Synthesis and biological evaluation of two agents for imaging estrogen receptor ß by positron emission tomography: challenges in PET imaging of a low abundance target.

INTRODUCTION: Independent measurement of the levels of both the estrogen receptors, ERα and ERß, in breast cancer could improve prediction of benefit from endocrine therapies. While ERα levels can be measured by positron emission tomography (PET) using 16α-[(18)F]fluoroestradiol (FES), no effective agent for imaging ERß by PET has yet been reported. METHODS: We have prepared the fluorine-18 labeled form of 8ß-(2-fluoroethyl)estradiol (8BFEE(2)), an analog of an ERß-selective steroidal estrogen, 8ß-vinylestradiol; efficient incorporation of fluorine-18 was achieved, but required very vigorous conditions. We have examined the biodistribution of this compound, as well as of Br-041, an analog of a known non-steroidal ERß-selective ligand (ERB-041), labeled with bromine-76. Studies were done in immature female rodents, with various pharmacological and endocrine perturbations to assess ERß selectivity of uptake. RESULTS: Little evidence of ERß-mediated uptake was observed with either [(18)F]8BFEE(2) or [(76)Br]Br-041. Attempts to increase the ERß content of target tissues were not effective and failed to improve biodistribution selectivity. CONCLUSIONS: Because on an absolute basis level, ERß levels are low in all target tissues, these studies have highlighted the need to develop improved in vivo models for evaluating ERß-selective radiopharmaceuticals for use in PET imaging. Genetically engineered breast cancer cells that are being developed to express either ERα or ERß in a regulated manner, grown as xenografts in immune-compromised mice, could prove useful for future studies to develop ER subtype-selective radiopharmaceuticals.

Small-animal PET of steroid hormone receptors predicts tumor response to endocrine therapy using a preclinical model of breast cancer.

UNLABELLED: Estrogen receptor-α (ERα) and progesterone receptor (PR) are expressed in most human breast cancers and are important predictive factors for directing therapy. Because of de novo and acquired resistance to endocrine therapy, there remains a need to identify which ERα-positive (ERα(+))/PR-positive (PR(+)) tumors are most likely to respond. The purpose of this study was to use estrogen- and progestin-based radiopharmaceuticals to image ERα and PR in mouse mammary tumors at baseline and after hormonal therapy and to determine whether changes in these imaging biomarkers can serve as an early predictive indicator of therapeutic response. METHODS: Mammary adenocarcinomas that spontaneously develop in aged female mice deficient in signal transducer and activator of transcription-1 (STAT1) were used. Imaging of ERα and PR in primary tumor-bearing mice and mice implanted with mammary cell lines (SSM1, SSM2, and SSM3) derived from primary STAT1-deficient (STAT1(-/-)) tumors was performed. Hormonal treatments consisted of estradiol, an ER agonist; letrozole, an aromatase inhibitor; and fulvestrant, a pure ER antagonist. Small-animal PET/CT was performed using (18)F-fluoroestradiol ((18)F-FES) for ER, (18)F-fluoro furanyl norprogesterone ((18)F-FFNP) for PR, and (18)F-FDG for glucose uptake. Tracer uptake in the tumor was quantified and compared with receptor concentration determined by in vitro assays of resected tumors. RESULTS: Primary STAT1(-/-) mammary tumors and implanted SSM2 and SSM3 tumors showed high (18)F-FES and (18)F-FFNP uptake and were confirmed to be ERα(+)/PR(+). Classic estrogen-induced regulation of the progesterone receptor gene was demonstrated by increased (18)F-FFNP uptake of estradiol-treated SSM3 tumors. Treatment with fulvestrant decreased (18)F-FFNP, (18)F-FES, and (18)F-FDG uptake and inhibited growth of SSM3 tumors but decreased only (18)F-FES uptake in SSM2 tumors, with no effect on growth, despite both tumors being ERα(+)/PR(+). Decreased (18)F-FFNP uptake by SSM3 tumors occurred early after initiation of treatment, before measurable tumor growth inhibition. CONCLUSION: Using small-animal PET, a profile was identified that distinguished fulvestrant-sensitive from fulvestrant-resistant ERα(+)/PR(+) tumors before changes in tumor size. This work demonstrates that imaging baseline tumoral (18)F-FES uptake and initial changes in (18)F-FFNP uptake in a noninvasive manner is a potentially useful strategy to identify responders and nonresponders to endocrine therapy at an early stage.

