of highly purified human follitropin and'thank'Ms. Ruth Lum for as- sistance in the preparation ofthis manuscript. This work was supported by National Institutes of ...
Proc. Natl. Acad. Sci. USA Vol. 76, No. 10, pp. 5153-5157, October 1979
Cell Biology
Rapid changes in the synthesis of specific ovarian granulosa cell proteins induced by human choriogonadotropin (hormones/cellular differentiation/two-dimensional electrophoresis)
THOMAS D. LANDEFELD, KENNETH L. CAMPBELL, AND A. REES MIDGLEY, JR. Department of Pathology, Reproductive Endocrinology Program, The University of Michigan, Ann Arbor,
Michigan 48109
Communicated by J. L. Oncley, June 18, 1979
ABSTRACT Total ovarian granulosa cell proteins were labeled in vitro with [35Simethionine and analyzed by two-dimensional polyacrylamide gel electrophoresis. Of the 100-150 distinct proteins resolved by this method, 1-2% demonstrated an observable change in their synthesis during the early phases of luteinization after hormone administration. Two specific roteins first appeared as earl as 30 min after treatment with human choriogonadotropin, while four additional new proteins became apparent at 3 hr. These changes in the synthesis of specific proteins occurred far in advance of the morphological changes associated with luteinization, which occur 24-72 hr later, and thus may play an important role in this process of hormonally induced cell differentiation in the rat ovary.
The luteinization of granulosa cells in Graafian follicles represents a well-defined process of mammalian cellular differentiation. The events associated with this process are initiated by the hormones lutropin or choriogonadotropin and involve significant changes in the morphology and biochemistry of granulosa cells. Prior to treatment with choriogonadotropin, these cells are characterized by a small amount of cytoplasm relative to their prominent nuclei. Over the period of 24-72 hr after administration of hormone, these cells undergo marked hypertrophy, with the appearance of a much more abundant cytoplasm and very large nuclei. These changes are accompanied by cessation of granulosa cell proliferation and concomitant inhibition of DNA synthesis, supporting the hypothesis that luteal cells represent a product of stable differentiation. Transcriptional changes have often been implicated in the process of differentiation. In a few of these studies, the expected changes in specific protein synthesis have been defined by using the two-dimensional electrophoresis system reported by O'Farrell (1). This technique, utilizing isoelectric focusing in one dimension, followed by sodium dodecyl sulfate (NaDodSO4)/polyacrylamide gel electrophoresis in the other, permits the resolution of a much larger number of proteins than has been attainable with either technique alone. As demonstrated by using this approach, the number of proteins that undergo changes in association with differentiation is quite small, usually representing less than 5% of the total proteins (2-6). In this report we have analyzed the synthesis of specific proteins from expressed granulosa cells at various times after the administration of human choriogonadotropin (hCG). (This hormone was used because its action is almost identical to that of lutropin, and it is available in a more purified state.) We report the appearance of changes in the synthesis of two specific proteins as early as 30 min after hormone treatment, and, by 3 hr, significant changes in the synthesis of a group of four additional proteins. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
