BACKGROUND
spermatogenesis is a complex biological process involving a number of cellular events including mitosis, meiosis, cell migration, apoptosis and differentiation, which enable the germ cells to undergo several developmental stages, from spermatogonia to primary and secondary spermatocytes, round spermatids and eventually spermatozoa.
it takes approximately 35 days in mice.
to date, hundreds of hsp40s derived from various species have been identified, of which several proteins are involved in spermatogenesis  <cit> .
a human dnaj homologue, dnaja <dig> , binds to the pituitary tumor-transforming gene protein and then plays a role in spermatogenesis  <cit> .
evidence from dnaja1-mutant mice has shown that dnaja <dig> plays a critical role in spermiogenesis by regulating androgen receptor signaling.
the dnajb <dig>  gene encodes a dnaj protein highly expressed in spermatids and spermatozoa of both rodents and amphibians  <cit> , <cit> .
it may be involved in vesicle fusion and protein quality control by means of interaction with heat shock proteins  <cit> .
molecular chaperones belonging to the superfamily of heat shock proteins , their co-chaperones, and the ubiquitination/deubiquitination system may be involved in the production of high-quality spermatozoa  <cit> .
hsp40s generally function as co-chaperones in the hsp <dig> chaperone systems  <cit> - <cit>  that are involved in many processes in cells.
mammalian cells possess a large and diverse family of hsp40/ dnaj proteins, and they can be divided into three subgroups, type i, ii, and iii, according to the presence or absence of the gly/ phe-rich region and the cysteine repeats  <cit> .
dnajb <dig>   is a type ii hsp40/dnaj protein.
dnajb <dig>  is also known as tsarg <dig>   <cit> .
in a previous study, we first reported that dnajb <dig> was expressed abundantly in mouse testis and up-regulated in cryptorchidism  <cit> .
guan et al.
characterized dnajb <dig> in mature mouse testes and epididymal spermatozoa  <cit> .
to date, however, little is known about the time of origin and location of dnajb <dig> during mouse spermatogenesis, which is an important clue to clarifying its function.
this study intends to identify the expression pattern of dnajb <dig> progressively and constitutively during mammalian spermatogenesis.
<dig>
RESULTS
<dig>  dnajb <dig> mrna and dnajb <dig> protein strongly expressed in testis, lung, oviduct and trachea
we performed reverse transcription polymerase chain reaction  to study the expression profile of dnajb <dig> mrna in different mouse tissues.
the results showed that the dnajb <dig> gene was extremely strongly expressed in adult testis, lung, oviduct and trachea .
gapdh was expressed in all kinds of tissues.
we also performed immunoblotting to study the expression pattern of dnajb <dig> in different mouse tissues.
the results showed that dnajb <dig> was expressed in adult testis, lung, oviduct, and trachea , being consistent with the rt-pcr results.
by multi-tissue rt-pcr and western blot, we found that the dnajb <dig> gene and dnajb <dig> protein were expressed not only in the testis, but also in several other ciliated cell-containing tissues like the trachea, lung and oviduct.
in mammals, both sperm flagella and cilia are related to cell migration and movement.
therefore, we speculated that the dnajb <dig> gene may be associated with cell migration and movement.
<dig>  expression pattern of dnajb <dig> mrna and dnajb <dig> protein located at different developmental periods of mouse testis
the different developmental periods of 1- to 9-week-old mice testes were collected for total rna and protein preparations, which were subsequently subjected to real-time quantitative pcr   and immunoblotting  analyses, respectively.
as shown in figure 2a, in mouse testis, the expression of dnajb <dig> mrna was detected as early as postnatal week  <dig>  was developmentally up-regulated at postnatal week  <dig> when the round spermatids began to appear, and then was constant.
after postnatal week  <dig> its expression was constant at a high level.
gapdh was expressed at various development periods in mouse testis as a control.
we also performed immunoblotting to study the expression pattern of dnajb <dig> in these testes.
the expression of dnajb <dig> was undetectable until postnatal week  <dig> when elongated spermatids were apparent .
these data demonstrate that the expression of the dnajb <dig> gene was developmentally regulated and there was an apparent delay in dnajb <dig> protein translation.
dnajb <dig> expressed from postnatal week  <dig> of mouse testis suggested that dnajb <dig> was expressed in the spermatids and spermatozoa.
