Biotransformation of Phytosterols into Androstenedione-A Technological Prospecting Study.

Androstenedione (AD) is a key intermediate in the body's steroid metabolism, used as a precursor for several steroid substances, such as testosterone, estradiol, ethinyl estradiol, testolactone, progesterone, cortisone, cortisol, prednisone, and prednisolone. The world market for AD and ADD (androstadienedione) exceeds 1000 tons per year, which stimulates the pharmaceutical industry's search for newer and cheaper raw materials to produce steroidal compounds. In light of this interest, we aimed to investigate the progress of AD biosynthesis from phytosterols by prospecting scientific articles (Scopus, Web of Science, and Google Scholar databases) and patents (USPTO database). A wide variety of articles and patents involving AD and phytosterol were found in the last few decades, resulting in 108 relevant articles (from January 2000 to December 2021) and 23 patents of interest (from January 1976 to December 2021). The separation of these documents into macro, meso, and micro categories revealed that most studies (articles) are performed in China (54.8%) and in universities (76%), while patents are mostly granted to United States companies. It also highlights the fact that AD production studies are focused on "process improvement" techniques and on possible modifications of the "microorganism" involved in biosynthesis (64 and 62 documents, respectively). The most-reported "process improvement" technique is "chemical addition" (40%), which means that the addition of solvents, surfactants, cofactors, inducers, ionic liquids, etc., can significantly increase AD production. Microbial genetic modifications stand out in the "microorganism" category because this strategy improves AD yield considerably. These documents also revealed the main aspects of AD and ADD biosynthesis: Mycolicibacterium sp. (basonym: Mycobacterium sp.) (40%) and Mycolicibacterium neoaurum (known previously as Mycobacterium neoaurum) (32%) are the most recurrent species studied. Microbial incubation temperatures can vary from 29 °C to 37 °C; incubation can last from 72 h to 14 days; the mixture is agitated at 140 to 220 rpm; vegetable oils, mainly soybean, can be used as the source of a mixture of phytosterols. In general, the results obtained in the present technological prospecting study are fundamental to mapping the possibilities of AD biosynthesis process optimization, as well as to identifying emerging technologies and methodologies in this scenario.

The synthesis of steroids by Taenia crassiceps WFU cysticerci and tapeworms is related to the developmental stages of the parasites.

Taeniids tapeworms are hermaphroditic helminths that gradually develop testis and ovaries in their reproductive units. The larval stage of the tapeworms named cysticercus is a vesicle that contains the scolex and proliferates asexually in the abdominal cavity of mice. Once in the host, they evaginate, attach to the gut and develop into an adult organism, the tapeworm. We have previously reported reported that T. crassiceps ORF and solium cysticerci transform steroid precursors to androgens and estrogens. Taenia crassiceps WFU cysticerci can also synthesize corticosteroids. The aim of the present work is to investigate the relationship between steroid synthesis ability and the developmental stage of the parasite T. crassiceps WFU. To this purpose, cysticerci were obtained from the abdominal cavity of female mice, manually separated in invaginated (IC) and evaginated parasites (EC) and preincubated for 24 h in DMEM plus antibiotics/antimycotics. Next step consisted in incubation for different periods in the fresh media added with tritiated androstenedione (3H-A4) or progesterone (3H-P4) and incubated for different periods. Taenia crassiceps WFU tapeworms were recovered from the intestine of golden hamsters that had been orally infected with cysticerci. The worms were pre-cultured in DMEM plus FBS and antibiotics, and then incubated without FBS for different time periods, in the presence of 3H-A4 or 3H-P4. At the end of the experiments the media from cysticerci and tapeworms were analyzed by thin layer chromatography. Results showed that testosterone synthesis was significantly higher in the evaginated cysticerci and increased with time in culture. The invaginated and evaginated cysticerci also synthesized small quantities of 17ß-estradiol (E2) and estrone. The evaginated cysticerci synthesized twice more 3H-deoxycorticosterone (3H-DOC) than the invaginated parasites, the production increased significantly with time in culture. Taenia crassiceps WFU tapeworms synthesized significant quantities of 3H-testosterone and small amounts of estrone after only 3 h of culture in the presence of 3H-A4. The tapeworms also transformed 3H-P4 to 3H-DOC and increased its synthesis after 24 h in culture. In summary, our data show the pathways that T. crassiceps WFU cysticerci use to synthesize sexual steroids in both larval developmental stages and reveals the steroidogenic capacity of the tapeworms.

