corpora lutea ( cl ) appear very similar , in terms of structure , function and regulation , between old - world monkeys ( rhesus macaques ) and humans . humans and macaques have similar length menstrual cycles ( luteal phases of the cycles are approximately the same length ) , neither relies on a uterine signal to induce luteolysis in non - gravid cycles and both exhibit an absolute requirement for luteinizing hormone ( lh ) from the anterior pituitary to maintain luteal structure  function ( stouffer , 2006 ) . treatment of rhesus macaques with an antagonist of gonadotrophin - releasing hormone ( gnrh ; antide , to inhibit lh production ) results in rapid destruction of developed cl [ as evidenced by a dramatic drop in progesterone ( p ) secretion ] , but the functional lifespan of these cl can be rescued by pulsatile administration of exogenous lh ( duffy et al . , 1999b ) . primate cl are remarkably sensitive to local lh levels as macaque luteinized granulosa / luteal cells respond to acute lh stimulation by rapidly secreting p in vitro , and lh pulses are entrained to p pulses by mid - luteal phase in vivo ( molskness et al . , 1991 ; stouffer , 2003 ) . it is generally accepted that lh exerts its effects on targets , i.e. luteal cells , via the lh receptor ( lhr ) , a seven transmembrane domain g - protein - coupled receptor capable of activating adenylate cyclase in the ovary ( benyo and zeleznik , 1997 ; zeleznik and somers , 1999 ; priyanka and medhamurthy , 2007 ) , and in other cells / tissues ( ascoli et al . , 2002 ) . however , the exact mechanism(s ) by which lh exerts its luteotropic effects on the primate cl have yet to be elucidated . local autocrine / paracrine factors produced in response to lh may also be important in controlling cl structure  function . for example , it is hypothesized that locally produced p is the primary luteotropin in the primate cl , and alone may be sufficient to sustain luteal function , i.e. the so - called treatment of rhesus monkeys during the mid - luteal phase with trilostane ( trl ) , an inhibitor of 3-hydroxysteroid dehydrogenase ( 3-hsd ) , results in onset of premature luteolysis within 4 days of treatment ( duffy et al . , luteal phase length is reduced by approximately half , whereas endogenous lh levels remain relatively unchanged following trl administration . the so - called  genomic progesterone receptors ( pgrs ; ligand - activated transcription factors ) are present in macaque luteal cells and co - localize with 3-hsd , supporting the premise of the rothchild hypothesis ( stouffer and duffy , 1995 ) . but , receptors for other sex steroids [ e.g. estrogen receptor  ( esr2 ) and androgen receptor ( ar ) ] as well as mrnas for putative membrane - associated steroid receptors were also detected in primate cl ( duffy et al . genomic steroid hormone receptors are ligand - activated transcription factors that bind to regulatory elements on dna with the assistance of several co - factors to modify gene transcription ( lonard and omalley , 2005 ) . thus , the actions of lh on cl structure  function are likely a composite of direct lh receptor - mediated effects and indirect effects resulting from lh - regulated local factors , e.g. steroids including p. to investigate both the direct and indirect ( via local steroids including p ) effects of lh on the primate cl during the menstrual cycle , an in vivo model was developed in which ablation and replacement of lh ( duffy et al . , 1999b ) and steroid ablation / progestin replacement could be performed in rhesus monkeys during the same experimental protocol ( young and stouffer , 2004 ) . previous ablation / replacement experiments on the primate cl focused primarily on the effects of lh versus p on individual gene families , including proteases and caspases involved in tissue reorganization ( young et al . recently , attempts were made to broadly evaluate lh regulation of genes in the macaque cl ( yadav et al . ( 2005 ) either left rhesus monkeys untreated or administered a single bolus of antide in the mid - luteal phase , the cl were removed and total cdna hybridized to a limited ( 11 600 transcripts ) human spotted array . the resulting data successfully identified a novel group of genes , including the corticotropin - releasing hormone ( crh)/urocortin ( ucn)-receptor - binding protein system , that appear to be involved in the maintenance of cl structure / function ( xu et al . , 2007 ) . ( 2004 ) used the gnrh antagonist cetrorelix , administered to bonnet monkeys in the mid - luteal phase , to identify changes in mrna transcripts by differential display . seven transcripts , including mrna for low - density lipoprotein ( ldl ) receptor , were identified as changing in expression after lh withdrawal ( yadav et al . , 2004 ) . while these approaches were successful in identifying novel gene regulation by lh in the cl , further opportunities now exist for global ( genome - wide ) evaluation of gene activities in macaque tissues ( noriega et al . , 2007 ; parent et al . , 2008 ) including the rhesus cl ( bogan et al . , 2008b ) using the affymetrix rhesus macaque genome expression array . building on these validated studies , we utilized the rhesus array to further identify changes in gene expression in the macaque cl in response to lh and steroid ablation / replacement as described earlier . it was expected that lh - dependent changes in gene expression ( directly regulated by lh ) could be contrasted and compared with changes in the expression of genes that were steroid ( especially p ) dependent ( indirectly regulated by lh ) . the studies were performed