animals : five genetically modified mice , b6j - xy tg4 , were 
 generated from the jackson laboratory   by way of 
 the national institute for basic biology in okazaki , japan ( prof . k. morohashi ) . except for 
 the sry allele from mus m. domesticus poschiavinus 
 ( sry ) , the sry allele from the 129 
 inbred strain ( sry ) is inserted as a transgene ( tg ) into an 
 ectopic position in the genome . a 14.6 kb dna fragment containing the 
 sry gene was injected into the male pronucleus in 
 fertilized eggs obtained from a b6j - xx mated to b6j - xy hermaphrodites . b6j - xy tg 
 ( sry ) mice on an inbred c57bl/6n ( b6n ) ( purchased from 
 japan slc , inc . , hamamatsu , japan ) background were generated by backcrossing for more than 
 11 generations . their offspring having only the sry allele from mus m. 
 domesticus poschiavinus ( sry ) were selected and 
 are the subject of this paper . all mice were maintained in individual 40.5  20.5  18.5-cm 
 ventilated cages ( sealsafe plus mouse ; tecniplast s.p.a , buguggiate , italy ) under specific 
 pathogen - free conditions with controlled temperature ( 23  2c ) and humidity ( 50  10% ) on a 
 12-hr light / dark cycle at the kobe university life - science laboratory with ad 
 libitum access to water and a pellet diet ( dc-8 ; clea japan , inc . , this study was approved by the institutional animal care and use committee ( permission 
 # 25 - 06 - 03 ) and carried out according to the kobe university animal experimental 
 regulation . dissection , staging and genotyping of fetal mouse gonads : timed matings 
 were used for all embryonic experiments . the gonads and mesonephroi that were removed from the embryos between 
 11.5 and 12.5 dpc were precisely staged by counting the number of ts : 11.5 dpc is ~18 ts , 
 and 12.5 dpc is ~30 ts . the sexual phenotype of 
 weaning - age mice was determined by examining the distance from the anus to the genitalia 
 ( anogenital distance : agd ) . fetal sex and the presence of the 
 sry and sry alleles were 
 determined by multiplex pcr ( polymerase chain reaction ) . the chromosomal sex of each fetus 
 was confirmed by pcr using the primer pair 5-ggg act cca agc ttc aat ca and 5-tgg agg agg 
 aag aaa agc aa to detect the interleukin-3 ( il-3 ) region 
 as an internal control , and the primer pair 5-ccc ctt tct aag cag aca tc and 5-tca cac taa 
 cct cac agg cc to detect y - specific b10-derived sequences 
 ( yb-10 ) , a region located on the long arm of y chromosome . the amplified 
 products were electrophoresed in 2% agarose gel , dyed by ethidium bromide and visualized 
 with a uv transilluminator . sry alleles : in addition to the primer pairs described above , a region of 
 the sry hmg box was amplified from genomic dna using 5-tgg gac tgg tga caa 
 ttg tc and 5-gag tac agg tgt gca gct ct as primers and the pcr product digested using 
 mbo i. the pcr conditions were 30 cycles of 94c for 45 sec , 55c for 45 
 sec and 72c for 45 sec . the sry allele contains an 
 mbo i recognition site , resulting in two fragments of 23 and 130 bp , 
 while the sry allele lacks the mbo i 
 recognition site , resulting in an undigested 153 bp fragment . tissue preparation : genital tissues were fixed in 4% paraformaldehyde in 
 0.1 m phosphate buffer ( ph 7.4 ) for 24 hr at 4c . the specimens were dehydrated with an 
 ethanol series followed by xylene and then embedded in paraffin . then , 
 4-m - thick sections were cut by a sliding microtome ( sm2000r ; leica 
 microsystems , wetzlar , germany ) and placed on slide glasses ( platinum ; matsunami glass ind . , histological and immunohistochemical analyses : for the general 
 histological analyses , tissue sections were stained with hematoxylin and eosin ( he ; merck 
 ltd . , japan , tokyo , japan ) after their deparaffinization and hydration . to detect nr5a1 ( a 
 generous gift from dr . k. morohashi , kyushu university , japan ) , sox9 ( santa cruz 
 biotechnology , inc . , santa cruz , ca , u.s.a . ) and forkhead box l2 ( foxl2 ) ( affinity 
 bioreagents , golden , co , u.s.a . ) , we performed the following immunohistochemical protocol by 
 using anti - nr5a1 rabbit polyclonal antibody ( 1:16,000 ) , anti - sox9 rabbit polyclonal antibody 
 ( 1:300 ) and anti - foxl2 rabbit polyclonal antibody ( 1:500 ) . the sections were immersed in 
 absolute methanol and 0.5% h2o2 for 30 min at room temperature ( rt ) to 
 quench the endogenous peroxidase activity . they were then incubated with blocking one histo 
 ( nacalai tesque , inc . , kyoto , japan ) for 1 hr at rt for protein blocking and then incubated 
 with each primary antibody in phosphate - buffered saline ( pbs ) containing 0.05% tween-20 
 ( pbst ; ph 7.4 ) for 18 hr at 4c . after being washed with pbst , the sections were reacted 
 with dako envision+ system - hrp ( horseradish peroxidase ) labeled polymer ( envision+ ; dako 
 japan , tokyo , japan ) in tris - hcl buffer ( ph 7.6 ) for 1 hr at rt . immunoreactivity was then 
 detected by incubation with 3,3-diaminobenzidine solution ( envision+ kit / hrp[dab ] ; dako ) . finally , the 
 sections were placed in a graded series of ethanol , dehydrated with absolute ethanol , 
 cleared by xylene and coverslipped with eukitt ( o. kindler , gmbh , freiburg , germany ) . whole - mount immunohistochemistry : gonad / mesonephros complexes were fixed 
 in 4% paraformaldehyde in 0.1 m phosphate buffer ( ph 7.4 ) for 24 hr at 4c . the specimens 
 were dehydrated with 100% methanol for 8 hr at 4c ; then passed through three cycles of 
