Natural selection in the water: freshwater invasion and adaptation by water colour in the Amazonian pufferfish.

Natural selection and ecological adaptation are ultimately responsible for much of the origin of biodiversity. Yet, the identification of divergent natural selection has been hindered by the spatial complexity of natural systems, the difficulty in identifying genes under selection and their relationship to environment, and the confounding genomic effects of time. Here, we employed genome scans, population genetics and sequence-based phylogeographic methods to identify divergent natural selection on population boundaries in a freshwater invader, the Amazonian pufferfish, Colomesus asellus. We sampled extensively across markedly different hydrochemical settings in the Amazon Basin and use 'water colour' to test for ecological isolation. We distinguish the relative contribution of natural selection across hydrochemical gradients from biogeographic history in the origin and maintenance of population boundaries within a single species and across a complex ecosystem. We show that spatially distinct population structure generated by multiple forces (i.e. water colour and vicariant biogeographic history) can be identified if the confounding effects of genetic drift have not accumulated between selective populations. Our findings have repercussions for studies aimed at identifying engines of biodiversity and assessing their temporal progression in understudied and ecologically complex tropical ecosystems.

Subchronic oral toxicity study of furan in B6C3F1 Mice.

Furan is a heterocyclic organic compound formed during heat treatment for processing and preservation of various types of food. Rodent studies have previously shown that furan is a hepatocarcinogen. Those studies were conducted over a high dose range, which induced tumors at nearly 100% incidence at all doses. This ninety-day gavage study in mice was conducted to extend the dose to a lower range (0.0, 0.03, 0.12, 0.5, 2.0, and 8.0 mg/kg body weight [bw] per day) to identify a no-observed adverse effect level for hepatotoxicity and to characterize non-neoplastic effects, including those affecting clinical biochemistry, hematology, tissue morphology, and histopathology. The liver was the primary target organ with dose-dependent toxicity. Liver weights were increased at the 8.0 mg/kg bw dose in females only. Levels of the serum enzyme alanine transaminase, representative of liver damage, were increased three-fold at the highest dose. Histological changes in the liver were observed at 2.0 and 8.0 mg/kg bw in both sexes. Although clinical parameters were also altered for the kidney, these differences were not accompanied by histological changes. Based on these clinical biochemical and histological changes, a no-observed adverse effect level of 0.12 mg/kg bw per day of furan in mice is suggested.

Subchronic oral toxicity study of furan in Fischer-344 rats.

Rodent studies have shown that furan is a hepatocarcinogen. Previous studies conducted with high doses showed tumors at nearly 100% incidence at all doses. In this paper, a ninety-day gavage experiment conducted with lower doses (0.0, 0.03, 0.12, 0.5, 2.0, and 8.0 mg/kg bw) to identify a no-observed adverse effect level for hepatotoxicity and to characterize non-neoplastic effects including gross changes and histopathology, clinical biochemistry, hematology, and immunotoxicology is reported. As indicated by changes in serum biomarkers, increased liver weights and gross and histological lesions, the liver is the major target organ affected by furan. There were no changes in body weights, food consumption, or histology in other organs. Some of the serum electrolyte markers, including phosphorus, were altered. There was a significant increase in serum thyroxine and triidothyronine in males. This increase was not accompanied by histological thyroid changes. Immunophenotypic analysis showed that thymic lymphocyte maturation was altered in male rats. Although altered clinical biochemistry and hematological parameters were observed at a dose of > 0.5 mg/kg bw, mild histological lesions in the liver were observed at > 0.12 mg/kg bw. Based on this finding, a furan dose of 0.03 mg/kg bw was proposed as the no-observed adverse effect level for hepatic toxicity.

Toxicokinetics of p-tert-octylphenol in male and female Sprague-Dawley rats after intravenous, oral, or subcutaneous exposures.