at the oregon national primate research center ( onprc ) on the west campus of oregon health & science university ( ohsu ) . all procedures and protocols were approved by the onprc / ohsu animal care and use committee , in accordance with the national institute of health ( nih ) guidelines for care and use of laboratory animals . adult , female rhesus macaques ( macaca mulatta ) exhibiting regular menstrual cycles were selected for the study and were under the direct care of the onprc division of animal resources ( dar ) . monkeys ( n = 22 ) received one of the following treatments for 3 days beginning on day 9 of the luteal phase ( mid - luteal phase , the time of peak cl function ) . some females were left untreated to establish gene / protein expression in normal cl ( control group ; n = 4 ) . alternatively , ablation of lh was accomplished by treating females with a gnrh antagonist [ antide obtained from the contraceptive development branch , center for population research , national institute of child health and human development ( nichd ) , 3 mg / kg once per day , n = 5 ] . this treatment significantly lowers lh and p levels within 1 day , with baseline levels present by the end of the experimental period ( duffy et al . , 1999b ) . recombinant human lh ( 40 iu , three times daily ) was also administered in a pulsatile manner ( a + lh ; n = 4 ) , which returns systemic lh and p to control levels ( duffy et al . , 1999b ) . in addition , steroid production by the cl in response to lh replacement was blocked by oral administration of the 3-hsd inhibitor trl ( a + lh + trl ; 600 mg b.i.d , n = 4 ) , which suppresses systemic p levels and induces premature luteolysis at this dosage and duration of treatment in rhesus monkeys ( duffy et al . 1994 ) . actions of p were restored in a subset of trl - treated animals by treatment with a synthetic progestin , r5020 ( a + lh + trl + r5020 ; 2.5 ng / ml once per day , n = 5 ) , at a dosage and duration that prevents the indices of luteolysis brought on by trl treatment in macaque monkeys ( young and stouffer , 2004 ) . following the 3-day treatment interval , cl were surgically removed and half of each tissue was individually processed for total rna , and the other half for total protein as described previously ( peluffo et al . , 2005 ) . anesthesia of animals and lutectomy were performed by the surgical veterinary staff ( dar , onprc ) as described previously ( duffy et al . , 2000 ) . all rhesus macaque genome arrays were processed by the ohsu affymetrix microarray core ( amc ) , ohsu . the amc performed the rna expression profiling , amplification and hybridization of samples ( http://www.ohsu.edu/gmsr/amc/amc_technology.html#assay ) . following data normalization using the robust multichip average algorithm , the web - based genesifter software ( http://www.genesifter.net/web/ ) was used to identify transcripts from log2-transformed data that significantly changed expression between treatment groups by pairwise comparison analysis ( t - test , p < 0.05 ) , and for the entire experimental group by multiple comparison analysis procedures ( one - way anova , p < 0.05 ; see http://www.genesifter.net/web/webinars.php for further explanation of analytical methods by genesifter ) . the affymetrix rhesus genome array contains 52 024 rhesus probe sets for approximately 47 000 rhesus transcripts , plus several control probe sets ; array design is based on the baylor school of medicine s rhesus macaque whole - genome shotgun assembly and genbank sequence - tagged sites ( stss ) , expressed sequence tag and mrna sequences ( http://www.affymetrix.com/support/technical/datasheets/rhesus_datasheet.pdf ) . there are multiple probes sets ( transcripts ) for individual gene products on this array ( spindel et al . , 2005 ) . therefore , microarray data will be referred to as  transcripts , whereas real - time pcr data will be referred to as  mrna values . data files were submitted to the gene expression omnibus ( www.ncbi.nlm.nih.gov/geo : series gse12 281 ) . to validate the microarray results , selected genes were analyzed by real - time pcr to quantify changes in mrna levels that occur following lh and/or p ablation / replacement . the real - time pcr probes were designed as described previously ( xu et al . , 2006 ) and labeled with the 5 reporter dye fam and the 3 quencher dye mgbnfq . sequences for real - time probes and primers are listed in table  i , along with gene identifiers . all real - time studies were performed and analyzed as reported previously ( xu et al . , the data were subjected to one - way anova by the use of sigmastat software ( version 2.0 ) . when significant ( p < 0.05 ) , differences between treatment groups were analyzed by pairwise multiple comparison procedures ( tukey test or student  newman  keuls method as appropriate ) . real - time pcr primer and probe sequences used to validate microarray expression based on ncbi entrez gene terminology . immunoblot ( western blot ) analysis was performed to permit comparison of the patterns of protein levels of two selected gene products versus their mrna levels in individual cl . electrophoresis was performed using gradient gels ( 1020% liner with 4% stacking gel ; biorad laboratories , hercules , ca , usa ) with 20 g of protein in denaturing sds loading buffer ( 3% sds , 7 mm edta , 7 mm egta , 7% glycerol , 0.36 m tris , 3.6 mm dtt and 3.6% bromophenol blue ) per individual sample . after electrophoresis , proteins were transferred to immun - blot pvdf membranes ( 0.2 m ; biorad ) overnight at 4c . membranes were blocked with a neutral protein ( 