 chilling in 1% h2o2 dissolved in absolute methanol to 80c in a 
 freezer and thawing to 4c to break down the cell membranes and quench the endogenous 
 peroxidase activity . next , the specimens were rinsed with 50% methanol in 0.1 m pbs for 1 
 hr , followed by 0.1 m pbs alone for 1 hr , then incubated with 0.1% albumin serum in blocking 
 one histo ( nacalai tesque , inc . ) at 4c for 18 hr and reacted with anti - sry rabbit 
 polyclonal antibody ( a generous gift from dr . d. wilhelm , monash university , australia ; 
 1:800 ) or anti - sox9 rabbit polyclonal antibody ( 1:600 ) at 4c for 24 hr . the tissues were 
 washed with pbs three times for 2 hr each and incubated with a dako envision+ system - hrp 
 labeled polymer ( dako ) at 4c for 42 hr . after washing with pbs as described above , they 
 were incubated with 0.17% dab in 0.05 m tris - hcl buffer ( ph 7.6 ) at 4c for 2 hr . immunoreactivities were visualized by using 0.17% dab and 0.3% h2o2 in 
 the tris - hcl ( ph 7.6 ) . negative controls , in which the primary antibodies were replaced with 
 nonimmunized serum , did not show nonspecific staining . sequence analysis : the sry coding sequence ( cds ) was analyzed by direct 
 sequencing of pcr products by the capillary electrophoresis systems . a total of 100150 
 ng of genomic dna was extracted from liver tissue using a qiaamp dna 
 microkit ( qiagen , hilden , germany ) . genomic dna was used as a template for the primers 
 msry-933 : 5-gca tgg tga tgc tgt aag gaa and msry+1195 : 5-gtg ttg gca tag gta gga gaa aag 
 in a 50-l reaction . pcr was performed by conventional techniques using 
 prime star gxl dna polymerase ( takara bio , otsu , japan ) employing 35 cycles of 
 98c for 10 sec , 60c for 15 sec and 68c for 100 sec . the pcr product was assayed for 
 specificity on a 2% agarose gel ( the expected pcr product size was 2,152 bp ) and then 
 purified for sequencing using a qiaquick gel extraction kit ( qiagen ) . a total of 50 
 ng of purified sry cds pcr product was directly sequenced using the 
 primers for sry ( table 1table 1.primers used for sry sequencingnameforward / reverseprimer sequence ( 5 to 3 direction)msry-537forwardcgttacagggcaaaagaactcamsry-328forwardggaggagggataaatattttcttacamsry-97forwardagatcttgatttttagtgttcmsry+162forwardttccaggaggcacagagattmsry+320forwardcccacatgccatcacatacamsry+410forwardtatccccactgggcacctmsry+1083reversegtaagctgctggtcgtggaamsry+1132forwardagcatacaccataccaggagcamsry+1195reversegtgttggcataggtaggagaaaagthe first base of sry nucleotide sequence , adenine , is set as 
 position one . we used mus musculus y chromosome sequence from ncbi ( gene 
 i d : 21674 ) as a reference . ) . sequencing of pcr products was outsourced and analyzed on an abi3730xl dna 
 analyzer ( applied biosystems , foster city , ca , u.s.a . ) by fasmac co. , ltd . dna sequence analysis and alignments were performed using the gene studio computer program 
 with subsequent hand editing . genomic dna from at least three b6n - xy mice was 
 used as a pcr template and for the sequence reactions . the first base of sry nucleotide sequence , adenine , is set as 
 position one . we used mus musculus y chromosome sequence from ncbi ( gene 
 i d : 21674 ) as a reference . quantification of ovarian follicles : the number of follicles was counted 
 in individual tissue sections . if one follicle was present in several continuous sections by 
 using ocular micrometer under light microscopy , it was only counted in the first section in 
 order to avoid repetition . the means were calculated , and the data are presented as the 
 means  standard deviation ( sd ) . fertility test : each b6n - xy male or b6n - xy 
 female offspring ( 58 weeks of age ) was housed with a b6n - xx or b6n - xy mouse 
 ( 610 weeks of age ) that was known to be fertile . the presence or absence of copulation 
 plugs the female mouse that had 
 copulated was housed singly until the expected delivery day . if pregnancy was not confirmed 
 after 12 days of delivery , this process was repeated two more times . statistical analysis : statistical analyses were performed with excel 
 statistics 2012 ( ssri , ltd . , tokyo , japan ) . all data were analyzed by two - way analysis of 
 variance ( anova ) followed by tukey - kramer s post hoc test . sexual development of b6n - xy mice : when analyzing 
 b6n - xy mice , which had been established by a cross between a male 
 b6j - xy transgenic mouse with the 14.6 kb dna fragment containing the 
 sry males and a b6n - xx females , we found a broad range of 
 gonadal phenotypes , but typically four types of external and internal 
 genitalia  i.e . , genitalia with bilateral testes ( t / t ) , bilateral ovaries 
 ( o / o ) , bilateral ovotestes ( ot / ot ) and a testis on one side and an ovary on another ( o / t , 
 hermaphrodite)with the same genotype ( fig . 1afig . the distance between the anus and penis or 
 vagina ( agd ) is indicated . gross anatomy of isolated 
 internal genitalia and associated reproductive organs from adult b6n - xy 
 mice . t : testis ; o : ovary ; ot : ovotestis ( b ) frequency analysis of abnormal sex 
 differentiation determined by examining the internal genitalia of adult mice . ( c ) agd of adult b6n - xy 
 mice and control mice . ) . hermaphrodites and individuals with unilateral testis or ambiguous gonads had both 
 nipples and penises ( fig . notably , o / t mice 
 had a right ovary and left testis with extraordinary frequency at few years since 
 assignation from the jackson laboratory ( fig . 1a ) . 
