Seasonal Variation in Plasma Lipids, Lipoproteins, Apolipoprotein A-I and Vitellogenin in the Freshwater Turtle, Chrysemys Picta

Comparative Biochemistry and Physiology Part A 130Ž. 2001 253᎐269

Seasonal variation in plasma lipids, lipoproteins, apolipoprotein A-I and vitellogenin in the freshwater turtle, Chrysemys picta

Annemarie Duggana,U, Marina Paolucci b, Ann Tercyak c, Michael Gigliotti c, Donald Smallc, Ian Callarda

aDepartment of Biology, Boston Uniërsity, 5 Cummington Street, Boston, MA 02215, USA bFacolta di Scienze, Instituto Suore Battistine, Via Port ‘ansa, 11, Beneënto, Italy cDepartment of Biophysics, Boston Uniërsity School of Medicine, Boston, MA 02118, USA

Received 17 May 2000; received in revised form 11 April 2001; accepted 17 April 2001

Abstract

An analysis of plasma lipids and lipoprotein fractions was performed over the course of the annual ovarian cycle of the female turtle, Chrysemys picta. Determinations of total plasma triglycerides, cholesterol, vitellogenin and apolipoprotein A-IŽ. apoA-I were made. The lipid and protein composition of the lipoprotein fractionsw very low density lipoprotein Ž.VLDL , low density lipoprotein Ž. LDL , high density lipoprotein Ž. HDL and very high density lipoprotein Ž VHDL .x were also observed over the same period. Plasma triglyceride and vitellogenin levels were signiﬁcantly increased in the spring preovulatory period and fall recrudescent phase. Total plasma cholesterol levels were signiﬁcantly elevated only at the onset of the fall recrudescent phase and apoA-I levels were highest during the postovipositionrovarian arrest phase. The triglyceride content of VLDL was highest in preovulatory animals and there were apparent seasonal changes in the expression of apoA-I and apoE of HDLrVHDL. We conclude that the coordinate regulation of lipids and protein contributes to seasonal ovarian growth and clearance of lipids from plasma, both of which are most likely under hormonal control. ᮊ 2001 Elsevier Science Inc. All rights reserved.

Keywords: Apolipoprotein A-I; Hormonal regulation; Lipids; Lipoproteins; Turtle; Vitellogenin; Seasonal; Reproductive cycle

1. Introduction developing oocyte, occurs in all submammalian vertebrate classes, and requires the mobilization Vitellogenesis, the estrogen-dependent process and transport of large amounts of maternal pro- of yolk synthesis followed by deposition in the tein and lipid. The extent of this process in females as well as its estrogen-inducibility in males, makes vitellogenesis and the associated lipid U Corresponding author. Tel.: q 1-617-353-2346; changes an ideal model for the study of sex- fax: q1-617-353-2923. related differences in lipidsŽ. Callard et al., 1990 . E-mail address: [email protected]Ž. A. Duggan . Variations in lipids and lipid-transporting pro-

1095-6433r01r$ - see front matter ᮊ 2001 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 5 - 6 4 3 3Ž. 0 1 00364-6 254 A. Duggan et al. rComparatië Biochemistry and Physiology Part A 130() 2001 253᎐269 teins are known to contribute to malerfemale et al., 1992b; Paolucci and Callard, 1995. . On the differences in cardiovascular disease in the hu- basis of the apparent multi-hormonal regulation manŽ. Tikkanen, 1990 and estrogen appears to be of hepatic lipoprotein synthesis, we believe that cardioprotectiveŽ. Espeland, 1998 . A great deal of the turtle is a good model for an investigation of work has been carried out on lipid metabolism sex-hormone regulation of major lipid transport- using the avian model, with a focus on estrogen ing proteins of relevance to cardiovascular dis- induction of hepatic vitellogenin I and II synthe- ease. Here, we report seasonal variation in vari- sis and very low density lipoproteinŽ VLDL; ous protein and lipid components of plasma asso- Williams, 1979; Chan et al., 1980. and the associ- ciated with the annual vitellogenic cycle of the ated changes in the blood lipid spectrum. How- female turtle. ever, avian studies have not investigated the role of hormones other than estrogen on vitellogenesis, plasma lipids and lipoproteins, although the 2. Materials and methods role of thyroid hormones has received attention Ž.Schjeide et al., 1986 . We have chosen a chelo- 2.1. Animals and animal care nian reptile, the freshwater turtle, Chrysemys picta, as a model for the hormonal regulation of lipid Freshly caught adult female turtles ŽChrysemys mobilization and transport. This choice is based picta) were shipped from Lemberger Co. on its phylogenetic position in the central verte- Ž.Oshkosh, WI, USA each month from November brate group from which mammals and birds 1993 to July 1994, and September 1995 to August evolved and our current knowledge of the role of 1996. Upon arrival, animals were allowed to accli- steroid hormones in the regulation of vitellogene- mate for 2 days in the AAALAC-accredited sis and hepatic protein synthesis in this species aquarium facility of the Department of Biology at Ž.Callard et al., 1994 . Boston University. Animals were maintained on a As in other species, there is wide seasonal 12 h lightr12 h dark photoperiod at 24ЊC with variation in plasma vitellogenin and plasma lipids fresh running water and were fed turtle chow during the annual cycle of the turtleŽ Callard et ŽWardley reptile ‘Total essential nutrition floating al., 1978; Gapp et al., 1979. . Vitellogenin and food sticks’; Wardley Corp., Secaucus, NJ, USA. triglyceride levels in particular are highest during ad libitum. the spring and fall ovarian growth periods and After cooling on ice, animals were decapitated lowest during reproductive quiescence in the win- and blood was collected in heparinized tubes. ter. Furthermore, estrogen-induced vitellogenin Plasma was obtained after centrifugation at 800 may be decreased by progesterone and testos- =g for 20 min at 4ЊC. Addition of 1 mM terone in malesŽ. Ho et al., 1981 . In addition, phenylmethyl-sulfonyl fluorideŽ. PMSF , 1 mM estrogen induction of vitellogenin may require ethylenediaminetetraacetic acidŽ EDTA; dis- growth hormone in femalesŽ. Ho et al., 1982 . odium salt. , and 0.2 mM sodium azide to the With regard to plasma lipids per se, changes in plasma followed. The plasma was used immedi- cholesterol, triglycerides and phospholipids asso- ately for separation into lipoprotein fractions. The ciated with seasonal ovarian growth have also total protein content of plasma and lipoprotein been documented for lizards and snakesŽ Lance, fractions was determined using BSAŽ. Sigma as 1975; Dessauer and Fox, 1959. . The magnitude of standard according to Lowry et al.Ž. 1951 . At the changes observed in these squamate reptiles autopsy, the reproductive status of the females is similar to that observed at the onset of laying was determined on the basis of ovarian and follic- or in response to estradiol in hensŽ Schjeide et al., ular size, and the presencerabsence of eggs in 1986. . the oviduct. More recently we and others have pointed out the significant homology between the major lipid 2.2. Separation of lipoprotein fractions transporting proteins of the low density lipopro- teinŽ. VLDL and LDL fractions, apolipoprotein B Lipoprotein fractions were separated by ultra- and vitellogeninŽ. Baker, 1988; Perez et al., 1992a . centrifugation at 20ЊC using a swinging bucket Furthermore, we have demonstrated the presence rotorŽ. Sorvall; AH-627 in a Sorvall OTD-65 of apolipoproteins A-I and B in the turtleŽ Perez ultracentrifuge. The procedure of Mills et al. A. Duggan et al. rComparatië Biochemistry and Physiology Part A 130() 2001 253᎐269 255