3% nfm ) at 4c overnight and then incubated with the following primary antibodies according to the manufacturer s protocols : mouse anti - human secreted frizzled related protein-4 ( sfrp4 ) whole sera antibody ( final dilution 1:1000 ; abnova / affinity bioreagents h00006424-a01 ) or rabbit anti - human steroidogenic acute regulatory ( star ) protein ( final concentration 1 g / ml ; fisher / affinity bioreagents pa1 - 560 ) . all horseradish peroxidase - labeled secondary antibodies were from the same source ( zymed laboratories , invitrogen corp . , carlsbad , ca , usa ) , specific to the host animal of the primary antibody , and used at a final dilution of 1:2000 . for the quantification of protein levels from individual cl , a luminescent signal was generated using western blotting luminol reagent ( santa cruz biotechnology , inc . , santa cruz , ca , usa ) and detected with kodak x - omat film ( eastman kodak co. , rochester , ny , usa ) . densitometry analysis was performed using a gel documentation system and quantity one software ( bio - rad ) . to quantify protein levels , the blots were stripped by a gentle method ( successive pbs and tbst washes , abcam inc . , this method was confirmed for each antibody by incubation of stripped blots with only the secondary antibody and then developed for any luminescent signal as described earlier . blots were then blocked again overnight and re - probed for -actin levels ( tesone et al . , 2005 ) by incubation with the primary antibody rabbit anti - human -actin ( 0.8 g / ml ; abcam ab8227 ) , and secondary antibody as before . all data were subject to one - way anova ( similar to real - time pcr data ) and are presented as the intensity of protein of interest : -actin intensity . because of high background to signal ratio for sfrp4 blots containing + trl and + r5020 samples , for both -actin and sfrp4 samples , background intensity was subtracted from average intensity for all bands ( regardless of treatment group ) before normalization for sfrp4 expression . for presentation of western blot data , samples from individual cl were pooled by treatment group , and then pooled samples underwent electrophoresis and transfer as stated earlier for the detection of each protein of interest ( star , sfrp4 and -actin ) . following electrophoresis and protein transfer to pvdf membranes , star and -actin blots were developed via the westerndot 625 western blot kit ( invitrogen , molecular probes ) with the aforementioned primary antibodies according to the manufacturer s protocol ; sfrp4 was developed as described earlier . all rhesus macaque genome arrays were processed by the ohsu affymetrix microarray core ( amc ) , ohsu . the amc performed the rna expression profiling , amplification and hybridization of samples ( http://www.ohsu.edu/gmsr/amc/amc_technology.html#assay ) . following data normalization using the robust multichip average algorithm , the web - based genesifter software ( http://www.genesifter.net/web/ ) was used to identify transcripts from log2-transformed data that significantly changed expression between treatment groups by pairwise comparison analysis ( t - test , p < 0.05 ) , and for the entire experimental group by multiple comparison analysis procedures ( one - way anova , p < 0.05 ; see http://www.genesifter.net/web/webinars.php for further explanation of analytical methods by genesifter ) . the affymetrix rhesus genome array contains 52 024 rhesus probe sets for approximately 47 000 rhesus transcripts , plus several control probe sets ; array design is based on the baylor school of medicine s rhesus macaque whole - genome shotgun assembly and genbank sequence - tagged sites ( stss ) , expressed sequence tag and mrna sequences ( http://www.affymetrix.com/support/technical/datasheets/rhesus_datasheet.pdf ) . there are multiple probes sets ( transcripts ) for individual gene products on this array ( spindel et al . , 2005 ) . data files were submitted to the gene expression omnibus ( www.ncbi.nlm.nih.gov/geo : series gse12 281 ) . to validate the microarray results , selected genes were analyzed by real - time pcr to quantify changes in mrna levels that occur following lh and/or p ablation / replacement . the real - time pcr probes were designed as described previously ( xu et al . , 2006 ) and labeled with the 5 reporter dye fam and the 3 quencher dye mgbnfq . sequences for real - time probes and primers are listed in table  i , along with gene identifiers . all real - time studies were performed and analyzed as reported previously ( xu et al . , 2006 ) . the data were subjected to one - way anova by the use of sigmastat software ( version 2.0 ) . when significant ( p < 0.05 ) , differences between treatment groups were analyzed by pairwise multiple comparison procedures ( tukey test or student  newman  keuls method as appropriate ) . real - time pcr primer and probe sequences used to validate microarray expression based on ncbi entrez gene terminology . immunoblot ( western blot ) analysis was performed to permit comparison of the patterns of protein levels of two selected gene products versus their mrna levels in individual cl . electrophoresis was performed using gradient gels ( 1020% liner with 4% stacking gel ; biorad laboratories , hercules , ca , usa ) with 20 g of protein in denaturing sds loading buffer ( 3% sds , 7 mm edta , 7 mm egta , 7% glycerol , 0.36 m tris , 3.6 mm dtt and 3.6% bromophenol blue ) per individual sample . after electrophoresis , proteins were transferred to immun - blot pvdf membranes ( 0.2 m ; biorad ) overnight at 4c . membranes were blocked with a neutral protein ( 3% nfm ) at 4c overnight and then incubated with the following primary antibodies according to the manufacturer s protocols : mouse anti - human secreted frizzled related protein-4 ( sfrp4 ) whole sera antibody ( final dilution 1:1000 ; abnova / affinity bioreagents h00006424-a01 ) or rabbit anti - human steroidogenic acute regulatory ( star ) protein ( final concentration 1 g / ml ; fisher / affinity bioreagents pa1 - 560 ) . all horseradish peroxidase - labeled secondary antibodies were from the same source ( zymed laboratories , invitrogen corp . , carlsbad , ca , usa ) , specific to the host animal of the primary antibody , and used at a final dilution of 1:2000 . for the quantification of protein levels from individual cl , a luminescent signal was generated using western blotting luminol reagent ( santa cruz biotechnology , inc . , santa cruz , ca , usa ) and detected with kodak x - omat film ( eastman kodak co. , rochester , ny , usa ) . densitometry analysis was performed using a gel documentation system and quantity one software ( bio - rad ) . to quantify protein levels , the blots were stripped by a gentle method ( successive pbs and tbst washes , abcam inc . , this method was confirmed for each antibody by incubation of stripped blots with only the secondary antibody and then developed for any luminescent signal as described earlier . blots were then blocked again overnight and re - probed for -actin levels ( tesone et al . , 2005 ) by incubation with the primary antibody rabbit anti - human -actin ( 0.8 g / ml ; abcam ab8227 ) , and secondary antibody as before . all data were subject to one - way anova ( similar to real - time pcr data ) and are presented as the intensity of protein of interest : -actin intensity . because of high background to signal ratio for sfrp4 blots containing + trl and + r5020 samples , for both -actin and sfrp4 samples , background intensity was subtracted from average intensity for all bands ( regardless of treatment group ) before normalization for sfrp4 expression . for presentation of western blot data , samples from individual cl were pooled by treatment group , and then pooled samples underwent electrophoresis and transfer as stated earlier for the detection of each protein of interest ( star , sfrp4 and -actin ) . following electrophoresis and protein transfer to pvdf membranes , star and -actin blots were developed via the westerndot 625 western blot kit ( invitrogen , molecular probes ) with the aforementioned primary antibodies according to the manufacturer s protocol ; sfrp4 was developed as described earlier . normalized data files for individual arrays have been uploaded to the national center for biotechnology information s gene expression omnibus gene expression repository ( http://www.ncbi.nlm.nih.gov/geo/ series gse12281 ) . of the more than 47 000 transcripts represented on the rhesus genome arrays , the multiple comparison analysis of genesifter identified 2% of all transcripts on the array as changing in expression 2-fold between various treatments ( one - way anova , p < 0.05 ) ; these were then analyzed by the cluster analysis algorithm of genesifter ( for further description of cluster analysis , see http://www.genesifter.net/web/resources_cluster.php , and http://www.statsoft.com/textbook/stcluan.html for a description of the cluster analyses performed by the genesifter software ) . the results are presented as a heat map showing relative levels of expression in fig . the heat map is divided into two regions : region 1 ( above the line ) shows transcripts whose levels increased ( red ) above controls following treatment with antide , and region 2 depicts transcripts whose levels decreased ( green ) with antide treatment . the majority of transcripts were in the latter region , i.e. levels decreased in expression following antide treatment . most transcripts in both regions were restored to near - control levels by lh replacement ( see table  ii for examples of transcripts not restored by lh replacement , which compares the transcripts that differ significantly between the control and the antide+lh groups ) . many , but not all , of the antide - sensitive transcripts appear to be affected similarly ( either decreased or increased ) by steroid withdrawal ( trl ) , but several of these changes were not as pronounced . treatment with r5020 reversed many of the effects of trl , but not necessarily to the level observed with lh replacement . ( a ) cluster analysis by multiple comparison procedure of genesifter of all transcripts whose levels significantly ( p < 0.05 ) changed in expression 2-fold when compared with control cl . the heat map indicates the relative levels of the expression of each transcript , with red indicating increased expression and green indicating decreased expression . the line separates the two regions of different transcript expression : 1 : increasing expression with antide treatment ; 2 : decreasing expression with antide treatment . c , control ; a , antide ; l , a + lh ; t , a + lh + trl ; r , a + lh + trl + r5020 . ( b ) summary of pairwise comparison analysis of the number of transcripts identified by genesifter as significantly changing expression 2-fold ( student s t - test , p < 0.05 ) between indicated treatment groups . comparisons between complimentary ablation / replacement groups are indicated on the top , whereas pairwise comparisons between control and a + lh , or a + lh and a + lh + trl + r5020 treatments are depicted below . for example , 1415 transcripts differ significantly between the control and antide treatment groups , whereas only 36 differ significantly between the control and antide + lh treatment groups . the number of transcripts whose expression differs between control , antide and a + lh groups ( black brackets ) are greater than those between a + lh , a + lh + trl and a + lh + trl + r5020 groups ( dashed brackets ) . transcripts not restored to control levels by lh replacement of antide - treated cl ( 2-fold differences p < 0.05 ) effect of antide , transcript not restored to near control levels by lh replacement . the largest number of differentially expressed transcripts was associated with lh ablation / replacement , while steroid ablation / replacement affected fewer transcripts ( fig . hierarchical cluster analysis of all treatment groups by genesifter indicated that the control and a+lh groups were similar in gene expression , whereas the antide group was most related to the a + lh + trl and a + lh + trl + r5020 groups . because a relatively small number of transcripts differed 2-fold between the control and a + lh groups ( n = 36 , fig . 1b ) , the a + lh group can be considered a separate  control for the steroid ablation / replacement groups for the purpose of further analysis . pathways significantly [ as determined by z - score ( doniger et al . , 2003 ) and ontology abundance ] affected by treatments ( 2-fold ) are depicted by known gene ontologies in table  iii . many of these significant ontologies are associated with the production of steroids and steroid precursors ( e.g. sterol biosynthetic processes and cholesterol biosynthetic processes ) , and a number are related to the immune system / response ( immunoglobulin production , regulation of b cell activation , and others ) . ontologies significantly impacted by lh and steroid ablation / replacement z - score 3.0 , as determined by genesifter software analysis and 5 transcripts per ontology , with significant transcripts representing at least 10% of ontology on array . % determined by number of transcripts in ontology list divided by total number of transcripts in ontology on array . several genes ( n = 22 ) , representing transcripts identified from the microarray data set as expressed in the macaque cl , were chosen for validation by real - time pcr . these included novel genes with respect to luteal function , steroid hormone receptors , previously determined lh- and/or steroid - regulated genes , plus negative control transcripts that did not change in expression between groups . table  iv lists all validated genes and summarizes the results of microarray expression analysis and one - way anova values for real - time pcr data . the table  is divided into examples of gene transcripts whose levels were ( i ) not altered by treatments ( not regulated ) , ( ii ) lh - regulated independent of steroid influence and ( iii ) lh - regulated in a p or ( iv ) other steroid - dependent manner . typically , the patterns of mrna levels determined by real - time pcr resembled those from microarray analysis ( table  iv ) . real - time pcr validation of select differentially expressed genes control values ( sem ) are presented as a reference . direction of expression of validated genes by microarray analysis is compared with previous group ( e.g. a compared with c ) , except for genes whose expression did not differ from control . one - way anova values of significance are presented for real - time pcr validation . an example of a novel gene identified by microarray as being lh - regulated in the primate cl is interleukin 1 receptor antagonist ( il1rn ) , depicted in fig .  2 . the il1rn gene belongs to the  negative regulation of immune system process ontology based on known kyoto encyclopedia of genes and genomes  kegg pathways ( similar to table  iii , which displays the number of transcripts with known gene ontology pathways ) ; many of the transcripts in this ontology , including il1rn , were mainly affected by lh ablation / replacement , and to a lesser extent , by steroid ablation / p replacement ( fig . results from real - time pcr analysis were comparable with those provided by microarray analysis ( fig . 2b ) ; il1rn mrna levels increased following antide treatment , but this effect was prevented by lh replacement . treatment with trl with or without r5020 replacement was without effect . a list of regulated transcripts identified by genesifter from the kegg ontology  negative regulation of immune system process ( as in table  iii ) as significantly ( one - way anova , p < 0.05 ) affected by treatments is shown in panel a ( blue indicates minimum expression , whereas red indicates maximum ) . a comparison of interleukin 1 receptor antagonist ( il1rn ; mean  sem ) results is depicted in panel b , with white bars representing microarray values ( genesifter one - way anova , p < 0.05 ) , and black bars representing real - time pcr data ( one - way anova , p < 0.001 ; different letters signify treatment groups with significantly different values , p < 0.05 ) . regulated members of the  steroid biosynthetic process ontology , based on known kegg pathways ( similar to the results from table  iii ) , and two validated genes from this category are presented in fig . most of these transcripts are regulated similarly by lh and steroid ablation ( fig . while lh replacement typically prevented the decline induced by antide , progestin replacement with r5020 was less effective in preventing trl s effects , except in some instances ( e.g. stard4 , cyp19a1 ) . results from real - time pcr assays of the prolactin receptor ( prlr ; fig . levels of both mrnas were decreased by antide treatment , and lh replacement maintained levels similar to control . likewise , trl treatment resulted in mrna levels similar to antide , but r5020 replacement either did not affect mrna levels ( prlr ; fig . 