Diarylpropionitrile (DPN) enantiomers: synthesis and evaluation of estrogen receptor ß-selective ligands.

Two estrogen receptor (ER) subtypes, ERα and ERß, mediate the actions of estrogens in diverse reproductive and nonreproductive target tissues. ER subtype-selective ligands, which bind to and activate these subtypes differentially, have proved to be useful in elucidating which actions of estrogens proceed through ERα vs ERß. Some of these ligands show potential as novel therapeutic agents. Diarylpropionitrile (DPN), an ERß selective ligand that we developed, is a chiral molecule, but it has been studied almost exclusively as the racemic mixture (rac-DPN, 1). Herein we report the development of an efficient enantioselective synthesis of the two isomers, R-DPN (3) and S-DPN (2), and we have compared the in vitro ligand binding affinities, coactivator binding affinities, recruitment potencies, and cellular transcriptional potencies of these isomers. Both enantiomers show a very high affinity and potency preference for ERß over ERα, typically in the range of 80-300-fold. Although the enantioselectivity is only modest (3-4-fold), the R-enantiomer is the higher affinity and more potent isomer. While ERß can be effectively and selectively stimulated by rac-DPN or by either R-DPN or S-DPN, R-DPN might be the preferred member of this isomeric series for biological studies of ERß function.

Exploration of dimensions of estrogen potency: parsing ligand binding and coactivator binding affinities.

The estrogen receptors, ERα and ERß, are ligand-regulated transcription factors that control gene expression programs in target tissues. The molecular events underlying estrogen action involve minimally two steps, hormone binding to the ER ligand-binding domain followed by coactivator recruitment to the ER·ligand complex; this ligand·receptor·coactivator triple complex then alters gene expression. Conceptually, the potency of an estrogen in activating a cellular response should reflect the affinities that characterize both steps involved in the assembly of the active ligand·receptor·coactivator complex. Thus, to better understand the molecular basis of estrogen potency, we developed a completely in vitro system (using radiometric and time-resolved FRET assays) to quantify independently three parameters: (a) the affinity of ligand binding to ER, (b) the affinity of coactivator binding to the ER·ligand complex, and (c) the potency of ligand recruitment of coactivator. We used this system to characterize the binding and potency of 12 estrogens with both ERα and ERß. Some ligands showed good correlations between ligand binding affinity, coactivator binding affinity, and coactivator recruitment potency with both ERs, whereas others showed correlations with only one ER subtype or displayed discordant coactivator recruitment potencies. When ligands with low receptor binding affinity but high coactivator recruitment potencies to ERß were evaluated in cell-based assays, elevation of cellular coactivator levels significantly and selectively improved their potency. Collectively, our results indicate that some low affinity estrogens may elicit greater cellular responses in those target cells that express higher levels of specific coactivators capable of binding to their ER complexes with high affinity.

Quantification of ligand-regulated nuclear receptor corepressor and coactivator binding, key interactions determining ligand potency and efficacy for the thyroid hormone receptor.

