Down-regulation of mt1 melatonin receptors in rat ovary following estrogen exposure.

In this study, we have demonstrated that 2-[125I]-iodomelatonin binds specifically to rat ovarian granulosa cell (GC) membranes with high affinity (KD=83 pM; Bmax=3.28 fmol/mg protein). Using immunoblot analysis and an anti-mt1 melatonin receptor antibody, we have also detected mt1 melatonin receptors in rat ovary. Because melatonin has been reported to alter the steroidogenic responses of ovarian tissues to gonadotropins, a physiological role for intra-ovarian melatonin may exist. Thus, in order to investigate a possible intra-ovarian role for melatonin, we have used both an in vivo and in vitro model of follicular development. Treatment of immature (day 21) female rats with estradiol (E; 0.2 mg/d x 3 d; subcutaneous) was used to induce follicular growth. Membranes from both untreated (U) and E-treated animals' ovaries contained high-affinity 2-[125I]-iodomelatonin (I-MEL) binding sites (Kd=83 and 23 pM, respectively). Estradiol treatment in vivo caused a significant decrease (P<0.05) in binding of 2-[125I]-iodomelatonin to ovarian membranes with untreated animals' ovaries having a Bmax=3.28 fmol/mg protein vs. estradiol-treated animals' ovaries having a Bmax=0.92 fmol/mg protein. In addition, following Estradiol treatment, mt1 melatonin receptors in rat ovary were down-regulated (approximately 95%) using immunoblot analysis. Granulosa cells isolated from E-treated rats were further matured in vitro with testosterone (T) and the pituitary gonadotropin follicle-stimulating hormone (FSH). Granulosa cells were cultured with either T (10 ng/ml) or FSH (5.71 ng ovine FSH-20/ml) alone, or both FSH and T for 48 h. There was no statistically significant specific binding of 2-[125I]-iodomelatonin to GC membranes cultured with T or FSH alone. However, following a 48-h exposure to FSH and T in vitro specific 2-[125I]-iodomelatonin binding occurred with total 2-[125I]-iodomelatonin binding =3.15 [corrected] fmol/mg protein. Therefore, the existence of hormonally-regulated expression of high-affinity melatonin binding sites suggests that melatonin may have an important intra-ovarian physiological role.

Steroid sulfatase activity in the rat ovary, cultured granulosa cells, and a granulosa cell line.

Direct production of gonadal steroids from sulfated adrenal androgens may be an important alternative or complementary pathway for ovarian steroidogenesis. The conversion of sulfated adrenal androgens, present in serum at micromolar concentrations in adult women, into unconjugated androgens or estrogens requires steroid sulfatase (STS) activity. STS activity has not been characterized in the rat ovary. Substantial STS activity was present in homogenates of rat ovaries, primary cultures of rat granulosa cells, and a granulosa cell line, as determined by conversion of radiolabeled estrone sulfate (E1S) to unconjugated estrone. The potent inhibitor estrone sulfamate eliminated the STS activity. Using E1S as a substrate with microsomes prepared from a granulosa cell line, the K(m) of STS activity was approximately 72 microM, a value in agreement with previously published data for rat STS. Therefore, ovarian cells possess STS and can remove the sulfate from adrenal androgens such as dehydroepiandrosterone sulfate (DHEA-S). Using DHEA-S as a steroidogenic substrate represents an alternative model for the production of ovarian steroids versus the "two cell, two gonadotropin" model of ovarian estrogen synthesis, whereby thecal cells produce androgens from substrate cholesterol and granulosa cells convert the androgens into estrogens. The relative contribution of STS activity to ovarian steroidogenesis remains unclear but may have important physiological and pathophysiological implications.

Expression and function of estrogen receptor subtypes in granulosa cells: regulation by estradiol and forskolin.