IGF2 and IGF1R in pediatric adrenocortical tumors: roles in metastasis and steroidogenesis.

Deregulation of the IGF system observed in human tumors indicates a role in malignant cell transformation and in tumor cell proliferation. Although overexpression of the IGF2 and IGF1R genes was described in adrenocortical tumors (ACTs), few studies reported their profiles in pediatric ACTs. In this study, the IGF2 and IGF1R expression was evaluated by RT-qPCR according to the patient's clinical/pathological features in 60 pediatric ACT samples, and IGF1R protein was investigated in 45 samples by immunohistochemistry (IHC). Whole transcriptome and functional assays were conducted after IGF1R inhibition with OSI-906 in NCI-H295A cell line. Significant IGF2 overexpression was found in tumor samples when compared with non-neoplastic samples (P<0.001), significantly higher levels of IGF1R in patients with relapse/metastasis (P=0.031) and moderate/strong IGF1R immunostaining in 62.2% of ACTs, but no other relationship with patient survival and clinical/pathological features was observed. OSI-906 treatment downregulated genes associated with MAPK activity, induced limited reduction of cell viability and increased the apoptosis rate. After 24h, the treatment also decreased the expression of genes related to the steroid biosynthetic process, the protein levels of the steroidogenic acute regulatory protein (STAR), and androgen secretion in cell medium, supporting the role of IGF1R in steroidogenesis of adrenocortical carcinoma cells. Our data showed that the IGF1R overexpression could be indicative of aggressive ACTs in children. However, in vitro treatments with high concentrations of OSI-906 (>1µM) showed limited reduction of cell viability, suggesting that OSI-906 alone could not be a suitable therapy to abolish carcinoma cell growth.

Effects of Adiponectin Including Reduction of Androstenedione Secretion and Ovarian Oxidative Stress Parameters In Vivo.

Adiponectin is the most abundantly produced human adipokine with anti-inflammatory, anti-oxidative, and insulin-sensitizing properties. Evidence from in vitro studies has indicated that adiponectin has a potential role in reproduction because it reduces the production of androstenedione in bovine theca cells in vitro. However, this effect on androgen production has not yet been observed in vivo. The current study evaluated the effect of adiponectin on androstenedione secretion and oxidative stress parameters in a rodent model. Seven-week-old female Balb/c mice (n = 33), previously treated with equine gonadotropin chorionic, were assigned to one of four different treatments: Group 1, control (phosphate-buffered saline); Group 2, adiponectin 0.1 µg/mL; Group 3, adiponectin 1.0 µg/mL; Group 4, adiponectin 5.0 µg/mL. After 24 h, all animals were euthanized and androstenedione levels were measured in the serum while oxidative stress markers were quantified in whole ovary tissue. Female mice treated with adiponectin exhibited a significant reduction (about 60%) in serum androstenedione levels in comparison to controls. Androstenedione levels decreased from 0.78 ± 0.4 ng/mL (mean ± SD) in controls to 0.28 ± 0.06 ng/mL after adiponectin (5 µg/mL) treatment (P = 0.01). This change in androgen secretion after 24 hours of treatment was associated with a significant reduction in the expression of CYP11A1 and STAR (but not CYP17A1). In addition, ovarian AOPP product levels, a direct product of protein oxidation, decreased significantly in adiponectin-treated mice (5 µg/mL); AOPP (mean ± SD) decreased to 4.3 ± 2.1 µmol/L in comparison with that of the controls (11.5 ± 1.7 µmol/L; P = 0.0003). Our results demonstrated for the first time that acute treatment with adiponectin reduced the levels of a direct oxidative stress marker in the ovary as well as decreased androstenedione serum levels in vivo after 24 h.

Placental Aromatase Is Deficient in Placental Ischemia and Preeclampsia.

