Cell Tissue Res (2011) 344:355363 DOI 10.

1007/s00441-011-1151-4

REGULAR ARTICLE

Developmental expression of heat shock proteins 60, 70, 90, and A2 in rabbit testis
Yingjie Wu & Yangli Pei & Yinghe Qin

Received: 12 October 2010 / Accepted: 14 February 2011 # Springer-Verlag 2011

Abstract Currently, no reports exist concerning the expression patterns and developmental changes of heat shock proteins (HSPs) in the reproductive system of the male rabbit. In the present study, the testes of rabbits were collected at postnatal months 1, 2, 3, 4, 5, and 40. HSP60, HSC70, HSP90, and HSPA2 were detected by both Western blot and immunohistochemical methods. The expression levels of HSP60 and HSC70 showed no apparent change during the developmental progress. HSP90 increased at the second month; prior to the third month, HSPA2 was expressed at a low level. Immunohistochemistry localized HSP60 in the cytoplasm of all of the cell types in the testis and in the apical pole of the spermatids. The distribution pattern of HSC70 and HSP90 was similar, both being mainly located in the spermatids of stage VII-VIII and in the cytoplasm of the spermatogonium. HSPA2 staining was mainly observed in the cytoplasm of pachytene spermatocytes and spermatids in testes of 3-, 4-, 5-, and 40-month-old rabbits. These results provide a basic reference point for studying the functions of HSPs in the male rabbit reproductive system and should be beneficial for the future determination of the mechanisms of heat shock on male rabbit fertility. Keywords Heat shock protein . Localization . Testis . Development . Rabbit (Rex)

Introduction

fxn/definition

spermatogenesis
Yingjie Wu and Yangli Pei contributed equally to this work. This work was supported by the Natural Science Foundation of China (31001009) and Earmarked Fund for Modern Agro-industry Technology Research System (nycytx-44). Y. Wu : Y. Pei : Y. Qin (*) College of Animal Science and Technology, China Agricultural University, Beijing 100193, Peoples Republic of China e-mail: qinyinghe@cau.edu.cn

A wide variety of environmental stresses induce cells to synthesize rapidly a distinct set of proteins known as heat shock proteins (HSPs; Lindquist 1986; Welch 1992). Some of the HSPs have been shown to be involved in basic cellular functions such as trafficking and translocating proteins through membranes (Neuer et al. 2000; Craig et al. 1994). Recently, several HSPs have been demonstrated to be present and have important functions in the male testis (Huang et al. 2005; Paranko et al. 1996; Son et al. 2000; Gruppi and Wolgemuth 1993; Dix 1997). HSP60 has been detected in spermatogonia, primary spermatocytes, and Sertoli cells in the rat (Meinhardt et al. 1995) and human (Werner et al. 1997) testes. Results in mouse, rat, bull, and boar have shown that HSP70 protein (HSC70) is constitutively expressed in specific spermatogenic cell types during spermatogenesis (Allen et al. 1988; Raab et al. 1995; Kamaruddin et al. 2004; Huang et al. 2005). Another member of the HSP70 family, HSPA2 (HSP70-2), is highly expressed in pachytene spermatocytes (Allen et al. 1996; Son et al. 1999). Its deficiency in mice results in the arrest of spermatogenesis in meiotic prophase I and infertility (Dix et al. 1996a, 1996b). Other HSP70 family proteins, such as constitutive proteins grp75 and grp78 and the inducible proteins HSP70-1 and HSP70-3, have also been observed in the testis. In addition, two isoforms of HSP90 have been identified in the mouse and human (Minami et al. 1991; Hickey et al. 1989; Moore et al. 1989) and have been reported to be highly expressed in premeiotic spermatogenic cells (Lee 1990; Gruppi et al. 1991). The focus of studies of the expression pattern of HSPs in testes has been on mice, rats, humans, and large domestic animals. However, as yet, no data exist about the expression and cellular distribution of HSPs in the rabbit, which is an

