The shift from C-19 to C-21 steroid synthesis in spawning male common carp, Cyprinus carpio, is regulated by the inhibition of androgen production by progestogens produced by spermatozoa

There is a rapid shift in the steroidogenic pathway from androgen to progestogen production in spawning male common carp, Cyprinus carpio. Experiments were conducted to determine the mechanism regulating this shift using in vitro cultures of testicular fragments and isolated sperm of spermiating male carp. The levels of 11-ketotestosterone (11-KT) continually increased for 48 h with or without gonadotropin (GtH) stimulation, suggesting that 11-KT is the principal androgen produced by carp testes. Ovine prolactin (oPRL) enhanced GtH-stimulated 11-KT production, but by itself had no effect. Gonadotropin, carp pituitary extract, and pregnenolone all enhanced the production of 11-KT, testosterone (T), and 17 alpha-hydroxyprogesterone (17-P) in a dose-dependent manner. No 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P) was detected in response to any of these agents; 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one (17,20 alpha-P) was not measured. Both 17,20 beta-P and 17,20 alpha-P inhibited 11-KT production in a dose-dependent manner in the presence of either GtH, 17-P, or T. Isolated sperm and testicular fragment preparations both produced 17,20 beta-P and approximately tenfold more 17,20 alpha-P when incubated with 17-P. Only testicular fragments, however, produced 11-KT. We conclude that androgen synthesis occurs only within somatic cells of common carp testes. GtH, and perhaps PRL, stimulates the production of steroid precursors that, under normal physiological conditions, are metabolized to androgens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Androgens inhibit estradiol-17beta synthesis in Atlantic croaker (Micropogonias undulatus) ovaries by a nongenomic mechanism initiated at the cell surface

The presence of androgen receptors in the ovaries of several vertebrate species, including Atlantic croaker, suggests that androgens may have important roles in ovarian function. In the current study the effects of androgens on ovarian steroidogenesis in Atlantic croaker were investigated. Addition of 17beta-hydroxy-5alpha-androstan-3-one (DHT), 11-ketotestosterone (11-KT), or Mibolerone to ovarian incubations caused dose-dependent decreases in gonadotropin-stimulated in vitro estradiol production, which was not reversed by cotreatment with the antiandrogens, cyproterone acetate or 1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene. Androgen treatment also caused significant decreases in estradiol production in the presence of 17-hydroxyprogesterone, which suggests that the site of androgen action is downstream of this steroid in the steroidogenic pathway. The mechanism of androgen action on ovarian steroidogenesis was also investigated. Coincubation with actinomycin D did not reverse the inhibitory effect of the androgens, which suggests that the mechanism of androgen action is nongenomic. An androgen conjugated to bovine serum albumin (DHT-BSA), which does not enter the cell, also caused inhibition of estradiol production in vitro, indicating that the androgen is acting at the cell surface. In addition, time course experiments revealed that the androgen action is rapid; 5-min exposure to DHT was sufficient to cause a significant reduction in estradiol production. Finally, preliminary evidence was obtained for the existence of a high-affinity, low-capacity androgen binding site in croaker ovarian plasma membranes. These studies suggest that androgens can down-regulate estrogen production in croaker ovaries via a rapid, cell surface-mediated, nongenomic mechanism.     

Binding characteristics of estrogen receptor (ER) in Atlantic croaker (Micropogonias undulatus) testis: different affinity for estrogens and xenobiotics from that of hepatic ER.

An estrogen receptor (ER) was identified in cytosolic and nuclear fractions of the testis in a marine teleost, Atlantic croaker (Micropogonias undulatus). A single class of high affinity, low capacity, and displaceable binding sites was identified by saturation analysis, with a Kd of 0.40 nM in cytosolic extracts and a Kd of 0.33 nM in nuclear extracts. Competition studies demonstrated that the receptor was highly specific for estrogens (diethylstilbestrol > estradiol > estriol = estrone) and also bound several antiestrogens. Testosterone and 5alpha-dihydrotestosterone had much lower affinities for the receptor, whereas no displacement of specific binding occurred with 11-ketotestosterone or any of the C21 maturation-inducing steroids. A variety of xenoestrogens, including o,p'-dichlorodiphenyltrichloroethane (DDT), chlordecone (Kepone), nonylphenol, hydroxylated polychlorinated biphenyls (PCBs), and the mycotoxin zearalenone, bound to the receptor with relatively low binding affinities, 10(-3) to 10(-5) that of estradiol. A comparison of the binding affinities of various ligands for the testicular ER and the hepatic ER in this species revealed that the testicular ER was saturated at a lower [3H]estradiol concentration (1 nM vs. 4 nM). The binding affinities of several compounds, including testosterone and nafoxidine, exhibited marked differences for the two ERs; and most of the estrogens and xenoestrogens tested had higher binding affinities for the testicular receptor. Minor amounts of estradiol (0.12 ng/g tissue/h) were produced by testicular tissue fragments incubated in vitro, and estradiol was detected in male Atlantic croaker plasma. The identification of a testicular ER and evidence that estradiol is produced by the testes in croaker suggest that estrogens participate in the hormonal control of testicular function in teleosts.     

Immunohistochemical evidence for 11beta-hydroxylase (P45011beta) and androgen production in the gonad during sex differentiation and in adults in the protandrous anemonefish Amphiprion clarkii

To obtain basic information on the endocrine mechanisms underlying sex change in the protandrous anemonefish Amphiprion clarkii, we examined the immunolocalization of the steroidogenic enzyme cytochrome 11beta-hydroxylase (P45011beta), which is involved in 11-ketotestosterone (11-KT) production, and analyzed the ability of gonads to produce steroid hormones throughout the sex differentiation process and at the breeding stage. Immunopositive reactions against P45011beta appeared in sexually undifferentiated gonads at 30 days post hatching (dph). The number of immunopositive cells continued to increase during ovarian differentiation (from 60 to 180 dph) and throughout the formation of ambisexual gonads with both ovarian and testicular tissue until 270 dph. In the male phase, strongly immunopositive cells were observed in the cellular interstices of both testicular and ovarian tissues. P45011beta was localized only in the theca cells enclosing developed oocytes in the female phase. In-vitro 11-KT production in the gonads gradually increased with testicular differentiation (before, during, and after differentiation). Production of 11-KT in the gonads was higher in the male phase than during testicular differentiation or in the female phase. Our results suggest that androgen is involved in testicular differentiation during sex differentiation and spermatogenesis.     

Developmental effects of estrogenic chemicals are predicted by an in vitro assay incorporating modification of cell uptake by serum

Many estrogenic chemicals found in the environment (xenoestrogens) show a lower affinity for plasma estrogen binding proteins relative to the natural estrogens such as estradiol. These binding proteins, which include alphafetoprotein in rats and mice, sex hormone binding globulin in humans, and albumin in all species, regulate estrogen uptake into tissues. Therefore, the in vivo estrogenic potency relative to estradiol of xenoestrogens that show lower binding to these serum proteins will thus be underestimated in assays that compare the potency of xenoestrogens to estradiol and do not take serum binding into account. We have examined the effects of the binding components in serum on the uptake of a number of xenoestrogens into intact MCF-7 human breast cancer cells. Since most estrogenic chemicals are not available in radiolabeled form, their uptake is determined by competition with [³H]estradiol for binding to estrogen receptors (ER) in an 18-h assay. Serum modified access (SMA) of cell uptake of xenoestrogens is calculated as the RBA in serum-free-medium ÷ the RBA in serum, and the bioactive free fraction of xenoestrogen in serum is then also calculated. We predicted the concentration of two xenoestrogens, bisphenol A and octylphenol, required to alter development of the prostate in male mouse fetuses. Whereas octylphenol was predicted to be a more potent estrogen than bisphenol A when tested in serum-free medium, our assay predicted that bisphenol A would be over 500-times more potent than octylphenol in fetal mice. The finding that administration of bisphenol A at a physiologically relevant dose predicted from our in vitro assay to pregnant mice from gestation day 11 to 17 increased adult prostate weight in male offspring relative to controls (similar to the effect of estradiol), while the same doses of octylphenol did not alter prostate development, provided support for our hypothesis.     

Effects of sex steroids and antihormones on growth, adhesiveness and receptors of L-929 cells cultured in serum-containing and serum-free media.

