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Erratum to: Breast Cancer Res Treat (2012), 134:569­581, DOI 10.1007/s10549-012-2090-9. Uunfortunately, authors could not find the original film from which the figure was drawn. Therefore, as suggested by the Editor, they have repeated the relative experiment, and ask to publish this new figure as a correction. The authors apologize for any inconvenience that it may cause.

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ERα function is crucial for the development of normal mammary gland as well as in the process of progression of breast cancer cells. Signals that target receptor levels contribute to regulate estrogens effects in the cells. An intricate cross-regulation has been documented between ERα and TGF-ß down-stream molecules: SMAD2, SMAD3, and SMAD4, that can bind ERα and regulate their signaling. Thus, identification of natural anticancer drugs able to influence the latter molecule might provide alternative choices for breast cancer treatment. Taking into account our previous published data we wanted to study the effect of 5-Methoxypsoralen (bergapten) on ERα and on TGF-ß pathway. We reported that bergapten, a coumarin containing compound, effectively depletes ERα in MCF-7 breast cancer sensitive cells and in tamoxifen-resistant clone. The decrease of ERα protein after bergapten treatment results from the ubiquitine-proteasome pathway as demonstrated by the use of MG-132. IP experiments with ER antibody, demonstrated that the protein has physical interaction with SMAD4 and poly-ubiquitine and the amount of ubiquitinated receptor, linked to SMAD4, is greater under bergapten. The crucial role played by SMAD4, in this process, emerges from the observation that in breast cancer cells, silencing of SMAD4, resulted in increased expression of endogenous ERα in both control and bergapten-treated cells, compared to wild- type cells. The same results were confirmed in siRNA TGF-ß RII cells. The results suggest a novel negative regulation of ERα by TGF-ß/SMAD4 in breast cancer cells and indicate that the SMAD4 protein is involved in the degradation of ERα induced by bergapten. We propose that bergapten may efficiently act as a natural antitumoral agent, able to deplete ERα from breast cancer tamoxifen-sensitive and resistant cells, thereby retraining the effect of membrane signals targeting ERα and in such way its mitogenic potentiality.

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Human estrogen receptors alpha and beta are crucially involved in the regulation of mammary growth and development. Normal breast tissues display a relative higher expression of ER beta than ER alpha, which drastically changes during breast tumorogenesis. Thus, it is reasonable to suggest that a dysregulation of the two estrogen receptor subtypes may induce breast cancer development. However, the molecular mechanisms underlying the potential opposing roles played by the two estrogen receptors on tumor cell growth remain to be elucidated. In the present study, we have demonstrated that ER beta overexpression in breast cancer cells decreases cell proliferation and down-regulates ER alpha mRNA and protein content, along with a concomitant repression of estrogen-regulated genes. Transient transfection experiments, using a vector containing the human ER alpha promoter region, showed that elevated levels of ER beta down-regulated basal ER alpha promoter activity. Furthermore, site-directed mutagenesis and deletion analysis revealed that the proximal GC-rich motifs at -223 and -214 are critical for the ER beta-induced ER alpha down-regulation in breast cancer cells. This occurred through ER beta-Sp1 protein-protein interactions within the ER alpha promoter region and the recruitment of a corepressor complex containing the nuclear receptor corepressor NCoR, accompanied by hypoacetylation of histone H4 and displacement of RNA-polymerase II. Silencing of NCoR gene expression by RNA interference reversed the down-regulatory effects of ER beta on ER alpha gene expression and cell proliferation. Our results provide evidence for a novel mechanism by which overexpression of ER beta through NCoR is able to down regulate ER alpha gene expression, thus blocking ER alpha's driving role on breast cancer cell growth.

