Article

Lipid Transport in the Developing Bovine Follicle: Messenger RNA Expression Increases for Selective Uptake Receptors and Decreases for Endocytosis Receptors

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

Differences in rates of steroid production and secretion will, eventually, determine the developmental rates of ovarian follicles. The major supply of cholesterol, the precursor for steroid and androgen biosynthesis, to ovarian cells is from circulating lipoproteins via membrane receptors from the low density lipoprotein receptor (LDL) superfamily. This occurs by either endocytosis, which has been described for very low density lipoprotein receptors (VLDLr), for LDL receptors (LDLr), and by the selective uptake pathway described for the scavenger receptor class B type 1 receptor (SRB1) and the recently described ovarian receptor, lipoprotein receptor-related protein 8 (LRP8). In this study, the mRNA expression of these four cholesterol receptors in bovine ovarian cells was determined at different stages of follicular development. In small antral follicles, mRNA expression of the endocytosis receptors was higher than in large antral follicles. Expression of LRP8 mRNA increased linearly with follicular size together with an increase in LDL, VLDL, and cholesterol concentrations in the follicular fluid. SRB1 mRNA expression tended to increase with follicular diameter. Because different mRNA expression patterns were found for the two types of receptor, this may imply different regulation of cholesterol supply at different stages of follicular development. Accumulation of LDL and VLDL particles in the follicular fluid of large antral follicles may enhance cholesterol availability for the intense steroidogenic activity that is essential at these stages.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... Due to their large molecular weight, very low density lipoproteins (VLDL) and LDL cannot transverse the intact blood-follicle barrier, in contrast with high density lipoproteins (HDL), which subsequently is the predominant lipoprotein in bovine FF ( Figure 5; Grummer and Carroll, 1988). As the follicle grows, the concentration of LDL and VLDL has been shown to increase (Argov et al., 2004). The HDL contain relatively more FA and less cholesterol, hence the enrichment of the LDL fraction in the developing follicle may increase the sensitivity to insulin and increase the estradiol production and secretion (Hodgen, 1982;Wiltbank et al., 2011). ...
... The HDL contain relatively more FA and less cholesterol, hence the enrichment of the LDL fraction in the developing follicle may increase the sensitivity to insulin and increase the estradiol production and secretion (Hodgen, 1982;Wiltbank et al., 2011). Whether the lower PUFA content of larger follicles can be attributed to relative differences in LDL and HDL (Argov et al., 2004) or may indicate a selective use of polyunsaturated FA during oocyte maturation (Zeron et al., 2002;, needs further investigation. ...
... In contrast, most probably because of smaller amounts of 18:3n-3, 20:4n-6 and 22:6n-3 in the basal diet of dairy cows and the lower basal concentration of these FA in the blood plasma (Chapter 5.2), 18:3n-3, 20:4n-6 and 22:6n-3 are more successfully transferred into all blood lipid classes, and hence are mirrored in both FF and milk fat. Edwards, 1974;Grummer and Carroll, 1988;Argov et al., 2004). ...
... Recently, we reported the differential expression of lipoprotein receptors in theca and granulose cells and corpora lutea from bovine ovaries [17]. Expression of mRNA encoding lowdensity lipoprotein receptor (LDLr), very-low-density lipoprotein receptor (VLDLr) and lipoprotein receptorrelated protein 8 (LRP8) were affected by follicular size, stage of follicular development [18] and seasonality [19]. Perhaps seasonal alterations, e.g. ...
... Total RNA was isolated using TRI reagent (10 mL/g tissue) as previously described [17] and according to the manufacturer's protocol (MRC, Cincinnati, OH, USA). For each sample, first-strand cDNAwas synthesized from 5 mg of total RNA using oligo (dT) 18 as the primer in the presence of MLV reverse transcriptase (Fermentas Inc., Hanover, MD, USA) for 1 h at 42 8C. The cDNA was purified from the polymerase chain reaction (PCR) mix using a High Pure PCR product purification kit (Roche Diagnostics GmbH, Marburg, Germany). ...
... The GAPDH primer was amplified for 30 cycles under the same conditions in a different tube. Bovine follicular granulosa cells, which express all lipoprotein receptor genes [18], served as a positive control for the entire PCR amplification process. The PCR products were separated by electrophoresis on a 1.5% agarose gel, stained with ethidium bromide, and quantified using Gel-Pro Analayzer TM version 3.0 (Media Cybernetics, LP, Silver Spring, MD, USA). ...
Article
Reduced reproductive performance of dairy cows during the summer is often associated with elevated temperature. Semen collected and cryopreserved during the summer may be of low quality and might contribute to the compromised fertility of dairy cows during this season. The present study examined the association between seasonality, semen quality and its potential to survive cryopreservation. A comparison between semen collected during the summer (July to August) and that collected during the winter (November to December) revealed the summer semen to be inferior, as reflected by low motility and high mortality of sperm. Furthermore, samples that were defined as good quality had changes in lipid concentration and fatty-acid composition in both the seminal plasma and cell compartment. In particular, semen collected during the summer had reduced levels of polyunsaturated arachidonic acid (20:4; P<0.05) and decreased levels of linoleic acid (18:2; P<0.05) in the cell compartment; corresponding reductions in cholesterol (P<0.06) and fatty-acid concentrations (P<0.001) were detected in seminal plasma of semen collected during the summer. In addition, we provided the first evidence for the existence of a very-low-density lipoprotein receptor (VLDLr) in bovine sperm, suggesting a mechanism for sperm utilization of extracellular lipids. Interestingly, the expression of VLDLr was three-fold greater in samples collected during the winter than in those collected in the summer (P<0.01) and was negatively associated with saturated fatty-acid concentration (P<0.018) but not with that of cholesterol. An opposite pattern was noted for samples obtained during the summer; mRNA expression of VLDLr was negatively associated with cholesterol concentration (P<0.01) but not with that of saturated fatty acids. Such modifications associated with extracellular lipid utilization and fatty-acid composition might explain, in part, the reduced quality of summer semen.
... The uptake of LDL is mediated via an endocytotic pathway (Golos et al. 1986) and the 2-macroglobulin receptor (Chu & Pizzo 1994, Ireland et al. 2004). The uptake of lipoprotein particle remnants and HDL is mediated via the scavenger receptor, class B, type I and LRP receptors (Acton et al. 1996, Reaven et al. 1998, Argov et al. 2004). While intact LDL is known to stimulate luteal steroidogenesis (Pate & Condon 1989, Brannian et al. 1995), oxidatively modified LDL (oxLDL) prepared in a laboratory setting has been reported to inhibit luteal progesterone production (Carroll et al. 1992). ...
... The LH surge induces an intense synthesis of progesterone in follicular thecal and granulosal layers, requiring a supply of serum lipoproteins as steroidogenic substrates in some mammalian species (Carroll et al. 1992, Brannian et al. 1995). Steroidogenic tissue is capable of utilizing circulating lipids via expression of diverse ligands and receptors (Umans et al. 1999, Argov et al. 2004). Progesterone possesses anti-mitogenic and anti-apoptotic properties that are mediated via progesterone receptors (Peluso & Pappalardo 1998, Quirk et al. 2004). ...
... These lipoproteins have been shown to support synthesis of progesterone by supplying cholesterol from LDL (Bao et al. 1995) but also by cholesterol-dependent and -independent actions of HDL (Azhar et al. 1998, Ragoobir et al. 2002). Follicle cells express receptors of the LDL superfamily, including very low density lipoprotein (VLDL) receptors, LDL receptors and lipoprotein receptor-related protein (LRP) receptors (Argov et al. 2004). The uptake of LDL is mediated via an endocytotic pathway (Golos et al. 1986) and the 2-macroglobulin receptor (Chu & Pizzo 1994, Ireland et al. 2004). ...
Article
Full-text available
The role of endogenously oxidized low density lipoprotein (oxLDL) in follicular steroidogenic regulation is unknown. Information may be important in order to elucidate ovulatory dysregulation in disordered lipid metabolism. To obtain specific data, we studied the effect of polar phospholipids (PL) isolated from oxLDL with different endogenous levels of lipohydroperoxides (LHP) on the thecal expression of mRNA encoding steroidogenic enzymes and cyclooxygenase 2 (COX-2), and on the thecal production of superoxide and progesterone. Large (preovulatory) bovine follicles were used and analyses of thecal fragments from single follicles were performed by radioimmunoassays, chemiluminescence assays and quantitative RT-PCR. Basal concentration of mRNA for several lipoprotein receptors exceeded by about 10-times the basal level of mRNA encoding steroidogenic enzymes, suggesting that preovulatory theca receptors may favour uptake of oxLDL. PL (5-11 pmol phosphorus/ml) decreased (up to 0.5-times the control) progesterone synthesis, production of superoxide and levels of P450 cholesterol side chain cleavage (P450 scc), 3beta-hydroxysteroid dehydrogenase and COX-2 mRNA. Abundance of COX-2 transcripts in thecal tissue incubated with forskolin depended on the progesterone/17beta-oestradiol ratio of the follicle fluid, i.e. the previous microenvironment in vivo. PL effects were mimicked by the platelet-activating factor (PAF). WEB 2086, a PAF receptor blocker, did not always abolish these responses, suggesting that the effects were not mediated solely by this receptor. PAF interfered dose-dependently with LH-induced responses, indicating interference with LH signalling. PL from mildly oxidized LDL (0.5 nmol/ml LHP) tended to exert greater effects than PL from oxLDL containing 1.5 nmol/ml LHP. In consideration of the known physiologic role of progesterone, COX-2 and possibly superoxide, these results provide evidence for a potential of PL from oxLDL to induce ovulatory dysregulation and suggest that the extent of the LDL oxidation seems to be important for interfering with thecal responses to the preovulatory LH surge.
... Previous studies have shown that LRP8 participates in transmitting Reelin signal from extracellular to intracellular signaling processes (Hiesberger et al., 1999). The LRP8 gene is a component of the selenium delivery pathway to spermatogenic cells of the mouse (Olson et al., 2007) and may have a role in cholesterol supply for steroid biosynthesis, which enables folliculogenesis and selection processes to occur in bovines (Argov et al., 2004). In the formation process of eggs, a mass of lipids, proteins, vitamins, and minerals is needed. ...
... Gallus gallus LRP8 was first reported (Novak et al., 1996) in brains and ovary of chickens as a member of the low-density lipoprotein receptor homolog with 8 ligand-binding repeats and has the typical o-linked sugar region and Phe-Asp-Asn-Pro-Val-Tyr internalization signal as a low-density lipoprotein receptor gene family member. As a receptor of apolipoprotein E in mammals, LRP8 may inhibit platelet aggregation and deliver cholesterol to steroidogenic cells (Riddell et al., 1999;Argov et al., 2004).The LRP8 gene has a role in follicular growth and spermatogenesis by paracrine interaction (Fayad et al., 2007;Olson et al., 2007). ...
Article
Full-text available
Low-density lipoprotein receptor-related protein 8 (LRP8), a member of the low-density lipoprotein receptor gene family with a role in clusterin processing, was investigated as a candidate gene for egg quality-related traits. One SNP from C to T at position 1623 of the open reading frame of LRP8 was identified and genotyped by a high-throughput genotyping method, matrix-assisted laser desorption-ionization time-of-flight mass spectrometry in 747 egg-type dwarf layers from 44 sire families. There were no significant differences among genotypes for any interior egg traits measured, except for yolk color, in which color was deeper for the TT genotype than CC or CT (P < 0.05). For shell traits, strength and thickness were greater for TT than CC (P < 0.05), with CT intermediate and not different from either. Shape index was lower for CT than either TT or CC, which did not differ, whereas for shell color, CT was intermediate to the homozygotes, which differed (CC > TT). The present results indicated that LRP8, as a new member of eggshell matrix protein, may be a candidate gene associated with eggshell traits.
... In cattle, HDL is the major lipoprotein in the plasma and follicular fluid [99,100], and α-Toc is mainly located in HDL among lipoproteins [37]. Rajapaksha et al. [101] sequenced bovine SCARB1 cDNA, which contains 509 amino acids. ...
... Rajapaksha et al. [101] sequenced bovine SCARB1 cDNA, which contains 509 amino acids. The changes in SCARB1 mRNA levels were evaluated in developing bovine ovarian cells; however, the relationship between the mRNA level and α-Toc concentration in follicular fluid is unknown [99,100]. By contrast, Higuchi et al. [37] clarified that the upregulation of SCARB1 mRNA in neutrophils in cattle supplemented with α-Toc and the cellular α-Toc contents were decreased after anti-SRBI treatment. ...
