Fractionation of nucleosomes by salt elution from micrococcal nuclease-digested nuclei.

The solubilization of nucleosomes and histone H1 with increasing concentrations of NaCl has been investigated in rat liver nuclei that had been digested with micrococcal nuclease under conditions that did not substantially alter morphological properties with respect to differences in the extent of chromatin condensation. The pattern of nucleosome and H1 solubilization was gradual and noncoordinate and at least three different types of nucleosome packing interactions could be distinguished from the pattern. A class of nucleosomes containing 13--17% of the DNA and comprising the chromatin structures most available for micrococcal nuclease attack was eluted by 0.2 M NaCl. This fraction was solubilized with an acid-soluble protein of apparent molecular weight of 20,000 daltons and no histone H1. It differed from the nucleosomes released at higher NaCl concentrations in content of nonhistone chromosomal proteins. 40--60% of the nucleosomes were released by 0.3 M NaCl with 30% of the total nuclear histone H1 bound. The remaining nucleosomes and H1 were solublized by 0.4 M or 0.6 M NaCl. H1 was not nucleosome bound at these ionic strengths, and these fractions contained, respectively, 1.5 and 1.8 times more H1 per nucleosome than the population released by 0.3 M NaCl. These fractions contained the DNA least available for micrococcal nuclease attach. The strikingly different macromolecular composition, availability for nuclease digestion, and strength of the packing interactions of the nucleosomes released by 0.2 M NaCl suggest that this population is involved in a special function.

Identification of histone H2b as a heat-shock protein in Drosophila.

Total cell polypeptides synthesized, in cultured Drosophila cells under control (25 degrees C) and heat-shock (37 degrees C) conditions have been compared in two different two-dimensional polyacrylamide gel electrophoresis systems which, together, resolve polypeptides having a wide range of isoelectric points, including the most basic polypeptides of the cell. The electrophoresis of basic proteins showed that the most prominent basic polypeptide synthesized in heat shock comigrated with histone H2b. This heat-shock polypeptide was identified as histone H2b by two criteria: (a) it comigrated with authentic histone H2b in Triton-urea-acetic acid acrylamide gel electrophoresis after solubilization from nuclei with acid; and (b) partial proteolysis peptide maps of the basic heat-shock protein and histone H2b were identical. The synthesis of histone H2b was induced threefold in heat shock, whereas synthesis of the other histones was reduced from two- to tenfold. The noncoordinate synthesis of histones in Drosophila in heat shock provides an interesting system in which to investigate transcriptional and translational controls of histone synthesis as well as assembly of histones into chromatin.

Vimentin: a phosphoprotein under hormonal regulation.

Past studies of norepinephrine-stimulated protein phosphorylation in intact C-6 glioma cells had identified a 58,000 molecular weight, 5.7 isoelectric point protein (58K-5.7) as a cyclic AMP-dependent phosphoprotein and had shown that 58K-5.7 was one of the most abundant proteins of the nuclear fraction. Initial experiments of present studies showed that the 58K-5.7 protein remained with the nuclear ghost, or matrix structure, after removal of chromatin. Based on the size, acidity, abundance, nonsolubilization by nonionic detergent and salt, and solubilization by urea, the hypothesis was advanced that the 58K-5.7 protein was the vimentin-type intermediate filament protein. The hypothesis was tested by two types of immunochemical experiments. Antisera against hamster vimentin reacted selectively with only the 58K-5.7 protein in polyacrylamide gels of urea-solubilized cellular residues (i.e., nonionic detergent and 0.6 M salt-insoluble material) as determined by immunoautoradiography. Antisera against the pure 58K-5.7 protein of C-6 cells bound selectively to a fibrous array of cellular material typical of vimentin filaments as determined by indirect immunofluorescence. It is concluded that the 58K-5.7 protein is vimentin.

Presence of histones in Aspergillus nidulans.

Five major histone proteins have been extracted from chromatin isolated from purified nuclei of the fungus, Aspergillus nidulans. These proteins had chromatographic properties which were similar to reference calf thymus histones and were purified to electrophoretic homegeneity by gel chromatography of Bio-Gel P10, Bio-Gel P60, and Sephadex G-100. Electrophoresis of these proteins in three different systems (urea-starch, urea-acetic acid polyacrylamide, and discontinuous SDS polyacrylamide) showed that the A. nidulans histones H3 and H4 were nearly identical to calf thymus H3 and H4 with respect to net charge and molecular weight criteria, whereas the fungal histones H1, H2a and H2b were similar but not identical to the corresponding calf thymus histones. Amino acid analysis of A. nidulans histones H2a, H2b, and H4 showed them to be closely related to the homologous calf thymus histones. The mobility patterns of A. nidulans ribosomal basic proteins in three different electrophoretic systems were distinctly different from those of the fungal histones.

