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Division of Endocrinology, Department of Internal Medicine, University of Virginia Health Sciences Center (H.A.L., J.C.G., R.N.D., J.D.V.), Charlottesville, Virginia 22908; and the Department of Biological Sciences, University of Maine (H.A.L.), Orono, Maine 04469
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In cultured maturing (porcine) granulosa cells, FSH stimulates progesterone biosynthesis; this trophic effect is augmented markedly by the intrafollicular peptide insulin-like growth factor I (IGF-I) (4, 5, 6). Supplying LDL to granulosa cells significantly increases trophic hormone-stimulated progesterone production (4). In addition, IGF-I and FSH both increase the number of LDL surface receptors, the cellular uptake and degradation of LDL, and cholesterol’s utilization in steroidogenesis (4, 7). In single cell studies, FSH is able to increase the population of cells capable of accumulating LDL, possibly due to an increase in LDL receptor synthesis (8). Previous nonquantitative studies suggested that FSH might also stimulate the accumulation of LDL receptor messenger RNA (mRNA) (9).
Studies in nongonadal cells have shown that LDL receptor mRNA regulation occurs at both the transcriptional and posttranscriptional levels and is subject to sterol negative feedback suppression (10, 11, 12, 13, 14). Human luteinized granulosa cells also exhibit sterol-induced feedback suppression of LDL receptor gene expression that is partially overridden by treatment with human CG or a cAMP analog (15).
Here, we investigated whether and how FSH and/or IGF-I individually and jointly regulate the expression of the LDL receptor gene as an extension of their previously reported effects on the receptor protein in ovarian cells. In the current study, we tested whether FSH and IGF-I alone or in combination can increase granulosa cell LDL receptor mRNA accumulation, and whether this hormone-facilitated expression is sensitive to LDL feedback suppression. To evaluate posttranscriptional control, the stability of the LDL receptor mRNA in granulosa cells was assessed in control vs. hormone-treated cells. We then cloned a 1076-bp fragment upstream of the translational start site of the porcine LDL receptor gene for use in transient transfection studies to further evaluate hormone and sterol regulation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture
Granulosa cells were isolated from small and medium-sized (1–5
mm) follicles of immature swine by fine needle aspiration, as
previously described (16). Cells were plated in bicarbonate-buffered
MEM with antibiotics (penicillin, streptomycin,
Amphotericin, and nystatin) supplemented initially with
3% FCS to permit cell anchorage. For mRNA accumulation studies,
approximately 4–5 x 107 viable granulosa cells were
plated in 10-cm culture dishes (Corning, Corning, NY). Granulosa cells
were allowed to attach to culture dishes at 37 C and 5%
CO2 for 16–18 h. After the attachment period, cells were
maintained in serum-free MEM with the indicated treatments or
vehicle.
mRNA stability studies
mRNA half-life studies employed the RNA polymerase II inhibitor,
DRB (17). Attached granulosa cells were treated with vehicle or FSH
plus IGF-I in serum-free MEM for an initial 48 h. After this
initial treatment period, spent medium was replaced with fresh medium
containing vehicle or FSH plus IGF-I with either 100 µM
DRB or an equivalent amount of DMSO (vehicle). DRB (100
µM) or vehicle was then added to cultures every 6 h.
Consistent with other studies (18), our parallel experiments using
[3H]uridine incorporation showed that repeated addition
of DRB was necessary to effectively suppress RNA synthesis. Total RNA
was isolated after 1, 2, 4, 6, 9, 12, 18, and/or 24 h and analyzed
by ribonuclease (RNase) protection assays.
RNA isolation and RNase protection assays
Granulosa cells from two or three culture dishes were pooled for
RNA isolation for each treatment in each experiment. Total RNA was
isolated with Tri-Reagent (Molecular Research Center, Inc., Cincinnati, OH). RNase protection assays were performed
with either 10 µg granulosa cell total RNA or nontarget yeast RNA and
saturating amounts of antisense porcine LDL receptor and human 18S
ribosomal RNA riboprobes as previously described (9, 19).