The expression and function of estrogen receptor ERalpha/beta subtypes and ERbeta variants in granulosa cells have been determined using several integrated approaches:, Western blotting, indirect immunofluorescence, RT-PCR, and transient transfection assays. Each of these approaches has provided specific details concerning the dynamics of ER expression, ER functional activity, and estradiol (E) regulation of target genes in granulosa cells. Specifically, the studies presented herein document that messenger RNAs (mRNAs) encoding ERbeta and its splice variants, as well as mRNA encoding ERalpha, are expressed in granulosa cells of immature rats before and during culture in serum-free medium. The results also provide the first documentation that functional (DNA binding and transcriptionally active) ER is present in cultured granulosa cells and that its ability to bind consensus estrogen response element (ERE) oligonucleotide and to transactivate an ERE promoter-reporter construct is associated with the level (type?) of receptor protein as well as the stage of granulosa cell differentiation. Using a labeled ERE consensus oligonucleotide and antibodies specific for ERbeta and ERalpha, we show that ERbeta but not ERalpha was detected (supershifted in electrophoretic mobility shift assays) in extracts of granulosa cells cultured overnight (0 h) in defined medium alone. When the cells were cultured with FSH and testosterone (T) to stimulate their differentiation, ERbeta binding activity, as well as immunoreactive ERbeta as determined by Western blot analyses, decreased progressively from 24 to 48 h and was undetectable by 72 h. ERbeta mRNA was low, and ERbeta binding activity was not observed in luteinized granulosa cells. ERalpha DNA binding activity was not observed in any of the granulosa cell cultures, although low levels of immunoreactive ERalpha were detected by Western blot analyses. Immunofluorescent analyses documented that ERbeta, as well as ERalpha, were localized to granulosa cell nuclei and that the intensity of nuclear staining was related to agonist stimulation and differentiation: forskolin increased, whereas E decreased immunostaining for ERbeta and ERalpha at 48 h. When an ERE-E1b-luciferase vector was transfected into granulosa cells of unprimed rats, basal luciferase activity was low but increased by forskolin (3-4x) and by E (2x), responses to both agonists being blocked by the ER antagonist, ICI. When the same vector was transfected into differentiated granulosa cells (cultured for 48 h with FSH/T), forskolin alone increased activity. Collectively, these results show that ERbeta protein is preferentially expressed in immature granulosa cells, is functionally active (binds DNA), can transactivate (either as a homodimer or heterodimer with ERalpha) ERE-containing promoter constructs, and might be associated with increased expression of the endogenous gene encoding c-Jun.

Hormone induction of progesterone receptor (PR) messenger ribonucleic acid and activation of PR promoter regions in ovarian granulosa cells: evidence for a role of cyclic adenosine 3',5'-monophosphate but not estradiol.

Expression of progesterone receptor (PR) mRNA in granulosa cells of ovarian preovulatory follicles is induced by LH (1, 2) and is essential for ovulation (3). Although 17beta-estradiol (E) can induce PR mRNA and activate PR promoter-reporter constructs in other cell types, the effects of E in granulosa cells appear to be indirect. We show herein that E alone does not induce the expression of PR mRNA in preovulatory granulosa cells. Rather, induction of PR mRNA depends on the differentiation of granulosa cells in response to E and a physiological amount of FSH followed by exposure to agonists (elevated levels of LH, FSH, and forskolin) that markedly increase cAMP. Induction of PR mRNA by forskolin is blocked by the A-kinase inhibitor H89 and cycloheximide but not by the E antagonist, ICI 164,384. These results indicate that phosphorylation and synthesis of some regulatory factor(s) other than or in addition to the estrogen receptor (ER) are essential for transactivation of the PR gene. When distal and proximal PR promoter-reporter constructs that are responsive to E in other cell types were transiently transfected into differentiated granulosa cells, forskolin, but not E, induced activity. Likewise, when a vector containing the consensus vitellogenin B1 gene estrogen response element (ERE) was transfected into differentiated granulosa cells, forskolin, but not E, induced activity. Using electrophoretic mobility shift assays, the consensus ERE was shown to bind ERbeta, the predominant subtype present in rat granulosa cells, and ERalpha, the predominant subtype present in luteal cells, whereas the putative ERE-like region (ERE3) of the proximal PR promoter did not bind either ER subtype. Although the identity of the specific factors binding to the ERE3 site remain to be determined, mutation of this region abolished forskolin-induced activity of ERE3-PR-CAT constructs. The GC-rich region of the distal PR promoter bound Sp1 and Sp3 but not C/EBPalpha/beta, indicating that factors binding to ERE3 interact synergistically with Sp1/Sp3 to confer increased responsiveness of the distal promoter to forskolin. Taken together, these results indicate that activation of the A-kinase pathway leads to the phosphorylation of some transcription factor(s) other than or in addition to ER that is (are) critical for the transactivation of the PR gene and that this mechanism is selectively activated in differentiated granulosa cells possessing a preovulatory phenotype.