METHODS AND MATERIALS Animal Model. Twenty-four-day-old Holtzman female rats were primed with estradiol-173 and highly purified human follitropin. The estradiol was administered subcutaneously in four injections (9 a.m. and 9 p.m. for 2 days) with a dose regimen of 0.5 mg, 0.5 mg, 1.0 mg, and 2.0 mg. The human follitropin was administered simultaneously with the estradiol at a dose of 1 jig per injection. Five international units (IU) of hCG was then administered intravenously (T = 0), and the animals were sacrificed at the indicated times by decapitation. The details of this model system were chosen as being optimal on the basis of a series of studies involving morphological examination, hormone binding, and cell viability. Cell Incubation and Expression. The ovaries were removed and incubated according to the method of Campbell and Midgley (7). Briefly, this involved incubating the ovaries in a 6.8 mM ethylene glycol bis(f,-aminoethyl ether)-N,N,N',N'tetraacetic acid (EGTA) solution for 15 min at 37°C under 95% 02/5% C02, followed by a similar incubation in 0.5 M sucrose for 5 min. The granulosa cells were expressed (8) into media (Earle's basal salts/10 mM Hepes/1% bovine serum albumin) and then centrifuged at 100 X g for 5 min. The cells were washed once, resuspended in media, and aliquoted to tubes for incubation. The incubation media contained 300 yCi (1 Ci = 3.7 X 10l° becquerels) of [a5S]methionine (Amersham, specific activity 800-1000 Ci/mmol) in a total volume of 1 ml. Incubation was performed at 37°C under 95% 02/5% CO2 for 1 hr. Processing of Cells. The cells were diluted with media containing unlabeled methionine (15 mg/ml) and centrifuged at 13,000 rpm for 2 min in an Eppendorf centrifuge. The pellet was resuspended in 1.2 ml of medium (containing no bovine serum albumin) and 0.2 ml was removed for quantification of protein and incorporation. The remaining cells were centrifuged and the pellet was resuspended in sonication buffer (1). Sonication was performed on ice with six bursts of 10 sec each at 15-sec intervals. The sample was then lyophilized and prepared in lysis buffer for two-dimensional electrophoresis. The samples removed for quantification were precipitated with 10% trichloroacetic acid, heated at 95°C for 15 min, cooled, and centrifuged at 15,000 rpm for 20 min. The pellets were washed once with 10% trichloroacetic acid and then dissolved in 0.1 M NaOH. This suspension was neutralized with 0.1 M HC1 and aliquots were taken for protein and radioactivity determinations. Two-Dimensional Electrophoresis. Electrophoresis was performed as described by O'Farrell (1), with minor modifications. The isoelectric focusing gel was 12.5 X 0.22 cm with a pH gradient of 4.9-8.5. Protein spots were revealed by autoradiography using Kodak XR-5 x-ray film. Abbreviations: hCG, human choriogonadotropin; NaDodSO4, sodium dodecyl sulfate; pI, isoelectric point.
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Quantification of Proteins on Gel. Proteins were quantified described by Wilson et al. (9). A spot of interest, as revealed in the autoradiogram, was cut from the gel and placed in a scintillation vial containing 10 ml of a cocktail consisting of OCS (Amersham), NCS (Amersham), and 4 M NH40H (44:5:1). The vial was shaken for 48 hr and then the radioactivity was measured in an Isocap 300 liquid scintillation counter (Searle). Pieces of gel similar in size to the eluted protein spots but containing no visible signs of radioactivity, were excised and eluted, and their radioactivities were measured for background deas
terminations.
Despite the low radioactivity counts for many of the spots, when the results were expressed as the percent of radioactivity applied, the reproducibility was quite good among gels and samples. Total radioactivity present in the NaDodSO4 gels was determined by using two methods. First, the gel was cut into 1 X 1 cm pieces and the radioactivity was eluted as described above. When totaled, these recovery values ranged from 58% to 76% of the radioactivity originally applied to the isoelectric focusing gel. It is assumed that the losses are due to proteins not entering the isoelectric focusing or NaDodSO4 gels, to losses in the NaDodSO4 equilibration buffer, and to peptides that ran off either gel. These sources of radioactive losses have been discussed by Peterson and McConkey (2, 10). Recoveries were also examined by incubating the total gel (1 X 1 cm pieces) in 200 ml of 30% H202 at 550C for 48 hr (11). Radioactivities of 0.05- to 0.1-ml aliquots were measured in 10 ml of ACS (Amersham). These recoveries were similar to those determined by the other method (50-70%).
