermatogenic cell differentiation and the delayed recovery process observed in human testes.

To address these questions, we performed a dose–response, time–course and hormone–effect study of the recovery of spermatogenesis after irradiation in three strains of rats. We performed all studies comparing BN and SHR as these strains are most amenable to future genetic studies as recombinant inbred rats between these two strains are already available to identify quantitative trait loci responsible for the differences in radiation sensitivity (Tabakoff et al., 2009) and their genomes have been sequenced (Gibbs et al., 2004; Atanur et al., 2010). In addition, dose-response studies were performed on SD rats as this strain is the most resistant and is most widely used in toxicological studies.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. Author contributions
  10. References

Animals and irradiation exposure

Brown-Norway (BN/SsNHsd) and Sprague–Dawley (Hsd:Sprague–Dawley SD) rats were obtained from Harlan Laboratories; SHR (SHR/NCrl) rats were obtained from Charles River Laboratories. We obtained the rats at 7 weeks of age and allowed them to acclimatize in our facility for 1 week prior to use. Rats were housed under standard lighting (12 h light, 12 h dark) and were given food and water ad libitum. All procedures were approved by the University of Texas MD Anderson Cancer Center Institutional Animal Care and Use Committee.

Rats were irradiated as described previously (Shetty et al., 2000). Briefly, they were anaesthetized and affixed to an acrylic board with surgical tape; then the lower part of the body was irradiated by a 60Co gamma ray unit (Eldorado 8; Atomic Energy Canada Ltd., Ottawa, ON, Canada). The field extended distally from a line about 6 cm above the base of the scrotum. Different doses (2.7 Gy to 12.5 Gy) were given at a dose rate of approximately 1 Gy/min; dose ranges were chosen for each strain based on the sensitivity observed previously (Abuelhija et al., 2012). Testis tissue was harvested at various times between 10 and 40 weeks after irradiation (Table 1). Each dose and time point represents the mean and standard error of between 3 and 10 rats.

Table 1. Radiation doses, analysis times and strains analysed
Rat strainDoses (in Gy) used and time of tissue harvest after irradiation
10 weeks15 weeks20 weeks30 weeks40 weeks
Brown-Norway2.7, 3.0, 3.3, 3.6, 4.0, 4.5, 5.0, 7.5, 10.03.3, 4.0, 4.5, 5.0, 7.5,3.3, 4.0, 7.5 
Spontaneously hypertensive rats4.0, 4.5, 5.0, 5.7, 6.5, 7.5, 10.04.0, 4.5, 5.0, 6.5, 7.56.5, 7.57.57.5
Sprague–Dawley5.0, 6.5, 7.5 8.5, 9.7, 11.0, 12.5   

Hormone treatment

Hormone suppressive treatment was performed with the GnRH antagonist (GnRH-ant) acyline (National Institute of Child Health and Human Development) and the androgen receptor-antagonist flutamide starting immediately after radiation and continuing until tissue harvest, Acyline was dissolved in water and administered as weekly subcutaneous injections of 1.5 mg/kg (Porter et al., 2006). Flutamide was administered by subcutaneous implantation of four 5-cm-long Silastic capsules calculated to deliver 20 mg/kg/day (Porter et al., 2009). Each treatment group (time and dose point) consisted of a minimum of four rats.

Intratesticular interstitial fluid and tissue processing

Rats were killed by an overdose of a ketamine–acepromazine mixture. Each testis was surgically excised and weighed with the tunica albuginea intact. The right testis was fixed overnight in Bouin's fluid.

Interstitial tubule fluid was collected from the left testis as we had performed previously (Abuelhija et al., 2012) using a modification of methods described earlier (Rhenberg, 1993; Porter et al., 2006). Briefly, the testis was suspended by silk sutures and centrifuged for 30 min at 60 × g at 4 °C, and the weight of the fluid collected was determined. The remaining weight of the testis parenchymal tissue was measured after removing the tunica albuginea. The tissue was then homogenized in water for sperm head counts.