L-929 cells contain distinct steroid hormone receptors for glucocorticosteroids, for androgens and for estrogens. We studied the effects of different hormones at physiological concentrations on androgen and estrogen receptor protein accumulation and on cell multiplication. The cells were cultured in steroid-free serum-containing medium, either in Petri dishes or in suspension cultures, and in serum-free medium in Petri dishes. The presence of androstanolone (30 nM) in suspension cultures decreased the concentration of estradiol receptor-binding sites in the cytoplasmic fraction. This decrease was progressive following 3, 5 or 10 days of suspension culture in the presence of the androgen; simultaneously a parallel increase in cell multiplication and DNA was observed. The estradiol receptor decrease was approx. 50% after 10 days of treatment and was unaltered after a further 5 days. It was verified that the low androstanolone concentration in the medium did not provoke the translocation of the estradiol receptor into the nucleus. Progesterone 50 nM also decreased the cytoplasmic estradiol binding sites but had no influence on cell growth and no cytoplasmic progesterone receptor could be found. Diethylstilbestrol (30 nM) did not decrease the concentration of androgen receptor.Cell multiplication was stimulated after several days of suspension culture in the presence of either diethylstilbestrol, estradiol or androstanolone at a concentration of 10–30 nM. The specific anti-hormones, tamoxifen and cyproterone acetate, inhibited selectively the growth effects of estrogens and androgen, respectively. L-929 cells could be cultured for a long period of time in serum-free medium in Petri dishes. Cell adhesiveness was increased in the presence of 40 nM androstanolone or 40 nM estradiol, as well as cell multiplication. Dexamethasone had a negative effect on cell adhesiveness and cell growth. The experimental data suggest that at low concentrations the different steroids operated each through its own receptor and were active on cell growth even in serum-free medium.     

Two sex steroid receptors in SC-115 mammary tumor cells

The growth of the SC-115 mammary carcinoma in mice is androgen dependent. Estrogens antagonize the androgen effect. The high affinity binding of androgens and estrogens has been studied in soluble extracts of the tumor, of primary culture cells and clone MI₁ cells.Results indicate that two distinct specific steroid hormone-binding sites (termed ‘receptors’) are found in all cytosol fractions. The androgen-receptor (A) binds testosterone, androstanolone, cyproterone (an anti-androgen), progesterone and estradiol, but only very weakly non-steroidal diethylstilbestrol. The estrogen-receptor (E) binds estrogenic substances such as estradiol and diethylstilbestrol, but no androgen. The apparent K_{D, eq} for A and E receptors of [³H]androstanolone and [³H]estradiol respectively, is identical (-0.5-1 nM at 4 °C). The affinity of estradiol for the A-receptor, when measured against [³H]androstanolone binding, indicates a K_i = 17.5 nM. The concentration of binding sites is of the order of 0.1 pmole/mg protein (somewhat higher for A than for E receptor) in MI₁ cell cytosol. Studies by ultracentrifugation through glycerol-Tris gradients (low salt medium) reveal the macromolecular nature of the cytosol A and E receptors (7–7.5 S). Evidence is presented of the transfer of the A and the E receptors to nuclei after incubation of tumor slices as well as of clone MI₁ cells with the corresponding hormones.Experiments suggest that the two different binding sites are present on two separated macromolecular moieties. After incubation at 37 °C of tumor slices with 10–20 nM [³H]testosterone, or with 10 nM [³H]estradiol, the corresponding radioactive hormone-receptor complexes are, as expected, found in the nuclear KCl extracts. In parallel experiments, where slices are incubated with non-radioactive hormones at the same concentration and the nuclear KCl extracts subsequently treated by radioactive steroids, no available androgen-binding sites are found in the nuclei after exposure to estradiol, nor estrogen-binding sites after exposure to testosterone.Therefore, in the same cell, two receptors are present which bind androgens and estrogens with high affinity, and one given hormone (estradiol) can be specifically bound (with different affinities) by two different receptors which, however, discriminate a synthetic analog (diethylstilbestrol). The data may give some molecular background for interpreting responses to the same hormone which may differ at various concentrations, for studying effects of analogs, and for analysing the control of tumor growth by antagonistic steroids.     