RESULTS As described below, the conditions for labeling were examined in preliminary experiments to optimize incubation times,
of labeled precursor, media, and number of cells. Specific activity, defined as the incorporation of radioactive label per tig of trichloroacetic acid-insoluble protein, was used as a criterion for evaluation. Amino acid incorporation into proteins of nonluteinized and fully luteinized cells was demonstrated to be linear from 15 min to 3 hr. Despite this, short-term incubations (1 hr) were utilized in this study to minimize the possible role of protein degradation and label recycling (12, 13). To increase the specific activities of the proteins, large amounts of [asS]methionine were utilized, because linearity of incorporation had been observed from 10 to 250 ,uCi/ml. The use of this amount of label resulted in specific activities for total cellular proteins ranging from 0.03 to 0.10 AOCi/jig. Incorporation was completely blocked by cycloheximide at 30,gg/ml or unlabeled methionine at 1.5 mg/ml and was insignificant in ovarian fragments or complete medium M-199 (which contains methionine at 30 ,tg/ml). Because various times were examined after hCG treatment in vivo, we also analyzed the specific activities of incorporated proteins at these time intervals (Table 1). Because a reproducible pattern of incorporation was not observed, no evidence was obtained for alterations in amino acid uptake. Protein Pattern. A typical autoradiogram of total labeled granulosa cell proteins is shown in Fig. 1. This general pattern of 100-150 proteins was quite reproducible in a number of experiments. We found that longer exposures (n130 days) resulted in the appearance of additional proteins. However, because none of these new proteins appeared to be responsive to the hormonal stimulation, and because this longer exposure time significantly increased background radioactivity, autoradiograms of shorter exposure are presented. The isoelectric focusing gel contained a pH gradient of 4.9-8.5, and the 10%
amount
Proc. Natl. Acad. Sci. USA 76 (1979)
Biology: Landefeld et al.
Table 1. Incorporation of [35Slmethionine into trichloroacetic acid-insoluble material during 1-hr incubations at various times after hCG treatment in vivo
Time, hr*
Mean specific activity + SEM, cpm/ng protein
29.1 ± 4.4 0 21.1 i 3.0 0.5 25.1 j:3.7 3 28.7 2.8 24 * Hours after hCG treatment in vivo.
polyacrylamide/NaDodSO4 gel resolved proteins of molecular weight ranging from 10,000 to 90,000. Because of the inherent reproducibility of the method it was possible to use the isoelectric points (pl) and molecular weights routinely to identify proteins. For the purposes of identification in this paper, each protein spot has been given a two-part number. The first part represents the approximate pI, while the second part represents the approximate molecular weight X 10-3; e.g., 7.5/14 = pI 7.5, molecular weight 14,000. Several marker proteins were used to standardize the gels, including bovine serum albumin, ovalbumin, carbonic anhydrase, and lysozyme. The values for pI and molecular weight were in good agreement with published values obtained by other means (e.g., gel filtration, ultracentrifugation). Protein Identification. Proteins were classified into three major groups on the basis of qualitative examination (Fig. 1). These groups included (A) those proteins that were present in significant amounts at T = 0 and increased continuously over the time period examined, (B) those that were present in significant amounts at T = 0 but remained at those levels throughout the treatment period, and (C) those not detectable at T = 0 and whose amounts varied during the treatment period in response to the hormone. Two proteins in group A were identified as actin and tubulin by the comigration of purified standards as well as by the pI and molecular weight coordinates. These proteins were chosen because they represent major structural cellular proteins and have been located in a number of two-dimensional systems. As seen in Table 2, these proteins were present in significant amounts at T = 0 and increased over the time period examined, reaching nearly 1% of the total activity of labeled proteins by 24 hr. Group B represents proteins that were present in lesser amounts at T = 0 than actin and tubulin but still were major species, as shown in the box in Fig. 1. In contrast to group A proteins, these proteins remained at a nearly constant amount throughout the treatment time as indicated in Table 2. The similarity of the percent standard errors, relative to the means Table 2. Recovery of group A and group B proteins from two-dimensional gels % total radioactivity* recovered at indicated times after hCG treatment Meant Pro0 hr 0.5 hr 3 hr 24 hr + SEM Group tein 0.512 0.778 0.890 0.560 0.600 0.680 0.077 0.085 0.079 0.083 ± 0.0017 B 0.079 0.083 0.080 0.081 ± 0.0009 0.290 0.298 0.285 0.30 i 0.0078 0.102 0.109 0.100 0.105 ± 0.0021 * Total radioactivity present in these gels was 3.5-5.0 X 105 cpm. t The relative percent standard errors, (SEM/mean) X 100, for group B proteins a-d were 2.1, 1.1, 2.6, and 2.0, respectively.