Ž.1984 was followed with some modifications. All was removed and the aqueous layer was extracted reagents used were of analytical grade. The salt again with ;2.5 ml of the ‘theoretical lower density intervals used were those currently used phase’Ž. chloroformrmethanolrwater ᎏ 85:14:1 . for human lipoprotein analysis. Details are con- The organic layers were then combined. tained in Paolucci and CallardŽ. 1995 . Briefly, to obtain the VLDL fraction, a solution of sodium 2.5. Folch extraction of lipoprotein fractions chlorideŽ. NaCl; ds1.006 grml containing PMSF Ž.0.01% and 1 mM EDTA Ž. 3 ml was carefully From lipoprotein fractions, 500 ␮l aliquots were layered on top of the plasmaŽ. 9 ml . Samples were added to 10 ml of chloroformrmethanol ᎏ 2:1. then centrifuged at 105 000=g for 22 h. The AcidrsalineŽ. 2 ml wash was then added. After VLDL fraction was collected by aspiration with a mixing and removal of the organic layer, the Pasteur pipette. The density of the remaining upper phase was washed again with ;6mlofthe plasma was raised to 1.063 grml by addition of theoretical lower phase. The organic layers were solid potassium bromideŽ. KBr and ultracen- combined. trifuged again as above. After collection of the In all samplesŽ whole plasma and lipoprotein LDL fraction, the density of the remaining plasma fractions. , organic layers were evaporated under a was increased to 1.21 grml by addition of solid gentle stream of nitrogen and resuspended to a KBr and ultracentrifuged at 105 000=g for 40 h. volume of 1.0 ml of chloroformrmethanol ᎏ 2:1. The resultant HDL fraction was collected and the Following the evaporation of solvent from the density of the remainder was raised to 1.25 grml aliquots, total lipid weights were determined by by again adding solid KBr. After a final ultracen- microgravimetry using a Cahn microbalance. trifugation at 105 000=g for 22 h, the VHDL fraction was collected. All fractions were dialyzed 2.6. Lipid analysis( of whole and fractionated against buffer containing 0.1 M NaCl, 2 mM plasma) EDTA, 0.01% sodium azide, 0.01% PMSF, pH 7.5 to remove salt. Dialysis was carried out at 4ЊC for Triglyceride content was determined using a 48 h with several changes. colorimetric semi-enzymatic assayŽ Sigma 320- UV. . 2.3. SDS-polyacrylamide gel electrophoresis Total cholesterol was determined by a colorimetric enzyme assayŽ Allain et al., 1974; Warnick, Lipoprotein fractions were run on SDS-poly- 1986. from which unesterified cholesterol and acrylamide gradient gelsŽ. 5᎐20% under denatur- cholesterol ester were determined according to ing conditions. A standard amountŽ. 20 ␮gof Small et al.Ž. 1991 . protein was loaded per lane. Gels were stained Determination of phospholipids was performed with Coomassie Blue R-250 in acetic acid and according to the method of BartlettŽ. 1959 . methanol. The following wide range molecular weight markersŽ. Sigma were used: myosin Ž 205 2.7. Gas᎐liquid chromatography of fatty acids kDa.Ž. ; beta-galactosidase 116 kDa ; phosphory- lase b Ž.97 kDa ; fructose-6-phosphate kinase Ž 84 Lipoprotein fractions were extracted according kDa.Ž. ; albumin 66 kDa ; glutamic dehydrogenase to the Folch method; transmethylated and hydro- Ž.55 kDa ; ovalbumin Ž. 45 kDa ; glyceraldehyde-3- lyzed by the method of Morrison and SmithŽ. 1964 . phosphate dehydrogenaseŽ. 36 kDa ; carbonic an- Conversion of free fatty acids into their methyl hydraseŽ. 29 kDa ; trypsinogen Ž. 24 kDa ; trypsin esters allows for their separation on the basis of inhibitorŽ. 20 kDa ; and alpha-lactalbumin Ž 14 molecular weight and degree of unsaturation. The kDa. . methylated fatty acids were separated on a 6-foot 5% DEGSrChromosorb W column and were 2.4. Folch extraction of whole plasma compared to authentic methyl ester standards Ž.Supelco . Quantitation by automatic integration A 200-␮l aliquot of whole plasma was added to of the peak areas yielded percent distribution of 4 ml of chloroformrmethanol ᎏ 2:1. Washing individual fatty acids. From these values, mol% was performed using 0.8 ml of 0.9% NaCl in 0.01 was calculated for each lipid fraction. The fol- N HCl. After vigorous mixing, the organic layer lowing fatty acids were measured in the lipopro- 256 A. Duggan et al. rComparatië Biochemistry and Physiology Part A 130() 2001 253᎐269