Identification of the novel player deltaEF1 in estrogen transcriptional cascades.

Although many genes are regulated by estrogen, very few have been shown to directly bind the estrogen receptor complex. Therefore, transcriptional cascades probably occur in which the estrogen receptor directly binds to a target gene that encodes another transcription factor that subsequently regulates additional genes. Through the use of a differential display assay, a transcription factor has been identified that may be involved in estrogen transcriptional cascades. This report demonstrates that transcription factor deltaEF1 is induced eightfold by estrogen in the chick oviduct. Furthermore, the regulation by estrogen occurs at the transcriptional level and is likely to be a direct effect of the estrogen receptor complex, as it does not require concomitant protein synthesis. A putative binding site was identified in the 5'-flanking region of the chick ovalbumin gene identifying it as a possible target gene for regulation by deltaEF1. Characterization of this binding site revealed that deltaEF1 binds to and regulates the chick ovalbumin gene. Thus, a novel regulatory cascade that is triggered by estrogen has been defined.

Noncoordinate histone synthesis in heat-shocked Drosophila cells is regulated at multiple levels.

Transferring Drosophila tissue culture cells from 25 to 37 degrees C (heat shock) causes histone protein synthesis to become noncoordinate. To determine the level at which this is controlled, the synthesis, degradation, and translation of individual histone mRNAs was studied under both heat shock and control conditions. The increased synthesis of histone H2b protein during heat shock appears to be controlled primarily at the level of translation. During heat shock, H2b mRNA is transcribed at about the same level as in the control. However, H2b mRNA is more stable under heat shock than under control conditions and is predominantly found in polysomes. The reduction in synthesis of H2a, H3, and H4 protein during heat shock appears to be controlled at both the transcriptional and translational levels. Although transcription of H2a, H3, and H4 mRNAs is reduced during heat shock, like H2b mRNA, they are more stable. However, unlike H2b mRNA, these mRNAs are not predominantly associated with polysomes during heat shock. Regulation of H1 synthesis during heat shock is completely different from that of the other histones. During heat shock, H1 mRNA is not transcribed, and unlike all of the other Drosophila mRNAs studied to date, its mRNA is not stable in heat-shocked cells. Results from in vitro translation studies support the conclusion that noncoordinate synthesis of the core histone proteins during heat shock is controlled at the level of translation.

Regulation of the chicken ovalbumin gene by estrogen and corticosterone requires a novel DNA element that binds a labile protein, Chirp-1.

Because induction of the chicken ovalbumin (Ov) gene by steroid hormones requires concomitant protein synthesis, efforts have focused on defining the binding site in the Ov gene for a labile transcription factor. Previous gel mobility shift studies identified one such site in the steroid-dependent regulatory element (SDRE) between -900 and -853. To ascertain whether estrogen and glucocorticoid affect the binding of this labile protein, genomic footprinting of the Ov gene was done by treating primary oviduct cell cultures with dimethyl sulfate. Several alterations that include steroid-dependent protection of guanine residues -889 and -885 and hypersensitivity of adenine residues -892 and -865 were observed. Of particular importance, the in vivo footprinting data are corroborated by two functional studies, one with linker-scanning mutations and the other with point mutations. Ten-base-pair linker-scanning mutations between -900 and -878 severely reduced the induction by estrogen and glucocorticoid. Likewise, point mutations of the protected guanine residues profoundly attenuated the response to these steroid hormones. In addition, in vitro binding activity correlated with in vivo functional activity. For example, mutant A4e shows no transcriptional activity in response to steroid hormones, and a corresponding oligomer does not bind protein in vitro. In contrast, mutant A4c is fully active in both contexts. These data support the contention that the ovalbumin gene is regulated by a steroid hormone-induced transcriptional cascade that culminates in the binding of chicken ovalbumin induced regulatory protein or protein complex (Chirp-I) to a DNA element from -891 to -878 in the SDRE.

Dephosphorylation of S6 and expression of the heat shock response in Drosophila melanogaster.