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Materials and methods Animals and tissue collection The procedures used in this study were approved by the Chinese Association for Laboratory Animal Sciences. Male Rex rabbits aged 1, 2, 3, 4, 5, and 40 months (n=3 for each age) were killed with an overdose of pentobarbital sodium salt (50 mg/kg i.v., Merck, Darmstadt, Germany). These ages took into account any changes in pre-puberty (1 month old), early puberty (2 and 3 months old), puberty (4 and 5 months old), and senility (40 months old) during rabbit development. The testis from one side of each rabbit was collected, fixed in Bouins solution, and embedded in paraffin for immunohistochemistry staining, whereas the testis from the other side was immediately frozen in liquid nitrogen for Western blot analysis. Immunohistochemistry The immunohistochemical localization of HSP60, 70, 90, or A2 was respectively performed by using mouse monoclonal antibodies from Abcam (Cambridge, UK: ab13532, ab47455, ab13492, and ab55290). The testis

Fig. 1 Detection of HSPs by Western blot during testicular development (IB immunobot). HSP60, HSC70, HSP90, and HSPA2 were detected in the testes of rabbits at months 1 (lane 1), 2 (lane 2), 3 (lane 3), 4 (lane 4), 5 (lane 5), and 40 (lane 6). -Actin protein was blotted as a reference

important animal model for studying the influence of heat shock and the function of HSPs because of its high sensitivity to hyperthermia. Therefore, this study is aimed at determining and comparing the cellular distributions of these HSPs in rabbit testes at various ages in order to help elucidate their possible functions.

Fig. 2 Stages 18 of the seminiferous epithelium cycle based on the tubular morphology system. a Stage 1 showing type A spermatogonia (A), pachytene primary spermatocytes (P), leptotene spermatocytes (L), and round spermatids (R). b Stage 2 presenting type A spermatogonia (A), leptotene spermatocytes (L), pachytene spermatocytes (P), Sertoli cells (S), and elongating spermatids (E). c Stage 3 containing type A spermatogonia (A), zygotene spermatocytes (Z), and further developing elongated spermatids (E). d Stage 4 showing zygotene spermatocytes (Z), meiotic figures (M), elongated spermatids (E), and Sertoli cells (S). e Stage 5 containing type A spermatogonia

(A), type B spermatogonia (B), pachytene spermatocytes (P), newly formed round spermatids (R), and elongated spermatids (E). f Stage 6 presenting type B spermatogonia (B), pachytene spermatocytes (P), round spermatids (R), and elongated spermatids (E). g Stage 7 with type B spermatogonia (B), pachytene spermatocytes (P), round spermatids (R), elongated spermatids (E), and residual bodies (Rb). h Stage 8 showing type A spermatogonia (A), type B spermatogonia (B), pachytene spermatocytes (P), pachytene/leptotene spermatocytes (PL), round spermatids (R), elongated spermatids (E), and residual bodies (Rb). Bar 15 m

Cell Tissue Res (2011) 344:355363 Fig. 3 Localization of HSP60 protein in the rabbit testes during development. a At 1 month. b At 2 months. c At 3 months. d At 4 months e At 5 months. f At 40 months. g Negative control with antiserum. HSP60 was detected by immunohistochemistry (brown; pSg prespermatogonium, Ley Leydig cell, Sg spermatogonium, S Sertoli cell, P pachytene spermatocyte, R round spermatid, L(Z) leptotene or zygotene spermatocyte). Bar 50 m

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sections were dewaxed and rehydrated through a graded series of ethanol to a final wash in water. Antigen retrieval was performed by microwaving the sections for 16 min (44 min) in 0.01 M sodium citrate buffer (pH 6.0). Nonspecific binding and endogenous peroxidase activity were blocked with 10% normal goat serum and 3% hydrogen peroxide in phosphate-buffered saline (PBS; pH 7.4). Sections were then incubated overnight with the four antibody subtypes at 4C, respectively. Sections were washed in PBS (35 min) and incubated with biotinylated goat anti-mouse IgG (Zymed, San Francisco, Calif., USA; dilution 1:200) for 2 h at room temperature. After three washes in PBS, avidin-biotin complex (Zymed) was added for 2 h, and the peroxidase activity was detected by using DAB (diaminobenzidine; Sigma-Aldrich, Saint Louis, Mo., USA) for 2 min. Finally, the sections were counterstained with hematoxylin and observed under a microscope (Leica Microsystems, Wetzlar, Germany).