Fig. 1. A. Duggan et al. rComparatië Biochemistry and Physiology Part A 130() 2001 253᎐269 257 tein fractions: myristicŽ. 14:0 ; palmitic Ž. 16:0 ; Ž.Berkeley, CA, USA using purified vitellogenin palmitoleicŽ. 16:1 ; stearic Ž. 18:0 ; oleic Ž. 18:1 ; prepared by Seung Jae Won in this laboratory. linoleicŽ. 18:2 ; linolenic Ž. 18:3 ; and arachidonic The standard curve was generated using purified Ž.20:4 acids. turtle vitellogenin diluted in carbonate buffer. Standard curves were linear from 1.875 to 10 2.8. Enzyme-linked immunosorbent assay() ELISA ␮grml and the average r 2 was 0.979. The detec- for determination of plasma apoA-I leëls tion limit of these assays was 1.64 ␮grml. We have previously published an RIA for turtle vitel- These assays were carried out using an anti- logeninŽ. Gapp et al., 1979 . serumŽ. raised in rabbit to chicken apoA-I kindly provided by Dr. David WilliamsŽ SUNY, Stony 2.10. Electron microscopic analysis of lipoprotein Brook. which was shown to cross-react with turtle particles apoA-IŽ. Paolucci and Callard, 1995 . Using round bottom ELISA platesŽ. Corning , wells were coated Lipoprotein fractions were dialyzed at 4ЊC with 100 ␮l of diluted standard or plasma in against a buffer containing 120 mM ammonium carbonate bufferŽ. 0.1 M Na23 CO . Plates were acetate, 3 mM ammonium carbonate, 1 mm covered and incubated overnight at 4ЊC. After EDTA, pH 7.4. Following the protocol described incubation, wells were washed with PBS᎐Tween in Forte et al.Ž. 1968 , lipoprotein samples were 20 and blocked with 200 ␮l 1.5% gelatinŽ Bio- negatively stained with an equal volume of 2% Rad. in PBS for 2 h at room temperature. Fol- sodium phosphotungstate solution, pH 7.4. A drop lowing another wash, 100 ␮l of primary antibody of this mixture was applied to Formvarrcarbon- Ž.anti-chicken apoA-I diluted in PBS were added coated copper gridsŽ 200 mesh; Electron Micros- to the wells. The plates were incubated at room copy Sciences. and allowed to dry. The stained temperature for 2 h, washed, and then 100 ␮lof preparations were examined using a Philips 410 secondary antibodyŽ goat anti-rabbit conjugated electron microscope at 60 kV. The sizes of parti- to alkaline phophatase; Sigma. diluted in PBS cles were measured and calculated for each frac- were added to the wells. After an additional in- tion. cubation for 2 h at room temperature, plates were washed and developed with 100 ␮lof p- nitrophenylphosphate solutionŽ. Sigma added to 3. Results each well. Using a plate readerŽ EL-312; Fisher Biotech. , the colorimetric readings were taken at 3.1. Whole plasma lipid and protein analysis( total 405 nm. The standard curve was generated using protein, ïtellogenin and apolipoprotein A-I) purified turtle apoA-I diluted in carbonate buffer. Standard curves were linear from 75 to 600 ngrml 3.1.1. Proteins: seasonal äriation in plasma total and the average r 2 was 0.983. The detection limit protein, apoA-I and ïtellogenin leëls of the assays was 60.9 ngrml. Plasma total protein levels varied little over the cycle until the ovarian recrudescent period in the 2.9. ELISA for determination of plasma ïtellogenin early fallw 153.5 mgrml; P-0.02, compared to all leëls other times of the year except for late fall Ž.November and Decemberx . Total protein de- The same procedure as above was employed clined thereafter as animals enter into the inac- with the exception of the following: rabbit anti- tive period in the late fallw 96.4 mgrmlŽ Novem- turtle vitellogenin antisera was raised by Babco ber.Ž. and 127.9 mgrml Decemberx Ž. Fig. 1a᎐c.

Fig. 1.Ž. a᎐e Comparison of whole plasma total protein, apoA-I, vitellogenin, and lipids during the annual reproductive cycle. Plasma samples were taken from several periods of the annual female reproductive cycleŽ winter; ns9, preovulatory; ns5, postovulatory; ns6, postoviposition; ns13, late summerrearly fall; ns5, late fall; ns4. . Panel A indicates seasonal variation in total plasma protein using the Lowry methodŽ. 1951 . Panels B᎐C demonstrate seasonal changes in plasma apoA-I and vitellogenin levels as determined by ELISA. Panels D᎐E show changes in plasma triglyceride and cholesterol levelsŽ using colorimetric enzyme kits from Sigma. over the course of the annual cycle. Values are reported as mean"S.E. Statistical signiﬁcance was assessed using one-way ANOVA followed by multiple comparison tests using Sigma Stat software. 258 A. Duggan et al. rComparati¨e Biochemistry and Physiology Part A 130() 2001 253᎐269

Plasma apoA-I levels were 36.6 and 35.6 mgrdl logenin levels declined steadilyw 15.6 mgrml in the winter and preovulatory periods, respec- Ž.Žpost-ovulatory , 11.2 mgrml postoviposition .x to tively, and rose significantly in the postoviposition ;1.5 mgrml in late summer. Values increased period to 64.7 mgrdl; Ž.P-0.05 during summer. during ovarian recrudescence in the late fall, Ž P By the early fall, apoA-I levels declinedŽ 16.3 -0.05.Ž 16.8 mgrml, November and 16.2 mgrml, mgrdl. , followed by an increase during ovarian December. . recrudescence in NovemberŽ.Ž 33.9 mgrdl P) 0.05. and further decreased later on in December 3.1.2. Lipids: seasonal äriation in plasma Ž.Ž.12.7 mgrdl; P)0.05 Fig. 1b . In addition, the triglyceride and cholesterol leëls amount of apoA-I in relation to total plasma Plasma triglyceride levels were 77.6 mgrdl in protein was higher in the postoviposition period JanuaryrFebruary and increased maximally to compared to all other times of the year. The ratio 211.6 mgrdl prior to ovulation in spring Ž P- of apoA-I to total protein Ž.=1000 for all times of 0.02. . Triglycerides then declined significantly the year was as follows: winterŽ. 4.9 ; preov. Ž. 4.0 ; during the summer monthsŽ. 48.8 mgrdl; P-0.03 postov.Ž. 6.13 ; postovip. Ž. 8.6 ; early fall Ž. 1.08 ; late prior to a second increase during the fall period fallŽ.Ž. Nov. 3.52 ; and late fall Ž.Ž. Dec. 1.0 . of ovarian recrudescenceŽ. 176.7 mgrdl; P)0.05 . Compared to the winterŽ. 2.5 mgrml , plasma Levels again declined at the onset of late fall vitellogenin levels were significantly higher in the inactivityŽ.Ž. 43.1 mgrdl; P)0.05 Fig. 1d . period immediately prior to ovulationŽ 22.9 Plasma cholesterol levels remained quite steady mgrml; P-0.001. . After ovulation occurs, vitel- throughout the yearŽ. 149.2᎐194.5 mgrdl except

