Maternal diet composition alters serum steroid and free fatty acid concentrations and vaginal ph in mice

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1 75 Mternl diet composition lters serum steroid nd free ftty cid concentrtions nd vginl ph in mice JJWhyte 1, A P Alexenko 2, A M Dvis 1,2, M R Ellersieck 3, E D Fountin 1 nd C S Rosenfeld 1 Deprtments of 1 Biomedicl Sciences, 2 Animl Sciences, Christopher S. Bond Life Sciences Center, 3 Food Systems nd Bioengineering, Agriculture Experiment Sttion-Sttistics, University of Missouri-Columi, Columi, Missouri 65211, USA (Requests for offprints should e ddressed to C S Rosenfeld; Emil: rosenfeldc@missouri.edu) Astrct We exmined the effects of three mternl diets (very high ft (VHF), low ft (LF), nd control (Purin 5015)) on serum steroids, free ftty cids (FFA), nd vginl ph in Ntionl Institutes of Helth Swiss mice. Femles were fed (VHF, nz33; LF, nz33; 5015, nz48) from 4 to 16 weeks of ge. Following reeding, femle serum ws collected t 0. 5 (pre-implnttion, erly diestrus) or 8. 5 (post-implnttion, mid-diestrus) dys post-coitus (dpc). The serum concentrtions of 17-estrdiol, testosterone, progesterone, nd FFA were nlyzed t oth collection points, nd vginl ph t 0. 5 dpc. Striking differences in steroids nd FFA were oserved t 0. 5 dpc mong the groups. Estrdiol ws higher in the VHF (14. 1G3. 0 pg/ml), compred with LF mice (5. 2G2. 3 pg/ml; P%0. 05). In contrst, 0. 5 dpc testosterone ws lower in the VHF (10. 5G3. 0 pg/ml) versus the LF group (32. 7G8. 4 pg/ml; P%0. 05). At 8. 5 dpc, progesterone ws higher in the VHF (89. 6G6. 7 ng/ml) versus the 5015 group (60. 1G4. 9 ng/ml; P%0. 05). VHF mice hd higher FFA concentrtions t 0. 5 dpc (1. 0G0. 2 mmol/l) thn LF nd control mice (0. 5G0. 1 nd 0. 6G0. 1 mmol/l respectively; P%0. 05). At 8. 5 dpc, VHF femles hd higher serum FFA (0. 8G0. 1 mmol/l) thn LF nd control femles (0. 4G0. 1 nd 0. 6G0. 1 mmol/l; P%0. 05). Men vginl ph of VHF femles (6. 41G0. 09) ws lower thn 5015 femles (6. 76G 0. 10; P%0. 05). These diet-induced ltertions in serum steroid nd FFA concentrtions might ffect severl reproductive processes, including preferentil fertiliztion y one clss of sperm over the other nd sex is in pre- nd post-implnttionl emryonic development. Journl of Endocrinology (2007) 192, Introduction For severl yers, this lortory hs een interested in the effects mternl diet cn hve on offspring sex rtio Rosenfeld et l Mny pst nd present reports hve suggested tht diet, prticulrly one enriched with either sturted or unsturted ftty cids, cn lter serum steroid concentrtions in vriety of species, including rodents, food nimls, nd humns (Tlver et l. 1985, Hilkivi-Clrke et l. 1996, Woods et l. 1996, Hilkivi-Clrke et l. 1997, Dorgn et l. 2003). Thus, s potentil side, we were interested in exmining the effects of three diets: very high ft/low ft (VHF, LF; Reserch s, New Brunswick, NJ, USA), nd Purin 5015 chow-sed diet (Purin, St Louis, MO, USA; see Tle 1) on serum steroid concentrtions nd free ftty cids. While the mechnisms underlying diet-induced ltertion of offspring sex rtio re likely complex, ny diet-induced differences in steroid concentrtion my help to elucidte how diet skews offspring sex rtio. ry ft cn influence the expression of enzymes tht metolize sex steroid hormones (Zhou et l. 2005, Dieudonne et l. 2006). Adipose tissue is n importnt site of steroid hormone iosynthesis (Siiteri 1987, Belnger et l. 2002, Simpson 2003, Dieudonne et l. 2006). Moreover, ovrinderived D4 ndrostenedione nd testosterone cn e romtized in dipose tissue to estrone nd estrdiol respectively (Lmrinoudki et l. 2006). An dditionl potentil mechnism of dietry influence on sex steroid concentrtion reltes to the sttus of cholesterol s precursor to steroid hormones. cn influence serum cholesterol (Menotti 1999) nd high cholesterol is correlted with high serum ndrogen nd estrogen concentrtions (Shelley et l. 1998, Kumgi et l. 2001). Pst studies in rodents, cttle, nd humns hve indicted tht diet might underpin chnges in serum hormonl concentrtions, including testosterone nd estrogen (Killen et l. 1989, Wynn & Wynn 1993, Dorgn et l. 1996, Hilkivi-Clrke et l. 2002, Dorgn et l. 2003, Fernndez- Twinn et l. 2003). Femle rts fed diet enriched with n-3 polyunsturted ftty cids hd 48% increse in serum concentrtions of 17-estrdiol compred with rts fed diet enriched with n-6 ftty cids (Hilkivi-Clrke et l. 2002). Similrly, femle rts fed low protein diet hd significnt increse in 17-estrdiol compred with those fed control diet (Fernndez-Twinn et l. 2003). Nutritionlly restricted heifers hve decresed serum estrogen concentrtions t dy 200 of pregnncy compred with lrger, non-food restricted heifers (Killen et l. 1989). A high sturted ft diet induces n increse in estrogen, estrone, nd dehydroepindrosterone sulfte concentrtions in women (Dorgn et l. 1996, Ngt et l. Journl of Endocrinology (2007) 192, DOI: /JOE /07/ q 2007 Society for Endocrinology Printed in Gret Britin Online version vi Downloded from Bioscientific.com t 01/10/ :58:05AM vi free ccess

