Identification of maturation-inducing steroid in sturgeons via comprehensive analyses of steroids produced during oocyte maturation.

Although 17α, 20ß-dihydroxy-4-pregnen-3-one (DHP) and 17α, 20ß, 21-trihydroxy-4-pregnen-3-one (20ß-S) have been identified as maturation-inducing steroids (MIS) in several teleosts, to date, no MISs have been identified in sturgeons. As it remains possible that an unidentified steroid is an MIS in sturgeons, this study aimed to identify a sturgeon MIS via comprehensive analyses and maturation-inducing (MI) assay of C21 steroids. In vivo and in vitro comprehensive analyses of C21 steroids revealed that serum DHP concentrations were rapidly elevated in the oocyte maturation phase and the DHP production level was notably high among C21 steroids. MI assay indicated that the MI activity of DHP, 17α-hydroxyprogesterone (17OHP), a precursor of DHP, 17α, 20α-dihydroxy-4-pregnen-3-one (αDHP), and 20ß-S was high among C21 steroids, but the MI activity of these steroids were similar. In the C21 steroids produced in ovarian follicles during oocyte maturation, 17OHP, αDHP, and unidentified compounds had a low production level, and 20ß-S was suggested to be metabolized from DHP after oocyte maturation. Against this background, this study concluded that DHP is a steroid that possesses strong MI activity and is highly produced during oocyte maturation. Although this study could not identify an MIS in sturgeons by fractionation of plasma and subsequent bio assay, it was suggested that DHP is a major MIS in sturgeons.

Characterization of human adrenal cytochrome P450 11B2 products of progesterone and androstenedione oxidation.

Cytochrome P450 (P450) 11B1 and 11B2 both catalyze the 11ß-hydroxylation of 11-deoxycorticosterone and the subsequent 18-hydroxylation of the product. P450 11B2, but not P450 11B1, catalyzes a further C-18 oxidation to yield aldosterone. 11-Oxygenated androgens are of interest, and 11-hydroxy progesterone has been reported to be a precursor of these. Oxidation of progesterone by purified recombinant P450 11B2 yielded a mono-hydroxy derivative as the major product, and co-chromatography with commercial standards and 2-D NMR spectroscopy indicated 11ß-hydroxylation. 18-Hydroxyprogesterone and a dihydroxyprogesterone were also formed. Similarly, oxidation of androstenedione by P450 11B2 yielded 11ß-hydroxyandrostenedione, 18-hydroxyandrostenedione, and a dihydroxyandrostenedione. The steady-state kinetic parameters for androstenedione and progesterone 11ß-hydroxylation were similar to those reported for the classic substrate 11-deoxycorticosterone. The source of 11α-hydroxyprogesterone in humans remains unresolved.

CYP17A1 exhibits 17αhydroxylase/17,20-lyase activity towards 11ß-hydroxyprogesterone and 11-ketoprogesterone metabolites in the C11-oxy backdoor pathway.

Cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17A1) plays a pivotal role in the regulation of adrenal and gonadal steroid hormone biosynthesis. More recent studies highlighted the enzyme's role in the backdoor pathway leading to androgen production. Increased CYP17A1 activity in endocrine disorders and diseases are associated with elevated C21 and C19 steroids which include 17α-hydroxyprogesterone and androgens, as well as C11-oxy C21 and C11-oxy C19 steroids. We previously reported that 11ß-hydroxyprogesterone (11OHP4), 21-deoxycortisol (21dF) and their keto derivatives are converted by 5α-reductases and hydroxysteroid dehydrogenases yielding C19 steroids in the backdoor pathway. In this study the 17α-hydroxylase and 17,20-lyase activity of CYP17A1 towards the unconventional C11-oxy C21 steroid substrates and their 5α- and 3α,5α-reduced metabolites was investigated in transfected HEK-293 cells. CYP17A1 catalysed the 17α-hydroxylation of 11OHP4 to 21dF and 11-ketoprogesterone (11KP4) to 21-deoxycortisone (21dE) with negligible hydroxylation of their 5α-reduced metabolites while no lyase activity was detected. The 3α,5α-reduced C11-oxy C21 steroids-5α-pregnan-3α,11ß-diol-20-one (3,11diOH-DHP4) and 5α-pregnan-3α-ol-11,20-dione (alfaxalone) were rapidly hydroxylated to 5α-pregnan-3α,11ß,17α-triol-20-one (11OH-Pdiol) and 5α-pregnan-3α,17α-diol-11,20-dione (11K-Pdiol), with the lyase activity subsequently catalysing to conversion to the C11-oxy C19 steroids, 11ß-hydroxyandrosterone and 11-ketoandrosterone, respectively. Docking of 11OHP4, 11KP4 and the 5α-reduced metabolites, 5α-pregnan-11ß-ol-3,20-dione (11OH-DHP4) and 5α-pregnan-3,11,20-trione (11K-DHP4) with human CYP17A1 showed minimal changes in the orientation of these C11-oxy C21 steroids in the active pocket when compared with the binding of progesterone suggesting the 17,20-lyase is impaired by the C11-hydroxyl and keto moieties. The structurally similar 3,11diOH-DHP4 and alfaxalone showed a greater distance between C17 and the heme group compared to the natural substrate, 17α-hydroxypregnenolone potentially allowing more orientational freedom and facilitating the conversion of the C11-oxy C21 to C11-oxy C19 steroids. In summary, our in vitro assays showed that while CYP17A1 readily hydroxylated 11OHP4 and 11KP4, the enzyme was unable to catalyse the 17,20-lyase reaction of these C11-oxy C21 steroid products. Although CYP17A1 exhibited no catalytic activity towards the 5α-reduced intermediates, once the C4-C5 double bond and the keto group at C3 were reduced, both the hydroxylation and lyase reactions proceeded efficiently. These findings show that the C11-oxy C21 steroids could potentially contribute to the androgen pool in tissue expressing steroidogenic enzymes in the backdoor pathway.

11α-Hydroxyprogesterone, a potent 11ß-hydroxysteroid dehydrogenase inhibitor, is metabolised by steroid-5α-reductase and cytochrome P450 17α-hydroxylase/17,20-lyase to produce C11α-derivatives of 21-deoxycortisol and 11-hydroxyandrostenedione in vitro.

11α-Hydroxyprogesterone (11αOHP4) and 11ß-hydroxyprogesterone (11ßOHP4) have been reported to be inhibitors of 11ß-hydroxysteroid dehydrogenase (11ßHSD) type 2, together with 11ß-hydroxytestosterone and 11ß-hydroxyandrostenedione, and their C11-keto derivatives being inhibitors of 11ßHSD1. Our in vitro assays in transiently transfected HEK293 cells, however, show that 11αOHP4 is a potent inhibitor of 11ßHSD2 and while this steroid does not serve as a substrate for the enzyme, the aforementioned C11-oxy steroids are indeed substrates for both 11ßHSD isozymes. 11ßOHP4 is metabolised by 11ßHSD2 yielding 11-ketoprogesterone with 11ßHSD1 catalysing the reverse reaction, similar to the reduction of the other C11-oxy steroids. In the same model system, novel 11αOHP4 metabolites were detected in its conversion by steroid-5α-reductase (SRD5A) types 1 and 2 yielding 11α-hydroxydihydroprogesterone and its conversion by cytochrome P450 17A1 (CYP17A1) yielding the hydroxylase product, 11α,17α-dihydroxyprogesterone, and the 17,20 lyase product, 11α-hydroxyandrostenedione. We also detected both 11αOHP4 and 11ßOHP4 in prostate cancer tissue- ∼23 and ∼32 ng/g respectively with 11KP4 levels >300 ng/g. In vitro assays in PC3 and LNCaP prostate cancer cell models, showed that the metabolism of 11αOHP4 and 11ßOHP4 was comparable. In LNCaP cells expressing CYP17A1, 11αOHP4 and 11ßOHP4 were metabolised with negligible substrate, 4%, remaining after 48 h, while the steroid substrate 11ß,17α-dihydroxyprogesterone (21dF) was metabolised to C11-keto C19 steroids yielding 11-ketotestosterone. Despite the fact that 11αOHP4 is not metabolised by 11ßHSD2, it is a substrate for SRD5A and CYP17A1, yielding C11α-hydroxy C19 steroids as well as the C11α-hydroxy derivative of 21dF-the latter associated with clinical conditions characterised by androgen excess. With our data showing that 11αOHP4 is present at high levels in prostate cancer tissue, the steroid may serve as a precursor to unique C11α-hydroxy C19 steroids. The potential impact of 11αOHP4 and its metabolites on human pathophysiology can however only be fully assessed once C11α-hydroxyl metabolite levels are comprehensively analysed.

