rats were maintained in rooms with controlled temperature and light at 23  3c and 14 h 
 light/10 h dark cycle ( lights on at 0500 h ) .
adult female rats showing at least two regular 
 four - day estrous cycles were mated .
the day of parturition was defined as day 0 ( d0 ) of lactation and the number of pups was adjusted to eight on d1 .
pups were 
 removed from their dam on d21 of lactation and mid - lactational forced weaning was performed on d10 .
all experiments were performed according to the guidelines for 
 animal experiments of kitasato university and approved by the committee for laboratory animals , care and use at school of veterinary medicine , kitasato 
 university .
inguinal mammary tissues were collected from rats on d12 , 21 , 22 , 23 , or two days after forced weaning on d10 .
the tissues of d21 ( weaning day ) rats were 
 harvested at 0 or 6 h after pup removal .
the tissue samples were snap - frozen in liquid nitrogen and stored at 80c until rna extraction .
total rna was extracted 
 using trizol reagent ( invitrogen , carlsbad , ca , usa ) and then reverse - transcribed into cdna using a high capacity cdna reverse transcription kit ( applied 
 biosystems , foster city , ca , usa ) .
reverse transcription - pcr ( rt - pcr ) for gnrh - r or ribosomal protein l19 ( rpl19 ) was performed 
 using premix taq ( ex taq version 2.0 ; takara bio , shiga , japan ) for 35 or 22 cycles , respectively , at 94c for 30 sec , 58c for 30 sec , 
 and 72c for 60 sec , with an initial denaturing step at 94c for 2 min and a final elongation step at 72c for 7 min .
primers used in the current studytargetprimer ( 5-3)gnrh - rforward : aatcatcttcgccctcacacreverse : agcacgggtttagaaaagcagnrh - r exon 1forward : ccgtccttggagaaatatggreverse : agcggcatgacgattagagtgnrh - r exon 2forward : tcttcaggatgatctacctagccreverse : cctgatgaaggactcgtgtggnrh - r exon 3forward : ccaagaataatatcccaagagcareverse : tcccgtatatgggtttcagcrpl19forward : ggaagcctgtgactgtccatreverse : ccatgagaatccgcttgttt .
amplified products were separated by 2% agarose gel electrophoresis and detected by ethidium bromide staining .
inguinal mammary tissues were also collected from rats each day from d20 to 24 for western blot analysis .
the tissues of d21 ( weaning day ) were harvested at 0 or 
 6 h after pup removal .
the samples 
 containing 20 g of protein were electrophoresed on 12% polyacrylamide gels ( bio - rad , hercules , ca , usa ) and transferred onto polyvinylidene fluoride membranes 
 ( bio - rad ) .
membranes were blocked with 5% skim milk ( wako pure chemicals , osaka , japan ) for 1 h at room temperature and then incubated with primary antibodies : 
 anti - gnrh receptor , mouse monoclonal antibody ( 1:200 ; acris antibodies gmbh , herford , germany ) , or anti--actin mouse monoclonal antibody ( 1:1,000 ; c4 , santa cruz 
 biotechnology , santa cruz , ca , usa ) , overnight at 4c .
the gnrh - r antibody was raised by immunization of the n - terminal 129 amino acid peptide of human gnrh - r , 
 which shows 82.8% homology with rat gnrh - r and less than 40% homology with other rat proteins .
after washing , the membranes were incubated with 
 peroxidase - conjugated goat igg fraction to mouse igg ( 1:20,000 ; icn pharmaceuticals , aurora , oh , usa ) for 2 h at room temperature .
immunoreactive protein was 
 detected with ecl plus western blotting detection reagents or ecl prime western blotting detection reagents ( ge healthcare , little chalfont , uk ) .
the signal was 
 detected by exposure of the membrane to an x - ray film for 5 , 10 , or 15 min with an imagequant las 4000 digital imaging system ( ge healthcare ) .
rt - pcr examination for gnrh - r mrna showed no amplification in the mammary tissues of lactation d20 , a day before weaning ( fig . 

