mice were maintained and used for experimentation according to the guidelines of the institutional animal care and use committees of the university of california , davis and the middlebury college animal facility .
the hei10 , mlh3 , rnf212 , spo11 and sycp1 mutant lines and primer sequences for genotyping were previously described .
tissues from adult mice were sonicated in ripa buffer , protein concentration was measured by the bradford assay and 100200 g of protein was separated by sds - page . after protein transfer to nitrocellulose membranes ( waterman )
, blots were incubated overnight with the following antibodies : mouse monoclonal anti - ccnb1ip1/hei10 ( ab118999 abcam , 1:2000 dilution ) , rabbit polyclonal anti - ccnb1ip1/hei10 ( this study , 1:2000 ) , or mouse anti - tubulin ( biolegend , 625902 , 1:2,000 ) .
secondary antibodies ( 1:10,000 dilution ) were goat anti - rabbit or anti - mouse iggs conjugated to horseradish peroxidase ( hrp ; southernbiotech , 4050 - 05 and 1031 - 05 , respectively ) .
a polyclonal antibody against mouse hei10/ccnb1ip1 was raised in rabbits against a mixture of two c - terminal peptides .
antibodies were purified from serum using protein a / g spin columns ( ge healthcare ) .
testes and ovaries were dissected from freshly killed animals and processed for surface spreading as described .
for all quantification , images from at least two animals ( 25 ) were analyzed .
all cytological analyses were performed by two observers ; the second observer was blind to which group / genotype was being analyzed .
immunofluorescence staining was performed as described , using the following primary antibodies with incubation overnight at room temperature : mouse anti - sycp3 ( sc-74568 santa cruz , 1:200 dilution ) , rabbit anti - sycp3 ( sc-33195 santa cruz , 1:300 ) , guinea pig anti - syce1 ( 1:2000)(generously provided by chist hg , karolinska institutet ) , guinea pig anti - rnf212 ( 1:50 ) , rabbit anti - rnf212 ( 1:200 ) , rabbit anti - msh4 ( ab58666 abcam , 1:100 ) , mouse monoclonal anti - ccnb1ip1/hei10 ( ab118999 abcam , 1:150 ) , rabbit polyclonal anti - ccnb1ip1/hei10 ( this study , ) , mouse anti - mlh1 ( 1:50 , 550838 bd pharmingen ) , mouse monoclonal anti-h2ax ( 05 - 636 millipore , 1:500 ) , mouse monoclonal anti - cdk2 ( sc-6248 santa cruz , 1:200 ) , guinea pig anti - h1 t ( a gift from m.a .
slides were subsequently incubated with the following goat secondary antibodies for 1 h at 37 c : anti - rabbit 488 ( a11070 molecular probes , 1:1000 dilution ) , anti - rabbit 568 ( a11036 molecular probes , 1:2000 ) , anti - mouse 555 ( a21425 molecular probes , 1:1000 ) , anti - mouse 594 ( a11020 molecular probes , 1:1000 ) , anti - mouse 488 ( a11029 molecular probes , 1:1000 ) , and anti - guinea pig fluorescein isothiocyanate ( 106 - 096 - 006 fitc , jackson labs , 1:200 ) .
for chiasma counts , air - dried preparations of diakinesis / metaphase i  stage cells were prepared as described and stained with dapi .
testes were fixed in formalin , embedded in paraffin , sectioned and processed using the apoptag plus peroxidase in situ apoptosis detection kit ( chemicon ) . immunolabeled chromosome spreads and dapi - stained diakinesis / metaphase i nuclei were imaged using a zeiss axioplan ii microscope with 63 plan apochromat 1.4 objective and exfo x - cite metal halide light source .
images were captured by a hamamatsu orca - er ccd camera and processed using volocity ( perkin elmer ) and photoshop ( adobe ) software packages .
sim analysis was performed using a nikon n - sim super - resolution microscope system and nis - elements 2 image processing software .
msh4-rnf212 colocalization was determined using nis - elements and co - foci were confirmed by visual inspection .
testes sections were imaged using an axiovert 200 microscope and axiocammrc camera using axiovision 4.4 software .
mice were maintained and used for experimentation according to the guidelines of the institutional animal care and use committees of the university of california , davis and the middlebury college animal facility .
the hei10 , mlh3 , rnf212 , spo11 and sycp1 mutant lines and primer sequences for genotyping were previously described .
