life expectancy has increased such that today 's women live on average a third of their lives in a postmenopausal stage . studying menopause has become of great importance due to its association with serious health risks such as osteoporosis , cardiovascular disease , ovarian cancer , and alzheimer 's disease . because women are expected to live a third of their lives after menopause , this translates to approximately three decades of increased risk for such disorders . in women , 17-estradiol ( e2 ) promotes osteoblast proliferation and collagen synthesis , blunts the effects of parathyroid hormone ( pth ) to resorb calcium from bone , and directs   1,25 dihydroxy - vitamin d ( 1,25(oh)2d3 ) action to the intestines for calcium and phosphate absorption .   accelerated loss of bone mineral density ( bmd ) has been associated with the loss of estrogen following menopause [ 6 , 7 ] . osteoporosis is a disease characterized by deterioration of bone tissue that leads to bone fragility and increased susceptibility to fractures [ 8 , 9 ] . there are an estimated 10 million cases of osteoporosis in the united states , of which 80% are women . it is estimated that by the year 2025 , annual costs associated with osteoporotic fractures will rise to $ 25.3 billion . therefore , studies aimed at understanding the biology of postmenopausal osteoporosis are of extreme importance . the most common and widely accepted animal model for studying the effects of ovarian failure on bone physiology is the ovariectomized ( ovx ) rodent . however , one drawback to the ovx animal is that it can not reproduce the effects of natural onset of menopause since removal of ovarian function is abrupt . the perimenopause may be associated with altered hormone levels and declines in bone mass . because the ovx animal can not mimic that process , an animal model capable of reproducing both peri- and postmenopause could provide better information regarding postmenopausal osteoporosis . an ovary - intact mouse model of menopause has been developed using the chemical 4-vinylcyclohexene diepoxide ( vcd ) . vcd has been shown to selectively destroy the smallest preantral ( primordial and primary ) follicles in ovaries of mice and rats by accelerating the natural process of follicular atresia [ 1215 ] . because vcd does not target larger follicles , the animal continues to ovulate normally until no more follicles are available for recruitment . thus , ovarian follicular depletion in the vcd - treated mouse ( vcd mouse ) is gradual . as with women undergoing perimenopause , vcd - treated mice show increased levels of fsh , declining levels of estrogen , and irregular estrous cycles   as they become follicle - depleted .   additionally , following ovarian failure residual ovarian tissue therefore , the vcd - treated mouse is relevant for studies related to both perimenopausal and postmenopausal stages . there has been an ongoing controversy as to whether residual ovarian tissue in postmenopausal women is steroidogenic [ 1922 ] . this is important to resolve because , in the face of declining 17  -estradiol , androgens produced by residual ovarian tissue could impact postmenopausal health in a negative or positive manner . previous studies reported that dispersed cells collected from residual ovarian tissue in vcd - treated mice were capable of producing androstenedione in vitro   and that residual ovarian tissue expresses the enzymatic machinery necessary to synthesize androgens de novo . further , a previous paper has demonstrated that retaining residual ovarian tissue in young vcd - treated follicle - depleted mice protects them from accelerated bone loss following ovarian failure . therefore , the purpose of this study was to determine in aged follicle - depleted mice whether retaining residual ovarian tissue also influences bone loss relative to ovariectomized and normally cycling animals . cycling female c57bl/6hsd mice ( 24 retired breeders ; aged 912 months ) were purchased from harlan sprague - dawley , inc . , animals were housed in polycarbonate plastic cages , kept at 22  2c on 12l : 12d cycles and fed   ad libitum . all animals were allowed to acclimate to the animal facilities for 1 week before the start of the experiment . each animal was assigned randomly to one of three groups : ( 1 ) vehicle control , ( 2 ) vcd , or ( 3 ) ovariectomy . all experiments and methods were approved by the university of arizona institutional animal care and use committee ( iacuc ) and conformed to the guide for the care and use of experimental animals . animals in the vcd group received vcd ( 160  mg / kg / d ; i.p . ; sigma - aldrich , st . louis , mo , n = 8 mice ) dissolved in sesame oil ( vehicle ) to induce ovarian failure . animals in the control group ( con ) received   sesame oil only ( i.p ; sigma - aldrich ; n = 8 mice ) . mice were weighed and dosed daily for 15 days and estrous cyclicity was monitored by daily vaginal cytology in both groups . on - going estrous cyclicity was confirmed in the vehicle - treated controls ( con ) , and only vcd - treated mice underwent ovarian failure . to match the age at the onset ovarian failure and therefore the duration of exposure to estrogen deficiency , ovariectomy was performed in a separate group of untreated mice at the average time when mice in the vcd group experienced ovarian failure ( day 35 after the onset of dosing in con and vcd mice ) . animals ( n = 8) were