Contribution of collagen stable isotope biogeochemistry to the paleobiology of extinct endemic vertebrates from Tenerife (Canary Islands, Spain).

The paleodiet and paleoenvironmental context of two extinct species from Tenerife island, one giant rat Canariomys bravoi and one giant lizard Gallotia goliath, have been investigated using carbon and nitrogen isotopic compositions of fossil bone collagen. Preliminary to this study, a calibration of the isotopic variations of bone collagen from modern Rat Rattus rattus, Rabbit Oryctolagus cuniculus and Lizard Gallotia galotti relative to environmental conditions on Tenerife Islands has been attempted. No clear relationship could be found between collagen delta13C and delta15N values and aridity; the only relevant factors seem to be seashore proximity for rat, and the relative amount of C3 and CAM plants. It seems that anthropic activities have interfered with the expected relationships between collagen isotopic compositions and environmental conditions. Most fossil specimens yielded well preserved collagen. The isotopic composition of giant rat and giant lizard collagen suggest a purely C3 environment, possibly more humid than today on Tenerife. Large ranges of nitrogen isotopic compositions, especially within giant rats, may be due to local environmental conditions. Further work is needed in order to provide more valuable paleobiological information in order to better understand the role of environmental factors in the evolution and extinction of insular endemic species on Tenerife.

Regulation of PTP1D mRNA by peptide growth factors in the human endometrial cell line HEC-1-A.

OBJECTIVE: To assess, in the human endometrial cell line HEC-1-A, the presence of protein tyrosine phosphatase 1D (PTP1D) and the possible regulation of its mRNA expression by mitogens such as forskolin (an agent that increases intracellular cyclic adenosine monophosphate [cAMP] levels), epidermal growth factor (EGF), and insulin-like growth factor-I (IGF-I). METHODS: Cells were grown to confluence and maintained in serum-free media for 24 hours before treatment. Cells were exposed to forskolin, EGF, and IGF-I for increasing time periods (0, 1, 3, 6, and 24 hours), and PTP1D mRNA expression was determined by Northern blot analysis. In addition, cells were incubated with increasing doses of forskolin (final concentrations: 1, 5, 10, 20, and 30 mumol/L) for 6 hours. RESULTS: When treated with the various mitogens, cells increased their stimulation of PTP1D mRNA expression in a time- and dose-dependent fashion. Specifically, forskolin, EGF, and IGF-I induced maximal mRNA expression at 6, 3, and 6 hours, respectively. Expression induced by forskolin, EGF, and IGF-I was five, three, and six times control levels, respectively. At a dose of 10 mumol/L, forskolin induced PTP1D mRNA expression almost two times higher than control values. CONCLUSION: These data suggest that in human endometrial carcinomas, cAMP, EGF, and IGF-I may regulate the expression of PTP1D mRNA, which may, in turn, play a role in uncontrolled cell proliferation and neoplastic transformation.

Epidermal growth factor activates protein kinase C in the human endometrial cancer cell line HEC-1-A.

Previous studies from this laboratory have shown that epidermal growth factor (EGF) and the tumor promoter, phorbol myristate acetate (PMA), are mitogenic in the endometrial cancer cell line HEC-1-A. Since the effects of EGF have been shown to be mediated by the protein kinase C (PKC) pathway transduction system, we examined the possibility that the EGF-responsive signal in the endometrial cancer cell line HEC-1-A involves protein kinase C activation. HEC-1-A cells were grown to confluency in 100-mm dishes and maintained in a serum-free medium for 24 hr prior to treatment. The cells were treated with EGF at varying time intervals (0.25 to 60 min) and concentrations (0.1 to 200 ng/ml). The cells were then lysed, homogenized, and centrifuged at 105,000g for 1 hr at 4 degrees C. The supernatant was chromatographed on DEAE-Sephacel columns. The membranous pellet was resuspended in 5 ml of lysis buffer containing 1% Nonidet P-40 and also chromatographed on DEAE-Sephacel columns separately. The eluates were collected and assayed for protein kinase C activity by determining the amount of 32P transferred from [gamma-32P]ATP onto histones in the presence of the phospholipids, phosphatidylserine, and diolein. Our results show that the cytoplasmic and membrane fraction of the HEC-1-A cell line contained phosphotransferase activity which displayed kinetic characteristics typical of the protein kinase C enzyme. The optimal incubation time for protein kinase C activation in the cytosol by EGF was 5 min (30-fold stimulation). The protein kinase C activity was increased when the cell lines were incubated with increasing concentrations of EGF. Enzyme saturation was seen at a concentration of 10 ng/ml of EGF (4.5-fold stimulation). Western blot analysis confirmed the presence of the PKC enzyme in the cytosol and membranes of our cancer cell line. These results suggest that EGF, at least partially, exerts its effects on the endometrial adenocarcinoma cell line by activating protein kinase C through increased breakdown of phosphatidyl inositol (PI). The PI cascade appears to be an important signal transduction system mediating the growth stimulatory effects of EGF on endometrial carcinoma.