Lunar synchronization of testicular development and steroidogenesis in rabbitfish

Lunar synchronization of testicular development in the golden rabbitfish, Siganus guttatus, was assessed by measuring changes in sperm motility and conditions in the seminal plasma, and by in vitro production of steroid hormones in testicular fragments and sperm preparations. The duration and percentage of sperm motility was low 1 week before spawning (the new moon), but increased significantly on the day of spawning (the first lunar quarter). During the first lunar quarter, the osmolality decreased, but Ca(2+) concentration increased in the seminal plasma. These results suggest that spermiation occurs rapidly towards the specific lunar phase. Testicular fragments and sperm preparations were incubated with human chorionic gonadotropin (hCG) and two precursor steroid hormones, 17alpha-hydroxyprogesterone (17alpha-OHP) and testosterone (T), during the two lunar phases. The production of 11-ketotestosterone (11-KT) increased significantly when the testicular fragments were incubated with hCG at the first lunar quarter, while incubation of sperm preparations with 17alpha-OHP during the same moon phase resulted in a significant increase in 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP) production in the medium. These results suggest that 11-KT is produced in the somatic cells of the testis under the influence of gonadotropin, and that sperm can convert 17alpha-OHP to DHP. Additionally, steroidogenic activity was considered to increase toward the specific lunar phase. The synchronous increase in testicular activity supports the hypothesis that lunar periodicity is a major factor for the testicular development of S. guttatus.     

Ovarian steroidogenesis and the role of sex steroid hormones on ovarian growth and maturation of the Japanese eel

Three sex steroid hormones, estradiol-17β (E2), 11-ketotestosterone (11-KT), and 17α,20β-dihydroxy-4-pregnen-3-one (DHP), are well established as primary estrogen, androgen, and progestin, respectively, in teleost fish. Japanese eel, Anguilla japonica, would be a suitable candidate to study ovarian steroid physiology of fish because the ovarian growth and steroidogenesis is dormant under laboratory condition but can be induced by administration of exogenous gonadotropic reagents. In this review, we summarized our work on the function and production of sex steroid hormones in the ovary of the Japanese eel during ovarian growth and oocyte maturation artificially induced by treatment with extract of salmon pituitary. In vitro and in vivo assays suggest that 11-KT and E2 play primary roles in previtellogenic and vitellogenic growth of oocytes, respectively, whereas DHP is essential for induction of final oocyte maturation. We also reviewed the correlation between ovarian steroidogenesis to produce these sex steroid hormones, serum titers and gene expression.     

11-Ketotestosterone induces silvering-related changes in immature female short-finned eels, Anguilla australis.

The developmental transition from a residential, immature 'yellow' eel to a migratory, maturing adult 'silver' eel is accompanied by many morphological changes that appear to be under endocrine control. High circulating levels of the teleost, and usually male-specific, androgen 11-ketotestosterone (11-KT) are found in migrating female short-finned eels, Anguilla australis. We examined the role of this steroid in silvering by implanting immature, female short-finned eels either with blank vehicles or with vehicles containing 11-KT. Six weeks after they had received the implants, eels treated with 11-KT had developed 'chisel-shaped' snouts and black pectoral fins with tapered ends, and the size of their eyes had increased significantly. 11-KT treated eels had a thicker dermis than control eels and an epidermis with fewer or no mucous cells. Ventricular mass at the end of the experiment was two-fold larger than in control eels. 11-KT treated eels also had larger livers and gonads. Ovaries contained predominantly cortical alveolus stage III oocytes, as opposed to the smaller gonads of control eels containing previtellogenic stage II oocytes. All of these changes correspond to changes during the developmental transition from yellow to silver eels in the wild. This demonstrates that silvering in eels is under endocrine control and that the presumed male-specific steroid 11-KT is capable of inducing silvering-related changes in a female teleost. We discuss how species-specific responses to 11-KT may differ depending on tissue-specific androgen receptor abundance and how a dual demand on liver function can explain the apparently positive effects of 11-KT on liver growth.     