Article
Full-text available
Levels of alpha-tocopherol (α-Toc) decline gradually in blood throughout prepartum, reaching lowest levels (hypovitaminosis E) around calving. Despite numerous reports about the disease risk in hypovitaminosis E and the effect of α-Toc supplementation on the health of transition dairy cows, its risk and supplemental effects are controversial. Here, we present some novel data about the disease risk of hypovitaminosis E and the effects of α-Toc supplementation in transition dairy cows. These data strongly demonstrate that hypovitaminosis E is a risk factor for the occurrence of peripartum disease. Furthermore, a study on the effectiveness of using serum vitamin levels as biomarkers to predict disease in dairy cows was reported, and a rapid field test for measuring vitamin levels was developed. By contrast, evidence for how hypovitaminosis E occurred during the transition period was scarce until the 2010s. Pioneering studies conducted with humans and rodents have identified and characterised some α-Toc-related proteins, molecular players involved in α-Toc regulation followed by a study in ruminants from the 2010s. Based on recent literature, the six physiological factors: (1) the decline in α-Toc intake from the close-up period; (2) changes in the digestive and absorptive functions of α-Toc; (3) the decline in plasma high-density lipoprotein as an α-Toc carrier; (4) increasing oxidative stress and consumption of α-Toc; (5) decreasing hepatic α-Toc transfer to circulation; and (6) increasing mammary α-Toc transfer from blood to colostrum, may be involved in α-Toc deficiency during the transition period. However, the mechanisms and pathways are poorly understood, and further studies are needed to understand the physiological role of α-Toc-related molecules in cattle. Understanding the molecular mechanisms underlying hypovitaminosis E will contribute to the prevention of peripartum disease and high performance in dairy cows.
... After selection, the DF expresses higher levels of mRNAs for LHCGR and enzymes that are required for progestin and androgen synthesis, such as hydroxy-delta-5-steroid dehydrogenase, 3 beta-and steroid delta-isomerase (HSD3B2), cytochrome P450, family 11, subfamily A, polypeptide 1 (CYP11A1), steroidogenic acute regulator (StAR), and CYP17A1, than the unrecruited follicles [6,7,34]. Moreover, the concentrations of apolipoproteins, such as LDL and VLDL, increase in the follicular fluids of medium-and large-sized antral follicles (4-10 mm) compared to small antral follicles (2-4 mm) [35]. Collectively, these reported observations and our present data suggest that LRP8 contributes to cholesterol uptake in GC during follicular growth and dominance, at which time the cholesterol requirement for steroidogenic activity is increased. ...
... The downregulation or absence of LRP8 contrasts markedly with the increased cholesterol requirements during ovulation and luteinization [38,39]. In contrast to LRP8, we show that LDLR and VLDLR, which are already known to mediate cholesterol uptake in the ovary [31,35,40], are expressed in bovine GC of OFs 24 h post-hCG and in the CL. ...
Article
Full-text available
The low-density lipoprotein (LDL) receptor-related protein 8 (LRP8) is a member of the LDL receptor family that participates in endocytosis and signal transduction. We cloned the full-length bovine LRP8 cDNA in granulosa cells (GC) of the dominant follicle (DF) as well as several LRP8 mRNA splicing variants, including a variant that contains a proline-rich cytoplasmic insert (A759-K817) that is involved in intracellular signaling. Expression of the A759-K817 variant was analyzed in the GC of follicles at different developmental stages: the small follicle (SF; 2-4 mm), the DF at Day 5 (D5) of the estrus cycle, ovulatory follicles (OF) 24 h after hCG injection, and corpora lutea (CL) at D5. RT-PCR analysis showed that expression was predominant in the GC of DF compared to other follicles and CL (P<0.0001), whereas the expression of other related receptors, such as LDLR and VLDLR, did not show differences. Temporal analyses of follicular walls from the OF following hCG treatment revealed a decrease in LRP8 mRNA expression starting 12 h post-hCG treatment (P<0.0001). LRP8 protein was exclusively localized to the GC, with higher levels in the DF than in the SF (P<0.05). RELN mRNA, which encodes an LRP8 ligand, was highly expressed in the theca of the DF as compared to the OF (P<0.004), whereas MAPK8IP1 mRNA, which encodes an LRP8 intracellular interacting partner, is expressed in the GC of the DF. These results demonstrate the differential expression patterns of LRP8, RELN, and MAPK8IP1 mRNAs during final follicular growth and ovulation, and suggest that a RELN/LRP8/MAPK8IP1 paracrine interaction regulates follicular growth.
... Tissue profiles have demonstrated the highest expression of SR-BI in the ovary and adrenal of the rat (10), and in situ hybridization have revealed that expression is restricted to the theca compartment of developing follicles (24). In rat (11) and bovine (2) ovaries, transcript abundance appears to increase as follicular development ensues. In the rat ovary, SR-BI expression is upregulated by folliculogenetic stimuli, in particular, the combination of luteinizing hormone (LH) and insulin, which induces theca cell steroid synthesis (12). ...
Article
Full-text available
Ovarian follicles luteinize after ovulation, requiring structural and molecular remodeling along with exponential increases in steroidogenesis. Cholesterol substrates for luteal steroidogenesis are imported via scavenger receptor-BI (SR-BI) and the low-density lipoprotein (LDL) receptor from circulating high-density lipoproteins and LDL. SR-BI mRNA is expressed in pig ovaries at all stages of folliculogenesis and in the corpus luteum (CL). An 82-kDa form of SR-BI predominates throughout, is weakly present in granulosa cells, and is robustly expressed in the CL, along with the less abundant 57-kDa form. Digestion of N-linked carbohydrates substantially reduced the SR-BI mass in luteal cells, indicating that differences between forms is attributable to glycosylation. Immunohistochemistry revealed SR-BI to be concentrated in the cytoplasm of follicular granulosa cells, although found mostly at the periphery of luteal cells. To examine receptor dynamics during gonadotropin-induced luteinization, pigs were treated with an ovulatory stimulus, and ovaries were collected at intervals to ovulation. SR-BI in granulosa cell cytoplasm increased through the periovulatory period, with migration to the cell periphery as the CL matured. In vitro culture of follicles with human chorionic gonadotropin induced time-dependent upregulation of 82-kDa SR-BI in granulosa cells. SR-BI and LDL receptor were reciprocally expressed, with the latter highest in follicular granulosa cells, declining precipitously with CL formation. We conclude that luteinization causes upregulation of SR-BI expression, its posttranslational maturation by glycosylation, and insertion into luteal cell membranes. Expression of the LDL receptor is extinguished during luteinization, indicating dynamic regulation of cholesterol importation to maintain elevated steroid output by the CL.
... High levels of expression of NLRC5 mRNA have been reported in immune organs and mucosal tissue in pigs (Yang et al. 2016). Low-density lipoprotein receptor-related protein 8 is produced by LRP8, has been used therapeutically to treat human skeletal disease (Yang and Williams 2017), and had significant effects on spermatogenesis in mice (Olson et al. 2007) and folliculogenesis in the bovine (Argov et al. 2004). In poultry, LRP8 is considered to be a candidate gene associated with eggshell traits in chicken (Yao et al. 2010). ...
Article
Full-text available
Haemonchus contortus is a common, intractably pathogenic and economically important gastrointestinal nematode for goat producers worldwide, especially in tropical and subtropical regions. The objective of this study is to identify single nucleotide polymorphisms (SNPs) of 12 candidate goat genes mainly related to the innate immune response associated with fecal egg counts (FECs) of Haemonchus contortus in goat as an indicator of the level of parasite infection. Phenotypic data including FEC and blood traits were recorded in 189 native goats from China and 191 ones from Bangladesh, respectively. Bangladeshi goats had significantly (P < 0.01) lower FEC compared to that of Chinese goats, suggesting higher susceptible and infection rates in Chinese goat populations. FEC was significantly positive correlated with body weight (r = 0.64, P < 0.01) and hemoglobin (r = 0.49, P < 0.01) value, but negative with pack cell volume (r = − 0.63, P < 0.05) in goats. Genotyping of SNPs was performed using a matrix-assisted laser desorption ionization time of flight mass spectrometry assay and a generalized linear model was used to evaluate the association between each SNP and goat FEC trait. Eleven novel SNPs in the NLRC3, NLRC5, HIP1, and LRP8, out of 46 variants from these 12 genes, were significantly associated with FEC of goats with a nominal significance level of P < 0.05. Of these 11 SNPs, linkage disequilibrium were revealed among SNPs in LRP8 (r² = 0.87 to 1), between SNPs in NLRC3, NLRC5, and HIP1 (r² = 0.96 to 0.99), respectively. Further, haplotypes within NLRC3, NLRC5, and HIP1 were significantly associated (P < 0.001) with FEC. In artificial challenge trail, quantitative real-time PCR exposed that the relative expression of mRNA was higher in the resistant group for NLRC3 (P < 0.01), LRP8 and HIP1 (P < 0.001) but lower in the resistant group for NLRC5 (P < 0.0001), compared to the susceptible group. The possible SNP markers and genes identified in this study could be potentially used in marker-assisted selection for breeding local goats breeds resistant to gastrointestinal nematode parasite particularly for Haemonchus contortus, and then for improving health and productivity of goat.
... Some of the LH-induced gene were SCARB1, PAPPA, EGR1, ADAMTS1, TNFAIP6, STAT3, and TIMP1, which were also confirmed by RT-qPCR analyses. It has been previously shown that the expression of SCARB1, which is responsible for cellular uptake of high-density lipoproteins in steroidogenic tissues, increases as the bovine follicle size increases, using abattoir-collected ovaries 40 . Moreover, Scarb1-null mice are infertile despite normal ovarian morphology, estrus cycles, progesterone levels, and number of ovulated follicles 41 . ...
Article
Full-text available
Abstract Ovulation is triggered by gonadotropin surge-induced signaling cascades. To study the role of extracellular signal-regulated kinase 1/2 (ERK1/2) in bovine ovulation, we administered the pharmacological inhibitor, PD0325901, into the preovulatory dominant follicle by intrafollicular injection. Four of five cows treated with 50 µM PD0325901 failed to ovulate. To uncover the molecular basis of anovulation in ERK1/2-inhibited cows, we collected granulosa and theca cells from Vehicle and PD0325901 treated follicles. Next-generation sequencing of granulosa cell RNA revealed 285 differentially expressed genes between Vehicle and PD0325901-treated granulosa cells at 6 h post-GnRH. Multiple inflammation-related pathways were enriched among the differentially expressed genes. The ERK1/2 dependent LH-induced genes in granulosa cells included EGR1, ADAMTS1, STAT3 and TNFAIP6. Surprisingly, PD0325901 treatment did not affect STAR expression in granulosa cells at 6 h post-GnRH. Granulosa cells had higher STAR protein and theca cells had higher levels of STAR mRNA in ERK1/2-inhibited follicles. Further, both granulosa and theca cells of ERK1/2-inhibited follicles had higher expression of SLC16A1, a monocarboxylate transporter, transporting substances including β-hydroxybutyrate across the plasma membrane. Taken together, ERK1/2 plays a significant role in mediating LH surge-induced gene expression in granulosa and theca cells of the ovulating follicle in cattle.
... We found that the expression levels of ApoB, ApoE, and MTTP decreased, and LDLR was significantly elevated in the hepatocytes after decreasing the SREBP-1c expression. The elevated expression of LDLR could inhibit the ApoB secretion and regulate the assembly of VLDL by blocking the secretion of VLDL [34]. The accumulation of TG in the liver is the most direct cause of fatty liver. ...
Article
Full-text available
Background: Fatty liver is a major metabolic disorder that occurs during early lactation in high-producing dairy cows. Sterol regulatory element-binding protein-1c (SREBP-1c) is an important transcription factor that regulates lipid synthesis by regulating the expression of lipid metabolism genes. Methods: In this study, we reduced the expression of SREBP-1c by adenovirus-mediated SREBP-1c with a low expression vector (AD-GFP-SREBP-1c) to study the effects of SREBP-1c on lipid deposits in bovine hepatocytes. The expression levels and enzyme activities of SERBP-1c and its target genes were determined by real-time PCR, western blot, and ELISA. Results: These results showed that Ad-GFP-SREBP-1c could inhibit SREBP-1c expression. The expression of the lipid synthesis enzyme acetyl-CoA carboxylase (ACC) was down-regulated. The expression levels of the lipid oxidation enzymes long-chain fatty acyl-COA synthetase (ACSL-1), carnitine palmitoyltransferase І (CPT-І), carnitine palmitoyltransferase II (CPT- II), and β-hydroxyacyl-CoA-DH (HADH) were significantly elevated. Furthermore, the expression levels of factors involved in the assembly and transport of very low-density lipoproteins (VLDLs), such as apolipoprotein B100 (ApoB), apolipoprotein E (ApoE), and microsomal triglyceride transfer protein (MTTP) were decreased comparison with the negative control and the blank control groups, but the low-density lipoprotein receptor (LDLR) was elevated. The concentrations of TG (triglyceride) and VLDL were also reduced. Conclusion: These data suggest that low SREBP-1c expression can decrease lipid synthesis, increase lipid oxidation, and decrease the TG and VLDL content in bovine hepatocytes. © 2014 S. Karger AG, Basel.
... Recently, the LRP8 gene has been studied in different species. In bovines, Argov et al. (2004) suggested that LRP8 might have a role in cholesterol supply for steroid biosynthesis, which enables folliculogenesis and selection processes to occur. In mouse, Olson et al. (2007) reported that the Lrp8 gene was a component of the selenium delivery pathway to spermatogenic cells. ...