This study was undertaken to characterize the toxicokinetics of p-tert-octylphenol (OP), a weak estrogenic compound, in male and female rats. Male and female Sprague-Dawley rats were given a single dose of OP either by oral gavage (50, 125 or 250 mg/kg), by intravenous (iv) injection (2, 4, or 8 mg/kg), or by subcutaneous (sc) injection (125 mg/kg). In a repeated dosing experiment, rats were given OP (oral) daily (25, 50, or 125 mg/kg) for 35 d (female) or 60 d (male). Blood and tissue samples were collected and analyzed for OP content using gas chromatography with detection by mass spectrometry. Blood OP concentrations were generally higher in female than male rats following a single oral or sc administration but were similar following a single iv injection. Tissue OP concentrations were also higher in female than male rats following oral exposure, consistent with the faster metabolism of OP observed in male rat liver microsomes. After subchronic administration, blood OP concentrations were higher at the end of exposure for female (33 d) (2.26-fold, not significant) and male (57 d) (3.47-fold) rats than single dosing but there was no change in the tissue OP concentrations. Gender differences in tissue OP concentrations may contribute, in part, to gender differences in the toxicity of OP in rats. The fact that OP was found in all reproductive tissues confirms its potential for direct endocrine-like effects.

The aromatase inhibitor fadrozole and the 5-reductase inhibitor finasteride affect gonadal differentiation and gene expression in the frog Silurana tropicalis.

Aromatase (cyp19) and the 5alpha- and 5beta-reductases (srd5alpha and srd5beta) are important enzymes for vertebrate sexual development. We investigated the effects of inhibition of cyp19 by fadrozole (FAD), and srd5alpha and srd5beta by finasteride (FIN) during anuran larval development. Chronic exposures of Silurana (Xenopus) tropicalis from Nieuwkoop-Faber stage 12 until stage 60 were performed using either 2 microM FAD or 25 microM FIN. Histological analysis of exposed metamorphic frogs revealed that both treatments induced intersex individuals (presence of testicular oocytes). FAD treatment resulted in 55% male, 30% female and 15% intersex, while FIN treatment produced 27% male, 53% female and 20% intersex. Real-time RT-PCR analysis of hepatic sex steroid- and thyroid hormone-related gene expression demonstrated that FAD-induced intersex animals had higher srd5alpha1, srd5alpha2 and eralpha mRNA levels than control and FAD males. In contrast, FIN-induced intersex had low srd5alpha1, srd5alpha2, srd5beta and dio3 and high dio2 mRNA levels. FIN-treated males exhibited high trbeta, dio2 and a lower dio3 mRNA levels. We conclude that chemically induced intersex animals display different gene expression profiles than non-exposed animals and that, although morphologically similar, intersex animals produced by different chemicals have different endocrine pathophysiologies.

Determination of p-tert-octylphenol in blood and tissues by gas chromatography coupled with mass spectrometry.

A sensitive and reproducible procedure using gas chromatography coupled with mass spectrometry is described for the determination of p-tert-octylphenol (OP), a persistent degradation product of alkylphenol ethoxylates that binds to the estrogen receptor in blood and tissues. The first step involved the extraction of blood (200 microL) or tissue homogenate (400 microL) with methyl tert-butyl ether, including p-tert-butylphenol (BP) as internal standard. After extraction, the sample was evaporated to dryness with a gentle stream of nitrogen at 45 degrees C, and OP and BP were derivatized with an acetylation reaction involving acetic anhydride and catalyzed by pyridine. Samples were then analyzed by a gas chromatograph equipped with a mass spectrometer (single ion monitoring) with a Varian VF-5ms capillary column. The limit of detection and the limit of quantification of the method in blood were 4.6 and 15.5 ng/mL, respectively. The linearity and reproducibility of the method were acceptable, with coefficients of variation of approximately 10% for blood and ranging between 9% and 27% for tissues. This method was applied to the determination of unchanged OP in blood and tissues obtained from Sprague-Dawley rats after oral and IV OP administration.

Effects of PNU157706, a dual 5alpha-reductase inhibitor, on gene expression in the rat epididymis.