Fig. 2. Electron microscopic analysis of lipoprotein fractions isolated by sequential ultracentrifugation as described in Section 2.Ž. a VLDLŽ.Ž.Ž.Ž.Ž.Ž.Ž 56᎐93 nm , b LDL 31᎐41 nm , c HDL 10᎐20 nm , d VHDL estimated 5᎐10 nm . . A. Duggan et al. rComparatië Biochemistry and Physiology Part A 130() 2001 253᎐269 259 for a significant increase during ovarian recrudes- 3.2.2. Electrophoretic analysis of lipoprotein fractions cence in the early fallŽ. 278.8 mgrdl; P-0.05 . VLDL, LDL, HDL, VHDL using SDS-PAGE oër Cholesterol levels declined again during late fall the annual reproductië cycle to a level of 99.3 mgrdl Ž.P-0.05 in December VLDL: Of the major proteins observed, a 350- Ž.Fig. 1e . kDa band thought to be apoB and a 66-kDa Ž.putative albumin band were always present. A 3.2. Analysis of plasma lipoprotein fractions less distinct 55-kDaŽ. putative apoA-IV band was most obviously present after ovulationŽ luteal 3.2.1. Electron microscopic analysis of lipoprotein phase. and when animals were entering reproduc- fractions tive quiescenceŽ. panel F; November . A smaller EM analysis was performed only on fractions 14-kDa band, though faint, was seen throughout obtained during the month of December. VLDL the cycle and possibly represents apoC. particle size ranged from 56 to 93 nm, compared LDL: The LDL fraction was dominated by a to LDLŽ.Ž. 31᎐41 nm , HDL 10᎐20 nm and VHDL strong apoB band, with less obvious albumin and particle size was estimated as 5᎐10 nm; although, 28 kDaŽ. putative apoA-I bands which were vari- the images obtained for VHDL were not clear ably present. Ž.Fig. 2 . HDL: This fraction was dominated by an in-

Fig. 3.Ž. a᎐f Electrophoretic analysis of lipoprotein fractions VLDL, LDL, HDL, VHDL using SDS-PAGE over the annual reproductive cycle. Lipoprotein fractions were isolated by sequential ultracentrifugation of pooled plasma obtained in winter, preovulatory, postovulatory, postovipositionrsummer, early and late fall stages as described in Section 2. 260 A. Duggan et al. rComparati¨e Biochemistry and Physiology Part A 130() 2001 253᎐269 variant apoA-I band. Marked changes in protein Clear albumin bands were seen at all times, as composition in the HDL fraction occurred after was apoA-I, with the exception of the periovula- ovulation in the luteal phaseŽ. Fig. 3c and post- tory periodŽ. Fig. 3b and fall Ž. Fig. 3f . As seen in oviposition phaseŽ. Fig. 3d . In the postovulatory the HDL fraction and in the VHDL fraction, period especially, 76, 66Ž.Ž. albumin , 55 apoA-IV , apoE was also prominent in luteal and postovipo- 34Ž. putative apoE kDa bands were prominent, as sition animals; this pattern held true in subse- well as a 14-kDa bandŽ. putative apoC or apoA-II . quent periods of the reproductive cycle, but the In the postovipositionrsummer period, the com- bands were less prominent. position of HDL appeared to be especially rich in apoA-I. In the early fallŽ. October , the intensity 3.2.3. Percent distribution of lipid components in of the apoE and albumin bands was higher than lipoprotein fractions o¨er the seasonal cycle the summer and in late fall. Triglycerides were highest in the VLDL frac- VHDL: This fraction mirrored the HDL frac- tionŽ. 38᎐62% especially in April Ž. 58% and De- tion in the luteal phase particularlyŽ. Fig. 3c . cemberŽ. 62% . In May, levels dropped to 38%

Table 1 Percent distribution of lipid components in lipoprotein fractionsŽ. VLDL᎐VHDL over the annual reproductive cycle

April May June July December Ž.Preov Ž Periov . Ž Postov . Ž Postovip . Ž Quiescent .

VLDL TG 58 38 57 55 62 PL 18 23 26 22 17 CE 10 19 8 14 16 UC 12 20 8 9 5

Total lipidŽ. mg 6.6 1.0 0.4 1.0 0.3 % Distrib. lipid 37.8 7.8 7.9 14.6 4.0

LDL TG 27 13 16 10 12 PL 21 23 31 33 23 CE 42 51 38 30 41 UC 9 8 14 17 14

Total lipidŽ. mg 3.0 3.9 6.0 6.3 10.3 % Distrib. lipid 41.3 64.0 68.6 71.9 85.7

HDL TG 17 9 15 17 11 PL 31 38 48 49 59 CE 44 42 27 23 22 UC 7 14 9 8 8

Total lipidŽ. mg 2.5 2.7 1.8 0.9 1.1 % Distrib. lipid 16.8 25.0 22.1 9.8 9.5

VHDL TG 27 27 Not 35 40 PL 39 38 Done 38 37 CE 26 30 21 19 UC 7 9 5 4

Total lipidŽ. mg 0.3 0.5 1.0 0.3 % Distrib. lipid 4.1 2.6 3.7 0.9

Lipoprotein fractions of pooled plasma obtained in preovulatory, periovulatory, postovulatory, postoviposition and quiescent Ž.winter stages were isolated by sequential ultracentrifugation, extracted and total lipid weights were determined as described in Section 2. A. Duggan et al. rComparati¨e Biochemistry and Physiology Part A 130() 2001 253᎐269 261

Ž.P-0.05 . For all months, fractional triglyceride 9 and 17% of the HDL fraction and between 27 percentage declined from VLDLŽ. 38᎐62% to and 40% of the VHDL fractionŽ Table 1 and Fig. LDLŽ. 10᎐27% . Triglycerides constituted between 4..