3b ) , or only partially maintained mrna at control levels ( sc4mol ; fig . steroid biosynthetic process ( similar to table  iii ) as significantly ( one - way anova , p < 0.05 ) affected by treatments is shown in panel a ( blue indicates minimum expression , whereas red indicates maximum expression ) . real - time pcr results for prlr ( panel b ; one - way anova , p < 0.01 ) and sc4mol ( panel c ; one - way anova , p = 0.01 ) are compared with microarray results ( p < 0.05 ) . the expression of transcripts for genomic steroid hormone receptors as measured by microarray was low , and the pattern did not change throughout the experimental groups ; examples include the pgr ( table  iv and fig . 4a ) , the ar and estrogen receptor  ( ar and esr2 , respectively ; table  iv ) . the only putative steroid hormone receptor whose transcript levels differed significantly with treatment , based on microarray and real - time pcr analyses , was the non - classical progestin receptor : progesterone receptor membrane component 1 ( pgrmc1 ; fig . this transcript was highly expressed , but the pattern of mrna expression differed between real - time pcr data and the microarray for both a + lh and a + lh + trl treatment groups ( fig . microarray and real - time pcr expression patterns for the genomic pgr ( panel a ; genesifter and real - time pcr one - way anova , p > 0.6 ) and p receptor membrane component 1 ( pgrmc1 ; panel b ; genesifter one - way anova , p < 0.05 ; real - time pcr one - way anova , p < 0.01 ) . statistics for pgr and pgrmc1 are depicted for real - time pcr results ( mean  sem ) , with white bars representing microarray values , and black bars representing real - time data . two gene products , star protein and sfrp4 , were chosen to determine whether there was any correlation between their mrna and protein levels . 5a ) , whereas expression of mrna for the other gene was moderate ( sfrp4 ; fig . again , the patterns of expression for mrna data from real - time pcr matched microarray analysis for both genes . the mrna levels for star were markedly decreased by antide treatment , but maintained near - control levels by lh replacement , and comparable results were obtained by trl treatment and r5020 replacement ( fig . notably , the pattern of star protein levels between treatment groups was similar to that of mrna levels ( fig . in contrast , sfrp4 mrna levels increased 6-fold with antide treatment , and replacement of lh reduced , but did not completely restore mrna to control levels ; trl treatment with or without r5020 replacement had a minimal effect when measured by real - time pcr ( fig . although the levels of protein for sfrp4 changed significantly across treatment groups ( fig . 6b ; p < 0.001 ) , the expression pattern of the protein did not match that of the mrna . protein levels were unchanged between lh ablation and replacement groups , whereas steroid ablation by trl treatment lowered the expression levels , and r5020 replacement had no further effect . the western blots for pooled samples for star and sfrp4 are shown in fig . star mrna expression ( panel a ; white bars indicate microarray data , black bars indicate real - time data : see the legend of fig .  2 for more details on microarray and real - time data ; one - way anova , p < 0.001 ) and protein levels ( panel b ; one - way anova , p < 0.001 ; means  sem ) in individual cl normalized to -actin as compared between treatment groups . see the legend of fig .  5 for details . the pattern of sfrp4 protein expression , although significant ( one - way anova , p < 0.001 ) , does not match the expression of sfrp4 mrna ( real - time pcr one - way anova , p < 0.02 ) . representative immunoblots for star , sfrp4 and -actin loading control for pooled samples of different treatment groups . 5a and b , star protein expression matches mrna expression patterns , whereas sfrp4 protein expression does not match mrna expression . this study provides a genome - wide analysis of macaque luteal transcripts that are either directly or indirectly regulated by lh . all normalized data files have been uploaded to the national center for biotechnology information s gene expression omnibus gene expression repository ( http://www.ncbi.nlm.nih.gov/geo/ ; series gse12281 ) , which allows for further analyses to be performed by independent investigators . the current microarray analysis confirmed earlier reports of lh - regulated transcripts in macaque luteal tissue . for example , our preliminary analysis of lh - regulated genes in the macaque cl from antide - treated monkeys identified crhbp mrna as markedly up - regulated by lh withdrawal ( xu et al . , 2005 ) . this evidence led to the detailed characterization of a novel local system in the macaque cl  the crh / ucn receptor - binding protein system  that may modulate lh action and hence regulate primate luteal structure  function ( xu et al . , 2007 ) . the current results expand our understanding of the control of crhbp expression and , possibly , action , with the finding that steroid withdrawal and r5020 replacement mimicked the effects of antide treatment and lh replacement . thus , crhbp appears indirectly regulated by lh via steroid hormone production , particularly the local actions of p. also , the current results confirm and extend the evidence from differential gene display in the bonnet monkey cl that lh withdrawal markedly suppressed the expression of ldl receptor mrna , a key membrane component for