Evaluation of spermatogenesis

For histological analysis, the fixed right testis was embedded in glycol methacrylate plastic (JB4; Polysciences Inc., Warrington, PA, USA), and 4 μm sections were cut and stained with periodic-acid Schiff's and hematoxylin. To evaluate the recovery of spermatogenesis from irradiation, we scored a minimum of 200 seminiferous tubules in one section from each animal for the most advanced germ-cell stage present in each tubule. Unless otherwise stated, we computed the tubule differentiation index (TDI), which is the percentage of tubules containing three or more cells that had reached type B spermatogonial stage or later (Meistrich & van Beek, 1993). To obtain a more complete description of the stages of differentiation present in the testis, we also determined the percentages of tubules with three or more cells reaching the leptotene spermatocyte stage or later (TDI-spermatocyte) or the round spermatid stage or later (TDI-spermatids), or with 10 or more cells reaching the elongating or elongated spermatid stage (TDI-late spermatids).

We counted all type A spermatogonia, which includes the stem, chains of undifferentiated and differentiating spermatogonia to type A4 (Chiarini-Garcia et al., 2003) and Sertoli cells, in atrophic seminiferous tubule cross-sections of irradiated rat testes at 1000 × magnification (n = 3–7/group). For samples with almost complete seminiferous tubule atrophy, cells were counted using systematic random sampling (Stereo Investigator version 8.0 software; MicroBrightField, Inc., Williston, VT, USA), by counting A spermatogonia and Sertoli cells in 300 randomly selected 100 μm × 80 μm fields. In samples with few atrophic seminiferous tubules, these tubules were identified visually using light microscopy, and all cells in the tubules were counted. A minimum of 500 Sertoli cells were counted per testis. Results were presented as A spermatogonia per 100 Sertoli cells.

Testicular sperm production was evaluated by counting sonication-resistant sperm heads, which represent nuclei of step 12–19 spermatids, in testicular homogenates. An aliquot of the homogenate of the left testis was sonicated, and the sperm heads were counted in a hemacytometer using phase contrast optics (Meistrich & van Beek, 1993).

Hormone assays

Serum testosterone and interstitial fluid testosterone (IFT) concentrations were measured using a coated-tube radioimmunoassay kit (Coat-A-Count Total Testosterone, Cat No. TKTT1; Siemens, Los Angeles, CA, USA) similar to procedures described previously (Shetty et al., 2000; Porter et al., 2006). Rat serum follicle-stimulating hormone (FSH) was measured by radioimmunoassay, and luteinizing hormone (LH) was measured by a sensitive two-site sandwich immunoassay. Both FSH and LH were measured by the University of Virginia, Center for Research in Reproduction, Ligand Assay and Analysis Core, using previously described methods (Gay et al., 1970).

Statistical analysis

Results were presented as either mean ± SEM calculated from untransformed data or, in the case of sperm head counts, testosterone and LH as the mean ± SEM calculated from log-transformed data obtained from individual rats. The statistical significance of differences between two groups was determined using the t-test with < 0.05 being considered significant.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. Author contributions
  10. References

Spermatogenesis at 10 weeks after irradiation (dose-response)

To identify the doses that induce the declines in spermatogenic recovery and the accumulation of testicular interstitial fluid, which had been correlated with the block to recovery (Porter et al., 2006), BN, SHR and SD rats were given different ranges of doses of radiation depending on the sensitivity of the strain, and tissue was harvested 10 weeks later. Radiation reduced the testicular parenchymal weights in all the strains in a dose-responsive manner, with a steep initial decline, corresponding to the major phase of germ cell loss, followed by a shallower slope reaching 15–20% of control at high doses (Fig. 1A). The steep decline occurred in BN rats at doses below 3 Gy, but 5–6 Gy were required in SHR and SD rats to complete the steep decline.

image

Figure 1. Weights of testis parenchymal tissue and interstitial fluid of BN, SHR and SD rats 10 weeks after irradiation. (A) Testis weights relative to those of unirradiated controls of same strain. Control values were 1.50, 1.27 and 1.66 g for BN, SHR and SD respectively. (B) Increase in interstitial fluid weights from unirradiated control levels.