Transforming growth factor-beta negatively modulates proliferation and c-fos expression of the human endometrial adenocarcinoma cell line HEC-1-A.

Growth factor regulation of normal and cancerous cell proliferation has been well-documented and may be mediated by proto-oncogene activity. The purpose of this study was to assess changes in proliferation and mitogen-induced c-fos mRNA expression of an endometrial carcinoma cell line, HEC-1-A, in response to TGF-beta, a potent growth-inhibitory peptide. HEC-1-A cells were incubated in the presence or absence of TGF-beta. Mitogen-stimulated cells were additionally treated with epidermal growth factor (EGF). Changes in proliferation were measured by [3H]thymidine uptake assays. Alterations in EGF-induced c-fos expression following TGF-beta pretreatment were assessed by Northern blot using a 32P-labeled human c-fos probe. Finally, chloramphenicol acetyltransferase assays were performed to evaluate c-fos promoter activity in response to treatment conditions. Basal and EGF-stimulated proliferation was inhibited by TGF-beta in a dose- and time-dependent manner. TGF-beta also reversibly decreased EGF-induced c-fos mRNA expression in a dose- and time-dependent manner. Sequences in the c-fos promoter that were stimulated by EGF showed suppressed activity when preincubated with TGF-beta. These results show that TGF-beta negatively modulates EGF-induced c-fos expression, which may be related to the observed inhibition of carcinoma cell proliferation.

IRS-I expression on the luteinized rat ovary: IGF-I and cyclic AMP effects on IRS-I tyrosine phosphorylation.

The expression of insulin receptor substrate-I (IRS-I) mRNA was demonstrated in rat luteal cells by Northern blot analysis, in situ hybridization as well as by reverse transcriptase polymerase chain reaction. Western blot with a polyclonal anti IRS-I antibody showed the presence of a 183 kDa protein which corresponds to the size of IRS-I reported in other tissues. Further studies were performed to determine whether human chorionic gonadotropin (hCG) can interact with the insulin-like growth factor-I (IGF-I) signaling pathway to increase tyrosine phosphorylation of IRS-I. While hCG alone was ineffective in stimulating the phosphorylation of IRS-I, IGF-I mediated phosphorylation of IRS-I was increased by prior exposure to hCG. These results were further confirmed by the immunoprecipitation of IRS-I from the lysate of hCG- and IGF-I-treated luteal cell cultures followed by Western blotting with anti-phosphotyrosine antibody. Similarly, pretreatment with forskolin also increased IGF-I stimulated IRS-I phosphorylation. The increased tyrosine phosphorylation of IRS-I seen in response to IGF-I stimulation following treatment with either hCG or forskolin was not due to an increase in IRS-I content. Furthermore, IGF-I receptor tyrosine kinase activity was not affected by forskolin, suggesting that the increase in IRS-I tyrosine phosphorylation was not the result of an increase in its activity. Thus, we conclude that hCG/LH and IGF-I signaling pathways 'cross-talk' to increase the levels of IRS-I tyrosine phosphorylation. The observed increase in IRS-I tyrosine phosphorylation may be the result of an increase in the stability of the phosphorylated form of IRS-I.

cAMP and PMA enhance the effects of IGF-I in the proliferation of endometrial adenocarcinoma cell line HEC-1-A by acting at the G1 phase of the cell cycle.