The epididymis is the site of sperm maturation and storage. 5alpha-Reductases (types 1 and 2) are key enzymes in this tissue because they convert testosterone to dihydrotestosterone (DHT), the main androgen regulating epididymal functions. Examining the consequences of inhibiting DHT formation is likely to provide important information regarding the regulation of epididymal functions, yet few inhibitor studies have focused on this tissue. To understand better DHT-mediated regulation of epididymal gene expression, we employed a dual 5alpha-reductase inhibitor and cDNA microarrays to examine the effects of 5alpha-reductase inhibition on gene expression in the initial segment, caput, corpus, and cauda epididymidis. Inhibition of epididymal 5alpha-reductase activity by PNU157706 was confirmed by in vitro enzyme assays. Rats were treated with 0, 0.1, 1.0 or 10 mg/kg per day PNU157706 for 28 days. The weights of DHT-dependent tissues, including the epididymis, were decreased following treatment. The effect of treatment on gene expression was dose-dependent and highly segment-specific. The initial segment responded uniquely in that a similar number of genes increased and decreased in expression compared with the other segments where the majority of affected genes decreased in expression. Some of the more dramatically affected genes were involved in signal transduction as well as fatty acid and lipid metabolism, regulation of ion and fluid transport, luminal acidification, oxidative defense and protein processing and degradation. These are essential processes contributing to the formation of an optimal luminal microenvironment required for proper sperm maturation. These results provide a novel insight into the DHT-dependent mechanisms that control epididymal functions.

Oral (gavage), in utero and postnatal exposure of Sprague-Dawley rats to low doses of tributyltin chloride. Part 1: Toxicology, histopathology and clinical chemistry.

Tributyltin (TBT) is a biocide that contaminates foods, especially shellfish. TBT is an endocrine disrupter in several marine species and is neurotoxic and immunotoxic in mammals. We have examined the effects of exposure to low doses of tributyltin chloride (TBTC) from day 8 of gestation until adulthood. Pregnant rats were gavaged daily with 0, 0.025, 0.25 or 2.5 mg TBTC/kg body weight from day 8 of gestation until weaning. Stomach contents of suckling pups contained undetectable levels of TBT and dibutyltin (DBT) levels were detectable only in the highest TBTC dose used, indicating negligible lactational transfer to pups. Post weaning, pups were gavaged daily with the same dose of TBTC administered to their mothers and sacrificed on post-natal days (PND) 30 (males and females), 60 (females) and 90 (males). TBTC had no effects on dams' body weights, food consumption, litter size, sex ratio or survival of pups to weaning. However, all doses of TBTC significantly affected parameters of the growth profile of the pups (mean body weights, average slope, curvature) and the ratio of weekly food consumption to weekly body weight gain indicated enhanced food conversion to body mass in females but a decreased conversion in males. Liver, spleen and thymus weights were also affected by TBTC. In male pups dosed at 2.5 mg/kg/day, reduced serum thyroxine levels were evident, indicating that the thyroid is a target for TBTC toxicity. No histopathological lesions were seen in the liver but elevated serum alanine aminotransferase, gamma-glutamyl transferase and amylase indicated hepatotoxicity. Significant decreases in liver weights in female pups exposed to 0.025 mg/kg/day TBTC were observed at PND 60. Decreases in spleen and thymus weights also pointed towards toxic effects of TBTC on the immune system. The 0.025 mg/kg/day TBTC should have been a no affect dose and yet this dose caused significant effects on growth profiles, decreased liver weights and elevated serum GGT levels in females.

The mammalian testis accumulates lower levels of organochlorine chemicals compared with other tissues.

Tissues were obtained from three separate experiments in order to quantify the tissue distribution of organochlorine chemicals that are thought to be potential reproductive toxicants in males: 1) Sprague Dawley rats received 1 microCi of 14C-Aldrin or 14C-Dieldrin (20.6 microCi/micromole) i.p. once a week for three weeks. One week and four weeks after the last injection, tissues were harvested and stored at -80 degrees C. Tissue 14C levels were quantified by scintillation spectrometry. 2) Cis- or trans-nonachlor (0, 0.25, 2.5, 25 mg/kg body weight) were administered daily in corn oil to male rats by gavage for 28 days. Tissues were harvested and frozen at -80 degrees C on the 29th day. Organochlorine residues were extracted and quantified by gas chromatography with electron capture detection. 3) Technical grade toxaphene (0, 0.1, 0.4 or 0.8 mg/kg body weight) was ingested daily by female cynomolgus monkeys of reproductive age for 18 months prior to being mated with control males. Dosing continued during pregnancy and lactation. Their infants received toxaphene via breast milk, and upon weaning, they ingested the same dose as their mothers for 48 to 49 weeks until, at 77 to 80 weeks of age, tissues were harvested and stored at -80 degrees C. Organochlorine residues were extracted and quantified as previously stated. In all three experiments, organochlorine residues in the testis were lower than in most of the other reproductive tract and nonreproductive tract tissues we examined. For example, testicular aldrin and dieldrin levels were <5% the epididymal content; testicular cis- and trans-nonachlor were <25% the epididymal content and, testicular toxaphene levels were <15% of the epididymal content. The reasons for the low degree of accumulation by the testis in comparison with other tissues are unknown. However, the lower testicular content may afford germ cells some protection from the potentially toxic effects of these chemicals.

