Allan J. Syms

Differential effects of androgens and glucocorticoids on regulation of androgen receptor concentrations and cell growth.

This report describes androgen induced up-regulation of intracellular androgen receptor concentrations in the DDT1MF-2 cell line derived from the hamster ductus deferens and the R3327H-G8-A1 line derived from the Dunning prostate adenocarcinoma. Within 6 h of exposure to androgens the receptor concentration is increased approximately 2-fold. Incorporation of dense amino-acids and subsequent analysis by sucrose density gradient centrifugation indicates that the observed increase in receptors is possibly due to de novo androgen receptor synthesis. In both cell lines this increase is inhibited by 30-50% within 6 h and completely during a subsequent 18 h period by the potent glucocorticoid triamcinolone acetonide (TA). TA also inhibits the growth of both cell lines and antagonizes the stimulatory effect of androgens. Flow cell cytometry studies indicate that TA blocks the cells in the G1 phase of the cell cycle. This event may be associated with regulation of androgen receptor concentrations.

Autocrine regulation of growth: I. Glucocorticoid inhibition is overcome by exogenous platelet derived growth factor

The ductus deferens smooth muscle tumor cell line (DDT1MF-2) expresses c-sis protooncogene mRNA transcripts which encode at least one subunit of the potent mitogenic agent, platelet derived growth factor (PDGF). These cells also synthesize and secrete a protein which is immunologically identical to this growth factor. Therefore, PDGF is implicated in the autocrine regulation of DDT1MF-2 cell proliferation. While androgens also stimulate proliferation and induce an augmentation in androgen receptor levels in DDT1MF-2 cells, glucocorticoids inhibit both events and arrest cells in the G1 phase of the cell cycle. Addition of PDGF overcomes the glucocorticoid cell cycle arrest, but does not diminish the suppressive action on androgen receptor concentration. These findings are consistent with a mechanism by which glucocorticoids regulate DDT1MF-2 cell proliferation through modulation of PDGF expression which is independent of the glucocorticoid effects on androgen receptor concentrations.

Proliferation of a highly androgen-sensitive ductus deferens cell line (DDT1MF-2) is regulated by glucocorticoids and modulated by growth on collagen

SummaryProliferation of the hamster ductus deferens cloned tumor cell line (DDT1MF-2) in monolayer culture is markedly stimulated by androgens in a dose dependent fashion. Furthermore, growth on collagen confers upon these cells a greater dependence on this class of hormones, such that testosterone (10 nM) induces a 15-fold elevation in cell number compared to controls. Addition of either dexamethasone (10 nM) or triamcinolone acetonide (TA; 10 nM) dramatically blocks this stimulation by reversibly arresting the cells in the G1 phase of the cell cycle as assessed by flow cell cytometry. Associated with the decreased growth rate is a change from a rounded to a more flattened morphology that may also implicate cell shape in the regulation of proliferation.These steroid effects presumably are mediated through specific receptor proteins for which dihydrotestosterone (DHT) and TA bind with equilibrium dissociation constants (Kd) of 0.3 and 1.0 nM, respectively. Moreover, not only do androgens increase growth rate but treatment with 1 nM [3H]DHT also results in an elevation in androgen receptor concentration from 1.6 to 3.6 f mol/μg DNA in 7 h. Simultaneous treatment with 10 nM TA, however, reduces this increase by 53%. Inasmuch as neither progesterone nor estradiol-17β display similar inhibitory activity, this effect also seems to be glucocorticoid specific. These observations may be important in elucidating the mechanism of androgen action and should provide some insight into the role of glucocorticoids in regulating the growth of androgen dependent tissues.

Androgen stimulated elevation in androgen receptor levels is inhibited by the synthetic glucocorticoid triamcinolone acetonide.

