Heat Shock Factors and Heat Shock Proteins in the Germ Line Against Heat Stress

Heat-shock response is characterized by the induction of a set of HSPs and is a fundamental response in all organisms to protect themselves from heat attack [103]. The HSP family comprises molecular chaperones that regulate protein folding and degradation under stress conditions to maintain cellular homeostasis and survival. These HSPs are expressed at euthermic body temperatures (approximately 37°C) and are found to have distinct locations and functional properties under conditions of stress. In addition to heat-induced oxidative stress, a number of other stimuli are known to induce HSPs, including energy depletion, hypoxia, acidosis, ischemia/ reperfusion, and viral infection. The HSP70 protein was first reported to be induced in response to heat shock in isolated germ cells [125].

Studies of oxidative stress suggest that HSP promoter activity and protein accumulation may be uncoupled [126[ . Data from previous studies demonstrate that both transcriptional and posttranscriptional regulatory steps are required for HSP production [126, 127]. Increased temperature and stress can activate HSPs, which are regulated by HSFs [128]. The expression of most HSPs, including testis-specific isoforms, is generally high, but controversy exists regarding whether heat-shock genes are activated in germ cells in response to heat shock [129]. Numerous studies have demonstrated that the cytoprotective functions of HSPs can be largely attributed to the suppression of apoptosis [130, 131], whereas several HSPs have the opposite functions in the germ cells.

A unique pattern of HSP70 expression has been revealed during spermatogenesis in mice and rats. HSP70 and HSP90 were expressed in germ cells, Sertoli cells, and Leydig cells in the testes during neonatal and early developing stages and in sper-matocytes and round spermatids after puberty. Further, the HSP90 protein was faintly expressed in spermatogonia during this period. In the degenerative condition, all HSP proteins were markedly expressed in germ cells after heat stress [132]. A previous study has demonstrated high HSP70-2 expression during meiosis in mice [133]. An HSP related to HSP70, Hsc70t, is expressed in late spermatids during spermiogenesis [134]. Hsc70t has been shown to be essential for spermatogenesis [135]. In contrast, HSP60 was expressed in Leydig cells [132, 136] during the neonatal and prepuberty stages, and in spermatogonia and primary spermatocytes [132, 137], in the mitochondria of spermatogonia and primary spermatocytes in stages I-V and IX-XIV [136], and mature spermatozoa [137]. HSP70-1, HSP110, and HSP27 mRNAs were highly induced in interstitial Leydig cells after heat shock, whereas these mRNAs were hardly expressed in cells within seminiferous tubules containing germ cells and somatic Sertoli cells [52]. In the rat testis, HSP105 was found to form complexes with p53 in the cytoplasm of germ cells at scrotal temperatures, while it dissociated from such complexes under heat-shock conditions such as spermatocyte apoptosis [138]. Germ cell death appeared to include p53-dependent mechanisms, and p53 is essential to maintain cellular integrity and appropriate number of germ cells during spermatogenesis [72]. Thus, one might speculate that the increased expression of HSP105 can affect the functional status of p53 in the testes of transgenic males and induce spermatocyte apoptosis. Among the small HSPs, HSP32 (HO-1) is mainly expressed in Leydig cells in response to heat stress [107, 117[. In human testes, HSP27 expression was strong in the cytoplasm of Sertoli cells, spermatogonia, and Leydig cells; moderate in the spermatocytes; weak in the spermatids; and absent in the spermatozoa [ 139] . HSP27 and HSP90 are mainly present in the cytoplasm of Sertoli cells, spermatogonia, spermatocytes, and spermatids, and their expressions in the nucleus were found to increase under heat stress [140]. HSP25 was neither expressed in germ cells nor somatic cells on all the days examined. HSP20 was strongly expressed in the heart and slightly in the testis of a 9-week-old rat, and the expression was localized in spermatocytes and round spermatids [141]. However, the precise role and regulation of HSPs in the testis is unknown.

