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D M Robins

Publications and source records attributed to D M Robins.

At least 19 recordsLinked to original sources
PubMed2001

The androgen receptor (AR) amino-terminus imposes androgen-specific regulation of AR gene expression via an exonic enhancer.

Androgen and glucocorticoid receptor (AR, GR), two closely related members of the nuclear receptor superfamily, can recognize a similar cis-acting DNA sequence, or hormone response element (HRE). Despite this apparent commonality, these receptors regulate distinct target genes in vivo. The AR gene itself is regulated by AR but not GR in a variety of cell types, including osteoblast-like cells, as shown here. To understand this specificity, we first identified the DNA sequences responsible for androgen-mediated up-regulation of AR messenger RNA. A 6.5-kb region encompassing exon D, intron 4, and exon E of the AR gene contains four exonic HREs and exhibits cell type-specific, AR-mediated transcriptional enhancement when placed upstream of a heterologous promoter and reporter gene. A 350-bp fragment consisting of just exons D and E exhibits the same cell- and androgen-specificity as the 6.5-kb region, as well as the native AR gene. Consistent with a role for the exonic HREs, androgen regulation via this intragenic enhancer requires the HREs as well as a functional receptor DNA binding domain. A panel of AR/GR chimeric receptors was used to test which AR domains (amino-terminal, DNA binding or ligand binding) confer androgen-specific regulation of the 350-bp enhancer. Only chimeric receptors containing the amino-terminus of AR induced reporter gene activity from the AR gene enhancer. Further, a constitutively active AR consisting of only the AR amino-terminus and DNA binding domain (AA phi) retained the capacity to activate the internal responsive region, unlike a constitutively active chimera harboring the GR amino-terminus and AR DNA binding domain (GA phi). Thus, the AR amino terminus is the sole determinant for androgen-specific regulation of the AR gene internal enhancer. These findings support a model in which the amino termini of ARs bound to HREs within the AR gene interact with an exclusive auxiliary factor(s) to elicit androgen-specific regulation of AR messenger RNA. This is the first example of androgen-specific response in which the necessary and sufficient distinguishing capacity resides within the AR amino terminus.

Androgens↗
PubMed2000

Oct-1 preferentially interacts with androgen receptor in a DNA-dependent manner that facilitates recruitment of SRC-1.

Gene regulation by steroid hormone receptors depends on the particular character of the DNA response element, the array of neighboring transcription factors, and recruitment of coactivators that interface with the transcriptional machinery. We are studying these complex interactions for the androgen-dependent enhancer of the mouse sex-limited protein (Slp) gene. This enhancer has, in addition to multiple androgen receptor (AR)-binding sites, a central region (FPIV) with a binding site for the ubiquitous transcription factor Oct-1 that appears crucial for hormonal regulation in vivo. To examine the role of Oct-1 in androgen-specific gene activation, we tested the interaction of Oct-1 with AR versus glucocorticoid receptor (GR) in vivo and in vitro. Oct-1 coimmunoprecipitated from cell lysates with both AR and GR, but significant association with AR required both proteins to be DNA-bound. This was confirmed by sensitivity of the protein association to treatment with ethidium bromide or micrococcal nuclease. Addition of DNA to micrococcal nuclease-treated samples restored interaction, even when binding sites were on separate DNA molecules, suggesting association was due to direct protein-protein interaction and not indirect tethering via the DNA. AR/GR chimeras revealed that interaction of the N and C termini of AR was required to communicate the DNA-bound state that enhances interaction with Oct-1. Protease digestion assays of hormone-bound receptors revealed further conformational changes in the ligand binding domain of AR, but not GR, upon DNA binding. Furthermore, these conformational changes led to increased interaction with the coactivator SRC-1, via the NID 4 domain, suggesting DNA binding facilitates recruitment of SRC-1 by the AR-Oct-1 complex. Altogether, these results suggest that the precise arrangement of binding sites in the Slp enhancer ensures proper hormonal response by imposing differential interactions between receptors and Oct-1, which in turn contributes to SRC-1 recruitment to the promoter.

Amino Acid Motifs↗
PubMed2000

Functional interaction of human Cdc37 with the androgen receptor but not with the glucocorticoid receptor.