2 76 J J WHYTE nd others. Mternl diet lters serum steroids in mice 2005). A controlled clinicl tril reveled tht girls fed low ft (LF) diet exhiited higher serum testosterone concentrtions during the lutel phse of the cycle ut lower estrdiol concentrtions (Dorgn et l. 2003). In the light of these previous studies, we hve exmined the serum concentrtions of testosterone, estrogen, progesterone, nd free ftty cids (FFA) in femle mice fed predominntly lrd diet (which skews the offspring sex rtio to mles), LF/high crohydrte diet (which skews the rtio towrds femles), nd chow-sed control diet (which gives rise to roughly equivlent numers of mle nd femle pups). Our underlying hypothesis ws tht diet might lter vrious serum steroid concentrtions, which in turn my influence the reproductive trct environment, such s vginl ph, t copultion or during emryonic development to fvor the success of one sex over the other. Mterils nd Methods Animl reeding nd diet conditions NIH Swiss mice (Mus musculus) were red nd mintined in the University of Missouri s Animl Science Reserch Center. All the experiments were crried out ccording to the NIH Guidelines for the Cre nd Use of Lortory Animls nd were pproved y the University of Missouri s Animl Cre nd Use Committee. Mice were housed in polysulfone cges (Uni Cge, Silom Springs, AR, USA; dimensions: 18. 4cm W!29. 2cm D!12. 7 cm H). During non-reeding periods, four femle mice were housed per cge, nd during reeding, two femles were housed with one mle. AllmiceusedinthisstudywereNIHSwissndwererednd ssigned to diet groups s descried previously (Rosenfeld et l. 2003). In Decemer 2004 (Study 1), 8 to 10-week-old P1 NIH Swiss femle mice (Hrln, Mdison, WI, USA) were red to stud mles. After wening t 21 dys, 18 femle offspring were mintined on regulr chow diet, Purin 5015 (5015), until they were 30 dys of ge nd then rndomly ssigned to three groups nd fed d liitum the control Purin 5015 (nz10), low ft (LF; nz4), or very high ft (VHF; nz4) diets throughout the study. The femles were red t 20 weeks of ge to stud NIH Swiss mles (z12-week-old). A replicte study ws initited 2 weeks fter eginning the first to provide totl of 13 mothers ech in the LF nd VHF dietry groups nd 28 mothers in the 5015 dietry group. To increse the smple size in ech diet group, two dditionl experiments of identicl design were initited in Octoer 2005 (Study 2; nz6 femles per group, 16 weeks on diets) nd Decemer 2005 (Study 3; nz14 femles per group, 16 weeks on diets) to provide n overll totl of 33 femles in the LF nd VHF diet groups, nd 48 femles in the 5015 diet groups. Conception ws ssessed y the presence of coitl plug (the dy of the coitl plug eing scored s 0. 5 dys post-coitus (dpc)), nd femles were ssessed to e pregnnt y the presence of either pre-implnttionl emryos in the oviduct (0. 5dpc)or implnted conceptuses in the uterine wll (8. 5 dpc). Vginl ph ws determined t 0. 5 dpc in ll femles s descried in the next Journl of Endocrinology (2007) 192, section. Femles were then rndomly ssigned to one out of the two study groups. In the first group, pregnnt femles (VHF, nz15; LF, nz14; 5015, nz23) were killed fter ph determintion y CO 2 inhltion nd susequent cervicl disloction. Collection of serum for steroid hormone nd FFA concentrtions ws performed immeditely. In the second group (VHF, nz11; LF, nz17; 5015, nz17), vginl ph ws determined, nd femles were housed in seprte cges nd mintined on their ssigned diet until 8. 