tissues from adult mice were sonicated in ripa buffer , protein concentration was measured by the bradford assay and 100200 g of protein was separated by sds - page . after protein transfer to nitrocellulose membranes ( waterman )
, blots were incubated overnight with the following antibodies : mouse monoclonal anti - ccnb1ip1/hei10 ( ab118999 abcam , 1:2000 dilution ) , rabbit polyclonal anti - ccnb1ip1/hei10 ( this study , 1:2000 ) , or mouse anti - tubulin ( biolegend , 625902 , 1:2,000 ) . secondary antibodies ( 1:10,000 dilution ) were goat anti - rabbit or anti - mouse iggs conjugated to horseradish peroxidase ( hrp ; southernbiotech , 4050 - 05 and 1031 - 05 , respectively ) .
a polyclonal antibody against mouse hei10/ccnb1ip1 was raised in rabbits against a mixture of two c - terminal peptides .
antibodies were purified from serum using protein a / g spin columns ( ge healthcare ) .
testes and ovaries were dissected from freshly killed animals and processed for surface spreading as described . for all quantification , images from at least two animals ( 25 ) were analyzed .
all cytological analyses were performed by two observers ; the second observer was blind to which group / genotype was being analyzed .
immunofluorescence staining was performed as described , using the following primary antibodies with incubation overnight at room temperature : mouse anti - sycp3 ( sc-74568 santa cruz , 1:200 dilution ) , rabbit anti - sycp3 ( sc-33195 santa cruz , 1:300 ) , guinea pig anti - syce1 ( 1:2000)(generously provided by chist hg , karolinska institutet ) , guinea pig anti - rnf212 ( 1:50 ) , rabbit anti - rnf212 ( 1:200 ) , rabbit anti - msh4 ( ab58666 abcam , 1:100 ) , mouse monoclonal anti - ccnb1ip1/hei10 ( ab118999 abcam , 1:150 ) , rabbit polyclonal anti - ccnb1ip1/hei10 ( this study , ) , mouse anti - mlh1 ( 1:50 , 550838 bd pharmingen ) , mouse monoclonal anti-h2ax ( 05 - 636 millipore , 1:500 ) , mouse monoclonal anti - cdk2 ( sc-6248 santa cruz , 1:200 ) , guinea pig anti - h1 t ( a gift from m.a .
slides were subsequently incubated with the following goat secondary antibodies for 1 h at 37 c : anti - rabbit 488 ( a11070 molecular probes , 1:1000 dilution ) , anti - rabbit 568 ( a11036 molecular probes , 1:2000 ) , anti - mouse 555 ( a21425 molecular probes , 1:1000 ) , anti - mouse 594 ( a11020 molecular probes , 1:1000 ) , anti - mouse 488 ( a11029 molecular probes , 1:1000 ) , and anti - guinea pig fluorescein isothiocyanate ( 106 - 096 - 006 fitc , jackson labs , 1:200 ) . coverslips were mounted with prolong gold antifade reagent ( molecular probes ) .
for chiasma counts , air - dried preparations of diakinesis / metaphase i  stage cells were prepared as described and stained with dapi .
testes were fixed in formalin , embedded in paraffin , sectioned and processed using the apoptag plus peroxidase in situ apoptosis detection kit ( chemicon ) .
immunolabeled chromosome spreads and dapi - stained diakinesis / metaphase i nuclei were imaged using a zeiss axioplan ii microscope with 63 plan apochromat 1.4 objective and exfo x - cite metal halide light source .
images were captured by a hamamatsu orca - er ccd camera and processed using volocity ( perkin elmer ) and photoshop ( adobe ) software packages .
sim analysis was performed using a nikon n - sim super - resolution microscope system and nis - elements 2 image processing software .
msh4-rnf212 colocalization was determined using nis - elements and co - foci were confirmed by visual inspection .
testes sections were imaged using an axiovert 200 microscope and axiocammrc camera using axiovision 4.4 software .
 crossover recombination facilitates accurate segregation of homologous chromosomes during meiosis1,2 . in mammals , poorly characterized regulatory processes ensure every pair of chromosomes obtains at least one crossover , even though the majority of recombination sites yield non - crossovers3 . 
 designation of crossovers involves selective localization of sumo - ligase rnf212 to a minority of recombination sites where it stabilizes pertinent factors , such as muts4 . here 
 we show ubiquitin - ligase hei10/ccnb1ip15,6 is essential for this crossover / non - crossover differentiation process . in hei10 mutant mice , 
 rnf212 localizes to most recombination sites and dissociation of rnf212 and muts from chromosomes is blocked . 
 consequently , recombination is impeded and crossing - over fails . in wild - type mice , hei10 accumulates at designated crossover sites suggesting a late role to implement crossing - over . like rnf212 
 , dosage - sensitivity indicates hei10 is a limiting factor for crossing - over . 
 we suggest sumo and ubiquitin play antagonistic roles during meiotic recombination that are balanced to effect differential stabilization of recombination factors at crossover and non - crossover sites . 