<dig>  in situ hybridization of dnajb <dig> in mouse testis at different developmental periods
the cellular localization of dnajb <dig> mrna expression was determined by in situ hybridization of mouse testis sections.
the specific tawny hybridization signal in the cytoplasm was distributed inside the tubules from week  <dig> , increased in week  <dig>  and then had a stable expression.
such a distribution is characteristic of the stage-specific expression of dnajb <dig> during mouse spermatogenesis, and is compatible with the qpcr of dnajb <dig> in the mouse seminiferous tubule.
the positive signals correspond to the cytoplasm of spermatocytes and round spermatids located along the spermatogonia in the basement membrane distant from the boundary of the tubules.
under identical conditions, the control probe did not give any signal.
<dig>  immunohistochemical detection of dnajb <dig> in mouse testis at different developmental periods
the seminiferous epithelium from different developmental periods of mouse testis contained different spermatogenic cells.
there were only primitive type a spermatogonia in the seminiferous epithelium of 0-week-old and 1-week-old mice.
type a and type b spermatogonia appeared in the seminiferous epithelium of 8-day-old mouse testis.
preleptotene spermatocytes were the smallest germ cells in sections of 10-day-old mouse testis.
pachytene spermatocytes were observed in the testis of 14-day-old mice.
round spermatids began to appear in the seminiferous epithelium of 18-day-old mouse testis  <cit> .
subcellular localization and distribution of the dnajb <dig> protein at defined stages of spermatogenesis were revealed.
the dnajb <dig> immunohistochemical signal was first seen very weakly in the round spermatid cytoplasm of 3-week-old mice; the signal intensity of dnajb <dig> increased as spermiogenesis proceeded.
a distinct expression pattern of dnajb <dig> was restricted to the spermatid layer in the mice from week  <dig>  the dnajb <dig> immunosignal was present in the cytoplasm of spermatids and flagella of mature spermatozoa that were about to be released into the tubular lumen , consistent with the wb result of dnajb <dig> in different developmental periods of mouse testis.
no immunosignal was detected in any other spermatogenic or somatic cells in the testicular sections.
the negative control using pre-immune serum was free of any signal.
notably, there is a discrepancy between the immunoblotting and immunohistochemical results.
the wb data indicated that the expression of dnajb <dig> was undetectable until postnatal week  <dig>  the dnajb <dig> immunohistochemical signal was first seen very weakly in 3-week-old mice, the signal intensity increasing in postnatal week  <dig>  although round spermatids began to appear in 18-day-old mouse testis, they comprised only 1% of the seminiferous epithelium cells , rising to 4% in 20-day-old mouse testis.
immunoblotting did not detect dnajb <dig> protein in postnatal week  <dig> mice.
however, a very weak immunohistochemical signal was observed in cross sections of seminiferous epithelium in 3-week-old mice.
as shown above, dnajb <dig> proteins were expressed in testis, trachea, lung and oviduct.
we chose testis as a target tissue to investigate the function of dnajb <dig> in spermatogenesis.
the results of the sequential expression profile in mouse testis showed that the dnajb <dig> protein was expressed distinctly at postnatal week  <dig>  more importantly, the positioning study confirmed the expression of dnajb <dig> in the cytoplasm of spermatids and the flagella of mature spermatozoa.
the location of dnajb <dig> in the flagella of mature spermatozoa indicates its function in sperm motility.
the flagellum is a major part of the spermatozoon.
the core of the flagellum is a microtubular structure called the axoneme.
during spermiogenesis, the axoneme develops from one of the two centrioles to form the flagellum.
as the axoneme grows, accessory components including mitochondria, outer dense fibers, a fibrous sheath and an annulus are added to the flagellum to form the sperm middle, principal and end pieces.
in a previous study, yang c found that rsp <dig>  a homologous protein of dnajb <dig> in chlamydomonas, may function as a co-chaperone in the assembly of radial spokes.
in a further study, knockdown of rsp <dig> influenced flagellar motility, and this motility defect could be rescued by rsp <dig> without the j domain  <cit> , <cit> .
guan j considered that dnajb <dig> was localized to the radial spokes of the mouse axoneme  <cit> .
using immunoelectron microscopy analyses of human sperm, we have found that dnajb <dig> is not only localized on the radial spokes of the axoneme in human sperm flagella, but also in other flagellar structures .
our results validate the views of yang c and guan j. therefore, we suggest that dnajb <dig> influences sperm motility.