5 dpc. At this point, femles were killed for serum collection s descried ove. Vginl ph determintion Vginl ph mesurements were performed y using n MI-413 Needle Comintion ph microelectrode (Microelectrodes Inc., Bedford, NH, USA) with electrode dimeter of 16 G needle nd BASIC ph meter (Denver Instruments, Denver, Colordo, USA). Mice were immoilized in restriner mde from 50 ml Flcon centrifuge tue (Becton Dickinson nd Compny, Frnklin Lkes, NJ, USA). Ech mouse ws plced inside the tue nd fixed with the cp from the sme tue with 15 mm dimeter hole in it, therey llowing ccess to the til nd the vginl opening. Presented dt re verges from t lest five independent mesurements on ech mouse. Blood collection nd serum preprtion A non-heprinized whole lood smple ws otined y crdic puncture, nd the microfuge tues contining the whole lood were plced on ice for 30 min to permit clotting. The smples were centrifuged t 7500 g for 20 min. The resulting serum frction ws collected nd trnsferred to new utoclved microcentrifuge tue nd centrifuged t 7500 g for 3 min to remove further trces of clotted lood. Aliquots of serum for 17-estrdiol, progesterone, nd testosterone enzyme immunossy (EIA) were stored t K20 8C, nd liquots of serum for FFA determintion were stored t K80 8C. Estrdiol ssys Circulting serum concentrtions of 17-estrdiol were nlyzed y using modified EIA procedure initilly descried y Perry et l. (2004). Duplicte smples (50 ml) were extrcted with 2 ml methyl-tert-utyl ether (HPLC grde; Fisher Chemicl Co., Fir Lwn, NJ, USA) for 2 min on vortexer. Smples were frozen in dry ice methnol th. The solvent frction ws decnted into 12!75 mm orosilicte glss tues nd dried under strem of nitrogen gs. Extrcts were reconstituted in 50 ml estrdiol-free control serum. Estrdiol EIA kits nd regents were otined from Dignostic Systems Lortories, Inc. (DSL; Wester, TX, USA) nd Cymn Chemicl (Ann Aror, MI, USA). The ssys were performed ccording to the mnufcturer s instructions. All extrcted smples were run in duplicte on 96-well plte nd mesured on Bio-Tek Synergy HT plte reder t 450 (DSL) or 412 nm (Cymn) sornce (Bio-Tek Downloded from Bioscientific.com t 01/10/ :58:05AM vi free ccess

3 Mternl diet lters serum steroids in mice. J J WHYTE nd others 77 Instruments Inc., Winooski, VT, USA). Estrdiol concentrtions in smples were determined sed on 17-estrdiol stndrd curve (DSL: pg/ml; r 2 Z0. 98; Cymn: pg/ml; r 2 Z0. 97). Concentrtions of 17-estrdiol in two duplicte control stndrds (250G100 nd 1000G 350 pg/ml; DSL) were determined within the specified mrgin of error. Intr- nd inter-ssy coefficients of vrition for the Cymn 17-estrdiol ssy were 3. 3nd7. 6% respectively. Intr- nd inter-ssy coefficients of vrition for the DSL 17-estrdiol ssy were 4. 6 nd 11. 2% respectively. Progesterone nd testosterone ssys Concentrtions of free progesterone nd testosterone were determined y EIA with unextrcted serum smples. Progesterone nd testosterone EIA kits nd regents were otined from Dignostic Systems Lortories, Inc. Ech ssy ws run ccording to the mnufcturer s protocols. All the serum smples (50 ml) were run in duplicte nd mesured on Bio-Tek Synergy HT plte reder t 450 nm sornce (Bio-Tek Instruments Inc.). Serum progesterone concentrtions were determined sed on five-point progesterone stndrd curve ( ng/ml; r 2 Z0. 99). Concentrtions of progesterone in two duplicte control stndrds (1G0. 3 nd 7G2 ng/ml; DSL) were determined within the specified mrgin of error. Testosterone concentrtions in smples were determined sed on six-point testosterone stndrd curve ( pg/ml; r 2 Z0. 99). Concentrtions of testosterone in two duplicte control stndrds (50G15 nd 250G50 pg/ml; DSL) were determined within the specified mrgin of error. Intr- nd inter-ssy coefficients of vrition for the testosterone ssy were 3. 0 nd 10. 5% respectively. Intrnd inter-ssy coefficients of vrition for the progesterone ssy were 3. 7 nd 12. 6% respectively. Serum free ftty cids Concentrtions of FFA were mesured in triplicte using n enzymtic ssy nd ccording to the mnufcturer s instructions (Wko Chemicls, Richmond, VA, USA) nd other pulished methods (Prks et l. 2005). Concentrtions of FFA were determined sed on four-point FFA stndrd curve (0 1 mmol/l; r 2 Z0. 99). The intr- nd inter-ssy coefficients of vrition were nd 9. 9% respectively. Sttisticl nlyses The results re expressed s the mengs.e.m. Effects of diet on serum steroids nd FFA were exmined y ANOVA s 2!3 fctoril rrngement with time of serum collection nd diet s fctors respectively. Effect of diet on vginl ph ws tested y using one-wy ANOVA. Distriutions of continuous vriles were tested for normlity y use of the Kolmogorov Smirnov test. Endpoints with heterogeneous vrince were logrithmiclly trnsformed. Post hoc tretment effects were tested y using Fisher s lest significnt difference men seprtion test. Person s product moment correltion ws employed to determine the reltionship etween serum steroid concentrtions nd mternl weight nd emryo numer. The n used for the nlyses vried for the different nlyses ecuse for some smples there ws not enough serum to perform ll of the EIA for the steroids nd the FFA. Sttisticl significnce ws set t the level (SAS 1988). Results Vginl ph At 0. 