Fig. 4. Summary of seasonal variation in plasma lipids, apoproteins, and composition of plasma lipoproteins over the course of the turtle annual ovarian cycle. Bottom: The stages of the female ovarian cycle are shown. The striped bar indicates the two periods of ovarian growthŽ.Ž.Ž. preovulatory and fall recrudescent stages . Middle: See Fig. 1 for absolute values Plasma apoA-I mgrdl ; cholesterol Ž.mgrdl ; vitellogenin Ž mgrml . ; and triglyceride Ž. mgrdl values vary seasonally. The ratio of plasma apoA-I to total plasma protein is shown as described in Section 3. Seasonal variation in the distribution of lipid and protein in VLDL, LDL, HDL, TriglycerideŽ. TG , PhospholipidŽ. PL , Cholesterol Ester Ž CE . and Unesterified Cholesterol Ž UC . . Top: Relative values of estradiol and progesterone are shown. Values are arbitrary: estradiolŽ. E2 , pgrml Ž peak ;1.0 ng . ; progesterone Ž. P , ngrml Ž peak ;3.0 ngrml . ; based on Callard et al.Ž. 1978 . 262 A. Duggan et al. rComparatië Biochemistry and Physiology Part A 130() 2001 253᎐269 ostovulatory observed in palmitoleic 16:1 , stearic 18:0 , oleic 18:1 , Ž. Ž. Ž. Ž. Ž. w Ž. Ž.Ž.Ž. x 3 from preovulatory, periovulatory, and postovulatory stages were isolated by sequential ultracentrifugation, extracted, and transmethylated. s n Ž. Ž.Ž.Ž.Ž. Ž.Ž. Ž.Ž. Ž.Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž. Ž . Ž . Ž . VLDL LDL HDL VLDL LDL HDL VLDL LDL HDL Ž. Ž. Ž. Lipoprotein fractions 16:1 and linoleic 18:2 acids. Mol.% distribution of fatty acids in lipoprotein fractions VLDL, LDL, HDL : analysis of samples from April preovulatory , May periovulatory and June p Table 2 Fatty acid MonthLipoprotein April May June fraction 14:016:016:118:018:118:2 0 018:3 18.50 2.0620:4 15.70 1.65 5.42 0.68 51.03 19.90 4.12Methylated 0.59 fatty acids 12.70 were 4.43 then 1.17 column 0.69 0 separated and 1.13 0 theŽ. percent 0.68 4.74 distribution 47.07 of 22.37 2.62 0.21 individual 1.97 fatty 0.77 1.51 acids 12.36 myristic 0.90 14:0 6.88 , palmitic 1.36 Ž. 16:0 0.44 , 40.70 palmitoleic 20.40 0.44 5.91 1.70 4.11 6.28 0.27 0.91 0 8.72 0 0.43 6.69 0.52 43.60 0.94 20.33 2.23 0.47 9.20 1.03 8.01 2.81 1.10 8.40 12.70 1.02 2.40 45.50 20.70 0 1.42 0 1.76 0 0 5.97 8.47 1.11 2.48 11.19 7.21 1.48 0.82 38.50 21.17 1.19 2.34 6.66 7.00 0.29 10.91 10.98 0.92 1.36 1.28 0.44 0 42.13 22.97 0.44 0 2.98 0.70 5.05 13.70 11.80 0.85 0.78 0.61 7.60 42.27 0.55 23.90 0 3.03 1.05 0 0 13.16 4.01 5.40 0 1.19 0.68 2.73 36.67 6.12 3.41 0.39 13.66 2.39 6.06 6.48 1.09 1.29 0.61 0.66 1.06 1.06 13.10 2.04 1.70 0.44 1.43 0.74 0.42 0.42 1.43 0.77 linoleic 18:2 , linolenic 18:3 and arachidonic 20:4 was calculated as described in Section 2. Values are shown as mean and S.E. Significant changes were A. Duggan et al. rComparatië Biochemistry and Physiology Part A 130() 2001 253᎐269 263

