©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Male Germ Cell-specific Alteration in Temperature Set Point of the Cellular Stress Response (*)

(Received for publication, May 4, 1995; and in revised form, June 6, 1995)

Kevin D. Sarge (§)

From theDepartment of Biochemistry, Chandler Medical Center, University of Kentucky, Lexington, Kentucky 40536-0084

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Heat shock factor (HSF), a transcriptional regulator with heat-activatable DNA binding ability, mediates the stress-induced expression of eukaryotic heat shock protein genes. Previous results from this laboratory demonstrated that a preparation of mixed male germ cell types from mouse testis exhibited a lower temperature threshold for activation of HSF1 DNA binding relative to other mouse cell types (Sarge, K. D., Bray, A. E., and Goodson, M. L. (1995) Nature 374, 126). The purpose of the present study was to determine whether the phenomenon of reduced HSF1 activation temperature is common to all testis cell types, both somatic and germ cell types, or whether it is a special property of male germ cells. The results show that a purified population of pachytene spermatocytes, one of the male germ cell types, exhibits a profile of reduced HSF1 activation temperature identical to that observed for the mixed germ cell preparation, with a threshold HSF1 activation temperature of 35 °C. Activation of HSF1 DNA binding in male germ cells by incubation at 38 °C is accompanied by the classic cellular stress response parameters of heat-induced HSF1 phosphorylation and increased expression of the hsp72 stress protein. In contrast, a preparation of somatic testis cell types exhibits HSF1 activation only at temperatures of 42 °C and above, a profile identical to that observed for mouse liver cells and mammalian cell lines. These results demonstrate that the phenomenon of reduced HSF1 activation temperature is a unique property of male germ cell types within the mammalian testis and demonstrate that HSF1 activated at this lower temperature threshold is fully capable of mediating a productive cellular stress response in these cell types.


INTRODUCTION

Cells exposed to elevated temperature and other environmental stress conditions respond by rapidly inducing the expression of heat shock proteins (hsps), (^1)which function to protect cells from the harmful effects of stress conditions on cellular proteins(1, 2, 3) . The stress-induced expression of hsps, called the cellular stress response, is mediated by heat shock transcription factor 1 (HSF1). HSF1 exists in unstressed cells in a monomeric non-DNA binding form, which is converted to the trimeric DNA binding form following exposure of cells to elevated temperature and other stress conditions (4, 5, 6) (reviewed in (7, 8, 9, 10) ). The HSF1 protein also undergoes heat-induced phosphorylation, which has been suggested to be important for maximal transcriptional activation function of this factor(4, 11) .

In most mammalian cell types, activation of HSF1 DNA binding occurs only at temperatures of 42 °C and above. However, previous results from this laboratory showed that HSF1 activation temperature is not identical in all cell types within an organism. These results demonstrated that a preparation of mixed male germ cell types from mouse testis exhibited an HSF1 activation temperature that is significantly lower than that observed in mouse liver cells(12) . Two hypotheses can be put forward to explain the phenomenon of reduced HSF1 activation temperature in testis. The first possibility is that it is somehow related to the lower basal growth temperature experienced by cell types within this tissue. A unique feature of the male gonads of many species is their location outside the main body cavity, so that testis cells have a significantly lower growth temperature relative to cells of other tissues. In the mouse, testis temperature is tightly regulated at 30 °C, 7 °C lower than the body cavity temperature at which other tissues such as liver are maintained(13) . Therefore, the lowered HSF1 activation temperature in male germ cells suggests the existence of a relationship between normal growth temperature and the temperature set point for HSF activation in a particular cell type (12) . This possibility is consistent with the hypothesis that HSF is not activated in response to absolute temperature experienced by the cell but rather in response to a change in temperature(14, 15, 16) . A second, alternative possibility is that reduced HSF1 activation temperature may have evolved as a unique property of male germ cells, somehow required for the normal function or development of these specialized cell types.

The purpose of the present study was to discriminate between these two possibilities by comparing the temperature set point of HSF1 activation in the two major classes of cell types present in testis, the male germ cell types (represented by purified pachytene spermatocytes) and somatic cell types (interstitial somatic cell preparation). The results demonstrate that the phenomenon of reduced HSF1 activation temperature in testis is a property unique to male germ cell types within this tissue, as it is exhibited by pachytene spermatocytes but not by somatic testis cell types. HSF1 DNA binding is induced in somatic testis cell types at temperatures of 42 °C and above, a temperature profile identical to that observed for mouse liver cells and mammalian cell lines(12, 17) . In contrast, the threshold temperature for HSF1 activation in pachytene spermatocytes is 35 °C, with maximal HSF1 DNA binding induced by treatment at 38 °C. The results also show that incubation of male germ cells at 38 °C results in heat-induced HSF1 phosphorylation and increased synthesis of the hsp72 stress protein, demonstrating the ability of HSF1 DNA binding activated at this lower temperature to mediate a productive cellular stress response.