 the frequency of these phenotypes in the growing stages ( pre - sm : pre - sexual maturation at 
 the ages of 2 weeks ; sm : sexual maturation at the ages of 9 weeks ; post - sm : post - sexual 
 maturation at the ages of 18 weeks ) showed that over 36% of our b6n - xy mice 
 displayed normal sex differentiation ( t / t individuals ) ( fig . the boundary of these phenotypes seemed 
 ill - defined , and some b6n - xy individuals had ambiguous gonads . although the 
 b6n - xy testes and accessory reproductive glands looked similar to those of 
 the b6n - xy , some b6n - xy testes were smaller . the ovaries and 
 accessory reproductive glands looked similar to those of the b6n - xx . the agd of these 
 animals was the same as that of the b6n - xx and shorter than that of the b6n - xy 
 mice . the distance between the anus and penis or 
 vagina ( agd ) is indicated . gross anatomy of isolated 
 internal genitalia and associated reproductive organs from adult b6n - xy 
 mice . t : testis ; o : ovary ; ot : ovotestis ( b ) frequency analysis of abnormal sex 
 differentiation determined by examining the internal genitalia of adult mice . gonadal descent was detected in t / t , ot / ot and o / t individuals , but not in o / o mice , even 
 in the post - sm period . the body weights and agds in mice with gonadal descent were 
 significantly higher than those in mice without gonadal descent ( data not shown ) . histological and immunohistological findings : histopathology was evaluated 
 using cross - sections of gonads stained with he by light microscopy . to investigate the 
 etiology of sex reversal , we examined the expression of nr5a1 , which is a critical regulator 
 of sexual development and reproduction , the 
 testicular sertoli cell marker sox9   and the 
 ovarian somatic cell marker foxl2 . because the 
 b6n - xy t / t individual testes showed normal cell arrangements with 
 well - developed seminiferous tubules consisting of spermatogonia , spermatocytes , spermatids 
 and sperm according to each sexual development stage , the gonads contained no foxl2-positive 
 cells , but only sox9-positive cells ( fig . 2.general histology and immunohistochemistry in the b6n - xy mice in each 
 sexual developmental stage . ( a ) hematoxylin and eosin staining ( he ) and sox9 
 immunostaining on transversal sections at pre - sexual maturation ( pre - sm ) , sm and 
 post - sm in t / t individuals . the testis shows a luminal structure and normal cell 
 arrangements including elongated spermatids in the wall of seminiferous tubules in the 
 sm and post - sm ( a  c ) . clusters of leydig cells are present in the intertubular space 
 ( arrows , a  c ) . ( b ) abnormal structures and immunoreactivities of the testis in 
 o / t individuals . deformed seminiferous tubules and foxl2-positive oocyte - like cells 
 are seen in the lumen of the seminiferous tubules in the pre - sm ( asterisk , g and k ) . detachment of sox9-positive sertoli cells ( arrowheads ) is also seen in the degenerated 
 seminiferous tubules in the sm and post - sm ( l  m ) . sperm masses are present in the 
 seminiferous tubules without spermatogonia , spermatocytes , spermatids and sertoli 
 cells ( j ) . ( c ) degenerated 
 structures and immunoreactivities of the ovary in o / t individuals . slight primordial 
 follicles and primary oocytes were observed in the pre - sm stage ( o ) , vesicular 
 follicles , corpus luteum and blood leakage ( large arrows ) in the sm stage ( p ) , and 
 secondary and vesicular follicles and corpus luteum in the post - sm stage ( q ) . 