Fractional percentage phospholipid values did fractional distribution of lipids, fatty acids and not show large changes from month-to-month. total protein within the lipoprotein fractions However, phospholipid values for HDL were Ž.VLDL, LDL and HDL during the major phases highest in JuneŽ. 48% , July Ž. 49% and December of the annual ovarian cycleŽ springrovarian re- Ž.59% . Cholesterol ester, the largest component crudescence, ovulation; summerrovarian arrest of total cholesterol, increased from VLDL to and atresia; fallrrecrudescence; winterrovarian LDLŽ. 8᎐19% vs. 30᎐51% . The cholesterol ester arrest. were determined. constituted between 22 and 44% of the HDL In summary, whole plasma analysis revealed fraction and 19᎐30% of the VHDL fraction. Free concordance between vitellogenin and triglyc- cholesterolŽ. unesterified constituted 4᎐20% of eride values, both showing significant peaks in the the total cholesterol, depending on time of year. spring preovulatory period and the fall ovarian In May, free cholesterol was highestŽ. 20% in recrudescent phase. Total cholesterol values were VLDL, whereas it was highest in LDLŽ. 17% in only increased significantly at the beginning of July. The cholesterol ester component of the HDL the fall ovarian recrudescent phase. In contrast, fraction generally declined from April to Decem- apoA-I values were significantly increased during berŽ. 44, 42, 27, 23, 22%, respectively . In addition, the postovipositionrovarian arrest phase. It is of it is clear that the majority of lipid was distributed interest that total protein was highest when vtg in the LDL fraction over the course of the year and apoA-I were lowest. Ž.41.3, 64.0, 68.6, 71.9, 85.7% . Analysis of the fractional distribution of apoproteins indicated that apoB was primarily 3.2.4. Mol.% distribution of fatty acids in lipoprotein associated with the LDL fraction and apoA-I and fractions: analysis of samples from April, May and E with HDLŽ. VHDL as in other vertebrates June Ž.Babin, 1987 . Some significant seasonal changes Of all the fatty acids analyzedw myristicŽ. 14:0 , were noted in apoA-I and E quantity and dis- palmiticŽ. 16:0 , palmitoleic Ž. 16:1 , stearic Ž. 18:0 , tribution as discussed below. The fractional lipid oleicŽ. 18:1 , linoleic Ž. 18:2 , linolenic Ž. 18:3 and composition of the apoprotein fractions were arachidonicŽ. 20:4 acidsx , oleic, palmitic, palmi- within the range found for other terrestrial verte- toleic and linoleic predominated. The only fatty bratesŽ. amphibians, birds, mammals as are acids which significantly differed between April lipoprotein particle sizes and fatty acid composi- and June were palmitoleic and linoleic acidsŽ Ta- tionŽ. Chapman, 1980 . ble 2. . The vitellogenin pattern reported here is simi- Significant changes in palmitoleic acidŽ. 16:1 lar to that reported by Gapp et al.Ž. 1979 and is and linoleic acidŽ. 18:2 were observed in LDL and correlated with increasing plasma estradiol levels HDL fractions between AprilŽ. preovulatory , May during spring and fall ovarian recrudescenceŽ Cal- Ž.Žperiovulatory and June postovulatory periods . . lard et al., 1978. . This is in accordance with the With respect to palmitoleic acid, its percentage known effect of estradiol on vtg induction in this decreased significantly in both the LDL Ž.P-0.05 Ž.Ho et al., 1982 and other species such as the and HDL Ž.P-0.01 fractions between April and birdŽ. Kudzma et al., 1979 . In the chicken ovary, May, and between April and June. However, vtg and VLDLŽ. a triglyceride-rich lipoprotein are mol% of linoleic acid increased significantly delivered to the oocyte in an endocytotic, recep- between April and June in LDL Ž.P-0.05 and in tor-mediated processŽ Opresko and Wiley, 1987; the HDL fraction; it also increased significantly Evans and Burley, 1987. . Although the concomi- between April and May, and between April and tant rise in triglyceride with vitellogenin observed June Ž.P-0.05 . has been previously noted for reptilesŽ Dessauer and Fox, 1959.Ž and birds Kudzma et al., 1979; Jackson et al., 1977. , the role of estradiol in 4. Discussion mobilizing triglycerides is not well-understood. However, some studies have indicated that estra- This is the first study of a vitellogenic species in diol is capable of both increasing hepatic VLDL which vitellogenin, lipids and apolipoprotein lev- synthesis and decreasing hepatic lipase activity els were determined in whole plasma during a thereby resulting in a net increase in circulating seasonal reproductive cycle. Furthermore, the triglyceride levelsŽ. Weinstein et al., 1986 . Early 264 A. Duggan et al. rComparatië Biochemistry and Physiology Part A 130() 2001 253᎐269 studiesŽ. Hahn, 1967 suggested a direct effect of the turtle, and an apoE cDNA has been cloned estradiol on lipolysisrlipid mobilization in a lizard, from the zebrafishŽ. Babin et al., 1997 . Limited Uta stansburiana. However, this may also be an amino acid analysis of the putative turtle apoE indirect effect involving growth hormone, a known supports this identityŽ. Duggan et al., 1997 . In lipolytic agent in mammalsŽ. Isaksson et al., 1985 . mammals, apoE is a ligand for the LDL-receptor Earlier studies from this laboratory demonstrated which mediates cholesterol uptake in all tissues a significant increase in growth hormone-secret- via internalization of the entire LDL particle ing cell types in the pituitary of estradiol-treated Ž.Brown and Goldstein, 1986 . It remains to be lizardsŽ. Callard et al., 1972 . Furthermore, we determined if the putative apoE of the turtle is have demonstrated that growth hormone is essen- capable of binding to the LDL-receptor. In fact, tial for estradiol-induced vitellogenesis in lizards mammalian apoE has been shown to bind the Ž.Ž.Callard et al., 1972 and turtles Ho et al., 1982 . chicken VLDLrvtg-receptorŽ. Nimpf et al., 1988 , This may be due in part to a growth hormone indicating that the receptors of the LDL-R super- requirement for normal hepatic estrogen receptor family are capable of binding apoE when present. synthesisŽ. Riley and Callard, 1988 . In addition, ApoE is also a component of HDL in some growth hormone levels in the female frog, Rana animalsŽ. Swaney et al., 1977; Mahley et al., 1977 esculenta, are well-correlated with vitellogenesis, and is thought to redirect HDL-cholesterol back i.e. peaks of growth hormone occurred during the to the liverŽ. Koo et al., 1985 for degradation. reproductively active period and fall ovarian re- However, apoA-I is also considered the major crudescenceŽ. Mosconi et al., 1994 . We have also ligand involved in directing peripheral cellular determined that growth hormone is involved in derived cholesterol to the liver in a process known the regulation of plasma apoA-I levelsŽ Duggan as ‘reverse cholesterol transport’Ž. RCT . Apo A-I and Callard, in preparation. . has recently been shown to mediate HDL choles- As with vitellogenin, triglycerides are taken up terol uptake via scavenger receptor class B type I in large quantities by yolking ovarian follicles Ž.SR-BI . This receptor has been found to bind Ž.George et al., 1987 and make up a large portion apoA-I and selectively take up cholesterol in the of the energy reserves of yolk. An increase in liver and steroidogenic tissues primarilyŽ Acton et plasma estrogen induces triglyceride-rich VLDL al., 1996. . We have identified a SR-BI in turtle and vtg, both of which are taken up by the same tissues including ovarian folliclesŽ Duggan and oocyte receptorŽ. Nimpf et al., 1988 , the Callard, in preparation. . Although both the apoE- VLDLrvtg-receptorŽ. 95 kDa , thereby providing and apoA-I-mediated cholesterol uptake pro- energy to the developing embryo. ApoB of VLDL cesses are likely to be important in cholesterol and vtg are the ligands for this receptor in the distributionrredistribution in the turtle, we have chickenŽ. Nimpf et al., 1988 . Molecular character- thus far quantitated only apoA-I levels. ization of this 95-kDa receptor, a member of the The electrophoretic analysis of lipoprotein LDL-R superfamily, demonstrates a cluster of fractions at each stage of the reproductive cycle eight ligand-binding repeats, a signature charac- demonstrated seasonal variation in the expression teristic of the mammalian VLDL-RŽ Bujo et al., of certain apoproteins. Although apoB is the 1994. . A 130-kDa variant of the VLDL-receptor principal protein component of LDL at all times has been reportedŽ. Heegard et al., 1995 and a of the year, it does not show any major changes in membrane protein of similar molecular weight quantity, based on gel electrophoretic analyses. Ž.;130 kDa has been identified in turtle oocyte This protein was previously described by Perez et and liver membrane extracts using antisera to the al.Ž. 1992b and is presumably the putative ligand chicken VLDLrvtg-receptorŽ Duggan and for the LDL-receptor involved in cholesterol de- Callard, in preparation. . livery to peripheral tissues. Reptiles may differ from birds inasmuch as the The protein components of HDL are the most turtle expresses apolipoprotein EŽ Paolucci and variable over the course of the cycle. ApoA-I is Callard, 1995.Ž in addition to apoA-I and B Perez the main apoprotein most consistently associated et al., 1992b. . It has been considered that apoE is with HDL in AprilŽ. preovulatory . As noted above, expressed only in mammals due to its apparent immunoassay of apoA-I in whole plasma suggests absence in birdsŽ. Hermier et al., 1985 , but we highest levels in postoviposited animalsŽ. July . have previously presented evidence for apoE in A protein tentatively identified as apoE A. Duggan et al. rComparatië Biochemistry and Physiology Part A 130() 2001 253᎐269 265