cholesterol uptake into primate luteal cells ( yadav et al . , 2004 ) . however , the current analysis also identified many novel transcripts whose expression is lh - regulated in the macaque cl . for example , mrna for the il1rn , a suppressor of the immune system , that was first detected in the ovaries of rats ( kol et al . , 1999 ) and later reported to increase interleukin-1-stimulated prostaglandin ( e , f2 ) release from human luteal cells ( miceli et al . this lh action appears independent of local steroid action , since lh treatment restored il1rn mrna to control levels and steroid withdrawal did not mimic the effect of antide treatment . unexpectedly , prlr mrna expression in the macaque cl was down - regulated by lh withdrawal . unlike in some non - primate species [ e.g. rodents ( herz et al . , 1986 ) ] , prl and prl - like hormones are not considered major luteotropic hormones for the primate cl ( stouffer , 2006 ) . however , a recent microarray analysis ( bogan et al . , 2008b ) of gene expression in the macaque cl throughout the menstrual cycle discovered appreciable prlr expression that declined at luteolysis ; moreover , the expression pattern for prlr mrna and protein were comparable . there are high levels of prolactin ( -like ) proteins circulating during pregnancy and lactation ( bachelot and binart , 2007 ) . the possibility proposed by knobil and colleagues ( richardson et al . , 1985 ) that prlr ligands could contribute to the maintenance of luteal function in primates , perhaps during pregnancy and lactation , warrants re - evaluation . notably , a small but significant number ( n = 36 , fig .  1b and table  ii ) of antide - sensitive transcripts in the macaque cl were not affected by lh replacement . of particular interest is the expression of ldl receptor ( discussed earlier ) , a key component in cholesterol entrance into the cell , and prostaglandin f receptor ( ptgfr ) , a potent , local and perhaps physiologic luteolytic factor in primates ( yadav et al . , 2004 ; bogan et al . , 2008a ) . expression of both ldl receptor mrna and ptgfr declined markedly with antide treatment , and this effect was not significantly altered by lh replacement . although the lh - replacement regimen restored the expression of many antide - sensitive gene products , and this regimen prevents the functional  structural regression of the cl in antide - treated monkeys ( duffy et al . , 1999b ) , it is possible that our lh protocol was sub - optimal for a cohort of lh - regulated genes . alternatively , there is evidence of gnrh receptor expression ( chakrabarti et al . , 2008 ) and gnrh effects ( siler - khodr et al . , 2004 ) in primate ovaries and cl . though it is currently controversial whether gnrh serves as an important local factor in primates , as it does in rodents ( gupta and flaws , 2005 ) , we can not rule out that antide affects luteal gnrh signaling and action . indeed , the antide - sensitive , lh - insensitive gene products could be markers of local gnrh action in the primate cl . of the approaching 1500 lh - sensitive transcripts in the macaque cl , less than one - third of this cohort was affected similarly by steroid ablation , and even fewer by progestin replacement ( fig . this evidence implies that many lh - regulated genes are independent of steroid action ; their expression may be regulated directly by lh signaling pathways , or indirectly by other lh - regulated local factors ( e.g. crh / ucn , vegf , pgs ) or lh - regulated receptors / binding proteins for other hormones or local factors ( prlr , crhbp ) in the cl . that progestin replacement only restored transcript levels for some genes may again ( as for lh replacement ) nevertheless , this regimen prevents endocrine and histological indices of functional and structural luteolysis ( young and stouffer , 2004 ) , in the macaque cl , and unequivocally restored mrna levels of select steroid - sensitive genes ( see what follows ) . it is possible that the numbers or types of cells expressing some of these steroid - sensitive transcripts changed after 3 days of treatment . previous histological analyses of antide- and a + lh + trl - treated cl revealed that steroidogenic cells were smaller in volume , and the numbers of immune cells were greater , compared with control , a+lh and a + lh + trl + r5020 treatments ( young and stouffer , 2004 ) ; but these observations were qualitative . alternatively , it is possible that by mid - late luteal phase ( day 912 ) , the cl is not as responsive to progestin as luteal tissue from the early - to - mid - luteal phase . we reported previously that the percent of pr - positive cells and pr mrna levels in the macaque cl decline after mid - luteal phase ( hild - petito et al . , 1988 ; duffy et al . , 1999b ) . moreover , there is evidence that gene expression in the cl in response to steroid withdrawal and progestin replacement differs between the early and mid - late luteal phase [ e.g. adamts-1 , a disintegrin and metalloproteinase with thrombospondin repeats-1 ( young et al . , 2004 ) ] . this differential response could also be related to changes in the ratio of pr a : b isoforms as the luteal lifespan progresses ( duffy et al . , 1997 ) . the current analysis also detected appreciable mrna levels for the novel membrane progestin receptor pgrmc1 in macaque luteal tissue . a physiologic role for pgrmc1 in the cl has yet to be defined , but it could play a role in mediating the lh - like , luteotropic effects of p by preventing apoptosis of luteinized cells ( engmann et al . , 2006 ) . finally , since the 3-hsd inhibitor , trl , markedly