 moreover , blood leakage and intrafollicular shedding ( large arrows ) of cells were also 
 seen ( u , v ) . oocyte ( arrowhead ) and follicular epithelium ( arrow ) slightly positive 
 for sox9 ( r ) and positive for foxl2 ( s ) were seen in the pre - sm . clusters of 
 sox9-positive cells ( small arrows ) were observed at sm ( t ) . * primordial follicles ; 
 * * primary follicles ; * * * secondary follicles ; * * * * vesicular follicles ;  corpus 
 luteum . ) , indicating that the testes were normal and thus that there was no failure of the 
 testis - determining pathway . while some seminiferous tubular structures in the testis of o / t 
 individuals had abnormal vacuoles and no sperm ( fig . 2b ) , the pre - sm testis showed foxl2-positive oocyte - like cells , but without a 
 pellucid zone or follicular epithelium ( fig . the number of ovarian follicles was quite small in the ovaries of the o / t mice , and some 
 follicles exhibited blood leakage or intrafollicular shedding of cells , while sox9-positive 
 cells were observed in some of the seminiferous tubule - like structures ( fig . 2c ) . many secondary ovarian follicles as well as primary ones 
 were observed in the pre - sm b6n - xy females ( o / o mouse ) , but no vesicular 
 ovarian follicles were detected ( fig . 3afig . 3.representative general histology and number of follicles related to sexual 
 development in o / o individuals . ( a ) growing follicles ( primordial ~ secondary ) are 
 more abundant in the xy ovary than in the b6n - xx at pre - sm ( 2 weeks age ) 
 ( a and d ) . these follicles are rare in the sm and post - sm stages ( b , c and e , f ) . ( b ) the number of follicles at 
 each stage at 2 , 9 and 18 weeks of age . total ovarian follicles in 
 b6n - xy rapidly decreased between 2 to 9 weeks of age . ) . these follicles were rare in the sm and post - sm sexual developing stages ( fig . the measurement of the number of ovarian 
 follicles revealed a rapid decrease of germ cells at between 2 to 9 weeks of age ( fig . general histology and immunohistochemistry in the b6n - xy mice in each 
 sexual developmental stage . ( a ) hematoxylin and eosin staining ( he ) and sox9 
 immunostaining on transversal sections at pre - sexual maturation ( pre - sm ) , sm and 
 post - sm in t / t individuals . the testis shows a luminal structure and normal cell 
 arrangements including elongated spermatids in the wall of seminiferous tubules in the 
 sm and post - sm ( a  c ) . clusters of leydig cells are present in the intertubular space 
 ( arrows , a  c ) . ( b ) abnormal structures and immunoreactivities of the testis in 
 o / t individuals . deformed seminiferous tubules and foxl2-positive oocyte - like cells 
 are seen in the lumen of the seminiferous tubules in the pre - sm ( asterisk , g and k ) . j ) . 
 detachment of sox9-positive sertoli cells ( arrowheads ) is also seen in the degenerated 
 seminiferous tubules in the sm and post - sm ( l  m ) . sperm masses are present in the 
 seminiferous tubules without spermatogonia , spermatocytes , spermatids and sertoli 
 cells ( j ) . ( c ) degenerated 
 structures and immunoreactivities of the ovary in o / t individuals . slight primordial 
 follicles and primary oocytes were observed in the pre - sm stage ( o ) , vesicular 
 follicles , corpus luteum and blood leakage ( large arrows ) in the sm stage ( p ) , and 
 secondary and vesicular follicles and corpus luteum in the post - sm stage ( q ) . 
 moreover , blood leakage and intrafollicular shedding ( large arrows ) of cells were also 
 seen ( u , v ) . oocyte ( arrowhead ) and follicular epithelium ( arrow ) slightly positive 
 for sox9 ( r ) and positive for foxl2 ( s ) were seen in the pre - sm . clusters of 
 sox9-positive cells ( small arrows ) were observed at sm ( t ) . * primordial follicles ; 
 * * primary follicles ; * * * secondary follicles ; * * * * vesicular follicles ;  corpus 
 luteum . representative general histology and number of follicles related to sexual 
 development in o / o individuals . ( a ) growing follicles ( primordial ~ secondary ) are 
 more abundant in the xy ovary than in the b6n - xx at pre - sm ( 2 weeks age ) 
 ( a and d ) . these follicles are rare in the sm and post - sm stages ( b , c and e , f ) . ( b ) the number of follicles at 
 each stage at 2 , 9 and 18 weeks of age . total ovarian follicles in 
 b6n - xy rapidly decreased between 2 to 9 weeks of age . the results of immunoreactive analysis in the genital ridges at 11.5 dpc are shown in fig . 4.immunoreactivity of nr5a1 , sox9 and foxl2 of the indicated genotypes in the genital 
 ridges at 11.5 dpc . ( a ) immunoexpression of nr5a1 is detected in the epithelium and 
 internal somatic cells , but not in the germ cells . nr5a1 immunoexpression shows no 
 difference between b6n - xy and b6n - xy and also no bilateral 
 difference . ( b ) sox9-positive cells ( arrowheads ) are observed scattered randomly in 
 the b6n - xy gonads , but not in the b6n - xx gonads . ( c ) in this stage , no 
 foxl2-positive cells are seen in both the b6n - xy and b6n - xx gonads . however , faint staining is seen in each b6n - xy gonad .. the immunoexpression of nr5a1 in the genital ridges of b6n - xy mice was 