Western blot analysis Western blots were performed as described previously (Wu et al. 2010). Briefly, the rabbit testes were homogenized in protein lysis buffer. The protein concentrations were determined by using a BCA kit (Vigorous, Beijing), and equal amounts (100 g) of protein were loaded and separated by electrophoresis on 12% sodium-dodecyl-sulfate polyacrylamide gels. Separated proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, Calif., USA) and blocked overnight at 4C with 5% fat-free milk in TRIS-buffered saline solution (TBS, 0.05 M, pH 7.4). Thereafter, the antibodies for HSP60, 70, 90, A2, and actin (Ambion, Austin, Tex., USA; 1:10,000) diluted in 1% fat-free milk solution were added respectively, followed by a 2-h incubation at room temperature. Horseradish-peroxidaseconjugated goat anti-mouse IgG antibody (Zymed; 1:5,000) was added, and the membranes were incubated for a further

358 Fig. 4 Localization of HSC70 protein in the rabbit testes during development. a At 1 month. b At 2 months. c At 3 months. d At 4 months. e At 5 months. f At 40 months. HSC70 was immunostained brown (pSg prespermatogonium, Sg spermatogonium, Ley Leydig cell, S Sertoli cell, P pachytene spermatocyte, R round spermatid, E elongated spermatid, Roman numerals spermatogenic stages). Bar 50 m

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2 h at room temperature. The targeted protein bands were visualized by using an enhanced chemiluminescence detection system (Millipore, Billerica, Mass., USA).

expressed before 2 months but was significantly elevated at 3 months and sustained at 4, 5, and 40 months (Fig. 1). Development of postnatal rabbit testis

Results Detection of expression of HSP60, 70, 90, and A2 in developing testes by Western blot Western blots with specific antibodies showed that HSP60, A2, 90, and HSC 70 were expressed in all developmental stages in the rabbit testes. No obvious change in the expression levels of HSP60 and HSC70 occurred among these stages (Fig. 1). However, the protein level of HSP90 appeared to increase in the rabbit testis at 2 months and decreased thereafter (Fig. 1, lane 2). HSPA2 was marginally

In the testes of the newborn rabbit, the seminiferous cords contain two types of cells: round prespermatogonia and columnar Sertoli cells. By the end of the 7th week, prespermatogonia differentiate into spermatogonia, and characteristic type A and B spermatogonia are present. Spermatocytes are seen at the end of the 8th week. Spermatids appear in the 16 week, together with tubular lumen formation (Gondos et al. 1973; Morton 1988). In the rabbit, approximately 48 days are required for a committed type A spermatogonium to differentiate into mature spermatozoa. Eight cellular associations of developing male gametes are recognized in histologic sections of seminifer-

Cell Tissue Res (2011) 344:355363 Fig. 5 Localization of HSP90 protein in the rabbit testes during development. a At 1 months. b At 2 months. c At 3 months. d, e At 5 months (boxed area in d shown at higher magnification in e). f At 40 months. Positive HSC70 staining is brown (pSg prespermatogonium, Sg spermatogonium, S Sertoli cell, P pachytene spermatocyte, R round spermatid, P(L) pachytene or leptotene spermatocyte, E elongated spermatid, Roman numerals spermatogenic stages). Bar 50 m (a-d, f), 30 m (e)

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ous tubules. These eight associations make up the spermatogenic cycle of the rabbit (Fig. 2). Immunolocalization of HSP60 in developing rabbit testes Immunohistochemical staining of rabbit testes with an antibody directed against HSP60 revealed that HSP60 was localized in the cytoplasm of prespermatogonia (germ cells in 1-month-old testis), spermatogonia, spermatocytes, and Sertoli cells within the seminiferous tubules and Leydig cells in the interstitium postnatally at 1 (Fig. 3a), 2 (Fig. 3b), 3 (Fig. 3c), 4 (Fig. 3d), and 5 (Fig. 3e) months of age. From the 4th month and onward, at which time spermatids were produced in the testis, immunostaining for HSP60 was additionally observed in spermatids (see also below). Interestingly, HSP60 seemed to be concentrated in the cytoplasm of spermatogonia in the testis of 40-monthold rabbits (Fig. 3f). Other germ cells of this age, including