INTRODUCTION: Preeclampsia is a maternal hypertensive disorder with uncertain etiology and a leading cause of maternal and fetal mortality worldwide, causing nearly 40% of premature births delivered before 35 weeks of gestation. The first stage of preeclampsia is characterized by reduction of utero-placental blood flow which is reflected in high blood pressure and proteinuria during the second half of pregnancy. In human placenta androgens derived from the maternal and fetal adrenal glands are converted into estrogens by the enzymatic action of placental aromatase. This implies that alterations in placental steroidogenesis and, subsequently, in the functionality or bioavailability of placental aromatase may be mechanistically involved in the pathophysiology of PE. METHODS: Serum samples were collected at 32-36 weeks of gestation and placenta biopsies were collected at time of delivery from PE patients (n = 16) and pregnant controls (n = 32). The effect of oxygen tension on placental cells was assessed by incubation JEG-3 cells under 1% and 8% O2 for different time periods, Timed-mated, pregnant New Zealand white rabbits (n = 6) were used to establish an in vivo model of placental ischemia (achieved by ligature of uteroplacental vessels). Aromatase content and estrogens and androgens concentrations were measured. RESULTS: The protein and mRNA content of placental aromatase significantly diminished in placentae obtained from preeclamptic patients compared to controls. Similarly, the circulating concentrations of 17-ß-estradiol/testosterone and estrone/androstenedione were reduced in preeclamptic patients vs. controls. These data are consistent with a concomitant decrease in aromatase activity. Aromatase content was reduced in response to low oxygen tension in the choriocarcinoma JEG-3 cell line and in rabbit placentae in response to partial ligation of uterine spiral arteries, suggesting that reduced placental aromatase activity in preeclamptic patients may be associated with chronic placental ischemia and hypoxia later in gestation. CONCLUSIONS: Placental aromatase expression and functionality are diminished in pregnancies complicated by preeclampsia in comparison with healthy pregnant controls.

Role of macrophage secretions on rat polycystic ovary: its effect on apoptosis.

Polycystic ovarian syndrome is the most common endocrine disorder among women of reproductive age. Little is known about its etiology, although the evidence suggests an intrinsic ovarian abnormality in which endocrine, metabolic, neural and immune factors would be involved. In this work, the effects of macrophage (MO) secretion on ovarian apoptosis in a polycystic ovary syndrome rat model (PCO rat) induced by estradiol valerate are studied. Spleen MO secretions were used to stimulate ovaries and ovarian interstitial and granulosa cells from both PCO and control rats. Ovarian hormones and prostaglandin E2 (PGE2) were measured by RIA; ovarian mRNA levels of Bax, Bcl2 and NFkB by RT-PCR; and ovarian inducible nitric oxide synthase (iNOS) by western blot. The number of apoptotic cells was evaluated by TUNEL. In the PCO ovary, the MO secretions from PCO rats increased the Bax and NFkB mRNA expressions and increased TUNEL staining in both granulosa and theca cells. In addition, the PCO MO secretions produced a decrease of nitric oxide release, iNOS protein level and PGE2 content in the PCO ovary, and it also induced an increase of androstenedione production by PCO interstitial cells, in comparison with control MO secretions. Considering these results and knowing that testosterone stimulates tumour necrosis factor-α production by PCO MO modifying ovarian response by increasing androstenedione, it is reasonable to suggest that the increase of androgens stimulated in ovarian cells by PCO MO secretions could in turn stimulate the cytokine production from MO, thus maintaining an apoptotic vicious cycle in the PCO ovary.

Comparison of steroidogenic pathways among normoandrogenic and hyperandrogenic polycystic ovary syndrome patients and normal cycling women.

AIM: To compare the corticosteroidogenic enzyme activities between normal cycling non-polycystic ovary syndrome (PCOS), and normoandrogenic PCOS (NA-PCOS) and hyperandrogenic PCOS (HA-PCOS) patients. METHODS: This cohort study was conducted at Julio Muller University Hospital and Tropical Institute of Reproductive Medicine and Menopause, and enrolled 114 non-PCOS women and 355 PCOS patients. The steroidogenic enzyme activities were measured using the serum steroid product/precursor molar ratio. RESULTS: In the Δ5 pathway the 17,20 lyase activity was equally low in the NA-PCOS and HA-PCOS women compared with the non-PCOS women (P < 0.01 and P < 0.001, respectively). In the Δ4 pathway, the 17,20 lyase activity was higher only in the HA-PCOS group (P < 0.001). The 17-hydroxylase activity was the same in PCOS and non-PCOS subjects (P > 0.05). The 3ß-hydroxysteroid dehydrogenase II (3ß-HSDII) activity was higher in the conversion of dehydroepiandrosterone into androstenedione in the HA-PCOS than in the NA-PCOS (P < 0.05) and the non-PCOS patients (P < 0.01). The aromatase activity was lower in the HA-PCOS than in the NA-PCOS (P < 0.05) patients and non-PCOS subjects (P < 0.01). In HA-PCOS subjects, the 17,20 lyase activity was related to insulin, estradiol, total testosterone concentrations and free androgen index in the Δ5 pathway. 3ß-HSDII showed weak correlation with estradiol in the HA-PCOS group. Anthropometric parameters had little impact, if any, on the steroidogenic enzyme activities. CONCLUSION: The NA-PCOS and HA-PCOS patients demonstrated different enzyme activities, and the results provided new directions for future studies including PCOS patients with different phenotypes.

