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Department of Experimental Radiation Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
Address all correspondence and requests for reprints to: Gunapala Shetty, Department of Experimental Radiation Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030. E-mail: sgunapal{at}mdanderson.org.
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Abstract |
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Introduction |
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In mice, testicular descent occurs at the age of 2–3 wk. The testosterone level in the testis is very low in immature mice around 10–20 d and then gradually rises after testicular descent (7), which correlates with the timing of spermatogenic degeneration in jsd mutant mice. Although it was demonstrated that testosterone inhibits spermatogonial differentiation in these mice, it is not known whether the scrotal environment, with its lower temperature of 32 C compared with the body temperature of 37 C is unfavorable for spermatogonial differentiation. In the present study we surgically cryptorchidized the jsd mutant mice on a mixed C3H-B6 background. We were surprised to find that cryptorchidism maintained and restored spermatogonial differentiation in young and adult mice, respectively.
We analyzed these effects in a similar rat model system to determine whether the results could be generalized. We chose irradiated LBNF1 rats because, like jsd mutant mice, they have a testosterone-dependent block in spermatogonial differentiation (8). We found that cryptorchidism did stimulate spermatogonial differentiation in the irradiated rats, indicating that the mechanism of inhibition of spermatogonial differentiation is similar in the two models.
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Materials and Methods |
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The jsd mutant mice on a hybrid background were initially developed by crossing C3H mice with mutant jsd-carrying B6 mice (3). The mice used for the present study arose from a stock in which these hybrid mice were crossed with another hybrid stock produced by crossing testicular feminized (Tfm) mice on a B6 background (obtained from Jackson Laboratory) with C3H mice; the mice we used lacked the Tfm mutation.
Jsd mice were genotyped by PCR analysis of crude tail DNA from 4- to 5-wk-old animals, as described earlier (3) except that the buffer used for the PCR mixture was modified. Thus, in the 25-µl reaction mixture the concentrations of each of the deoxy-NTPs and MgCl2 (Sigma Chemical Co., St. Louis, MO) were increased to 1 and 4.5 mM, respectively. It also contained 22 mM Tris-HCl, 11 mM ammonium sulfate, 6.7 mM mercaptoethanol (electrophoresis grade; Fisher, Fair Lawn, NJ), 4.5 µM EDTA, and 0.01% BSA (Pharmacia, Piscataway, NJ). The D1Mit 415 and D1Mit 181 loci encoding microsatellite DNA sequences close to the jsd locus were used for screening (5). The genotype in some cases was confirmed using two other closer microsatellite markers, D1Mit 334 and D1Mit 215. In this method the fluorescently labeled PCR products obtained from the above procedure were analyzed using a 3730xL DNA fragment analyzer (Applied Biosystems, Foster City, CA).
Adult LBNF1 (F1 hybrids of Lewis and Brown-Norway) male rats were obtained from Harlan Sprague Dawley (Indianapolis, IN). All rats were acclimatized for at least a week before the initiation of experiments, at which time they were 9–12 wk of age.
Rats were irradiated as described earlier (9). A single dose of 5 Gy was administered at a dose rate of 0.96 Gy/min. Control animals received only anesthesia.
Cryptorchidism of mice and rats
A minimum of five mice were used for analysis in all the experimental groups except in the third experiment where n = 2. In all cases the positions of the cryptorchidized testes were verified at the time of killing, and animals were excluded from analysis if the testes had descended.
Three experiments involving cryptorchidism of jsd mutant mice were performed. In the first experiment jsd/+ and jsd mutant mice were bilaterally cryptorchidized at wk 4 after birth, at which time the testes had just descended and the jsd mutant mice were showing almost normal spermatogenesis. Surgical cryptorchidism was created using a slight modification of procedures reported earlier (10, 11). Briefly, mice were anesthetized with an im injection of 1.6 ml/kg of a mixture of ketamine (37.5 mg/ml), xylazine (1.9 mg/ml), and acepromazine (0.39 mg/ml). A cut was made along the skin in the lower abdominal region, and the adipose tissue of the caput epididymides on each side was sutured to the inner peritoneal wall, pushing the testes into the abdomen. The testes on both sides were placed slightly above the urinary bladder in this manner without cutting the gubernaculum. Mice were killed 8 wk later for histological and hormone analysis, at the age of 12 wk.