the expression of dnajb <dig> in spermatids indicates the protein may participate in spermiogenesis.
spermiogenesis is the final stage of spermatogenesis.
during this morphogenetic process, shaping and condensation of the nucleus as well as the formation of the acrosome and the tail take place.
spermiogenesis takes about 2– <dig> weeks in mice.
at this stage, the haploid germ cells do not divide, but morphogenesis occurs  <cit> .
these dramatic changes require a stringent, well-coordinated and unique system that regulates specific patterns of gene and protein expression  <cit> .
several hsp <dig> proteins are involved in spermiogenesis.
dnaj protein msj- <dig>  is a molecular chaperone expressed in spermatids and spermatozoa  <cit> .
mubpy and msj- <dig> label the cytoplasmic side of the acrosomal membrane and the centrosome in mouse spermatozoa, and could have a key role during mouse fertilization  <cit> .
dja <dig> mice revealed a primary defect of sertoli cells in maintaining spermiogenesis at steps  <dig> and  <dig>  <cit> .
more thorough work is needed to elucidate the function and mechanism of dnajb <dig> in spermiogenesis.
<dig>
CONCLUSIONS
in this study, we characterized the profile of the dnajb <dig> gene and dnajb <dig> protein in different mouse tissues at different developmental periods.
we revealed a spatiotemporal association of dnajb <dig>  a type ii hsp <dig>  with the testis and elucidated that dnajb <dig> is expressed in the cytoplasm of spermatids and the flagella of mature spermatozoa.
we suggest that dnajb <dig> participates in the process of spermiogenesis and sperm movement.
<dig> methods
<dig>  animals
balb/c mice were purchased from shanghai, china.
animals were housed in a standard animal facility under controlled temperature  and photoperiod  with free access to water and mouse chow.
all animal experiments were approved by the ethics committee of citic-xiangya reproductive and genetic hospital.
<dig>  rna isolation and rt-pcr
the multi-tissues collected from adult balb/c mice were submerged in trizol  immediately to stabilize the rna.
testes collected from mice at postnatal weeks  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> were submerged in trizol  immediately to stabilize the rna.
we treated rna samples with dnase i   to ensure the elimination of any genomic dna contamination, then verified the purity of the rna samples by means of electrophoresis and measuring od values with a nano drop  <dig> spectrophotometer  operated by nd-1000 v <dig> .
<dig> software.
the rna samples were quantified and the first-strand cdna was synthesized using a revertaid™ first strand cdna synthesis kit .
the cdna samples were subsequently subjected to rt-pcr with dnajb <dig>  and gapdh primers .
pcr was carried out with pfu dna polymerase  .
gapdh was also amplified with an equivalent amount of cdna template as a control.
for the primer sequences, see table  <dig>
<dig>  real-time quantitative pcr
the cdna synthesis was performed at 37°c for 60 min after heat inactivation at 95°c for 10 min.
pcr was performed using 1 × sybr green pcr master mix  on a real-time pcr system .
cycling conditions were as follows: 2 min at 50°c, 10 min at 95°c, and 40 cycles of 15 s at 58°c, plus 1 min at 60°c.
commercial software  was used to calculate ΔΔct relative expression values for all the genes studied, normalized to the gapdh endogenous control.
for the primer sequences, see table  <dig>
<dig>  in situ hybridization
we used gene expression images measured using in situ hybridization .
to measure the expression of a target gene, ish uses dig-labeled dna sequences that are complementary to the target gene dnajb <dig> rna.
these dna probes are cloned and applied to each mouse testis section.
the complementary probes hybridize to the target rna sequence inside the cells, while the non-bound probes are washed away.
dnajb <dig> probe sequences: 5′-ctccatggacaactggctacgtcttccatg-3′, 5-caccctcgcttccgcagggagcatgacaac-3′, 5′-ttcatcttctttgacatccagttccccacc-3′.
this dig labeling captures the spatial pattern of expression of a target gene across the testis.
preparation of riboprobe and ish digoxigenin-11-utp-labeled riboprobe  was used according to the manufacturer’s instructions .
sections  were deparaffinized and rehydrated, washed in pbs-t  and digested with proteinase k .
the reaction was then blocked with a stop-solution containing 2 mg/ml glycine in pbs-t. after two washes with pbs-t, the sections were post-fixed with 4% formaldehyde in pbs-t for 30 min.