Biotransformation into 11α-hydroxyprogesterone glucosides by glucosyltransferase.

Recently, studies on the steroidal hormone activity in the brain have attracted attention, and the influences of the varied glucosides and their artificial derivatives have been discussed; additionally, it has been suggested that glucosides are the synthetic precursors of glucuronide as a label molecule. However, glucosides are formed with 11α-hydroxyprogesterone (1), which is important as a blood pressure regulator, but anti-androgen activity remains unknown. Using UDP-glucosyltransferase, glucoside synthesis was successful in linking ß-d-glucopyranose and ß-d-laminaribiose to 11α oxygen of 1 at a high conversion ratio, and full assignment structure was analyzed for the two glucosides by high-resolution quadrupole-time flight electrospray ionization-mass spectrometry, 1D (1H and 13C) NMR and 2D (COSY, ROESY, HSQC-DEPT and HMQC) NMR. Furthermore, the bioactivity of 1 and two 11α-hydroxyprogesterone glucosides [11α-(ß-d-glucopyranosyl)oxyprogesterone, 2, and 11α-(ß-d-laminaribiosyl)oxyprogesterone, 3] was tested in vitro. On rotenone-induced PC12 cells, the two 11α-hydroxyprogesterone glucosides (2 and 3) showed superior neuroprotective effects and increased cellular ATP levels compared with those of 1.

An efficient biotransformation of progesterone into 11α-hydroxyprogesterone by Rhizopus microsporus var. oligosporus.

Rhizopus microsporus var. oligosporus is a fungus that belongs to the Mucoraceae family that is used for the preparation of some soy-fermented foods. Microbial biotransformation of progesterone by R. microsporus var. oligosporus afforded some monohydroxylated and dihydroxylated metabolites. The main product was purified using chromatographic methods and identified as 11α-hydroxyprogesterone on the basis of its spectroscopic features. Time course studies by high-performance thin-layer chromatography demonstrated that this fungi efficiently hydroxylated progesterone at the 11α-position for 3 days with a yield of 76.48%, but beyond this time, the microorganism transformed 11α-hydroxyprogesterone into dihydroxylated metabolites. 11α-Hydroxyprogesterone is widely used as a precursor in the synthesis of hydrocortisone and other steroidal anti-inflammatory agents.

Protein Engineering of the Progesterone Hydroxylating P450-Monooxygenase CYP17A1 Alters Its Regioselectivity.

The CYP171 enzyme is known to catalyse a key step in the steroidogenesis of mammals. The substrates progesterone and pregnenolone are first hydroxylated at the C17 position, and this is followed by cleavage of the C17-C20 bond to yield important precursors for glucosteroids and androgens. In this study, we focused on the reaction of the bovine CYP17A1 enzyme with progesterone as a substrate. On the basis of a created homology model, active-site residues were identified and systematically mutated to alanine. In whole-cell biotransformations, the importance of the N202, R239, G297 and E305 residues for substrate conversion was confirmed. Additionally, mutation of the L206, V366 and V483 residues enhanced the formation of the 16α-hydroxyprogesterone side product up to 40 % of the total product formation. Furthermore, residue L105 was found not to be involved in this side activity, which contradicts a previous study with the human enzyme.

The in vitro metabolism of 11ß-hydroxyprogesterone and 11-ketoprogesterone to 11-ketodihydrotestosterone in the backdoor pathway.