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As shown previously (Abuelhija et al., 2012), there were large increases (~0.2 g) in testicular interstitial fluid in BN rats at 5 Gy of irradiation but small or negligible increases in SHR and SD rats. In all strains, the increases in interstitial fluid were dose responsive (Fig. 1B). The increase reached a maximum at 3.3 Gy in BN, but 5.7 Gy and 10–11 Gy were required in SHR and SD respectively, to reach maximal levels, which were less than that observed in BN.

To assess whether the radiation-induced decline in spermatogenesis was quantitatively different in the different strains, the dose response of the recovery of spermatogenesis from surviving stem cells was assessed by the percentage of tubules showing differentiated cells (TDI) in histological sections (Fig. 2A) and the numbers of late spermatids produced (Fig. 2B). Control rats showed differentiation in 100% of the tubules and 2 × 108 late spermatids per testis. Both parameters showed dose-responsive declines, with the BN rats being most sensitive to irradiation, SHR showing intermediate sensitivity and SD displaying the most resistance. It was noted that doses that reduced the TDI to between 30 and 60% of control reduced late spermatid counts to between 0.05 and 1% of control.

image

Figure 2. Recovery of spermatogenesis at 10 weeks after various doses of radiation. (A) Tubule differentiation index (TDI), defined as percentage of tubules differentiating to the B spermatogonial stage or beyond. (B) Testicular sperm production: numbers of sonication-resistant late spermatids per testis. The dashed lines indicate the control values.

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The atrophic tubules showing no differentiated cells at 10 weeks after irradiation were then examined to determine whether the absence of differentiated cells was a consequence of killing of all stem spermatogonia or a block in spermatogonial differentiation. In the sensitive BN strain, atrophic tubules were observed at all doses tested, but in the resistant strains they could only be observed after 5.0 Gy in SHR and after 6.5 Gy in SD rats. At the lower doses of radiation, atrophic tubules in all the strains contained between 2.5 and 2.8 type A spermatogonia per 100 Sertoli cells (Fig. 3A). Increasing the radiation exposure produced a dose-responsive reduction in the numbers of type A spermatogonia in the three strains resulting in about 0.5 type A spermatogonia per 100 Sertoli cells after 10 Gy of irradiation. This reduction suggests that stem cells were killed at these higher doses of radiation. However, the presence of some type A spermatogonia in atrophic tubules demonstrated that a block in spermatogonial differentiation (Meistrich & Shetty, 2003) also contributed to the failure of spermatogenesis to recover.

image

Figure 3. Numbers of type A spermatogonia per 100 Sertoli cells in non-repopulating tubules of (A) BN, SHR and SD rats 10 weeks after irradiation (dose response), and (B) BN and SHR rats at longer periods of time after different doses of irradiation (time course).

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Recovery of spermatogenesis after irradiation (time course)

To identify strains with permanent or reversible blocks in spermatogonial differentiation, we examined recovery at times longer than 10 weeks. In BN rats, there was no significant histological recovery of spermatogonial differentiation between 10 and 20 weeks after irradiation with the doses (≥3.3 Gy) that were tested (Fig. 4C). The numbers of late spermatids remained low (≤105) (Fig. 4E); values in the 104–105 range were occasionally observed despite the lack of histological evidence of differentiation in the testis and may have represented sperm heads retained in the testis. The lack of recovery can be attributed to a continued block in spermatogonial differentiation and not a loss of stem cells, as the numbers of type A spermatogonia did not show any decrease between weeks 10 and 20 (Fig. 3B).

image

Figure 4. Time courses of changes in (A, B) absolute testis weights, (C, D) tubule differentiation indices and (E, F) sperm head counts of BN (A, C, E) and SHR (B, D, F) rats after different doses of radiation. The dashed lines indicate the control values. (*) indicates significantly different from value at 10 weeks (< 0.05, t-test).