rt - pcr was performed with total rna isolated from mammary tissues of three rats each on 
 day 21 before weaning and 6 h after weaning on days 21 , 22 , and 23 .
the primer sets for gnrh - r and rpl19 
 mrna were designed to yield 251-bp and 264-bp fragments . ) .
gnrh - r mrna was dramatically increased over time until two days after weaning on d23 ( fig .
next , the 
 expression was decreased to trace levels on d26 and 29 ( fig .
this indicates that the expression of gnrh - r is 
 well - regulated and that the cessation of suckling stimuli triggers this expression .
ikeda et al . found that gnrh mrna was expressed in the mouse 
 mammary tissues during the lactating and involution periods , but did not detect gnrh - r mrna by pcr .
did not determine the regulation of gnrh - r mrna expression during the narrow period just after weaning .  
rt - pcr was performed with total rna isolated from mammary tissues of three rats each on 
 day 21 before weaning and 6 h after weaning on days 21 , 22 , and 23 .
the primer sets for gnrh - r and rpl19 
 mrna were designed to yield 251-bp and 264-bp fragments .
we further confirmed the expression of gnrh - r in mammary tissues with different sets of primers in forced weaned rats .
the gnrh - r gene consists 
 of three exons and encodes 984 base pairs through exons 13 in rodents [ 33 , 34 ] .
mammary tissues were collected from a forced weaned rat after two days and from a lactating rat .
( a ) primers for exons 13 of the gnrh - r gene were used .
rt - pcr was performed on dnase - treated total 
 rna isolated from the mammary tissues on lactation day 12 ( d12 ) and 2 days after forced weaning on day 10 ( fw ) .
amplification of gnrh - r mrna ( lane 14 ; exon 
 1 , lane 58 ; exon 2 , lane 912 ; exon 3 ) was performed using templates with or without reverse transcription reaction ( rt ) to prevent the amplification of 
 genomic dna .
three primer sets specific to exons 1 , 2 , and 3 were designed to amplify 306- , 201- , and 271-bp amplicons , respectively .
( b ) rpl19 was used as 
 an internal control for the two rats used ( lane 1316 ) . ) .
this result again revealed that the cessation of suckling induces gnrh - r mrna expression in the mammary tissues and that full - length mrna is 
 synthesized .  
( a ) primers for exons 13 of the gnrh - r gene were used .
rt - pcr was performed on dnase - treated total 
 rna isolated from the mammary tissues on lactation day 12 ( d12 ) and 2 days after forced weaning on day 10 ( fw ) .
amplification of gnrh - r mrna ( lane 14 ; exon 
 1 , lane 58 ; exon 2 , lane 912 ; exon 3 ) was performed using templates with or without reverse transcription reaction ( rt ) to prevent the amplification of 
 genomic dna .
three primer sets specific to exons 1 , 2 , and 3 were designed to amplify 306- , 201- , and 271-bp amplicons , respectively .
( b ) rpl19 was used as 
 an internal control for the two rats used ( lane 1316 ) .
we previously reported that the expression of annexin a5 is dramatically increased in the epithelial cells of mammary tissues .
we predicted that gnrh acts locally in the mammary gland during post - lactational involution .
we also published several reports regarding the 
 relationship between gnrh and annexin a5 in various tissues .
currently , the physiological function of annexin a5 is unknown , but it is 
 thought to be involved in apoptosis and tissue remodeling .
the present data support that gnrh affects degeneration and tissue remodeling in the mammary epithelium 
 after lactation . to confirm the translation of gnrh - r mrna
, western blotting analysis using a gnrh - r antibody against the n - terminal peptide sequence was performed .
the results 
 confirmed two immunoreactive bands , approximately 60 kda and 30 kda proteins , in the lactating and post - lactating mammary tissues .
( a , b ) western blotting was performed with mammary tissue samples on lactation 
 day 20 ( d20 ) and 6 h after weaning on day 21 ( d21 + 6h ) , day 22 ( d22 ) , day 23 ( d23 ) , and day 24 ( d24 ) .