Porcine LDL receptor promoter cloning
The porcine LDL receptor promoter region was cloned using PCR
techniques. An oligonucleotide primer corresponding to sequence within
the partially cloned porcine LDL receptor complementary DNA (cDNA) for
exon 17 (9) was synthesized (5'-GTGGTCTTCTGGTAGACAGGGTTG-3') and used
as a downstream primer with rapid amplification of cDNA 5'-ends
(5'-RACE; Clontech Marathon cDNA amplification kit, Palo
Alto, CA). The RNA for cDNA synthesis was derived from healthy porcine
corpus luteum. This strategy yielded initial sequence information in
the 5'-end of the LDL receptor mRNA. Several DNA libraries were
constructed by digesting aliquots of genomic porcine DNA with different
restriction enzymes (SspI, ScaI, or
PvuII). The DNA restriction fragments were ligated to
adapters that served as a template for an upstream oligonucleotide
primer (Clontech Marathon cDNA kit). Two downstream
oligonucleotide primers for PCR were designed corresponding to the
sequence of the LDL receptor gene (exon 1) as elucidated from 5'-RACE.
Genomic PCR products were subjected to Southern hybridization with LDL
receptor cDNA sequences, and positive bands were subcloned and
sequenced. LDL receptor promoter sequences were identified by LDL
receptor cDNA sequence overlap and comparison of the sequence homology
with published promoter sequences for the LDL receptor (human, hamster,
rat, mouse) (20, 21, 22). The longest isolated LDL receptor promoter
fragment spans 1076 bp upstream of the translational start site and 11
bases into the coding region of exon 1. Both strands were fully
sequenced at least twice, and the sequence has been submitted to
GenBank (accession no. AF022657). Additional oligonucleotide primers
were also used with two other batches of porcine genomic DNA to verify
that the same fragment(s) could be amplified from nonadapter-ligated
DNA. DNA was sequenced manually and/or by an automated ABI model DNA
sequencer (Biomolecular Research Facility, University of Virginia,
Charlottesville, VA) and an ABI stretch model sequencer (DNA Sequencing
Facility, University of Maine, Orono, ME).
The promoterless luciferase vector contains a gene for firefly luciferase that targets to the cytoplasm (Luc IAV, Promega Corp., Madison, WI) followed by a polyadenylation tract (TK poly A, Stratagene, La Jolla, CA). LDL receptor promoter 5'-deletional fragments were prepared by PCR. All oligonucleotide primers contained engineered restriction enzyme digestion sites to allow for directional cloning upstream of the start codon for the luciferase gene. Promoter DNA fragments included -1076, -753, -455, -255, and -139 bp upstream through +11 bp downstream of the LDL receptor translational start site. Porcine LDL receptor promoter luciferase reporter gene constructs are denoted pLDLR/luciferase. All constructs were verified by DNA sequencing.
Transfection of porcine granulosa cells
Granulosa cells were plated in bicarbonate-buffered MEM plus
antibiotics supplemented with 3% FCS (or LDS as indicated) to permit
cell anchorage. For transfection studies, 8 x 106
viable cells were allowed to attach to culture dishes (3.5 cm) at 37 C
and 5% CO2 for 39–43 h, with a medium change after the
first 24 h. This longer attachment period compared with mRNA
studies was found to increase cell retention on culture dishes during
and after the transfection period. Transfection medium (1 ml/well) was
prepared in serum-free MEM without antibiotics using 2 µg total
plasmid DNA and 12 µl lipofectamine. After a 6-h transfection period,
the medium was replaced with serum-free MEM containing antibiotics and
the indicated hormone treatment or vehicle. At the end of the treatment
period (indicated in figures), cells were rinsed with D-PBS, lysed, and
stored at -70 C until assayed. Light production by lysates was
measured using the Luciferase Assay System (Promega Corp.,
Madison, WI) and a Turner TD-20e luminometer (Turner Designs,
Sunnyvale, CA). Protein concentrations from lysate supernatants were
measured using Bio-Rad protein dye reagent (Bio-Rad Laboratories, Inc., Hercules, CA). As previously reported, porcine granulosa
cells do not express most viral promoters (23); therefore, results were
normalized to protein concentrations. Studies with 5'-deletional
fragments were performed using equimolar concentrations of
pLDLR/luciferase constructs adjusted to a total of 2 µg DNA using the
promoterless luciferase construct.