In the second experiment the normal (jsd/+) and jsd mutant mice were unilaterally cryptorchidized at 12 wk of age, at which time spermatogenic degeneration was completed in jsd mutant mice. Only the right testis was cryptorchidized after a procedure similar to the one described above; the left testis left in the scrotum served as control. Mice were killed 8 wk later, at the age of 20 wk, for analysis.
In the third experiment unilateral cryptorchidism of the right testis was performed in old jsd mutant mice at the age of 62 wk, and mice were killed 8 wk later.
Unirradiated and irradiated rats (n = 4 in each group) were unilaterally cryptorchidized following a similar procedure described for the mice. The rats were anesthetized with a mixture of ketamine (61.5 mg/ml) and xylazine (7.7 mg/ml) at a dose of 0.2–0.3 ml/rat. At 4 wk after irradiation the right testis was cryptorchidized without cutting the gubernaculum, and the left testis in the scrotum served as control. Rats were killed for analysis 8 wk later.
Histological analysis
In bilaterally cryptorchidized mice, whenever the sizes or locations of the left and right testes were different, both testes were weighed and fixed in Bouin’s fluid for histology. Otherwise, only the left testis was fixed in this experiment and the right was used for intratesticular testosterone (ITT) measurement. In mice unilaterally cryptorchidized during wk 12–20 and in unilaterally cryptorchidized rats, a portion of both left and right testes was fixed for histology, and the remaining portion of each was weighed and used for ITT measurement. The most advanced germ cell types were recorded in approximately 100–150 or 200–300 tubules in each mouse or rat testis section, respectively, and tubules were categorized as either differentiating (containing germ cells at stage B spermatogonia or beyond) or not. The tubule differentiation index (TDI) was taken to be the percentage of tubules that were differentiating.
Hormone measurements
At the time of killing, the mice were anesthetized with sodium pentobarbital, and blood was collected by cutting the axillary vein. The serum was separated and stored at–20 C until the analysis of testosterone and/or gonadotropins. The right testis from bilaterally cryptorchidized mice and a portion of both scrotal and cryptorchid testis from the unilaterally cryptorchid mice were weighed and frozen in liquid nitrogen for ITT measurement. From the unilaterally cryptorchidized rats, a portion of weighed testis from both sides was collected on ice before being homogenized for ITT analysis. Either the whole homogenate (in rats) or the supernatant (in mice) was used for the assay of ITT using a DSL-4000-coated tube RIA kit as described earlier (3, 9, 12). The lower limit of detection for testosterone by this method was 0.041 ng/ml.
Serum levels of FSH and LH were measured using immunofluorometric assays (Delfia, Wallac OY, Turku, Finland) as previously described (13, 14). The minimum levels of detection of LH and FSH by this method are 0.04 ng/ml and 0.1 ng/ml, respectively. All the experimental samples from the study were analyzed for FSH and LH in a single assay except those from the jsd mutant controls, which were run in a different assay and normalized to the present assay.
Statistical analysis
The data were represented either as the arithmetic mean ± SEM or averages and upper and lower limits calculated on log-transformed data as indicated in tables. The differences between the groups were first analyzed by one-way ANOVA. If the differences were significant (P < 0.05), a Dunnett’s post hoc test for multiple comparisons was performed to determine the significance of differences between the experimental groups and the control groups. In experiments where only two groups were compared, a Student’s t test was performed to analyze the significance of the difference. In comparisons of the values for the left and right testis of the same animal, a paired-samples t test was performed; otherwise, an independent-samples t test was carried out. A computer-assisted statistics program (SPSS, Inc., Chicago, IL) was used.
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Results |
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In normal heterozygous (jsd/+) males, cryptorchidism did not reduce the percentage of tubules showing differentiation, although the testis weighed less and displayed varying degrees of loss of spermatogenesis at later stages (Table 1
, Fig. 1, A and C). We were surprised to find that in cryptorchidized jsd mutant testes 98% of the tubules showed differentiation at wk 12 compared with 14% in age-matched control jsd mutant testes (Fig. 1, B and D). The distribution of tubules showing spermatocytes, round spermatids, and late spermatids (elongating and condensed) as the most advanced germ cell types in these cryptorchidized jsd mutant testes were 49 ± 11, 30 ± 9, and 14 ± 7%, respectively (n = 12 testes). The respective values for the bilaterally cryptorchidized normal testes (jsd/+) were 52 ± 16, 15 ± 9, and 28 ± 18% and were not significantly different from the corresponding values for cryptorchidized jsd mutant testes. In jsd mutant mice six of 12 cryptorchidized testes showed late spermatids (Fig. 1D); in the cryptorchidized normal littermate controls a similar fraction, three of six testes, had stages up to condensed spermatids. Thus, the degree of spermatogenesis did not appear different in the normal and jsd mutant mice after bilateral cryptorchidism during wk 4–12.