The potency and efficacy of ligands for nuclear receptors (NR) result both from the affinity of the ligand for the receptor and from the affinity that various coregulatory proteins have for ligand-receptor complexes; the latter interaction, however, is rarely quantified. To understand the molecular basis for ligand potency and efficacy, we developed dual time-resolved fluorescence resonance energy transfer (tr-FRET) assays and quantified binding of both ligand and coactivator or corepressor to the thyroid hormone receptor (TR). Promoter-bound TR exerts dual transcriptional regulatory functions, recruiting corepressor proteins and repressing transcription in the absence of thyroid hormones (THs) and shedding corepressors in favor of coactivators upon binding agonists, activating transcription. Our tr-FRET assays involve a TRE sequence labeled with terbium (fluorescence donor), TRbeta.RXRalpha heterodimer, and fluorescein-labeled NR interaction domains of coactivator SRC3 or corepressor NCoR (fluorescence acceptors). Through coregulator titrations, we could determine the affinity of SRC3 or NCoR for TRE-bound TR.RXR heterodimers, unliganded or saturated with different THs. Alternatively, through ligand titrations, we could determine the relative potencies of different THs. The order of TR agonist potencies is as follows: GC-1 approximately T 3 approximately TRIAC approximately T 4 >> rT 3 (for both coactivator recruitment and corepressor dissociation); the affinities of SRC3 binding to TR-ligand complexes followed a similar trend. This highlights the fact that the low activity of rT 3 is derived both from its low affinity for TR and from the low affinity of SRC for the TR-rT 3 complex. The TR antagonist NH-3 failed to induce SRC3 recruitment but did effect NCoR dissociation. These assays provide quantitative information about the affinity of two key interactions that are determinants of NR ligand potency and efficacy.

Dual-mode fluorophore-doped nickel nitrilotriacetic acid-modified silica nanoparticles combine histidine-tagged protein purification with site-specific fluorophore labeling.

We present the first example of a fluorophore-doped nickel chelate surface-modified silica nanoparticle that functions in a dual mode, combining histidine-tagged protein purification with site-specific fluorophore labeling. Tetramethylrhodamine (TMR)-doped silica nanoparticles, estimated to contain 700-900 TMRs per ca. 23 nm particle, were surface modified with nitrilotriacetic acid (NTA), producing TMR-SiO2-NTA-Ni2+. Silica-embedded TMR retains very high quantum yield, is resistant to quenching by buffer components, and is modestly quenched and only to a certain depth (ca. 2 nm) by surface-attached Ni2+. When exposed to a bacterial lysate containing estrogen receptor alpha ligand binding domain (ERalpha) as a minor component, these beads showed very high specificity binding, enabling protein purification in one step. The capacity and specificity of these beads for binding a his-tagged protein were characterized by electrophoresis, radiometric counting, and MALDI-TOF MS. ERalpha, bound to TMR-SiO2-NTA-Ni++ beads in a site-specific manner, exhibited good activity for ligand binding and for ligand-induced binding to coactivators in solution FRET experiments and protein microarray fluorometric and FRET assays. This dual-mode type TMR-SiO2-NTA-Ni2+ system represents a powerful combination of one-step histidine-tagged protein purification and site-specific labeling with multiple fluorophore species.

Raloxifene and ICI182,780 increase estrogen receptor-alpha association with a nuclear compartment via overlapping sets of hydrophobic amino acids in activation function 2 helix 12.

The basis for the differential repressive effects of antiestrogens on transactivation by estrogen receptor-alpha (ERalpha) remains incompletely understood. Here, we show that the full antiestrogen ICI182,780 and, to a lesser extent, the selective ER modulator raloxifene (Ral), induce accumulation of exogenous ERalpha in a poorly soluble fraction in transiently transfected HepG2 or stably transfected MDA-MB231 cells and of endogenous receptor in MCF7 cells. ERalpha remained nuclear in HepG2 cells treated with either compound. Replacement of selected hydrophobic residues of ERalpha ligand-binding domain helix 12 (H12) enhanced receptor solubility in the presence of ICI182,780 or Ral. These mutations also increased transcriptional activity with Ral or ICI182,780 on reporter genes or on the endogenous estrogen target gene TFF1 in a manner requiring the integrity of the N-terminal AF-1 domain. The antiestrogen-specific effects of single mutations suggest that they affect receptor function by mechanisms other than a simple decrease in hydrophobicity of H12, possibly due to relief from local steric hindrance between these residues and the antiestrogen side chains. Fluorescence anisotropy experiments indicated an enhanced regional stabilization of mutant ligand-binding domains in the presence of antiestrogens. H12 mutations also prevent the increase in bioluminescence resonance energy transfer between ERalpha monomers induced by Ral or ICI182,780 and increase intranuclear receptor mobility in correlation with transcriptional activity in the presence of these antiestrogens. Our data indicate that ICI182,780 and Ral locally alter the ERalpha ligand binding structure via specific hydrophobic residues of H12 and decrease its transcriptional activity through tighter association with an insoluble nuclear structure.