5 dpc, femle NIH Swiss mice in the VHF diet group hd significntly lower vginl ph (6. 41G0. 09; nz16) compred with the 5015 control group (6. 76G0. 10; nz19; ANOVA, P%0. 05). However, this sttisticl difference did not extend to the LF (6. 61G0. 08; nz19) diet group. Mternl ody weights As ws oserved in previous study (Rosenfeld et l. 2003), the mice tolerted the different diet conditions well, including the high lrd content of the VHF diet. Mternl ody weights over the 16-week dietry tretment period re displyed in Fig. 1. The gretest increse in ody weight over time ws seen in VHF-fed femles, ut this difference ws only significnt in Study 3 (Tle 1;ANOVA;P%0. 05). Despite the lck of mternl ody weight difference mong diet tretments, in generl, ody composition ws visily different during dissection, with VHF-fed femles hving lrger mounts of viscerl ft. Serum estrdiol Men concentrtions of extrcted serum estrdiol t 0. 5dpcin mice fed the VHF diet (14. 1G3. 0 pg/ml; Fig. 2) were significntly higher thn mice fed the LF (5. 2G2. 3 pg/ml; P%0. 05) nd 5015 control (8. 2G2. 3 pg/ml; P%0. 05) diets. At 8. 5 dpc, the reltive concentrtions of extrcted serum estrdiol in mice-fed VHF nd LF diets ppered to e inverted compred with the 0. 5 dpc mens, ut did not differ sttisticlly t this collection time point (Fig. 2). Extrcted serum estrdiol t 0. 5 dpc ws correlted with reeding mternl ody weight (g) when the three diet groups for ll the study times were nlyzed (rz0. 55; nz44; P%0. 05). No correltion with serum estrdiol ws detected for reeding mternl ody weight t 8. 5dpc. Serum testosterone Mice fed LF diet hd significntly higher men serum testosterone concentrtions t 0. 5 dpc (32. 7G8. 4 pg/ml) compred with oth VHF (10. 5G3. 0 pg/ml) nd 5015 control (12. 9G3. 4 pg/ml)-fed mice (Fig. 2; ANOVA; P%0. 05). In contrst, t 8. 5 dpc (Fig. 2), VHF-fed mice hd significntly higher serum testosterone concentrtions (77. 8G27. 8 pg/ml) compred with 5015 control-fed mice (51. 3G40. 7pg/ml; ANOVA; P%0. 05). However, Journl of Endocrinology (2007) 192, Downloded from Bioscientific.com t 01/10/ :58:05AM vi free ccess

4 78 J J WHYTE nd others. Mternl diet lters serum steroids in mice Femle mss (g) Weeks on diet testosterone concentrtions in LF-fed mice t 8. 5 dpc (62. 3G pg/ml) did not differ from the other two groups. As ws the cse with serum estrdiol, serum testosterone t 0. 5 dpc ws negtively correlted with reeding mternl ody weight (g) when the three dietry groups for ll study times were nlyzed (rzk0. 38; nz43; P%0. 05). No reltionship existed etween 8. 5 dpc serum testosterone concentrtion nd mternl reeding weight. Serum estrdiol/testosterone (E/T) rtio The rtio of extrcted serum estrdiol to serum testosterone in individul mice ws on verge greter thn one for femles on the VHF diet t 0. 5 dpc (23. 3G11. 6; nz12) nd less thn Figure 1 Chnges in men mternl ody weight (GS.E.M.) of mice until reeding on the VHF (:; nz23), LF (-; nz28), nd 5015 control (%; nz35) diets. The dt re from three studies, with femles mintined on diets for 16 weeks strting t 30 dys of ge. Significnt weight difference of VHF femles from LF nd control groups is denoted y sterisks (ANOVA; P!0. 05). Tle 1 Effect of control (5015), LF, nd VHF diets on emryo numer nd mternl weight (g) of femle mice ged 20 weeks t reeding (mengs.e.m.) Mternl weight (g; 20 weeks) Breeding dte Study 1 (July 2005) G0. 7(nZ22) LF 29. 1G0. 9(nZ10) VHF 32. 5G1. 4(nZ9) Study 2 (Ferury 2006) G3. 6(nZ4) LF 26. 8G0. 7(nZ4) VHF 32. 5G4. 0(nZ4) Study 3 (April 2006) G5. 6(nZ9) LF 30. 0G2. 2(nZ14) VHF 47. 0G11. 8(nZ10) Comined G0. 7(nZ35) LF 29. 2G0. 5(nZ28) VHF 38. 6G2. 3(nZ23) Three identicl study designs conducted on different dtes re nlyzed seprtely nd in comintion. Emryo numer counts include oth 0. 5nd 8. 5 dpc emryos collected. Lowercse letters denote significnt differences mong dietry tretments (ANOVA; P%0. 05). Journl of Endocrinology (2007) 192, one for femles on the LF diet t 0. 5 dpc (0. 2G0. 1; nz9). The men LF femle E/T rtio ws significntly lower thn oth the VHF nd the 5015 rtios (ANOVA; P%0. 05) t 0. 5 dpc. The men E/T rtios for the three dietry tretments t 8. 5 dpc differed significntly (ANOVA; P%0. 05), ut in contrst to the 0. 5 dpc results, the E/T rtio in individul mice ws generlly less thn one for femles on the VHF diet (0. 