pre-hybridization with hybridization solution consisting of 50% formamide, 50 μg/ml heparin, 500 μg/ml yeast trna,  <dig> % tween  <dig>  and 5× standard sodium citrate  was carried out for 1 h at 37°c.
hybridization was performed with 15% riboprobe in hybridization solution overnight at 37°c.
the sections were rinsed with pbs-t and washing solution , incubated at room temperature  with horse serum  and denatured at 95°c for 5 min by applying the probe solution to them and hybridized with anti-digoxigenin-fab-antibody  for 1 h at r.t. finally, the sections were incubated in a 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium liquid-substrate system .
control experiments were performed using the corresponding sense crna .
two specimens from each developmental stage were examined.
<dig>  protein preparation
the multi-tissues collected from adult balb/c mice were lysed in ripa buffer  supplemented with 1 × protease inhibitor cocktail  in a dounce homogenizer.
testes collected from mice at postnatal weeks  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> were lysed in ripa buffer.
lysates were sonicated to disrupt genomic dna and then centrifuged at  <dig>  g for 10 min at 4°c to remove tissue debris.
protein quantification was measured by a bcatm protein assay kit .
we usually used 50–100 μg of proteins for immunoblotting.
the clarified lysates were mixed with 5 × sds sample buffer  and boiled for 5 min at 95°c.
then these protein samples were kept at −20°c for later use.
<dig>  immunoblotting
the protein samples were analyzed by western blotting with antibodies against dnajb <dig> and beta-actin, respectively.
the multi-tissue proteins from adult balb/c mice and the testes proteins from different stages of balb/c mice were separated by sds–page on a 12% polyacrylamide gel and transferred to pvdf membrane .
western blotting analysis was performed as described previously  <cit> .
the membranes were blocked with tbst  containing 5% nonfat dry milk for 4 h at r.t. and incubated with the primary antibodies in blocking buffer overnight at 4°c.
after washing with tbst, the blots were incubated with the secondary antibodies in blocking buffer for 1 h at r.t. both antibodies were conjugated to horseradish peroxidase.
the membranes were detected with lumigen™ ps- <dig> detection kit  according to the manufacturer’s directions and exposed to x-ray film .
the primary antibodies used in this study were tsarg <dig>  goat polyclonal antibody  , and mouse anti-beta-actin monoclonal antibody  .
the secondary antibodies were hrp-conjugated donkey anti-goat igg antibody   and hrp-conjugated goat anti-mouse igg antibody  .
<dig>  immunohistochemistry
mouse testes were collected from adult balb/c mice and balb/c mice at postnatal weeks  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig>  and immediately fixed in freshly made 4% paraformaldehyde  solution for 4 h at r.t. after fixation, tissues were embedded in paraffin wax, and 5–6-μm sections were prepared and kept at 4°c until further use.
after deparaffinization and rehydration, the sections were boiled in  <dig>  m sodium citrate  in a microwave oven for 10 min to retrieve the antigens.
the following staining procedure was performed as described previously  <cit> .
after a brief rinse in 1 × pbs, the sections were blocked with 5% bsa in 1 × pbs  for 30 min at r.t and then incubated with the primary antibody diluted in blocking buffer overnight at 4°c.
the primary antibodies used in this experiment were tsarg <dig>  goat polyclonal antibody  , and goat pre-immune sera.
after washing in 1 × pbs, the sections were incubated with hrp-conjugated donkey anti-goat igg antibody   diluted in blocking buffer for 1 h at r.t. after counterstaining with hematoxylin, the sections were dehydrated, mounted in resin and examined with an olympus bx <dig> microscope .
images were captured using a hamamatsu orca-hr digital camera  with a microcolor filter operated by cri rgb color merge software .
competing interests
the authors declare that they have no competing interests.
authors’ contributions
lwn participated in the design of the study, carried out all the experimental procedures and wrote the manuscript.
lg conceived and supervised the study, and revised the manuscript.
both authors read and approved the final manuscript.
acknowledgments
we would like to thank prof. lu gx and the institute of reproduction and stem cell engineering for providing the hamamatsu orca-hr digital camera and cri rgb color merge software.
we also thank prof. xing xw and the anonymous reviewers for helpful comments.
this work was supported by the mechanism study of a novel mouse gene dnajb <dig> and its human homolog dnajb <dig> during spermiogenesis from the projects of national natural science foundation .