Phosphorylation of thyroid hormone receptor-associated nuclear receptor corepressor holocomplex by the DNA-dependent protein kinase enhances its histone deacetylase activity.

It is well documented that unliganded thyroid hormone receptor (TR) functions as a transcriptional repressor of specific cellular target genes by acting in concert with a corepressor complex harboring histone deacetylase (HDAC) activity. To fully explore the cofactors that interact with the transcriptionally repressive form of TR, we biochemically isolated a multiprotein complex that assembles on a TR.retinoid X receptor (RXR) heterodimer in HeLa nuclear extracts and identified its polypeptide components by mass spectrometry. A subset of TR.RXR-associated polypeptides included NCoR, SMRT, TBL1, and HDAC3, which represent the core components of a previously described NCoR/SMRT corepressor complex. We also identified several polypeptides that constitute a DNA-dependent protein kinase (DNA-PK) enzyme complex, a regulator of DNA repair, recombination, and transcription. These polypeptides included the catalytic subunit DNA-PKcs, the regulatory subunits Ku70 and Ku86, and the poly(ADP-ribose) polymerase 1. Density gradient fractionation and immunoprecipitation analyses provided evidence for the existence of a high molecular weight TR.RXR.corepressor holocomplex containing both NCoR/SMRT and DNA-PK complexes. Chromatin immunoprecipitation studies confirmed that unliganded TR.RXR recruits both complexes to the triiodothyronine-responsive region of growth hormone gene in vivo. Interestingly, DNA-PKcs, a member of the phosphatidylinositol 3-kinase family, was found to phosphorylate HDAC3 when the purified TR.RXR.corepressor holocomplex was incubated with ATP. This phosphorylation was accompanied by a significant enhancement of the HDAC activity of this complex. Collectively, our results indicated that DNA-PK promotes the establishment of a repressive chromatin at a TR target promoter by enhancing the HDAC activity of the receptor-bound NCoR/SMRT corepressor complex.

Central nervous system inflammation is a hallmark of pathogenesis in mouse models of GM1 and GM2 gangliosidosis.

Mouse models of the GM2 gangliosidoses [Tay-Sachs, late onset Tay-Sachs (LOTS), Sandhoff] and GM1 gangliosidosis have been studied to determine whether there is a common neuro-inflammatory component to these disorders. During the disease course, we have: (i) examined the expression of a number of inflammatory markers in the CNS, including MHC class II, CD68, CD11b (CR3), 7/4, F4/80, nitrotyrosine, CD4 and CD8; (ii) profiled cytokine production [tumour necrosis factor alpha (TNF alpha), transforming growth factor (TGF beta 1) and interleukin 1 beta (IL1 beta)]; and (iii) studied blood-brain barrier (BBB) integrity. The kinetics of apoptosis and the expression of Fas and TNF-R1 were also assessed. In all symptomatic mouse models, a progressive increase in local microglial activation/expansion and infiltration of inflammatory cells was noted. Altered BBB permeability was evident in Sandhoff and GM1 mice, but absent in LOTS mice. Progressive CNS inflammation coincided with the onset of clinical signs in these mouse models. Substrate reduction therapy in the Sandhoff mouse model slowed the rate of accumulation of glycosphingolipids in the CNS, thus delaying the onset of the inflammatory process and disease pathogenesis. These data suggest that inflammation may play an important role in the pathogenesis of the gangliosidoses.

Glycosphingolipid lysosomal storage diseases: therapy and pathogenesis.