The role of androst-5-ene-3ß,17ß-diol (androstenediol) in cell proliferation in endometrium of women with polycystic ovary syndrome.

Women with polycystic ovary syndrome (PCOS) show high prevalence of endometrial hyperplasia and adenocarcinoma. Endometrial proliferation is increased, evaluated by high levels of Ki67 (cell cycle marker) and low levels of p27 (negative regulator of cell cycle). Nevertheless, endometrial changes in cyclin D1 (positive regulator of cell cycle) in PCOS-women are not described. Androst-5-ene-3ß,17ß-diol (androstenediol), steroid with estrogenic activity present in endometria, could be related to increased endometrial cell proliferation. The objective of this study was to determine protein content of cyclin D1 and androstenediol levels in endometria from PCOS and control-women and to evaluate the possible mechanism favoring cell proliferation associated with hormonal characteristics of patients. Therefore, cyclin D1 protein content in PCOS-women and control-endometrial tissue were assessed by western blot and immunohistochemistry. The androstenediol levels were evaluated by ELISA. To further analyze the effect of steroids (androstenediol, 17ß-estradiol, testosterone) in cell proliferation, levels of proteins cyclin D1, p27 and Ki67 were evaluated in an in vitro model of stromal endometrial cells T-HESC and St-T1b. An increase in cyclin D1 and androstenediol was observed in tissues from PCOS-women relative to control group (p<0.05). In the in vitro model, androstenediol exerted increase in cyclin D1 (p<0.05) and a decrease in p27 protein level (p<0.05), while Ki67 in St-T1b cells increased under this stimulus (p<0.05). Testosterone produces opposite effects in the levels of the above markers (p<0.05). Therefore, the hormonal imbalance associated with this syndrome could alter endometrial tissue homeostasis, promoting cell proliferation. Androstenediol is a molecule that could be involved by stimulating proliferation, whereas testosterone elicits a role of cell cycle repressor.

Adiponectin and its receptors in the ovary: further evidence for a link between obesity and hyperandrogenism in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS), characterized by ovarian androgen excess, is the commonest endocrine disorder in women. Obesity increases androgen synthesis, a phenomenon attributed to the accompanying hyperinsulinemia. Our hypothesis was that adipokines, fat cell-derived hormones, play a direct role in modulating ovarian androgen secretion. Therefore, the aims of this study were to explore the effects of adipokines (in particular, adiponectin) on ovarian steroidogenesis and compare the expression of adiponectin receptors in ovaries from women with and without PCO. Sections of archived human ovaries (nine from women with normal ovaries and 16 with PCOS, classified histologically, with reference to menstrual history and ultrasound) were analysed by quantitative morphometry and the proportion of positive-labelling cells compared. In addition, studies of androgen production in relation to adipokine function in primary bovine theca cell culture were also performed. A significantly lower proportion of theca cells expressed adiponectin receptors 1 and 2 (AdipoR1, AdipoR2) in polycystic ovaries than in normal ovaries. In cultured theca cells, adiponectin suppressed androstenedione production and gene expression of LH receptor and key enzymes in the androgen synthesis pathway. Moreover, knockdown of genes for AdipoR1 and AdipoR2 was associated with increased androstenedione secretion by bovine theca cells. These results provide evidence for a direct link between fat cell metabolism and ovarian steroidogenesis, suggesting that disruption of adiponectin and/or its receptors plays a key role in pathogenesis of hyperandrogenism in PCOS.

Metabolism of dehydroepiandrosterone sulfate and estrone-sulfate by human platelets.