EXPERIMENTAL PROCEDURES

Experimental Animals

C3HF/SED mice were obtained from Massachusetts General Hospital (Boston, MA) and maintained under a controlled light cycle (14 h light:10 h dark). These studies were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Preparation of Pachytene Spermatocytes and Interstitial Cells from Mouse Testes

Pachytene spermatocytes were isolated by centrifugal elutriation of enzymatically dissociated testes of adult C3HF/SED strain mice. Testes of six mice were decapsulated by removal of the tunica albuginea and then placed in Dulbecco's modified Eagle's medium (DMEM) containing 1 mM sodium pyruvate, 6 mM sodium L-lactate, 0.5 mg/ml collagenase, and 0.25 mg/ml trypsin. Enzymatic disruption of seminiferous tubules was carried out at 32 °C for 30 min with gentle inversion mixing every 5 min. After allowing debris to settle, the supernatant was removed and the dissociated cells collected by centrifugation at 500 g for 3 min. Cells were then resuspended and subjected to elutriation as described previously(18) . After elutriation, fractions containing pachytene spermatocytes were collected by centrifugation and resuspended in 12 ml of DMEM containing 1 mM sodium pyruvate and 6 mM sodium L-lactate. The purity of the pooled pachytene spermatocytes was 86%, with the remaining cells consisting of round spermatids. For preparation of testis interstitial cells, decapsulated adult mouse testes were incubated in a DMEM enzymatic digestion solution identical to that described above for preparation of germ cells, except that trypsin was not added to this solution. In order to minimize contamination of the interstitial cells with germ cells, care was taken to gently tease the seminiferous tubules apart to avoid tearing them. After incubation at 32 °C for 30 min with gentle inversion mixing every 5 min, seminiferous tubules were allowed to settle, and the dissociated interstitial cells in the supernatant were collected by centrifugation at 500 g for 3 min and resuspended in DMEM. Microscopic examination of the interstitial cell preparation detected no apparent contamination with male germ cells. For heat treatment in vitro, interstitial cells or purified pachytene spermatocytes were incubated at various temperatures for 60 min and subjected to gel shift analysis as described below. In experiments examining heat-induced hsp expression in germ cells, the initial 60-min heat treatments were followed by incubation at 32 °C for 120 min to allow cells to recover and synthesize hsps.

Native Gel Mobility Shift and Western Blot Analysis

Whole cell extracts of pachytene spermatocytes and interstitial somatic testis cells were prepared by Dounce homogenization of cell pellets in 5 volumes of Buffer C (20 mM Hepes (7.9), 0.42 M NaCl, 25% glycerol, 1.5 mM MgCl(2), 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride). After centrifugation of the extract at 12,000 g to pellet insoluble material, the native gel mobility shift assay was performed as described previously (4) with a self-complementary consensus HSE oligonucleotide (5`-CTAGAAGCTTCTAGAAGCTTCTAG-3`), which contains four perfect inverted 5`-NGAAN-3` repeats after annealing. For experiments involving addition of antibodies, 1 µl of a 1:250 dilution of specific HSF1 or HSF2 polyclonal antibody was added to aliquots of extract and incubated at 22 °C for 10 min before gel shift analysis. For Western blot analysis, mixed germ cells or purified pachytene spermatocytes were boiled in 2 Laemmli buffer (125 mM Tris-HCl (pH 6.8), 20% glycerol, 4% sodium dodecyl sulfate, 200 mM dithiothreitol), electrophoresed on an 8% SDS-PAGE gel, and then blotted to nitrocellulose using a Bio-Rad semidry transfer apparatus. The blots were then probed with HSF1 polyclonal antibodies or 3a3 monoclonal antibody as described previously(4) .