 compared to that in the b6n - xy males and females . nr5a1 positivity was detected 
 in the epithelium and internal somatic cells but not in the germ cells , and no difference 
 was found in the staining properties ( fig . although sox9 was expressed in a diffusely - arranged manner in both the right and left gonads 
 of the b6n - xy , the level of sox9 expression in the b6n - xy gonads 
 was much lower than that in the b6n - xy(fig . in addition , a bilateral difference or very few sox9-positive cells were 
 demonstrated in b6n - xy gonads ( data not shown ) . by contrast , no immunopositive 
 cells were detected in the b6n - xx ( fig . no 
 foxl2 immunoreactivity was detected in the genital ridges of both b6n - xy and 
 b6n - xx , but extremely - weak reactivity was seen in the b6n - xy(fig . immunoreactivity of nr5a1 , sox9 and foxl2 of the indicated genotypes in the genital 
 ridges at 11.5 dpc . ( a ) immunoexpression of nr5a1 is detected in the epithelium and 
 internal somatic cells , but not in the germ cells . nr5a1 immunoexpression shows no 
 difference between b6n - xy and b6n - xy and also no bilateral 
 difference . ( b ) sox9-positive cells ( arrowheads ) are observed scattered randomly in 
 the b6n - xy gonads , but not in the b6n - xx gonads . ( c ) in this stage , no 
 foxl2-positive cells are seen in both the b6n - xy and b6n - xx gonads . spatiotemporal expression of sry : we found no expression at 11 ts in 
 either the b6n - xy or b6n - xy gonads ( fig . 5a and 5hfig . 5.spatiotemporal expression of sry in embryonic urogenital ridges from 11 ts to 30 ts 
 in b6n - xy(a  g ) and b6n - xy(h  o ) mice . dotted lines show 
 outlines of the gonads ( genital ridges ) in each sample . the comparison of the 
 expression pattern between different sry alleles reveals an early and brief expression 
 profile in b6n - xy mice and a delayed and prolonged expression profile in 
 b6n - xy mice . the structures present in the gonad ( urogenital ridge ) 
 explants are shown ( p ) . ) . weakly positive cells were observed chiefly in the central region of the 13 ts 
 b6n - xy gonad ( fig . 5b ) . getting to 
 15 ts , positive cells were expanded toward both the craniad and the caudal region , and the 
 expression level in the central region was stronger than that in both acral regions ( fig . 5c ) . from 16 ts to 21 ts , strongly positive cells 
 were seen throughout the gonad ( fig . 5d ) . sry 
 expression began to decrease from the central region after 23 ts , and then , it became 
 limited by 25 ts ( fig . few positive cells 
 were observed in the thickening gonads at 26 ts ( fig . briefly , sry expression of b6n - xy gonads starts from the 
 central region at 1213 ts , rises toward a peak at 1721 ts and then falls to a low level at 
 2327 ts ( fig . spatiotemporal expression of sry in embryonic urogenital ridges from 11 ts to 30 ts 
 in b6n - xy(a  g ) and b6n - xy(h  o ) mice . dotted lines show 
 outlines of the gonads ( genital ridges ) in each sample . the comparison of the 
 expression pattern between different sry alleles reveals an early and brief expression 
 profile in b6n - xy mice and a delayed and prolonged expression profile in 
 b6n - xy mice . the structures present in the gonad ( urogenital ridge ) 
 explants are shown ( p ) . on the other hand , sry expression of b6n - xy was not detected beyond 13 ts 
 ( fig . 5h and 5i ) . weakly positive cells were 
 wholly observed in the 15 ts b6n - xy gonad ( fig . , positive cells were seen throughout the gonad , though the 
 expression level was weak around the caudal region ( fig . 5l and 5 m ) , and it was reduced beyond 28 ts ( fig . although there were few positive cells from 
 the craniad to the central region at 30 ts , many strongly positive cells remained in the 
 caudal region ( fig . sry expression 
 of b6n - xy gonads starts from the central region at 1415 ts , rises toward a 
 peak at 1926 ts and then falls to a low level after 28 ts ( fig . spatiotemporal expression of sox9 : weakly positive cells emerged chiefly 
 in the central region of the 15 ts b6n - xy gonad ( fig . 6.spatiotemporal expression of sox9 expression in embryonic urogenital ridges from 15 
 ts to 30 ts in b6n - xy(a  g ) and b6n - xy(h dotted 
 lines show outlines of the gonads ( genital ridges ) in each sample . sox9 expression was 
 delayed in b6n - xy mice ; the staining observed in the 
 b6n - xy gonads at the 16 and 17 ts stages ( h and i ) shows a delay of 2 
 ts compared to the b6n - xy mice . in addition , no increase of sox9 positive 
 cells was observed at either acral region even after 22 ts ( l  n ) . ) . at 17 ts , positive cells were seen throughout the gonad , though the expression 
 level was significantly low around the caudal region ( fig . by the 20 ts stage , sox9 expression was seen throughout 
 the gonad ( fig . an increase in positive 
 cells and gonadal thickening were observed after 22 ts , and testis cords including positive 
 cells were observed after 26 ts ( fig . briefly , sox9 expression of b6n - xy gonads starts from the central region at 
 1415 ts and expands to all regions at 18 ts , and then , the number of sox9-positive cells 
 increases , causing gonadal thickening and testis cords formation ( fig . spatiotemporal expression of sox9 expression in embryonic urogenital ridges from 15 