Influence of season and low-level oestradiol immunoneutralization on episodic LH and testosterone secretion and testicular steroidogenic enzymes and steroidogenic acute regulatory protein in the adult ram.

The regulation of LH-dependent and -independent increases in testosterone secretion by key proteins in the testes of adult rams was investigated. Serial blood samples were collected from groups of four control and passively immunized (oestradiol antiserum for 3 weeks) rams and the animals were gonadectomized in either the non-breeding season (April) or the breeding season (September). LH pulse frequency and basal (interpulse) concentrations were several times greater (P < 0.01) in the breeding season than in the non-breeding season. Neither of these parameters nor LH pulse amplitude were affected by oestradiol immunization. Parameters of testosterone episodic secretion and response to an injection (i.v.) of 15 micrograms NIH-LH-S25 were also greater (P < 0.05) in the breeding season and, with the exception of pulse frequency, in immunized rams versus controls. Substrate utilization established that testosterone biosynthesis was predominantly via the 5-ene pathway. Increases in blood testosterone concentration in the breeding season were associated with a fivefold higher (P < 0.01) activity of cytochrome P450 17alpha-hydroxylase/C-17,20 lyase (P450(17alpha)) and a 65% higher (P < 0.05) relative amount of mRNA for cytochrome P450 cholesterol side-chain cleavage enzyme complex (P450scc) in the testis. Of the steroidogenic enzyme activities examined, only that for 17beta-hydroxysteroid dehydrogenase (17beta-HSD) tended to be increased by oestradiol immunization. Blood concentrations of cholesterol lipoproteins and expression of the testicular low density lipoprotein receptor were not affected by season or immunization. The amount of steroidogenic acute regulatory protein (StAR) mRNA was 65% higher (P < 0.01) in the breeding season and 20% higher (P < 0.01) in immunized rams versus controls. These results indicate that greater LH stimulation may increase testosterone biosynthesis in the breeding season by increasing StAR mRNA (and presumably delivery of cholesterol to P450scc) and the activity of P450(17alpha), and possibly that of P450scc (activity not measured). More moderate increases in StAR mRNA and 17beta-HSD activity may explain, in part, the increases in testosterone secretion with oestradiol immunization.

Examination of selected food additives and organochlorine food contaminants for androgenic activity in vitro.

In order to produce a reporter gene assay for androgenic chemicals, a constitutive expression vector coding for the human androgen receptor and a reporter construct containing the firefly luciferase coding sequence under transcriptional control of the androgen responsive MMTV promoter were cotransfected into the androgen-insensitive human PC-3 prostate carcinoma cell line and stable transfectants selected. One colony of transfectants, PC-3 LUCAR+, was characterized further. 5alpha-Dihydrotestosterone (DHT) enhanced luciferase activity in a linear fashion for up to 3 days of culture. The Kd for DHT activation was within the range of 25.0-60.0 pM (r2 values >0.95). Flutamide competitively inhibited DHT activation (mean Ki value of 0.89 microM). Progesterone, estradiol, dexamethasone, and hydrocortisone were weak agonists (100-fold less effective than DHT) and diethylstilbestrol was without effect. The effects of organochlorine food contaminants (0, 0.1, 1.0, and 10.0 microM) on luciferase activity in PC-3 LUCAR+ cells were determined after exposure to the chemical for 18 h in the presence and absence of DHT (50 pM). 1,1-dichloro-2,2-bis(p-chlorophenyl)-ethylene (p,p'-DDE) induced luciferase activity in the absence of DHT (100 microM p,p'-DDE equivalent to 50 pM DHT), but in the presence of DHT (50 pM), p,p'-DDE acted antagonistically. 2,3,7,8-Tetrachlorodibenzo-p-dioxin, kepone, butylated hydroxyanisole, and butylated hydroxytoluene all partially inhibited activation by DHT (50 pM) but alone had little or no effect. Toxaphene at 10 microM induced luciferase activity in the absence of DHT but decreased cell viability. Alpha- and delta-Hexachlorocyclohexanes (HCH) at 10 microM antagonized the DHT effect, but beta-HCH and gamma-HCH mirex, photomirex, oxychlordane, cis- and trans-nonachlor were without effect. Thus, of the chemicals tested, some interact with the human androgen receptor in vitro as agonists, others as antagonists, and some as partial agonists/antagonists.