A
0.480 Actin Tubulin 0.440 0.080 a b 0.080 0.320 c 0.107 d
Proc. Natl. Acad. Sci. USA 76 (1979)
Cell Biology: Landefeld et at.
IEF
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NaDodSO4
t -,
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FIG. 1. Autoradiogram of a two-dimensional gel containing total ovarian granulosa cell proteins labeled in vitro with [35S]methionine. The isoelectric focusing (IEF) gel consisted of a pH gradient from 8.5 (on the left) to 4.9 (on the right). The origin of the NaDodSO4 gel is at the top and proteins were resolved with molecular weights of approximately 90,000 to 10,000. This gel, containing approximately 2 X 106 cpm and 25 ,gg of protein, was exposed to Kodak XR-5 film for 24 hr. Ac, actin; T, tubulin. See text for meaning of other labels.
(2.1, 1.1, 2.6, and 2.0) reflects the reproducibility of the system and also the recoveries of these proteins. Group C proteins are those of primary interest because hormone treatment significantly influenced their synthesis. These proteins cover a wide range of pI values and molecular weights and occur in Fig. 1 as a large area covering almost the entire lower half of the NaDodSO4 gels. These proteins are better illustrated in Fig. 2, which shows a similar area from autoradiograms of the four time points examined (T = 0, 0.5 hr, 3 hr, 24 hr). These autoradiograms have been equalized by maintaining a constant value for the product of cpm applied to the gels and time of autoradiography. In comparing the autoradiograms from T = 0 and T = 0.5 hr, it is evident that at least two proteins were present at T = 0.5 hr that were not seen at T = 0, protein 1 (8.4/23) and protein 2 (6.5/31). Examination Table 3. Recovery of group C proteins from two-dimensional gels Proteins recovered at indicated times after hCG treatment* 24 hr 3 hr 0 hr 0.5 hr Protein 0.19 0.65 ND 0.20 0.43 0.16 ND 0.18 0.30 ND ND 0.93 0.24 ND ND 0.56 0.13 ND ND 0.75 0.25 0.21 ND ND ND, not detectable above background. * Total radioactivity was 3.5-5.0 X 105 cpm. Proteins are expressed relative to protein b of group B. 1 2 3 4 5 6
of the 3-hr time point reveals the appearance of at least four proteins (3-6, arrows) that were not evident at the earlier time points. These proteins ranged in pI from 7.4 to 8.5 and in molecular weight from 23,000 to 40,000. Autoradiograms of labeled proteins from control experiments (i.e., 3 hr after saline injection) were similar to those of T = 0. Quantitative data for these proteins are presented in Table 3. The results have been expressed relative to protein b of group B because this protein remained fairly constant throughout the times examined. Protein 1, first observed at 0.5 hr, demonstrated approximately a 3-fold increase by 3 hr but had fallen to original amounts by 24 hr. Protein 2 had not increased from 0.5 to 3 hr but did increase about 2.5-fold by 24 hr. Of those proteins that first appeared at 3 hr, proteins 3-5 exhibited higher amounts of radioactivity than the proteins that first became apparent at 0.5 hr (proteins 1 and 2). By 24 hr, the radioactivity associated with these proteins fell to 20-40% of the amounts observed at 3 hr. In contrast, protein 6 (8.5/40), initially present in amounts similar to proteins 1 and 2, increased slightly over this same time period. The amount of radioactivity in these proteins ranged from 0.015 to 0.075% of the radioactivity applied to the gel and was 80-250 cpm after subtraction of background (nt30 cpm). The reproducibility of the recovery of group C proteins was demonstrated by an average percent standard error of the mean of 19.5. This number was determined from the six proteins at all the time points at which they appeared and is defined as (sum of the SEMs/mean) divided by the total number of determinations.
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Cell Biology: Landefeld et al.
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Proc. Natl. Acad. Sci. USA 76 (1979)
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