RESULTS

Reduced Temperature Threshold for HSF1 Activation in Male Germ Cells

Previous results from this laboratory demonstrated that HSF1 DNA binding is induced at significantly lower temperatures in a preparation of mixed male germ cell types relative to other mouse cell types(12) . This result suggested the possibility that the growth temperature experienced by a particular cell type in vivo plays a primary role in determining the HSF1 activation temperature in that cell type. In order to directly test this hypothesis, the temperature profiles of HSF1 activation in purified preparations of different cell types present in the testis, both somatic and germ cell types, were determined.

First, HSF1 activation temperature in male germ cells was examined, using a purified population of pachytene spermatocytes. Aliquots of the pachytene spermatocytes were incubated at various temperatures in vitro and then subjected to gel shift analysis using an oligonucleotide containing an HSF binding site. The results of this experiment, shown in Fig.1A, indicate that these purified pachytene spermatocytes, like the preparation of mixed male germ cells examined previously(12) , exhibit a reduced HSF1 activation temperature profile. Low levels of HSF1 DNA binding are induced by treatment at 36 °C, with high levels at 38 and 40 °C, followed by diminishing levels at 42 and 44 °C. In order to precisely define the temperature profile for HSF1 activation in pachytene spermatocytes, a similar analysis was performed using a 1 °C temperature course. The results, shown in Fig.1B, indicate that the threshold temperature for HSF1 activation in these cells is approximately 35 °C. Gel shift analysis in conjunction with polyclonal antibodies specific to the HSF1 and HSF2 polypeptides confirms that the HSF DNA binding activity induced in male germ cells by treatment at 38 °C, like that induced in 42 °C-treated human and mouse cell lines(4, 5) , is composed of HSF1 and not HSF2 (Fig.1C).


Figure 1: Temperature profile of HSF1 activation in pachytene spermatocytes. A, purified pachytene spermatocytes were incubated at the temperatures indicated for 60 min and then subjected to gel shift assay using a radioactively labeled HSE-containing oligonucleotide. The position of the heat-induced HSF DNA binding activity is indicated (HSF). NS represents a nonspecific DNA binding activity, and F represents the free probe. B, temperature threshold for HSF1 activation in pachytene spermatocytes. Purified pachytene spermatocytes were incubated at the temperatures indicated for 60 min and then subjected to gel shift assay using a radioactively labeled HSE-containing oligonucleotide. C, heat-induced HSF DNA binding activity in pachytene spermatocytes is composed of HSF1. Extracts of pachytene spermatocytes incubated at 38 °C were subjected to gel shift analysis either alone (N) or after preincubation with specific HSF1 antibodies (1) or HSF2 antibodies (2) .



Next, the temperature profile for HSF1 activation in somatic cell types of mouse testis was determined. Heat treatment and gel shift analysis was performed on a preparation of mixed somatic testis cell types present in the interstitial space between the seminiferous tubules. The results of this analysis, shown in Fig.2, demonstrate that these somatic testis cell types exhibit activation of HSF1 DNA binding at temperatures of 42 °C and above, a profile very similar to that observed previously for mouse liver cells and mammalian cell lines(12, 17) . Taken together, the results shown in Fig.1and Fig. 2demonstrate that the phenomenon of reduced HSF1 activation temperature in testis is unique to the male germ cell types within this tissue.


Figure 2: Temperature profile of HSF1 activation in somatic testis cells. Interstitial somatic testis cells were incubated at the temperatures indicated for 60 min and then subjected to gel shift analysis. The position of the heat-induced HSF DNA binding activity is indicated (HSF). NS represents a nonspecific DNA binding activity, and F represents the free probe.



Heat-induced HSF1 Modification and hsp70 Protein Synthesis in Male Germ Cells

Previous studies showed that incubation of mouse and human cell lines at 42 °C causes an increase in phosphorylation of the HSF1 protein in these cells, which results in decreased mobility of this protein on SDS-PAGE gels(4, 11) . Therefore, to determine whether HSF1 activated in male germ cells by incubation at the lower threshold temperature of 38 °C also undergoes heat-induced covalent modification, Western blot analysis was performed on extracts of germ cells incubated at 32 or 38 °C, using HSF1 polyclonal antibodies (4) . The results, shown in Fig.3A, demonstrate that the HSF1 protein in male germ cells increases in size from a set of bands of 70-74 kDa in 32 °C-treated cells (representing different HSF1 basal phosphorylation states(4) ) to a relatively discrete band of 84 kDa in 38 °C-treated cells. These heat-induced changes in HSF1 size between 32 and 38 °C-treated male germ cells are very similar to those previously observed for HSF1 between control (37 °C) and heat-shocked (42 °C) mouse NIH 3T3 cells(4) .