Paediatric neurodegenerative diseases are frequently caused by inborn errors in glycosphingolipid (GSL) catabolism and are collectively termed the glycosphingolipidoses. GSL catabolism occurs in the lysosome and a defect in an enzyme involved in GSL degradation leads to the lysosomal storage of its substrate(s). GSLs are abundantly expressed in the central nervous system (CNS) and the disorders frequently have a progressive neurodegenerative course. Our understanding of pathogenesis in these diseases is incomplete and currently few options exist for therapy. In this review we discuss how mouse models of these disorders are providing insights into pathogenesis and also leading to progress in evaluating experimental therapies.

Enhanced survival in Sandhoff disease mice receiving a combination of substrate deprivation therapy and bone marrow transplantation.

Sandhoff disease is a lysosomal storage disorder characterized by G(M2) ganglioside accumulation in the central nervous system (CNS) and periphery. It results from mutations in the HEXB gene, causing a deficiency in beta-hexosaminidase. Bone marrow transplantation (BMT), which augments enzyme levels, and substrate deprivation (using the glycosphingolipid biosynthesis inhibitor N-butyldeoxynojirimycin [NB-DNJ]) independently have been shown to extend life expectancy in a mouse model of Sandhoff disease. The efficacy of combining these 2 therapies was evaluated. Sandhoff disease mice treated with BMT and NB-DNJ survived significantly longer than those treated with BMT or NB-DNJ alone. When the mice were subdivided into 2 groups on the basis of their donor bone marrow-derived CNS enzyme levels, the high enzyme group exhibited a greater degree of synergy (25%) than the group as a whole (13%). Combination therapy may therefore be the strategy of choice for treating the infantile onset disease variants.

A nuclear receptor corepressor modulates transcriptional activity of antagonist-occupied steroid hormone receptor.

Synthetic steroid hormone antagonists are clinically important compounds that regulate physiological responses to steroid hormones. The antagonists bind to the hormone receptors, which are ligand-inducible transcription factors, and modulate their gene-regulatory activities. In most instances, a steroid receptor, such as progesterone receptor (PR) or estrogen receptor (ER), is transcriptionally inactive when complexed with an antagonist and competitively inhibits transactivation of a target steroid-responsive gene by the cognate hormone-occupied receptor. In certain cellular and promoter contexts, however, antagonist-occupied PR or ER acquires paradoxical agonist-like activity. The cellular mechanisms that determine the switch from the negative to the positive mode of transcriptional regulation by an antagonist-bound steroid receptor are unknown. We now provide strong evidence supporting the existence of a cellular inhibitory cofactor that interacts with the B form of human PR (PR-B) complexed with the antiprogestin RU486 to maintain it in a transcriptionally inactive state. In the presence of unliganded thyroid hormone receptor (TR) or ER complexed with the antiestrogen 4-hydroxytamoxifen, which presumably sequesters a limiting pool of the inhibitory cofactor, RU486-PR-B functions as a transcriptional activator of a progesterone-responsive gene even in the absence of hormone agonist. In contrast, hormone-occupied TR or ER fails to induce transactivation by RU486-PR-B. Recent studies revealed that a transcriptional corepressor, NCoR (nuclear receptor corepressor), interacts with unliganded TR but not with liganded TR. Interestingly, coexpression of NCoR efficiently suppresses the partial agonistic activity of antagonist-occupied PR-B but fails to affect transactivation by agonist-bound PR-B. We further demonstrate that RU486-PR-B interacts physically with NCoR in vitro. These novel observations suggest that the inhibitory cofactor that associates with RU486-PR-B and represses its transcriptional activity is either identical or structurally related to the corepressor NCoR. We propose that cellular mechanisms that determine the switch from the antagonistic to the agonistic activity of RU486-PR-B involve removal of the corepressor from the antagonist-bound receptor so that it can effect partial but significant gene activation.

Development of male contraceptive vaccine--a perspective.