The present study was undertaken to determine whether endometrial cancer cell line HEC-1-A differ from nontransformed cells, in that the cAMP and protein kinase C pathways may enhance IGF-I effects in mitogenesis by acting at the G1 phase of the cell cycle instead of G0. Immunofluorescence staining of HEC-1-A cells using the proliferating cell nuclear antigen (PCNA) monoclonal antibody and flow cytometric analysis determined that HEC-1-A cells do not enter the G0 phase of the cell cycle when incubated in a serum-free medium. Approximately 51% of the cells were in G1, 12% were in S and 37% in G2 phase of the cell cycle prior to treatment. Forskolin and phorbol-12-myristate 13-acetate (PMA) were used to stimulate cAMP production and protein kinase C activity, respectively. IGF-I, forskolin and PMA each increased (P < 0.01) [3H]-thymidine incorporation in a dose and time dependent manner. The interaction of forskolin and PMA with IGF-I was then determined. Cells preincubated with forskolin or PMA followed by incubation with IFG-I incorporated significantly more (P < 0.01) [3H]-thymidine into DNA than controls or any treatment alone. It is concluded that forskolin and, to a lesser extent, PMA exert their effect at the G1 phase of the cycle to enhance IGF-I effects in cell proliferation.

Protein kinase C-dependent and -independent pathways mediate epidermal growth factor (EGF) effects in human endometrial adenocarcinoma cell line KLE.

The role of EGF in the proliferation of the poorly differentiated endometrial adenocarcinoma cell line (KLE) was examined. EGF (10 ng/ml) and the tumor promoter, protein kinase C agonist, phorbol 12-myristate 13-acetate (PMA) were potent stimulators (P < 0.01) of DNA synthesis in this cell line as determined by [3H]thymidine incorporation into DNA. Staurosporine, a protein kinase C inhibitor, partially blocked the EGF effects on [3H]thymidine incorporation. Similarly, downregulation of protein kinase C also failed to completely abolish EGF effect on DNA synthesis and cell division. These results suggest that in the KLE cell line EGF stimulation of cell growth is exerted through both protein kinase C-dependent and -independent pathways.

Epidermal growth factor receptor expression in three different human endometrial cancer cell lines.

BACKGROUND: The aim of the current study was to establish the characteristics of the epidermal growth factor (EGF) receptor in endometrial cell lines to determine the possible relation of EGF to endometrial cancer. METHODS: Three different cell lines were used: RL95-2 (derived from a moderately differentiated adenosquamous carcinoma), HEC-I-A (from a moderately differentiated adenocarcinoma), and KLE (from a poorly differentiated adenocarcinoma). The binding of (125-I) EGF to these cell lines and the stimulatory effect of EGF on (3H) thymidine incorporation into DNA were examined. RESULTS: EGF receptor was present in all three cell lines. The binding of 125-I-labeled EGF was saturable and of high affinity. Scatchard analysis of the competitive binding data for KLE, HEC-I-A, and RL95-2 revealed linear plots, indicating a single class of binding sites with almost identical equilibrium dissociation constants (0.34 nM, 0.23 nM, and 0.20 nM, respectively). Other peptides, such as insulin-like growth factor (IGF) I and II and insulin, did not compete for the receptor. RL95-2 cells bound significantly more EGF (P < 0.005) than did the HEC-I-A and KLE cell lines. EGF increased 3H-thymidine incorporation in all three cell lines. CONCLUSION: Because EGF receptors are expressed by all three cell lines at markedly different levels and because EGF stimulates 3H-thymidine incorporation into DNA in the three cell lines, the current study suggests that EGF may play a role in the promotion of growth endometrial adenocarcinoma.

Mitogenic activity of growth factors in the human endometrial adenocarcinoma cell lines HEC-1-A and KLE.

Endometrial adenocarcinoma is the most common gynecologic malignancy occurring in the United States. Evidence is accumulating that links peptide growth factors with malignant proliferation. Epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) are known mitogens for endometrial adenocarcinoma in vitro. However, the biological activity of other growth factors in this malignancy is unclear. This study was undertaken to determine the influence of growth factors on the mitogenic activity of the human endometrial adenocarcinoma cell lines HEC-1-A and KLE. Incubation with EGF, IGF-I, insulin-like growth factor II (IGF-II), or insulin stimulated a time-dependent mitogenic response in both cell lines, with the peak response occurring at 24 hr for HEC-1-A and 48 hr for KLE. After two doubling intervals, the number of HEC-1-A cells was increased 3.5-fold by EGF (100 ng/ml), 2.7-fold by IGF-I (100 ng/ml), 2.3-fold by IGF-II (100 ng/ml), and 2.2-fold by insulin (1000 ng/ml) when compared to untreated controls (P < 0.05). The number of KLE cells was increased 2.6-fold by EGF (100 ng/ml), 2.3-fold by IGF-I (100 ng/ml), 2.1-fold by IGF-II (100 ng/ml), and 2.0-fold by insulin (1000 ng/ml) when compared to untreated controls (P < 0.05). Similar results were obtained when DNA content was measured. PDGF failed to stimulate any mitogenic response in either cell line at all concentrations tested (0.1-100 ng/ml). These findings suggest that EGF, IGF-I, IGF-II, and insulin may play a regulatory role in the proliferation of endometrial adenocarcinoma.