Ž.Paolucci and Callard, 1995; Duggan et al., 1997 instancesŽ Nabulsi et al., 1993; Nicosia et al., appears in both the HDL and VHDL fractions 1992. . The strong level of apoE expression as part after ovulationŽ. June and remains in VHDL af- of HDL and VHDL after ovulation when estra- ter ovipositionŽ. July . ApoE is always present in diol levels fall may help clear the bloodstream of the VHDL fraction and is found in the HDL cholesterol via the RCT pathway. Whether these fraction in preovulatory, postovulatory and fall seasonal changes in apoA-I and E reflect underly- Ž.October animals when ovaries are again growing. ing endocrine regulatory mechanisms cannot be However, total plasma apoA-I varies seasonally determined with certainty at present. However, showing maximal values after egg-laying and differential sensitivity of apoA-I, apoE, and vitel- minimal values by late summerrearly fall when logenin gene regulatory elements to estradiol, plasma steroid values are lowŽ Callard et al., progesterone and testosterone may be involved in 1978. . The observed apoA-IrapoE changes may cyclic changes in lipid transporting proteins and be related to a role for these proteins in main- the maintenance of lipid homeostasis. taining plasma cholesterol levels in the period Apart from the three major apolipoproteins following ovulation. It is at this time, when ovar- Ž.A-I, B, E , distinct bands are found at 14, 55, 66, ian uptake of lipids and growth declines, that ;76 kDa in HDLrVHDL at most times of the recycling of excess cholesterol to the liver may be year with the exception of winter, the quiescent necessary. Associated with this, apoA-I is con- period. Although the identity of these bands is sidered to be involved in SR-BI recognition not known, it is likely that they represent apoA-II thereby serving to reduce circulating cholesterol. Ž.14 kDa , a protein similar to human apoA-IV Ž 55 Clearly, there is a seasonality in the amount of kDa.Ž. , albumin 66 kDa and a protein similar to lipid substrate other than vitellogenin which is apoJŽ. 76 kDa . The apolipoprotein distribution in supplied to the oocyte which increases syn- trout lipoprotein fractionsŽ. Babin, 1987 is some- chronously with vitellogenin in both spring and what similar to that reported here in the turtle. fallŽ. Gapp et al., 1979, and this paper . This Trout VLDL and LDL contain an apoB-like pro- difference is reflected in yolk lipid content: large tein of 240 kDa, a 76-kDa proteinŽ. apoJ? , a oocytes destined to ovulate in spring have choles- 25-kDa proteinŽ. apoA-I and a 13-kDa protein terol values that are 50% of those of the largest Ž.apoA-II or apoC; primarily in VLDL . Trout growing follicles in fallŽ 2.1 vs. 4.5 mgrg wet wt.; HDL is comprised primarily of apoA-IŽ. 25 kDa Duggan, unpublished. when plasma cholesterol is and apoA-IIŽ. 13 kDa in addition to small amounts highest. Possibly the preferential inflow of triglyc- of 55-kDa proteinŽ a possible analog of human eride in the spring prior to ovulation dilutes yolk apoA-IV. . It has been determined that the cutoff cholesterol content. density of 1.063 grml does not completely sepa- We believe that the levels of apoA-I and apoE rate LDL from HDL in trout plasma; a pheno- are reflective of the pattern of supply of lipids to menon that has also been reported in plasma the ovary, and it is likely that their receptors from chickensŽ. Hermier et al., 1985 , guinea pigs Ž.SR-BI and the VLDLrvtg-R must be regulated Ž.Chapman and Mills, 1977 and chimpanzees appropriately. ApoE may be down-regulated by Ž.Chapman et al., 1984 . The constituents of trout estrogen. In birds, apoE may be suppressed to VHDL include 20, 55 and 76 kDa proteins; the enhance rapid lipid deposition in growing ovarian latter two share similarity to turtle HDLrVHDL. follicles. This would slow delivery of lipids to The distribution of lipids, as well as their asso- competing peripheral tissues via the LDL-recep- ciated transport proteins, between VLDL, LDL, tor and ensure that lipid is delivered preferen- HDL and VHDL fractions of turtle plasma is of tially to the ovaryŽ see discussion below related to interest from a comparative viewpoint. Triglyc- VLDL targeting to the avian ovarian follicle, erideŽ. TG , a primary source of energy, is the Walzem et al., 1999. . In preovulatory animals major lipid component of VLDLŽ as is the case Ž.April , when lipid delivery to the ovary in the with humans and other vertebrates. at all times of turtle is essential and maximal and correlates the year. Apart from DecemberŽ ovarian quies- with elevated plasma estradiol levels, it appears cence.Ž , TG content is highest in April preovula- that apoE is also suppressed to some degree tory; 58%. and lowest immediately prior to ovula- compared to other times of the year. ApoE sup- tionŽ. May; 38% . This may be related to high pression by estrogen has been reported in other ovarian LDL-R expression ensuring an appropri- 266 A. Duggan et al. rComparatië Biochemistry and Physiology Part A 130() 2001 253᎐269 ate nutrient mix for embryonic development. Pre- between VLDLŽ. 37.8% and LDL Ž. 41.3% frac- liminary Western blots using turtle ovarian tissue tions. As for the hen, triglyceride accounts for extracts demonstrate that the VLDLrvtg-R ho- most of the distributed lipidŽ percent TG relative molog is expressed to a higher degree in ovarian to total lipid. in the VLDL fraction. follicular membranes from the preovulatory pe- In order to determine the fatty acid spectrum riod vs. postovulatoryrpostoviposition period. associated with lipid metabolism in the turtle, we Ž.Duggan, in preparation After ovulation, triglyc- analyzed each lipoprotein fraction for fatty acids erides rebound again in the VLDL fractionw 57% over the course of the seasonal cycle. Of all the Ž.Junex and 55%Ž. July, postovip. . In December, fatty acids analyzedŽ 14:0, myristic acid; 16:0, triglycerides of VLDL remain elevatedŽ. 62% , palmitic acid; 16:1, palmitoleic acid; 18:0, stearic presumably due to ovarian quiescence. Unesteri- acid; 18:1, oleic acid; 18:2, linoleic acid; 18:3, fied cholesterolŽ. UC is highest in the VLDL linolenic acid; and 20:4, arachidonic acid. , the fraction in the periovulatory stageŽ. May . In an- principal fatty acids found were 16:0, 16:1, 18:1, other reptile, the watersnake, the unesterified 18:2. This is true in the sharkŽ. Mills et al., 1977 , cholesterolŽ. UC content of VLDL is also much troutŽ.Ž Chapman et al., 1978 and bullfrog Suzuki higher than most other vertebratesŽ Chapman, et al., 1977. . In addition, the primary fatty acids 1980. . found in TG of VLDL of the laying hen are 16:0, Cholesterol esterŽ. CE , the storage form of 18:1 and 18:2Ž. Gornall and Kuksis, 1973 ; a spec- cholesterol, is clearly highest in LDLŽ the major trum similar to that found in human VLDLŽ Skipsi carrier of cholesterol for delivery to peripheral et al., 1967. . Thus, it appears that the fatty acid tissues. through much of the year. Cholesterol composition of turtle lipoproteins is similar to ester is presumably delivered to the ovary in the that reported for many other species. As can be preovulatory period, as a nutrient for embryonic seen, there are few seasonal changes in the fatty development and for steroidogenesis. Interest- acid spectrum found in the different fractions ingly, CE is the smallest lipid component of LDL over the course of the year. However, significant in the laying henŽ. Evans et al., 1977 , indicating differences in palmitoleic acidŽ. 