spermatocytes at various phases and spermatids, appeared to be negative for immunnostaining for HSP60 (Fig. 3f). No immunostaining was observed in rabbit testis sections probed with antiserum alone (Fig. 3g), indicating the specificity of the antibody. Immunolocalization of HSC70 in developing rabbit testes The immunohistochemical results revealed that HSC70 staining was located in the cytoplasm of spermatogonia throughout the stages examined (Fig. 4). However, no apparent staining was detected in the Leydig cells (Fig. 4d). In the 1- and 2-month-old rabbits, HSC70 staining was clear and was widely distributed in the germ cells. Spermatogonia, Sertoli cells, and round spermatids were stained specifically in the 3-, 4-, and 5-month-old rabbit (Figs. 4c-d). Staining in the 40-month testis was comparatively weak (Fig. 4f). In addition, elongated spermatids in

360 Fig. 6 Localization of HSPA2 protein in rabbit testes during development. a At 2 months. b At 3 months. c, d At 4 months (boxed area in c shown at higher magnification in d). e At 5 months. f At 40 months. Immunostaining for HSPA2 appears brown (P pachytene spermatocyte, Sg spermatogonium, R round spermatid, L(Z) leptotene or zygotene spermatocyte). Bar 50 m (a-c, e, f), 30 m (d)

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rabbits of 4 (Fig. 4d; see also below), 5 (Fig. 4e), and 40 (Fig. 4f) months of age were also positively stained with HSC70, but staining was mainly restricted to the seminiferous tubule at the spermatogenic stages of VII and VIII (Fig. 4f). Immunolocalization of HSP90 in developing rabbit testes Positive staining for HSP90 was observed in the prespermatogonia in the 1-month-old testis (Fig. 5a) and spermatogonia in the 2-month-old testis (Fig. 5b). However, only a few of the spermatogonia expressed HSP90 in the 3month-old (Fig. 5c), 5-month-old (Fig. 5d, e), and 40month-old (Fig. 5f) testis. Spermatocytes and round spermatids, seemed not to express HSP90 (Fig. 5c-f). Comparably, elongated spermatids in the seminiferous tubules at stage VII and VIII showed positive staining for HSP90 (Fig. 5d, f; see also below).

Immunolocalization of HSPA2 in developing rabbit testes No positive staining was found for HSPA2 in the testes of 1- and 2-month-old rabbits (Fig. 6a). Strongly positive staining was observed in the cytoplasm of pachytene spermatocytes and round spermatids in the testes of rabbits aged 3 (Fig. 6b), 4 (Fig. 6b-f; see also below), 5 (Fig. 6e), and 40 (Fig. 6f) months. No staining of HSPA2 was detected in the spermatognia, Sertoli, and Leydig cells. Immunolocalization of HSPs in developing spermatids in rabbit testis In rabbits aged 4 months, HSP60 immunostaining was observed in spermatids, with the intriguing property of being specifically localized at the apical pole of the round spermatids and at the head of the elongated spermatids (Fig. 7a). Elongated spermatids were also immunopositive for

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361 Fig. 7 Localization of HSP60 (a), HSC70 (b), HSP90 (c), and HSPA2 (d) proteins in the spermatids of the 4-month-old rabbit. Positive signals are immunostained in brown (P pachytene spermatocyte, R round spermatid, E elongated spermatid, L(Z) leptotene or zygotene spermatocyte). Bar 30 m

HSC70 (Fig. 7b), HSP90 (Fig. 7c), and HSPA2 (Fig. 7d). Round spermatids however appeared to be immunonegative for HSP90 (Fig. 7c; see also Fig. 5c-f). Leptotene and zygotene spermatocytes in some of the seminiferous tubules showed positive staining for HSPA2 (Fig. 7d).