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Psoralens (5-MOP and 8-MOP), a class of naturally occurring compounds, in combination with ultraviolect light are potent modulators of epidermal cell growth and differentiation. For a long time, photo-chemotherapy has been used in the treatment of psoriasis where it can reduce the number of cycling keratinocytes and decrease the IGF-1 receptors. However, the molecular mechanism of PUVA therapy remains unclear. In this study, we have evaluated, for the first time, in MCF-7 and SKBR-3 breast cancer cells the effects of 5-MOP (Bergapten), independently of its photoactivation, on the signalling pathways involved in cell cycle arrest and in apoptosis. Drug treatment induced a block in the G0/G1 phase and increased mRNA and protein levels of p53 and p21waf. These data correlate with a functional activation of caspase 8/caspase 9 together with DAPI staining and DNA ladder. Bergapten can transactivate p53 gene promoter in these cells and site-direct mutagenesis studies showed that the binding sequence of the nuclear factor NF-Y on p53 promoter is required for 5-MOP responsiveness. Besides, Bergapten increases NF-Y nuclear translocation through p38 MAPK activation. The same treatment impairs the PI3Kinase/AKT survival signal, in hormone-dependent MCF-7 cells even in the presence of IGF-I/E2 mitogenic factors. Here, we demonstrated that Bergapten, independently on the exposure to UV, generates membrane signalling pathways able to address apoptotic responses in breast cancer cells and to counteract the stimulatory effect of IGF-I/E2 on estrogen-receptor positive MCF-7 cell growth and progression.

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In the present study, we demonstrate that elevated levels of the progesterone receptor (PR)-B isoform in breast cancer cells induces down-regulation of estrogen receptor (ER) alpha mRNA and protein content, causing concomitant repression of the estrogen-regulated genes insulin receptor substrate 1, cyclin D1, and pS2, addressing a specific effect of PR/PR-B on ERalpha gene transcription. ERalpha gene promoter activity was drastically inhibited by PR-B overexpression. Promoter analysis revealed a transcriptionally responsive region containing a half-progesterone response element (PRE) site located at -1757 bp to -1752 bp. Mutation of the half-PRE down-regulated the effect induced by PR/PR-B overexpression. Moreover chromatin immunoprecipitation analyses revealed an increase of PR bound to the ERalpha-regulatory region encompassing the half-PRE site, and the recruitment of a corepressor complex containing nuclear receptor corepressor (NCoR) but not silencing mediator of retinoid and thyroid hormone receptor and DAX1, concomitantly with hypoacetylation of histone H4 and displacement of RNA polymerase II. Furthermore, NCoR ablation studies demonstrated the crucial involvement of NCoR in the down-regulatory effects due to PR-B overexpression on ERalpha protein and mRNA. We also demonstrated that the ERalpha regulation observed in MCF-7 cells depended on PR-B expression because PR-B knockdown partially abrogates the feedback inhibition of ERalpha levels after estrogenic stimulus. Our study provides evidence for a mechanism by which overexpressed PR-B is able to actively repress ERalpha gene expression.

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In the present study, the molecular mechanism underlying the up-regulatory effect of estradiol (E2) on mouse insulin receptor substrate-1 (IRS-1) promoter was investigated in CHO cells on which the same promoter had first been functionally characterized. The mouse IRS-1 promoter bears four consensus half Estrogen Responsive Elements (ERE) sequences and thirteen AP-1- and ten Sp1-binding elements. We performed molecular dissection of this promoter gene providing 3' different deleted constructs, containing the same AP-1 rich region with a progressively increased number of ERE half sites located downstream. None of these constructs was responsive to E2, while a downstream region (nt -1420 to -160) rich in GC elements was induced by E2. However, the latter region lost its intrinsic E2 responsiveness when the whole IRS-1 promoter was mutated for deletion in all four ERE half sites. Deletion analysis of the ERE half sites demonstrated that only ERE located at the position -1500 to -1495, close to the GC-rich region, was able to maintain the induced activatory effect of E2 on the IRS-1 gene. Electrophoretic mobility shift and chromatin immunoprecipitation assays identified the region containing the half ERE/Sp1 (nt -1500 to -1477) as the one conferring E2 responsiveness to the whole promoter. This effect occurs through the functional interaction between E2/ERalpha and Sp1.