A basic ribosomal phosphoprotein of 30,000 molecular weight was rapidly dephosphorylated in cultured Drosophila melanogaster cells heat shocked at 37 degrees C. The protein was associated with the 40S ribosomal subunit and had an electrophoretic mobility similar to that of purified rat liver protein S6 on basic two-dimensional polyacrylamide gels as well as a similar partial proteolysis peptide map. In logarithmically growing cultures, this D. melanogaster S6 protein appeared to have a single phosphorylated species consisting of 30 to 40% of the total cellular S6. Thus, the nearly complete dephosphorylation of this protein observed in heat shock involves a large fraction of the cellular S6. The significance of this dephosphorylation in the expression of the heat shock response was investigated by examining the phosphorylation status of S6 in recovery from heat shock and in response to chemical inducers of the heat shock response. During recovery from a 30-min heat shock, the recovery of normal protein synthesis was almost complete in 2 to 4 hr, whereas there was no significant rephosphorylation of S6 for 8 h. Two chemical inducers of the heat shock response, canavanine and sodium arsenite, induced the synthesis of heat shock proteins in D. melanogaster cells. Sodium arsenite also caused an inhibition of normal protein synthesis similar to that observed in heat shock. Neither agent, however, caused significant dephosphorylation of S6. These results suggest that the dephosphorylation of S6, although invariably observed in heat-shocked cells, may in some cases be dissociated from both the induction of heat shock protein synthesis and the turnoff of normal protein synthesis which occur in a heat shock response.

Estrogen opposes the apoptotic effects of bone morphogenetic protein 7 on tissue remodeling.

Interactions between estrogen and growth factor signaling pathways at the level of gene expression play important roles in the function of reproductive tissues. For example, estrogen regulates transforming growth factor beta (TGFbeta) in the uterus during the proliferative phase of the mammalian reproductive cycle. Bone morphogenetic protein 7 (BMP-7), a member of the TGFbeta superfamily, is also involved in the development and function of reproductive tissues. However, relatively few studies have addressed the expression of BMP-7 in reproductive tissues, and the role of BMP-7 remains unclear. As part of an ongoing effort to understand how estrogen represses gene expression and to study its interactions with other signaling pathways, chick BMP-7 (cBMP-7) was cloned. cBMP-7 mRNA levels are repressed threefold within 8 h following estrogen treatment in the chick oviduct, an extremely estrogen-responsive reproductive tissue. This regulation occurs at the transcriptional level. Estrogen has a protective role in many tissues, and withdrawal from estrogen often leads to tissue regression; however, the mechanisms mediating regression of the oviduct remain unknown. Terminal transferase-mediated end-labeling and DNA laddering assays demonstrated that regression of the oviduct during estrogen withdrawal involves apoptosis, which is a novel observation. cBMP-7 mRNA levels during estrogen withdrawal increase concurrently with the apoptotic index of the oviduct. Furthermore, addition of purified BMP-7 induces apoptosis in primary oviduct cells. This report demonstrates that the function of BMP-7 in the oviduct involves the induction of apoptosis and that estrogen plays an important role in opposing this function.

Identification of topoisomerase I as the cytotoxic target of the protoberberine alkaloid coralyne.

Protoberberine alkaloids (coralyne and its derivatives), which exhibit antileukemic activity in animal models, have been shown to be potent inducers of topoisomerase (topo) I-DNA cleavable complexes using purified recombinant human DNA topo I. Different from the structurally similar benzophenanthridine alkaloid nitidine (a dual poison of both topos I and II), coralyne and its derivatives have marginal poisoning activity against DNA topo II. Yeast cells expressing human DNA topo I are shown to be specifically sensitive to killing by coralyne derivatives and nitidine, suggesting that cellular DNA topo I is their cytotoxic target. Two human camptothecin-resistant cell lines, CPT-K5 and A2780/CPT-2000, which are known to express highly camptothecin-resistant topo I, are only marginally resistant to coralyne derivatives and nitidine. Purification of human topo I from Escherichia coli cells overexpressing CPT-K5 recombinant topo I has demonstrated similar marginal cross-resistance to nitidine. It seems possible to develop coralyne and nitidine derivatives as new topo I-targeted therapeutics to overcome aspects of camptothecin-related resistance.

Induction of DNA topoisomerase II-mediated DNA cleavage by beta-lapachone and related naphthoquinones.