Production and characterization of WEG-1, an epidermal growth factor/transforming growth factor-alpha-responsive mouse uterine epithelial cell line.

Uterine epithelial cells (UEC) isolated from 6-week-old CF-1 mice were immortalized using retroviral-mediated transfection of SV40 large T-antigen. One line, WEG-1, retained epithelial morphology and reacted with antibodies to cytokeratins 18, 19, laminin, fibronectin, and beta-catenin. In addition, WEG-1 cells displayed strong nuclear immunoreactivity to SV40 large T-antigen, confirming integration of the retrovirus vector and expression of this gene. WEG-1 cells were negative for nonepithelial markers such as desmin and factor 8. WEG-1 cells did not proliferate in serum-free medium; however, addition of 0.5% FBS supported proliferation to the same extent as 10% FBS. Addition of 50 ng/ml epidermal growth factor to medium containing 0.5% charcoal-stripped FBS restored proliferation comparable with 0.5% whole FBS. Epidermal growth factor or transforming growth factor-alpha (50 ng/ml), but not transforming growth factor-beta, leukemia-inhibiting factor, or fibroblast growth factor, induced the secretion of three proteins (M(r) approximately to 158K, 148K, and 36K). Comparison of protein secretions of WEG-1 cells and UEC showed shared as well as distinct bands. Like UEC, WEG-1 cells secreted PGF2a and PGE2 and expressed PG GH synthase-2. Unlike UEC, WEG-1 cells showed no apical/basal preference for either uptake of methionine or secretion of proteins. The absence of immunoreactive E-cadherin or zona occludens-1 was consistent with the absence of cell polarity in WEG-1 cells. Primary UEC, which polarize in vitro, do not support blastocyst attachment. WEG-1 cells, although not polarized in vitro, also exhibited delayed blastocyst attachment compared with nonuterine cell lines, suggesting that WEG-1 cells partially retained some aspects of UEC function relevant to embryo attachment. WEG-1 cells expressed messenger RNA for Muc-1, an UEC mucin suggested to have antiadhesive properties. Furthermore, WEG-1 cells did not display high affinity heparin binding sites, an activity associated with embryo attachment. WEG-1 cells may provide a model for studying various aspects of UEC function and murine embryo attachment.

Effects of hormones and protein kinase inhibitors on expression of steroidogenic enzyme promoters in electroporated primary rat granulosa cells.

Previous studies have shown that inhibitors of protein tyrosine kinases, tyrphostins, can markedly attenuate the steady-state levels of mRNAs of hormone-induced genes expressed in ovarian cells. To further elucidate the mechanism of tyrphostin action, rat granulosa cells were electroporated with chimeric expression vectors containing the promoters of two key steroidogenic genes, cholesterol side chain cleavage cytochrome P450 (CYP11A; P450scc) and aromatase cytochrome P450 (CYP19; P450arom), ligated to the CAT reporter gene. The electroporation method of transfection documents that the respective promoter-reporter constructs, -379sccCAT and -534aromCAT, can confer greater than 10-fold FSH/cAMP responsiveness to the reporter genes expressed in naive granulosa cells. Furthermore, the electroporation approach allows transfection of DNA into small numbers of cells and facilitates the assay of expression in cells isolated from follicles at advanced stages of differentiation. In naive granulosa cells, the functional activities of -379sccCAT, -534aromCAT, and -169 alpha CGCAT were abolished by the A-kinase specific inhibitor, H89, supporting the notion that activation of protein kinase A is obligatory for transcriptional activation of the promoter regions within these genes. Similar inhibitory effects were also observed for tyrphostin AG18, thus implicating a tyrosine kinase in the regulation of the steroidogenic genes. As a result of eCG/hCG treatments, a gradual loss of transfection efficiency accompanied by decreasing forskolin induction of CAT expression was observed in the differentiating granulosa-lutein cells. Although the reason(s) for the apparent loss in the ability of hormones to regulate chimeric gene expression remains to be determined, cell and promoter refractoriness to hormone treatment appears to reflect a fundamental change in the mechanism of promoter activation in the differentiated cells compared to the naive granulosa cells.

Analysis of cytochrome P-450 side-chain cleavage gene promoter activation during trophoblast cell differentiation.