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To investigate the link existing between androgens and human breast cancer, the hormonal milieu present in pre- and post-menopausal women has been translated in an in vitro model utilizing a hormone dependent breast cancer cell line MCF-7 exposed to DHEA, DHEAS, androstenediol, T, DHT with or w/o E(2). DHEAS and androstenediol stimulate the growth of MCF-7 cell line but reduce cell proliferation induced by E(2) (1 nM). T and DHT (1-100 nM) instead inhibit MCF-7 cell proliferation independently on E(2) presence. When we focused our study on the most powerful androgen, DHT alone (100 nM) consistently inhibits MCF-7 cell proliferation by 50% of the basal growth rate and counteracts E(2) proliferative action by 68%. These data correlate well with cell cycle analysis showing an enhanced number of cells in G(0)/G(1) phase after 6 days of DHT treatment. Upon prolonged DHT exposure, Western blotting analysis shows a markedly increased AR content, while immunohistochemistry indicates that it was mostly translocated into the nucleus. So we assumed that the enhanced activation of the AR might inhibit MCF-7 cells proliferation. This assumption is corroborated by the fact that the inhibitory effects induced by DHT on MCF-7 cell proliferation are abrogated in the presence of hydroxyflutamide. Therefore to better investigate the role of AR in inhibiting E(2) action at genomic level, MCF-7 cells were transiently cotransfected with the reporter plasmid XETL carrying firefly luciferase sequence under the control of an estrogen responsive element and the full length AR or with an AR carrying a mutation (Cis 574-->Arg 574) which abolishes its binding to DNA. The over-expression of the AR markedly decreases E(2) signalling which furthermore appears inhibited by simultaneous exposure to DHT but reversed by addition of hydroxyflutamide. The inhibitory effect was no longer noticeable when MCF-7 cells were cotransfected with XETL and the mutant AR. Taken together these data demonstrate that gonadal androgens antagonize MCF-7 proliferation induced by E(2). This seems to be related to the inhibitory effects of the over-expressed AR on E(2) genomic action.

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This study demonstrates how the potentiating effects of E2 on insulin signaling in ER-positive breast cancer cells are consequent to an enhanced IRS-1 expression [corrected]. It induces an increase of both PI-3K/AKT and ERK1/2 activities. A direct action of E2 in the regulating mouse IRS-1 gene is also investigated in both Chinese hamster ovary and MCF-7 cells that are transfected with mouse IRS-1 regulatory sequences. The authors have reported, for the first time, how E2 induction of IRS-1 mRNA was correlated with a direct positive regulatory role of E2 on the IRS-1 promoter. This effect seems to be not strictly related to the cell type.

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Phytoestrogens are a chemically diverse group of compounds made by plants that can have estrogenic effects in animals. Both tumorigenic and antitumorigenic effects have been reported. Although estrogens stimulate the growth of many breast tumors, there is a negative correlation between the incidence of breast cancer and the phytoestrogen-rich diet of certain Asian populations. To begin to resolve this paradox, we have analyzed the estrogenic properties of genistein and quercetin, two flavonoid phytoestrogens particularly abundant in soybeans. Trans-activation experiments with a transfected reporter gene for nuclear estrogen receptors (ER) show strong activation of the endogenous ER alpha by both phytoestrogens in two MCF7 human breast cancer cell lines. This is supported by the observation that the two phytoestrogens induce the down-regulation of ER alpha mRNA and protein levels. Using chimeric proteins consisting of the hormone binding domains of ER alpha and ER beta fused to the Gal4 DNA binding domain, we have established that genistein and quercetin are full estrogenic agonists of both ER isoforms. Ligand binding experiments with purified ER alpha and ER beta confirm that the two phytoestrogens are ER ligands. At concentrations that are sufficient to obtain substantial transcriptional activity, they stimulate the proliferation of two ER alpha-dependent breast cancer cell lines. At high concentrations, such as those reached with a soy-rich diet, genistein and quercetin are strong cytotoxic agents that even kill ER-independent HeLa cells. Thus, the mode of action of phytoestrogens and the balance between being risk or chemopreventive factors for breast cancer may depend on the dietary load.