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In contrast, SHR rats showed progressive recovery at all doses up to 7.5 Gy. With doses up to 5 Gy, sperm production approached control levels by 15 weeks after irradiation (Fig. 4F). After higher doses, the percentage of tubules with differentiated cells was less than 7% at 10 weeks after irradiation, but steadily increased reaching 60% by 20 weeks after 6.5 Gy and by 30 weeks after 7.5 Gy (Fig. 4D). Although most tubules showed differentiation, it was generally only to the B spermatogonial or spermatocyte stages (Fig. 5). After 6.5 Gy appreciable differentiation to the round or late spermatid stages was observed in only one rat at 15 weeks (out of four examined) and one at 20 weeks (out of five). After 7.5 Gy, few tubules progressed to the round spermatid stage and almost none to the late spermatid stage, indicating a block at a later stage of differentiation. Sperm production measured in the contralateral testis, surprisingly, appeared to slightly increase at 15 weeks after 7.5 Gy but then remained low at later times (Fig. 4F). Furthermore, after 7.5 Gy (Fig. 5B), there was no further histological recovery of spermatogenesis between 30 and 40 weeks.

image

Figure 5. Recovery of progression of spermatogenesis as measured by the percentage of tubules with morphologically differentiated cells reaching indicated stage of differentiation or beyond for SHR rats at various times after (A) 6.5 Gy or (B) 7.5 Gy.

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Hormone analyses

To determine whether differences in testosterone or FSH levels might be related to the differential induction of the block in spermatogonial differentiation in the strains, hormone analyses were performed on the three strains of rats before and after irradiation. Serum testosterone showed a modest trend towards reduction at 10 weeks after irradiation in all strains (Fig. 6A), but this was only significant in SHR and SD rats. IFT levels were unaffected by the radiation in all three strains (Fig. 6B). Serum FSH levels significantly increased by about twofold 10 weeks after radiation in all strains (Fig. 6C) as expected owing to the germ cell loss that occurs. LH levels (data not shown) also appeared to be elevated by irradiation.

image

Figure 6. Hormones levels in BN, SHR and SD rats measured 10 weeks after different doses of radiation. (A) Serum testosterone. (B) Intratesticular fluid testosterone. (C) Serum FSH. * indicates values in SHR are significantly different from those in BN. # indicates values in SHR are significantly different from those in SD. $ indicates values in SD are significantly different from those in BN (< 0.05, t-test).

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The levels of serum testosterone, IFT and serum FSH levels in SHR rats were significantly higher than the corresponding values in BN rats both in unirradiated rats and after nearly all dose (Fig. 6) and time points (Fig. 7). The values in SD rats were generally intermediate between those of the other two strains (Fig. 6A–C). Although testosterone and FSH were previously shown to contribute to the spermatogonial differentiation block in LBNF1 rats (Shetty et al., 2006), the greater sensitivity of BN rats than of SHR or SD to induction of a spermatogonial block by radiation cannot be attributed to higher levels of testosterone or FSH.

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Figure 7. Hormone levels in BN and SHR rats without hormone suppression (filled symbols) and after hormone suppression (open symbols) at different times after 7.5-Gy irradiation. (A) Serum testosterone. (B) Intratesticular fluid testosterone. (C) Serum FSH. LOD indicates limit of detection of the assay, (L) Indicates undetectable values of some, but not all samples in the group.

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Suppression of hormone levels and spermatogenesis recovery

To confirm that the action of testosterone and/or FSH was involved in the radiation-induced block of spermatogonial differentiation in these strains, we examined the effects of hormone suppression on spermatogenic recovery in BN and SHR rats at different times after 7.5 Gy (Fig. 7) and at 10 weeks after 5 and 10 Gy (data not shown). Hormone suppression decreased serum testosterone to below the limits of detection in both strains (Fig. 7A). IFT levels were reduced in BN rats to ~1 ng/mL and were reduced even more in SHR rats (Fig. 7B). However, these residual levels of intratesticular testosterone would not be expected to have significant effects on spermatogenesis because the rats were also treated with flutamide. The suppressive treatment also markedly reduced serum FSH levels to about 1 ng/mL in all groups of rats (Fig. 7C) and reduced LH to undetectable levels (data not shown).