16:1 and linoleic that the turtle is more compositionally similar to acidŽ. 18:2 content between preovulatory and pos- humans and other mammals in this regard. After tovulatory periods were observed. These differ- ovulation, the content of CE in the HDL fraction ences may be attributed to changes in dietŽ feed- in particular is decreasedw postovulatoryŽ. 27% ing periods. since one of these fatty acids is and postovipositionŽ. 23% vs. preovulatory Ž. 44% essentialŽ. linoleic and must be obtained through and periovulatoryŽ. 42%x . These periods after the diet. Nevertheless, the distribution of palmi- ovulationŽ. postovulatoryrpostoviposition corre- toleic acid differs significantly between AprilŽ pre- spond with increases in plasma apoA-I levels. ovulatory.Ž and June postovulatory . in LDL and Since SR-BI levels are increased at this time of HDL fractions. Since reptiles are considered year, a functional relationship is suggested. poikilotherms, seasonal changes in temperature When peripheral VLDLs are hydrolyzed, the may influence the degree of saturation of fatty remaining apolipoproteins and phospholipids are acids, as this group possesses more unsaturated transferred to HDL making these fractions phos- fatty acids than do endothermsŽ. Sheridan, 1994 . pholipid-rich. HDL has the highest percent com- Interestingly, the concentration of palmitoleic position of phospholipidŽ. June, July, December acidw in plasma phospholipidsŽ. PCx in humans is which appears to resemble values of other species inversely correlated with the concentration of such as the human, laying hen and bullfrogŽ and LDL-cholesterolŽ. Zak et al., 1998 . In the turtle most other vertebrates, Chapman, 1980. . in May, the amount of cholesterol in the LDL According to ChapmanŽ. 1980 , lipoprotein pro- fraction is the highest when palmitoleic acid con- files in amphibians and reptiles ‘are characterized tent in all of the fractions is considerably lower. by having relatively high concentrations of LDL With regard to the distribution of linoleic acid and HDL, with LDL predominating’. Our data Ž.18:2 between preovulatory and postovulatory demonstrate that in the female turtle, the ma- periods, it appears that its content in all fractions jority of lipid is distributed in the LDL fraction, Ž.VLDL, LDL, HDL in April are considerably as in the dogfish. Only in preovulatory animals lower than in MayrJune. In April, triglyceride Ž.April is the amount of lipid distributed evenly levels are highest and it appears there is an A. Duggan et al. rComparatië Biochemistry and Physiology Part A 130() 2001 253᎐269 267 inverse relationship between TG levels and Acknowledgements linoleic acid concentration in the fractions, a phenomenon which has also been reported in humansŽ. Zak et al., 1998 . The authors wish to sincerely thank Melanie Finally, the electron microscopic analysis of Rie for her assistance in both handling the ani- turtle lipoprotein fractions demonstrated distinct mals and sample preparation. This work was sup- differences in particle diameter among VLDL ported by NIHRR 06633 to IPC. Ž.Ž.Ž.56᎐93 nm , LDL 31᎐41 nm , HDL 10᎐20 nm , VHDLŽ. 5᎐10 nm . It is worth noting that the References particle diameters of these particles are within the range of that found in the human. It would be of interest to compare the particle sizes found Acton, S., Rigotti, A., Landschulz, K., Yu, S., Hobbs, here for a December sample with fractions ob- H.H., Krieger, M., 1996. Identification of scavenger receptor SR-BI as a high-density lipoprotein recep- tained during ovarian growth and lipid mobiliza- tor. ScienceŽ. Wash DC 271, 518᎐520. tion. For example, since the TG content of VLDL Allain, C.A., Poon, L.S., Chan, C.S.G., Richmond, W., is higher in DecemberŽ. 62% than at other times Fu, P.C., 1974. Enzymatic determination of total w x of the year i.e. MayŽ. 38% , it is possible that the serum cholesterol. Clin. Chem. 20, 470. diameter of VLDL is larger at this time of year. Babin, P.J., 1987. Apolipoproteins and the association In the case of the laying hen, the size of VLDL of egg-yolk proteins with plasma high density particles decreases by half in response to estrogen lipoproteins after ovulation and follicular atresia in Ž.Walzem et al., 1999 . This may be associated the rainbow trout Ž.Salmo gairdnerii . J. Biol. Chem. with a shift from ‘generic’ VLDLŽ. larger to 262, 4290᎐4296. ‘yolk-targeted’ VLDLŽ. smaller which allows the Babin, P.J., Thisse, C., Durliat, M., Andre, M., Aki- avian ovary to take up VLDL much more effi- menko, M.A., Thisse, B., 1997. Both apolipoprotein E and A-I genes are present in a nonmammalian cientlyŽ. Walzem et al., 1999 . These estrogen-in- vertebrate and are highly expressed during embry- duced smaller VLDL particles are resistant to onic development. Proc. Natl. Acad. Sci. USA 94, lipoprotein lipase and thus are targeted directly 8622᎐8627. to the ovary. However, in the female turtle, estro- Baker, M.E., 1988. Is vitellogenin an ancestor of gen does not appear to change the size of VLDL apolipoprotein B-100 of human low density lipopro- particles as occurs in the laying henŽ R. Walzem, tein and human lipoprotein lipase? Biochem. J. 255, personal communication. . 1057᎐1060. This study has demonstrated seasonal changes Bartlett, G.R., 1959. Phosphorous assay in column in several lipid parameters in both whole plasma chromatography. J. Biol. Chem. 234, 466᎐468. and lipoprotein fractions relative to the reproduc- Brown, M.S., Goldstein, J.L., 1986. A receptor medi- tive cycle of the female turtle. In addition, the ated pathway for cholesterol homeostasis. Science 232, 34᎐47. seasonal variation in the protein content of Bujo, H., Hermann, M., Kaderli, M.O. et al., 1994. lipoproteins and one of the major cholesterol-re- Chicken oocyte growth is mediated by an eight lig- distributing proteins, apoA-I, are reported. Since and binding repeat member of the LDL receptor information on reptilian lipid metabolism is scarce family. EMBO J. 13, 5165᎐5175. in comparison to other speciesŽ. Chapman, 1980 , Callard, I.P., Banks, S.H., Banks Jr., W.L., 1972. Hep- this study provides a ‘link’ between avian and atic protein and nucleic acid content in Dipsosurus mammalian lipid metabolic strategies. It is appar- dorsalis following hypophysectomy and treatment ent that the reproductive success of the female with estradiol 17-B and growth hormone. Comp. turtle, as with the laying hen, is dependent upon Biochem. Physiol.Ž. B 41, 503᎐510. the coordinate regulation of lipids and protein Callard, I.P., Lance, V., Salhanick, A.R., Barad, D., and is under hormonal control. To ascertain the 1978. The annual ovarian cycle of Chrysemys picta: correlated changes in plasma steroids and parame- possible role of hormones on lipid metabolism in ters of vitellogenesis. Gen. Comp. Endocrinol. 35, the turtle, studies in our laboratory have been 245᎐257. underway to determine the effects of exogenous Callard, I.P., Riley, D., Perez, L., 1990. Vitellogenesis sex steroids on the same parameters reported in reptiles as a model for mammalian sex-differenti- here in both female and male turtlesŽ Duggan ated hepatic protein synthesis. J. Exp. 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