Increased circulating 11ß-hydroxyprogesterone (11OHP4), biosynthesised in the human adrenal, is associated with 21-hydroxylase deficiency in congenital adrenal hyperplasia. 17α-hydroxyprogesterone levels are also increased, with the steroid's metabolism to dihydrotestosterone in the backdoor pathway contributing to hyperandrogenic clinical conditions. In this study we investigated the in vitro biosynthesis and downstream metabolism of 11OHP4. Both cytochrome P450 11ß-hydroxylase and aldosterone synthase catalyse the biosynthesis of 11OHP4 from progesterone (P4) which is converted to 11-ketoprogesterone (11KP4) by 11ß-hydroxysteroid dehydrogenase type 2, while type 1 readily catalysed the reverse reaction. We showed in HEK-293 cells that these C11-oxy C21 steroids were metabolised by steroidogenic enzymes in the backdoor pathway-5α-reductase (SRD5A) and 3α-hydroxysteroid type 3 (AKR1C2) converted 11OHP4 to 5α-pregnan-11ß-ol,3,20-dione and 5α-pregnan-3α,11ß-diol-20-one, while 11KP4 was converted to 5α-pregnan-3,11,20-trione and 5α-pregnan-3α-ol-11,20-dione (alfaxalone), respectively. Cytochrome P450 17α-hydroxylase/17,20-lyase catalysed the hydroxylase and lyase reaction to produce the C11-oxy C19 steroids demonstrated in the conversion of alfaxalone to 11-oxy steroids demonstrated in the conversion of alfaxalone to 11ketoandrosterone. In LNCaP cells, a prostate cancer cell model endogenously expressing the relevant enzymes, 11OHP4 and 11KP4 were metabolised to the potent androgen, 11-ketodihydrotestosterone (11KDHT), thus suggesting the C11-oxy C21 steroids contribute to the pool of validating the in vitro biosynthesis of C11-oxy C19 steroids from C11-oxy C21 steroids. The in vitro reduction of 11KP4 at C3 and C5 by AKR1C2 and SRD5A has confirmed the metabolic route of the urinary metabolite, 3α,20α-dihydroxy-5ß-pregnan-11-one. Although our assays have demonstrated the conversion of 11OHP4 and 11KP4 by steroidogenic enzymes in the backdoor pathway yielding 11KDHT, thus suggesting the C11-oxy C21 steroids contribute to the pool of potent androgens, the in vivo confirmation of this metabolic route remains challenging.

Hormonal induction of ovulation using Ovaprim™ [(D-Arg6, Pro9NEt)-sGnRH+domperidone] and its impact on embryonic development of wild-caught Longspine scraper, Capoeta trutta (Heckel, 1843).

Knowledge of gamete quality is a prerequisite for developing techniques to fertilize eggs and rear offspring for hatchery production. Our objective was to develop assisted reproductive techniques, via hormonal induction of final oocyte maturation (FOM), for Longspine scraper, Capoeta trutta. Fish were administered injections of salmon gonadotropin-releasing hormone analogue containing anti-dopaminergic drug (Ovaprim™) or saline (control). Effects of Ovaprim on induction of ovulation, gamete quality, embryonic development, and larval survival were later examined with serum steroid hormone levels and ovarian histology. The saline group failed to spawn, whereas Ovaprim accelerated FOM and induced spawning. Fish treated with Ovaprim showed an increase in gonadosomatic index, egg diameter, and wet weight relative to controls. Average absolute fecundity, relative fecundity, fertilization, and hatching rates were 8823 eggs/spawn, 53 eggs/g body weight, 95%, and 91%, respectively. Serum 17α,20ß-dihydroxy-4-pregnen-3-one (DHP) levels were significantly enhanced by ∼4-fold in Ovaprim-treated fish compared to the saline-injected fish, while 17ß-estradiol levels declined upon FOM in hormone treated fish. Embryonic development closely resembled the teleost scheme, despite variations in timing. Larval survival at 6 and 12days post-hatch were 98% and 95%, respectively. Results suggest that Ovaprim is efficient for inducing spawning in C. trutta for stock enhancement or hatchery purposes.

Screening for strains with 11α-hydroxylase activity for 17α-hydroxy progesterone biotransformation.

Various corticosteroids are prepared by using 11α,17α-diOH-progesterone (11α,17α-diOH-PROG) as an important intermediate and raw material. Hence, strains that can improve the yields of 11α,17α-diOH-PROG should be screened. Cunninghamella elegans CICC40250 was singled out from five common 11α hydroxylation strains. The reaction parameters of 11α,17α-diOH-PROG production were also investigated. C. elegans CICC40250 could efficiently catalyze the hydroxylation of 17α-hydroxy progesterone (17α-OH-PROG) at C-11α position. This strain could also effectively convert 11α,17α-diOH-PROG at high substrate concentrations (up to 30g/L). After the coenzyme precursor glucose was added, the rate of 11α,17α-diOH-PROG formation reached 84.2%, which was 11.4% higher than that of the control group. Our study established a simple and feasible mechanism to increase 11α,17α-diOH-PROG production levels. This mechanism involves C. elegans CICC40250 that can be efficiently applied to induce the biotransformation of 17α-OH-PROG with a hydroxylation biocatalytic ability.

Dual estrogenic regulation of the nuclear progestin receptor and spermatogonial renewal during gilthead seabream (Sparus aurata) spermatogenesis.