The aim of the present research was to study the uptake of DHEAS, and to establish the intracrine capacity of human platelets to produce sex steroid hormones. The DHEAS transport was evaluated through the uptake of [(3)H]-DHEAS in the presence or absence of different substrates through the organic anion transporting polypeptide (OATP) family. The activity of sulfatase enzyme was evaluated, and the metabolism of DHEAS was measured by the conversion of [(3)H]-DHEAS to [(3)H]-androstenedione, [(3)H]-testosterone, [(3)H]-estrone and [(3)H]-17beta-estradiol. Results indicated the existence in the plasma membrane of an OATP with high affinity for DHEAS and estrone sulphate (E(1)S). The platelets showed the capacity to convert DHEAS to active DHEA by the steroid-sulfatase activity. The cells resulted to be a potential site for androgens production, since they have the capacity to produce androstenedione and testosterone; in addition, they reduced [(3)H]-estrone to [(3)H]-17beta-estradiol. This is the first demonstration that human platelets are able to import DHEAS and E(1)S using the OATP family and to convert DHEAS to active DHEA, and to transform E(1)S to 17beta-estradiol.

Macrophage secretions modulate the steroidogenesis of polycystic ovary in rats: effect of testosterone on macrophage pro-inflammatory cytokines.

AIMS: The macrophage secretions' effect on ovarian steroidogenesis is investigated in a polycystic ovary syndrome rat model (PCO rat). The influence of testosterone environment on the expression of macrophage pro-inflammatory cytokines that participate in ovarian steroidogenesis is studied. MAIN METHODS: PCO rats were induced by estradiol valerate. Spleen macrophages were cultured with and without testosterone (10(-6) M) and their secretions were used to stimulate ovaries from PCO and control rats. Ovarian hormones released and ovary mRNA levels of P450 aromatase and 3ß-hydroxysteroid dehydrogenase were measured by radioimmunoassay and RT-PCR, respectively. The tumor necrosis factor alpha (TNFα) and nitric oxide (NO) levels in macrophage culture medium, along with the TNFα, interleukin (IL)-6, IL-10 and androgen receptors (AR) mRNA levels in macrophage cells were determined. KEY FINDINGS: Macrophages from PCO rats released more TNFα and NO, expressed higher TNFα and IL-6, lower AR, and no change in IL-10 mRNA levels than control macrophages. TNFα, IL-6 and AR changes were greater after macrophage testosterone treatment. Macrophage secretions from PCO rats stimulated androstenedione and decreased estradiol release and ovarian mRNA P450 aromatase expression in PCO rats compared to macrophage secretions from control rats. These effects were greater when macrophages from PCO rats were treated with testosterone. Ovarian progesterone response was unchanged. SIGNIFICANCE: The differential steroidogenic ability of macrophage secretions from PCO rats is associated to the in vitro testosterone environment. Testosterone, probably acting on macrophage AR, induces a greater release of TNFα, modifying ovarian response by increasing androstenedione and slightly decreasing estradiol without affecting progesterone.

Involvement of the mesenteric ganglia on androstenedione, noradrenaline and nitrite release using a testis ex vivo system.

The autonomic nerve fibres converge to the testis along two major pathways, the superior spermatic nerve (SSN) and the inferior spermatic nerve (ISN). The object of this work was to evaluate whether the addition of noradrenaline (NA) in the ganglionic compartment of two ex vivo systems: superior mesenteric ganglion (SMG)-SSN-testis, inferior mesenteric ganglion (IMG)-ISN-testis modulate androstenedione (A2), NA and nitrite release and to determine whether there are secretory differences between the right and the left testis. Each gonad with its respective ganglion was transferred into a cuvette with two compartments and incubated in a Dubnoff metabolic shaker. The testis incubation liquids were collected and analysed for NA by HPLC, A2 by RIA and nitrites by the Griess method. When NA is added to the IMG, A2 and NA release diminishes and nitrite increases in the left testis, while in the right gonad, A2 and NA increase and nitrite decreases. When NA was administered to the SMG, A2 and NA increase and nitrite diminishes in the left gonad, but they show opposite fluctuations in the right testis. These ex vivo systems appear to be excellent models for studying the sympathetic ganglionic control of the testis though A2, NA and nitrite release from the male gonad. It is evident that a better knowledge about the role of catecholamines and nitric oxide in the testis physiology may facilitate the understanding of some reproductive diseases.

Androstenedione acts on the coeliac ganglion and modulates luteal function via the superior ovarian nerve in the postpartum rat.