Concentrations of energy substrates in oviduct fluid in unilaterally ovariectomised pigs.

Nichol et al (1992, Journal of Reproduction and Fertility, 96, 699-707) identified a pre- to post-ovulatory decrease (approx 1 mM) in the amount of glucose in pig oviduct fluid. The present studies investigated whether the decrease was due to metabolism by embryos and/or oviduct tissues, and also whether there was a local influence of the ovary on the oviduct fluid content of energy substrates. Unilaterally ovariectomised pigs were used, in which, through compensation, oviducts that contained twice the normal number of embryos could be compared with oviducts which contained no embryos. Following unilateral ovariectomy and after two oestrous cycles of normal duration, surgery was performed 88 hours after the beginning of standing heat to obtain oviduct fluid samples, just before embryonic entry into the uterus. Luminal fluid samples from the ampulla and ampullary-isthmic junction from oviducts with and without an adjacent ovary were assayed for glucose, pyruvate and lactate concentrations. No significant differences were found between the glucose, pyruvate and lactate concentrations in fluids from the ampulla or ampullary-isthmic junction from oviducts containing embryos compared with absence of embryos (P > 0.05). Therefore, the post-ovulatory decrease was not due to the presence of embryos or to a local effect of the ipsilateral ovary. Consequently, pig oviduct fluid concentrations of glucose, lactate and pyruvate are seemingly regulated by systemic mechanisms.

Effects of in utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on serum androgens and steroidogenic enzyme activities in the male rat reproductive tract.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been shown to impair reproductive function of males in animal models, possibly due to a reduction in serum androgen levels. Thus, TCDD may alter the testosterone biosynthetic pathway in the testis or the conversion of testosterone to 5alpha-dihydrotestosterone (DHT) in androgen target tissues. Pregnant Sprague Dawley rats were gavaged with TCDD (0, 0.2 or 1.0 microg/kg) on day 15 of gestation only. TCDD caused a reduction in the body weight gain of the dams in both dose groups and a significant reduction in litter size in the higher dose group. Litters delivered normally and TCDD exposed male offspring grew at the same rate as controls. Males were sacrificed at 15, 30, 45, 60, 90 and 120 d of age. Steroidogenic enzyme activities were determined in testicular microsomes and androgen target tissue nuclear fractions. Serum androgens were measured by radioimmunoassay (RIA). At 30 d of age, rats exposed to 1.0 microg/kg TCDD exhibited lower 17-hydroxylase activity (P < 0.05) and lower caput-corpus epididymal weights (P < 0.05). At 45 d of age, the same treatment resulted in testicular 3beta-HSD, 17beta-HSD and 5alpha-reductase activities that were significantly greater (P < 0.05) but, conversely, serum androgens were one quarter the values evident in controls (P < 0.05). At the other ages, no differences were observed in serum androgens and, with the exception of lower 17beta-HSD activity at 90 d of age (P < 0.05), no other differences in testicular steroidogenic enzyme activities were found. 5Alpha-reductase activities in the androgen target tissues were also unchanged. Histological examination of testes showed that the spermatogenic profile was identical to controls at all ages.

Oviduct fluid pH in intact and unilaterally ovariectomized pigs.