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Transient postnatal hypothyroidism in male rats induces a prolonged proliferation of immature Sertoli cells. This change in Sertoli cell replication at young ages is coincident with enhanced and prolonged aromatase activity that leads to a marked increase in the conversion of androgens into estrogens. Both events are drastically inhibited by tri-iodothyronine (T(3)) replacement either in vivo or in vitro. This study, after the immunolocalization of aromatase in cultured rat Sertoli cells, examined the effects elicited by T(3) on this enzyme, by simultaneously investigating three functional levels of aromatase: mRNA expression, protein content, and enzymatic activity. The immunolocalization of cytochrome P450 aromatase (P450 arom) was shown in the cytoplasm of cultured Sertoli cells from 15- and 21-day-old rats. Western blot analysis revealed an enhancement of aromatase protein content upon stimulation with N(6),2'-O-dibutyryladenosine-3':5'-cyclic monophosphate ((Bu)(2)cAMP) that was clearly down-regulated by T(3). The presence of a functional P450 arom protein in purified Sertoli cells was confirmed by the measurement of [(3)H]H(2)O released after incubation with [1 beta-(3)H]androst-4-ene-3,17-dione. With 100 nM T3, a decrease in both P450 arom mRNA levels and aromatase activity was observed. The aromatase enzymatic activity was strongly stimulated by (Bu)(2)cAMP and markedly down-regulated by T(3). In contrast, the strong increase in aromatase mRNA upon (Bu)(2)cAMP stimulation was apparently unaffected by T(3) administration. This paper shows how the identification of an altered transcript induced by T(3) coding for putative truncated and inactive aromatase protein might explain such a decrease in aromatase activity in T(3)-treated cells. On the basis of these results, it is concluded that at least two mechanisms could be involved in the down-regulatory effect of T(3) on aromatase activity in prepuberal Sertoli cells. The first mechanism is linked to a possible direct modulatory role for T(3) in the regulation of the aromatase promoter, whilst the second one is represented by the induction of altered transcripts coding for truncated and inactive aromatase proteins.

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Cross-talk between steroid hormones and polypeptide growth factors regulates the growth of hormone-responsive breast cancer cells. For example, in the MCF-7 human breast cancer cell line, insulin up-regulates estrogen receptor (ER) content and binding capacity. Since the insulin receptor (IR) substrate 1 (IRS-1) is one of the core signaling elements transmitting mitogenic and metabolic effects of insulin, we investigated whether IRS-1 is also required for the insulin-induced function of the ER. The effects of insulin on the ER were compared in MCF-7 cells and MCF-7-derived cell lines with decreased levels (by approximately 80%) of IRS-1 due to the expression of IRS-1 antisense RNA. The severe IRS-1 deficiency in MCF-7 cells was associated with (1) reduced mitogenic response to 20 ng/ml insulin and 10% calf serum (CS), but not to 1 nM estradiol (E2); (2) loss of insulin-E2 synergism; (3) up-regulation of ER protein expression and binding capacity; and (4) loss of insulin-induced regulation of ER tyrosine phosphorylation. In conclusion, the data confirm the existence of the IR-ER cross-talk and suggest that IRS-1-dependent signaling may contribute to the negative regulation of the ER expression and function in MCF-7 cells.