Increased cellular cholesterol upregulates high density lipoprotein binding to rat luteal cells.

Rat luteal cells preferentially utilize cholesterol derived from high density lipoproteins (HDL) as a substrate for steroid hormone synthesis. The uptake of cholesterol from HDL by these cells is in contrast to nonsteroidogenic cells, which export cholesterol to HDL. A previous study demonstrated that HDL binding to luteal cell membranes was increased in conjunction with in vivo cholesterol depletion or cholesterol loading of the ovary induced by pharmacological agents. These results suggest a biphasic regulation of the HDL receptor in luteinized rat ovaries. In the present studies, the in vitro regulation of HDL binding in rat luteal cells by increased intracellular cholesterol was examined. Cultured luteal cells were incubated with increasing doses of low density lipoproteins (LDL) for 2 days after which the cellular sterol content and the effects on progesterone production and HDL binding were measured. As expected, the LDL treatment increased total cellular sterol content in a dose-dependent manner, resulting in a 2.1-fold increase over control at a dose of 1 mg LDL/ml. Increased cellular cholesterol was accompanied by a comparable increase in progesterone secretion. These results suggest that exogenous cholesterol was utilized by these cells. The LDL treatment also increased the binding of HDL to the cells in a dose-dependent manner to a maximum of 2.2-fold over control. The effect of increased cellular sterol on HDL binding was also examined using a more polar cholesterol derivative, 25-hydroxycholesterol. Cells were cultured for 2 days in media containing 0.3-40 micrograms/ml 25-hydroxycholesterol in the presence of 100 micrograms/ml aminoglutethimide, an inhibitor of cholesterol metabolism. The HDL binding to luteal cells exhibited dose-dependent up-regulation by 25-hydroxycholesterol with a 5.8-fold increase in binding at the maximum dose tested. Equilibrium binding studies using cells treated with 10 micrograms/ml 25-hydroxycholesterol revealed a 2.1-fold increase in the number of HDL binding sites on the luteal cells without affecting the binding affinity. From the results of this study, it is concluded that HDL binding in rat luteal cells is up-regulated by an increase in the intracellular cholesterol level.

Studies on rat luteal cell response to insulin-like growth factor I (IGF-I): identification of a specific cell membrane receptor for IGF-I in the luteinized rat ovary.

Insulin-like growth factor-I (IGF-I) has been shown to stimulate biosynthesis of progesterone and other differentiated functions of granulosa cells. The presence of the IGF-I receptor messenger RNA and IGF-I receptor as well as the role of IGF-I in the amplification of gonadotropin action in luteinized rat ovarian cells were assessed in the present study. Rat ovarian luteal tissue were obtained on day 6 of pseudopregnancy. After collagenase dispersion, luteal cells were cultured with or without IGF-I for 72 h in a serum-free medium in the absence or presence of human CG (100 ng/ml), 8-bromo-cAMP (1 mM), and 25-OH cholesterol (30 micrograms/ml). IGF-I alone increased (P less than 0.01) progesterone secretion 47% over controls. When combined with hCG the increase in progesterone secretion was enhanced 6-fold (P less than 0.001) over controls. The effects of 8 bromo-cAMP on progesterone secretion also was amplified (P less than 0.001) when IGF-I was present in the medium. When 25-OH cholesterol, a diffusable substrate for steroidogenesis, was included in the incubation medium along with IGF-I, progesterone secretion was enhanced significantly (P less than 0.01) over controls and either treatment alone. Binding studies performed with ovarian luteal cell membranes in the ovary revealed a dissociation constant of 0.94 nM for the IGF-I receptor. Autoradiograms from affinity labeling studies revealed a band corresponding to 132 Kd which is characteristic for the alpha-subunit of the type I IGF receptor. Northern blot analysis showed the presence of a major transcript at approximately 11 kilobases, which agrees with the size of the IGF-I receptor messenger RNA. In conclusion, we provide evidence for the fact that IGF-I regulates differentiated functions of the corpus luteum through interaction with specific high affinity IGF-I receptors.

Characterization of epidermal growth factor receptor in normal and neoplastic human endometrium.