 spermatogenesis is a complex biological process involving a number of cellular events including mitosis, meiosis, cell migration, apoptosis and differentiation, which enable the germ cells to undergo several developmental stages, from spermatogonia to primary and secondary spermatocytes, round spermatids and eventually spermatozoa. it takes approximately 35 days in mice. to date, hundreds of hsp40s derived from various species have been identified, of which several proteins are involved in spermatogenesis. a human dnaj homologue, dnaja, binds to the pituitary tumor-transforming gene protein and then plays a role in spermatogenesis. evidence from dnaja1-mutant mice has shown that dnaja plays a critical role in spermiogenesis by regulating androgen receptor signaling. the dnajb gene encodes a dnaj protein highly expressed in spermatids and spermatozoa of both rodents and amphibians,. it may be involved in vesicle fusion and protein quality control by means of interaction with heat shock proteins. molecular chaperones belonging to the superfamily of heat shock proteins, their co-chaperones, and the ubiquitination/deubiquitination system may be involved in the production of high-quality spermatozoa. hsp40s generally function as co-chaperones in the hsp chaperone systems - that are involved in many processes in cells. mammalian cells possess a large and diverse family of hsp40/ dnaj proteins, and they can be divided into three subgroups, type i, ii, and iii, according to the presence or absence of the gly/ phe-rich region and the cysteine repeats. dnajb is a type ii hsp40/dnaj protein. dnajb is also known as tsarg. in a previous study, we first reported that dnajb was expressed abundantly in mouse testis and up-regulated in cryptorchidism. guan et al. characterized dnajb in mature mouse testes and epididymal spermatozoa. to date, however, little is known about the time of origin and location of dnajb during mouse spermatogenesis, which is an important clue to clarifying its function. this study intends to identify the expression pattern of dnajb progressively and constitutively during mammalian spermatogenesis.  dnajb mrna and dnajb protein strongly expressed in testis, lung, oviduct and trachea we performed reverse transcription polymerase chain reaction to study the expression profile of dnajb mrna in different mouse tissues. the results showed that the dnajb gene was extremely strongly expressed in adult testis, lung, oviduct and trachea. gapdh was expressed in all kinds of tissues. we also performed immunoblotting to study the expression pattern of dnajb in different mouse tissues. the results showed that dnajb was expressed in adult testis, lung, oviduct, and trachea, being consistent with the rt-pcr results. by multi-tissue rt-pcr and western blot, we found that the dnajb gene and dnajb protein were expressed not only in the testis, but also in several other ciliated cell-containing tissues like the trachea, lung and oviduct. in mammals, both sperm flagella and cilia are related to cell migration and movement. therefore, we speculated that the dnajb gene may be associated with cell migration and movement. expression pattern of dnajb mrna and dnajb protein located at different developmental periods of mouse testis the different developmental periods of 1- to 9-week-old mice testes were collected for total rna and protein preparations, which were subsequently subjected to real-time quantitative pcr and immunoblotting analyses, respectively. as shown in figure 2a, in mouse testis, the expression of dnajb mrna was detected as early as postnatal week was developmentally up-regulated at postnatal week when the round spermatids began to appear, and then was constant. after postnatal week its expression was constant at a high level. gapdh was expressed at various development periods in mouse testis as a control. we also performed immunoblotting to study the expression pattern of dnajb in these testes. the expression of dnajb was undetectable until postnatal week when elongated spermatids were apparent. these data demonstrate that the expression of the dnajb gene was developmentally regulated and there was an apparent delay in dnajb protein translation. dnajb expressed from postnatal week of mouse testis suggested that dnajb was expressed in the spermatids and spermatozoa. in situ hybridization of dnajb in mouse testis at different developmental periods the cellular localization of dnajb mrna expression was determined by in situ hybridization of mouse testis sections. the specific tawny hybridization signal in the cytoplasm was distributed inside the tubules from week, increased in week and then had a stable expression. such a distribution is characteristic of the stage-specific expression of dnajb during mouse spermatogenesis, and is compatible with the qpcr of dnajb in the mouse seminiferous tubule. the positive signals correspond to the cytoplasm of spermatocytes and round spermatids located along the spermatogonia in the basement membrane