Article
Full-text available
Abstract 1. The low density lipoprotein receptor-related protein 8 (LRP8), a member of the low density lipoprotein receptor (LDLR) gene family, participates in the supplying of lipid during follicular development. The objective of the study was to identify and characterise the LRP8 gene in goose. 2. A 2867 bp fragment that covered the complete coding region (CDS) of goose (Anser cygnoides) LRP8 gene was cloned. It encoded a protein of 917 amino acid residues containing a 24-amino acid signal peptide and 5 functional domains. The goose LRP8 showed high nucleic acid and amino acid identities with those in other species. 3. Similarly to duck LRP8 gene, two splice variants of LRP8, LRP8-1 (containing eight ligand-binding repeats) and LRP8-2 (containing 7 ligand-binding repeats), were identified in goose. 4. Semi-quantitative RT-PCR analysis indicates that the LRP8-1 transcript is expressed in heart, liver, spleen, lung, kidney, breast muscle, duodenum, hypothalamus, pituitary and ovary, negligible or absent in sebum and oviduct, and the LRP8-2 transcript is widely expressed in all examined tissues. 5. A total of 7 SNPs were identified in the coding region of the goose LRP8 gene.
Article
Summer heat stress (HS) is a major factor in decreased reproductive performance in high-producing dairy cattle, possibly by affecting the steroidogenic capacity of ovarian follicles and ovarian follicular dynamics. In the present study, mRNA expression of cholesterol receptors was determined in bovine ovarian cells. Two endocytotic receptors (very-low-density lipoprotein receptor (VLDLr) and low-density lipoprotein receptor (LDLr)), and two selective-uptake receptors (scavenger receptor class B type 1 receptor (SRB1) and the lipoprotein-receptor-related protein 8 (LRP8)) were evaluated. Ovarian follicles in four diameter categories were evaluated from cows under non-heat stress (NHS) and HS conditions. As follicle size increased, expression of mRNA in NHS cows increased for the selective-uptake receptors, SRB1 and LRP8, and decreased (P<0.004) for the endocytotic receptors, LDLr and VLDLr. In contrast, in HS cows, mRNA expression did not significantly change (with increasing follicle diameter) for either receptor type. With increasing follicle diameter, cholesterol and fatty acid concentrations in the follicular fluid did not change in HS cows, whereas in NHS cows, cholesterol increased (P<0.008) and fatty acid decreased (P<0.0001). These changes paralleled those in the different lipoprotein fractions in the follicular fluid. In follicles from HS cows, the altered mRNA expression patterns for the endocytotic and selective-uptake receptors caused changes in the regulation of cholesterol supply at critical stages of folliculogenesis, which may play a role in the low turnover rates of ovarian follicles during the summer.
Article
Full-text available
Present study was focus to compare LDL receptor mRNA expression, total cholesterol, triglyceride, lipoprotien levels and abdominal fat weight in genetically fat and lean chickens. Genetically lean (Rugao) and fat (Anka) chickens were reared in the same environmental condition, at 12 weeks of age samples of liver tissue were collected and abdominal fat weight was determined. Similarly, total cholesterol, triglycerides and high density lipoprotein were assayed using a commercial enzymatic kit, very low density lipoprotein and low density lipoprotein were estimated using the Friedewald equation. Total RNA from liver tissues were isolated using the standard Trizol methods and then total RNA was reverse transcribed by moloney murine Leukemia virus. Semi-quantitative RT-PCR was developed to quantify the levels of LDL receptor mRNA expression. The level of LDL receptor expression was significantly (p< 0.05) difference between lean and fat chicken. In addition, lean and fat chickens were significantly differed on triglyceride, very low density lipoprotein and abdominal fat weight. The expression of LDL receptor mRNA in liver of fat chicken was negatively correlated with abdominal fat weight. However, in lean chicken was negatively correlated with total cholesterol, triglyceride, lipoprotein concentration, abdominal fat weight and percentage of abdominal fat weight. In addition, within two breeds LDL receptor mRNA expression in liver was negatively correlated with low density lipoprotein, abdominal fat weight and percentage of abdominal fat weight.
Article
Full-text available
Different fatty acid (FA) sources are known to influence reproductive hormones in cattle, yet there is little information on how dietary FAs affect oocyte quality. Effects of three dietary sources of FAs (supplying predominantly palmitic and oleic, linoleic (n-6) or linolenic (n-3) acids) on developmental potential of oocytes were studied in lactating dairy cows. A total of 12 Holstein cows received three diets containing rumen inert fat (RIF), soyabean or linseed as the main FA source for three periods of 25 days in a Latin-square design. Within each period, oocytes were collected in four ovum pick-up sessions at 3-4 day intervals. FA profiles in plasma and milk reflected profiles of dietary FA sources, but major FAs in granulosa cells were not affected. Dietary FA source did not affect plasma concentrations of leptin, insulin, IGF1, GH, or amino acids. RIF led to a higher proportion of cleaved embryos than soya or linseed, but blastocyst yield and embryo quality were not affected. It is concluded that the ovary buffers oocytes against the effects of fluctuations in plasma n-3 and n-6 FAs, resulting in only modest effects on their developmental potential.
Article
This study was designed to evaluate the effect of nutritional supplementation offered during the pre- and postpartum periods on serum cholesterol, triglycerides and total lipids of Canchim beef cows and their relationship with folliculogenesis. Thirty cows with predicted calving date between September and October, kept in pastures of Brachiaria brizantha cv. Marandú together with their calves, were randomly distributed into three experimental groups: the first received only a mineral mixture (Control Group, CG); the second group received a concentrate with 16%crude protein/kg dry matter (DM) and 3000 kcal digestible energy/kg DM offered for 45 days prepartum and 120 days postpartum (PREG); the third group received the concentrate from parturition until the 120th day postpartum (POSG). Consumption was estimated at 1% of body weight, and each cow received approximately 4.0 kg/day (fresh weight) of supplement. Blood samples were taken and an ultrasound examination of the ovaries was performed twice a week until the 60th day postpartum. The body condition score (BCS) and the weight of the cows were recorded at 15-day intervals from calving until the 60th day postpartum. Data are presented as mean+/-SEM. Mean weight and BCS at calving were, respectively, 448+/-54.9 kg and 6.2+/-0.25 (PREG); 432+/-71.1 kg and 5.5+/-0.69 (POSG); and 434+/-66.4 kg and 5.5+/-0.69 (CG). Total cholesterol (TC), triglycerides (TRIG) and total lipids (TLIP) were measured using colorimetry until the 60th day postpartum. TC averages were PREG 186+/-62.6 mg/dL, POSG 159+/-43.1mg/dL and CG 133+/-35.1mg/dL (P<0.05). For TRIG, the means were PREG 29+/-11.3mg/dL (P<0.05), POSG 24+/-8.1mg/dL and CG 26+/-12.1mg/dL (P>0.05). Serum concentrations of TLIP were PREG 588+/-145.6 mg/dL, POSG 512+/-137.6 mg/dL and CG 452+/-122.4 mg/dL (P<0.05). The first dominant follicle (DF) was identified on Day 21+/-10.3 (PREG), 36+/-28.5 (POSG) and 51+/-32.8 (CG) after calving. The difference between PREG and CG was significant (P<0.05). TC was positively correlated with the calving to first estrus interval (P<0.05). Results showed that nutritional supplementation before parturition assured good body condition at calving and suggested that it was effective at increasing cholesterol availability to maintain ovarian follicle function and to favor earlier resumption of ovarian activity.
Article
Low-density lipoprotein receptor-related protein 8 (LRP8) is a member of the low-density lipoprotein receptor gene family that functions in body lipoprotein homeostasis. In this study, reverse transcription-polymerase chain reaction, rapid amplification of cDNA ends, and real-time PCR were performed to characterize the duck LRP8 gene. The cDNA of duck LRP8 contained a 14-bp 5' UTR, a 2754-bp open reading frame, and a 189-bp 3' UTR. The duck LRP8 encoded a protein of 917 amino acid residues composed of five functional domains and resembling other members of the LDLR family, and it displayed high nucleotide and amino acid homology to the LRP8 sequences in other avian species. The mRNA expression level of LRP8 was greater in duck extra-hepatic adipose tissue than in the liver. The peak expression values of LRP8 in both liver and adipose tissues occurred at week 1 and were significantly higher than the values observed during any other week (p < 0.05). Differences in the expression patterns of LRP8 mRNA from weeks 2 to 8 of growth were observed in different organs. A consistent low expression was observed in the liver, and fluctuating expression was observed in the subcutaneous adipose tissue (up- and then down-regulated) and abdominal adipose tissue (down-, then up-, then down-regulated). These findings suggest that LRP8 might play more important roles in regulating lipid metabolism in extra-hepatic adipose tissues than in the liver during early growth after hatching in the duck.
Article
The steroidogenic capacity of several tissues has been shown to be regulated by lipoproteins. However, the ability of the various lipoprotein classes to affect steroid production in general and estradiol-synthesis in particular has not been established in bovine ovarian cells. In previous studies, we described alterations in the lipoprotein profiles in follicles of different sizes and corresponding changes in lipoprotein-receptor gene expression. In the present study, the effect of low-density lipoprotein (LDL)-enriched medium on aromatization competence of whole ovarian follicles was determined in vitro. Gene expression of aromatase increased and that of selective-uptake receptors decreased in the presence of LDL. These results suggest a role for LDL availability in folliculogenesis.
Article
Finely regulated fatty acid (FA) metabolism within ovarian follicles is crucial to follicular development and influences the quality of the enclosed oocyte, which relies on the surrounding intra-follicular environment for its growth and maturation. A growing number of studies have examined the association between the lipid composition of follicular compartments and oocyte quality. In this review, we focus on lipids, their possible exchanges between compartments within the ovarian follicle and their involvement in different pathways during oocyte final growth and maturation. Lipidomics provides a detailed snapshot of the global lipid profiles and identified lipids, clearly discriminating the cells or fluid from follicles at distinct physiological stages. Follicular fluid appears as a main mediator of lipid exchanges between follicular somatic cells and the oocyte, through vesicle-mediated and non-vesicular transport of esterified and free FA. A variety of expression data allowed the identification of common and cell-type-specific actors of lipid metabolism in theca cells, granulosa cells, cumulus cells and oocytes, including key regulators of FA uptake, FA transport, lipid transformation, lipoprotein synthesis and protein palmitoylation. They act in harmony to accompany follicular development, and maintain intra-follicular homeostasis to allow the oocyte to accumulate energy and membrane lipids for subsequent meiotic divisions and first embryo cleavages.
Article
Full-text available
Abstract 1. Two splice variants of duck LRP8 were identified, one containing 8 ligand-binding repeats (LRP8-1) and the other containing only 7 repeats (LRP8-2). The two transcripts share ~71-91% nucleic acid identity and ~65-94% amino acid identity with their counterparts in other species. A phylogenetic tree based on amino acid sequences shows that duck LRP8 proteins are closely related to those of chicken, turkey and zebra finch. 2. The semi-quantitative reverse transcription polymerase chain reaction (RT-PCR )analysis indicates that the two transcripts are expressed in all the examined tissues, and the LRP8-1 transcript is more highly expressed in hypothalamus, ovary and pituitary gland than in other detected tissues. 3. Six single nucleotide polymorphisms (SNPs) were identified in the coding region. Association analysis demonstrated that the c.528C > T genotypes were associated with egg production (EP) (EP210d, EP300d and EP360d), age at laying the first egg (AFE) and body weight at sexual maturity (BWSM). The c.1371A > G genotypes were associated with egg production (EP210d, EP300d and EP360d). 4. The haplotypes of c.528C > T and c.1371A > G were associated with EP (EP210d, EP300d and EP360d), yolk weight (YW), albumen weight (AW), egg weight (EW), BWSM and the first egg weight (FEW). 5. Duck LRP8 gene was associated with some reproductive traits and is an important candidate gene for the genetic selection of improved reproductive traits.
Article
Very low density lipoprotein receptor (VLDLR)-mediated endocytosis of plasma lipoproteins into the ovary is essential for ovarian follicle development. Two splice variants of VLDLR have been identified in several species, yet little is known about their distinctive roles in ovarian developing follicles. In the present study, the full-length cDNAs of two splice isoforms of VLDLR were obtained from geese (Anser cygnoide) ovaries using the RACE method. The longer isoform (TypeIVLDLR) is 3141 base pairs (bp) and contains five conserved structural domains, while the other (TypeIIVLDLR) lacks 90 bp encoding for the O-linked sugar domain. TypeIIVLDLR was predominantly expressed in the ovary, with greater amounts of mRNA in theca and granulosa cells from early stages of follicle development but decreased during vitellogenesis. However, there was mimimal expression of the TypeIVLDLR gene in theca cells and expression was almost undetectable in granulosa cells throughout follicle development. Yolk VLDL concentrations decreased as stage of development advanced while yolk triglyceride and cholesterol concentrations increased in a follicular size-dependent manner. The significant correlations between transcripts of TypeIIVLDLR and yolk lipids supported its important role on yolk lipid deposition. In addition, in vitro experiments suggested that exogenous cholesterol, 25-hydroxycholesterol and mevinolin (a highly potent competitive inhibitor of HMG-CoA) treatment could significantly alter TypeIIVLDLR gene expression in granulosa cells from both pre-hierarchical and pre-ovulatory follicles. Collectively, data from the present study indicate that TypeIIVLDLR is more important for the transport of plasma lipoproteins into developing follicles than TypeIVLDLR, and provide new evidence about the influence of steroids in modulating VLDLR gene expression in ovarian cells.