Studies in teleosts suggest that progestins have crucial functions during early spermatogenesis. However, the role of the different progestin receptors in these mechanisms is poorly understood. In this work, we investigated the expression pattern and hormonal regulation of the classical nuclear progestin receptor (Pgr) in the gilthead seabream at three different stages of spermatogenesis: the resting (postspawning) phase, onset of spermatogenesis, and spermiation. Immunolocalization experiments using a seabream specific Pgr antibody revealed that the receptor was expressed in Sertoli and Leydig cells, and also in a subset of spermatogonia type A, throughout spermatogenesis. Short-term treatment of testis explants with 17ß-estradiol (E2) increased pgr mRNA expression at all stages, while the progestin 17α,20ß-dihydroxy-4-pregnen-3-one (17,20ßP) had the opposite effect. At the resting stage, Sertoli cell Pgr expression was positively correlated with the occurrence of proliferating spermatogonia type A in the tubules, and both processes were incremented in vitro by E2 likely through the estrogen receptor alpha (Era) expressed in Sertoli and Leydig cells. In contrast, treatment with 17,20ßP downregulated Pgr expression in somatic cells. The androgen 11-ketotestosterone (11-KT) upregulated pgr expression in Leydig cells and promoted the proliferation of mostly spermatogonia type B, but only during spermiation. No relationship between the changes in the cell type-specific expression of the Pgr with the entry into meiosis of germ cells was found. These data suggest a differential steroid regulation of Pgr expression during seabream spermatogenesis and the potential interplay of the E2/Era and 17,20ßP/Pgr pathways for the maintenance of spermatogonial renewal rather than entry into meiosis.

The metabolic fate and receptor interaction of 16α-hydroxyprogesterone and its 5α-reduced metabolite, 16α-hydroxy-dihydroprogesterone.

16α-hydroxyprogesterone (16OHP4) is not well characterised in terms of metabolism and receptor interaction. We therefore investigated its metabolism by adrenal CYP11B and peripheral steroidogenic enzymes, SRD5A and AKR1C2. UHPLC-MS/MS analyses identified novel steroids: the biosynthesis of 4-pregnen-11ß,16α-diol-3,20-dione catalysed by CYP11B2; the 5α-reduction of the latter and 16OHP4 catalysed by SRD5A yielding 5α-pregnan-11ß,16α-diol-3,20-diovne and 5α-pregnan-16α-ol-3,20-dione (16OH-DHP4); and 16OH-DHP4 converted by AKR1C2 to 5α-pregnan-3α,16α-diol-20-one. Receptor studies showed 16OHP4, 16OH-DHP4, progesterone and dihydroprogesterone (DHP4) were weak partial AR agonists; 16OHP4, 16OH-DHP4 and DHP4 exhibited weak partial agonist activity towards PR-B with DHP4 also exhibiting partial agonist activity towards PR-A. Data showed that while the 5α-reduction of P4 decreased PR activation significantly, 16OHP4 and 16OH-DHP4 exhibited comparable receptor activation. Although the clinical relevance of 16OHP4 remains unclear the elevated 16OHP4 levels characteristic of 21OHD, CAH, PCOS, prostate cancer, testicular feminization syndrome and cryptorchidism likely contribute towards these clinical conditions, inducing receptor-activated target genes.

Biologically active recombinant carp LH as a spawning-inducing agent for carp.

Currently, spawning is induced in carp species by carp pituitary extract (CPE) and a combination of synthetic agonist of GnRH combined with a dopamine antagonist. The main goal of this study was the production of recombinant gonadotropins (GtHs) on a large scale to serve as an alternative to currently used agents. We produced carp (c) recombinant (r) Lh as a single chain in the methylotrophic yeast Pichia pastoris Lha subunit was joined with Lhb subunit with a flexible linker of three glycine-serine repeats and six Histidines to form a mature protein, the ß-subunit formed the N-terminal part and the α-subunit formed the C-terminal part. The ability of the rcLh to elicit biological response was tested by in vivo stimulation of estradiol (E2) and 17α,20ß-dihydroxy-4-pregnen-3-one (DHP) and by its in vivo potency to induce ovulation and spawning induction. rcLh tested in this work significantly enhanced both E2 and DHP secretion in a dose-dependent manner similar to the results obtained with CPE. E2 levels showed a moderate rise following the priming injection and a subsequent decrease during the rest of the trial. DHP levels were only increased after the resolving injection, approximately 5 h before spawning. At the highest dose of rcLh (350 µg/kg BW), the recombinant protein was more efficient than CPE in terms of both spawning success and fertilization rate. It is shown here that rcLh can elicit the secretion of DHP in vivo and actually trigger spawning. These novel findings introduce the potential of utilizing recombinant gonadotropins in aquaculture.