placed under ketamine / xylazine anesthesia (  ketaject  , phoenix pharmaceutical , inc , st . , shenandoah , ia , usa ) , and the right and left flanks were shaved , and skin was cleaned with povidone - iodine and 70% ethanol . incisions were made through the skin , fascia , and abdominal wall , and the ovaries were identified and externalized . a ligature was placed at the ovary / oviduct boundary before each ovary was removed . the muscle wall and skin were closed using 6 - 0 and 3 - 0 braided silk suture , respectively . tissumend ii surgical glue ( veterinary products laboratories , phoenix , az , usa ) was applied over the incision to prevent reopening of the wound . animals were euthanized by co2 inhalation followed by cervical dislocation on day 181 after the beginning of experiment . skeletons were fixed in 10% neutral buffered formalin for 48  h and transferred to 70% ethanol for 24  h. lumbar bmd was determined in vivo by dual - energy x - ray absorptiometry ( dxa ) using a piximus absorptiometer ( ge lunar , madison , wi ) .   after performing quality control measurements , animals were placed under anesthesia ( ketamine / xylazine mix ) and scanned .   for each dxa scan , the region of interest ( roi ) was manually set to include lumbar vertebrae 24 . vertebrae were evaluated using a desktop ct imaging system ( ct40 ; scanco medical ag , bassersdorf , switzerland ) equipped with a 10-mm focal spot microfocus x - ray tube and images acquired with a 12  m isotropic voxel size , as previously described . morphometric variables describing bone microstructure were computed using direct 3d methods , including bone volume fraction ( bv / tv , % ) , trabecular number ( tb.n , mm ) , trabecular thickness ( tb.th , m ) , trabecular separation ( tb.sp , m ) , connectivity density ( connd , mm ) , and the structure model index ( smi ) , a measure of the plate- versus - rod - like nature of the trabecular structure . data were compared using anova followed by a fischer 's plsd ( protected least significant difference ) post hoc test . statistical tests were conducted using statview for windows ( version 5.0 , sas institute , inc . , cary , nc ) with the significance level set at p < .05 for all tests .   an observation was considered a trend with p  .09 but > .05 . retired breeders ( 912 months old ) were dosed daily with vcd to induce premature ovarian failure . the average day of ovarian failure was d34.8  1.3 ( range : d3139 ) after the onset of dosing . thus , the ovaries from ovx mice were removed on d35 after the onset of dosing to match ovarian failure in vcd - treated mice . estrous cyclicity was determined in vehicle control - treated animals to confirm ovarian cyclicity .   throughout the month preceding the end of the study ( d151  d181 ) , cycling controls had estrous cycles of 5.08  0.34 days ( range : 37 days ; estrus to estrus ) . lumbar bone mineral density ( bmd ) of aged vcd - treated and ovariectomized ( ovx ) mice was monitored by dxa and compared to that in age - matched cycling controls ( figure 1 ) . lumbar bmd was not different between vcd - treated animals and age - matched cycling controls at any of the measured time points .   however , relative to cycling and vcd - treated animals , ovx had higher ( p < .05 ) lumbar bmd at the two time points preceding ovarian failure , but was not different from the other groups at any other time point .   in order to obtain information regarding the amount of bone density gain or loss within each group , all data was compared to their respective baseline bmd ( figure 1 ; con : 0.051  0.002  g / cm ; vcd : 0.051  0.001  g / cm ; ovx : 0.052  0.001 as expected for animals without ovarian failure , lumbar bmd in cycling control mice did not change ( p > .05 ) over time until the end of the study ( range : 0.051  0.002 to 0.048  0.003 however , both vcd - treated and ovx animals experienced a decline ( p < .05 ) from baseline in bmd starting on d59 ( age : ~1114 months ; ~1 month after ovarian failure or surgery in vcd and ovx mice ) and continuing to the end of the study ( vcd : 0.051  0.001 to 0.043  0.004 g / cm , 16% loss ; ovx : 0.052  0.001 to 0.044  0.002 trabecular bone microarchitecture of the fifth lumbar vertebral body was assessed by ct after sacrifice on d181 , after the onset of dosing ( age : 18.8 months ; figure 2 ) . there were no differences in any of the variables measured between cycling controls and vcd - treated mice ( p > .05 , figure 2 ) .   however , ovx mice had lower ( p < .05 ) bv / tv% and tbth and trends for decreased connd ( p = .09 ) and increased smi when compared to the cycling control group ( figure 2 ) . had lower ( p < .05 ) tbth and a trend for decreased connd ( p = .06 ) compared to vcd - treated mice .   two - dimensional frontal planar images of the vertebral bodies of ovariectomized animals showed obvious loss of integrity when compared to images from vertebrae of cycling control - treated and vcd - treated animals ( figure 3 ) . this study was designed to determine whether retaining residual ovarian tissue influences the rate or extent of ovarian failure - induced bone loss in aged , follicle - depleted mice .   in order to investigate the effect of ovarian failure on bone integrity , lumbar bmd of aged vcd - treated and ovariectomized mice was evaluated by dxa and ct and compared to that in age - matched cycling animals . there were no differences in lumbar bmd between vcd - treated and cycling control animals throughout the study . lumbar bmd of cycling controls did not differ over time until the end of the study ( age : 17.1 months ) .   