( c , d ) western blotting was performed on the mammary tissues from d21 ( on 
 weaning ) and d23 , and the anterior pituitary tissue .
-actin is used as an internal control . ) . a previous study that also used a gnrh - r monoclonal antibody against the n - terminal 129 amino acid residues , which differed from the antibody used in 
 the present study , detected an approximately 60-kda protein in the rat pituitary gland .
gnrh - r in mice and rats is a 
 seven - transmembrane , g - protein - coupled receptor of 327 amino acid residues with two or three n - terminal glycosylation sites and can be detected at 5570 kda by 
 sds - page .
this band was increased after weaning and reached 
 a peak on d23 , coinciding with mrna results .
interestingly , there was another 30-kda immunoreactive band , which decreased after weaning . although the sequence of 
 the 30-kda protein is not known and was observed even during lactation while there was no detectable level of gnrh - r mrna , the gnrh - r variant may have been 
 present
alternatively , impairment of post - translational glycosylation may produce a gnrh - r 
 with a lower apparent molecular weight on sds - page .
future studies 
 are needed to investigate the post - transcriptional or post - translational modification of gnrh - r in the mammary gland .  
( a , b ) western blotting was performed with mammary tissue samples on lactation 
 day 20 ( d20 ) and 6 h after weaning on day 21 ( d21 + 6h ) , day 22 ( d22 ) , day 23 ( d23 ) , and day 24 ( d24 ) .
( c , d ) western blotting was performed on the mammary tissues from d21 ( on 
 weaning ) and d23 , and the anterior pituitary tissue .
time - specific expression of gnrh - r in the mammary gland after pup removal strongly suggests that the expression is related to changes in the endocrine milieu 
 after lactation , specifically the cessation of massive prolactin release .
we previously 
 reported that prolactin suppresses annexin a5 expression in the corpus luteum , suggesting negative control of gnrh function by prolactin .
experimental suppression 
 of prolactin release caused luteal regression , and a gnrh antagonist was shown to inhibit the process of apoptosis . 

further studies are needed to clarify the role of gnrh in apoptosis and the signaling pathways involved in the post - lactational mammary gland . in summary , we detected a post - lactational increase in gnrh - r expression in rat mammary tissues .
these results suggest that local gnrh is involved in the 
 involution of mammary tissues after weaning to induce epithelial apoptosis and tissue remodeling .
conflict of interests : none of the authors have any potential conflicts of interest associated with this research .
   gonadotropin - releasing hormone ( gnrh ) is a neurohormone of the hypothalamus controlling pituitary gonadotropin secretion and hence gametogenesis . 
 while it has 
 also been believed that gnrh is synthesized and functions in various peripheral tissues , the expression of gnrh receptor ( gnrh - r ) in peripheral tissues is not 
 well - described . 
 we previously found that annexin a5 , which is increased in the pituitary gonadotropes by gnrh , is dramatically increased in rat mammary 
 epithelial cells after weaning , suggesting that local gnrh is responsible for this increase . 
 annexin a5 is a member of the annexin family of proteins and is 
 thought to be involved in various regulatory mechanisms , including apoptosis . in the present study , we examined gnrh - r expression in the mammary tissues after 
 weaning . 
 although gnrh - r mrna was not detected in the mammary tissues during lactation , it was dramatically increased after weaning . 
 forced weaning at 
 mid - lactation ( day 10 ) also promoted the expression of gnrh - r transcripts in mammary tissues within 2 days . 
 furthermore , western blotting analysis with 
 anti - gnrh - r showed that the expression of an immuno - positive 60-kda protein , whose size was equivalent to that of rat gnrh - r , was confirmed to increase after 
 weaning . 
 these findings clarified the induction of gnrh - r in the mammary tissues after weaning and suggest that gnrh is involved in the involution and tissue 
 remodeling of post - lactating rat mammary tissues . 
Materials and Methods
Results and Discussion