Cdc37 is a molecular chaperone closely associated with the folding of protein kinases. Results from studies using a yeast model system showed that it was also important for activation of the human androgen receptor (AR). Based on results from the yeast model system (Fliss, A. E., Fang, Y., Boschelli, F., and Caplan, A. J. (1997) Mol. Biol. Cell 8, 2501-2509), we initiated studies to address whether AR and Cdc37 interact with each other in animal cell systems. Our results show that Cdc37 binds to AR but not to glucocorticoid receptors (GR) synthesized in rabbit reticulocyte lysates. This binding occurs via the ligand-binding domain of the AR in a manner that is partially dependent on Hsp90 and the presence of hormone. Further studies using the yeast system showed that Cdc37 is not interchangeable with Hsp90, suggesting that it functions at a distinct step in the activation pathway. Expression of a dominant negative form of Cdc37 in animal cells down-regulates full-length AR but has very little effect on an AR truncation lacking the ligand-binding domain or full-length GR. These results reveal differences in the mechanisms by which AR and GR become active transcription factors and strengthen the notion that Cdc37 has a wider range of polypeptide clients than was realized previously.

Binding Sites↗
PubMed2000

Design, synthesis, and pharmacological characterization of 4-[4, 4-dimethyl-3-(4-hydroxybutyl)-5-oxo-2-thioxo-1-imidazolidinyl]- 2-iodobenzonitrile as a high-affinity nonsteroidal androgen receptor ligand.

4-[4, 4-Dimethyl-3-(4-hydroxybutyl)-5-oxo-2-thioxo-1-imidazolidinyl]-2-+ ++trif luoromethylbenzonitrile (RU 59063) is a prototype of a new class of high-affinity nonsteroidal androgen receptor (AR) ligands. The search for a radioiodinated AR ligand prompted us to synthesize 4-[4, 4-dimethyl-3-(4-hydroxybutyl)-5-oxo-2-thioxo-1-imidazolidinyl]-2-i odo benzonitrile (DTIB) wherein the trifluoromethyl group of RU 59063 was substituted with the similarly hydrophobic iodine atom. DTIB displayed subnanomolar binding affinity (K(i) = 0.71 +/- 0.22 nM) for the rat AR in competitive binding assays. Additionally, DTIB demonstrated potent agonist activity, comparable to that of the natural androgen 5alpha-dihydrotestosterone (DHT), in a cell-based functional assay (cotransfection assay). DTIB represents a new lead for the development of high-affinity radioiodinated AR radioligands.

Androgens↗
PubMed2000

Mouse complement components C4 and Slp act synergistically in a homologous hemolytic C4 assay.

Goals of the present study were to compare the hemolytic activities of mouse C4 and Slp in a homologous system and to study a possible interaction between these proteins during complement activation. As reagents for mouse C4 and Slp, we used serum of C4(- / -) knockout C57BL / 6 (C4(-) / Slp(-)) mice and sensitized rabbit erythrocytes as target cells. Sera to be tested contained none, either of the two or both proteins. We found that C4(-) / Slp(+) serum has some hemolytic C4 activity, but less than C4(+) / Slp(-) serum. Comparing C4 activities of C4(+) / Slp(-) and C4(+) / Slp(+) sera, we found a threefold enhanced activity in double-positive serum. Hemolytic C4 levels of mixtures of solely C4- and Slp-sufficient sera did not overlap with expected C4 levels, but rather these sera showed synergy. This explains the enhanced activity of double-positive serum. Similar results were observed for total complement activation. In conclusion, Slp has measurable, but poor C4 activity as compared with mouse C4. Using our homologous system, we showed that the enhanced classical pathway activity of double-positive sera is most probably based on synergy between C4 and Slp. Our results answer an old question as to why C4(+) / Slp(+) mice have higher complement levels than C4(+) / Slp(-) mice.

Animals↗
PubMed1999

AML3/CBFalpha1 is required for androgen-specific activation of the enhancer of the mouse sex-limited protein (Slp) gene.