Androgens stimulate sex change in protogynous grouper,Epinephelus coioides: spawning performance in sex-changed males

We examined the efficacy of androgens (1.0 mg/kg body mass), testosterone (T), 11-ketotestosterone (11-KT), 17alpha-methyltestosterone (MT), testosterone propionate (TP) or androgen mixture (T, MT and TP in an equal ratio), for induction of sex change in protogynous orange-spotted grouper, Epinephelus coioides. The spawning performance in sex-changed males was also investigated. MT and androgen mixture at a dose of 1.0 mg/kg BW induced a sex transition and completion of spermatogenesis up to the functional male phase. The androgen mixture was most effective. Significantly, higher plasma T levels were found in MT and androgen mixture groups compared to control and other androgen implantation (T, TP or 11-KT) groups. We found that plasma levels of estradiol-17beta (E2) or 11-KT were not different among treated groups. Sex-changed males could successfully fertilize mature eggs. Fertilization and hatching rates were of 23.5-70.4% and 8.4-44.6%, respectively. The data demonstrated that induction of sex change by exogenous androgens in groups could apply to the aquaculture field for seed production.     

Testosterone and 11-ketotestosterone have different regulatory effects on electric communication signals of male Brachyhypopomus gauderio

The communication signals of electric fish can be dynamic, varying between the sexes on a circadian rhythm and in response to social and environmental cues. In the gymnotiform fish Brachyhypopomus gauderio waveform shape of the electric organ discharge (EOD) is regulated by steroid and peptide hormones. Furthermore, EOD amplitude and duration change on different timescales and in response to different social stimuli, suggesting that they are regulated by different mechanisms. Little is known about how androgen and peptide hormone systems interact to regulate signal waveform. We investigated the relationship between the androgens testosterone (T) and 11-ketotestosterone (11-KT), the melanocortin peptide hormone α-MSH, and their roles in regulating EOD waveform of male B. gauderio. Males were implanted with androgen (T, 11-KT, or blank), and injected with α-MSH before and at the peak of androgen effect. We compared the effects of androgen implants and social interactions by giving males a size-matched male stimulus with which they could interact electrically. Social stimuli and both androgens increased EOD duration, but only social stimuli and 11-KT elevated amplitude. However, no androgen enhanced EOD amplitude to the extent of a social stimulus, suggesting that a yet unidentified hormonal pathway regulates this signal parameter. Additionally, both androgens increased response of EOD duration to α-MSH, but only 11-KT increased response of EOD amplitude to α-MSH. Social stimuli had no effect on EOD response to α-MSH. The finding that EOD amplitude is preferentially regulated by 11-KT in B. gauderio may provide the basis for independent control of amplitude and duration.     

Estrogen and androgen regulation of sex hormone binding globulin secretion by a human liver cell line

Both estrogens and androgens have been shown to stimulate sex hormone binding globulin (SHBG) secretion in vitro in the hepatocellular carcinoma cell line, Hep G2, in contrast to the expected inhibition by androgens from in vivo studies. However, such in vitro stimulation was only demonstrated at high steroid doses, generally in serum-containing medium, with added Phenol Red. In the present study, Hep G2 cells were grown in serum-free medium, without Phenol Red, under the influence of testosterone (T) (0, 0.5-500 nM) and ethinyl estradiol (EE2) (0, 50 pM-500 nM). Levels of secreted SHBG and albumin were correlated with androgen receptors in cytosolic (ARc) and nuclear (ARn) fractions and with DNA levels. In the presence of increasing T levels, SHBG levels fell to 39% of control values at 5 nM T (P = 0.047), rising to 97% of control at 500 nM. Conversely, incubation with EE2 produced a rise in SHBG secretion of more than 100% at 0.5 nM (P less than 0.02) which was sustained to 50 nM (P less than 0.005). DNA levels did not change with the addition of testosterone or EE2, with the exception of a 15% reduction at 5 nM EE2 (P less than 0.05). Albumin levels in the medium were not significantly altered by either steroid. However, in response to T, androgen receptor (AR) levels were reduced in cytosolic (42% of control) and nuclear (22%) fractions at 5 nM, and these changes in ARc and ARn correlated with SHBG levels over the range of T concentrations (P = 0.04 and P = 0.017, respectively). Nuclear estrogen receptor (ER) increased over 10-fold at 5 and 50 pM EE2 (P less than 0.001) and maintained 50 nM (P less than 0.001). Cytosolic ER was reduced at 0.5 and 5 nM but recovered at 50 nM, correlating with SHBG levels (P less than 0.001). These findings are consistent with the hypothesis that estrogens and androgens regulate SHBG synthesis in man by direct, specific, probably receptor-mediated effects on hepatocytes. Hep G2 cells grown in serum-free medium are a suitable experimental system for further study of this phenomenon.     