suppresses production of other steroids relying on p as a precursor , those steroid - sensitive transcripts that were not restored by r5020 replacement could be altered by androgen or estrogen withdrawal . the macaque cl expresses ar and er ( particularly er ) during its lifespan ( duffy et al . ( 2000 ) reported that er mrna levels decreased around luteolysis and that steroid withdrawal at mid - luteal phase ( day 6 ) increased er expression , whereas r5020 replacement returned expression to control levels . further studies are needed at earlier stages of luteal development and function , and while replacing estrogen or androgen , as well as progestin , to clarify the lh- and steroid - regulated gene processes in the primate cl . nevertheless , the changes in mrna levels of several genes in response to lh withdrawal and replacement were mimicked by steroid withdrawal and progestin replacement . for example , star expression exhibited a similar pattern following either withdrawal / replacement regimen ( fig . 5 ) , strongly suggesting that the changes evoked by lh were indirect and are due to lh - stimulated p production by the tissue . abundant levels of star protein are expressed in the developing and developed human cl , and these levels decline between the mid- and late - luteal phase ( devoto et al . likewise , expression of other enzymes in the ontology  steroid biosynthetic processes ( fig . 3 ) , sc4mol and cyp19a1 , exhibited a similar pattern following either lh or steroid withdrawal / replacement regimen . thus , p may regulate a select number of key genes that are critical for luteal structure  function around the onset of luteolysis at mid - late luteal phase . as noted from our initial analyses of mrna and protein levels , it will be important to follow genomic analyses with evaluations of the translation and activity of gene products . the patterns of mrna and protein expression were similar for star , suggesting that transcriptional regulation is a key part of progestin regulation of star production . however , the patterns of mrna and protein levels for secreted frizzled related protein 4 ( sfrp4 ) were not comparable , suggesting that regulation of sfrp4 can occur at the translational level . this concept is consistent with previous evidence ( young and stouffer 2004 ; peluffo et al . , 2005 ) that lh / progestin regulation of proteases ( caspases , mmps ) can occur at either / both the mrna , protein or enzyme activity level . further analysis of the actions of lh and steroids at the transcriptional , translational and post - translational levels is needed to understand the regulation of luteal structure  function in primates . in summary , this microarray experiment broadly defines the changes in gene expression in the primate cl that are regulated by lh around the onset of luteolysis in the menstrual cycle . the data suggest that lh regulates the expression of many genes involved in the maintenance of cl structure  function independently of steroid action . however , steroid withdrawal and r5020 replacement indicate that progestin is involved in the regulation of key pathways that regulate the cl lifespan ( stouffer , 2003 ) , and this may involve actions via a non - classical pr . also , steroid - sensitive genes whose activity was not restored by r5020 replacement could be regulated by androgens or estrogens . further studies on lh / steroid withdrawal and replacement during cl development in the early - luteal phase and analysis of the patterns of both mrna and protein expression in macaque cl are needed . during revision of this manuscript , a microarray analysis of a limited sample of primate cl after short - term lh ablation was published online ( priyanka et al . , 2008 ) . comparisons of similar studies will help to elucidate the relative roles of lh and steroids on gene expression in the primate cl during the menstrual cycle . this work was supported by several grants from the national institutes of health ( nih ) [ r01 hd20869 to r.l.s . , r01 hd42000 to j.d.h . , u54 hd18185 ( molecular and cellular biology core ) , p51 rr00163 ] ; plus a national research service award ( nrsa ) institutional fellowship [ t32 hd007133 to cvb ] .
this study was designed to provide a genome - wide analysis of the effects of luteinizing hormone ( lh ) versus steroid ablation / replacement on gene expression in the developed corpus luteum ( cl ) in primates during the menstrual cycle . on days 911 of the luteal phase 
 , female rhesus monkeys were left untreated ( control ) or received a gnrh antagonist antide ( a ) , a + lh , a + lh + the 3-hydroxysteroid dehydrogenase inhibitor trilostane ( trl ) or a + lh + trl + a progestin r5020 . on day 12 of the luteal phase 
 , cl were removed and samples of rna from individual cl were hybridized to affymetrix rhesus macaque total genome microarrays . the greatest number of altered transcripts was associated with the ablation / replacement of lh , while steroid ablation / progestin replacement affected fewer transcripts . 
 replacement of lh during antide treatment restored the expression of most transcripts to control levels . 
 validation of a subset of transcripts revealed that the expression patterns were similar between microarray and real - time pcr . 
 analyses of protein levels were subsequently determined for two transcripts . 
 this is the first genome - wide analysis of lh and steroid regulation of gene transcription in the developed primate cl . 
 further analysis of novel transcripts identified in this data set can clarify the relative role for lh and steroids in cl maintenance and luteolysis .