Progesterone measurements
Progesterone concentrations in culture medium were
measured by RIA using antibody-coated tubes manufactured by ICN Biomedicals, Inc. (Costa Mesa, CA). The RIA has a sensitivity of
0.15 ng/ml and less than 1% cross-reactivity with other relevant
steroid hormones (16).
Statistics
Individual experiments were performed using separate batches of
200–300 ovaries and were performed at least 3 times unless indicated.
For transfection studies, duplicate wells were used to generate a mean
value for each treatment in each experiment. To adjust for
nonhomogeneous variances in the LDL receptor mRNA and transfection
data, raw values were transformed by multiplying by a factor of 10 and
converting each to its natural logarithm value.
Progesterone concentrations were transformed directly to
natural logarithm values. Transformed values were subjected to ANOVA
followed by Tukey’s highest significant difference multiple
comparison test. P < 0.05 was considered significant.
mRNA stability data were also transformed before analysis. Data for
0 h of DRB treatment were normalized to the average control or
average FSH plus IGF-I value to adjust for different starting mRNA
levels and were then transformed to their natural logarithm. Linear
regression was performed on the transformed data from five experiments
(no LDL) or three experiments (plus LDL). Half-life ranges were
estimated by the equation t1/2 =
0.693/(Kd ± SE) (24). All
statistical comparisons were performed using Systat software version
7.0 (Systat, Inc., Evanston, IL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, LDL receptor
mRNA accumulation in swine granulosa cells was significantly
stimulated by 48-h treatment with FSH (25 ng/ml), IGF-I (100 ng/ml), or
their combination (P < 0.01 vs. control, no
LDL). These hormone concentrations yield maximal
progesterone production in culture (9) (Jayes, F.
L., and J. D. Veldhuis, unpublished). Incubation of cells
with LDL (50 µg/ml) substantially suppressed basal and
hormone-stimulated LDL receptor mRNA accumulation relative to the same
treatment without LDL (P < 0.05; reduction range,
54–75%). In the presence of LDL, the combination of FSH and IGF-I was
the only treatment able to significantly increase LDL receptor mRNA
accumulation above basal levels (P < 0.05).
Progesterone concentrations from the cultures in Fig. 1
are shown in Table 1 and confirm hormone
efficacy. In the absence of LDL, corresponding mRNA and
progesterone concentrations followed the same pattern of
increase. In the presence of LDL substrate, progesterone
significantly increased in all hormone treatments, whereas mRNA, which
is sensitive to sterol feedback repression, only increased with the
combination of FSH and IGF-I.
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To examine the contribution of transcription to the observed changes in
the LDL receptor mRNA levels, we cloned and sequenced a region of the
porcine LDL receptor gene promoter. Figure 2 shows the cloned nucleotide sequence
for 1076 bases of the 5'-region of the LDL receptor gene upstream of
the translational start site denoted +1. The transcriptional start
site(s) is estimated from 5'-RACE products to be at or near nucleotide
-114. Two regions of high homology between the porcine sequence and
the human sequence were identified. Included in the more 3'-homologous
region was a TATA-like element and 3 repetitive sequences needed for
regulated expression of the human promoter (25). One notable difference
between the pig and the human was the lack of 1 nucleotide in repeat 2
(the sterol regulatory element) of the porcine sequence at -148 to
-149. Manual and automated sequencing of both DNA strands from
multiple fragments generated by PCR using three different pools of
porcine genomic DNA showed only 15 instead of the 16 nucleotides
observed in the human and rodents (20, 21, 22). The second region of high
homology (bases -565 to -599) contains a putative serum response
element in the human promoter (26). Immediately before repeat 1 in the
porcine sequence is an additional motif that resembles repeats 1 and
3.
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shows the effects of FSH and IGF-I on transcription initiated from the
-1076-bp promoter fragment as measured by luciferase reporter gene
activity normalized for protein and expressed relative to vehicle
control. Figure 3, a and b, shows that FSH stimulates reporter gene
activity in a dose-dependent manner. IGF-I had no significant effect on
promoter activity at any time point sampled. In Fig. 3a, cells were
initially cultured in 3% FCS for 39–43 h, then changed to serum-free
medium for transfection and treatment. In Fig. 3b, granulosa cells were
initiated in 3% LDS before transfection to determine whether
hormone-stimulated increases in promoter activation observed at 4
h were due to lipoprotein withdrawal when cells were changed to
serum-free medium. After the attachment period, these cells were
treated identically to those allowed to attach in the presence of FCS.