A complex 120-base pair enhancer, derived from the mouse sex-limited protein (Slp) gene, is activated solely by the androgen receptor (AR) in specific tissues, although it contains a hormone response element recognized by several steroid receptors. The generation of this transcriptional specificity has been ascribed to the interactions of the receptor with tissue-specific nonreceptor factors bound to accessory sites within the enhancer. Protein-DNA interaction assays revealed two factors binding the 5' part of the enhancer that differ widely in abundance between cells showing AR-specific activation of the Slp element compared with those that also permit activation by glucocorticoid receptor (GR). The factor designated B formed a complex centered on the sequence TGTGGT, a core motif recognized by members of the AML/CBFalpha transcription factor family. This complex was competed by a high affinity binding site specific for AML/CBFalpha and was specifically supershifted by an antibody to AML3/CBFalpha1, placing factor B within the AML3/CBFalpha1 subclass. Interestingly, this factor was shown to bind to a second site in the 3' part of the enhancer, positioned between the two critical AR binding sites. Transfection studies revealed that AML1-ETO, a dominant-negative AML/CBFalpha construct, abrogated AR induction of the enhancer, but not of simple hormone response elements. Furthermore, overexpression of AML3/CBFalpha1 could rescue the AML1-ETO repression. Finally, glutathione S-transferase-AML/CBFalpha fusion proteins demonstrated direct interaction between AML/CBFalpha and steroid receptors. Although this interaction was equivalent between AML1/CBFalpha2 and AR or GR, AML3/CBFalpha1 showed stronger interaction with AR than with GR. These data demonstrate that AML3/CBFalpha1 is functionally required for hormonal induction of the Slp enhancer and that direct, preferential protein-protein interactions may contribute to AR-specific activation. These results demonstrate an intriguing role of AML3/CBFalpha1 in steroid- as well as tissue-specific activation of target genes.

Animals↗
PubMed1999

Sex-limited protein: in vitro and in vivo functions.

Mouse complement component C4 exists in two isoforms, C4 and a protein with expression restricted to male animals called sex-limited protein (Slp). Although Slp is about 95% homologous to C4, it is generally believed to be non-functional, at least in conventional haemolytic complement assays. In a previous study, however, we showed that Slp is haemolytically active in a C1-inhibitor (C1INH)-regulated, EDTA-resistant mouse complement activation pathway. To study other possible implications of this finding, we generated constitutively expressing Slp-transgenic mice. The transgene was crossed into otherwise Slp-deficient C57Bl/6J and NZB mice. Members of the third backcross generation of C57Bl/6J mice were tested for functional Slp and classical and alternative complement pathway activities (CH50 and AP50 levels, respectively). Slp-transgenic C57Bl/6J mice showed enhanced CH50, but normal AP50 levels when compared with non-transgenic littermates. To discover a possible protective role for Slp in spontaneous systemic lupus erythematosus (SLE) in NZBxNZW (NZBxW) mice, the third backcross generation of Slp-transgenic NZB mice was mated with NZW mice and the development of SLE in the female offspring was followed. In these introductory experiments, Slp-transgenic NZBxW animals presented with a significantly extended life span. Our results imply that Slp is a mouse complement component with functions which partially resemble some of those of human C4A.

Animals↗
PubMed1998

Multiple receptor domains interact to permit, or restrict, androgen-specific gene activation.

A critical problem within transcription factor families is how diverse regulatory programs are directed by highly related members. Androgen and glucocorticoid receptors (AR, GR) recognize a consensus DNA hormone response element (HRE), but they activate target genes with precise specificity, largely dependent on the promoter and cell context. We have assessed the role of different receptor domains in hormone-specific response by testing chimeras of AR and GR for their ability to activate the androgen-specific enhancer of the mouse sex-limited protein (Slp) gene. Although all of the mutant receptors activated simple HREs, only a few activated the androgen-specific element. One component shared by receptors functional on the AR-specific target was the AR DNA binding domain. Activation was not due to differential DNA affinity but rather to the AR DNA binding domain escaping suppression directed at the GR DNA binding domain in this enhancer context. A further mechanism increasing specific activation was cooperation of receptors at multiple and weak HREs, which was accentuated in the presence of both the AR N terminus and ligand binding domain. These domains together increased recognition of weak HREs, as demonstrated by in vitro DNase I footprinting and transactivation of mutant enhancers. Further, AR N-terminal subdomains reported to interact directly with the ligand binding domain relieved an inhibitory effect imposed by that domain. Therefore, functions intrinsic to AR augment steroid-specific gene activation, by evading negative regulation operating on the domains of other receptors and by enhancing cooperativity through intra- and inter-receptor domain interactions. These subtle distinctions in AR and GR behavior enforce transcriptional specificity established by the context of nonreceptor factors.