Article
Cholesterol is an important substrate for the synthesis of ovarian sex hormones, which has an important influence on follicular development. Cholesterol in follicular fluid is mainly taken from plasma. High-density lipoprotein (HDL) and low-density lipoprotein (LDL) play an important role in ovarian cholesterol transport. Knockout of related receptors in mammalian HDL and LDL pathways have resulted in reduced or absent fertility, leading us to further believe in the importance of cholesterol homeostasis in the ovary. However, little is known about ovarian cholesterol metabolism and its complex homeostasis regulation. This review retrospects the literature on the role of cholesterol metabolism in female fertility and follicular cell development.
Article
To assess the expression and regulation of antilipoprotein D (ApoD) and antilipoprotein E (ApoE) in human endometrium. Endometrial biopsies from healthy, regularly cycling women were collected during the late proliferative and mid-secretory phase. mRNA gene expression of ApoD and ApoE was determined using real-time PCR in whole tissue, in isolated stromal (ESC), epithelial (EEC) and CD45(+) leukocytes (EIC), as well as after hormonal stimulation of ESC and EEC in vitro. Protein expression was analyzed using immunohistochemistry. ApoD and ApoE mRNA was expressed in all cell types examined. A rise in ApoD mRNA expression was seen in whole endometrium, ESC, and EEC in the secretory phase, as well as after hormonal stimulation of ESC and EEC in vitro. ApoE mRNA was significantly upregulated in whole endometrium of secretory phase biopsies, while its expression was not altered by progesterone in vitro. Immunohistochemistry of whole endometrial tissue localized ApoD mainly in ESC and EEC. While ApoE was localized slightly in ESC, it was particularly noted on the surface of secretory phase endothelial cells. We demonstrate for the first time the cell-type and cycle dependent expression of ApoD and ApoE within human endometrium, suggesting their role in endometrial modulation.
Article
Full-text available
Sterol-dependent regulation of the low density lipoprotein (LDL) receptor promoter has been localized previously to a 16-base pair sequence, designated repeat 2, in the 5'-flanking region of the gene. In the current study, we show that the central 10 nucleotides of repeat 2 are crucial for the sterol regulatory activity. This sequence includes an octamer, designated sterol regulatory element 1 (SRE-1), which was identified previously in the promoter of the gene for 3-hydroxy-3-methylglutaryl coenzyme A synthase, a sterol-regulated enzyme of cholesterol biosynthesis. We made a series of single-base substitutions within a 1471-base pair fragment of the intact LDL receptor promoter, introduced the mutant plasmids into hamster cells by transfection, and measured mRNA levels in the absence and presence of sterols. Substitutions within the 10-base pair sequence in repeat 2 largely prevented the induction of transcription which occurs in the absence of sterols. None of these point mutations affected transcription in the presence of sterols. Like an enhancer, the SRE-1 in repeat 2 functioned in an orientation-independent manner. We interpret these findings to indicate that the SRE-1 of the LDL receptor promoter is a conditional positive element that cooperates with other elements to enhance transcription in the absence of sterols and loses its function in the presence of sterols.
Article
Full-text available
This paper describes a rapid two-step procedure for the purification of the low density lipoprotein receptor from bovine adrenal cortex membranes. After solubilization with nonionic detergents, the receptor adheres tightly to a DEAE-cellulose column at pH 6. Following elution from DEAE-cellulose, detergent is removed, leaving the receptor in a soluble form. The receptor is then subjected to affinity chromatography on low density lipoprotein coupled to Sepharose 4B. The receptor is eluted with suramin, a newly-found inhibitor of low density lipoprotein-receptor interactions. This procedure yields a single protein with a molecular weight of 164,000. The same protein is also isolated when the crude DEAE-cellulose fraction is applied to an immunoaffinity column containing a monoclonal antibody directed against the receptor. The 164,000-dalton receptor protein has an acidic isoelectric point of 4.6, which rises to 4.8 after extensive treatment with neuraminidase. The purified receptor retains all of the binding properties of the receptor of intact cells and crude membranes.
Article
Full-text available
Article
Full-text available
The free and esterified fatty acid composition of pig serum and pig ovarian follicular fluid (POFF) samples were determined at three different developmental stages of the follicle. The most striking difference between pig serum and POFF was that the contents of free arachidonic (20:4 ω6) and free docosatetraenoic (22:4 ω6) acids were ~4 times higher in POFF than in serum. The high content of 20:4 ω6 in POFF may relate to the synthesis of prostaglandins. Free fatty acid (FFA) concentrations of POFF also decreased with follicular maturation. The concentration of POFF-FFA from large sized folicles was similar to that of pig serum. Variations in the esterified fatty acid profile with respect to follicle development were particularly noticeable in the phosphatidylcholine fraction. The decrease of palmitic (16:0) and oleic (18:1) acids and the increase of stearic (18:0) acid and polyunsaturated fatty acids (mainly 20:4 ω6) in ovarian fluid as follicles increased in size from <2 to >6 mm suggest that fatty acid chain elongation and desaturation increase with follicular maturation.
Article
Full-text available
Although the 'selective' pathway is a major cholesteryl ester (CE) uptake pathway used by steroidogenic cells, essentially nothing is known about the itinerary of the CE once it is extracted from lipoproteins at the cell surface. In the current report we have begun to trace 'selective' pathway internalized-CE using both native and reconstituted human (h) high density lipoproteins (hHDL3) with variously labeled and tagged CEs to provide information from either a biochemical or morphological perspective. It appears that the amount of hHDL3-CE that is internalized and processed through the 'selective' pathway is directly related to the amount of cholesterol used for steroidogenesis at any given time point. There is a time-related correlation between the level of the Bt2cAMP-stimulated cell steroidogenic response, the level of conversion of freshly obtained hHDL3-CE into progestins, increases in hHDL3-derived CE internalization, hHDL3-CE hydrolysis, re-esterification and/or storage. None of this processing takes place in non-stimulated (non-Bt2cAMP treated) cells which do not secrete progestins despite the availability of hHDL3 as a cholesterol source. The data suggest that the 'selective' pathway has a special role in steroidogenic cells-one of providing sufficient cholesterol to fuel the required production of steroid hormones.
Article
Full-text available
Two cell surface molecules, the low density lipoprotein (LDL) receptor and the LDL receptor-related protein (LRP)/alpha 2-macroglobulin receptor, have been found to have a role in the removal of apoE-rich lipoproteins. To further study this process and to assess the relative contributions of these proteins to the removal of chylomicron remnants, we used an anti-LDL receptor antibody, activated alpha 2-macroglobulin and the 39-kDa receptor-associated protein (RAP) as inhibitors of chylomicron remnant removal in intact mice. The advantage of this study is that we were able to use the same litters of animals and batches of lipoprotein for the experiment. In cultured Chinese hamster ovary cells, the anti-LDL receptor antibody inhibited remnant uptake and degradation about 80% as well as did unlabeled remnants. It did not affect activated alpha 2-macroglobulin uptake or degradation. Activated alpha 2-macroglobulin did not inhibit remnant uptake in normal cells but is reported to block uptake in cells that lack LDL receptors. Chylomicron remnants blocked activated alpha 2-macroglobulin uptake as effectively as unlabeled activated alpha 2-macroglobulin. This confirmed that the two ligands are cross-competitors but suggests that the LDL receptor is the primary mediator of remnant uptake in cultured cells that express the LDL receptor. In vivo, pretreatment with the anti-LDL receptor antibody decreased remnant uptake 5 min after injection by one-third. The antibody did not affect the removal of activated alpha 2-macroglobulin. Activated alpha 2-macroglobulin had a small, but reproducible effect on remnant removal, decreasing it by about 7% at 5 min. Injection of chylomicron remnants affected activated alpha 2-macroglobulin removal slightly. A similar pattern, but somewhat greater effect was seen on the hepatic uptake of the ligands. Anti-LDL receptor antibody reduced chylomicron remnant uptake by about half, and activated alpha 2-macroglobulin reduced it by about 15%. Studies with RAP provided results generally similar to those with activated alpha 2-macroglobulin, although RAP appears to bind to a site not recognized by either remnants or activated alpha 2-macroglobulin in addition to sharing a site with these ligands. Together, these results add support for the hypothesis that, although both receptors can play a role in chylomicron remnant removal, in the normal mouse in vivo the LDL receptor plays a substantially greater role, making the role of the LRP difficult to appreciate. The same is true in cells that express LDL receptors.(ABSTRACT TRUNCATED AT 400 WORDS)
Article
Full-text available
The scavenger receptor, class B, type I (SR-BI) binds HDL and mediates the selective transfer of cholesteryl esters from HDL to cultured cells. The tissue distribution of SR-BI in mice suggests that this receptor may deliver HDL-cholesterol to the liver and to nonplacental steroidogenic tissues. To examine the role of SR-BI in vivo, we determined its tissue and cell type-specific expression pattern and regulation in rats. High levels of immunodetectable SR-BI were present in the adrenal gland, ovary, and liver. In pregnant animals, the mammary gland also expressed high levels of the protein. SR-BI was localized by immunofluorescence to the surfaces of steroidogenic cells in the zona fasciculata and zona reticularis of the adrenal gland and to the corpus luteal cells of the ovary. High-dose estrogen treatment dramatically reduced SR-BI in the liver and increased SR-BI in the adrenal gland and corpus luteal cells of the ovary. These estrogen-induced increases in SR-BI in the adrenal gland and ovary were accompanied by enhanced in vivo uptake of fluorescent lipid from HDL. The administration of human chorionic gonadotropin induced a dramatic increase in SR-BI in the steroidogenic Leydig cells of the testes. These findings suggest that SR-BI mediates physiologically relevant uptake of cholesterol from HDL to nonplacental steroidogenic tissues in vivo.
Article
Full-text available
The class B, type I scavenger receptor, SR-BI, binds high density lipoprotein (HDL) and can mediate selective uptake of HDL cholesteryl esters by cultured cells. The high levels of expression of SR-BI in steroidogenic tissues and the importance of selective uptake from HDL as a source of cholesterol for steroidogenesis raised the possibility that SR-BI may participate in cholesterol delivery to steroidogenic tissues in vivo. We have used immunoblotting and immunohistochemical methods to show that SR-BI is specifically expressed in a distinctive pattern on the surfaces of steroid-producing cells in the murine adrenal gland's cortex and that its expression in vivo is induced by adrenocorticotropic hormone and suppressed by glucocorticoids. Thus, expression of SR-BI protein is coordinately regulated with adrenal steroidogenesis. These data provide strong support for the hypothesis that SR-BI is a physiologically relevant HDL receptor that provides substrate cholesterol for steroid hormone synthesis.
Article
Full-text available
This study examined the mechanisms by which calcium soaps of fatty acids and bovine somatotropin (bST) affect production and reproduction of high producing cows. Calcium soaps of fatty acids were fed at 2.2% dry matter, and 500 mg of Zn-sometribove (Monsanto Inc., St Louis, MO) were injected subcutaneously every 14 d from 10 to 150 d in milk (DIM). Production of fat-corrected milk was increased by 3.5 kg/d when calcium soaps of fatty acids were fed, by 6.1 kg/d when bST was administered, and by 7.4 kg/d when calcium soaps of fatty acids were fed and bST was administered. Body weight was similar for cows on all treatments until 85 DIM after which cows that were treated with bST had lower body weights. Body condition scores decreased more for cows treated with bST and began increasing later and more slowly. Treatment with bST resulted in more cows that experienced first ovulation after 30 DIM, and more cows on the control treatment exhibited first estrus before 35 DIM. Days open were greater when bST was administered. After the first artificial insemination, conception rates were similar for cows on the control treatment and for cows fed calcium soaps of fatty acids; conception rates after the first artificial insemination were low for all cows treated with bST. Pregnancy rates at 120 and 150 DIM were decreased by bST. Number of DIM to first ovulation, number of DIM to first estrus, and days open were negatively correlated with glucose and cholesterol concentrations in plasma. Production of fat-corrected milk was correlated with days open and with concentrations of triglycerides in plasma, nonesterified fatty acids, and cholesterol. Increased production had different effects on reproduction when induced by calcium soaps of fatty acids or bST treatment. Some of the adverse effects of bST treatments were alleviated by calcium soaps of fatty acids.