 ts to 30 ts in b6n - xy(a  g ) and b6n - xy(h dotted 
 lines show outlines of the gonads ( genital ridges ) in each sample . sox9 expression was 
 delayed in b6n - xy mice ; the staining observed in the 
 b6n - xy gonads at the 16 and 17 ts stages ( h and i ) shows a delay of 2 
 ts compared to the b6n - xy mice . in addition , no increase of sox9 positive 
 cells was observed at either acral region even after 22 ts ( l  n ) . by contrast , typically , sox9 expression was not detected in b6n - xy gonads at 
 16 ts ( fig . 6h ) , though expression in a small 
 number of cells was sometimes seen in the central region ( data not shown ) . weakly positive 
 cells started to expand toward both acral regions from 17 ts to 20 ts , but the number of 
 positive cells in b6n - xy gonads increased more moderately than that in the 
 b6n - xy gonads ( fig . the 
 number of positive cells continued to rise in the central region , and no increase was 
 observed at either acral region after 22 ts ( fig . 6l and 
 6 m ) . thickening was observed only in the center of the gonads at 26 ts ( fig . although the gonads had developed to the 
 same level as the b6n - xy gonads at 30 ts , they formed no testis cords and 
 contained very few positive cells at either acral region . moreover , the coelomic epithelium 
 invaginated into the gonads at 30 ts , and weakly positive cells were eccentrically located 
 at the mesonephric side ( fig . , then , 
 the sox9 expression of b6n - xy gonads starts from the central region at 1617 
 ts , and then , it expands to all regions by 20 ts . the number of positive cells in 
 b6n - xy gonads increased more moderately than that in the b6n - xy 
 gonads . gonads at 30 ts have less positive cells at both acral regions , which results in no 
 formation of testis cords ( fig fertility : all fertility - tested b6n - xy female mice were 
 infertile . although all ( four ) b6n - xy females that had been placed with 
 b6n - xy mated as evidenced by the presence of copulation plugs , none of them 
 were pregnant on day 12 after copulation . in contrast , some of the b6n - xy 
 males that had functional seminiferous tubes mated with b6n - xx , and most of 
 them carried the pregnancy to full term . to date , no pups have been obtained from 
 b6n - xy ( o / o individuals ) . sry cds sequence : to determine whether our b6n - xy mouse had 
 a normal mus . domesticus - derived sry allele , we sequenced 
 sry cds and found the same sequence as previously 
 reported for sry . 
 while sry is composed of 395 amino acids , sry is composed of 230 
 amino acids because of the point mutation creating a tag stop codon in cag repeats . in 
 addition , the q - rich domain of sry consists of 21 blocks of glutamine residues , 
 and that of sry consists of 8 blocks . in recent years , not only the sex - determining gene sry , but also some 
 other genes have been identified as important factors involved in sex determination and sex 
 differentiation . it is already known that knocking such genes out causes gonadal sex 
 reversal in mice . for example , xy gonadal sex 
 reversal has been described in cases of homozygous deletion of a number of 
 testis - determining genes , including sox9 [ 5 , 8 , 28 ] , fgf9 , 
 fgfr2 [ 4 , 25 ] , cbx2 ( m33 )   and wt1 ( + kts ) . a number of additional cases of xy sex reversal in the mouse depend on the 
 presence of a mus m. domesticus y chromosome and the b6j genetic 
 background : i.e. , so - called b6j - xy sex reversal [ 1 , 16 ] . b6j - xy mice carrying a mus m. domesticus 
 poschiavinus - derived y chromosome show sex reversal , which was one of the 
 b6j - xy sex reversal , and 75% of them develop as females ( ovaries ) while the 
 remainder develop ovotestes ; none develop a testis . however , when we analyzed offspring of b6n - xy mice in our 
 laboratory , they had a broad range of gonadal phenotypes that could be divided into four 
 types ( t / t , o / o , ot / ot and o / t ) , and the frequency of the t / t animals , which displayed 
 normal sex differentiation and were fertile , was over 30% . this implies that the 
 b6n - xy mice in our laboratory were somewhat different from those reported 
 initially . accordingly , sry cds sequence analysis was carried out to investigate 
 whether our b6n - xy mouse has a different sry allele from 
 mus m. domesticus poschiavinus . however , the results showed that our 
 sry was identical to that reported previously , which means that they have the same published 
 mus m. domesticus poschiavinus - derived sry allele . this 
 suggests that the gonadal phenotypic divergence between our present data and the results 
 published previously was not due to the sry cds . sry expression expands from the central region to the poles along the 
 anteroposterior axis of the xy gonad [ 6 , 40 ] , and testis cord formation subsequently proceeds in a 
 similar gradient manner . the study of bullejos 
 and koopman ( 2005 )   clearly indicates that a delay 
 of expression of sry relative to 
 b6j - sry underlies b6j - xy sex reversal , and 
 sry must act during a critical time window to appropriately activate 
 sox9 and effect male sex determination before the onset of the 