In mammals, the expression of classical HSPs is regulated by heat-shock factor-1 [142]. Four HSF genes (HSF1, HSF2. HSF3, and HSF4) have been identified in mammals [142-144[. Apparently heat stress is not the only stimulus to activate HSF. HSF1 is ubiquitously expressed and is the most effective transactivator of stress-induced HSP expression. HSF1 associates with multiple proteins during its activation and inactivation processes, suggesting that HSF activity is regulated at multiple levels [142]. H2O2 targets HSF1-mediated transcription, but not through inhibition of HSF1-binding ability [145]. HSF1 remains as a monomer in both the cytoplasm and nucleus in unstressed cells, but is converted to a trimer that can bind to a DNA sequence motif, the heat-shock element (HSE), which is translocated into the nucleus in response to heat shock [129] and induces a robust activation of heat-shock genes [146, 147]. This HSF1-mediated induction of HSP expression is required for protection of cells from various pathophysiological conditions such as neurodegenerative and other degenerative diseases and confers lifespan extension and thermotolerance [146, 147]. During the past few years, comprehensive analysis has revealed numerous HSFs, particularly HSF1, whose targets include HSP and non-HSP genes, the expressions of which are regulated differentially by HSF family members. HSFs are expressed in germ cells and studies using animal models have revealed their roles in spermatogenesis [50, 148]. HSF2 is found mainly as a dimer in unstressed cells, and for the most part, neither forms a trimer nor is translocated into the nucleus during heat shock. HSF2 is considered to function in development. In fact, HSF2 is associated with development of the brain and reproductive organs [148]. Recently, HSF2 was shown to positively regulate proteasome activity, leading to the decreased expression of p53 [149] . HSF3 represents a unique HSF that has the potential to activate only nonclassical heat-shock genes to protect the cells from detrimental stress [150]. HSF4 is found as a trimer in the nucleus in unstressed cells as it uniquely lacks the HR-C domain that suppresses trimer formation [143]. HSF4 expression is ubiquitous in all cell types. The roles of HSF2, HSF3, and HSF4 on spermatogenesis are currently under investigation. As a germ cell-specific HSF, the HSFY gene is localized in the AZFb region of the Y chromosome, whose deletion causes a severe alteration in spermatogenesis [151], but the regulation of this gene by heat and oxidative stress is unknown. Ferlin et al. reported that testicular

HSP90, HSPA4, HSF1, HSF2, and HSFY were transcriptionally upregulated in the presence of varicocele [152]. Taken together with the fact that varicocele increases testicular ROS [8, 54, 75, 91, 118, 153], these expressions are perhaps regulated, at least in part, by heat-induced oxidative stress.

Generally, HSF1 overexpression protects cells from heat stress-induced cell death, whereas germ cells actively die through apoptosis [50, 52] . However, in a previous study, it was observed that neither heat-induced HSF1 activation [52] nor active HSF1 overexpression results in expression of HSP70 in spermatogenic cells [50, 70]. Although HSF1 acts as a cell-survival factor in premeiotic germ cells [52], expression of a constitutively active form of HSF1 in the spermatocytes of transgenic mice promotes germ cell apoptosis [50, 52], but it is logical to think about the character of the germ cells. In addition, Widlak et al. reported that heat-activated HSF1 paradoxically downregulated the expression of HSP 70.2 and promotes apoptosis (Fig. 8.4) [154]. HSF1-induced apoptosis in spermatocytes might be a result of the lack of induction of the antiapoptotic protein, inducing HSP70i, which has previously been shown to confer resistance to apoptosis in somatic cells [70] . In the testis, apoptosis is common and is believed to play an important role in controlling the germ cell population and eliminating defective germ cells to produce functional spermatozoa. Thus, apoptosis protects the testis against harmful stress and functions to preserve future spermatogenesis, and a study using an ischemia/reperfusion model reported that inhibition of apoptosis by caspase inhibitors does not ameliorate spermatogenesis [79] .

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