2G0. 1; nz8) nd greter thn one for femles on the LF diet (5. 5G3. 8; nz11). At 0. 5 nd 8. 5 dpc, men E/T rtios for mice on the 5015 control diets were 3. 2G1. 7(nZ17) nd 1. 7G0. 6(nZ12) respectively. Serum progesterone Serum progesterone t 0. 5 dpc did not differ mong the dietry groups (Fig. 2), ut the concentrtion of this steroid ws inversely relted to mternl ody weight t the time of reeding (rzk0. 36, nz44, P%0. 05). At 8. 5 dpc, (Fig. 2), VHF-fed mice hd significntly higher serum progesterone concentrtions (89. 6G6. 7 ng/ml) compred with 5015 control-fed mice (60. 1G4. 9 ng/ml; ANOVA; P%0. 05). In contrst to the negtive correltion t 0. 5 dpc, significnt positive reltionship existed etween mternl ody weight t the time of reeding nd serum progesterone concentrtion t 8. 5 dpc (rz49, nz34, P%0. 05). Serum free ftty cids (FFA) Mice in the VHF diet group hd n elevted men concentrtion of FFA t 0. 5 dpc (Fig. 2; 1. 0G0. 2 mmol/l) compred with oth the LF nd the 5015 control diet groups (0. 5G0. 1 nd 0. 6G0. 0 respectively; ANOVA; P%0. 05). This sme pttern of concentrtions ws oserved in the different dietry groups t 8. 5 dpc, with significnt differences mong ll the three groups (Fig. 2; ANOVA; P%0. 05). No reltionship etween FFA nd mternl ody weight t reeding ws oserved t either 0. 5or8. 5 dpc (nz48 nd 37 respectively). Discussion Herein, we hve exmined the influence of diet on some reproductive vriles in mice, including serum concentrtions of sex steroids nd vginl ph, nd discovered tht diet cn influence these prmeters. The interctions etween diet nd circulting steroid hormone concentrtions were complex. The VHF diet incresed serum estrogen concentrtions reltive to the other dietry tretments t 0. 5 dpc, ut not t 8. 5 dpc. Previous studies hve shown tht serum estrogen concentrtions re highest t proestrus nd decline during pregnncy (McCormck & Greenwld 1974, Nelson et l. 1992, Wlmer et l. 1992; reviewed in Overpeck et l. 1978). The higher mount of estrdiol in the mice on the VHF diet compred with the other diet groups could e relted to the mount of ccumulted ody ft in the mice. Downloded from Bioscientific.com t 01/10/ :58:05AM vi free ccess

5 Mternl diet lters serum steroids in mice. J J WHYTE nd others 79 Estrdiol (pg/ml) Testosterone (pg/ml) 120 c 77 8 cd d Progesterone (ng/ml) c cd d 60 1 FFA (mmol/l) c d e Figure 2 Steroid hormone concentrtions (estrdiol, testosterone, nd progesterone) nd free ftty cid (FFA) concentrtions t 0. 5 (lck rs) nd 8. 5 dpc (open rs) in serum of femle NIH Swiss mice mintined on very high ft (VHF), low ft (LF), nd control (5015) diets for 16 weeks. Lowercse letters denote significnt differences mong dietry tretments within the 0. 5 nd 8. 5 dpc results (P%0. 05) for ech endpoint. Femle smple size is denoted within ech r nd the men concentrtion for ech group is denoted ove ech r. Adipose tissue is the mjor tissue site of conversion of ndrostenedione to estrone, nd estrdiol hs een reported to e elevted in oese humns nd non-humn primtes (reviewed in Siiteri 1987, Dieudonne et l. 2006). Mternl ody weight ws higher overll in the VHF mice in this study (Fig. 1) nd n erlier study (Rosenfeld et l. 2003), nd the extr viscerl ft in these mice proly provided n dditionl estrogen source. Anlogous dietry interventions exert similr effects on circulting estrogen concentrtions. Rts fed diet enriched with nk3 FFA hd higher estrogen concentrtions thn their counterprts fed norml, non-supplemented diet (Hilkivi-Clrke et l. 2002, Fernndez- Twinn et l. 2003). Conversely, nutritionlly restricted cttle hd lower estrogen concentrtions t dy 200 of pregnncy thn controls (Killen et l. 1989). A met-nlysis of studies in women suggested tht incresed dietry ft intke resulted in elevted serum estrdiol concentrtions (Wu et l. 1999, 2000). Finlly, Dorgn et l. (2003) noted tht 5-yer reduction in dietry ft in pre-puertl girls reduced serum estrdiol (mesured t the folliculr phse) ut incresed serum testosterone concentrtions (mesured t the lutel phse). Whether the mount of ccumulted ody ft is the min vrile in the ove studies is uncler. As reviewed y Lmrinoudki et l. (2006), there hve een conflicting reports in women s to whether high serum estrogens re positively correlted with circulting cholesterol nd triglyceride concentrtions. Species vrition nd interction of diet nd timing of the estrous or menstrul cycle might ccount for these seemingly disprte results. At 0. 5 dpc, the three groups (VHF, LF, nd 5015 controls) hd very different E/T rtios. The VHF group, in prticulr, hd reltively high estrdiol vlues nd somewht reduced testosterone vlues reltive to the 5015 controls, wheres the LF-fed mice hd elevted circulting testosterone concentrtions. Clerly, the reltive concentrtion of the two sex steroids is ffected y diet. As discussed ove, the low Journl of Endocrinology (2007) 192, Downloded from Bioscientific.com t 01/10/ :58:05AM vi free ccess