The pH of the oviduct lumen was measured at different stages of the estrous cycle in the ampulla and ampullary-isthmic junction (AIJ) of intact and unilaterally ovariectomized mated or nonmated pigs. The pH profile consisted of high frequency small peaks superimposed on low frequency large amplitude peaks. One animal examined at midcycle exhibited fluctuations in pH (peak to nadir; delta pH) of 0.3 and 0.7 units in the ampulla and AIJ, respectively, and the frequencies of the large peaks in these regions were 2.6 and 1.6 peaks.min-1, respectively. In six preovulatory unmated pigs, the delta pH (mean +/- SE) was 0.50 +/- 0.04 units in both regions and the large peak frequencies were 0.6 +/- 0.06 peaks.min-1. In one animal that was assessed during ovulation, the pH showed deviations of up to 0.4 pH units, which were probably due to the alkalinity of follicular fluid accompanying the ovulated eggs. In the ampullae of five unilaterally ovariectomized postovulatory-mated pigs, the delta pH in oviducts with and without an ipsilateral ovary was significantly lower than preovulatory (p < 0.05), but the large and the small peak frequencies were not significantly different. By contrast, the delta pH in the AIJ with an ipsilateral ovary (0.11 +/- 0.02 units) was significantly lower than before ovulation (0.54 +/- 0.04 units) and also when compared with the contralateral AIJ (0.36 +/- 0.06 units) (p < 0.05). The ovary also influenced the small peak frequency, which was significantly higher if the ipsilateral ovary was absent (10.7 +/- 1.5 vs. 14.9 +/- 1.6 peaks.min-1, respectively). Thus, oviduct fluid pH is controlled by both systemic and local mechanisms, and the ipsilateral ovary and (or) embryonic factors influence the pH profile of the oviduct.

Motility of spermatozoa in hydrosalpingeal and follicular fluid of pigs.

Hydrosalpinges were created to collect adequate volumes of fluid during pre-, peri- and postovulatory intervals from the ampulla, ampullary-isthmic junction and the isthmic-utero-tubal junction of the oviducts from Large White gilts that had exhibited at least two natural oestrous cycles. The accumulated fluids, follicular fluid and Butschwiler's medium were compared for their effects on various parameters of boar sperm motility using the CellSoft, computer-assisted, digital image analysis system. Sperm velocity (micron s-1 +/- SEM) was significantly higher (P < 0.05) in follicular fluid (84 +/- 3; n = 5) than in fluids from the ampulla during peri- and early postovulatory intervals, and from the isthmic-utero-tubal junction during pre- and early postovulatory intervals. It was also higher (P < 0.05) than in the fluid from the ampullary-isthmic junction during pre- and early postovulatory intervals; however, sperm velocity in follicular fluid was not significantly different from that in the periovulatory fluid from the ampullary-isthmic junction. The mean lateral head displacement (ALHmean) of spermatozoa was significantly greater in follicular fluid (3.9 +/- 0.3 microns; n = 5) than in fluid from the ampulla during peri- and early postovulatory intervals and from the isthmic-utero-tubal junction during pre- and early postovulatory intervals, and was also higher (P < 0.05) than in fluid from the ampullary-isthmic junction during the preovulatory period, but was not different from the peri- and postovulatory ampullary-isthmic junction fluids. The proportion of spermatozoa exhibiting circular motion was significantly higher (P < 0.05) in the periovulatory fluid from the ampullary-isthmic junction (24 +/- 3%) compared with fluids obtained during preovulatory and early postovulatory periods. Follicular fluid had no effect on the proportion of spermatozoa exhibiting circular motion. The average radius of sperm movement in circular trajectories was higher in follicular fluid than in the periovulatory fluids from the ampulla and ampullary-isthmic junction (P < 0.05). In hydrosalpingeal fluids collected 2-5 days after ovulation, the average radius of movement was greater in the ampulla fluid and ampullary-isthmic junction fluid than in fluid from the isthmic-utero-tubal junction (P < 0.05). These results demonstrate that follicular fluid and oviductal fluids have considerable influences on boar sperm motility. Furthermore, the immediate effect of periovulatory ampullary-isthmic junction fluid in increasing the percentage of spermatozoa swimming in circles (hyperactivated) is relevant, since it is at this time and within this region that fertilization occurs.

Rapid generation of stable cell lines expressing corticotropin-releasing hormone receptor for drug discovery.