distant from the boundary of the tubules. under identical conditions, the control probe did not give any signal. immunohistochemical detection of dnajb in mouse testis at different developmental periods the seminiferous epithelium from different developmental periods of mouse testis contained different spermatogenic cells. there were only primitive type a spermatogonia in the seminiferous epithelium of 0-week-old and 1-week-old mice. type a and type b spermatogonia appeared in the seminiferous epithelium of 8-day-old mouse testis. preleptotene spermatocytes were the smallest germ cells in sections of 10-day-old mouse testis. pachytene spermatocytes were observed in the testis of 14-day-old mice. round spermatids began to appear in the seminiferous epithelium of 18-day-old mouse testis. subcellular localization and distribution of the dnajb protein at defined stages of spermatogenesis were revealed. the dnajb immunohistochemical signal was first seen very weakly in the round spermatid cytoplasm of 3-week-old mice; the signal intensity of dnajb increased as spermiogenesis proceeded. a distinct expression pattern of dnajb was restricted to the spermatid layer in the mice from week the dnajb immunosignal was present in the cytoplasm of spermatids and flagella of mature spermatozoa that were about to be released into the tubular lumen, consistent with the wb result of dnajb in different developmental periods of mouse testis. no immunosignal was detected in any other spermatogenic or somatic cells in the testicular sections. the negative control using pre-immune serum was free of any signal. notably, there is a discrepancy between the immunoblotting and immunohistochemical results. the wb data indicated that the expression of dnajb was undetectable until postnatal week the dnajb immunohistochemical signal was first seen very weakly in 3-week-old mice, the signal intensity increasing in postnatal week although round spermatids began to appear in 18-day-old mouse testis, they comprised only 1% of the seminiferous epithelium cells, rising to 4% in 20-day-old mouse testis. immunoblotting did not detect dnajb protein in postnatal week mice. however, a very weak immunohistochemical signal was observed in cross sections of seminiferous epithelium in 3-week-old mice. as shown above, dnajb proteins were expressed in testis, trachea, lung and oviduct. we chose testis as a target tissue to investigate the function of dnajb in spermatogenesis. the results of the sequential expression profile in mouse testis showed that the dnajb protein was expressed distinctly at postnatal week more importantly, the positioning study confirmed the expression of dnajb in the cytoplasm of spermatids and the flagella of mature spermatozoa. the location of dnajb in the flagella of mature spermatozoa indicates its function in sperm motility. the flagellum is a major part of the spermatozoon. the core of the flagellum is a microtubular structure called the axoneme. during spermiogenesis, the axoneme develops from one of the two centrioles to form the flagellum. as the axoneme grows, accessory components including mitochondria, outer dense fibers, a fibrous sheath and an annulus are added to the flagellum to form the sperm middle, principal and end pieces. in a previous study, yang c found that rsp a homologous protein of dnajb in chlamydomonas, may function as a co-chaperone in the assembly of radial spokes. in a further study, knockdown of rsp influenced flagellar motility, and this motility defect could be rescued by rsp without the j domain,. guan j considered that dnajb was localized to the radial spokes of the mouse axoneme. using immunoelectron microscopy analyses of human sperm, we have found that dnajb is not only localized on the radial spokes of the axoneme in human sperm flagella, but also in other flagellar structures. our results validate the views of yang c and guan j. therefore, we suggest that dnajb influences sperm motility. the expression of dnajb in spermatids indicates the protein may participate in spermiogenesis. spermiogenesis is the final stage of spermatogenesis. during this morphogenetic process, shaping and condensation of the nucleus as well as the formation of the acrosome and the tail take place. spermiogenesis takes about 2– weeks in mice. at this stage, the haploid germ cells do not divide, but morphogenesis occurs. these dramatic changes require a stringent, well-coordinated and unique system that regulates specific patterns of gene and protein expression. several hsp proteins are involved in spermiogenesis. dnaj protein msj- is a molecular chaperone expressed in spermatids and spermatozoa. mubpy and msj- label the cytoplasmic side of the acrosomal membrane and the centrosome in mouse spermatozoa, and could have a key role during mouse fertilization. dja mice revealed a primary defect of sertoli cells in maintaining spermiogenesis at steps and. more thorough work is needed to elucidate the function and mechanism of dnajb in spermiogenesis.  