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). Furthermore, the percent distribution of tubules showing spermatocytes, round spermatids, or late spermatids as the most advanced germ cell types were 82 ± 5, 17 ± 5, and 1 ± 1%, respectively, in cryptorchidized right testis (Fig. 1F
). The respective values for the normal mouse testis unilaterally cryptorchidized at 12 wk of age were 62 ± 23, 21 ± 13, and 17 ± 11% (Fig. 1E). Although the percentage of tubules showing late spermatids in these cryptorchidized testes of jsd mutants was significantly lower (P < 0.001) than in cryptorchidized normal mice testes, the percentage of tubules showing spermatocytes and round spermatids as the most advanced germ cell types did not differ between the two.
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and 2). Consequently, the weights of the testes after bilateral cryptorchidism in jsd mutant and normal mice were not significantly different from each other. However, the testes weights of the unilaterally cryptorchidized adult normal mice remained significantly higher than that of the unilaterally cryptorchidized age matched jsd mutant testes. In a third experiment we tested whether spermatogonial differentiation could be stimulated in very old jsd mutant mice by cryptorchidism (n = 2). The right testes of the jsd mutant mice cryptorchidized at the age of 62 wk for 8 wk showed TDIs of 96.3 and 100%, the TDI being 0% in the contralateral scrotal testes. More than 96% of the tubules contained spermatocytes; one of the mice had round spermatids in 59% of the tubules. Germ cells more advanced than round spermatids were not observed in these cryptorchidized testes.
We demonstrated earlier that the hormonal control of genetically determined inhibition of spermatogonial differentiation in jsd mutant mice was largely similar to such an inhibition in irradiated rats. In both cases, testosterone inhibited spermatogonial differentiation. To further evaluate the similarity of these two models, we examined whether cryptorchidism could reverse the inhibition of spermatogonial differentiation in irradiated rats. In the rat testis cryptorchidized at 4 wk after irradiation for a period of 8 wk, the TDI was slightly but significantly increased to 2.1% from a value of 0.1% in the contralateral irradiated-only scrotal testis, and testis weight significantly increased (Table 3). In unirradiated rats that underwent a similar surgical procedure, the TDI fell to 14% in the cryptorchidized testis, and testis weight was significantly reduced. Spermatocytes were the most advanced germ cell types observed in these differentiated tubules of both irradiated and unirradiated cryptorchidized testes.
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), as we had observed earlier. The ITT levels did not vary between the bilaterally cryptorchidized and scrotal testis from jsd mutant mice at wk 12, although normal mice showed a significant increase in ITT concentration after bilateral cryptorchidism. However, in the unilaterally cryptorchidized jsd mutant mice killed at wk 20, the cryptorchidized right testis had significantly lower ITT concentration than the left scrotal testis, the concentration still being above physiological levels (Table 2). Such a variation in ITT levels was not observed between the left and right testes of unilaterally cryptorchidized normal mice of the same age. The serum testosterone levels remained constant and did not vary between the jsd mutant and heterozygous males with or without bilateral or unilateral cryptorchidism. Also the weight of the seminal vesicle, a marker for peripheral androgenic potency, did not show significant variation in the normal and jsd mutant mice, with and without cryptorchidism. However, unilaterally cryptorchidized normal mice had slightly heavier seminal vesicles than those of the jsd mutant mice of the same age with a similar surgery.
ITT levels were the same in the cryptorchidized and scrotal testis of irradiated rats (Table 3). However, in the unirradiated rats the ITT levels in the cryptorchidized testis were significantly lower than in the contralateral scrotal testis. The scrotal and cryptorchid testes of the unirradiated rats showed significantly lower ITT concentrations than irradiated rat testes at the same positions. Serum testosterone levels remained constant in the unirradiated and irradiated rats with or without unilateral cryptorchidism.