Recent studies have suggested that 3,4-dihydro-2,2-dimethyl-2H-naphtho[1,2-b]pyran-5,6-dione (beta-lapachone) inhibits DNA topoisomerase I by a mechanism distinct from that of camptothecin. To study the mechanism of action of beta-lapachone, a series of beta-lapachone and related naphthoquinones were synthesized, and their activity against drug-sensitive and -resistant cell lines and purified human DNA topoisomerases as evaluated. Consistent with the previous report, beta-lapachone does not induce topoisomerase I-mediated DNA breaks. However, beta-lapachone and related naphthoquinones, like menadione, induce protein-linked DNA breaks in the presence of purified human DNA topoisomerase IIalpha. Poisoning of topoisomerase IIalpha by beta-lapachone and related naphthoquinones is independent of ATP and involves the formation of reversible cleavable complexes. The structural similarity between menadione, a para-quinone, and beta-lapachone, an ortho-quinone, together with their similar activity in poisoning topoisomerase IIalpha, suggests a common mechanism of action involving chemical reactivity of these quinones. Indeed, both quinones form adducts with mercaptoethanol, and beta-lapachone is 10-fold more reactive. There is an apparent correlation between the rates of the adduct formation with thiols and of the topoisomerase II-poisoning activity of the aforementioned quinones. In preliminary studies, beta-lapachone and related naphthoquinones are found to be cytotoxic against a panel of drug-sensitive and drug-resistant tumor cell lines, including MDR1-overexpressing cell lines, camptothecin-resistant cell lines, and the atypical multidrug-resistant CEM/V-1 cell line.

Association between maternal genital mycoplasma colonization and histologic chorioamnionitis in preterm births.

OBJECTIVE: Genital mycoplasmas (GMs) can be associated with chorioamnionitis and preterm birth, but are viewed as commensal organisms with low virulence. We sought to determine if cervical GM colonization is associated with histologic chorioamnionitis independent of infection with other bacteria. STUDY DESIGN: Retrospective study of patients who delivered preterm, had cervical cultures for GMs, and placental cultures for bacteria other than GM. GM positive patients were compared to GM negative patients. Histologic grading of placentas was compared between GM negative patients with negative placental cultures (Group 1), GM positive patients with negative placental cultures (Group 2), GM negative patients with positive placental cultures (Group 3) and GM positive patients with positive placental cultures (Group 4). RESULTS: GM positive patients were less likely than GM negative patients to have placental cultures positive for other bacteria (39% versus 47%, P = 0.0071). Group 2 had higher rates of membrane inflammation compared to Group 1 (p = 0.0079), and no significant difference in rates of membrane inflammation compared to Groups 3 or 4 (p = 0.36, p = 0.18). GM positivity was independently associated with increased membrane inflammation and decreased inflammation in the chorionic plate. CONCLUSIONS: GM colonization is associated decreased inflammation of the chorionic plate, and increased inflammation of the membranes.

The COUP-adjacent repressor (CAR) element participates in the tissue-specific expression of the ovalbumin gene.

The ovalbumin (Ov) gene is an excellent model for the study of tissue-specific gene regulation as it is only active in the estrogen-stimulated oviduct. Previous studies have demonstrated that the negative regulatory element (NRE) in the Ov gene 5'-flanking region is responsible for silencing the gene in oviduct in the absence of steroids. Linker scanning analysis defined an element within the NRE designated the COUP-adjacent repressor (CAR) element as a repressor of Ov gene expression. However, the role of the CAR element in non-oviduct tissues has not been addressed. Using transient transfection analysis of various Ov 5'-flanking region constructs into the estrogen-responsive chicken hepatocyte cell line LMH/2A, we demonstrate that Ov gene expression is not induced by estrogen and that an active repressor element exists in the NRE. Deletion analysis indicates that the region from -134 to -87, which includes the CAR element, mediates this repression. Mutation of the CAR element relieves repression, leading to high levels of gene expression. These data support a model where the inhibition of Ov gene expression in non-oviduct cells is a combination of the lack of essential positive factors and the presence of an active repressor, which binds to the CAR element.

Regulation of expression of the chicken ovalbumin gene: interactions between steroid hormones and second messenger systems.