Amino Acid Substitution↗
PubMed1997

Regulatory capacity of an androgen-specific enhancer of the mouse Slp gene in transgenic mice.

Different steroid hormone receptors can activate transcription from the same hormone response element (HRE) in vitro, but in vivo the effects of each hormone on gene activity are distinct. To determine sequences mediating androgen-specific response in a physiological setting, we placed the androgen-responsive mouse sex-limited protein gene (Slp) enhancer before a tkCAT reporter in transgenic mice. The enhancer contains a consensus HRE plus accessory factor binding sites that act in concert to direct transcription in response to androgen. A 160 bp fragment, C'delta2, is responsive to several steroids in transfection; in transgenic mice, this enhancer was active in several tissues of male and female mice, in four of six transgenic lines. In striking contrast, C'delta9, a 120 bp sub-fragment of C'delta2 that responds only to androgen in transfection, showed activity in testes, prostate and kidney, where it was strongly androgen-inducible in females. However, expression was obtained in only one transgenic line. Multimerization of the C'delta9 enhancer conferred expression in prostate, but again in only one line. The greater penetrance of C'delta2 expression was not driven by glucocorticoids, as adrenalectomy had little effect, but may be dependent on the NF-kappaB-like element absent from the C'delta9 fragment. That two transgenic lines showed expression in androgen target sites driven by enhancers that are androgen-specific in vitro suggested that activation of this enhancer, when it could occur, was in response to androgen. The dramatically different behavior of the two related enhancer sequences underscores the importance of chromosomal context to the activity and specificity of regulatory elements.

Adrenal Glands↗
PubMed1997

Steroid hormone responsiveness of a family of closely related mouse proviral elements.

Regulation of the mouse sex-limited protein (Slp) gene in unusual in that hormone response is conferred by the 5' LTR of an upstream inserted provirus, dubbed the imposon (imp1). In a search for additional genes whose regulation has been affected by retrotransposition events, we isolated two partial proviral elements by stringent screening of a mouse genomic library. One clone (imp2) contained a portion of the envelope gene and a 3' LTR that was nearly identical to the 3' LTR of imp1; this similarity extended to insertion into a B1 repetitive element. The second proviral clone (imp3) contained a 5' LTR and associated coding sequences, but lacked its 3' LTR; the LTR of imp3 differed by 12% from the imp1 sequence. To assess potential hormone response, proviral enhancer regions cloned into reporter vectors were tested in transfection. The imp2 enhancer was similar in behavior to imp1, conferring both androgen and glucocorticoid induction in one fragment context and an androgen-specific response in another. In contrast, the imp3 enhancer allowed high expression in the absence of hormone and was less responsive to steroids in general and androgen in particular. These three proviral elements define a small family of steroid responsive proviruses in the mouse genome, and at least one member has had a lasting impact on an endogenous gene's regulation.

Animals↗
PubMed1997

Involvement of an octamer-like sequence within a crucial region of the androgen-dependent Slp enhancer.

Androgen dependence of the mouse sex-limited protein (Slp) gene is conferred by an enhancer encompassing a consensus hormone response element (HRE) and sites for several nonreceptor factors. The footprint IV (FPIV) region of the enhancer plays a key role in hormone- and tissue-specific response, both in vitro and in vivo. We characterized FPIV-binding factors by methylation interference analysis and UV cross-linking of several complexes evident in gel mobility-shift assays. The footprinting analysis revealed that distinct base contacts within the multiple nuclear protein-DNA complexes occurred primarily within a sequence similar to an octamer transcription factor (Oct-1) binding site. With additional data on approximate molecular weights from UV cross-linking, several plausible candidates were tested for their DNA binding and functional activity at FPIV. Oct-like protein binding in gel-shift assays with several cell and tissue extracts was evident using specific competitors and antibodies, but was lower in affinity for FPIV than for an Oct-1 consensus site. Site-directed mutation of the FPIV sequence to a consensus Oct-1 element within the Slp enhancer context increased Oct-1 binding in vitro, but greatly reduced hormonal induction in vivo. This suggested that Oct-1 is not directly involved in response, or alternatively, that Oct-1 bound to the lower-affinity site interacts with neighboring factors significantly differently than Oct-1 bound to a consensus sequence. A sequence overlapping the Oct-like element that was similar to a hepatic nuclear factor-4 (HNF-4) site showed no ability to bind HNF-4 in vitro, nor the related orphan receptor, chicken ovalbumin upstream promoter factor (COUP-TF). Intriguingly, however, expression of COUP-TF in transfection had a dramatic inhibitory effect on response of the androgen-specific enhancer (C' delta9), but did not affect other enhancer configurations that can also be induced by glucocorticoid (C 'delta2). This underscores that, despite extensive sequence identity of C' delta9 and C' delta2, components of the androgen-specific transcription complex differ significantly from that of one that is more generally steroid responsive.