Trophoblast giant cell differentiation is accompanied by transcriptional activation of the cytochrome P-450 side-chain cleavage (P450scc) gene. The Rcho-1 trophoblast cell line has the capacity to differentiate along the trophoblast giant cell lineage and has been used to study trophoblast-specific P450scc gene expression. In this report, P450scc gene promoter activities in trophoblast-specific P450scc gene expression. In this report, P450scc gene promoter activities in trophoblast cells have been mapped and the involvement of known modulators of steroid hydroxylase gene expression, the cyclic AMP/protein kinase A pathway and steroidogenic factor-1 (SF-1), evaluated. Comparisons were made with Y-1 adrenal and R2C Leydig cells. The cumulative results from transient and stable transfection experiments implicate the region between -428 and -511 bp of 5'-flanking DNA in the developmental activation of the P450scc promoter during trophoblast giant cell differentiation. Differences in basal activities of the P450scc promoter constructs were also observed in Y-1 adrenal and R2C Leydig cells; however, the magnitude of the differences was modest. Activators of the protein kinase A pathway stimulated P450scc promoter activity in Y-1 cells, whereas similar treatment of Rcho-1 trophoblast cells did not stimulate but actually inhibited P450scc promoter activity. The inhibitory activity was localized between -639 and -894 bp of the P450scc promoter. SF-1 mRNA and protein were detected in adrenal and gonadal cells but not in rat placenta or Rcho-1 trophoblast cells by Northern and Western blotting, respectively. Thus, P450scc gene activation during trophoblast cell differentiation involves an 83-bp region of its 5'-flanking DNA between -428 and -511 but does not appear to involve cyclic AMP-activated pathways or SF-1. In conclusion, the mechanism of P450scc gene activation during trophoblast cell differentiation appears different from the regulation of P450scc gene activation in other steroidogenic tissues.

Ovarian cell differentiation: a cascade of multiple hormones, cellular signals, and regulated genes.

During the development of preovulatory follicles, tonic levels of FSH (and steroid) induce expression of aromatase, the LH receptor, and RII beta in a coordinate manner. Despite the similar temporal increase in steady-state levels of mRNA encoding these proteins, the cis-acting DNA elements and trans-acting factors regulating each gene are distinct (Richards, 1993). Whereas the aromatase gene has a TATA motif and a single transcriptional initiation site (Fitzpatrick and Richards, 1993), both the LH receptor (Wang et al., 1992; Tsai-Morris et al., 1993) and RII beta (Kurten et al., 1992; Luo et al., 1992) genes have promoters that are GC rich, lack TATA motifs, and initiate transcription at multiple sites. The aromatase promoter appears to be regulated, in part, by SF-1, a CRE-like region, and possibly another or overlapping region binding an Ad3BP-like factor. The RII beta promoter has a region that binds several nuclear proteins, whose identity is not yet known. Likewise, the LH receptor promoter elements have yet to be clearly defined (Figures 2, 4, and 25; Kurten et al., 1992). FSH can also induce the expression of at least three immediate-early genes that encode novel kinases or kinase-like proteins (Figure 25). One of these is called serum-inducible kinase (snk) (Simmons et al., 1992), another is serum and glucocorticoid regulated kinase (sgk) (Webster et al., 1993), and a third is called pole kinase (Clay et al., 1993). Steady-state levels of snk and sgk mRNA are induced rapidly (within a few hours) by FSH in granulosa cells prior to the appearance of transcripts for aromatase, LH receptor, and RII beta (T. Alliston and J. S. Richards, in preparation). The functional role of these kinases in the initial response of granulosa cells to tonic (not surge) levels of FSH remains to be elucidated. The cellular signaling pathways mediating the effects of the LH surge appear equally or more complex (Fig. 25). Based on data presented herein, as well as on analyses of the cloned and expressed LH receptor (Guderman et al., 1992), it is clear that low concentrations of LH stimulate adenylyl cyclase, cAMP production, and activation of protein kinase A. Higher (surge) concentrations of LH also increase IP3 and activation of protein kinase C. GnRH has been used in several studies to examine the ability of the protein kinase C pathway to mimic effects of high LH.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in sodium and D-glucose transport between hamster and rat lungs.