Article
Full-text available
Steroidogenic cells in rats and mice obtain most of their cholesterol for steroid production and cholesteryl ester (CE) storage via the selective uptake pathway in which high density lipoprotein CE (HDL-CE) is taken into the cell without the uptake and degradation of the HDL particle. A number of recent studies show that the scavenger receptor, class B, type I (SR-BI) can mediate HDL-CE selective uptake in cultured cells and suggest that this receptor may be responsible for HDL-CE selective uptake in steroidogenic cells in vivo. In the current study we examine the relationship between SR-BI expression and HDL-CE selective uptake in the gonadotropin-primed, luteinized rat ovary and in the ovary that is desensitized by multiple gonadotropin treatments. Results from this study demonstrate a tight association between expression of SR-BI and measurements of HDL-CE selective uptake regardless of the steroidogenic state of the ovary. Thus, in the luteinized ovary (which is actively producing progestins), HDL-CE selective uptake is high, as is the expression of SR-BI. In the desensitized ovary (where CE content is reduced by 90% and progestin production is virtually absent), HDL-CE selective uptake and SR-BI are induced 2- to 3-fold compared with those in the luteinized ovary. These data argue that SR-BI can be regulated by the cholesterol status of the luteal cell independently of gonadotropic stimulation. Immunostaining at the light microscopic level showed strong expression of SR-BI specifically on the surface of luteal cells in the luteinized and desensitized ovary. Immunolocalization at the electron microscopic level showed that SR-BI was associated with microvilli and microvillar channels of the luteal cell surface. This result supports the hypothesis that microvilli and microvillar channels represent a cell surface compartment that is specialized for the selective uptake of lipoprotein cholesterol into steroidogenic cells.
Article
Full-text available
The plasma clearance of intestinally derived remnant lipoproteins by the liver is a process that likely involves three steps. Our model suggests that the initial rapid clearance by the liver begins with sequestration of the remnants within the space of Disse, where apolipoprotein E secreted by hepatocytes enhances remnant binding and uptake. Heparan sulfate proteoglycans (HSPG), which are also abundant in the space of Disse, mediate this enhanced binding. Next, the remnants undergo further processing in the space of Disse by hepatic and lipoprotein lipases, which may also serve as ligands mediating remnant uptake. The final step, endocytosis by hepatocytes, appears to be mediated, at least in part, by the low density lipoprotein (LDL) receptor and by the LDL receptor-related protein (LRP). Cell-surface HSPG play a critical role in remnant uptake, not only in the important initial sequestration or capture step in the space of Disse, but also as an essential or integral component of the HSPG-LRP pathway. In addition, HSPG appear to function alone as a receptor and display unique handling properties for specific isoforms of apolipoprotein E. —Mahley, R. W., and Z-S. Ji. Remnant lipoprotein metabolism: key pathways involving cell-surface heparan sulfate proteoglycans and apolipoprotein E. J. Lipid Res. 1999. 40: 1–16.
Article
Full-text available
Insulin-like growth factor I (IGF-I) and the gonadotropin, FSH, can synergize to stimulate progesterone production in primary cultures of maturing granulosa cells. These trophic hormones increase low density lipoprotein (LDL) receptor binding and internalization, and the utilization of LDL-borne cholesterol by granulosa cells. To determine whether and how IGF-I and FSH control the genomic expression of the LDL receptor, we evaluated their individual and concerted effects on LDL receptor messenger RNA (mRNA) accumulation, stability, and gene promoter activity in first passage monolayer (serum-free) cultures of porcine granulosa cells. Ribonuclease protection assays revealed that LDL receptor mRNA accumulation was increased by human recombinant IGF-I (100 ng/ml), FSH (25 ng/ml NIDDK oFSH-20), or their combination by 2.2-, 2.6-, and 4.6-fold, respectively (P < 0.01). Hormonally stimulated LDL receptor mRNA accumulation was suppressed by 54-75% by the concurrent addition of LDL substrate (50 microg/ml). The combination of FSH and IGF-I significantly prolonged the message half-life, even in the presence of LDL. Using a combination of rapid amplification of cDNA 5'-ends, PCR with adapter-ligated genomic DNA, Southern hybridization, and DNA sequencing, we isolated 1076 bp of the porcine LDL receptor gene upstream of the coding region. In transient transfection assays, with a pLDLR1076/luciferase plasmid construct, FSH, FSH plus IGF-I, or 8-bromo-cAMP (1 mM) treatment (but not IGF-I alone) increased luciferase reporter gene activity by 10- to 23-fold in porcine granulosa cells. Over time in serum-free culture, the basal activity of the LDL receptor gene promoter increased and eventually surpassed hormone-stimulated effects, but was suppressed by LDL substrate (by 75%) at 24 h. The foregoing stimulatory hormone effects and sterol repression were localized to a 116-bp region in the porcine promoter between -255 and -139 upstream of the translational start site. We conclude that the combination of FSH and IGF-I can induce accumulation of LDL receptor mRNA in cultured granulosa cells even in the presence of sterol negative feedback and can do so mechanistically by a combination of promoter activation and increased mRNA stability.
Article
Full-text available
The high density lipoprotein (HDL) receptor SR-BI (scavenger receptor class B type I) mediates the selective uptake of plasma HDL cholesterol by the liver and steroidogenic tissues. As a consequence, SR-BI can influence plasma HDL cholesterol levels, HDL structure, biliary cholesterol concentrations, and the uptake, storage, and utilization of cholesterol by steroid hormone-producing cells. Here we used homozygous null SR-BI knockout mice to show that SR-BI is required for maintaining normal biliary cholesterol levels, oocyte development, and female fertility. We also used SR-BI/apolipoprotein E double homozygous knockout mice to show that SR-BI can protect against early-onset atherosclerosis. Although the mechanisms underlying the effects of SR-BI loss on reproduction and atherosclerosis have not been established, potential causes include changes in (i) plasma lipoprotein levels and/or structure, (ii) cholesterol flux into or out of peripheral tissues (ovary, aortic wall), and (iii) reverse cholesterol transport, as indicated by the significant reduction of gallbladder bile cholesterol levels in SR-BI and SR-BI/apolipoprotein E double knockout mice relative to controls. If SR-BI has similar activities in humans, it may become an attractive target for therapeutic intervention in a variety of diseases.
Article
Full-text available
All members of the low density lipoprotein (LDL) receptor family contain at least one copy of the NPXY sequence within their cytoplasmic tails. For the LDL receptor, it has been demonstrated that the NPXY motif serves as a signal for rapid endocytosis through coated pits. Thus, it is generally believed that the NPXY sequences function as endocytosis signals for all the LDL receptor family members. The primary aim of this study is to define the endocytosis signal(s) within the cytoplasmic tail of LDL receptor-related protein (LRP). By using LRP minireceptors, which mimic the function and trafficking of full-length endogenous LRP, we demonstrate that the YXXL motif, but not the two NPXY motifs, serves as the dominant signal for LRP endocytosis. We also found that the distal di-leucine motif within the LRP tail contributes to its endocytosis, and its function is independent of the YXXL motif. Although the proximal NPXY motif and the proximal di-leucine motif each play a limited role in LRP endocytosis in the context of the full-length tail, these motifs were functional within the truncated receptor tail. In addition, we show that LRP minireceptor mutants defective in endocytosis signal(s) accumulate at the cell surface and are less efficient in delivery of ligand for degradation.
Article
Full-text available
The members of the low density lipoprotein (LDL) receptor gene family bind a broad spectrum of extracellular ligands. Traditionally, they had been regarded as mere cargo receptors that promote the endocytosis and lysosomal delivery of these ligands. However, recent genetic experiments in mice have revealed critical functions for two LDL receptor family members, the very low density lipoprotein receptor and the apoE receptor-2, in the transmission of extracellular signals and the activation of intracellular tyrosine kinases. This process regulates neuronal migration and is crucial for brain development. Signaling through these receptors requires the interaction of their cytoplasmic tails with the intracellular adaptor protein Disabled-1 (DAB1). Here, we identify an extended set of cytoplasmic proteins that might also participate in signal transmission by the LDL receptor gene family. Most of these novel proteins are adaptor or scaffold proteins that contain PID or PDZ domains and function in the regulation of mitogen-activated protein kinases, cell adhesion, vesicle trafficking, or neurotransmission. We show that binding of DAB1 interferes with receptor internalization suggesting a mechanism by which signaling through this class of receptors might be regulated. Taken together, these findings imply much broader physiological functions for the LDL receptor family than had previously been appreciated. They form the basis for the elucidation of the molecular pathways by which cells respond to the diversity of ligands that bind to these multifunctional receptors on the cell surface.
Article
Full-text available
The steroidogenic acute regulatory protein (StAR) is required for the movement of cholesterol from the outer to the inner mitochondrial membrane, the site of cholesterol side chain cleavage. Here we describe a novel form of regulation of StAR gene expression in steroidogenic cells. Treatment of Y-1 BS1 adrenocortical cells with either low density lipoprotein (LDL) or high density lipoprotein (HDL) increases expression of endogenous StAR mRNA and protein in a dose-dependent manner. Induction of StAR mRNA by lipoprotein requires basal cAMP-dependent protein kinase, since the inhibitor, R(p)-8-Br-cAMP, inhibited induction of StAR protein by LDL. Likewise, basal StAR expression or LDL induction of StAR protein was not detectable in Y-1 kin-8 cells which are deficient in cAMP-dependent protein kinase. Aminoglutethimide and ketoconazole were used to determine if side chain cleavage of lipoprotein-derived cholesterol is required for induction of StAR mRNA. Treatment with either drug alone induced StAR mRNA expression 1.5-3-fold, while induction of StAR in cells treated with either drug plus LDL, was equal to, or greater than, induction seen with either agent alone, suggesting that lipoprotein does not regulate StAR via generation of an oxysterol intermediate. Both LDL and HDL increased expression of a mouse -966 StAR promoter-reporter construct 1.5-2.5-fold, indicating that regulation occurs at the level of transcription. In contrast, neither lipoprotein was able to induce transcription from a -966 StAR promoter in which the steroidogenic factor-1 site at -135 was abolished, indicating that regulation of StAR transcription by lipoproteins requires steroidogenic factor-1. The regulation of StAR gene expression by lipoproteins may represent a positive feedback circuit which links cholesterol availability with steroidogenic output.
Article
Full-text available
The steroidogenic acute regulatory protein (StAR) gene controls the rate-limiting step in the biogenesis of steroid hormones, delivery of cholesterol to the cholesterol side-chain cleavage enzyme on the inner mitochondrial membrane. We determined whether the human StAR promoter is responsive to sterol regulatory element-binding proteins (SREBPs). Expression of SREBP-1a stimulated StAR promoter activity in the context of COS-1 cells and human granulosa-lutein cells. In contrast, expression of SREBP-2 produced only a modest stimulation of StAR promoter activity. One of the SREBP-1a response elements in the StAR promoter was mapped in deletion constructs and by site-directed mutagenesis between nucleotides -81 to -70 from the transcription start site. This motif bound recombinant SREBPs in electrophoretic mobility shift assays, but with lesser affinity than a low density lipoprotein receptor SREBP-binding site. An additional binding site for the transcriptional modulator, yin yang 1 (YY1), was observed within the SREBP-binding site (nucleotides -73 to -70). Mutation of the YY1-binding site increased the responsiveness of the StAR promoter to exogenous SREBP-1a, but did not alter the affinity for SREBP-1a binding in electrophoretic mobility gel shift assays. Manipulations that altered endogenous mature SREBP-1a levels (e.g. culture in lipoprotein-deficient medium and addition of 27-hydroxycholesterol) did not affect StAR promoter function, but influenced low density lipoprotein receptor promoter activity. We conclude that 1) the human StAR promoter is conditionally responsive to SREBP-1a such that promoter activity is up-regulated in the presence of high levels of SREBP-1a, but is unaffected when mature SREBP levels are suppressed; and 2) the human StAR promoter is selectively responsive to SREBP-1a.
Article
Full-text available
The aim of this study was to examine the function of granulosa cells and hormone concentrations in follicular fluid in bovine ovarian follicles during selection of the first dominant follicle. Ovaries were obtained from beef heifers on days 1-5 after ovulation: follicles > 4 mm in diameter were dissected and follicular fluid and granulosa cells were collected from individual follicles. Oestradiol production by granulosa cells after culture with testosterone was used to determine aromatase activity and responsiveness to gonadotrophins was determined by cAMP production after culture with FSH or LH. Concentrations of oestradiol, progesterone and insulin-like growth factor binding proteins (IGFBPs)-4 and -5 were measured in follicular fluid. Follicles were classified as largest or smaller (days 1 and 2), or dominant or subordinate (days 3-5). Aromatase activity was greater in granulosa cells from the largest follicle than in granulosa cells from smaller follicles on days 1, 3, 4 and 5 (P < 0.05). Responsiveness to LH was not detected in granulosa cells on day 1, but from day 2 to day 5 cells from the largest follicle were significantly more responsive than cells from smaller follicles (P < 0.05). Responsiveness to FSH was detected in granulosa cells from all follicles from day 1 onwards and did not differ between cells from the largest follicle or smaller follicles on any day. Follicular fluid concentrations of oestradiol and the ratio of oestradiol:progesterone were greater and concentrations of IGFBP-4 and -5 were lower in the largest follicle than in smaller follicles from day 2 to day 5 (P < 0.05). In conclusion, selection of the dominant follicle is associated with increased granulosa cell aromatase activity followed by increased cAMP response to LH and follicular fluid oestradiol concentrations, and decreased follicular fluid concentrations of IGFBP-4 and -5 within 2 days after ovulation.