Discussion In this study, the developmental presence and cellular distribution of HSP60, HSC70, HSPA2, and HSP90 in rabbit testes have been evaluated by immunohistochemistry and Western blot techniques. The results show that these HSPs have different expression patterns during the spermatogenic process. HSP60 is mainly expressed in the cytoplasm of Sertoli, Leydig cells, spermatogonia, and early primary spermatocytes. This expression pattern of HSP60 during rabbit spermatogenesis is in agreement with that previously observed in rat seminiferous epithelium (Meinhardt et al. 1995). An interesting result of this study is that HSP60 has only been observed in the spermatogonium, but not in spermatocytes and spermatids in the testis of senile (40month-old) rabbits. This is probably attributable to the decrease in mitotic activity of the spermatocytes in the testis of the aged animals. A similar correlation between the high levels of HSP60 expression and the mitotic activity of germ cells in spermatogenesis has previously been established in rodents (Meinhardt et al. 1995). An intriguing property of HSP60 is that it is specifically localized at the apical poles in the spermatids of the rabbit, hence revealing an inherent polarity within the spermatid nucleus. These results differ from those showing HSP60 immunoreactivity in boar, stallion, dog, and cat spermatozoa at mitochondrial positions (Volpe et al. 2008). However, they are in agreement with the acrosomal HSP60 localization observed in mouse spermatozoa (Asquith et al. 2004). Whether HSP60 imparts this polarity by itself or whether its localization is induced by other proteins remains to be determined, although Walsh et al. (2008) have identified HSPE1 (formerly known as HSP10) co-immunoprecipitating with HSP60 at the rostral aspect of the sperm head, an ideal position to mediate sperm-zona pellucida interactions. The HSP70 family is one of the HSPs that are most abundantly expressed in the testis (Sarge and Cullen 1997). Our results indicate that HSC70 staining is mainly located in the cytoplasm of prespermatogonia, spermatogonia, and

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spermatocytes. The condensed presence of HSC70 in the spermatogonia reveals its importance in the initial stages of spermatogenesis. Additionally, its expression level does not change at different ages. This is in agreement with the results in boar in which HSC70 is expressed in both immature and adult testes (Huang et al. 2005). Moreover, our data demonstrate the presence of HSC70 in rabbit spermatids. Although no direct evidence is available concerning the presence of HSC70 in spermatids, many investigations have demonstrated the localization of HSP70 immunolabeling in spermatozoa (Matwee et al. 2001; Spinaci et al. 2005; Volpe et al. 2008). HSP90 is expressed in the mouse (Moore et al. 1989), pig (Huang et al. 2005) and rat (Ohsako et al. 1995) testes. Lee (1990) and Gruppi et al. (1991) have detected high levels of HSP90 transcripts in mouse meiotic prophase spermatogenic cells by Northern blot analysis and in situ hybridization. In agreement with their results, our study has found that the expression of HSP90 is intense in prespermatogonia and spermatogonia in rabbit testes. Furthermore, our result demonstrates that the expression level of HSP90 reaches peak levels at postnatal month 2, a time at which germ cell proliferation stops, and meiosis commences in the rabbit testis (Ricken and Viebahn 2002). Thus, the HSP90 expression peak parallels the period of the increase in the number of spermatogonia. This result not only confirms the spermatogonia-localizing pattern of HSP90 shown by immunohistochemistry, but also strongly suggests that HSP90 plays an important role in the development and function of spermatogonia. In addition, our results have shown that the expression pattern of HSP90 in the rabbit testes is similar to that of HSC70. This is possibly explained by a previous study revealing that HSP90 co-precipitates with HSP70 in murine testis (Gruppi and Wolgemuth 1993). Immunostaining for HSP90 has also been detected at the heads of elongated spermatids. This is different from the findings in rat (Ohsako et al. 1995) but is in agreement with a previous study showing that HSP90 is expressed at the spermatozoa surface and becomes tyrosine-phosphorylated during sperm capacitation (Ecroyd et al. 2003). In this study, we have investigated the expression and localization of HSPs in rabbit testes under normal conditions. Our data show some general agreement with results from other species, but also some differences. This work is thus an important supplement to the diverse expression patterns of HSPs found in the gonads of male animals. As previously stated, the rabbit is a favorable animal model for the study of HSPs. This first approach regarding HSP expression in the rabbit testis has provided basic and important information for further elucidation of the influences of heat shock and the roles of HSPs in the reproduction of the rabbit.

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