6 80 J J WHYTE nd others. Mternl diet lters serum steroids in mice testosterone nd high estrdiol concentrtions in VHF femles t 0. 5 dpc my reflect n incresed conversion of ndrogen to estrogen in dipose tissue. The reltively higher testosterone concentrtions in the LF-fed femles t 0. 5 dpc is seemingly hrder to explin ut is consistent with Dorgn et l. (2003) findings tht girls fed reduced clorie diet hd higher testosterone concentrtions, suggesting tht reduced dipose tissue mss results in uildup of ndrogen. In serum collected t 8. 5 dpc, the E/T reltionship etween VHF nd LF groups ws reversed, in lrge prt ecuse of the incresed concentrtion of testosterone nd the reduction of estrdiol in the VHF-fed mice. The excess of circulting cholesterol, s would e nticipted in mice on diet rich in sturted ft, might ccount for the reltively high concentrtions of progesterone nd its downstrem metolite, testosterone (Arensurg et l. 1999) in the VHF group t 8. 5 dpc. It remins to e determined whether these differences in steroid concentrtions could influence the sex rtio of pups. Mle fetuses hve een proposed to e more sensitive to progesterone insufficiency thn femles (Flint et l. 1997). However, the concentrtion of this hormone did not differ significntly etween the VHF nd the LF groups t either 0. 5or8. 5 dpc. Moreover,vluesintheLFgroupt8. 5 dpc were not different from those mesured in the controls. Clerly, progesterone is n unlikely meditor of sex rtio skewing in this cse. Androgens re known to exert epigenetic effects on the developing fetus. For exmple, femle mice orn etween two mles (2M) tend to e msculinized nd hve more mle offspring (Vndenergh & Huggett 1995, Vom Sl et l. 1999). Dominnt women with elevted serum testosterone concentrtions re reported to hve significntly more sons thn sumissive femles with lower testosterone concentrtions (Grnt 1994, Singh & Zmrno 1997, Grnt & Frnce 2001), nd Jmes (1990) hs implicted high testosterone concentrtions in the mother round the time of conception s fctor likely to fvor sons. However, our dt show tht the mice on the LF diet unexpectedly hd the highest testosterone concentrtions t 0. 5 dpc nd hd roughly similr concentrtions to the VHF group t 8. 5 dpc. Since mture LF femles produce more femle thn mle pups, testosterone seems to e n unlikely meditor of the skewing towrds mle pups in mice on the VHF diet. Another difference etween mice on the VHF nd the LF diets ws the concentrtion of circulting FFA. At 8. 5 dpc, in prticulr, serum FFA ws higher in the VHF group, perhps not surprising outcome, considering tht these mice were consuming lmost sixfold more clories from ft thn the LF group. Little is known out the effects of FFA on the developing emryo nd whether there is ny sexul dimorphism with regrd to either their utiliztion or reltive toxicity. Nor is it cler whether FFA could influence the properties of X nd Y sperm in some selective mnner. The lower vginl ph of mice on the VHF diet might e n outcome of their higher circulting concentrtions of FFA or estrogen (Gorodeski et l. 2005). Moreover, the motility of X Journl of Endocrinology (2007) 192, nd Y sperm my e influenced y vginl ph (Ericsson 1976, Prtt et l. 1987), with more cid conditions providing n dvntge to Y-ering sperm. In hmsters, more mle irths occur if fertiliztion occurs lte in estrus, possily s the result of low vginl ph (Prtt et l. 1987). Studies in opossums nd humns hve implicted elevted dietry FFA, nd prticulrly the essentil polyunsturted ftty cids (PUFA), in incresing mle irths (Austd & Sunquist 1986). The presence of excessive lrd in the VHF diet, which leds to more mle irths, could conceivly provide spring effect on metolism of PUFA y mice in the VHF diet group, if indeed PUFA re importnt in controlling sex rtio, e.g. y ltering the production of reproductively significnt prostglndins (PGs), such s PGE or PGF 2 (Thtcher et l. 1995). To exmine the effects of PUFA, we re eginning study to determine whether diets differing in PUFA ut equivlent in totl ft clorie content re le to cuse chnge in sex rtio mong pups orn to NIH Swiss mice. To determine whether, indeed, potentil linkge exists etween steroid concentrtions t the time of conception nd ltertion of sex rtio, we collected ll of the 1-cell emryos t 0. 