The basal reporter gene activity was lower in cells attaching in the
presence of LDS (0.04–0.19 arbitrary light units (alu)/10 µg
protein) compared with cells in FCS (0.15–6.19 alu/10 µg
protein). In LDS and FCS cultures, the relative stimulations by FSH
were similar, as was IGF-I’s augmentation of (5 and 25 ng/ml)
FSH-stimulated promoter activity (163% and 146%, respectively, for
LDS and 163% and 144%, respectively for FCS).
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. The cAMP analog, 8-Br-cAMP (1
mM), alone stimulated the pLDLR1076/luciferase reporter
gene activity by 23 ± 6.5-fold at 4 h (P =
0.001 vs. control; n = 3), similar to maximal
stimulation with (25 ng/ml) FSH plus IGF-I (19 ± 2.0-fold). There
was no consistent effect of IGF-I on this 8-Br-cAMP stimulation at this
time point.
As granulosa cell LDL receptor mRNA is sensitive to sterol feedback
repression (27), as is the human LDL receptor promoter (28),
experiments were performed to evaluate the effects of sterol feedback
on the porcine promoter. Figure 4 shows
the results of granulosa cells transfected with the
pLDLR1076/luciferase construct and treated with vehicle or hormones,
with or without 50 µg/ml LDL. These time-course experiments reveal
the temporal patterns of basal activity, hormone stimulation, and LDL
responsiveness of the porcine promoter. Relative hormonal stimulation
was optimal at 4 h and decreased over time. This decrease in
hormone stimulation was apparently due to an increase in basal (vehicle
control, no LDL) promoter activity over time that eventually surpassed
hormone effects (Fig. 4, inset). Basal activity (raw values,
alu per 10 µg protein) in the control (no LDL) at 8, 16, and 24
h increased, on the average, 4.7-, 58.8-, and 74.1-fold vs.
the 4 h control value, respectively. Although no relative
stimulation was observed by IGF-I at any time point over basal
(including 48-h treatment; data not shown), luciferase activity (alu
per 10 µg protein) in IGF-I-treated cells increased at 8, 16, and
24 h by 2.0-, 17.2-, and 18.0-fold vs. that at 4
h, respectively. Relative FSH-stimulated responses decreased over time,
whereas the absolute luciferase activity (alu per 10 µg protein)
increased at 8, 16, and 24 h by 1.4-, 6.2-, and 5.3-fold
vs. that to the 4-h treatment, respectively. In contrast,
FSH- plus IGF-I-treated cells maintained a fairly constant luciferase
activity at 8, 16, and 24 h, with 1.0-, 1.2-, and 1.2-fold changes
vs. that at 4 h, respectively.
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). All basal and
hormone-stimulated promoter activities were localized to a 116-bp
region between -255 and -139 bp upstream of the translational start
site. LDL-mediated repression of the basal activity of the promoter
assayed at 24 h also resided in the -255 bp fragment (Fig. 5b).