Article
Full-text available
The concept that selective transfer of high density lipoprotein (HDL)-derived cholesteryl esters (CE) does not require lipoprotein internalization has been challenged recently by evidence that implicates HDL recycling during the selective uptake process. This has prompted us to examine the role of the low density lipoprotein receptor-related protein (LRP) in selective uptake. LRP is an endocytic receptor for lipoprotein lipase (LpL) and apolipoprotein E (apoE) ligands that are able to mediate selective uptake. We report that molecules that interfere with ligand binding to LRP, such as the receptor-associated protein (RAP), suramin, alpha(2)-macroglobulin, or lactoferrin, inhibit HDL-CE selective uptake by human primary adipocytes and SW872 liposarcoma cells by 35-50%. This partial inhibition of selective uptake from total HDL was not due to preferential inhibition of the HDL(2) or HDL(3) subfractions. Selective uptake by the scavenger receptor BI was not inhibited by RAP, excluding its involvement. Furthermore, in SW872 cells in which LRP was reduced to 14% of control levels by stable antisense expression, selective uptake was attenuated by at least 33%, confirming a role for LRP in this process. RAP, alpha(2)-macroglobulin, lactoferrin, and suramin (individually or in paired combinations) also attenuated selective uptake of HDL-CE by primary human adipocytes by about 40%. On the other hand, human skin fibroblasts express LRP abundantly but lack the capacity for selective uptake, demonstrating that other molecules are required. In SW872 cells, exogenous apoE or LpL can facilitate selective uptake but only the apoE-enhanced uptake can be inhibited by RAP, implicating apoE as a likely co-mediator. We discuss the possible mechanisms by which the endocytic receptor, LRP, can mediate selective uptake.
Article
Full-text available
Vascular smooth muscle cell (SMC) proliferation and migration are important events in the development of atherosclerosis. The low-density lipoprotein receptor–related protein (LRP1) mediates suppression of SMC migration induced by platelet-derived growth factor (PDGF). Here we show that LRP1 forms a complex with the PDGF receptor (PDGFR). Inactivation of LRP1 in vascular SMCs of mice causes PDGFR overexpression and abnormal activation of PDGFR signaling, resulting in disruption of the elastic layer, SMC proliferation, aneurysm formation, and marked susceptibility to cholesterol-induced atherosclerosis. The development of these abnormalities was reduced by treatment with Gleevec, an inhibitor of PDGF signaling. Thus, LRP1 has a pivotal role in protecting vascular wall integrity and preventing atherosclerosis by controlling PDGFR activation.
Article
Full-text available
Peroxisome proliferator-activated receptors (PPARs α, β, γ1, and γ2) are widely regarded as monitors of intracellular nonesterified fatty acid (NEFA) levels. As such, fatty acid binding to PPAR leads to changes in the transcription of many genes involved in lipid metabolism and storage. Although the composition of the intracellular NEFA pool is likely an important factor controlling PPAR activity, little information is available on factors affecting its composition. Accordingly, we have examined the effects of exogenous fatty acids on PPARα activity and NEFA pool composition in rat primary hepatocytes. Prior to the addition of fatty acids to primary hepatocytes, nonesterified unsaturated fatty acid levels are very low, representing ≤0.5% of the total fatty acid in the cell. The relative abundance of putative PPARα ligands in the NEFA pool is 20:4n-6 = 18:2n-6 = 18:1n-9 > 22:6n-3 > 18:3n-3/6 = 20:5n-3. Of these fatty acids, only 20:5n-3 and 22:6n-3 consistently induced PPARα activity. Metabolic labeling of primary hepatocytes indicated that both 14C-18:1n-9 and 14C-20:5n-3 are rapidly assimilated into neutral and polar lipids. Although the addition of 18:1n-9 had no effect on NEFA pool composition, 20:5n-3 mass increased >15-fold within 90 min. Changes in NEFA pool 20:5n-3 mass correlated with dynamic changes in the PPARα-regulated transcript mRNACYP4A. Metabolic labeling also indicated that a significant fraction of 14C-20:5n-3 was elongated to 22:5n-3. Cells treated with 22:5n-3 or 22:6n-3 led to a significant accumulation of 20:5n-3 in the NEFA pool through a process that requires peroxisomal β-oxidation and fatty acyl CoA thioesterase activity. Further analyses suggest that 20:5n-3 and 22:6n-3, but not 22:5n-3, are active ligands for PPARα. These studies suggest that basal fatty acid levels in the NEFA pool coupled with rates of fatty acid esterification, elongation, desaturation, peroxisomal β-oxidation, and fatty acyl thioestease activity are important determinants controlling NEFA pool composition and PPARα activity.
Article
The presence of high density lipoprotein-binding protein (HBP) mRNA in rat ovarian cells and its possible regulation by gonadotropin were examined in this study. RNA, isolated from pseudopregnant rat ovaries, was amplified by reverse transcriptase-polymerase chain reaction (PCR) with HBP-specific oligonucleotide primers. A distinct and prominent 576-basepair PCR product was generated. Sequence analysis revealed that the sequence homology of the PCR product and the corresponding human HBP cDNA sequence was greater than 90%. Using Northern blot analysis, we identified two species of HBP mRNA transcripts in the ovarian cells (i.e. a major one of 4.5 kilobases and a minor one of 6.0 kilobases). Cellular localization of HBP mRNA in the ovary was determined by in situ hybridization analysis. Prominent specific hybridization of the labeled antisense probe was observed in all cell types of the follicles and corpora lutea, whereas little hybridization was observed in the stroma and the connective tissues separatin...
Article
Quantitative changes were found in lipids in six categories of goat follicles: early antral; small antral; medium antral; large antral; pre-ovulatory; ovulatory. The total lipid content increased steadily up to medium antral follicles, a steep increase was observed in large antral follicles, and thereafter there was a decline. The total cholesterol content increased with the growth of follicles. The reverse was the case for free fatty acid content. Phospholipids and triglycérides formed the major component of lipids. Triglycerides and glycolipids were irregular during the growth of antral follicles. The physiological significance of these lipid changes is discussed in relation to follicular growth.
Article
The low-density Lipoprotein receptor-related protein (LRP) is a 4544-amino-acid membrane protein which closely resembles the LDL receptor in its arrangement of cysteine-rich motifs. Binding studies have suggested that one function of the molecule is as a receptor for ligands containing apolipoprotein E. We present here the sequence and structure of the promoter region of the LRP. These data show that the LRP contains no sterol regulatory element, and is not down-regulated by sterols like the LDL receptor. This lends further support to the identity of the LRP as a chylomicron remnant receptor.
Article
Follicular fluid (FF) lipoprotein content was evaluated in an in vitro fertilization/embryo transfer and gamete--intrafallopian--transfer program and correlated to follicular and oocyte maturation. Moreover, the in vitro progesterone response of granulosa-luteal cells from 10 patients to high-density lipoprotein (HDL) and to low-density lipoprotein (LDL) was assessed. Most FFs contained only HDL. Sixteen out of 97 FFs contained also very low levels of LDL and very-low-density lipoprotein (VLDL). The presence of LDL was associated to features of follicle and oocyte hypermaturity. LDL alone induced a much more potent increase of progesterone (P) release by granulosa-luteal cells than HDL alone, and the HDL partially reversed the potent effect of LDL. It is concluded that in late follicular phase HDL maintains P release by granulosa cells at a low rate and prevents a potent stimulation of P production by LDL which might cross the maturating blood-follicle barrier, until increasing passage of LDL in FF decreases the HDL:LDL ratio and the action of LDL becomes prominent.
Article
The hormonal regulation of thecal cell function was investigated with cells isolated at various stages of antral follicle development. Bovine thecal cells were isolated from small antral, medium antral, and large Graffian follicles (small, medium, and large ovarian follicles). Serum-free cultures of thecal cells were established and viable for a minimum of 6-8 days of culture. The purity of the thecal cell population was characterized cytochemically and was found to contain less than 5% endothelial cell and/or granulosa cell contamination. The steroidogenic capacity of this purified population of thecal cells in serum-free culture was examined through an analysis of androgen and progesterone production. Androgen production was high during the first 3 days of culture, then declined to undetectable levels. Production of androstenedione was approximately 10-fold higher than production of testosterone. Progesterone production remained relatively constant throughout the 8-day culture period. hCG was found to stimulate androgen production during days 1-3 of culture, but had a negligible effect on progesterone production. In contrast, hCG stimulated progesterone production during days 3-6 of culture, but had a negligible effect on androgen production. Insulin stimulated progesterone production during days 3-6 of culture, but had no effect on androgen or progesterone production during days 1-3 of culture. The minimum effective concentrations of hCG and insulin required to stimulate steroidogenesis of the thecal cells ranged from approximately 1-10 ng/ml. Addition of serum to the cultures decreased androgen production and suppressed the hormone responsiveness of the cells. Thecal cells in culture appear to alter their steroidogenic capacity from an androgen-producing cell to a progesterone-producing cell. Analysis of the developmental regulation of thecal cell function revealed that androgen production and hormone responsiveness were relatively constant in small, medium, and large follicles. In contrast, progesterone production and hormone responsiveness were highest in small follicles, intermediate in medium follicles, and lowest in large follicles. A more general analysis of the developmental regulation of thecal cell function examined the secretion of radiolabeled proteins. A large number of radiolabeled proteins were secreted by thecal cells, ranging in molecular mass from 5-500 kDa. Interestingly, insulin and hCG had no major effect on secretion of proteins by cells isolated from any of the stages of development examined.(ABSTRACT TRUNCATED AT 400 WORDS)
Article
The LDL receptor, which mediates the cellular uptake of cholesterol, is subject to classic end-product repression when cholesterol accumulates in the cell. We here show that the sensitivity to end-product repression depends upon a 42 bp element in the 5'-flanking region of the human LDL receptor gene. This sequence, designated sterol regulatory element 42 (SRE 42), contains two 16 bp direct repeats that exhibit positive and negative transcriptional activities. Cells transfected with a fusion gene containing SRE 42 inserted into the promoter of the herpes simplex viral TK gene produced abundant mRNA when grown without sterols. When sterols were present, the mRNA was reduced by 57%-95%, depending on the number of copies of SRE in the fusion gene. These transfection data plus DNAase I footprinting experiments suggest a model of end-product repression in which the end product (sterols) opposes the action of a positive transcription factor that binds to a discrete promoter element.
Article
Insulin synergistically amplified the stimulatory effect of low density lipoprotein (LDL) on progesterone biosynthesis by primary cultures of swine granulosa cells. The mechanisms subserving this facilitative interaction included the following: 1) insulin's synergism with LDL was profoundly attenuated by covalent modification of arginine residues in LDL by 1,2-cyclohexanedione treatment; 2) insulin increased by 2- to 6-fold the number of specific high affinity LDL receptors on granulosa cells, with no change in apparent binding affinity; 3) insulin augmented rates of [125I]iodo-LDL internalization and degradation without enhancing nonspecific bulk fluid-phase pinocytosis (assessed with [125I]iodo-polyvinylpyrollidone); 4) insulin increased by 2.5- to 3-fold granulosa cell content of free and esterified cholesterol (measured by fluorometry) in response to treatment with unlabeled LDL; 5) insulin stimulated the intracellular accumulation of free [3H]cholesterol and [3H]cholesteryl ester, and amplified [3H]progesterone secretion by granulosa cells exposed to [3H]cholesteryl linoleate-labeled LDL; and 6) insulin action was specific in that it was not mimicked by desoctapeptide insulin, epidermal growth factor, fibroblast growth factor, or relaxin. We conclude that insulin and LDL synergistically enhance progesterone biosynthesis by swine granulosa cells via specific mechanisms that depend upon 1,2,-cyclohexanedione-sensitive residues within LDL apoprotein. Insulin action results in significantly augmented binding, internalization, and degradation of LDL, which is accompanied by increased effectual delivery of cholesterol substrate into cellular sterol pools that participate in enhanced steroidogenesis.