 ovarian - determining pathway . their findings also showed that the expression of 
 sry reached a peak level only after the expression of 
 sry had all but ceased ; this represents a delay of 4 ts 
 ( approximately 9.6 hr ) , and the onset of sox9 expression is delayed by a 
 period corresponding to 6 ts ( approximately 14.4 hr ) compared with b6j - xy . in 
 contrast , the spatiotemporal expressions of sry and sox9 examined using whole - mount 
 immunohistochemistry in the present study showed that the onsets of both sry and sox9 
 expressions in the b6n - xy gonads were 23 ts later than those in 
 b6n - xy gonads . these results suggest that the onset of sry 
 expression is earlier than that reported previously , that is , our sry 
 expression seemed more similar to that in b6n - xy gonads . furthermore , it is 
 possible that such a small difference in timing of the sry expression underlies testicular 
 development in our b6n - xy mice . at the same time , even at 30 ts 
 b6n - xy gonads , sox9-positive cells were limited at the central region and 
 were not seen at the cranial and caudal regions , which results in no formation of testis 
 cords at both acral regions . sox9 expression normally persists throughout 
 life in sertoli cells , suggesting that sox9 executes and maintains their differentiation and 
 aspects of their function . it suggests that the 
 failure of expressing sox9 at both acral regions of the gonad leads to the formation of 
 ambiguous gonads seen in ot / ot individuals of b6n - xy . actually , it is 
 mentioned that testicular tissue in ovotestis was invariably concentrated in the mid region 
 of the gonad , with ovarian tissue distributed at the cranial and caudal poles . most studies on xy mice have not focused on histological and 
 immunohistological findings , so we provide indication of normality or abnormality of adult 
 tissues ( pre - sm , sm and post - sm ) in detail . in t / t individuals , the testes showed normal 
 cell arrangement and sperm formation , which is the histological evidence supporting 
 fertility in our xy male mice . on the other hand , the gonads in o / t 
 individuals showed abnormal structures , especially in the testes , where the seminiferous 
 tubular structures had abnormal vacuoles and no sperm . the pre - sm testes showed 
 foxl2-positive oocyte - like cells , but without a pellucid zone or follicular epithelium . foxl2 , a forkhead transcription factor , is expressed in fetal and adult ovarian follicular 
 cells and involved in ovarian development and maintenance . testicular oocytes are also observed in mrl / mpj xy mice with fertility , 
 although they are surrounded by a pellucid zone and follicular epithelium unlike in the 
 present case . the ovaries in our o / t individuals 
 had quite small follicles , and sox9-positive cells were observed in the seminiferous 
 tubule - like structures . our studies are the first to histologically show the expression and 
 localization of a female - related gene in the b6n - xy testes and a male - related 
 gene in the b6n - xy ovaries . the ectopic expressions of such transcriptional 
 factors as foxl2 in the testes and sox9 in the ovaries suggest that the normal 
 differentiation system in b6n - xy gonads is disturbed due to an imbalance 
 between the male and female pathway since foxl2 and sox9 oppose each other s actions and 
 thereby ensure the establishment and maintenance of the different female and male supporting 
 cell types , respectively . we have indicated that delayed sry and sox9 expressions disrupt normal testicular 
 development , as described previously . however , in 
 the b6n - xy gonads , nr5a1 expression , which regulates sry expression , was at 
 the same level as in the b6n - xy gonads . several studies suggest that the 
 c57bl/6j background is sensitized to disruptions to testis determination due to the 
 relatively delayed expression of testis - determining genes and higher levels of expression of 
 ovary - determining genes ; in addition , in most sex - reversing mouse mutants examined , b6j 
 increases the amount of ovarian tissue that forms in mutant xy embryos [ 11 , 34 , 44 ] . therefore , there may be some interactions between 
 autosomal factors specific to the b6j background and the domesticus - derived 
 y chromosome . first , there is the issue of the c57bl/6 
 background . because testicular tissue develops in dba/2j or 129s1/svimj ( 129 ) mice 
 containing the poschiavinus - derived y chromosome , sex reversal in 
 b6j - xy mice is attributable to the genetic background of b6j . previous studies of gene expression within b6j and 
 another non - sex - reversing strain , 129s1 , in developing testis showed that , compared to 
 129-xy gonads , b6j - xy gonads had a higher average expression of 
  female-related genes and a lower expression of  male-related genes [ 34 , 35 ] . moreover , the b6 mouse , a 
 common inbred strain of laboratory mouse , is classified into mus m. 
 musculus . however , commonly used classical inbred mouse strains have mosaic 
 genomes with sequences from different subspecific origins , and with regard to b6 , their 
 genomes are derived predominantly from the western european subspecies mus m. 