Androstenedione can affect luteal function via a neural pathway in the late pregnant rat. Here, we investigate whether androstenedione is capable of opposing to regression of pregnancy corpus luteum that occurs after parturition, indirectly, from the coeliac ganglion. Thus, androstenedione was added into the ganglionar compartment of an ex vivo coeliac ganglion-superior ovarian nerve-ovary system isolated from non-lactating rats on day 4 postpartum. At the end of incubation, we measured the abundance of progesterone, androstenedione and oestradiol released into the ovarian compartment. Luteal mRNA expression and activity of progesterone synthesis and degradation enzymes, 3ß-hydroxysteroid-dehydrogenase (3ß-HSD) and 20α-hydroxysteroid-dehydrogenase (20α-HSD), respectively, as well as the aromatase, Bcl-2, Bax, Fas and FasL transcript levels, were also determined. Additionally, we measured the ovarian release of norepinephrine, nitric oxide and luteal inducible nitric oxide synthase (iNOS) mRNA expression. The presence of androstenedione in the ganglion compartment significantly increased the release of ovarian progesterone, androstenedione and oestradiol without modifying 3ß-HSD and 20α-HSD activities or mRNA expression. The ovarian release of oestradiol in response to the presence of androstenedione in the ganglion compartment declined with time of incubation in accord with a reduction in the aromatase mRNA expression. Androstenedione added to the ganglion compartment decreased FasL mRNA expression, without affecting luteal Bcl-2, Bax and Fas transcript levels; also increased the release of norepinephrine, decreased the release of nitric oxide and increased iNOS mRNA. In summary, on day 4 after parturition, androstenedione can mediate a luteotropic effect acting at the coeliac ganglion and transmitting to the ovary a signaling via a neural pathway in association with increased release of norepinephrine, decreased nitric oxide release, and decreased expression of FasL.

Steroidal 5α-reductase inhibitors using 4-androstenedione as substrate.

The aim of this study was to determine the capacity of some progesterone derivatives, to inhibit the conversion of labeled androstenedione ([(3)H] 4-dione) to [(3)H]dihydrotestosterone ([(3)H]DHT) in prostate nuclear membrane fractions, where the 5α-reductase activity is present. The enzyme 5α-reductase catalyzes the 5α-reduction of 4-dione whereas the 17ß-hydroxysteroid dehydrogenase catalyzes the transformation of 4-dione to testosterone or 5α-dione to dihydrotestosterone (DHT). Moreover, we also investigated the role of unlabeled 5α-dione in these pathways. In order to determine the inhibitory effect of different concentrations of the progesterone derivatives in the conversion of [(3)H] 4-dione to [(3)H]DHT, homogenates of human prostate were incubated with [(3)H] 4-dione, NADPH and increasing concentrations of non-labeled 5α-dione. The incubating mixture was extracted and purified using thin layer chromatography. The fraction of the chromatogram corresponding to the standard of DHT was separated and the radioactivity determined. The results showed that the presence of [(3)H] 4-dione plus unlabelled 5α-dione produced similar levels of DHT as compared to [(3)H] 4-dione. On the other hand, the results indicated that 17α-hydroxypregn-4-ene-3,20-dione 5 and 4-bromo-17α-hydroxypregn-4-ene-3,20-dione 7b, were the most potent steroids to inhibit the conversion of [(3)H] 4-dione to [(3)H]DHT, showing IC(50) values of 2 and 1.6 nM, respectively.

Modulation of the noradrenergic activity index by neural stimulus, and its participation in ovarian androstenedione release during the luteal phase.

OBJECTIVE: To investigate the participation of catecholamines in the association between peripheral innervation and luteal steroidogenesis. DESIGN: Animal study. SETTING: University animal laboratory. ANIMAL(S): Six to eight virgin adult Holtzman-strain female rats in control and experimental groups on diestrus days 1 and 2. INTERVENTION(S): Removal of the coeliac ganglion-superior ovarian nerve-ovary system, with catecholaminergic agonist or antagonist added in the ganglion compartment (experimental group only). The control group received no treatment. MAIN OUTCOME MEASURE(S): Ovarian neurotransmitters and their catabolites measured by reverse-phase high-pressure liquid chromatography, and A(2) measured by radioimmunoassay. RESULT(S): On day 1, dopamine and catabolite increased whereas norepinephrine decreased, and the noradrenergic neuronal activity index was higher. On day 2, dopamine levels decreased, norepinephrine increased, and dopaminergic neuronal activity was higher. The release of A(2) was decreased by addition of norepinephrine to the ganglions on day 1, but was increased by the norepinephrine antagonist on day 2. Hence, norepinephrine increased A(2) release, and propranolol diminished it. CONCLUSION(S): Ganglionic activity is modified by noradrenergic stimulus, leading to different ovarian A(2) release profiles. The peripheral nervous system is a modulator in these homeostatic mechanisms.