17ß-HSD Type 12-Like Is Responsible for Maturation-Inducing Hormone Synthesis During Oocyte Maturation in Masu Salmon.

The maturation-inducing hormone 17α,20ß-dihydroxy-4-pregnen-3-one (DHP) was first identified in the amago salmon. Although carbonyl reductase-like 20ß-hydroxysteroid dehydrogenase (CR/20ß-HSD) was reported to convert 17α-hydroxyprogesterone (17OHP) to DHP in rainbow trout, we previously found that CR/20ß-HSD messenger RNA (mRNA) was not upregulated in stimulated granulosa cells from masu salmon, which suggested that DHP is synthesized by a different enzyme. Accordingly, the current study aimed to identify the specific 20ß-hydroxysteroid dehydrogenase (20ß-HSD) responsible for DHP production by granulosa cells during final oocyte maturation in masu salmon. RNA sequencing was performed on granulosa layers that were isolated from ovarian follicles at 1 month before ovulation and incubated with or without forskolin, which was used to mimic luteinizing hormone, and ∼12 million reads were obtained, which yielded 71,062 contigs of >100 bp. tBlastx analysis identified 1 contig (#f103496) as similar to 17ß-hydroxysteroid dehydrogenase type 12 (hsd17ß12); however, because the full-length #f103496 sequence was different from hsd17ß12, it was termed hsd17ß12-like (hsd17ß12l). We found that mammalian cells transfected with full-length hsd17ß12l exhibited considerable 20ß-HSD activity, as indicated by efficient conversion of exogenous 17OHP to DHP. In addition, we found that hsd17ß12l mRNA levels were consistently low in follicles during vitellogenic growth; however, the levels increased significantly during final oocyte maturation. The levels of hsd17ß12l mRNA were also considerably increased in granulosa layers in which 20ß-HSD activity was induced by salmon pituitary extract. Therefore, we suggest that hsd17ß12l, not CR/20ß-HSD, is the 20ß-HSD responsible for DHP production by granulosa cells in masu salmon during final oocyte maturation.

Involvement of pax2 in ovarian development and recrudescence of catfish: a role in steroidogenesis.

PAX2, a member of paired box family, is an essential transcription factor for the organ development in vertebrates including teleosts, yet no evidence has been shown for its involvement in reproduction. To study this, partial- and/or full-length cDNA of pax2 was isolated from the ovary of catfish, Clarias batrachus, along with its other Pax family members, pax1 and pax9 Tissue distribution and ontogeny expression analysis indicated the prevalence of pax2 but not pax1 and pax9 in ovary. Varied phase-wise expression during ovarian cycle and elevation of pax2 after human chorionic gonadotropin induction showed probable regulation by gonadotropins. Pax2 could be localized in various stages of oocytes and in follicular layer of vitellogenic and post-vitellogenic oocytes. To assess the functional significance of pax2, transient RNA silencing was performed using primary catfish ovarian follicle culture, in vitro, and in catfish, in vivo, through ovary-targeted injection of PEI-esiRNA. Pax2 siRNA treatment reduced the expression of various transcripts related to ovarian development like signaling molecules such as wnt4 and wnt5, estrogen receptors, several steroidogenic enzymes and transcription factors. These transitions in transcript levels might have been mediated by Pax2 acting upstream of wnt4/5 that may play a role in steroidogenesis and/or ovarian development along with ad4bp/sf-1 or by direct or indirect interaction with steroidogenic enzyme genes, which is evident from the change in the levels of serum estradiol-17ß but not 17α,20ß-dihydroxy-4-pregnen-3-one. Taken together, it seems that pax2 has a plausible role during ovarian development and/or recrudescence of catfish either directly or indirectly through Wnt signaling pathway.

Vitellogenin, sex steroid levels and gonadal biomarkers in wild Solea solea and Solea senegalensis from NW Mediterranean fishing grounds.