 domesticus , with the remaining sequences derived mostly from the japanese 
 subspecies mus m. molossinus . molossinus - derived autosomal factors in b6 may provide sensitivity to sex 
 reversal , because some other inbred strains , such as akr , that are classified into 
 mus m. domesticus do not show sex reversal . furthermore , although they derive from the same b6 strains , b6j and 
 b6n are genetically and phenotypically different because their breeding stocks have been 
 maintained separately for a long period of time , allowing genetic differences to accumulate 
 due to individual variety and genetic drift [ 30 , 
 49 ] . at the beginning of our breeding , b6j - xx were 
 mated to b6j - xy tg ( 129 ) mice over five generations , with the result that only 
 individuals with ovaries or hermaphrodites were produced , which is the same result as 
 reported in 1982 . however , b6n - xx were mated to b6j - xy tg ( 129 ) mice 
 subsequently , and the frequency of their progeny was the subject of this paper . thus , it was 
 suggested that the difference between b6j and b6n may be responsible for the phenotypes in 
 our b6n - xy mice . secondly , we would like to focus attention on the epigenetic factors upstream of 
 sry . jumonji domain - containing 1a ( jmjd1a ) ( also called 
 tsga / jhdm2a / kdm3a ) acts as a h3k9 demethylase , and jmjd1a - deficient mice 
 frequently show xy sex reversal . the phenotypes 
 of these animals have a broad range that is quite similar to that of our 
 b6n - xy mice . jmjd1a directly and positively controls sry 
 expression by regulating h3k9me2 marks , which reveals that certain epigenetic factors , such 
 as histone demethylation , have an important role in mammalian sex determination . interestingly , the generation and comparison of xy jmjd1a - deficient mice , 
 which carried the y chromosome from either cba ( y ) or b6 ( y ) on a 
 b6n autosomal background , revealed that the sex - reversal phenotype was dependent on not only 
 the loss of jmjd1a but also the genetic origin of the y chromosome combined 
 with the b6 background . in the case of the xy 
 female in humans , without a functional mutation in sry , pericentric 
 inversion of the y chromosome causes xy females : if sry is inverted and 
 relocated to the center of the heterochromatic region , this positional variegation will lead 
 to sry gene silencing , inducing xy female development [ 17 , 31 , 32 ] . these facts indicate that , even if 
 sry is intact , the interaction between sry and other 
 genes will cause sex reversal because sry expression is regulated in a 
 strictly temporal and spatial manner . finally , a recent study on b6j - xy mice showed that the non - coding congenic 
 region on chromosome 11 ( 110 region ) that protects against b6j - xy sex reversal 
 is located within the sox9 promoter and promotes the expression of 
 sox9 , thereby driving testis development within the b6j - y 
 background . xy  
 were heterozygous for this 110 region derived from mus m. domesticus 
 poschiavinus , and heterozygosity for this region was sufficient to prevent the 
 formation of complete embryonic b6j - y ovaries . however , heterozygosity for the 
 110 region does not result in fully normal embryonic testis , with the majority of gonads 
 showing some degree of ovarian formation . this 
 suggests that the sox9 promoter region of our b6n - xy mice has 
 a similar congenic region that protects against b6j - xy sex reversal to a 
 certain degree . by contrast , our b6n - xy progeny show a broad range of gonadal 
 phenotypes even in littermates , and the frequency of t / t progeny is as high as that of o / o 
 progeny . our b6n - xy mice may have a similar autosomal non - coding region that 
 prevents sex reversal in a dose - dependent manner . therefore , further experiments will be 
 needed to analyze the promoter regions of sry and sox9 or 
 the expression of sex - related genes affected by epigenetic regulations .
mammalian sexual fate is determined by the presence or absence of sex determining 
 region of the y chromosome ( sry ) in the  bipotential  gonads . 
 recent studies have demonstrated that both male and female sexual development are induced 
 by distinct and active genetic pathways . 
 breeding the y chromosome from mus m. 
 domesticus poschiavinus ( pos ) strains into c57bl/6j ( b6j ) mice 
 ( b6j - xypos ) has been shown to induce sex reversal ( 75% : bilateral ovary , 25% : 
 true hermaphrodites ) . 
 however , our b6n - xypos mice , which were generated by 
 backcrossing of b6j - xypos on an inbred b6n - xx , develop as males ( 36% : bilateral 
 testis with fertility as well as bilateral ovary ( 34% ) , and the remainder develop as true 
 hermaphrodites . here , we investigated in detail the expressions of essential sex - related 
 genes and histological features in b6n - xypos mice from the fetal period to 
 adulthood . 
 the onsets of both sry and sry - box 9 ( sox9 ) expressions as determined 
 spatiotemporally by whole - mount immunohistochemistry in the b6n - xypos gonads 
 occurred 23 tail somites later than those in b6n - xyb6 gonads , but earlier than 
 those in b6j - xypos , respectively . 
 it is possible that such a small difference 
 in timing of the sry expression underlies testicular development in our 
 b6n - xypos . 
 our study is the first to histologically show the expression and 
 ectopic localization of a female - related gene in the xypos testes and a 
 male - related gene in the xypos ovaries . 
 the results from these and previous 
 experiments indicate that the interplay between genome variants , epigenetics and 
 developmental gene regulation is crucial for testis development .