Serum LH levels were not significantly altered in normal and jsd mutant mice after bilateral or unilateral cryptorchidism (Tables 1 and 2). In normal mice FSH levels were significantly lower than that of the jsd mutants and were not altered after bilateral cryptorchidism. Bilateral cryptorchidism also did not change the FSH levels in jsd mutants. In the irradiated rats unilateral cryptorchidism did not change the LH and FSH levels significantly. However, FSH levels were significantly different between the irradiated and unirradiated rats after unilateral cryptorchidism.
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Discussion |
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We and others (3, 4) had observed earlier that testosterone inhibits spermatogonial differentiation in jsd mutant mice. We wondered whether cryptorchidism stimulated spermatogonial differentiation by lowering the levels and/or action of testosterone. In the present study no change was observed in the intratesticular and serum concentrations of testosterone in jsd mutant mice after bilateral cryptorchidism. Although the cryptorchidized testis showed lower ITT concentration than the scrotal testis in the unilaterally cryptorchidized 20-wk-old jsd mutant mice, these levels were still well above the physiological range and equal to that in noncryptorchidized 12-wk-old jsd mutant mice. It remains to be tested whether testicular androgen action is impaired in these mice upon cryptorchidism by some means, such as changes in contents of androgen-binding protein or androgen receptor (16, 17). However our recently obtained preliminary data showed that cryptorchidized jsd mutant testis expresses mRNA transcripts of Pem (Shetty, G., O. U. Bolden-Tiller, C. C. Y. Weng, and M. L. Meistrich, unpublished data), an androgen-dependent gene (18). In addition, the presence of late spermatids and the unchanged weight of the seminal vesicle after cryptorchidism suggest that testicular and peripheral androgenic function, respectively, were not impaired. The results imply that spermatogonia become sensitive to the inhibitory effect of testosterone only in the scrotal environment, but not when they are cryptorchidized.
Because local heating to the scrotum is known to cause changes in the testis (19) similar to that of cryptorchidism, we speculate that elevated abdominal temperature accounts for the effect of cryptorchidism on spermatogenesis (20, 21). Hence the results in the present study suggest that the maintenance or restoration of spermatogonial differentiation in jsd mutant mice is due to the elevation of temperature to which the testis is exposed. Thus the decline in spermatogonial differentiation with age in jsd mice is due to a combination of two factors: the descent of testis into the scrotum and the rise in testosterone levels. Both factors are required for the block because either lowering the testosterone or raising temperature restores differentiation. It would also be interesting to know whether the recovery of spermatogenesis by suppression of testosterone reported previously in these mice was a result of elevated testicular temperature. Several mechanisms might account for such an effect. For example, testosterone can alter vasomotion and blood flow in the testis (22, 23) of these mice.
The reported increase in estradiol levels in the cryptorchid rat and mouse testis (24, 25) raises the question of what role estradiol plays if it has a role to play in the stimulation of spermatogonial differentiation in cryptorchidized jsd testis. We have shown that estradiol does not inhibit spermatogonial differentiation in GnRH-treated irradiated rat testis (26) and have preliminary data to show that it actually can stimulate spermatogonial differentiation in irradiated rats and jsd mice (Shetty, G., G. A. Shuttlesworth, K. Porter, C. C. Y. Weng, and M. L. Meistrich, unpublished data). However, the stimulation by estradiol largely involves suppression of ITT levels, which were not observed here. Previous studies indicated that FSH may have a role in the inhibition of spermatogonial differentiation in irradiated rats (9, 26) but not in jsd mutant mice (3, 4). In the present study serum FSH levels were not changed after bilateral cryptorchidism in jsd mutants, further ruling out the participation of FSH in the inhibition of spermatogonial differentiation in these mice.