This paper reviews the recent advances that have occurred in the area of development of a male contraceptive vaccine. The vaccine candidates considered for review are hormone/hormone receptor-based proteins including luteinizing hormone-releasing hormone (LHRH)/LH, follicle stimulating hormone (FSH), as well as LH and FSH receptor proteins. The review also highlights the advances in our basic understanding of gonadotrophin action which have led to development of these vaccines. Focus is mainly on studies in the non-human primate which may be directly relevant to projected studies in the human. The data indicate that the vaccines are well tolerated by the primate (including the human based on limited data) and do not give rise to any known toxic symptoms or immediate health hazards. The response to the immunogen has been uniform and it may be possible to increase antibody titres as well as prolong the immune response by adding acceptable immune stimulators to the adjuvant cocktail and developing better immunization schedules or immunogen delivery systems. Contraceptive vaccines for the male are a feasible proposition and attention should now be focussed on evaluating carefully the bioefficacy of antibodies raised to recombinant ovine FSHbeta or FSH receptor protein fragments in both human and non-human primates. The advantage of the FSH/FSH receptor over the LHRH/LH-based vaccine lies in the fact that the former does not require an exogenous testosterone supplement to maintain accessory gland function, libido etc. The LHRH/LH-based vaccine results in azoospermia, while the FSH vaccine causes the production of low numbers of poor quality spermatozoa which are incapable of impregnating cycling females.

Analysis of the functional role of steroid receptor coactivator-1 in ligand-induced transactivation by thyroid hormone receptor.

The nuclear hormone receptors belonging to the steroid/thyroid/retinoid receptor superfamily are ligand-inducible transcription factors. These receptors modulate transcription of specific cellular genes, either positively or negatively, by interacting with specific hormone response elements located near the target promoters. Recent studies indicated that the hormone- occupied, DNA-bound receptor acts in concert with a cellular coregulatory factor, termed coactivator, and the basal transcription machinery to mediate gene activation. Consistent with this scenario, a number of nuclear proteins with potential coactivator function have been isolated. In the present study, we demonstrate that steroid receptor coactivator-1 (SRC-1), a recently isolated candidate coactivator, functions as a positive regulator of the thyroid hormone receptor (TR)-mediated transactivation pathway. In transient transfection experiments, coexpression of SRC-1 significantly enhanced ligand-dependent transactivation of a thyroid hormone response element (TRE)-linked promoter by human TRbeta. Our studies revealed that deletion of six amino acids (451-456) in the extreme COOH-terminal region of TRbeta resulted in a receptor that retained the ability to bind T3 but failed to be stimulated by SRC-1. These six amino acids are part of an amphipathic helix that is highly conserved among nuclear hormone receptors and contains the core domain of the ligand-dependent transactivation function, AF-2. In agreement with this observation, in vitro protein binding studies showed that SRC-1 interacted with a ligand binding domain peptide (145-456) of TRbeta in a T3-dependent manner, whereas it failed to interact with a mutant ligand binding domain lacking the amino acids (451-456). We demonstrated that a synthetic peptide containing the COOH-terminal amino acids (437-456) of TRbeta efficiently blocked the ligand-induced binding of SRC-1 to the receptor. These results suggest that the conserved amphipathic helix that constitutes the AF-2 core domain of TRbeta is critical for interaction with SRC-1 and thereby plays a central role in coactivator-mediated transactivation. We further observed that a heterodimer of TRbeta and retinoid X receptor-alpha (RXR alpha), either in solution or bound to a DR+4 TRE, recruited SRC-1 in a T3-dependent manner. The AF-2 of TR was clearly involved in this process because a TR-RXR heterodimer containing a mutant TRbeta (1-450) with impaired AF-2 failed to bind to SRC-1. Surprisingly, the RXR-specific ligand 9-cis-retinoic acid induced binding of SRC-1 to the RXR component of the TRE-bound heterodimer. This novel finding suggests that RXR, as a heterodimeric partner of TR, has the potential to play an active role in transcriptional regulation. Our results raise the interesting possibility that a RXR-specific ligand may modulate T3-mediated signaling by inducing additional interactions between TRE-bound TR-RXR heterodimer and the coactivator.

Ligand-dependent cross-talk between steroid and thyroid hormone receptors. Evidence for common transcriptional coactivator(s).