Beta 2-adrenergic stimulation of androgen production by cultured mouse testicular interstitial cells

The present studies characterized the beta-receptor subtype involved in androgen production by cultured mouse testicular interstitial cells and explored the possible stimulation of androgen release by alpha-adrenergic agonists. During a 3-hour incubation period, LH and a non-specific beta-adrenergic agonist, L-isoproterenol steadily increased androgen production with a similar time-course. Isoproterenol, epinephrine, norepinephrine and a specific beta 2-receptor agonist, salbutamol stimulated androgen release in a concentration-dependent manner. The concentrations of the agonists required for half-maximum stimulation (EC50) were approximately 1 nM (isoproterenol), 8 nM (epinephrine), 9 nM (salbutamol) and 2 microM (norepinephrine) giving an order of potency of isoproterenol greater than epinephrine = salbutamol much greater than norepinephrine. L- but not the D-isomer of isoproterenol induced androgen production. A non-selective beta-receptor antagonist, propranolol, abolished androgen production induced by isoproterenol. A selective beta 2-receptor antagonist ICI 118,551 inhibited the isoproterenol effect in a concentration-dependent manner with half-maximum inhibition (IC50) at approximately 23 nM. The beta 1-receptor antagonists, metoprolol and atenolol had no effect on isoproterenol-induced androgen release. The stimulatory effect of norepinephrine (an alpha- and beta-agonist) was completely (100%) abolished by propranolol, unaffected by the alpha-antagonist phentolamine and only partially (35%) inhibited by phenoxybenzamine. Phenoxybenzamine and the alpha 2-agonist, clonidine reduced basal androgen production. These studies indicate that androgen production by primary cultures of mouse testicular interstitial cells occurs exclusively via the beta 2-receptor subtype and that alpha-receptor agonists do not stimulate androgen release by these cells.     

11-Ketotestosterone induces silvering-related changes in immature female short-finned eels, Anguilla australis

The developmental transition from a residential, immature ‘yellow’ eel to a migratory, maturing adult ‘silver’ eel is accompanied by many morphological changes that appear to be under endocrine control. High circulating levels of the teleost, and usually male-specific, androgen 11-ketotestosterone (11-KT) are found in migrating female short-finned eels, Anguilla australis. We examined the role of this steroid in silvering by implanting immature, female short-finned eels either with blank vehicles or with vehicles containing 11-KT. Six weeks after they had received the implants, eels treated with 11-KT had developed ‘chisel-shaped’ snouts and black pectoral fins with tapered ends, and the size of their eyes had increased significantly. 11-KT treated eels had a thicker dermis than control eels and an epidermis with fewer or no mucous cells. Ventricular mass at the end of the experiment was two-fold larger than in control eels. 11-KT treated eels also had larger livers and gonads. Ovaries contained predominantly cortical alveolus stage III oocytes, as opposed to the smaller gonads of control eels containing previtellogenic stage II oocytes. All of these changes correspond to changes during the developmental transition from yellow to silver eels in the wild. This demonstrates that silvering in eels is under endocrine control and that the presumed male-specific steroid 11-KT is capable of inducing silvering-related changes in a female teleost. We discuss how species-specific responses to 11-KT may differ depending on tissue-specific androgen receptor abundance and how a dual demand on liver function can explain the apparently positive effects of 11-KT on liver growth.     

The androgen receptor in LNCaP cells contains a mutation in the ligand binding domain which affects steroid binding characteristics and response to antiandrogens

The human prostate tumor cell line LNCaP containd an abnormal androgen receptor system with broad steroid binding specificity. Progestagens, estradiol and several antiandrogens compete with androgens for binding to the androgen receptor in the cells to a higher extent than in other androgen sensitive systems. Optimal growth of LNCaP cells is observed after addition of the synthetic androgen R1881 (0.1 nM). In addition, estrogens, progestagens and several antiandrogens do not inhibit androgen responsive growth, but have striking growth stimulatory effects and increase EGF receptor level and acid phosphatase secretion. We have found that the androgen receptor in the LNCaP cells contains a single point mutation changing the sense of codon 868 (Thr to Ala) in the ligand binding domain. Expression vectors containing the normal or mutated androgen receptor sequence were transfected into COS or HeLa cells. Androgens, progestagens, estrogens and several antiandrogens bind the mutated androgen receptor proteon and activate the expression of an androgen-regulated reporter gene (GRE-tk-CAT), indicating that the mutation directly affects both binding specificity and the induction of gene expression. Interestingly, the antiandrogen casodex showed antiandrogenic properties in growth studies of LNCaP cells and did not induced reporter gene activity in Hela cells transfected with the mutant receptor. The mutated androgen receptor of LNCaP cells is therefore a useful tool in the elucidation of different levels of action of steroids and antisteroids.     