Specimens of Solea solea and Solea senegalenesis at different developmental stages were obtained from seven fishing grounds along the NW Mediterranean. Gonad development in males was classified into five stages, from early spermatogenesis to recovery, while four stages were considered in females, from growth to maturation. Vitellogenin (VTG) and sex steroid levels including an estrogen (estradiol, E2), two androgens (testosterone, T and 11-ketotestosterone, 11KT) and a progestin (17,20ß-dihydroxy pregn-4-en-3-one, 17,20ß-P or maturation inducing steroid, MIS) were analysed in plasma. Their levels were more clearly related to the developmental stage of the gonads than to the sampling site characteristics. In addition, enzyme activities in gonads, such as acetylcholinesterase (AChE) and carboxylesterase (CbE) were gender-dependent and higher in males than in females. Gonadal glutathione S-transferase (GST) activity was enhanced in the most anthropogenic impacted sites. VTG was absent in males and very low or undetectable in immature females, while mature females exhibited high VTG levels, clearly related to the gonado-somatic index. Sex steroid levels (ng/ml) varied in males and females regardless of the species. E2 levels in females ranged from 0.22 to 6.98 while in males ranged from 0.11 to 0.27. T varied from 0.12 to 0.93 in females and from 0.56 to 1.36 in males, while 11KT in females fluctuated from 0.03 to 0.57 and from 0.26 to 6.42 in males. Similarly, MIS in females ranged from 0.75 to 3.71 and from 1.12 to 5.61 in males. The lack of endocrine disturbances was confirmed by histological examination of the gonads. This study informs on basal sex hormone levels and enzyme activities during gonadal maturation of wild Solea spp. that can be useful in the identification and further remediation of possible pollution events.

Regulation of recombinant human insulin-induced maturational events in Clarias batrachus (L.) oocytes in vitro.

Regulation of insulin-mediated resumption of meiotic maturation in catfish oocytes was investigated. Insulin stimulation of post-vitellogenic oocytes promotes the synthesis of cyclin B, histone H1 kinase activation and a germinal vesicle breakdown (GVBD) response in a dose-dependent and duration-dependent manner. The PI3K inhibitor wortmannin abrogates recombinant human (rh)-insulin action on histone H1 kinase activation and meiotic G2-M1 transition in denuded and follicle-enclosed oocytes in vitro. While the translational inhibitor cycloheximide attenuates rh-insulin action, priming with transcriptional blocker actinomycin D prevents insulin-stimulated maturational response appreciably, albeit in low amounts. Compared with rh-insulin, human chorionic gonadotrophin (hCG) stimulation of follicle-enclosed oocytes in vitro triggers a sharp increase in 17α,20ß-dihydroxy-4-pregnen-3-one (17α,20ß-DHP) secreted in the incubation medium at 12 h. Interestingly, the insulin, but not the hCG-induced, maturational response shows less susceptibility to steroidogenesis inhibitors, trilostane or dl-aminoglutethimide. In addition, priming with phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) or cell-permeable dbcAMP or adenylyl cyclase activator forskolin reverses the action of insulin on meiotic G2-M1 transition. Conversely, the adenylyl cyclase inhibitor, SQ 22536, or PKA inhibitor H89 promotes the resumption of meiosis alone and further potentiates the GVBD response in the presence of rh-insulin. Furthermore, insulin-mediated meiotic maturation involves the down-regulation of endogenous protein kinase A (PKA) activity in a manner sensitive to PI3K activation, suggesting potential involvement of a cross-talk between cAMP/PKA and insulin-mediated signalling cascade in catfish oocytes in vitro. Taken together, these results suggest that rh-insulin regulation of the maturational response in C. batrachus oocytes involves down-regulation of PKA, synthesis of cyclin B, and histone H1 kinase activation and demonstrates reduced sensitivity to steroidogenesis and transcriptional inhibition.

Progesterone Metabolites Produced by Cytochrome P450 3A Modulate Uterine Contractility in a Murine Model.