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) suggested that a maximal stimulus by FSH plus
IGF-I could partially overcome the LDL suppression of mRNA accumulation
at 48 h in granulosa cell cultures, we tested whether LDL receptor
mRNA was stabilized by this hormone combination and whether LDL
affected mRNA half-life. First granulosa cells were treated with
vehicle or FSH plus IGF-I for 48 h, as described in Fig. 1, to
hormonally up-regulate the LDL receptor mRNA. After this initial
period, cells were treated with the RNA polymerase II inhibitor, DRB,
at 0 h and again every 6 h to stop transcription. As shown in
the top panel of Fig. 6, the
absolute half-life range with FSH plus IGF-I (16–25 h) was longer than
that of the vehicle control (4.9–6.1 h). The bottom panel
of Fig. 6 shows that the addition of 50 µg/ml LDL did not alter the
half-lives of FSH- plus IGF-I-treated (11–25 h) cells or control cells
(4.9–6.2 h). Dimethylsulfoxide-treated cultures not receiving DRB were
also carried through experiments. Dimethylsulfoxide-treated cultures
collected at 24 h (n = 2) showed that control and FSH- plus
IGF-I-treated cultures averaged 81% and 95% of 0 h values,
respectively, whereas cultures treated with LDL averaged 10% and 68%
of 0 h values, respectively. This observation also supports the
idea that LDL receptor mRNA accumulation in FSH- plus IGF-I-treated
cells is more resistant to sterol repression than that in
vehicle-treated granulosa cells. In two other experiments with IGF-I
alone, IGF-I increased the LDL receptor mRNA half-life by 1.3 to
3.2 h compared with the vehicle control value.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The kinetics of the sensitive luciferase reporter system may not necessarily parallel LDL receptor mRNA accumulation, as the latter depends on transcription and mRNA stability. The half-lives of the luciferase polyadenylated mRNA and protein in eukaryotic cells are relatively short, being approximately 2 and 3 h, respectively (30, 31), whereas the LDL receptor mRNA half-life was at least 5 h. In addition, transfected plasmid DNA may be more structurally accessible to regulatory proteins than endogenous chromosomal DNA facilitating transcription (32). Despite these concerns, important points can be derived from this reporter gene system in these untransformed cells. FSH with or without IGF-I can stimulate transcription of the LDL receptor promoter. Early promoter stimulation is observed even in the presence of exogenous LDL. Hormone activation and sterol responsiveness are localized to regions with known transcriptional regulation.
FSH and IGF-I interact to induce progestin biosynthesis in maturing human, rat, and pig granulosa cells in vitro (4, 33, 34). Although in vivo progesterone is not a major product of nonluteinized granulosa cells, progesterone production in culture is an indicator of cytodifferentiation and luteinization (35). Our cultured granulosa cells represent a model for the early stages of hormonally driven luteinization. In vivo, multiple trophic hormones, including FSH and IGF-I, coordinate changes in granulosa cell development over the course of the entire follicular phase. The terminal luteinization process results from the gonadotropin surge and subsequent follicle rupture. Thus, we speculate that LH, rather than FSH, may play a significant role in maintaining LDL receptor mRNA concentrations in fully luteinized cells.
Unlike other tissues, the ovarian follicle represents a unique
environment with respect to LDL availability. Before the LH surge, the
basement membrane between the thecal and granulosa cell layers prevents
the large LDL molecule (
2.2 million Da) from entering the follicular
fluid (2), and thus LDL is not readily available to granulosa cells for
steroidogenesis. Despite this lack of access to LDL substrate, LDL
binding and uptake occur in freshly isolated granulosa cells from
(mammalian) ovarian follicles before ovulation (36, 37). Recent
in vivo studies in PMSG-stimulated gilts show that LDL
receptor mRNA is increased in preovulatory follicles compared with that
in unstimulated follicles even before a hCG injection that mimics the
LH surge (19). Given these observations, we postulate that LDL receptor
mRNA expression is up-regulated in granulosa cells before ovulation in
an LDL-deficient environment in anticipation of later steroidogenic
needs. The LDL receptor mRNA could subsequently be stabilized further
at high levels by trophic hormones (FSH, IGF-I, and/or, most likely,
LH).
cAMP analogs increase granulosa cell LDL receptor mRNA accumulation in culture (27, 38). Our porcine granulosa cell luciferase-reported transfection studies also reveal potent cAMP analog stimulation of homologous LDL receptor promoter expression. In other transfection studies with human LDL receptor promoter-chloramphenicol acetyltransferase constructs, steroidogenic JEG-3 cells exhibited LDL feedback suppression (28), but not cAMP responsiveness (39). A contrast between the latter clonal cells and our primary culture cells may reflect a cell-specific transcription factor found in ovarian granulosa cells and/or the use of a more sensitive luciferase reporter system (10).