Stage-Specific Effects of Androgens and Estradiol-17beta on the Development of Late Primary and Early Secondary Ovarian Follicles of Coho Salmon (Oncorhynchus kisutch) In Vitro

An in vitro system was used to analyze the effects of sex steroids on the development of primary (late perinucleolar stage) and early secondary, previtellogenic (early cortical alveolus stage) ovarian follicles of coho salmon cultured for up to 21 days. Late perinucleolar-stage follicles increased significantly in size after 7 days of treatment with low concentrations of 11-ketotestosterone (11-KT), a nonaromatizable androgen. An androgen receptor antagonist (flutamide) inhibited this growth-promoting effect, and the highest concentration resulted in atresia of follicles, implicating androgens as survival factors at this stage. Testosterone (T) was less effective than 11-KT in promoting growth, but blocking aromatization with exemestane resulted in a growth response similar to that of 11-KT. Estradiol-17beta (E2) had no effect on growth at this stage. After 21 days of culture, E2 was the most potent steroid in increasing the number of follicles containing cortical alveoli and the number of cortical alveoli within those follicles. At the early cortical alveolus stage, low doses of E2 promoted growth and strongly stimulated synthesis of cortical alveoli, actions that were inhibited by an estrogen receptor antagonist (tamoxifen). 11-KT displayed moderate growth-promoting effects, and 11-KT and T stimulated moderate to substantial increases in abundance of cortical alveoli. This study shows that the predominant role of androgens is the promotion of growth of late perinucleolar-stage follicles, while E2 stimulates both the growth and accumulation of cortical alveoli in early cortical alveolus-stage follicles.     

Brain and gonadal aromatase activity and steroid hormone levels in female and polymorphic males of the peacock blenny Salaria pavo

In the peacock blenny Salaria pavo large males with well-developed secondary sexual characters establish nests and attract females while small “sneaker” males mimic female sexual displays in order to approach the nests of larger males and parasitically fertilize eggs. These alternative reproductive tactics are sequential, as sneakers irreversibly switch into nesting males. This transition involves major morphologic and behavioral changes and is likely to be mediated by hormones. This study focuses on the role of aromatase, an enzyme that catalyses the conversion of androgens into estrogens, in the regulation of male sexual polymorphism in S. pavo. For this, sex steroid plasma levels and aromatase activity (AA) in gonads, whole brain and brain macroareas were determined in sneakers, transitional males (i.e. sneakers undergoing the transition into nesting males), nesting males and females collected in the field. AA was much higher in ovarian tissue than in testicular tissue and accordingly circulating estradiol levels were highest in females. This supports the view that elevated AA and estradiol levels are associated with the development of a functional ovary. Transitional males are in a non-reproductive phase and had underdeveloped testes when compared with sneakers and nesting males. Testicular AA was approximately 10 times higher in transitional males when compared with sneakers and nesting males, suggesting high AA has a suppressive effect on testicular development. Nesting males had significantly higher plasma levels of both testosterone (T) and 11-ketotestosterone when compared with the other male morphs and previous studies demonstrated that these androgens suppress female-like displays in sneakers. In the brain, AA was highest in macroareas presumably containing hypothalamic nuclei traditionally associated with the regulation of reproductive behaviors. Overall, females presented the highest levels of brain AA. In male morphs AA increased from sneakers, to transitional males, to nesting males in all brain macroareas. These results suggest that the transition into the nesting male tactic is accompanied both by an increase in testicular androgen production and by a higher conversion of androgens into estrogens in the brain. The increase in androgen production is likely to mediate the development of male secondary sexual characters while the increase in brain AA may be related to the behavioral changes associated with tactic transition.