Human HEK293 cells that stably express the Epstein Barr nuclear antigen 1 (EBNA1) support the episomal replication of plasmids containing the Epstein Barr virus origin of replication (EBV oriP). A 293EBNA (293E) cell line expressing the human corticotropin-releasing hormone receptor subtype I (CRHR1) from an episomal plasmid was generated (293CR1s), analyzed, adapted to spinner culture, and scaled-up for production in less than 6 weeks. Forty-seven stable CHO cell lines transfected with CRHR1 were also isolated. Expression of the receptor in the best of these lines (as judged by CRH-induced cAMP production), CHO-R22, was compared to that in 293CR1s cells. Results indicate that the CRHR1 episomal expression vector in 293E cells (1) rapidly generates stable cell lines suitable for scale-up; (2) is stably maintained during 3 months in culture; (3) expresses high levels of CRHR1 mRNA; and (4) expresses significantly more CRHR1 than the CHO-R22 line. Coexpression of additional G protein alpha subunit (G alpha s) with CRHR1 in 293E cells converts a higher percentage of receptor to the agonist high-affinity G-protein-coupled state. Our data support the idea that using the EBV oriP-driven episomal system for gene expression results in greater production of protein in a relatively short period of time.

The effects of progesterone, 4,16-androstadien-3-one and MK-434 on the kinetics of pig testis microsomal testosterone-4-ene-5alpha-reductase activity.

The enzyme 3-oxo-steroid: NADP+ 4-oxidoreductase (EC 1.3.1.22; 5alpha-reductase) was assayed in testicular microsomes of pigs of 3, 20 and 24 weeks of age. The activity was very low in 3-week-old animals and approximately 10-fold higher in 5- and 6-month-old pigs. The pH optimum was 6.3 in 6-month-old animals, 5.7 in 5-month-old animals, but could not be reliably determined in 3-week-old animals. The kinetic parameters for 5alpha-reductase in testis microsomes from 6-month-old animals were; K((m)(app)), 8.0 micromol/l, V((max)(app)), 6.7 nmoles/90 min/mg protein. Progesterone was a competitive inhibitor of testosterone 5alpha-reduction with an apparent K((i)(app)) of 0.86 micromol/l. However, 4,16-androstadien-3-one (dienone), which undergoes 5alpha-reduction in the biosynthesis of the pheromonally active 16-androstenes, was a comparatively poor inhibitor with a K((i)(app)) of 4.9 micromol/l. Similarly, MK434, which is a selective inhibitor of the human type 2 5alpha-reductase, but which inhibits both types 1 and 2 in the rat, was also a poor competitive inhibitor of testosterone 5alpha-reductase in the pig testis (K((i)(app)), 3.1 micromol/l). It would appear from these studies that the pig testis microsomal 5alpha-reductase corresponds to a type 1 isozyme that is not capable of reducing dienone other than under conditions where the dienone concentration would be in considerable excess of testosterone. It is, therefore, probable that substrate-specific 5alpha-reductases exist in the pig testis for the 5alpha-reduction of testosterone and dienone.

Luteal and placental characteristics of carnivore gestation: expression of genes for luteotrophic receptors and steroidogenic enzymes.

Experiments were carried out to investigate the abundance of mRNA for luteotrophic receptors and steroidogenic elements in the ovaries and corpora lutea of mink during the embryonic diapause, peri-implantation and postimplantation pregnancy. The second aim was to determine whether the mink placenta synthesized progesterone. Homologous cDNA probes for the mink LH and prolactin receptors were generated by the polymerase chain reaction. Heterologous cDNA probes for steroidogenic acute regulatory protein (StAR), cytochrome P450 side chain cleavage (P450scc) and 3 beta-hydroxysteroid dehydrogenase-delta 4-delta 5 isomerase (3 beta HSD) were also used. The abundance of mRNA encoding the prolactin receptor was low during the period of embryonic diapause and increased concurrent with circulating progesterone. The abundance of LH receptor message reached peak values during the peri-implantation period followed by maintenance of a steady-state after implantation. The abundance of StAR and P450scc messages appeared not to vary during gestation, while that for 3 beta HSD was correlated with changes in circulating progesterone. There was no evidence of 3 beta HSD activity or transcripts in the placenta. These results indicate that prolactin and LH are necessary for activation of the corpus luteum during the period of embryonic diapause, and for its maintenance during postimplantation gestation. The mink placenta does not synthesize progesterone.

The role of pregnenolone-metabolizing enzymes in the regulation of oestradiol biosynthesis during development of the first wave dominant follicle in the cow.