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The effect of Tri-iodothyronine (T3) administration leading to the precocius differentiation of Sertoli cell in prepuberal rats has been previously shown. The functional maturation of Sertoli cells is associated with changes in androgen metabolism. We have recently demonstrated that T3 influences androgen metabolism in Sertoli cells by inhibiting aromatase activity and reduces drastically the ER contents in peripubertal hypothyroid rats. To better understand the role of T3 in modulating steroid action on Sertoli cells, we performed a time course study evaluating the in vitro effects of T3 and testosterone (T) on androgen (ARs) and estrogen (ERs) receptor content in Sertoli cells isolated from two weeks old Wistar rats. ARs and ERs basal levels did not change during the time course study indicating that the exposure to culture medium per se did not affect either receptor type. After 24 hrs of incubation with either T3 or T, a decrease of ERs in both nucleus and cytosol was observed. Such a decrease was augmented by the simultaneous administration of both hormones. ARs displayed a different temporal pattern in the two cellular compartments and exhibited an earlier rise in the cytosol induced by either T3 or T. At 36 hrs, ARs were significantly enhanced in both compartments in response to either T or T3 exposure while combined hormonal treatment caused an additive increase compared with the single treatment group. As a consequence of the opposite behaviour pattern displayed by ARs and ERs, the ratio between total ARs and ERs contents was increased after 24 hrs of exposure to hormonal treatment. To evaluate if treatments performed induced a functional maturation of Sertoli cells, transferrin levels in culture medium were measured. The increase of this protein paralleled that of ARs content as well as that of ARs/ERs ratio. This study demonstrates that thyroid hormone induces a progressive increase of (AR)/(ER) ratio in the differentiating Sertoli cells bringing them to a prevalent androgen dependency along their functional maturation.

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The effects of thyroid hormone on androgen metabolism in peripuberal Sertoli cells through the inhibition of estradiol production have been reported previously. It was our intention to investigate further the possible role of thyroid hormone on the interaction between testicular steroids and Sertoli cells by analyzing the effects of triiodothyronine (T3) on estrogen receptor content in 2-, 3- and 4- week-old euthyroid rats. Triiodothyronine treatment (3 micrograms/100 body wt per day) given during the last week prior to sacrifice resulted in reduced testicular growth in 2-week-old animals. Sertoli cells from all groups were cultured initially under basal conditions for the first 24 h and subsequently in the presence of testosterone and/or T3 for the additional 24 h. The in vitro addition of T3 induced a decrease of estrogen receptors (ERs) in 2- and 3-week-old animals that appeared more pronounced especially in the presence of T3 and testosterone. When T3 was tested in vivo we noticed that the decrease of ER content was even greater in all three groups under the in vitro influence of both T3 and testosterone. In 3-week-old animals a simultaneous assay of ERs in both nuclear and cytoplasmic compartments was performed. The ER concentrations in the nucleus were closely related to those of the cytoplasm. The in vivo administration of T3 was responsible for a greater decrease of ERs in the nucleus than in the cytosol. On the basis of these results, and in agreement with our previous data, we speculate that the effect of T3 in the maturational events of Sertoli cells could involve both estradiol production and ER content.

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Transforming growth factor beta (TGF-beta) is a potent growth inhibitor of non-malignant breast tissue, and TGF-beta resistance could play a role in tumorigenesis. Treatment of breast-tumor cells with anti-estrogens and progestins has been shown to correlate with an increase in the levels of secreted TGF-beta, suggesting that the growth inhibition observed with these (anti)hormones is mediated by this growth factor. In the present study we have investigated the effects of anti-estrogens and progestins on breast-tumor cell lines, which are either resistant or sensitive to TGF-beta. A hormone-independent variant of the MCF7 cell line is shown to have lost its sensitivity to TGF-beta during its progression towards an autonomous phenotype, but has preserved its sensitivity to anti-estrogens. In addition, evidence is presented showing that progestins and anti-estrogens inhibit proliferation, irrespective of the sensitivity to TGF-beta in variants of the T47D cell line. Therefore, we conclude that, although TGF-beta seems an important growth inhibitor for mammary epithelial cells, both progestins and anti-estrogens can inhibit cell proliferation independent of induced TGF-beta production.