Figure 3: Heat-induced HSF1 modification and hsp70 protein synthesis in male germ cells. A, heat-induced modification of HSF1 in male germ cells subjected to heat treatment in vitro. Extracts prepared from male germ cells incubated at 32 or 38 °C for 60 min were subjected to Western blot analysis using specific HSF1 polyclonal antibodies. B, heat-induced synthesis of hsp70 protein in male germ cells subjected to heat treatment in vitro. Male germ cells were incubated at 32 or 38 °C for 60 min and then further incubated at 32 °C for 120 min to allow cells to recover and translate hsp mRNA induced during the heat treatment. Extracts prepared from these cells were subjected to Western blot analysis using a monoclonal antibody (3a3), which detects both the constitutively expressed hsc70 protein (hsp73) and the heat-inducible hsp70 protein (hsp72).



HSF1 DNA binding activated in mammalian cells by treatment at 42 °C mediates the increased expression of heat shock protein genes(10) . Therefore, to determine whether the HSF1 binding activity induced in male germ cells by heat treatment at 38 °C functions to regulate hsp gene expression in these cells, extracts of germ cells incubated at 32 or 38 °C were subjected to Western blot analysis using a monoclonal antibody (3a3), which detects both the constitutively expressed hsc70 protein (hsp73) and the heat-inducible hsp70 protein (hsp72)(4) . The results, shown in Fig.3B, reveal an accumulation of heat-inducible hsp72 stress protein following incubation of germ cells at 38 °C, demonstrating the functional ability of the HSF1 DNA binding activity induced at this reduced temperature to mediate a productive cellular stress response in these cells. Previous studies, which employed higher heat shock temperatures than those used in this study, also observed heat-induced hsp expression in male germ cells (19, 20) , while other studies reached different conclusions(21, 22) . These discrepancies may be due to methodological differences in the condition of the cells exposed to heat treatment (intact testes, dissected seminiferous tubules, or dissociated cells) or to differences in the heat shock treatments employed in each study.


DISCUSSION

Previous results from this laboratory showed that HSF1 DNA binding is activated at a significantly lower temperature in mouse mixed male germ cells relative to mouse liver cells, indicating that the temperature set point for HSF1 activation does not have a fixed value in a given species and can vary in a tissue-dependent manner(12) . The results presented in this paper now reveal two important new features of the regulation of the cellular stress response in testis. First, the results demonstrate that the phenomenon of reduced HSF1 activation temperature in testis is specific to male germ cell types, as it is exhibited by purified pachytene spermatocytes but not by somatic cell types present in testis. Second, these results demonstrate that the HSF1 DNA binding activity induced in male germ cells at this lower threshold temperature is fully functional, capable of mounting a productive cellular stress response resulting in the increased expression of hsp72 stress protein in these cells.

What is the mechanism by which the threshold temperature of the cellular stress response is altered in male germ cells? It is unlikely that this phenomenon evolved primarily as an adaptive response to the lower relative growth temperature experienced by cell types within the testis, since somatic cell types present in this tissue do not exhibit a reduced HSF1 activation temperature. We hypothesize that unique features of the biochemical environment in male germ cells, or germ cell-specific regulatory factors, are responsible for altering the temperature set point for HSF1 activation in these cells. Heat-induced protein denaturation has been suggested to be the signal that triggers the conversion of HSF1 to the active, DNA binding form(23, 24, 25, 26) . Therefore, one possibility is that the reduced HSF1 activation temperature in male germ cells could be due to a lower relative thermal stability of one or more germ cell-specific proteins(27, 28, 29, 30, 31) .

The functional consequences of reduced HSF1 activation temperature in male germ cell types of the testis are not yet known. The fact that this phenomenon is restricted to male germ cell types suggests that an alteration in temperature set point of the cellular stress response is somehow required for the proper function or development of these specialized cell types. However, it is also possible that the reduced induction temperature of the cellular stress response may have negative consequences for male germ cell types. In mouse, testis temperature is tightly regulated at 30 °C, 7 °C below that of the main body cavity(13) . The importance of precise thermoregulation of testis is evidenced by the fact that elevations of scrotal temperature, caused by conditions such as varicocele, cryptorchidism, sauna use, or even the wearing of tight insulating clothing, dramatically inhibit spermatogenic cell functions and result in male infertility(32) . The mechanistic basis for heat-induced male infertility is unknown. One possibility is that it is due to relative thermolability of essential spermatogenic cell proteins. However, the results presented above showing induction of the cellular stress response in male germ cells within the same range of temperatures that inhibit spermatogenesis suggest the alternative possibility that heat-induced hsps may play a role in causing male infertility. Stress-induced hsps could inhibit one or more cellular processes essential to male germ cell development, thus leading to an inhibition of spermatogenesis. Further studies will be required to determine whether the cellular stress response plays a cytoprotective or inhibitory role in the negative effects of elevated temperature on spermatogenic cell function and the process of spermatogenesis.