5 dpc from the mice in the third study (nz14 dms one the LF diet nd nz10 for dms on the VHF diet). All of their emryos were cultured to the lstocyst stge nd the gender of ech ws determined y XY-FISH nlysis with proes from Open Biosystems (Huntsville, AL, USA). The preliminry results from this study suggest tht sex rtio skewing is lredy present t 0. 5 dpc with n incresed rtio of mle lstocysts in the VHF group nd more femle lstocysts in the LF group, s determined y the GENMOD procedure in SAS nd logit trnsformtion (odds rtio) (P%0. 01). Further studies will need to e performed to confirm these preliminry results nd determine whether the differences in the sex steroids nd FFA concentrtions t this time point in gesttion underpin this puttive skewing in sex rtio. In summry, the mount of ft in the diet fed to femle mice, prticulrly round the time of erly diestrus (conception) nd mid-diestrus (fter conceptus implnttion), cn influence the circulting concentrtions of sex steroids, FFA, nd vginl ph in those mice on the VHF diet. Acknowledgements The uthors re very grteful for the dvice nd support of Dr R Michel Roerts (University of Missouri-Columi). The uthors would like to thnk Dr Elizeth Prks (University of Minnesot, MN) for dvice relted to FFA determintion. Assistnce with niml husndry nd surgery ws provided y Dr Jime Riley, Mr Cory Weimer, nd Ms Emily Kern, University of Missouri-Columi, MO. This study ws supported y NIH Grnt Numer HD to Dr R Michel Roerts nd C S R. The uthors declre tht there is no conflict of interest tht would prejudice the imprtility of this scientific work. Downloded from Bioscientific.com t 01/10/ :58:05AM vi free ccess

7 Mternl diet lters serum steroids in mice. J J WHYTE nd others 81 References Arensurg J, Pyne AH & Orly J 1999 Expression of steroidogenic genes in mternl nd extremryonic cells during erly pregnncy in mice. Endocrinology Austd S & Sunquist M 1986 Sex-rtio mnipultion in the common opossum. Nture Belnger C, Luu-The V, Dupont P & Tchernof A 2002 Adipose tissue intrcrinology: potentil importnce of locl ndrogen/estrogen metolism in the regultion of diposity. Hormone nd Metolic Reserch Dieudonne MN, Smmri A, Dos Sntos E, Leneveu MC, Giudicelli Y & Pecquery R 2006 Sex steroids nd leptin regulte 11et-hydroxysteroid dehydrogense I nd P450 romtse expressions in humn predipocytes: sex specificities. Journl of Steroid Biochemistry nd Moleculr Biology Dorgn JF, Reichmn ME, Judd JT, Brown C, Longcope C, Schtzkin A, Formn M, Cmpell WS, Frnz C, Khle L et l Reltion of energy, ft, nd fier intkes to plsm concentrtions of estrogens nd ndrogens in premenopusl women. Americn Journl of Clinicl Nutrition Dorgn JF, Hunserger SA, McMhon RP, Kwiterovich PO Jr, Luer RM, Vn Horn L, Lsser NL, Stevens VJ, Friedmn LA, Ynovski JA et l nd sex hormones in girls: findings from rndomized controlled clinicl tril. Journl of the Ntionl Cncer Institute Ericsson RJ 1976 X nd Y spermtozo. Contrception, Fertilité, Sexulité Fernndez-Twinn DS, Oznne SE, Ekizoglou S, Doherty C, Jmes L, Gusterson B & Hles CN 2003 The mternl endocrine environment in the low-protein model of intr-uterine growth restriction. British Journl of Nutrition Flint AP, Alon SD & Jfr SI 1997 Blstocyst development nd conceptus sex selection in red deer Cervus elphus: studies of free-living popultion on the Isle of Rum. Generl nd Comprtive Endocrinology Gorodeski GI, Hopfer U, Liu CC & Mrgles E 2005 Estrogen cidifies vginl ph y up-regultion of proton secretion vi the picl memrne of vginl-ectocervicl epithelil cells. Endocrinology Grnt VJ 1994 Mternl dominnce nd the conception of sons. British Journl of Medicl Psychology Grnt VJ & Frnce JT 2001 Dominnce nd testosterone in women. Biologicl Psychology Hilkivi-Clrke L, Cho E & Onojfe I 1996 High-ft diet induces ggressive ehvior in mle mice nd rts. Life Science Hilkivi-Clrke L, Clrke R, Onojfe I, Rygd M, Cho E & Lippmn M 1997 A mternl diet high in n-6 polyunsturted fts lters mmmry glnd development, puerty onset, nd rest cncer risk mong femle rt offspring. PNAS Hilkivi-Clrke L, Cho E, Cnes A, DeAssis S, Olivo S, Helferich W, Lippmn ME & Clrke R 2002 ry modultion of pregnncy estrogen levels nd rest cncer risk mong femle rt offspring. Clinicl Cncer Reserch Jmes W 1990 The hypothesized hormonl control of humn sex rtio t irth: n updte. Journl of Theoreticl