Growth factors, including epidermal growth factor (EGF), have been implicated in the growth of several types of cancer. This study compares EGF receptors in normal and neoplastic endometrium. Membrane fractions were isolated from surgical specimens. Radioreceptor assays demonstrated the presence of receptors with a dissociation constant of 0.64 nmol/l in normal endometrium. Affinity cross-linking revealed receptor molecular weight of 150 to 170 kiloDaltons (KD). A survey of samples (n = 37) revealed progressive decrease of EGF receptors in cancers of increasing grade: Grade 1-2 adenocarcinoma decreased 34% from control (n = 6, P less than 0.01), whereas Grade 3 adenocarcinoma decreased 90% (n = 7, P less than 0.01) and sarcoma decreased by 72% (n = 3, P less than 0.01). The dissociation constant and molecular weight of the receptor in neoplastic endometrium did not differ significantly from normal. The inverse relationship with grade suggests receptor alteration or down regulation by hormones and/or growth factors.

Regulation of epidermal growth factor and insulin-like growth factor I receptors by estradiol and progesterone in normal and neoplastic endometrial cell cultures.

Growth factors are polypeptides which regulate cell proliferation through binding to specific receptor proteins. Normal and neoplastic human endometrium have been shown to express epidermal growth factor (EGF) and insulin-like growth factor I (IGF-1) receptors. Endometrial cell cultures were used to test modulation of EGF and IGF-1 receptors in response to steroid hormones. Endometrial gland and stroma cells were separated by enzymatic dispersion and were incubated in medium containing estradiol (10, 100, or 1000 pg/ml) or progesterone (1, 10, or 100 ng/ml) followed by radioligand assays. Normal endometrial cultures (n = 6) treated with estradiol demonstrated 40% less EGF binding than control cultures (P less than 0.05), while IGF-1 binding was unaffected. Stromal cells treated identically decreased in only one treatment group. Progesterone treatment stimulated a significant increase in EGF and IGF-1 receptors in gland cultures. Cultures derived from adenocarcinoma (n = 2) demonstrated decreased EGF binding compared with normal endometrium (P less than 0.05). Carcinoma cells treated with progesterone resulted in a dose-dependent increase in EGF binding over control (P less than 0.05). These data illustrate effects of steroid hormones upon growth factor receptors in human endometrium, and suggest involvement of growth factors in the regulation of normal and neoplastic endometrial growth.

Insulin-like growth factor I receptors in normal and neoplastic human endometrium.

Insulin-like growth factor I (IGF-I) binding sites were characterized in normal and neoplastic endometrium. The characteristics of the endometrial IGF-I receptor are similar to those reported for other tissues. The binding of 125I-IGF-I to the endometrial membranes is saturable and time, temperature, and pH dependent. The 125I-IGF-I binding activity to the membranes obtained from differentiated and undifferentiated adenocarcinoma as well as sarcoma of the endometrium was significantly higher (P less than 0.05) when compared to the binding activity of the membranes obtained from normal endometrium. The Scatchard analysis of the competitive binding data of both normal and neoplastic endometrium revealed linear plots. This indicated a single class binding site for IGF-I with equilibrium dissociation constants (Kd) of 5.0, 6.8, 6.94, and 6.88 nM for normal, differentiated, and undifferentiated adenocarcinoma, and sarcoma of the endometrium, respectively. Therefore, the differences observed in 125I-IGF-I binding between normal and neoplastic endometrial membranes was due to an increase in the number of IGF-I binding sites and not to a change in receptor binding affinity. Autoradiograms from affinity labelling studies revealed a band corresponding to Mr 132,000 subunit of the receptor which is characteristic of the type I receptor reported for other tissues. A dimer of the alpha subunit (Mr 263,000) was also observed in all four categories of endometrial tissue. Additionally, autoradiograms obtained from sarcoma of the endometrium revealed a Mr 40,000 band that was only displaced by IGF-I and IGF-II peptides but not by the monoclonal antibody alpha IR-3 to the type I receptor. These suggest that the band is representative of the IGF-I or IGF-II binding protein. A similar band was not observed in the other tissues. The results show that the human endometrium contains high affinity IGF-I or IGF-II binding sites. The fact that IGF-I binding activity was significantly higher for neoplastic endometrium suggests that IGF-I may play an important role on supporting the growth of this neoplastic tissue.

Regulation of rat luteal cell high density lipoprotein receptors: up-regulation in response to changes in intracellular cholesterol concentration.