Two cloned tumor cell lines derived from the rat prostate and hamster ductus deferens contain receptors for androgens and glucocorticoids. Androgens increase both the rate of proliferation in these cells and induce a doubling in the number of androgen receptors within 6 hours. This elevation in androgen receptors is dependent on protein synthesis. The glucocorticoid triamcinolone acetonide specifically inhibits both these androgen mediated events without altering the equilibrium dissociation constant (Kd) of the androgen receptor for either [3H]-methyltrienolone (Kd = 0.46 nM) or [3H]-dihydrotestosterone (Kd = 0.2 nM). These observations infer that androgen receptor up-regulation is an important facet of androgen action which may be modulated by glucocorticoids.

Glucocorticoid effects on growth, and androgen receptor concentrations in DDT1MF-2 cell lines.

The DDT1MF-2 smooth muscle tumor cell line contains receptors for and is differentially sensitive to androgens and glucocorticoids. Androgens stimulate and glucocorticoids inhibit growth. We now confirm that the latter involves the induction of a block in the G1 phase of the cell cycle. We have developed and characterized in vitro and in vivo a glucocorticoid resistant variant of this cell line, the DDT1MF-2-GR. Glucocorticoids specifically inhibit androgen induced androgen receptor augmentation in DDT1MF-2 cells, but not in the GR variant suggesting that growth inhibition is related to inhibition of androgen receptor augmentation. However, under optimal conditions for cell proliferation, when glucocorticoid inhibited growth is relieved by the exogenous addition of platelet derived growth factor, androgen receptor augmentation is still suppressed. Thus, androgen induced elevation in androgen receptor concentrations is not a prerequisite for cell proliferation. These results imply that in androgen responsive cells, although androgen stimulation of growth can be blocked by antagonism of androgen receptor mediated events, the antagonism can be bypassed by supplying the cells with exogenous growth factors. These results provoke speculation on how cells, which are dependent upon androgens for growth, become autonomous.

Autocrine regulation of growth: II. Glucocorticoids inhibit transcription of c-sis oncogene-specific RNA transcripts

The ductus deferens smooth muscle tumor cell line (DDT1MF-2) expresses c-sis proto-oncogene poly A+ RNA transcripts which are thought to encode at least one subunit of the potent mitogen platelet derived growth factor (PDGF). We have previously demonstrated that glucocorticoids block DDT1MF-2 cells in G0/G1 stage of the cell cycle, and that exogenously applied PDGF reinitiates cell cycle progression. In this paper we document that glucocorticoids act to inhibit cell cycle progression by inhibiting the expression of c-sis poly A+ transcripts, which we suggest are encoding a PDGF-like molecule for DDT1MF-2 cells.

Steroid regulation of receptor concentration and oncogene expression

The DDT1MF-2 cell line was derived from an estrogen/androgen-induced tumor of the hamster ductus deferens. This cell line contains receptors for both androgens and glucocorticoids and its proliferation is differentially sensitive to these classes of steroids. Androgens stimulate cell growth dramatically and augment intracellular androgen receptors, whereas glucocorticoids inhibit growth and prevent androgen receptor augmentation. Androgen receptor augmentation occurs by an androgen-dependent increase in receptor half-life and an increase in the rate of synthesis. Glucocorticoids, in the presence of androgens, reduce both the half-life and the rate of synthesis of androgen receptors. For comparison purposes, a glucocorticoid-resistant mutant (DDT1MF-2-GR) of this cell line has been developed. Unlike the wild type, in this variant glucocorticoids neither arrest cell growth nor inhibit androgen receptor augmentation. Glucocorticoids block growth of the wild type in the G1-phase of the cell cycle. This event can be overcome either by addition of exogenous platelet-derived growth factor (PDGF) or by the addition of concentrated conditioned medium from nonglucocorticoid-treated cells, however, androgen receptor augmentation remains inhibited. The reduced production of PDGF-like growth factors in the presence of glucocorticoids appears to be the result of a decrease in production of mature mRNA with homology to v-sis (the viral oncogene coding for PDGF-like proteins). The level of regulation appears to be posttranscriptional and does not occur in the DDT1MF-2-GR cells. Thus glucocorticoids regulate the autocrine growth of the DDT1MF-2 cells by a mechanism that can be uncoupled from the regulation of androgen receptor augmentation.