The chicken ovalbumin gene is subject to multihormonal regulation. Maximal expression of it requires not only the synergistic effects of estrogen and corticosterone, but also the permissive effects of insulin. In addition to effects on transcription, the stability of its message is greatly enhanced by estrogen. Furthermore, two signal transduction pathways involving protein kinases have been implicated in the regulation of the ovalbumin gene. To better define the role of second messengers on expression of the ovalbumin gene, the effects of the protein kinase-C (PKC) and the cAMP-dependent protein kinase (PKA) pathways on the endogenous levels of ovalbumin mRNA and the transcription of an ovalbumin fusion gene were investigated. Primary cultures of oviduct cells were treated with phorbol 12-myristilate 13-acetate (an activator of PKC) or with forskolin and 3-isobutyl-1-methylxanthine (an activator of PKA) alone, activators plus estrogen and corticosterone, or activators plus both steroids and insulin. The results indicate that phorbol 12-myristilate 13-acetate causes a dramatic destabilization of ovalbumin message, resulting in a reduction in ovalbumin mRNA levels. In contrast, the activators of the PKA system can substitute for insulin and, thereby, increase expression of the ovalbumin gene synergistically with the steroids. The effect of the activators of the PKA system is at the level of transcription. Thus, in chicken oviduct cell cultures, the PKA and PKC signal transduction pathways act in opposing ways to modulate the steroid-induced expression of the ovalbumin gene.

Ten years after: reclassification of steroid-responsive genes.

Although several hundred genes are directly or indirectly regulated by steroid hormones, significant gaps exist in our understanding of the relevant mechanisms, particularly for those genes that do not directly bind intracellular receptors or that exhibit delayed changes in transcription rates upon receptor binding. To assist in defining the mechanism of action of steroid hormones, we are proposing that a standard nomenclature be adopted for classifying steroid-responsive genes, based upon whether the receptors directly bind to the target genes and the kinetics of the response. Three categories are proposed: primary response genes, delayed primary response genes, and secondary response genes.

The MMTV LTR promoter is induced by progesterone and dihydrotestosterone but not by estrogen.

Induction of the mouse mammary tumor virus (MMTV) promoter by steroid hormones was examined in chick oviduct primary cell cultures transfected with MMTV-chloramphenicol acetyl transferase fusion constructs. Our results demonstrate that in this system glucocorticoids, progesterone and dihydrotestosterone are all able to stimulate MMTV transcription; induction by progesterone and dihydrotestosterone is not mediated by glucocorticoid receptors, since the specific glucocorticoid antagonist RU486 did not inhibit the response. In contrast, estrogen does not stimulate MMTV transcription, although estrogen does induce the endogenous ovalbumin gene in the same cells. While progesterone effects are mediated by the same response elements within the MMTV long terminal repeat that were originally characterized for the glucocorticoid receptor, an androgen response element has not yet been defined. Our data indicate that the MMTV long terminal repeat does not contain an estrogen response element.

Repression of the ovalbumin gene involves multiple negative elements including a ubiquitous transcriptional silencer.

Most eukaryotic genes are controlled by a complex array of cis-acting regulatory elements that modulate transcriptional activity. Two major regulatory elements reside in the chicken ovalbumin gene, a steroid-dependent regulatory element (SDRE, -892 to -780) and a negative regulatory element (NRE, -308 to -88). The SDRE is required for responsiveness to estrogen and glucocorticoid. The NRE appears to have the dual role of repressing transcription in the absence of steroids and of cooperating with the SDRE to activate transcription in the presence of steroids. The experiments described herein were designed to investigate the role of the NRE in repressing gene expression. Transfection of OvCAT fusion genes containing deletions in the NRE into primary oviduct cell cultures identified three elements (-308 to -256, -239 to -220, and -174 to -88) that repress transcription. Oligomers corresponding to portions of these elements also independently repress the viral thymidine kinase promoter. Interestingly, the element from -239 to -220 functions mechanistically as a silencer and shares sequence identity with silencers in other genes (TCTCTCCNA). Mobility shift studies indicated that all of the negative elements bind specific protein complexes from oviduct, none of which is appreciably affected by treatment with steroid hormones. However, oviduct-specific proteins bind to the regions from -280 to -252 and from -134 to -88, providing the first identification of potential tissue-specific elements in the ovalbumin gene. These results demonstrate that the region of DNA originally called the NRE is a multifunctional regulatory element that may be involved in several diverse regulatory activities.

Chicken egg white genes: multihormonal regulation in a primary cell culture system.