High density lipoprotein (HDL)-derived cholesterol is preferentially utilized by rat luteal cells for steroid synthesis. This process is mediated, at least in part, by the HDL receptor. The regulation of the HDL receptor by intracellular cholesterol concentration in the ovary was assessed in the present study. Superovulated rats were treated with 4-aminopyrozolo [3, 4-d] pyrimidine (4-APP), which inhibits lipoprotein synthesis by the liver, to determine the effects of reduced levels of intracellular cholesterol on HDL receptor expression. Pseudopregnant rats were treated with 4-APP or the vehicle on days 3, 4, and 5 of pseudopregnancy. On day 6 rats were killed and ovarian membranes isolated for binding studies. Treatment with 4-APP resulted in almost a 50% decrease in serum cholesterol level 24 h later. A similar effect was observed on the level of ovarian esterified cholesterol. The binding of apolipoprotein E-free HDL to ovarian plasma membranes increased up to 34%. The effects of increased intracellular cholesterol on HDL receptor binding was then determined. Pseudopregnant rats received two injections of aminoglutethimide or vehicle on day 5 of pseudopregnancy at 10 h intervals for a period of 20 h. Rats were decapitated 10 h after the second injection of aminoglutethimide, and [125I] HDL binding studies were performed in ovarian plasma membranes. The treatment increased ovarian free and esterified cholesterol 27% and 70%, respectively. The number of binding sites for HDL in ovarian membranes increased up to 31% in response to the treatment. The evidence suggests that the HDL receptor may play a dual role in HDL metabolism. The receptor may increase to promote influx of cholesterol when intraovarian cholesterol stores are reduced and to promote cholesterol efflux when stores of intraovarian cholesterol are increased. Thus, HDL receptor appears to play a crucial role in the regulation of ovarian cholesterol level.

Comparative role of retinol, retinoic acid and beta-carotene on progesterone secretion by pig corpus luteum in vitro.

Quadruplicate wells of pig luteal cells were incubated for 24 h in the presence of different concentrations of retinol, beta-carotene (0, 1 x 10(-5), 1 x 10(-6) and 1 x 10(-7) M) or retinoic acid (0, 1 x 10(-6), 1 x 10(-7) and 1 x 10(-8) M). In addition, the responsiveness of luteal cells to LH challenge was also evaluated. Progesterone was assayed in the media. Cell viability was estimated using trypan blue exclusion and showed over 95% viability. In the presence of LH, progesterone content in the medium was increased by 7-fold. As compared to their respective controls, all concentrations of retinoic acid and beta-carotene increased progesterone content in the media. The highest level of stimulation was observed with 1 x 10(-6) M-retinoic acid (5-fold increase) and 1 x 10(-7) M-beta-carotene (10-fold increase). Only 1 x 10(-5) M-retinol stimulated progesterone secretion (over 3-fold). Therefore, retinol, retinoic acid and beta-carotene stimulate progesterone secretion by pig luteal cells in vitro.

Relationships among dietary lipid intake, serum cholesterol and ovarian function in Holstein heifers.

Experiments were performed to determine whether serum concentrations of total cholesterol (TC) undergo cyclic changes during the estrous cycle of dairy heifers and to assess the relationship between serum concentrations of TC and ovarian steroid hormones. The effects of a hypercholesterolemic diet upon luteal progesterone secretion also were determined. Experiment 1 involved five dairy heifers exhibiting normal estrous cycles. Serum concentrations of TC, progesterone, testosterone and estradiol-17 beta were determined in blood samples collected throughout a complete estrous cycle. A transient decline in TC was observed during the luteal phase of all heifers beginning on d 2 and reached a nadir 6 d after estrus. Highest mean concentrations of TC occurred between d -2 and +2 (96.3 +/- 8.2 mg/dl), which were markedly higher (P less than .05) than the lowest mean concentrations (76.3 +/- 10.3 mg/dl) observed on d 6. Concentrations of serum TC were negatively correlated (r = -.40; P less than .01) with progesterone between d 2 and 9. Serum TC was not correlated with testosterone or estradiol-17 beta. In Exp. 2, seven cycling Holstein heifers were fed a control diet for 70 d (Stage I), a diet containing 15% whole sunflower seed as a source of supplemental dietary lipid for 70 d (Stage II) and then the control diet for 70 d (Stage III). Diets were isocaloric and isonitrogenous. All heifers were synchronized with prostaglandin F2 alpha after wk 5 in each of the three feeding stages.(ABSTRACT TRUNCATED AT 250 WORDS)