Although hormonal suppression in control and treated rats markedly decreased testicular parenchymal weights to about 7% of control in both strains at the various dose and time points (Fig 8A,D), which was also evident by the decrease in tubule diameter (compare Fig. 9A and C), it induced differentiation in a high percentage of tubules in irradiated rats of both strains (Figs 8B and 9C). In BN rats, irradiation with 5 Gy and above almost completely eliminated the differentiating spermatogenic cells (TDI < 2%); nevertheless, hormone suppression starting immediately after irradiation with 5 Gy restored the production of differentiated cells in 100% of tubules; however, with the low testosterone and FSH levels, differentiation could only proceed to the spermatocyte stage (Fig. 9D). There was incomplete recovery of spermatogonial differentiation at 10 weeks after 7.5 Gy, as only 88% of tubules showed differentiating cells, but recovery progressed with time so that by week 20 100% of tubules were differentiated (Fig. 8E). The higher dose of 10 Gy reduced the percentage of tubules showing differentiation at 10 weeks to 48%. In SHR rats, after the 7.5- and 10-Gy doses, which blocked all spontaneous recovery at 10 weeks after irradiation, hormonal suppression stimulated the production of differentiated cells in 100 and 90% of tubules respectively.

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Figure 8. Dose response and time course of changes in testis weights, differentiation in tubules and interstitial fluid in BN and SHR rats without hormone suppression (filled symbols) and after hormone suppression (open symbols). (A, D) Testis weights relative to unirradiated controls of same strain. (B, E) Percentage of tubules with differentiated cells. (C, F) Change in interstitial fluid weights from unirradiated control levels.

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image

Figure 9. Histology of BN rat testes 10 weeks after irradiation with 7.5 Gy without (A, B) or with (C, D) hormone suppression. (A) Irradiation produced atrophic tubules and interstitial oedema. (B) Most tubules contained only Sertoli cells (SC), but some contained a few type A spermatogonia (Spg). (C) Hormone suppression after irradiation induced recovery of spermatogenesis in nearly all tubules, except those marked with. (X). (D) The recovering tubules showed development to only the pachytene spermatocyte stage (p). (A, C) bar: 100 μm, (B, D) bar: 10 μm.

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Hormone suppression completely reversed the large increase in interstitial fluid accumulation observed in BN rats (Fig 8C,F). The modest increases in interstitial fluid observed in SHR rats after irradiation were also reversed by the hormone suppression.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. Author contributions
  10. References

The human testis is characterized by high sensitivity to and delayed recovery of spermatogenesis after moderate doses of radiation. Here, we compared three rat strains and found that the BN rats were also very sensitive to the gonadotoxic effects of radiation, but they showed no recovery of spermatogenesis. SD rats displayed the most resistance to radiation as high doses were required to produce severe gonadotoxic effects. However, SHR rats showed marked and prolonged gonadotoxic effects to doses of about 6 Gy, and may indicate that Wistar-derived rats might have some of the sensitivity characteristics similar to human testes.

This study clarifies the question of whether the data of our previous study (Abuelhija et al., 2012), showing that at 10 weeks after 5-Gy irradiation there was no recovery of spermatogenesis in BN rats, whereas in SHR and SD rats nearly all of the tubules contained differentiating germ cells, are a result of qualitative or quantitative differences between the strains. The dose-response studies showed that at 10 weeks after irradiation, BN rats failed to show recovery even after low doses (4 Gy) (Fig. 2), similar to the sensitivity described previously in LBNF1 rats (Kangasniemi et al., 1996b), which are F1 hybrids of BN and Lewis, another very sensitive strain (Abuelhija et al., 2012). At these low doses, the atrophic tubules are almost exclusively owing to a block in spermatogonial differentiation, as type A spermatogonia were present and their numbers were maintained in the atrophic tubules. In the resistant SHR and SD strains, low to intermediate doses (≤5 Gy) did not produce a significant block in spermatogonial differentiation. However, higher doses of irradiation induced radiation-induced blocks in spermatogonial differentiation, similar to that observed in BN rats at the lower doses, in SHR and SD rats after 6.5 and 8 Gy respectively. Thus, the major contribution to the differences in recovery of spermatogenesis between strains is the quantitative difference in their sensitivities to a radiation-induced block of spermatogonial differentiation.