A primary cell culture from estrogen-withdrawn chicken oviduct has been developed in which the genes for the major egg white proteins, ovalbumin and conalbumin, are induced by steroid hormones. The responsiveness of the isolated cells depends on the combination of enzymes employed to dissociate the oviduct; trypsin and pronase are essential. Administration of estradiol to cultured cells is insufficient to induce ovalbumin mRNA (mRNAov) to levels achieved in vivo. Both insulin and a second steroid (a glucocorticoid, progestin, or androgen) are required for the induction of the ovalbumin gene by estradiol to the extent reached in vivo. Although low levels of a progestin or androgen can synergize with estradiol to obtain an induction of mRNAov, we have demonstrated that a physiological concentration of corticosterone (10(-8) M) potentiates a large response to estradiol without causing an induction when added alone. When estradiol, corticosterone, and insulin are present in the culture medium, mRNAov increases from about 20 to 6500 molecules/cell by 55 h. With the same culture conditions, conalbumin mRNA increases from about 120 to 4300 molecules/cell by 52 h. After about 55 h, the level of egg white mRNAs decreases, and this reduction in mRNAov correlates with a diminished transcription of that gene. The data obtained with the cultured cells demonstrate that the induction of the ovalbumin gene requires the permissive effects of insulin and a second steroid (probably a glucocorticoid in vivo) to facilitate the response to estradiol.

A winged-helix family member is involved in a steroid hormone-triggered regulatory circuit.

A common theme emerging in eukaryotic gene regulation is that maximal gene induction requires several transcription factors acting in concert to regulate the activation of critical genes. Increasingly, nuclear receptors play key roles in orchestrating this regulation, often by integrating additional signaling pathways, through complex regulatory elements known as hormone response units. The ovalbumin gene contains one such unit, known as the steroid-dependent regulatory element. The binding of the chicken ovalbumin induced regulatory protein-I (Chirp-I) to this element occurs only in response to treatment with estrogen and glucocorticoid. Evidence presented herein demonstrates that Chirp-I has many features in common with the winged-helix (W-H) family of transcription factors. The binding sites for Chirp-I and for the W-H proteins have similar sequence recognition requirements. Northern blots establish that members of the W-H family are expressed in oviduct. Most convincing, the Chirp-I complex interacts with two different antibodies specific to W-H family members. The culmination of this work supports the hypothesis that Chirp-I is a member of the W-H family, and it lends credence to the idea that W-H proteins are essential components of some steroid hormone regulatory circuits.

Differential processing of the two subunits of human choriogonadotropin by granulosa cells. II. In vivo studies.

Using a well characterized preparation of hCG, consisting of a mixture of hCG labeled in the alpha-subunit with 125I and hCG labeled in the beta-subunit with 131I (see preceding paper), the hormone-specific preferential retention by granulosa cells in vivo of the radiolabel originally associated with the beta-subunit of hCG has been confirmed and extended. Additional studies have shown that this retention is peculiar to the granulosa cells. Luteal and interstitial/thecal elements of the ovary failed to show preferential accumulation of the label originally associated with the beta-subunit. Measurement of both radioactivities in crude subfractions of the ovarian tissues revealed that granulosa cells retain the excess of beta-subunit label in a plasma membrane/vesicular component. No such preferential retention of label was seen in any of the subfractions obtained from luteal or interstitial/thecal tissues. The radiolabeled components associated with the granulosa cells were shown to be mainly macromolecular by their precipitability with 13% trichloroacetic acid. Luteal tissue degraded the components associated with each label more rapidly than granulosa cells. In contrast, interstitial/thecal tissue degraded very little of the bound labeled components. The differential processing of individual hCG subunits by granulosa cells was shown not to result from different kinetics of binding of serum-borne hormone by two methods. Thus, changes over time in the ability of circulating hormone to bind to LH receptor in vitro were shown not to be a function of the hCG subunit having the label. Moreover, blockade of further radiolabel uptake by injection of a large excess of unlabeled hCG 30 min after radiolabel administration did not alter the rise in the ratio of beta-subunit label to alpha-subunit label normally observed in granulosa cells. The ability of kidney tissue to accumulate and metabolize hCG also varied with the physiological state. Within the limitations of following the radioiodides added to proteins rather than the peptides themselves, these studies demonstrate that differences exist in the metabolism of hCG by the various target cells of the ovary and that changes in processing occur during luteinization.