Our previous studies demonstrated that another member of the mammalian HSF family, HSF2, exhibited constitutive DNA binding activity in testis cell extracts(33) . However, in our present and past studies on the heat induction of HSF1 DNA binding activity in male germ cells, we have observed little HSF DNA binding activity in male germ cells incubated at temperatures of 32-34 °C(12) . The most likely explanation for this apparent contradiction is that it is due to heat lability of HSF2 DNA binding activity. Previous studies have shown that, in contrast to the heat-stable DNA binding exhibited by HSF1, HSF2 DNA binding activity is very temperature-sensitive and is rapidly destroyed by heat treatment(34, 35, 36, 37) .

Heat treatment of mammalian cells at 42 °C has been shown to cause an increase in phosphorylation of the HSF1 polypeptide(4, 11) . This heat-induced HSF1 modification has been suggested to be important for maximal transcriptional activation function of this factor(4) . The results presented in this paper demonstrate that heat treatment of male germ cells at 38 °C causes an increase in HSF1 phosphorylation that is similar to that observed in 42 °C-treated mouse and human cell lines(4) . Thus, it appears that the temperature set point of the mechanism that controls the activity of the kinase(s) responsible for heat-induced HSF1 phosphorylation, like the mechanism that controls the activation of HSF1 DNA binding, has been shifted to a lower temperature in male germ cells relative to other cell types. These results suggest that a common signal transduction pathway may be utilized for the regulation of both HSF1 DNA binding and HSF1 phosphorylation in mammalian cells, but this remains to be elucidated.


FOOTNOTES

*
This work was supported by funds from the National Science Foundation/EPSCoR (National Science Foundation Award OSR-9108764) and the March of Dimes Birth Defects Foundation (Award 5-FY95-0009). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, University of Kentucky, 800 Rose St., Lexington, KY 40536-0084.

^1
The abbreviations used are: hsp, heat shock protein; HSF1, heat shock factor 1; HSE, heat shock element; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

I thank William Wright, Deborah O'Brien, and Mitch Eddy for providing training in spermatogenic cell isolation procedures and Ok-Kyong Park-Sarge for excellent critical comments on the manuscript.