Ovaric physiology in the first oestral cycle: influence of noradrenergic and cholinergic neural stimuli from coeliac ganglion.

Both peripheral innervation and nitric oxide (NO) participate in ovarian steroidogenesis. The aims of the work were (1) to investigate whether ganglionic noradrenergic (NE) and cholinergic (Ach) stimulus modify the ovarian steroids and NO release and (2) to examine the effect of those stimuli on the mRNA expression of 3beta-HSD and P450 aromatase in the ovary. The experiments were carried out using the ex vivo coeliac ganglion-superior ovarian nerve-ovary (CG-SON-O) system of rats in the first oestral cycle. The system was incubated in a buffer solution for 120min, with the ganglion and ovary located in different compartments and linked by the SON. NE and Ach were added into the ganglion compartment. Both NE and Ach predominantly induced ovarian release of androstenedione and oestradiol while inhibited progesterone release. Ovarian NO release increased after ganglionic stimulation during proestrous while its secretion decreased during the diestrous. Noteworthily, 3beta-HSD and P450 aromatase expression were modulated by neural stimulation. In the follicular phase, Ach in CG increased 3beta-HSD and NE increased P450 aromatase. In the luteal phase both neurotransmitters increased 3beta-HSD and Ach increased P450 aromatase transcript levels. All above observations suggest that the preponderancy of an either noradrenergic or cholinergic effect would depend on the stage of the first oestral cycle in the rat. The ovarian response to noradrenergic and cholinergic stimuli on GC, via SON, is strongly linked to oestral-stage-specific ovarian structures and their secretion products.

Steroidogenic capacity of Trypanosoma cruzi trypomastigotes.

American Trypanosomiasis is caused by the hemoflagellate Trypanosoma cruzi (T. cruzi) and affects millions of persons causing variable degrees of digestive and heart disturbances. As far as we concerned, T. cruzi capacity to synthesize steroid hormones has not been investigated. Therefore, the aim of this work was to investigate the capacity of T. cruzi trypomastigotes to transform tritiated steroid precursors into androgens and estrogens. The T. cruzi Tulahuén strain was obtained from mice blood. The trypomastigotes were cultured for 6 and 24h in Dulbbeco's modified Eagle's medium plus FCS and antibiotics. Tritiated dehydroepiandrosterone or androstendione were added to the culture media and parasites were incubated for 6 or 24h. The cultures were centrifuged and ether extracted. The steroids were analyzed by thin layer chromatography (TLC) in two solvent systems. After incubation with 3H-androstenedione, T. cruzi trypomastigotes synthesized 3H-testosterone (T), 3H-17beta-estradiol (E2) and 3H-estrone (E1). Metabolism of 3H-DHEA by the parasites yielded 3H-androstendione and 3H-androstendiol at 6h of incubation. The recrystallization procedure further demonstrated the 3H-androstendiol and 3H-17beta-estradiol syntheses. Results indicate for the first time that T. cruzi trypomastigotes produce androgens and estrogens when incubated in the presence of steroid precursors and suggest the presence of active parasite steroidogenic enzymes.

Morphological and endocrine study of the ovarian interstitial tissue of viscacha (Lagostomus maximus maximus).

The morphological and endocrine aspects of the ovarian interstitial tissue of adult female viscachas were investigated to establish the probable function and the biological significance of this compartment in this rodent. Pregnant and nonpregnant adult female viscachas were used. The histological characteristics, histochemical properties, and ultrastructural features of the interstitial tissue were studied. A morphometric study was carried out to measure the relative area of lipid droplets. The progesterone and androstenedione levels in ovarian tissue as well as in serum were determined by radioimmunoassay. In this species, the histological observations showed an abundant interstitial tissue that contained a large amount of lipids. The cholesterol and its esters were present in nonpregnant females and were scarce in pregnant animals. The most ultrastructural differences were observed at mid-pregnancy. At this stage, the interstitial cells showed features that suggested higher steroidogenic activity. Furthermore, during mid-pregnancy, the relative area of lipid droplets was smaller. Both progesterone and androstenedione levels in ovarian tissue and serum were higher during pregnancy. Our results suggest that the interstitial tissue may be storage of precursor substances for the steroidogenesis via. These precursors are probably used when the endocrine requirements are high, that is, during the pregnancy. Thus, this compartment may contribute to the normal gestation of Lagostomus. However, the relation between the interstitial tissue and the pregnancy is complex, and further studies are needed to clearly establish it.