In nongonadal cells, sterol repression of human LDL receptor promoter trans-activation has been localized to a regulatory sequence designated the sterol regulatory element-I (SRE-I) (11, 40). SRE-I acts as a conditional enhancer that promotes LDL receptor gene expression in the absence of sterols, but suppresses transcription in the presence of sterols. Other known regulatory elements in the human promoter include one essential TATA box-like sequence and three 16-bp GC-rich imperfect repeats (41). With respect to the repeats, the SRE-I lies within repeat 2, whereas repeats 1 and 3 can bind the transcription factor Sp1. The SRE-binding protein can associate with the SRE-I and cooperates with Sp1 bound to repeat 3 to induce transcription in nongonadal cells (42). SRE-binding protein is regulated by sterols. Recent evidence suggests that repeat 3 may act independently of the SRE-I to induce transcription as well (43). We identified similar sequence elements within the porcine LDL receptor promoter -255-bp fragment. Indeed, all basal and hormone (FSH plus IGF-I)-stimulated activity in porcine granulosa cells was localized to the region between -255 and -139 containing the three repetitive sequences. Sterol repression of the promoter in granulosa cells was also represented within the same -255-bp fragment of the porcine promoter. Future studies will address which specific DNA elements are involved in FSH/IGF-I responses in this 116-bp region and whether the transcription factor Sp1 mediates trans- activation.
FSH or cAMP analogs can trans-activate the cytochrome P450scc gene promoter (23, 44, 45). In porcine granulosa cells, FSH and IGF-I trans-activation is mediated by a 30-bp GC-rich region of the porcine cytochrome P450scc promoter. IGF-I appears to involve Sp1 binding and a second unknown transcription factor, designated P2 (23, 46). Although our porcine LDL receptor promoter did not respond to IGF-I alone, IGF-I consistently augmented FSH-stimulated effects. Thus, it is plausible that the interaction between these two trophic factors involves cooperation between one or more trans-acting factors regulated by FSH or IGF-I.
Our estimation of the unstimulated LDL receptor mRNA half-life (4.9–6.1 h) in granulosa cells was longer than the approximately 1–3 h reported for nongonadal cells (10, 14, 47). The difference in half-life may be due to our use of the less toxic RNA polymerase II inhibitor, DRB, rather than actinomycin compounds in earlier studies (24), to different culture conditions, or to intrinsic differences between granulosa and nonsteroidogenic cells.
The intrafollicular IGF-I system amplifies gonadotropic hormone action in granulosa cells at multiple end points, including steroid production and follicular survival (48, 49). IGF-I alone or combined with FSH can induce steroidogenic enzyme activity, mRNA accumulation, and promoter trans-activation in cultured granulosa cells (4, 23, 50). We extend these finding to include IGF-I’s modulation of basal and FSH-stimulated LDL receptor gene expression. IGF-I alone stimulates LDL receptor mRNA accumulation and interacts with FSH to promote the accumulation and stability of specific message in the presence of repressive sterol substrate. IGF-I also augments FSH-stimulated LDL receptor promoter activity. Further characterization of DNA elements and cell-specific proteins regulating this FSH/IGF-I interaction will probably provide new insights into how multiple genes are coordinated by trophic factors in preovulatory follicles.
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Acknowledgments |
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Footnotes |
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Current address: Dr. Holly A. LaVoie, Box 5751 Murray Hall, University of Maine, Orono, Maine 04469.
1 This work was supported by the Lalor Foundation, NIH National
Research Service Awards HD-07382 and HD-08019 (to H.A.L.), NIH Grants
HD-16393 and HD-16806 (to J.D.V.), and NIH Grant P30-HD-28934 (to the
Center for Cellular and Molecular Reproduction). 
Received April 10, 1998.
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L. Devoto, L. K. Christenson, J. M. McAllister, A. Makrigiannakis, and J. F. Strauss III Insulin and insulin-like growth factor-I and- II modulate human granulosa-lutein cell steroidogenesis: enhancement of steroidogenic acute regulatory protein (StAR) expression Mol. Hum. Reprod., November 1, 1999; 5(11): 1003 - 1010. [Abstract] [Full Text] [PDF]
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 J. F. Strauss III and A. Dunaif Molecular Mysteries of Polycystic Ovary Syndrome Mol. Endocrinol., June 1, 1999; 13(6): 800 - 805. [Full Text]
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H. A. LaVoie, J. C. Garmey, and J. D. Veldhuis Mechanisms of Insulin-Like Growth Factor I Augmentation of Follicle-Stimulating Hormone-Induced Porcine Steroidogenic Acute Regulatory Protein Gene Promoter Activity in Granulosa Cells Endocrinology, January 1, 1999; 140(1): 146 - 153. [Abstract] [Full Text]
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, FSH plus IGF-I. The mRNA half-life
(t1/2) ranges are shown. Hormone treatment increased the
LDL receptor half-life, whereas LDL did not affect stability.