Steroid and thyroid hormone receptors exhibit striking structural and functional similarity, suggesting that these nuclear receptors may enhance transcription of target genes by similar mechanisms. To address this issue, we studied transcriptional interference between progesterone and thyroid hormone receptors in vivo and in vitro. We observed that transcriptional interference occurred in a ligand-dependent manner between progesterone receptor-B (PR-B) and thyroid hormone receptor (TR) alpha or beta in transient transfection experiments. Ligand-occupied TRalpha or TRbeta, but not the unliganded receptor, strongly suppressed transactivation of a progesterone-responsive reporter gene by endogenous PRs in human breast carcinoma T47D cells. Ligand-dependent inhibitory cross-talk also occurred between transfected PR-B and TRalpha or TRbeta and vice versa in CV1 cells. This phenomenon did not require DNA binding by the "interfering" receptor but required it to be hormone-bound, indicating that a transcriptionally active form of the interfering receptor is essential for the interfering effect. To analyze further the mechanism of the ligand-dependent cross-talk, we reproduced transcriptional interference between PR and TR in a cell-free transcription system. We observed that the addition of triiodothyronine-bound recombinant TRbeta or a ligand binding domain (LBD) peptide(145-456) inhibited specifically transcriptional activation of a progesterone-responsive gene by endogenous PRs in nuclear extracts of T47D cells, while the basal level of transcription from a minimal TATA-promoter or transcription from an adenovirus major-late promoter remained unaffected. These results indicated that a transactivation function within the LBD of the interfering receptor TRbeta was likely to interact with a mediator protein(s), termed coactivator, that is distinct from basal transcription factors and is critical for efficient PR-induced transactivation. This concept was reinforced by biochemical evidence that treatment of T47D extracts with immobilized TRbeta LBD depleted the extract of the coactivator function in a triiodothyronine-dependent manner and markedly impaired progesterone-induced transactivation of progesterone response element-linked genes. Deletion of six amino acids(451-456) in the extreme COOH terminus of TRbeta resulted in a receptor that retained the ability to bind thyroid hormone but failed to inhibit progesterone-dependent transcription. Interestingly, these six amino acids are present in a region that is highly conserved among various nuclear hormone receptors and contains a ligand-dependent transactivation function, AF-2. Based on these results, we propose that a limiting coactivator protein(s) interacts with the AF-2 of PR or TR and mediates transactivation by the ligand-bound receptor. This regulatory molecule(s) may therefore serve as a common functional link between the pathways of hormone-inducible gene activation by various members of the nuclear receptor superfamily.

Changes in testicular function following specific deprivation of LH in the adult male rabbit.

Sexually mature male rabbits actively immunized against highly purified ovine LH (oLH) were used as a model system to study the effects of endogenous LH deprivation (and therefore testosterone) on spermatogenesis as well as pituitary FSH secretion. Immunization against oLH generated antibody titres capable of cross-reacting and neutralizing rabbit LH and this resulted in a significant reduction (P < 0.01) in serum testosterone levels by 2-4 weeks of immunization. A significant increase in circulating FSH concentration (from a basal level of approximately 1 ng to 60-100 ng/ml; P < 0.01) was observed within 4-6 weeks of immunization, perhaps a consequence of the negative feedback effect of the lack of testosterone. The effect of LH deprivation on spermatogenesis assessed by DNA flow cytometry and histological analyses of testicular biopsy tissue revealed that lack of testosterone primarily results in a rapid reduction and complete absence of round (1C) and elongated (HC) spermatids. The immediate effect of LH/testosterone deprivation thus appears to be at the step of meiotic transformation of primary spermatocytes (4C) to 1C. A significant reduction (> 80%; P < 0.01) in the 4C population and a relative accumulation (> 90%; P < 0.01) in spermatogonia (2C) was also observed, suggesting a need for testosterone during the transformation of 2C to 4C. In all but one of the rabbits, both qualitative and quantitative recovery in spermatogenesis occurred during the recovery phase, even at a time when only a marginal increase in serum testosterone (compared with the preimmunization) levels was observed as a result of a rapid decline in the cross-reactive antibody titres. These results clearly show that LH/testosterone deprivation in addition to primarily affecting the meiotic step also regulates the conversion of 2C to 4C during spermatogenesis.