Article
Six multiparous Holstein cows ranging from 7 to 19 wk postpartum were in a switchback design to determine effects of feeding high grain diets on lipoprotein concentration and composition in blood. Percents concentrate, alfalfa haylage, and corn silage of control and high grain diets were 50, 83; 25, 9; and 25, 8 dry matter. Milk yield (kg/day), milk fat percentage, and fat yield (g/day) averaged 27.7, 3.58, 997 and 30.2, 2.45, 729 for control and high grain treatments. Very low density, low density, and high density lipoproteins were isolated by gel filtration of total lipoproteins obtained by ultracentrifugation. Lipoprotein concentrations in blood plasma of cows fed the control or high grain diets were similar. Triglyceride, phospholipid, cholesterol, and cholesterol ester contents of each lipoprotein class were not altered by treatment. High grain feeding increased octadecadienoic fatty acid content of low density and high density lipoprotein cholesterol esters and decreased octadecenoic and octadecatrienoic acids. The trend was similar for the very low density lipoprotein fraction. Phospholipid octadecenoic acid decreased in all lipoprotein fractions but only significantly for high density lipoproteins. It is unlikely that differences in lipid composition of plasma lipoproteins caused alterations in lipoprotein metabolism leading to fat depression in this and other similar studies.
Article
It will be interesting to see resolution of the seemingly paradoxical actions of follicular fluid extract on pituitary gonadotropin secretion - being selective in the suppression of tonic FSH levels, but effective in blocking the surge modes of both FSH and LH release by estrogen or GnRh action. Is inhibin activity derived from a single product of ovarian secretion? Is the hypothalamic release (pulse frequency) of GnRH altered by inhibin? Or is inhibin acting directly on the pituitary gonadotrophs, desensitizing them to GnRH- or estrogen-positive feedback? Perhaps these studies will clarify whether a single GnRH regulates both FSH and LH secretion. What role may inhibin have in polycystic ovarian disease or other endocrinopathies impairing the human HPO axis? Can we develop novel means to manipulate inhibin activity for fertility control or enhancement? Perhaps the answers will seed important insights into inhibin's role in male reproductive endocrinology as well. These are among the principal queries soon to be squarely addressed in the laboratory and the clinic. Such mysteries within the ovaries are likely to entertain us, clinical investigators and basic scientists alike, for the foreseeable future.
Article
The presence of high density lipoprotein-binding protein (HBP) mRNA in rat ovarian cells and its possible regulation by gonadotropin were examined in this study. RNA, isolated from pseudopregnant rat ovaries, was amplified by reverse transcriptase-polymerase chain reaction (PCR) with HBP-specific oligonucleotide primers. A distinct and prominent 576-basepair PCR product was generated. Sequence analysis revealed that the sequence homology of the PCR product and the corresponding human HBP cDNA sequence was greater than 90%. Using Northern blot analysis, we identified two species of HBP mRNA transcripts in the ovarian cells (i.e. a major one of 4.5 kilobases and a minor one of 6.0 kilobases). Cellular localization of HBP mRNA in the ovary was determined by in situ hybridization analysis. Prominent specific hybridization of the labeled antisense probe was observed in all cell types of the follicles and corpora lutea, whereas little hybridization was observed in the stroma and the connective tissues separating corpora lutea and follicles. As hCG has been shown to induce high density lipoprotein-binding activity in the rat ovary, we examined the possible regulation of ovarian HBP mRNA by this hormone. Administration of hCG caused a significant time-dependent increase in the steady state levels of HBP mRNA. The induction of HBP mRNA levels by hCG is specific for the ovary, as pretreatment with hCG had no effect on the HBP mRNA levels of the liver, heart, lung, or kidney. The present study, for the first time, shows conclusively the presence of HBP mRNA in the rat ovary and its induction by hCG, implicating a physiological role for HBP in the ovary.
Article
Insulin and insulin-like growth factors (IGFs) have direct effects on cultured ovarian cells. These effects include stimulation of granulosa cell mitogenesis, granulosa and luteal cell progesterone production, and thecal cell androgen production and appear similar among species. However, species differences exist with regard to insulin and IGF-I effects on granulosa cell estradiol production. In addition to endocrine effects of insulin and IGFs, IGFs are produced by granulosa, thecal, and luteal cells, allowing for an intraovarian autocrine and paracrine system. Granulosa, thecal, and luteal cells contain receptors for insulin and IGFs, and these receptors appear to mediate the effects of insulin and IGFs. Adding to the complexity of the regulatory role of IGFs is the presence of IGF-binding proteins (IGFBPs) within the ovary. These IGFBPs are produced by granulosa, thecal, and luteal cells, and their production is hormonally regulated. Evidence for a coherent mechanism by which insulin, IGFs, and IGFBPs interact and regulate ovarian function in vivo has yet to be found.
Article
We thank our colleagues Beth Duncan, Jay Horton, Axel Nohturfft, Jih-tung Pai, Juro Sakai, Jin Shimano, and Iichiro Shimomura for helpful comments in the preparation of this review. We thank Ravi Pathak for the immunofluorescence micrograph (Figure 3Figure 3). Our research is supported by grants from the National Institutes of Health (HL20948) and the Perot Family Foundation.
Article
The properties of the very-low-density lipoprotein (VLDL) receptor have been studied in Chinese hamster ovary (CHO) cells stably transfected with human VLDL-receptor cDNA and compared with those of the low-density lipoprotein (LDL) receptor expressed under the same conditions. Immunoblotting showed that the cells produced a mature VLDL receptor protein, of apparent Mr 123000 on non-reduced and 158000 on reduced gels, that was less extensively glycosylated than the LDL receptor. The VLDL receptor was more slowly processed than the LDL receptor, with only approx. 70% of the precursor being converted into the mature protein. Nevertheless, the majority of the receptor in the cells was in the mature form, and most of this was present on the cell surface. The human VLDL receptor bound rabbit very-low-density lipoprotein with beta electrophoretic mobility (betaVLDL), but not human LDL, and uptake through the receptor led to stimulation of oleate incorporation into cholesteryl esters. At 37 degrees C, the characteristics of VLDL-receptor-mediated uptake and degradation of betaVLDL were essentially the same as those mediated by the LDL receptor. However, the VLDL receptor apparently did not show the increase in affinity and decrease in binding of betaVLDL on cooling to 4 degrees C that was exhibited by the LDL receptor. Thus the overexpressed VLDL receptor in CHO cells appears to behave as a lipoprotein receptor with similar, but not identical, properties to the LDL receptor.
Article
Steroidogenic activity in the mature corpus luteum of most mammals depends upon provision of cholesterol from the circulating lipoproteins. In cattle, as in many species, high-density lipoprotein (HDL) is the major lipoprotein involved. The recent identification of the scavenger receptor SR-BI as an HDL-receptor allows control of this process to be investigated more closely. In this study, we have sequenced the bovine SR-BI HDL-receptor and examined changes in expression of the receptor mRNA during corpus luteum development in vivo and granulosa cell luteinization in vitro. Sequencing of the bovine HDL-receptor showed that it codes for a protein of 509 amino acids with close identity to hamster, mouse, rat and human sequences. Examination of the tissue distribution of the HDL-receptor mRNA showed high levels in adrenal cortex and corpus luteum and lower levels in spleen and liver. Using a semi-quantitative, reverse transcription-polymerase chain reaction technique levels of HDL-receptor mRNA were measured in corpora lutea from cattle at known stages of the oestrus cycle and in bovine granulosa cells luteinized in culture. Levels of HDL-receptor mRNA were low in isolated bovine granulosa cells, but increased 7-fold during corpus luteum development in vivo and 5-fold during granulosa cell luteinization in culture. Results show that luteinization of granulosa cells is associated with an increase in HDL-receptor RNA levels which, along with changes in steroidogenic enzyme activity, is likely to explain the marked increase in steroidogenic capacity which occurs during corpus luteum formation.
Article
The aim of this study was to define the role of sterol regulatory element binding protein (SREBP)-1c, the human homologue to ADD1 (adipocyte determination- and differentiation-dependent factor 1), in insulin-induced gene expression. Transfection studies using SREBP-1-deficient cells and a LDL receptor promoter fragment containing the ADD1/SREBP-1c binding side showed that the effects of insulin and PDGF were abolished compared to control cells and completely reconstituted by overexpressing ADD1/SREBP-1c. Overexpression of upstream activators of MAP kinases, like MEKK1 or MEK1, demonstrated that ADD1/SREBP-1c-mediated effects of insulin and PDGF might be linked to the MAP kinase cascade. The recombinant N-terminal domain of ADD1/SREBP-1c was phosphorylated predominantly on serine and slightly on threonine residues by MAP kinases ERK1 and ERK2 in vitro. This was reversible by alkaline phosphatase. We conclude that ADD1/SREBP-1c mediates gene regulatory effects of insulin as well as PDGF and that this signalling is linked to the MAP kinase cascade.
Article
To determine whether human luteal cells can utilize very low density lipoproteins (VLDL)-carried cholesterol for steroidogenesis, we investigated the expression of VLDL receptor mRNA in human ovarian tissues and progesterone production by human luteinized granulosa cells after the addition of VLDL. The production of progesterone in the presence of human chorionic gonadotrophin (HCG) was increased significantly (P < 0.05) by VLDL (2479 +/- 1477 ng/10(5) cells, mean +/- SD, n = 6) and low density lipoproteins (LDL) (2726 +/- 1287), in comparison with the level in the absence of these lipoproteins (1350 +/- 739). Northern blot analysis revealed that the levels of expression of VLDL and LDL receptor mRNA in granulosa cells were almost equal to those in whole ovarian tissue. VLDL receptor mRNA was abundant in granulosa cells of preovulatory follicles and cells of the corpus luteum. Preovulatory thecal cells and stromal cells expressed lower amounts of VLDL receptor mRNA than granulosa cells of preovulatory follicles and cells of the corpus luteum. From the present study, it might be suggested that VLDL is utilized for steroidogenesis in human luteinized granulosa cells.
Article
Layering of neurons in the cerebral cortex and cerebellum requires Reelin, an extracellular matrix protein, and mammalian Disabled (mDab1), a cytosolic protein that activates tyrosine kinases. Here, we report the requirement for two other proteins, cell surface receptors termed very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2). Both receptors can bind mDab1 on their cytoplasmic tails and are expressed in cortical and cerebellar layers adjacent to layers that express Reelin. mDab1 expression is upregulated in knockout mice that lack both VLDLR and ApoER2. Inversion of cortical layers and absence of cerebellar foliation in these animals precisely mimic the phenotype of mice lacking Reelin or mDab1. These findings suggest that VLDLR and ApoER2 participate in transmitting the extracellular Reelin signal to intracellular signaling processes initiated by mDab1.
Article
Ovarian androgen production is rate limiting for follicular maturation and can induce follicular atresia. Thus, it is important to define the actions of the intraovarian agents, such as apolipoprotein (apo) E, that modulate theca cell androgen production. Theca cell androgen production is stimulated at low concentrations and inhibited at higher concentrations of native apo E. The apo E peptide, acetyl-Y(LRKLRKRLLRDADDL)(2)C or acetyl-Y(141-155)(2)C, has low density lipoprotein (LDL) receptor and LDL receptor-related protein-binding activity, and it mimics the activity of native apo E in the theca-interstitial cell system. To define the role of members of the LDL receptor superfamily in the apo E peptide-mediated responses, we found that receptor-associated protein prevented the stimulation without altering the inhibition of androstenedione production. The apo E peptide (129-162), which has no LDL receptor-binding activity, did not stimulate androstenedione production. The apo E peptide acetyl-Y(141-155)(2)C did not stimulate androstenedione production when cell surface heparan sulfate proteoglycans were degraded with heparinase. The apo E peptide acetyl-Y(141-155)(2)C bound to heparin, a property of LDL receptor ligands, and in this complex the peptide had no effect on androstenedione production. These observations support the conclusion that apo E-mediated stimulation, but not inhibition, of ovarian theca cell androstenedione production was mediated by members of the LDL receptor superfamily.
Article
The high density lipoprotein (HDL) receptor, or scavenger receptor class B type I (SR-BI), is critical for cholesterol transport and a potential target for hypercholesterolemic drugs. Thus, elucidation of the mechanism underlying regulation of the HDL receptor SR-BI gene is essential. It has been previously shown that there is a correlation between depletion in ovarian cholesteryl ester content and increased HDL receptor SR-BI expression in response to hormonal stimulation. We wanted to determine whether the levels of mature sterol response element-binding protein-1a (SREBP-1a), a key protein in the transcriptional regulation of several genes by sterols, are affected under these conditions. Thus, Western blot analysis was carried out. Consistent with the possibility that SREBP-1a may be involved in the regulation of the HDL receptor SR-BI gene, we found that mature SREBP-1a levels increased up to 11-fold in the ovary after treatment with 50 U hCG. This increase in mature SREBP-1a protein levels correlated with a 30% decrease in ovarian cholesterol levels. These changes in both SREBP-1a and cholesterol levels preceded a 2-fold induction of HDL receptor SR-BI protein levels. To determine whether SREBP-1a could directly regulate the expression of the rat HDL receptor SR-BI gene, approximately 2.2 kb of the receptor SR-BI promoter were cloned and sequenced, and deletion analysis and mobility shift assays were performed. The results of these studies demonstrate that the rat HDL receptor SR-BI promoter contains two sterol response elements (pSRE and dSRE) through which SREBP-1a can bind and activate transcription of this gene. These motifs are similar to known SRE motifs reported for sterol-sensitive genes, and the pSRE is located between two Sp1 sites, similar to the SRE-1 motif in the low density lipoprotein receptor. The cysteine protease inhibitor N-acetyl-leucyl-leucyl-norleucinal, which inhibits SREBP degradation, enhanced the effect of SREBP-1a on the regulation of the rat HDL receptor SR-BI gene. It has previously been shown that tropic hormones such as hCG can also influence gene expression by increasing cAMP levels. Consistent with this fact, we have recently shown that steroidogenic factor-1 (SF-1) mediates cAMP activation of the HDL receptor SR-BI gene. Thus, we decided to examine whether SREBP-1a could cooperate with SF-1 to enhance transcription this gene. The results confirm that indeed both SF-1 and SREBP-1a synergize to induce HDL receptor SR-BI gene expression.