REFERENCES

  1. Morimoto, R. I., Tissieres, A., and Georgopoulos, C. (eds) (1990) Stress Proteins in Biology and Medicine , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  2. Craig, E. A., Gambill, B. D., and Nelson, R. J. (1993) Microbiol. Rev. 57,402-414 [Abstract]
  3. Hendrick, J. P., and Hartl, F.-U. (1993) Annu. Rev. Biochem. 62,349-384 [CrossRef][Medline] [Order article via Infotrieve]
  4. Sarge, K. D., Murphy, S. P., and Morimoto, R. I. (1993) Mol. Cell. Biol. 13,1392-1407 [Abstract]
  5. Baler, R., Dahl, G., and Voellmy, R. (1993) Mol. Cell. Biol. 13,2486-2496 [Abstract]
  6. Westwood, J. T., and Wu, C. (1993) Mol. Cell. Biol. 13,3481-3486 [Abstract]
  7. Sorger, P. K. (1991) Cell 65,363-366 [Medline] [Order article via Infotrieve]
  8. Kingston, R. E. (1991) in The Hormonal Control of Gene Transcription (Cohen, P., and Fouldes, J. G., eds) pp. 377-398, Elsevier Science Publishing Co., Inc., New York
  9. Lis, J., and Wu, C. (1992) in Transcriptional Regulation (McKnight, S. L., and Yamamoto, K. R., eds) pp. 907-930, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  10. Morimoto, R. I., Sarge, K. D., and Abravaya, K. (1992) J. Biol. Chem. 267,21987-21990 [Abstract/Free Full Text]
  11. Larson, J. S., Schuetz, T. J., and Kingston, R. E. (1988) Nature 335,372-375 [CrossRef][Medline] [Order article via Infotrieve]
  12. Sarge, K. D., Bray, A. E., and Goodson, M. L. (1995) Nature 374,126 [Medline] [Order article via Infotrieve]
  13. Harrison, R. G., and Weiner, J. S. (1948) J. Physiol. (Lond.) 107,48P
  14. Abravaya, K., Phillips, B., and Morimoto, R. I. (1991) Genes & Dev. 5,2117-2127
  15. Clos, J., Rabindran, R., Wisniewski, J., and Wu, C. (1993) Nature 364,252-255 [CrossRef][Medline] [Order article via Infotrieve]
  16. Liu, A. Y. C., Bian, H., Huang, L. E., and Lee, Y. K. (1994) J. Biol. Chem. 269,14768-14775 [Abstract/Free Full Text]
  17. Mosser, D. D., Kotzbauer, P. T., Sarge, K. D., and Morimoto, R. I. (1990) Proc. Natl. Acad. Sci. U. S. A. 87,3748-3752 [Abstract]
  18. Meistrich, M. L., Longtin, J., Brock, W. A., Grimes, S. R., and Mace, M. L. (1981) Biol. Reprod. 25,1065-1077 [Medline] [Order article via Infotrieve]
  19. Allen, R. L., O'Brien, D. A., and Eddy, E. M. (1988) Mol. Cell. Biol. 8,828-832 [Medline] [Order article via Infotrieve]
  20. Allen, R. L., O'Brien, D. A., Jones, C. C., Rockett, D. L., and Eddy E. M. (1988) Mol. Cell. Biol. 8,3260-3266 [Medline] [Order article via Infotrieve]
  21. Zakeri, Z. F., Welch, W. J., and Wolgemuth, D. J. (1990) J. Cell Biol. 111,1785-1792 [Abstract]
  22. Lemaire, L., and Heinlein, U. A. O. (1991) Life Sci. 48,365-372 [Medline] [Order article via Infotrieve]
  23. Kelley, P. M., and Schlesinger, M. J. (1978) Cell 15,1277-1286 [Medline] [Order article via Infotrieve]
  24. Hightower, L. E. (1980) J. Cell. Physiol. 102,407-427 [Medline] [Order article via Infotrieve]
  25. Mosser, D. D., Theodorakis, N. G., and Morimoto, R. I. (1988) Mol. Cell. Biol. 8,4736-4744 [Medline] [Order article via Infotrieve]
  26. Ananthan, J., Goldberg, A. L., and Voellmy, R. (1986) Science 232,522-524 [Medline] [Order article via Infotrieve]
  27. Hightower, L. E. (1991) Cell 66,191-197 [Medline] [Order article via Infotrieve]
  28. Erickson, R. P. (1990) Trends Genet. 6,264-269 [CrossRef][Medline] [Order article via Infotrieve]
  29. Wolgemuth, D. J., and Watrin, F. (1991) Mamm. Genome 1,283-288 [Medline] [Order article via Infotrieve]
  30. Hecht, N. B. (1992) in Cell and Molecular Biology of the Testis (Desjardins, C., and Ewing, L. L., eds) pp. 464-503, Oxford University Press, Oxford
  31. Eddy, E. M., Welch, J. E., and O'Brien, D. A. (1993) in The Molecular Biology of the Male Reproductive System (de Kretser, D. M., ed) pp. 181-232, Academic Press, New York
  32. Zorgniotti, A. W. (1989) Temperature and Environmental Effects on the Testis , Plenum Press, NY
  33. Sarge, K. D., Park-Sarge, O.-K., Kirby, J. D., Mayo, K. E., and Morimoto, R. I. (1994) Biol. Reprod. 50,1334-1343 [Abstract]
  34. Sarge, K. D., Zimarino, V., Holm, K., Wu, C., and Morimoto, R. I. (1991) Genes & Dev. 5,1902-1911
  35. Sistonen, L., Sarge, K. D., and Morimoto, R. I. (1994) Mol. Cell. Biol. 14,2087-2099 [Abstract]
  36. Murphy, S. P., Gorzowski, J. J., Sarge, K. D., and Phillips, B. (1994) Mol. Cell. Biol. 14,5309-5317 [Abstract]
  37. Mezger, V., Rallu, M., Morimoto, R. I., Morange, M., and Renard, J.-P. (1994) Dev. Biol. 166,819-822 [CrossRef][Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.