Participation of vasoactive intestinal polypeptide in ovarian steroids production during the rat estrous cycle and in the development of estradiol valerate-induced polycystic ovary.

Vasoactive intestinal polypeptide (VIP) stimulates estradiol and progesterone release from ovarian granulosa cells in vitro. Very little information is available as to the role VIP plays in the control of steroid secretion during reproductive cyclicity and in ovarian pathologies involving altered steroid secretion. In this study, we determined the involvement of VIP in regulating ovarian androgen and estradiol release during estrous cyclicity and estradiol valerate (EV)-induced polycystic ovarian development in rats. Our findings show that androgen and estradiol release from ovaries obtained during different stages of rat estrous cycle mimic cyclic changes in steroid release observed in vivo with maximal release occurring during late proestrus. VIP increased androgen release from ovaries of all cycle stages except late proestrus and estradiol release from all cycle stages. Increases in VIP-induced androgen and estradiol release were maximal at early proestrus. Inclusion of saturating concentrations of androstenedione increased magnitude of VIP-induced estradiol release at diestrus and estrus but not proestrus. Magnitude of VIP-induced androgen and estradiol release tended to be greater in the ovaries from EV-treated rats with polycystic ovary compared with estrous controls. At the tissue level, ovarian VIP concentration was cycle stage dependent with highest level seen in diestrus. Maximum concentration of VIP was found in EV-treated rats. Changes in VIP were inversely related to changes in ovarian nerve growth factor, a neuropeptide involved in ovarian androgen secretion. These results strongly suggest that intraovarian VIP participates in the control of estradiol secretion during the rat estrous cycle and possibly in the maintenance of increased ovarian estradiol secretory activity of EV-treated rats.

The celiac ganglion modulates LH-induced inhibition of androstenedione release in late pregnant rat ovaries.

BACKGROUND: Although the control of ovarian production of steroid hormones is mainly of endocrine nature, there is increasing evidence that the nervous system also influences ovarian steroidogenic output. The purpose of this work was to study whether the celiac ganglion modulates, via the superior ovarian nerve, the anti-steroidogenic effect of LH in the rat ovary. Using mid- and late-pregnant rats, we set up to study: 1) the influence of the noradrenergic stimulation of the celiac ganglion on the ovarian production of the luteotropic hormone androstenedione; 2) the modulatory effect of noradrenaline at the celiac ganglion on the anti-steroidogenic effect of LH in the ovary; and 3) the involvement of catecholaminergic neurotransmitters released in the ovary upon the combination of noradrenergic stimulation of the celiac ganglion and LH treatment of the ovary. METHODS: The ex vivo celiac ganglion-superior ovarian nerve-ovary integrated system was used. This model allows studying in vitro how direct neural connections from the celiac ganglion regulate ovarian steroidogenic output. The system was incubated in buffer solution with the ganglion and the ovary located in different compartments and linked by the superior ovarian nerve. Three experiments were designed with the addition of: 1) noradrenaline in the ganglion compartment; 2) LH in the ovarian compartment; and 3) noradrenaline and LH in the ganglion and ovarian compartments, respectively. Rats of 15, 19, 20 and 21 days of pregnancy were used, and, as an end point, the concentration of the luteotropic hormone androstenedione was measured in the ovarian compartment by RIA at various times of incubation. For some of the experimental paradigms the concentration of various catecholamines (dihydroxyphenylalanine, dopamine, noradrenaline and adrenaline) was also measured in the ovarian compartment by HPLC. RESULTS: The most relevant result concerning the action of noradrenaline in the celiac ganglion was found on day 21 of pregnancy resulting in the inhibition of androstenedione release from the ovarian compartment. In addition on day 15 of pregnancy, LH placed in the ovarian compartment led to an inhibition of the release of androstenedione, and this inhibitory effect was further reinforced by the joint action of noradrenaline in the celiac ganglion and LH in the ovary. The levels of catecholamines in the ovarian compartment showed differences among the experiments; of significance, the joint treatment of noradrenaline in the celiac ganglion and LH in the ovary resulted in a remarkable increase in the ovarian levels of noradrenaline and adrenaline when compared to the effect achieved by either one of the compounds added alone. CONCLUSION: Our results demonstrate that the noradrenergic stimulation of the celiac ganglion reinforces the LH-induced inhibition of androstenedione production by the ovary of late pregnant rats, and that this effect is associated with marked changes in the release of catecholamines in the ovary.