Article
Insulin and glucose may be limiting factors for ovarian function in dairy cows genetically selected for high milk yield. The effects of nutrition on the intrafollicular content of insulin and glucose were investigated in Israeli Holstein dairy cattle fed a basic total mixed ration and producing 34-39kg of milk daily. In experiment 1, carried out in 11 oestrus-synchronised cows, little variation in insulin concentration was found in plasma sampled during the luteal phase, but high variation was found in plasma sampled during the follicular phase. Therefore, in order to prevent confounding the effects of diet and of phase in cycle in the following experiments, experimental diets were fed during the luteal phase of synchronised oestrus cycles. In experiment 2, designed as Latin-Square, six cows received sequentially diets containing 17.1 (control) or 19.7% of crude protein, using two sources of supplementary protein, i.e. soyabean meal (SBM) and corn gluten meal (CGM), differing in ruminal degradability and leucine content. When dry matter intake was used as covariant, plasma insulin on day 16 was 29.5 and 26.4% higher in cows fed diets containing SBM and CGM than in the control (P<0.05). In experiment 3, 17 cows were individually fed the basic diet and then switched to isoenergetic diets containing SBM (n=5), CGM (n=6) or corn grain (CG, n=6) given from day 10 to 16 of the synchronised oestrus cycle. On the eve of day 16, and in the morning of day 17, they were administered PGF(2alpha) and the content of 26 largest follicles was aspirated by using the transvaginal ovum pick-up technique. Follicles were sorted into two classes (preovulatory and subordinate) according to oestradiol concentration and the progesterone:oestradiol ratio in follicular fluid (FF). Higher concentrations of insulin (0.282 versus 0.127ng/ml, P<0.0001) and of glucose (0.614 versus 0.386g/l, P<0.002), were found in FF from preovulatory follicles. The insulin concentration in the FF of cows fed the CG diet was 26% higher than in their counterparts fed CGM (P<0.04), SBM being intermediate. Dietary effects did not reach significance in subordinate follicles. The finding that preovulatory follicular status is associated with increased intrafollicular insulin and glucose suggests that insulin is involved in follicular maturation. The nutritional effect on intrafollicular glucose and insulin may have practical implications to optimise feeding in dairy cows during phases of the oestrus cycle.
Article
The present study utilizes in situ molecular hybridization and immunocytochemistry to investigate the follicular localization and expression of four thematically related sterol-metabolizing genes; low-density lipoprotein (LDL) receptor, steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage (CYP11A) enzyme, and cytochrome P450 17alpha-hydroxylase/C(17-20) lyase (CYP17). To this end, semiquantitative analyses were applied to individual nonatretic follicles (N=54) harvested from cycling gilts slaughtered on days 1, 3, 5, and 7 (N=3 per day) following withdrawal of the progesterone agonist, altrenogest. In situ and immunocytochemical signal intensities were assigned numerical values of 0-3 corresponding to the degree of expression of each mRNA and its corresponding protein. LDL receptor mRNA and protein content was undectable in theca and granulosa cells on days 1, 3, and 5, and then increased to low levels in theca cells on day 7. StAR message rose progressively in theca cells with follicular maturation, reaching a maximum on day 5, and then declining slightly on day 7 after the LH surge. In granulosa cells, small amounts of StAR mRNA and protein were detected on days 5 and 7. The amounts of CYP11A mRNA and protein were high in theca cells, and increased at each time point studied. Granulosa cells exhibited minimal CYP11A message on days 3, 5, and 7, while protein became detectable at low levels on day 7 only. Expression of CYP17 was localized exclusively in theca cells with protein and message content increasing unidirectionally to maxima on days 5 (RNA) and 7 (protein), respectively. Follicular fluid concentrations of androstenedione, and progesterone in contralateral ovaries correlated strongly and positively with accumulation of CYP17, and CYP11A proteins. In summary, these analyses demonstrate that preovulatory follicular development proceeds with the coordinate induction of pivotal genes and proteins that mediate the selective uptake, delivery and utilization of sterol substrate in granulosa and theca-cell steroidogenesis.
Article
Follicle dynamics and oocyte viability in Holstein primiparous and multiparous cows and the relationships between fertility and the biochemical and physical properties of oocyte membranes with season were examined. The conception rates of primiparous (n = 70 885) and multiparous (n = 143 490) cows differed, peaking in the winter and decreasing in the summer. The number of follicles 3-8 mm in diameter per ovary was higher in winter (19.6) compared with summer (12.0). However, in winter the percentage of ovaries with fewer than ten follicles per ovary was 16%, in contrast to 50% in summer. After aspiration of follicles, 7.5 oocytes per ovary were found in winter and 5.0 oocytes per ovary in summer. Cleavage to the two- to four-cell stage after chemical activation was greater in winter than in summer; this was enhanced at the morula stage and embryo development to the blastocyst stage was significantly higher in winter than in summer. Determination of the lipid phase transition in oocyte membranes revealed a shift of 6 degrees C between summer and winter. Fatty acid composition of phospholipids from follicular fluid, granulosa cells and oocytes indicated that there was a higher percentage of saturated fatty acids during the summer and that the percentages of mono-unsaturated and polyunsaturated fatty acids were higher in oocytes and granulosa cells during the winter. Oocytes and granulosa cells had similar fatty acid compositions, in contrast to follicular fluid. These results may explain the differences in the ability of oocytes to develop to the blastocyst stage at different seasons. Thus, temperature changes may lead to changes in membrane properties, which, in turn, can influence oocyte function and fertility.
Article
It has been proposed that in the liver, chylomicron remnants (lipoproteins carrying dietary lipid) may be sequestered before being internalized by hepatocytes. To study this, chylomicron remnants labeled with a fluorescent dye were perfused into isolated livers of LDL receptor-deficient (LDLR-deficient) mice (Ldlr(-/-)) and examined by confocal microscopy. In contrast to livers from normal mice, there was clustering of the chylomicron remnants on the cell surface in the space of DISSE: These remnant clusters colocalized with clusters of LDLR-related protein (LRP) and could be eliminated by low concentrations of receptor-associated protein, an inhibitor of LRP. When competed with ligands of heparan sulfate proteoglycans (HSPGs), the remnant clusters still appeared but were fewer in number, although syndecans (membrane HSPGs) colocalized with the remnant clusters. This suggests that the clustering of remnants is not dependent on syndecans but that the syndecans may modify the binding of remnants. These results establish that sequestration is a novel process, the clustering of remnants in the space of DISSE: The clustering involves remnants binding to the LRP, and this may be stabilized by binding with syndecans, eventually followed by endocytosis.
Article
Fat supplementation in the diet influences reproductive performance of lactating ruminants. Changes in the fat supply alter fatty acid composition and this can affect physical properties of cell membranes. This study examined the effect of rumen bypass polyunsaturated fatty acid (PUFA) supplementation on oocyte quality, chilling sensitivity, and lipid phase transition in oocytes of the sheep. Ewes were fed a diet supplemented with calcium soaps of fish oil for 13 weeks. More follicles and oocytes were found in the ovaries of ewes supplemented with PUFA than in control ewes. The number of high-quality oocytes was higher in ewes fed PUFA than in control ewes (74.3 and 57.0%, P < 0.05, respectively). Evaluation of phospholipid fatty acid composition indicated that PUFA were present in small proportions in oocytes, and eicosapentaenoic acid and docosahexaenoic acid were absent. Supplementation with PUFA increased the proportion of long chain unsaturated fatty acid in the plasma and cumulus cells phospholipids by 7.4 and 12.7%, respectively (P < 0.05). Integrity of oocyte membranes following chilling (16°C, 15 min) was improved by PUFA supplementation increasing from 62.5 to 90.0% (P < 0.05). Due to changes in the oocyte's fatty acid profile, physical properties of the membrane were changed and the midpoint temperature of lipid transition reduced by 11°C. These results suggest that supplementation of rumen bypass PUFA to ruminant diets can change fatty acid composition of follicle components and influence parameters such as number and quality of oocytes and their chilling resistance. Mol. Reprod. Dev. 61: 271–278, 2002.
Article
The effects of E2 on the high-density lipoprotein receptor (HDL-R) scavenger receptor class B type I (SR-BI) gene were examined. Four putative estrogen response element half-site motifs (ERE(1/2)) (-2176, -1726, -1622, and -1211, designated ERE(1/2)-1, 2, 3, and 4, respectively) were identified in the HDL-R SR-BI promoter. Transfection studies and mutation analysis demonstrated that E2 significantly increased HDL-R SR-BI promoter activity and that mutating ERE(1/2)-1, 2, and 4 resulted in a loss of E2 responsiveness. Both ER alpha and ER beta formed specific complexes with ERE(1/2)-1, 2, and 4 but did not bind ERE(1/2)-3 in vitro. Interestingly, ERE(1/2)-3 was the motif shown not to be important for E2-activation of the HDL-R SR-BI promoter in the mutational analysis studies. The influence of SREBP-1a (sterol regulatory element binding protein-1a) on E2 regulation of the HDL-R SR-BI gene was also examined. SREBP-1a was able to bind directly to the ERE(1/2) motifs and enhanced ER binding when both ER subtypes were present. ER alpha and beta also bound to a sterol response element motif, but they did not enhance SREBP-1a binding. Cotransfection studies demonstrated that the presence of the three factors, ER alpha, ER beta, and SREBP-1a, enhanced the overall luciferase activity produced from the HDL-R SR-BI promoter construct in the presence of only one of the factors. Interaction of SREBP-1a with both ERs was demonstrated using a mammalian two-hybrid assay. The data confirmed that E2 through the ERs can positively regulate the HDL-R SR-BI through binding and activation of three ERE(1/2) motifs and identified SREBP-1a as a potential coactivator of the E2-ER-dependent effects on the HDL-R SR-BI gene.
Article
Steroid hormones are synthesized using cholesterol as precursor, with a substantial portion supplied by the selective uptake of lipoprotein-derived cholesteryl esters. Adrenals express a high level of neutral cholesteryl ester hydrolase activity, and recently hormone-sensitive lipase (HSL) was shown to be responsible for most adrenal neutral cholesteryl ester hydrolase activity. To determine the functional importance of HSL in adrenal steroidogenesis, adrenal cells were isolated from control and HSL-/- mice, and the in vitro production of corticosterone was quantified. Results show that, even though adrenal cholesteryl ester content was substantially elevated in both male and female HSL-/- mice, basal corticosterone production was reduced approximately 50%. The maximum corticosterone production induced by dibutyryl cAMP, and lipoproteins was approximately 75-85% lower in adrenal cells from HSL-/- mice compared with control. There is no intrinsic defect in the conversion of cholesterol into steroids in HSL-/- mice. Dibutyryl cAMP-stimulated conversion of high-density lipoprotein cholesteryl esters into corticosterone was reduced 97% in HSL-/- mice. An increase in low-density lipoprotein receptor expression appears to be one of the compensatory mechanisms for cholesterol delivery in HSL-/- mice. These findings suggest that HSL is functionally linked to the selective pathway and is critically involved in the intracellular processing and availability of cholesterol for adrenal steroidogenesis.
Article
Lipoproteins in the plasma are the major source of cholesterol obtained by the ovarian theca and granulosa cells for steroidogenesis. In this study, we have identified mRNA expression in bovine theca and granulosa cells of two lipoprotein receptors, low density lipoprotein receptor (LDLr) and very low density lipoprotein receptor (VLDLr) in granulosa cells from small antral follicles through preovulatory follicles and in theca cells from large and medium sized antral follicles. In the corpus luteum (CL) both these receptors were found in the developing and differentiating stages whereas only mRNA for VLDLr was detected in the regression stage. This study also described for the first time, the presence of lipoprotein receptor related protein (LRP8) in granulosa cells from small antral follicles through preovulatory follicles and in theca cells from large and medium sized antral follicles. This may indicate a role of LRP8 in cholesterol delivery to steriodogenic cells. LRP8 was not detected in any of the CL stages. The roles of the LDLr superfamily in lipid transport to ovarian cells and its participation in follicular and CL development and regression is discussed.
Gonadotrophin responsiveness
  • Fm Rhodes
  • Aj Peterson
  • Pd Jolly
  • Argov
  • Al
Rhodes FM, Peterson AJ, Jolly PD. Gonadotrophin responsiveness, 484 ARGOV ET AL.