Biology Killen JH, Forrest DW, Byers FM, Schelling GT & Long CR 1989 Gondotropin-relesing hormone-induced luteinizing hormone relese in heifers: effect of nutrition during gesttion. Journl of Theoreticl Biology Kumgi S, Ki Y & Sski H 2001 Reltionship etween insulin resistnce, sex hormones nd sex hormone-inding gloulin in the serum lipid nd lipoprotein profiles of Jpnese postmenopusl women. Journl of Atherosclerosis nd Thromosis Lmrinoudki I, Christodoulkos G, Rizos D, Economou E, Argeitis J, Vlchou S, Crets M, Kouskouni E & Botsis D 2006 Endogenous sex hormones nd risk fctors for therosclerosis in helthy Greek postmenopusl women. Europen Journl of Endocrinology McCormck JT & Greenwld GS 1974 Progesterone nd oestrdiol-17et concentrtions in the peripherl plsm during pregnncy in the mouse. Journl of Endocrinology Menotti A 1999, cholesterol nd coronry hert disese. A perspective. Act Crdiologic Ngt C, Ngo Y, Shiuy C, Kshiki Y & Shimizu H 2005 Ft intke is ssocited with serum estrogen nd ndrogen concentrtions in postmenopusl Jpnese women. Journl of Nutrition Nelson JF, Felicio LS, Osterurg HH & Finch CE 1992 Differentil contriutions of ovrin nd extrovrin fctors to ge-relted reductions in plsm estrdiol nd progesterone during the estrous cycle of C57BL/6J mice. Endocrinology Overpeck JG, Colson SH, Hohmnn JR, Applestine MS & Reilly JF 1978 Concentrtions of circulting steroids in norml prepuertl nd dult mle nd femle humns, chimpnzees, rhesus monkeys, rts, mice, nd hmsters: literture survey. Journl of Toxicology nd Environmentl Helth Prks EJ, Schneider TL & Br RA 2005 Mel-feeding studies in mice: effects of diet on lood lipids nd energy expenditure. Comprtive Medicine Perry GA, Smith MF & Gery TW 2004 Aility of intrvginl progesterone inserts nd melengestrol cette to induce estrous cycles in postprtum eef cows. Journl of Animl Science Prtt NC, Huck UW & Lisk RD 1987 Offspring sex rtio in hmsters is correlted with vginl ph t certin times of mting. Behviorl nd Neurl Biology Rosenfeld CS, Grimm KM, Livingston KA, Brokmn AM, Lmerson WE & Roerts RM 2003 Striking vrition in the sex rtio of pups orn to mice ccording to whether mternl diet is high in ft or crohydrte. PNAS SAS 1988 SAS SAS/STAT User s Guide. Cry, NC: SAS Institute, Inc. Shelley JM, Green A, Smith AM, Dudley E, Dennerstein L, Hopper J & Burger H 1998 Reltionship of endogenous sex hormones to lipids nd lood pressure in mid-ged women. Annls of Epidemiology Siiteri PK 1987 Adipose tissue s source of hormones. Americn Journl of Clinicl Nutrition Simpson ER 2003 Sources of estrogen nd their importnce. Journl of Steroid Biochemistry nd Moleculr Biology Singh D & Zmrno RJ 1997 Offspring sex rtio in women with ndroid ody ft distriution. Humn Biology Tlver F, Prk CS & Willims GL 1985 Reltionships mong dietry lipid intke, serum cholesterol nd ovrin function in Holstein heifers. Journl of Animl Science Thtcher WW, Meyer MD & Dnet-Desnoyers G 1995 Mternl recognition of pregnncy. Journl of Reproduction nd Fertility. Supplement Vndenergh J & Huggett C 1995 Mother s prior intruterine position ffects the sex rtio of her offspring in house mice. PNAS Vom Sl F, Clrk M, Glef B, Drickmer L & Vndenergh J 1999 The intruterine position (IUP) phenomenon. In Encyclopedi of Reproduction, pp Eds E Knoil & J Neill. New York: Acdemic Press. Wlmer DK, Wron MA, Hughes CL & Nelson KG 1992 Lctoferrin expression in the mouse reproductive trct during the nturl estrous cycle: correltion with circulting estrdiol nd progesterone. Endocrinology Woods MN, Brnett JB, Spiegelmn D, Tril N, Hertzmrk E, Longcope C & Gorch SL 1996 Hormone levels during dietry chnges in premenopusl Africn Americn women. Journl of the Ntionl Cncer Institute Wu AH, Pike MC & Strm DO 1999 Met-nlysis: dietry ft intke, serum estrogen levels, nd the risk of rest cncer. Journl of the Ntionl Cncer Institute Wu AH, Strm DO & Pike MC 2000 RESPONSE re: met-nlysis: dietry ft intke, serum estrogen levels, nd the risk of rest cncer. Journl of the Ntionl Cncer Institute 92 78A 78. Wynn A & Wynn M 1993 The effects of food shortge on humn reproduction. Nutrition nd Helth Zhou Y, Lin S, Chng HH, Du J, Dong Z, Dorrnce AM, Brnds MW & Wng MH 2005 Genderdifferences of renl CYP-derived eicosnoid synthesis in rts fed high-ft diet. Americn Journl of Hypertension Received in finl form 24 Octoer 2006 Accepted 26 Octoer 2006 Mde ville online s n Accepted Preprint 30 Octoer Journl of Endocrinology (2007) 192, Downloded from Bioscientific.com t 01/10/ :58:05AM vi free ccess