in this study, we characterized the profile of the dnajb gene and dnajb protein in different mouse tissues at different developmental periods. we revealed a spatiotemporal association of dnajb a type ii hsp with the testis and elucidated that dnajb is expressed in the cytoplasm of spermatids and the flagella of mature spermatozoa. we suggest that dnajb participates in the process of spermiogenesis and sperm movement. methods animals balb/c mice were purchased from shanghai, china. animals were housed in a standard animal facility under controlled temperature and photoperiod with free access to water and mouse chow. all animal experiments were approved by the ethics committee of citic-xiangya reproductive and genetic hospital. rna isolation and rt-pcr the multi-tissues collected from adult balb/c mice were submerged in trizol immediately to stabilize the rna. testes collected from mice at postnatal weeks and were submerged in trizol immediately to stabilize the rna. we treated rna samples with dnase i to ensure the elimination of any genomic dna contamination, then verified the purity of the rna samples by means of electrophoresis and measuring od values with a nano drop spectrophotometer operated by nd-1000 v. software. the rna samples were quantified and the first-strand cdna was synthesized using a revertaid™ first strand cdna synthesis kit. the cdna samples were subsequently subjected to rt-pcr with dnajb and gapdh primers. pcr was carried out with pfu dna polymerase. gapdh was also amplified with an equivalent amount of cdna template as a control. for the primer sequences, see table  real-time quantitative pcr the cdna synthesis was performed at 37°c for 60 min after heat inactivation at 95°c for 10 min. pcr was performed using 1 × sybr green pcr master mix on a real-time pcr system. cycling conditions were as follows: 2 min at 50°c, 10 min at 95°c, and 40 cycles of 15 s at 58°c, plus 1 min at 60°c. commercial software was used to calculate ΔΔct relative expression values for all the genes studied, normalized to the gapdh endogenous control. for the primer sequences, see table  in situ hybridization we used gene expression images measured using in situ hybridization. to measure the expression of a target gene, ish uses dig-labeled dna sequences that are complementary to the target gene dnajb rna. these dna probes are cloned and applied to each mouse testis section. the complementary probes hybridize to the target rna sequence inside the cells, while the non-bound probes are washed away. dnajb probe sequences: 5′-ctccatggacaactggctacgtcttccatg-3′, 5-caccctcgcttccgcagggagcatgacaac-3′, 5′-ttcatcttctttgacatccagttccccacc-3′. this dig labeling captures the spatial pattern of expression of a target gene across the testis. preparation of riboprobe and ish digoxigenin-11-utp-labeled riboprobe was used according to the manufacturer’s instructions. sections were deparaffinized and rehydrated, washed in pbs-t and digested with proteinase k. the reaction was then blocked with a stop-solution containing 2 mg/ml glycine in pbs-t. after two washes with pbs-t, the sections were post-fixed with 4% formaldehyde in pbs-t for 30 min. pre-hybridization with hybridization solution consisting of 50% formamide, 50 μg/ml heparin, 500 μg/ml yeast trna, % tween and 5× standard sodium citrate was carried out for 1 h at 37°c. hybridization was performed with 15% riboprobe in hybridization solution overnight at 37°c. the sections were rinsed with pbs-t and washing solution, incubated at room temperature with horse serum and denatured at 95°c for 5 min by applying the probe solution to them and hybridized with anti-digoxigenin-fab-antibody for 1 h at r.t. finally, the sections were incubated in a 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium liquid-substrate system. control experiments were performed using the corresponding sense crna. two specimens from each developmental stage were examined. protein preparation the multi-tissues collected from adult balb/c mice were lysed in ripa buffer supplemented with 1 × protease inhibitor cocktail in a dounce homogenizer. testes collected from mice at postnatal weeks and were lysed in ripa buffer. lysates were sonicated to disrupt genomic dna and then centrifuged at  g for 10 min at 4°c to remove tissue debris. protein quantification was measured by a bcatm protein assay kit. we usually used 50–100 μg of proteins for immunoblotting. the clarified lysates were mixed with 5 × sds sample buffer and boiled for 5 min at 95°c. then these protein samples were kept at −20°c for later use. immunoblotting the protein samples were analyzed by western blotting with antibodies against dnajb and beta-actin, respectively. the multi-tissue proteins from adult balb/c mice and the testes proteins from different stages of balb/c mice were separated by sds–page on a 12% polyacrylamide gel and transferred to pvdf membrane. western blotting analysis was performed as described previously. the membranes were blocked with tbst containing 