The time-course studies addressed whether this block was reversible at later times in the different strains. The block was not reversible at all between 10 and 20 weeks in BN rats even at doses as low as 3.3 or 4 Gy (Fig. 4A). Based on results with LBNF1 rats, which were followed for 60 weeks to demonstrate the permanence of the block, we suggest that no recovery will occur with BN rats even after longer periods of time (Kangasniemi et al., 1996b). Furthermore, in LBNF1 rats, the incomplete block produced at 3.5 Gy became even more severe between 10 and 60 weeks, with spermatogonial differentiation steadily declining to a complete block; a similar decline occurred in BN rats after 3.3 Gy (Fig. 4C).

In contrast, in the more resistant SHR rats, doses above 5 Gy were required to produce a block in spermatogonial differentiation at 10 weeks after irradiation. This block that was observed after doses of 6.5 and 7.5 Gy was reversible, as demonstrated by the progressive increase in the number of spermatogonia in the atrophic tubules at 15 and 20 weeks (Fig. 3B), and in differentiating tubules at 20 and 30 weeks after irradiation (Fig. 4D). Thus, there is a qualitative difference between strains, as the more resistant strains, like SHR, showed a delayed but progressive recovery of spermatogonial differentiation, whereas the block in spermatogonial differentiation in the sensitive strains like BN and LBNF1 was permanent.

Although the block in spermatogonial differentiation in SHR rats was reversed at later post-irradiation times, there still was a prolonged decrease in spermatogenesis as exemplified by the reduction in testis weights, later differentiated cells and sperm production at doses >5 Gy (Figs 4B,F & 5). The reversible, but incomplete recovery in the SHR strain appears to be similar to that previously reported in SD rats (see Introduction). In SHR rats, the presence of appreciable numbers of tubules containing B spermatogonia and spermatocytes at 20 weeks after 7.5-Gy irradiation, but almost no spermatids at weeks 30 and 40, demonstrates that the absence of late stage germinal cells is a result of a decreased efficiency or even a block in development to later differentiation steps, and not just a result of the delay in the initiation of spermatogonial differentiation. It is highly unlikely that these differentiated germ cells were arrested in development as the spermatogonia were mitotically active and, when present, the later cells were arranged according to the stages of the cycle of the seminiferous epithelium. We believe that a damaged somatic environment, as previously observed to produce the block in spermatogonial differentiation in LBNF1 rats (Zhang et al., 2007), is unable to properly support spermatogenic cell differentiation. Hence, the recovery observed between 15 and 30 weeks may be owing to restoration of a favorable somatic environment, like that which occurs when hormones are suppressed, but a mechanism for this spontaneous recovery is not known.

As doses of ≥5.7 or 6.5 Gy were necessary to produce a block in spermatogonial differentiation in resistant strains like SHR and SD respectively (Fig. 2A), the possible role of stem cell killing could also be considered as a cause of the atrophic tubules at higher doses. However, as the numbers of type A spermatogonia were still maintained in the atrophic tubules at doses up to 5.7 Gy or 6.5 Gy for SHR and SD, respectively (Fig. 3A), the block in spermatogonial differentiation must be the principal cause of tubular atrophy at these doses. However, at higher doses, there was a decline in the numbers of A spermatogonia (Fig. 3A), suggesting that stem cell killing is also a cause of tubular atrophy, but cannot be the only cause as A spermatogonia were still observed. These results are consistent with direct counts of isolated type A spermatogonia, the putative stem cells, in SD rats, which indicated that although there was a transient loss of these cells after doses as low as 2 Gy, 6 Gy was required to cause a more prolonged loss of the these stem cells for 26 days (Erickson, 1976).