The purpose of this study was to characterize phloridzin- and amiloride-sensitive transport across blood-gas barrier of hamster and rat lungs. Air spaces of isolated perfused lungs were instilled with a solution containing 22Na or L-[3H]glucose, D-[14C]glucose, and fluorescein isothiocyanate-labeled dextran. Apparent permeability-surface area products (PS) were calculated. Phloridzin (Na(+)-dependent D-glucose transport inhibitor) had no effect on D-glucose or sodium transport out of air spaces in hamster lungs. In contrast, in rat lungs, phloridzin decreased PS for D-glucose by 89% and that for Na by 28%. Trapping of 14CO2 in vascular samples was measured to estimate metabolism. Unlabeled air space D-glucose increased appearance of perfused D-[14C]glucose in air spaces of rat lungs. We conclude that Na(+)-dependent D-glucose transport is important for D-glucose uptake in rat lungs but not in hamster lungs. In hamster lungs, amiloride (Na+ transport inhibitor) also decreased PS for sodium, but drugs known to stimulate sodium transport in rat lungs had no effect. Thus, species differences in active transport processes exist in the distal air spaces of mammalian lungs.

Steroidogenic factor-1 binding and transcriptional activity of the cholesterol side-chain cleavage promoter in rat granulosa cells.

The cholesterol side-chain cleavage cytochrome P450 (CYP11A; P450scc) gene is expressed in rat ovarian follicles in response to gonadotropins (FSH/LH) and cAMP. To identify functional regions within the rat P450scc promoter, 894 basepairs (bp) of 5'-flanking sequence and 5'-deletions (at -379, -101, -73, and -38 bp) were linked to the human GH reporter gene and transfected into cultured rat granulosa cells. cAMP inducibility of the rat promoter was localized to a region (between -73/-38 bp) that contains one of two AGGT/CC/TA motifs, designated SCC1 (-51/-43 bp) and SCC2 (-79/-71 bp), within the rat promoter. One of the nuclear proteins in granulosa cells that binds to SCC1 was identified as the orphan receptor, steroidogenic factor-1 (SF-1). In contrast, multiple protein-DNA complexes formed with SCC2, only one of which was clearly identified as SF-1. Nuclear extract binding was sequence specific; SCC1 bound SF-1 more strongly than did SCC2. Thus, the two AGGT/CC/TA motifs of the rat promoter appear to differ structurally and functionally. Furthermore, because the expression of SF-1 mRNA precedes hormonal/cAMP induction of P450scc mRNA and is not regulated in vitro by cAMP, the functional role of SF-1 in transcriptional regulation of the P450scc gene, including its induction by cAMP, is not entirely clear and is probably dependent on other factors and/or the modification (phosphorylation?) of SF-1.

Effects of microgravity or simulated launch on testicular function in rats.

Testes from flight rats on COSMOS 2044 and simulated-launch, vivarium, or caudal-elevation control rats (5/group) were analyzed by subjective and quantitative methods. On the basis of observations of fixed tissue, it was evident that some rats had testicular abnormalities unassociated with treatment and probably existing when they were assigned randomly to the four treatment groups. Considering rats without preexisting abnormalities, diameter of seminiferous tubules and numbers of germ cells per tubule cross section were lower (P less than 0.05) in flight than in simulated-launch or vivarium rats. However, ratios of germ cells to each other or to Sertoli cells and number of homogenization-resistant spermatids did not differ from values for simulated-launch or vivarium controls. Expression of testis-specific gene products was not greatly altered by flight. Furthermore, there was no evidence for production of stress-inducible transcripts of the hsp70 or hsp90 genes. Concentration of receptors for rat luteinizing hormone in testicular tissue and surface density of smooth endoplasmic reticulum in Leydig cells were similar in flight and simulated-launch rats. However, concentrations of testosterone in testicular tissue or peripheral blood plasma were reduced (P less than 0.05) in flight rats to less than 20% of values for simulated-launch or vivarium controls. Thus spermatogenesis was essentially normal in flight rats, but production of testosterone was severely depressed. Exposure to microgravity for greater than 2 wk might result in additional changes. Sequelae of reduced androgen production associated with microgravity on turnover of muscle and bone should be considered.

Regulation of testosterone production in the adjuvant-induced arthritic rat.