5% nonfat dry milk for 4 h at r.t. and incubated with the primary antibodies in blocking buffer overnight at 4°c. after washing with tbst, the blots were incubated with the secondary antibodies in blocking buffer for 1 h at r.t. both antibodies were conjugated to horseradish peroxidase. the membranes were detected with lumigen™ ps- detection kit according to the manufacturer’s directions and exposed to x-ray film. the primary antibodies used in this study were tsarg goat polyclonal antibody, and mouse anti-beta-actin monoclonal antibody. the secondary antibodies were hrp-conjugated donkey anti-goat igg antibody and hrp-conjugated goat anti-mouse igg antibody. immunohistochemistry mouse testes were collected from adult balb/c mice and balb/c mice at postnatal weeks and and immediately fixed in freshly made 4% paraformaldehyde solution for 4 h at r.t. after fixation, tissues were embedded in paraffin wax, and 5–6-μm sections were prepared and kept at 4°c until further use. after deparaffinization and rehydration, the sections were boiled in  m sodium citrate in a microwave oven for 10 min to retrieve the antigens. the following staining procedure was performed as described previously. after a brief rinse in 1 × pbs, the sections were blocked with 5% bsa in 1 × pbs for 30 min at r.t and then incubated with the primary antibody diluted in blocking buffer overnight at 4°c. the primary antibodies used in this experiment were tsarg goat polyclonal antibody, and goat pre-immune sera. after washing in 1 × pbs, the sections were incubated with hrp-conjugated donkey anti-goat igg antibody diluted in blocking buffer for 1 h at r.t. after counterstaining with hematoxylin, the sections were dehydrated, mounted in resin and examined with an olympus bx microscope. images were captured using a hamamatsu orca-hr digital camera with a microcolor filter operated by cri rgb color merge software. competing interests the authors declare that they have no competing interests. authors’ contributions lwn participated in the design of the study, carried out all the experimental procedures and wrote the manuscript. lg conceived and supervised the study, and revised the manuscript. both authors read and approved the final manuscript. acknowledgments we would like to thank prof. lu gx and the institute of reproduction and stem cell engineering for providing the hamamatsu orca-hr digital camera and cri rgb color merge software. we also thank prof. xing xw and the anonymous reviewers for helpful comments. this work was supported by the mechanism study of a novel mouse gene dnajb and its human homolog dnajb during spermiogenesis from the projects of national natural science foundation.
this study intends to identify the expression pattern of dnajb during mammalian spermatogenesis. hundreds of hsp40s derived from various species have been identified, of which several proteins are involved in spermatogenesis. in a previous study, we cloned mouse dnajb which is up-regulated in cryptorchidism. dnajb is a type ii hsp40/dnaj protein. based on the spatiotemporal expression observed in the cytoplasm of spermatids and sperm flagella, we suggest that dnajb participates in spermiogenesis and the motility of mature spermatozoa. rt-pcr and western blot revealed that the dnajb gene and dnajb protein were expressed not only in the testis but also in several other ciliated cell-containing tissues like the trachea, lung and oviduct. quantitative pcr showed that the expression of dnajb mrna in testis was detectable as early as postnatal week and sharply increased from postnatal week western blotting and immunohistochemistry determined that the dnajb protein, which was located in the cytoplasm of spermatids and the sperm flagellum, was detectable from postnatal week 
 hundreds of hsp40s derived from various species have been identified, of which several proteins are involved in spermatogenesis. dnajb is a type ii hsp40/dnaj protein. in a previous study, we cloned mouse dnajb which is up-regulated in cryptorchidism. to date, however, little is known about the localization and functions of dnajb during spermatogenesis. this study intends to identify the expression pattern of dnajb during mammalian spermatogenesis.  rt-pcr and western blot revealed that the dnajb gene and dnajb protein were expressed not only in the testis but also in several other ciliated cell-containing tissues like the trachea, lung and oviduct. quantitative pcr showed that the expression of dnajb mrna in testis was detectable as early as postnatal week and sharply increased from postnatal week western blotting and immunohistochemistry determined that the dnajb protein, which was located in the cytoplasm of spermatids and the sperm flagellum, was detectable from postnatal week  based on the spatiotemporal expression observed in the cytoplasm of spermatids and sperm flagella, we suggest that dnajb participates in spermiogenesis and the motility of mature spermatozoa. dnajb13spermatidflagellaspermiogenesismotility 