The block in spermatogonial differentiation in irradiated LBNF1 rats was previously shown to be mediated by the action of testosterone and also to some extent by FSH (Shetty et al., 2006). This inhibitory action of the hormones is in contrast with the situation in normal rats, in which spermatogonial differentiation is qualitatively independent of both testosterone and FSH (Huang & Nieschlag, 1986). Here, we show that hormones were also responsible for the spermatogonial block in BN rats, as the production of differentiated cells in all tubules could be restored by hormone suppression for 10 weeks after 5-Gy irradiation (Fig. 8B). The lack of sensitivity of SHR or SD rats to the radiation-induced block in spermatogonial differentiation at 5 Gy cannot be a result of lower levels of testosterone and FSH as irradiated rats of these strains actually had higher levels of these hormones than did BN (Fig. 6), or of the absence of the hormone dependence of the block in spermatogonial differentiation, which was demonstrated in 7.5-Gy irradiated SHR rats (Fig. 8B). Thus, the differences between the sensitive and resistant strains appear to be a result of differences in the dose required to render the testis sensitive to this block of spermatogonial differentiation.

Another factor that may be involved in this block in spermatogonial differentiation appears to be the accumulation of testicular interstitial fluid as irradiation of LBNF1 rats dramatically increased testicular interstitial fluid at the time the block in spermatogonial differentiation occurred and hormonal treatments to restore spermatogonial differentiation reduced interstitial fluid (Porter et al., 2006) However, other sensitive (Lewis) or intermediate (Wistar–Kyoto) strains also had low interstitial fluid accumulation, indicating that fluid accumulation could not be the cause of the block in those strains (Abuelhija et al., 2012). In this study, dose responsive increases in interstitial fluid levels (Fig. 1B), although lower in magnitude in SHR and SD than in BN, occurred in all three strains at doses corresponding to the decline in spermatogonial differentiation (Fig. 2A). This result and the reversal of the radiation-induced fluid increase in BN and SHR rats with hormone suppression (Fig. 8C,F) further support a correlation between increases in interstitial fluid and the block in spermatogonial differentiation in these strains.

Finally, the demonstration of a delay in the recovery of spermatogonial differentiation (15–20 weeks) in SHR rats irradiated with 7.5 Gy (Fig. 4D) and an even longer delay in the production of late spermatids in the testis (30–40 weeks) appears to provide a rat model for the prolonged delays in recovery of human spermatogenesis after radiation and other cytotoxic exposures. The relative roles of stem cell renewal/spermatogonial differentiation, which show differences between rodents and primates (Ehmcke & Schlatt, 2006), and the changing ability of the somatic environment to support spermatogenic cell differentiation in the delayed recovery phenomenon is not known. Further studies at longer times are needed in SHR, other Wistar-derived strains or SD rats (Pinon-Lataillade et al., 1991) to determine if the recovery continues to progress and will lead to increases in epididymal sperm counts and to investigate the mechanisms underlying the delay or block.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. Author contributions
  10. References

This work was supported by grants from the National Institutes of Health, USA (ES-008075 to MLM and Cancer Center Support Grant CA-016672 to M.D. Anderson Cancer Center) and the Florence M. Thomas Professorship in Cancer Research (to MLM). We thank Dr. Min S. Lee (Contraception and Reproductive Health Branch, National Institute of Child Health and Human Development) for providing the Acyline and Walter Pagel of M.D. Anderson Cancer Center for editorial assistance.

Author contributions

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. Author contributions
  10. References

MA, GS & MLM, conception and design: MA & CCW, collection of data; MA & MLM, data analysis and manuscript writing.

References

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  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. Author contributions
  10. References
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