Androgens↗
PubMed1997

Repression of cAMP-induced expression of the mouse P450 17 alpha-hydroxylase/C17-20 lyase gene (Cyp17) by androgens.

In primary cultures of mouse Leydig cells, testosterone represses the cAMP-induced de novo synthesis of P450 17 alpha-hydroxylase/C17-20 lyase (P450c17) protein and the accumulation of P450c17 mRNA, via an androgen receptor (AR)-mediated mechanism. To examine the mechanism by which androgens repress the cAMP-induced expression of the mouse Cyp17 gene, constructs containing 5'-flanking sequences of the mouse Cyp17 linked to the chloramphenicol acetyltransferase (CAT) reporter gene were cotransfected into MA-10 tumor Leydig cells with a mouse AR expression plasmid. In the presence of dihydrotestosterone, the cAMP-induced expression of a reporter construct containing -1021 bp of Cyp17 promoter sequences was repressed. In contrast, no repression by dihydrotestosterone was observed when the -1021 bp Cyp17-CAT construct was cotransfected with a human AR expression plasmid missing the second zinc finger of the DNA-binding domain, indicating that DNA binding is involved in AR-mediated repression of Cyp17 expression. Analysis of deletions -346 bp of 5'-flanking region of the mouse Cyp17 promoter are sufficient to confer androgen repression of the cAMP-induced expression of Cyp17. Deoxyribonuclease I footprinting analysis indicated that the AR interacts with sequences between -330. and -278 bp of the Cyp17 promoter. This region overlaps with the previously identified cAMP-responsive region located between -346 and -245 bp of the Cyp17 promoter. These results suggest that AR-mediated repression involves binding of the AR to sequences in the cAMP-responsive region of the Cyp17 promoter, possibly interfering with the binding of the protein(s) that mediate cAMP induction of Cyp17.

Animals↗
PubMed1997

Two distinct mechanisms elicit androgen-dependent expression of the mouse sex-limited protein gene.

The mouse sex-limited protein (Slp) gene is expressed in liver and kidney of adult males and is testosterone-inducible in females, indicative of androgen dependence. Analysis of mRNA levels and chromatin configuration reveals that this androgen regulation is achieved by distinct means in the two tissues. In the liver, Slp expression requires pituitary function, and specifically, as shown by others, a pulsatile pattern of GH secretion that is itself determined by androgen. After hypophysectomy, Slp synthesis cannot be reestablished in liver by testosterone, although mRNA decline can be slowed. In contrast, in the kidney Slp mRNA is directly induced by androgen in hypophysectomized mice. In vivo footprinting was used to examine the role of the Slp enhancer, which directs androgen-specific transcription in transfection and contains a factor-binding site, FPIV, whose protection in vivo has been correlated with Slp expression. In kidney, FPIV was protected in intact males and hypophysectomized mice supplemented with testosterone, but not in females or untreated hypophysectomized mice, corroborating FPIV's association with androgen-driven transcription. Surprisingly, protection of FPIV also occurred in liver of hypophysectomized males treated with testosterone, despite the lack of Slp expression. Thus androgen directly affects the Slp enhancer in kidney, where steroid is sufficient for gene activation, as well as in liver, where chromatin remodeling occurs in response to androgen, although GH is clearly required for expression. This may indicate that both GH and androgen signal transduction pathways target the Slp enhancer to elicit precise gene regulation.

Animals↗
PubMed1996

Hsp90 regulates androgen receptor hormone binding affinity in vivo.