OBJECTIVE: We seek to characterize the effect of progesterone metabolites on spontaneous and oxytocin-induced uterine contractility. STUDY DESIGN: Spontaneous contractility was studied in mouse uterine horns after treatment with progesterone, 2α-hydroxyprogesterone, 6ß-hydroxyprogesterone (6ß-OHP), 16α-hydroxyprogesterone (16α-OHP), or 17-hydroxyprogesterone caproate (17-OHPC) at 10(-9) to 10(-6) mol/L. Uterine horns were exposed to progestins (10(-6) mol/L), followed by increasing concentrations of oxytocin (1-100 nmol/L) to study oxytocin-induced contractility. Contraction parameters were compared for each progestin and matched vehicle control using repeated measures 2-way analysis of variance. In vitro metabolism of progesterone by recombinant cytochrome P450 3A (CYP3A) microsomes (3A5, 3A5, and 3A7) identified major metabolites. RESULTS: Oxytocin-induced contractile frequency was decreased by 16α-OHP (P = .03) and increased by 6ß-OHP (P = .05). Progesterone and 17-OHPC decreased oxytocin-induced contractile force (P = .02 and P = .04, respectively) and frequency (P = .02 and P = .03, respectively). Only progesterone decreased spontaneous contractile force (P = .02). Production of 16α-OHP and 6ß-OHP metabolites were confirmed in all CYP3A isoforms tested. CONCLUSION: Progesterone metabolites produced by maternal or fetal CYP3A enzymes influence uterine contractility.

Effects of the anti-androgen cyproterone acetate (CPA) on oocyte meiotic maturation in rainbow trout (Oncorhynchus mykiss).

In the present study, we aimed at characterizing the effect of cyproterone acetate (CPA), an anti-androgenic compound, on oocyte meiotic maturation in a freshwater teleost fish species, the rainbow trout (Oncorhynchus mykiss). Fully-grown post-vitellogenic ovarian follicles were incubated in vitro with CPA, luteinizing hormone (Lh) or a combination of CPA and Lh. Incubations were also performed using a combination of Lh and testosterone (T). The occurrence of oocyte maturation (i.e., resumption of the meiotic process) was assessed by monitoring germinal vesicle breakdown (GVBD) after a 72h in vitro incubation. The effect of CPA on the production of 17,20ß-dihydroxy-4-pregnen-3-one (17,20ßP), the natural maturation-inducing steroid (MIS), was quantified by radioimmunoassay. Our results show that CPA dramatically inhibits Lh-induced oocyte maturation and MIS synthesis. We also observed a synergistic effect of Lh and T on oocyte maturation in highly competent oocytes (i.e., able to resume meiosis after stimulation by low doses of Lh). Our results also show that a combination of CPA and Lh inhibits phosphorylation of extracellular signal-regulated kinase (Erk), kinases that are associated with oocyte maturation in many species. As a whole, our results indicate that CPA has a potential to alter meiotic maturation in rainbow trout. Further analyses are, however, needed to determine the mechanisms by which this anti-androgen interferes with the meiotic process. Furthermore, the present study provides a framework for better understanding of the ecological consequences of exposure to anti-androgens and resulting meiotic maturation abnormalities observed in trout.

Neurones in the preoptic area of the male goldfish are activated by a sex pheromone 17α,20ß-dihydroxy-4-pregnen-3-one.

Pheromones are interesting molecules given their ability to evoke changes in the endocrine state and behaviours of animals. In goldfish, a sex pheromone, 17α,20ß-dihydroxy-4-pregnen-3-one (17,20ß-P), which is released by preovulatory females, is known to trigger the elevation of luteinising hormone (LH) levels, as well as reproductive behaviour in males. Interestingly, when 11-ketotestosterone (11-KT) is implanted into adult female fish, LH levels increase in response to the pheromone at any time of the day, which is normally a male-specific response. However, the neural mechanisms underlying the male-specific information processing of 17,20ß-P and its androgen dependence are yet unknown. In the present study, we focused on the preoptic area (POA), which plays important roles in the regulation of reproduction and reproductive behaviours. We mapped activity in the POA evoked by 17,20ß-P exposure using the immediate-early gene c-fos. We found that a population of ventral POA neurones close to kisspeptin2 (kiss2) neurones that appear to have important roles in reproduction was activated by 17,20ß-P exposure, suggesting that these activated neurones are important for the 17,20ß-P response. Next, we investigated the distribution of androgen receptor (ar) in the POA and its relationship with 17,20ß-P-responsive and kiss2 neurones. We found that ar is widely expressed in the ventral POA, whereas it is only expressed in approximately 10% of 17,20ß-P-activated neurones. On the other hand, it is expressed in almost 90% of the kiss2 neurones. Taken together, it is possible that ar expressing neurones in the ventral POA, most of which were not labelled by c-fos in the present study, may at least partly account for androgen effects on responses to primer pheromones; the ar-positive kiss2 neurones in the ventral POA may be a candidate. These results offer a novel insight into the mechanisms underlying male-specific information processing of 17,20ß-P in goldfish.