During the luteal phase in the cow, a first-wave dominant follicle grows to reach ovulatory size, but then ceases to grow, becomes no longer dominant and enters a phase of slow regression. During this growth transition, the concentration of oestradiol has been shown to decrease in follicular fluid. The objective of this study was to determine if follicular fluid oestradiol concentrations are regulated by the activity of three major steroidogenic enzymes, namely P450-aromatase (P450-arom), 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) and 17 alpha-hydroxylase C-17,20 lyase cytochrome P450 enzyme (P450-17 alpha) measured in granulosa and theca cells isolated from individual first-wave dominant follicles. Follicle growth and state of dominance was assessed by ultrasonography and follicles were classified as growing-dominant (GD, n = 6), non-growing-dominant (NGD, n = 8) or non-growing-non-dominant (NGD, n = 6). Mean follicular fluid concentrations of oestradiol were higher in GD than in NGD or NGND follicles (511 +/- 98 versus 136 +/- 16 and 20 +/- 11 nmol/l respectively). Oestradiol was not correlated with P450-arom in any of the three groups. In GD follicles, oestradiol was positively correlated with pregnenolone concentration but neither was correlated with granulosa or theca 3 beta-HSD activity or with theca P450-17 alpha activity. In NGD follicles, oestradiol was negatively correlated with theca 3 beta-HSD activity and pregnenolone was negatively correlated with granulosa 3 beta-HSD activity. In NGND follicles, oestradiol was positively correlated, and pregnenolone was negatively correlated with theca 3 beta-HSD and P450-17 alpha activities. These studies demonstrated that pregnenolone supply is the principal regulating factor of oestradiol output during follicle dominance and during the loss of dominance but that the levels of P450-17 alpha and 3 beta-HSD activity become rate-limiting when the follicle is no longer dominant.

Differential effects of trilostane and cyanoketone on the 3 beta-hydroxysteroid dehydrogenase-isomerase reactions in androgen and 16-androstene biosynthetic pathways in the pig testis.

3 beta-Hydroxysteroid dehydrogenase-isomerase (3 beta-HSD-I) activity in the pig testis is responsible for the conversion of dehydroepiandrosterone (DHA) to 4-androstenedione and also for the conversion of 5,16-androstadien-3 beta-ol (andien-beta) to 4, 16-androstadien-3-one (dienone). Therefore, 3 beta-HSD-I plays an essential role in the biosynthesis of hormonally and pheromonally active steroids. Previous studies from this laboratory have suggested that the 3 beta-HSD-I reactions in the androgen and 16-androstene biosynthetic pathways may be catalysed by different enzymes with selective substrate specificities [3, 4]. The aim of the present studies was to investigate the reactions further by examining the effects of two classical steroidal inhibitors of 3 beta-HSD-I, trilostane (WIN 24540) and cyanoketone (WIN 19578), on the kinetic parameters of the 3 beta-HSD-I reactions in immature (< 3 weeks) pig testis microsomes. In kinetic analyses of the conversion of DHA to 4-androstenedione, both trilostane and cyanoketone caused increases in the Km(app) for DHA which at the highest concentration used, were 15-fold the control Km(app) of 1.4 mumol/l. No effect on the Vmax(app) (6.55 +/- 0.74 nmol/h/mg protein) was observed, demonstrating that competitive inhibition was evident. Slope and intercept replots confirmed the competitive nature of the inhibition and Ki(app) values of 0.16 mumol/l for trilostane and 0.20 mumol/l for cyanoketone were respectively 9 and 7-fold lower than the Km(app) value. In contrast, trilostane and cyanoketone had no effect on the Km(app) for andien-beta (0.26 mumol/l). The Vmax(app) (1.12 nmol/h/mg protein) was decreased by 40-50% only by trilostane at the highest concentration used, demonstrating a very low affinity for the andien-beta active site. Ki(app) values for trilostane and cyanoketone, obtained from slope and intercept replots were, respectively 1.1 and 1.6 mumol/l, which were 4 and 6-fold greater than the Km(app) for andien-beta. Therefore, trilostane and cyanoketone were powerful competitive inhibitors of the conversion of DHA to 4-androstenedione but were weak non-competitive inhibitors of the conversion of andien-beta to dienone. The selective effects of trilostane and cyanoketone on the 3 beta-HSD-Is involved in the androgen and 16-androstene biosynthetic pathways strongly suggest that the reactions are catalysed by separate enzymes, or at least separate, non-interacting active sites on a single enzyme.