The regulation of human androgen receptor (AR) by the molecular chaperone Hsp90 was investigated using the yeast Saccharomyces cerevisiae as a model system. These studies were performed in strains expressing a conditional temperature-sensitive mutant allele of the hsp82 gene, which encodes Hsp90 protein. At the restrictive temperature in the mutant, there is a decrease in hormone-dependent transactivation by the AR, although steady state levels of AR protein are unchanged. Quantitative hormone binding studies at the permissive temperature revealed the presence of both high affinity and low affinity hormone binding states. At the restrictive temperature in the hsp82 mutant, the high affinity state was abolished, and only the low affinity state was observed. The change in hormone binding affinity was further investigated by a competition assay with the anti-androgen hydroxyflutamide. Under permissive conditions, hydroxyflutamide competes poorly for the synthetic androgen R1881, but under restrictive conditions in the hsp82 mutant strain, hydroxyflutamide was shown to be a potent competitive inhibitor. Our findings indicate that Hsp90 participates in the activation process by maintaining apoAR in a high affinity ligand binding conformation which is important for efficient response to hormone.

Androgen Antagonists↗
PubMed1996

Contextual dependence of steroid receptor function on an androgen-responsive enhancer.

The enhancer of the mouse sex-limited protein (Slp) gene includes a consensus hormone response element (HRE) that interacts with several auxiliary elements for steroid induction. The 160-bp fragment. C' delta 2, confers response to androgen or glucocorticoid in transfection, while a 120-bp subfragment, C' delta 9, is activated only by androgen in some cells. Site-directed mutants were tested to identify elements affecting differential response of androgen or glucocorticoid receptors (AR, GR). While most mutations of C' delta 2 affected induction by either steroid similarly, disruptions of the consensus HRE or an octamer-like sequence were more severe for GR than AR activity. An HRE half-site was critical to androgen-specific induction of C' delta 9 but had little impact in the nonspecific C' delta 2 context. In DNase I footprinting, full-length AR and GR bound similarly to the consensus HRE but dissimilarly to nonconsensus sites. Intriguingly, NF-kappa B bound the region of C' delta 2 absent from C' delta 9. Expression of I kappa B decreased response of C' delta 2, but not C' delta 9, confirming a permissive role of NF-kappa B in steroid activation. In this case, different factors may associate with receptors in the presence of NF-kappa B than those that confer androgen specificity in NF-kappa B's absence, suggesting that exclusion of some factors from a specific transcription complex is as crucial as inclusion of others. This dissection of C' delta 2 and C' delta 9 in vitro reveals subtle distinctions in AR and GR interactions that may underlie specific hormonal response in vivo.

Androgens↗
PubMed1995

In vivo footprinting of an androgen-dependent enhancer reveals an accessory element integral to hormonal response.

A hormonally responsive enhancer that is specifically activated by androgens resides 2 kilobases upstream of the transcription start site of the mouse sex-limited protein (Slp) gene. We have previously shown that strong androgen induction in transfection requires a consensus hormone response element as well as several nonreceptor factor binding sites within this complex enhancer. To determine which accessory elements are required for androgen-dependent transcription, we have examined binding of nuclear proteins to the enhancer both in vitro and in vivo. In vitro footprinting assays demonstrated that multiple factors present in mouse liver and kidney nuclear extracts bound the enhancer, with tissue-specific but not sex-dependent differences in pattern. In contrast, examination of DNA sites occupied in liver chromatin identified a footprint (FPIV) that is well protected in males but sensitive to DNase I in females. FPIV was occupied in males in other sites of Slp expression, such as kidney, but not in tissues lacking expression, such as lung. FPIV protection was induced in females treated with androgen, abrogated in castrated males, and absent in immature mice, implying hormonal and developmental regulation of FPIV binding. Protection of the hormone response element, in contrast to FPIV, was not obvious but was discerned by analysis of densitometry data. Together with results from in vivo protein-DNA interactions determined for other steroid-dependent enhancers, this suggests that in some cases receptor may permit transcriptional activation by altering chromatin structure to allow access to other factors, which may not necessitate tight binding of receptor itself. This further emphasizes the crucial role of the nonreceptor factors in hormone response. The ubiquitous transcription factor Oct-1 forms complexes with an octamer motif present within FPIV by gel shift analysis with liver and kidney extracts, making Oct-1 an intriguing candidate for partnership in androgen regulation.

Androgens↗