Serum testosterone concentrations are reduced in men with rheumatoid arthritis and in rats with adjuvant-induced arthritis, a common model for rheumatoid arthritis. To understand the mechanism responsible for this reduction, testosterone production by testicular cells and Percoll-purified Leydig cells from nonarthritic and arthritic rats was studied. Leydig cells in crude interstitial cell preparations from arthritic rats secreted significantly less testosterone in response to human chorionic gonadotropin (hCG) stimulation than cells from nonarthritic animals. In contrast, no differences in hCG and dibutyryl cyclic adenosine monophosphate-stimulated testosterone production by Percoll-enriched Leydig cells from arthritic and nonarthritic animals were observed. To determine whether a secretory product from testicular macrophages was important to this reduction, macrophages from arthritic and nonarthritic animals were cultured. The conditioned media from these cultures were added to cultures of interstitial cells from nonarthritic animals. Nonarthritic rat testicular macrophage-conditioned medium had no significant effect on testosterone production. In contrast, conditioned medium from arthritic rat testicular macrophages significantly reduced testosterone production. These results suggest that testicular macrophages secrete a factor that may be important in the regulation of testosterone production in the adjuvant-induced arthritic rat.

Testicular dysfunction in the adjuvant-induced arthritic rat.

Adjuvant-induced arthritis, an autoimmune disease similar to rheumatoid arthritis, was used to investigate possible mechanisms of immune system modulation of the reproductive system. This laboratory previously reported that arthritic male rats have reduced serum testosterone and elevated serum LH concentrations. In the experiments described here, serum prolactin levels were not significantly different in arthritic animals compared with non-injected control animals. Neither reduced food consumption of arthritic rats nor the injection vehicle appear to cause a reduction of serum testosterone. Serum corticosterone was significantly elevated in the arthritic group compared with both the non-injected or the vehicle-injected control animals. Testicular cells from arthritic animals secrete significantly less testosterone in vitro compared with cells from non-injected control animals, both basally and in response to dbcAMP and hCG. In summary, the reduced serum testosterone of arthritic animals appears to be the result of a testicular dysfunction.

Evidence for regulation of sodium transport from airspace to vascular space by cAMP.

Evidence has been accumulating that regulation of the rate of solute and fluid removal from the alveolar spaces may play an important role in the prevention and/or resolution of alveolar pulmonary edema. In this study, the isolated perfused rat lung was used to investigate the effects of an adenosine 3',5'-cyclic monophosphate (cAMP) analogue or a phosphodiesterase inhibitor on active sodium transport from airspace to vascular space. Three tracers were instilled into the airways of isolated Krebs-Ringer bicarbonate solution (KRB)-perfused rat lungs. The appearance of tracers in the single-pass perfusate was measured, and the apparent permeability-surface area products (PS) were calculated for each tracer at each sample time based on Fick's first law of diffusion. After steady-state PS values had been reached, a cAMP analogue or phosphodiesterase inhibitor was added to the perfusate. Both agents caused significant increases in the PS for 22Na. In another group of experiments, a cAMP analogue was added to the perfusate, followed by the subsequent addition of a sodium transport inhibitor and the resultant large decrease in the PS for 22Na. These data are consistent with the regulation of active sodium transport across the intact mammalian alveolar epithelium by a cAMP-mediated process leading to removal of sodium from the alveolar spaces, with anions and water following passively.

Effect of adjuvant-induced arthritis on serum luteinizing hormone and testosterone concentrations in the male rat.

Male Lewis rats were made arthritic by injecting 1 mg Mycobacterium butyricum suspended in Freund's incomplete adjuvant into their right hind footpad. Arthritic and non-arthritic animals were sacrificed on days 18, 21, 24 or 27 after the injection of the adjuvant. Body weight, left and right hind paw volume, thymus weight, and serum luteinizing hormone (LH) and testosterone concentrations were determined on each day. Adjuvant injection resulted in a significant enlargement in the left and right hind paws on days 18 through 27. In contrast, body and thymus weights were reduced significantly in the arthritic rats compared to the non-arthritic animals. Serum concentrations of testosterone were also reduced significantly in arthritic rats on days 18, 21 and 24 after the injection of the adjuvant. However, by day 27 serum testosterone concentrations recovered to near control values. Serum concentrations of LH in the arthritic animals were elevated on days 18 through 27. These results demonstrate that serum testosterone concentrations were reduced in rats with adjuvant-induced arthritis. The reduction in serum testosterone is probably not the result of an impaired hypothalamic-pituitary axis.