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In a long-term experiment MCF-7 cells showed an exponential proliferation rate in fetal calf serum while they become quiescent in growth-factor- and steroid-free serum. The mitogenic activity of this cell line was increased by oestradiol and insulin, exposure to both hormones showing a synergic effect. These mitogen-mediated effects are inhibited by genistein a phytoestrogen which, by itself at the doses used, did not affect either the basal proliferation rate or the total protein content. Immunoblotting and Scatchard analysis reveal respectively that insulin increases the oestrogen receptor (ER) content and its binding capacity. The presence of genistein decreases the ER content and completely abolishes the binding capacity of this protein. Insulin is able to increase progesterone receptor (PR) levels while, in the presence of genistein, the above-reported effect was completely abolished. On the basis of the latter data the authors suggest that insulin is able to affect the phosphorylation status of ER and PR, inducing functional changes in both proteins, although this was in the absence of their natural ligands. Indeed, the presence in the medium of a tyrosine kinase inhibitor such as genistein could substantially decrease both ER and PR levels in these cells.

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A possible role of tri-iodothyronine (T3) on the interplay between testicular steroids and Sertoli cells has been investigated on the basis of previous findings demonstrating a direct inhibitory influence of T3 on aromatase activity and oestradiol production in peripuberal Sertoli cells. In this context, the present study was focused on the effects of T3 on oestrogen receptor (ER) and androgen receptor (AR) contents in the cytosol and nucleus of Sertoli cells isolated from 2-, 3- and 4-week-old euthyroid, hypothyroid and hypothyroid treated rats. Hypothyroidism was induced by the oral administration of 0.025% methimazole (MMI) from birth until the rats were killed at 2, 3 and 4 weeks of age. Half of the MMI-treated animals were injected i.p. with L-tri-iodothyronine (T3; 3 micrograms/100 g body weight) during the last week before death. Sertoli cells from all groups were initially cultured under basal conditions for the first 24 h and subsequently in the presence of testosterone with or without T3 for an additional 24 h. Hypothyroidism was associated with severe impairment of body as well as testicular growth. Euthyroid ERs showed an elevated Kd (0.76 nM) which was similar in the different age groups investigated. The in vitro addition of T3 or testosterone induced a decrease in ER content and this decrease was greater after exposure to both hormones. In 2- and 3-week-old hypothyroid rats, ER content was markedly increased and was reversed in euthyroid rats when T3 was given in vivo. When ERs were assayed in the Sertoli cell nucleus and cytoplasm of 2- and 3-week-old animals, a strong relationship in ER content in the two cellular compartments was observed. Neither of the hormones tested seemed to affect the AR content in the nucleus significantly, while the in vitro addition of testosterone or T3 or both hormones together augmented the ARs in the cytosol to a greater extent, resulting in an increase in their total (cytosolic and nuclear) content in the cells. The present data suggest that T3 down-regulates ERs and up-regulates ARs in peripuberal Sertoli cells. The additive effect of testosterone and T3 in up-regulating ARs could possibly involve a role for T3 in influencing the androgen responsiveness of the Sertoli cells during spermatogenesis.

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The inhibitory effect of triiodothyronine (T3) given in early postnatal life on Sertoli cell proliferative activity, leading to their precocious terminal differentiation, has been demonstrated previously. However, data concerning the role of thyroid hormone on androgen metabolism of Sertoli cells during the same period are still lacking. In this study we performed a time-course investigation on the effects of T3 treatment on testosterone metabolism in Sertoli cells isolated from 2-, 3- and 4-weeks-old euthyroid rats. Triiodothyronine (3 micrograms/100 g body wt) was given ip., during the last week prior to sacrifice. Sertoli cells from all animal groups initially were cultured under basal conditions during the first 24 h and subsequently in the presence of testosterone (0.5 mumol/l) with or without T3 (1 nmol/l) for an additional 24 h. This treatment given to 2-week-old animals resulted in reduced testicular growth. As far as androgen metabolism is concerned, T3 in vivo and in vitro treatment in 2- and 3-week-old animals induced a lowering of dihydrotestosterone + 3 alpha-diol with an enhancement of the two other 5 alpha-reduced androgens. The effect was much less pronounced in the oldest group. In both 2- and 3-week-old treated rats a marked reduction of oestradiol was observed, which indicates an inhibition of aromatase activity, mainly in younger animals. This enzyme has been reported to be extremely active in Sertoli cells of rats (of the same strain) between the age of 5 and 20 days, but it decreases rapidly thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)