A scrotal temperature that is 5–7 C lower than abdominal temperature, depending on the species, has been shown to be a requirement for normal spermatogenesis. Thus it was indeed surprising that cryptorchidism, which has detrimental effects on spermatogenesis (27, 28), stimulated spermatogenesis in the present models. That can be explained by the fact that the block in spermatogenesis in jsd mutant mice and irradiated rats is at the spermatogonial stage. The extent of germ cell differentiation in animals with surgical cryptorchidism depends on the species and strain (21). In B6 mice, the testis was severely affected by cryptorchidism, showing only very few spermatocytes; the histological picture was found to be similar to that of adult jsd mutant mice (2). In contrast, cryptorchidism had only a minimal effect in MRL/MpJ mice, with only the elongated spermatids being lost. Cryptorchid C3H/He mice showed a good number of early and late spermatocytes. In the present study the effect of cryptorchidism on the control jsd/+ mice on mixed C3H-B6 background appears comparable to that found in the MRL/MpJ mice. One of the factors responsible for the resistance of germ cells to high temperature may be the expression of heat-shock proteins (21, 29). The resistance of the germ cells of C3H-B6 mice used in the present study to severe damage by cryptorchidism may be a result of relatively high levels of heat-shock proteins produced by these cells.
The detrimental effect of cryptorchidism could have been also minimized in the present study because we placed the testes in the lower abdomen just above the bladder without cutting the gubernaculum. Perhaps an intermediate temperature between the scrotum and upper abdomen can alleviate the inhibitory effects of testosterone on spermatogonial development and largely sustain spermatocyte and spermatid development in normal and jsd mice of this hybrid background.
As was the case in jsd mutant mice, cryptorchidism stimulated spermatogonial differentiation in irradiated rats, suggesting further similarity in how spermatogonial differentiation is inhibited in these two models. The inability to dramatically increase spermatogonial differentiation in these irradiated rats by producing cryptorchidism may be due to the species- and/or strain-specific sensitivity of spermatogonial differentiation to high abdominal temperature. This is evident from the low TDI observed in the cryptorchidized unirradiated rat testis.
The model proposed earlier (8) to explain the testosterone-dependent changes in the relationship between the Sertoli cells and the spermatogonia in determining differentiation/apoptosis of spermatogonia in jsd mutant mice could be further extended to accommodate the present results. The reversal of the jsd phenotype by cryptorchidism suggests that the wild-type jsd gene is involved in the maintenance of spermatogonial differentiation at scrotal temperature. One possibility is that the jsd mutation renders the gene nonfunctional in spermatogonia at scrotal temperature (32 C) and hence becomes susceptible to testosterone-dependent changes in a differentiation or apoptotic factor produced by the Sertoli cells. However, in this case the mutant gene becomes functional at and slightly below abdominal temperature (35–37 C), restoring spermatogonial differentiation. The cloning of the jsd gene, which is underway, would be important for the elucidation of the role of this gene in regulating spermatogonial differentiation (30). The other possibility assumes changes in the testosterone-dependent factors produced by the Sertoli cells at scrotal and abdominal temperatures, because spermatogonial differentiation proceeds in jsd mutants at the scrotal temperature only in the absence of testosterone. Thus according to this model a Sertoli-cell gene responsible for the survival and differentiation of jsd mutant spermatogonia is inhibited by testosterone at 32 C but becomes constitutively expressed at 35–37 C even in the presence of testosterone. Conversely, a Sertoli-cell gene responsible for the apoptosis of spermatogonia in jsd mutant mice could be dependent on testosterone at 32 C but become totally repressed at 35–37 C. In this model, Sertoli cell genes up-regulated or down-regulated by testosterone would be regulated in the opposite direction by elevated temperature. However, the specificity of these hormonal effects depends on the dependence or receptivity of spermatogonia with the jsd mutation, but not with the wild-type allele, to one or more testosterone-dependent differentiation or apoptotic signals originating from the Sertoli cells.
There are several genes whose expression in the testis is reduced at elevated temperatures. Cold-inducible RNA-binding protein (CIRP) (31) and the RNA-binding protein 3 (Rbm3) (32, 33) are known to be expressed in the germ cells and Sertoli cell respectively at 32 C but not at 37 C. It is not yet known how the expressions of these proteins are controlled by testosterone.
The present findings along with an understanding of the underlying mechanism may be helpful in restoring fertility in patients with similar genetic or toxicological bases for infertility. For example, in such cases temporary and moderate testicular heating can produce one wave of spermatogenesis and produce late spermatids for intracytoplasmic sperm injection. It will be important to elucidate the mechanism by which the elevation of temperature enhances spermatogonial differentiation to determine whether this invention will be applicable to humans.
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Acknowledgments |
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Footnotes |
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Abbreviations: ITT, Intratesticular testosterone; TDI, tubule differentiation index.
Received July 24, 2003.
Accepted for publication September 12, 2003.
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