Effect of aging and cold temperature storage of hamster ova as assessed in the sperm penetration assay

The penetration of zona pellucida-free hamster ova by human spermatozoa has been used to quantitate sperm penetration potential. However, since mammalian eggs in vitro have limited viability, the effect of in vitro aging on the ability of hamster ova to be penetrated by human spermatozoa was examined. Zona-free ova maintained at room temperature (25 degrees C) lost their ability to be subsequently penetrated with a half-life of 50.1 +/- 8.8 minutes. This was partly the result of removing the zona pellucida by trypsin digestion, since zona-free oocytes in the presence of trypsin inhibitor or zona pellucida-intact oocytes had half-lives of 99.1 +/- 15.2 and 120.5 +/- 17.4 minutes, respectively. Reduction in penetration rates associated with ovum aging did not appear to be due to loss of viability and could be completely prevented by maintaining the ova on ice (4 degrees C). In the presence of TEST-yolk buffer at 4 degrees C, ova retained (100%) their ability to be penetrated for up to 24 hours and were morphologically indistinguishable from fresh ova. These observations show that ovum aging in vitro at 25 degrees C is much greater than previously anticipated. This may result in artifactually low and variable scores in the penetration bioassay.

Glucocorticoids induce a 29 000 Mr protein in DDT1 MF-2 smooth muscle cells but not in the DDT1 MF-2 GR glucocorticoid resistant variant

We have demonstrated that glucocorticoids induce in DDT1 MF-2 cells by a glucocorticoid mediated mechanism the synthesis of a methionine-cysteine rich protein of 29 000 Mr (p29). Induction of p29 is not observed in DDT1 MF-2 GR glucocorticoid resistant variants which have only 7% of glucocorticoid receptor site per cell compared to wild type cells. Increased synthesis of p29 is specific to glucocorticoids since neither androgens, estrogens, progesterone nor the glucocorticoid antagonist dexamethasone mesylate are effective inducers. Stimulation of p29 synthesis in wild type cells is observed at 10−10 M triamcinolone acetonide, reaching a maximum at a concentration of 1 × 10−8 M. The induction of p29 is not a function of glucocorticoid arrest of DDT1 MF-2 cells since DDT1 MF-2 cells promoted to re-enter the cell cycle by 50 ng/ml platelet derived growth factor (PDGF) continue synthesis of p29. Finally, increased levels of p29 translation products are observed in cell free translation assays carried out utilizing poly A− RNA transcripts isolated from glucocorticoid treated cells. These data suggest that the glucocorticoid stimulation of p29 synthesis is a transcriptional and/or RNA processing event controlled by glucocorticoid receptor complexes.

Studies on human spermatozoa with round head syndrome**Supported in part by a grant from the Shell Companies Foundation, Incorporated.††Presented at the Fortieth Annual Meeting of The American Fertility Society, April 2 to 7, 1984, New Orleans, Louisiana.

The ability of spermatozoa with round head syndrome to penetrate zona pellucida-free hamster ova and to undergo nuclear decondensation was studied in three infertile patients. Whereas spermatozoa from pregnancy-proven donors bound to hamster ova and achieved a penetration rate of 100%, those with round heads did not bind to or penetrate any ova. While spermatozoa nuclear decondensation occurs after ovum penetration and indicates a positive result, it has now been demonstrated that this phenomenon can also be induced by incubating spermatozoa with crushed hamster ova. In addition, nuclei from normal and round-headed spermatozoa decondense in the presence of these ova to a similar degree. These observations suggest that infertility related to round head sperm morphology is associated with an inability to interact with and penetrate the oolemma, rathern than dysfunctional spermatozoan changes following penetration.