in comparison , within 1 month from ovarian failure , both vcd - treated and ovx mice experienced a decline in bone mineral density that remained stable until the end of the study . trabecular bone microarchitecture , assessed by ct did not differ between aged vcd - treated mice and age - matched cycling animals .   however , ovx animals showed deteriorated trabecular architecture relative to both age - matched cycling controls and vcd - treated mice . in particular , ovx mice had decreased bone volume fraction and trabecular thickness , along with trends for decreased connectivity density and a more rod - like architecture .   compared to trabecular microarchitecture values reported in a previous study , the 18.8-month - old cycling controls in the present study had 51.4% lower bone volume , 45.1% lower trabecular number , 84.8% greater trabecular separation , and 74.4% lower connectivity density than the 3-month - old mice of the same strain . therefore , the lack of differences in trabecular microarchitecture may be explained by age - dependent loss of bone integrity in the cycling control group . a previous study conducted in young mice supports the hypothesis that retaining residual ovarian tissue can afford protection against accelerated bone loss following ovarian failure .   in that study , young ovx mice showed reduced lumbar bmd when compared to cycling animals 1 month after ovariectomy while bmd from vcd - treated , follicle - depleted animals did not differ until approximately 3 months after vcd - induced ovarian failure . results from that study also showed that despite observing significant trabecular deterioration in both mouse models of ovarian failure , changes in the ovx group were more dramatic than those observed in vcd - treated mice . one proposed mechanism for this protection is the production of androgens by residual ovarian tissue . androgens derived from residual steroidogenesis may act directly on androgen receptors previously identified in bone tissue   or be converted to estrogens by aromatase expressed in bone [ 2730 ] before acting on bone cells to protect against accelerated bone loss . increased production of androstenedione has been shown in young vcd - treated , follicle - depleted mice [ 16 , 23 ] .   also , young vcd - treated mice have been shown to have levels of androstenedione that are similar to those in cycling mice , but higher than those observed in age - matched ovx mice . however , the present dxa data using aged animals suggests that this benefit may decrease with advancing age . previous work showed that circulating androstenedione declines with age in cycling vehicle - treated mice . this decline , although not completely understood , could be due to a decline in residual ovarian function as it has also been observed in aging cycling women   as well as in older postmenopausal women [ 32 , 33 ] .   a decline in residual ovarian function and circulating androstenedione may explain the diminished protection against bone loss observed in aged , follicle - depleted mice in the present experiment compared to that previously observed in young , follicle - depleted mice . previous studies have demonstrated that the presence of steroidogenically active residual ovarian tissue ( versus removal by ovx ) seems to play a beneficial role by retarding bone loss following ovarian failure in young mice .   surprisingly , this protection was not as obvious in the present study , in which aged vcd and ovx animals had similar progression to significant bone loss when bmd was monitored by dxa . no differences in trabecular bone architecture were identified between aged vcd - treated and cycling control mice , suggesting that cycling controls had already undergone age - dependent loss of bone integrity at the time of measurement ( age : 18.8 months ) . however , there were significant detrimental effects of ovarian failure on trabecular bone of ovx animals compared to cycling and vcd - treated mice . therefore , these observations suggest that retaining residual ovarian tissue is less protective in aged follicle - depleted mice than in young . these findings further support the vcd - treated mouse as a useful model for studies related to menopause and osteoporosis .
previous work showed that retaining residual ovarian tissue protects young mice from accelerated bone loss following ovarian failure . 
 the present study was designed to determine whether this protection is also present in aged animals . 
 aged ( 912 months ) c57bl/6hsd female mice were divided into : con ( vehicle ) , vcd ( 160  mg / kg ; 15d ) , or ovx ( ovariectomized ) . 
 lumbar bmd was monitored by dxa and ct used to assess vertebral microarchitecture . 
 bmd was not different between vcd and con at any time point but was lower ( p < .05 ) than baseline , starting 1 month after ovarian failure in vcd and ovx mice .   following ct analysis there were no differences between con and vcd , but ovx mice had lower bone volume fraction , trabecular thickness , and a trend for decreased connectivity density . 
 these findings provide evidence that retention of residual ovarian tissue may protect aged follicle - depleted mice from accelerated bone loss to a lesser extent than that observed in young mice .