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The aim of the present study was to investigate the influence of thyroid hormones on androgen metabolism in Sertoli cells isolated from 3- and 4- week-old rats. Hypothyroidism was induced by the oral administration of 0.025% methimazole (MMI) from birth until the rats were killed at 3 and 4 weeks of age. Half of the MMI-treated animals were injected i.p. with L-triiodothyronine (T3 3 micrograms/100 g body weight) during the last week before death. Sertoli cells from all groups were initially cultured under basal conditions for the first 24 h and subsequently in the presence of testosterone with or without T3 for an additional 24 h. Hypothyroidism was associated with severe impairment of body as well as testicular growth. Indeed, body and testicular weights were similar in 4-week-old hypothyroid animals to those in 3-week-old control rats. Testosterone metabolism in Sertoli cells isolated from 3- and 4-week-old hypothyroid rats was mainly expressed by the lowering of 5 alpha-dihydrotestosterone + androstane 3 alpha, 17 beta-diol and an enhanced formation of 5 alpha-reduced steroids with poor androgenic properties (e.g. 5 alpha-androstane, 3, 17 alpha-dione (androstanedione), 5 alpha-androstane, 3-ol-17-one (androsterone)). Treatment of the same group of animals with T3 in vivo and in vitro did not influence the pattern of 5 alpha-reductase steroids substantially. The most striking finding in the Sertoli cells of 3-week-old hypothyroid rats was the dramatic enhancement of oestradiol formation which persisted to a lesser extent 1 week later.(ABSTRACT TRUNCATED AT 250 WORDS)

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Progesterone (P), 17-OH-progesterone (17-OH-P), androstenedione (A), dehydroepiandrosterone (DHEA), testosterone (T), 5 alpha-dihydrotestosterone (5 alpha-DHT), and 17 beta-estradiol (E2) were measured by RIA in plasma and testes of 114 males of the oviparous lizard Podarcis s. sicula raf, a species that displays annual hibernating cycles. Hormones were determined each month from January until December, except for August. Testosterone peaked at 174.8 ng/ml of plasma after emergence (March), while 5 alpha-DHT and A peaked in April. Plasma DHEA increased during hibernation. During the refractory period there were progressive increases in P and E2 plasma levels. The testicular peak of T, in March, coincided with that observed in plasma. The striking increases in testicular T and A in early July occurred at a time when plasma androgen concentrations were low. 5 alpha-DHT increased in April when spermatogenesis with spermiation occurred and then decreased alongside a second peak of T. There is an apparent separation of plasma and testicular androgen concentrations during the reproductive cycle.

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The influence of hypothyroidism on testicular steroidogenesis was investigated by evaluating the production of testosterone and its precursors by isolated testes from adult male rats. Animals were made hypothyroid starting from the 4th week of life either by daily oral administration of 0.1% methimazole (MMI) or by surgical thyroidectomy (TZ). Half of the thyroidectomized rats were i.p. injected with 3 gamma T3/100 g body weight on alternate days during the last three weeks before sacrifice. Hypothyroidism is associated with a severe retardation of body growth, which appears more marked in thyroidectomized than in MMI treated rats; no significant variations in testis weight are observed. Administration of T3 does not completely restore body weight. A significant decrease in the "in vitro" production of testosterone and its precursors by testes isolated from hypothyroid rats is observed. This effect is more evident in thyroidectomized rats where a marked drop in the "in vitro" production of some testosterone delta 4 precursors is associated with the increase in DHEA/delta 4 ratio. T3 injection to thyroidectomized rats only partially restores the "in vitro" testosterone production. Results suggest that as the degree of hypothyroidism became more severe, the rate of testosterone production decreases and testicular steroidogenesis changes from the delta 4 to delta 5 metabolic pathway as a consequence of the impairement of 3-beta-ol-dehydrogenase activity.

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