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Analysis of mutational signatures can reveal the underlying molecular mechanisms of the processes that have imprinted the somatic mutations found in a cancer genome. Here, we present a pan-cancer mutational signatures analysis of single base substitutions (SBS) and small insertion and deletions (ID) in pediatric cancers encompassing 537 whole genome sequenced tumors from 20 molecularly defined cancer subtypes. We identified only a small number of mutational signatures active in pediatric cancers when compared to the previously analyzed adult cancers. Further, we report a significant difference in the proportion of pediatric tumors which show homologous recombination repair defect signature SBS3 compared to prior analyses. Correlating genomic alterations with signature activities, we identified an association of TP53 mutation status with substitution signatures SBS2, SBS8, SBS13 and indel signatures ID2 and ID9, as well as chromothripsis associated with SBS8, SBS40 and ID9. This analysis provides a systematic overview of COSMIC v.3 SBS and ID mutational signatures active across pediatric cancers, which is highly relevant for understanding tumor biology as well as enabling future research in defining biomarkers of treatment response.

Most of the 160 million river buffalo in the world are in Asia where they are used extensively, both as a food source and for draught power. Only recently have investigations begun exploring the buffalo genome for variation that might influence health and productivity of these economically important animals. This paper describes the sequence variability of the toll-like receptor 5 (TLR5) gene, which recognizes bacterial flagellin and is a key player in the immune system. TLR5 is comprised of a single exon that is 2577 bp and codes 858 amino acids. We examined single-nucleotide polymorphisms (SNPs) located within the coding region. Overall, 17 SNPs were discovered, seven of which are non-synonymous. Our study population yielded four different haplotypes. We examined predicted protein domain structure and found that river buffalo, swamp buffalo, and African Forest buffalo shared the same protein domain structure and are more similar to each other than they are to cattle and American bison, which are similar to each other. PolyPhen 2 analysis revealed one amino acid substitution in the river buffalo population with potential functional significance.

Dispersal plays a crucial role in many aspects of species' life histories, yet is often difficult to measure directly. This is particularly true for many insects, especially nocturnal species (e.g. moths) that cannot be easily observed under natural field conditions. Consequently, over the past five decades, laboratory tethered flight techniques have been developed as a means of measuring insect flight duration and speed. However, these previous designs have tended to focus on single species (typically migrant pests), and here we describe an improved apparatus that allows the study of flight ability in a wide range of insect body sizes and types. Obtaining dispersal information from a range of species is crucial for understanding insect population dynamics and range shifts. Our new laboratory tethered flight apparatus automatically records flight duration, speed, and distance of individual insects. The rotational tethered flight mill has very low friction and the arm to which flying insects are attached is extremely lightweight while remaining rigid and strong, permitting both small and large insects to be studied. The apparatus is compact and thus allows many individuals to be studied simultaneously under controlled laboratory conditions. We demonstrate the performance of the apparatus by using the mills to assess the flight capability of 24 species of British noctuid moths, ranging in size from 12-27 mm forewing length (similar to 40-660 mg body mass). We validate the new technique by comparing our tethered flight data with existing information on dispersal ability of noctuids from the published literature and expert opinion. Values for tethered flight variables were in agreement with existing knowledge of dispersal ability in these species, supporting the use of this method to quantify dispersal in insects. Importantly, this new technology opens up the potential to investigate genetic and environmental factors affecting insect dispersal among a wide range of species.

1. The metabolism of a model xenobiotic plant metabolite S-benzyl-N-malonyl-L-cysteine (BMC) administered to rat at 10 mg/kg has been studied using a combination of radio-t.l.c. and h.p.l.c. 2. The major route of excretion for the administered 14C was via the urine (79% in 3 days). 3. The major metabolite was hippuric acid. The extent of biotransformation of BMC indicated the lability of the N-malonyl bond whose hydrolytic removal initiated a metabolic sequence which involved the action of C-S lyase to produce benzyl thiol. 4. A comparison of the findings from this study with those from experiments with N-acetyl-S-benzyl-L-cysteine and S-benzyl-L-cysteine is made to support the metabolic pathway proposed.

There are several models for the CYP2D6 active site with the characteristics of their substrates and inhibitors well defined. Imipramine possesses such characteristics and is both a substrate and an inhibitor of the CYP2D6 enzyme. Possible synthetic strategies to avoid interaction with the enzyme have been investigated, including: attenuation of basicity; and alteration of rigidity and length of the alkyl chain. Imipramine inhibited the 1'-hydroxylation of bufuralol (10 microM), an in vitro marker of CYP2D6 activity, in a CYP2D6 cell line (IC50 = 2.4 microM). Inhibitory potency was attenuated by the removal of the basic centre; imipramine N-oxide had no inhibitory effect on bufuralol 1'-hydroxylation. However, removal of this basic centre, as a strategy to decrease CYP2D6 interaction, may well have a detrimental effect on pharmacological efficacy. Both an increase and decrease in the N-N carbon chain length [2C,4C] caused a reduction in inhibitory potency. In addition, introduction of a carbonyl adjacent to the amino dibenzyl moiety into 2C, 3C and 4C compounds brought about a further reduction in inhibitory potency. These data demonstrate that changes to the molecule, distal to the basic centre, can attenuate the affinity of the molecule for CYP2D6 and are in keeping with the known characteristics of the enzyme.

The acyclic cytosine nucleoside analog cytallene [1-(4'-hydroxy-1',2'-butadienyl)cytosine], which has both (+)- and (-)-enantiomers, was evaluated for its anti-hepatitis B virus (HBV) activity in 2.2.15 cells and was found to have potent activity against HBV DNA synthesis. The R-(-)-enantiomer was found to be the more active of the cytallene enantiomers, with a 50% inhibition concentration against HBV synthesis (HBIC50) of 0.08 microM. Its antiviral activity could be reversed by deoxycytidine (dC) and less efficiently by cytidine. Upon removal of the R-(-)-enantiomer from culture medium, the synthesis of HBV DNA could reinitiate, which suggested that the antiviral action is reversible. The R-(-)-enantiomer was also found to be more cytotoxic than the S-(+)-enantiomer. The degree of cytotoxicity varied among the cell lines, with a 50% inhibition of cell growth at greater than 10 microM. The R-(-)-enantiomer had no effect on HBV RNA synthesis and mitochondrial DNA synthesis at a concentration of 10 times or more than the HBIC50. The two enantiomers cannot be deaminated by dC deaminase, and they can be phosphorylated by cytoplasmic dC kinase. The R-(-)-enantiomer of cytallene is the first acyclic cytosine analog with potent inhibitory activity against HBV similar to those of other L-(-)-ddC analogs.

Extensive remodeling of the follicular extracellular matrix occurs during the process of ovulation. This remodeling involves the breakdown of collagen, which is regulated, in part, by the action of the metalloproteinase collagenase and its associated inhibitors. In the present study, follicular metalloproteinase inhibitors were characterized to determine whether they were serum-borne or of ovarian origin, possibly a tissue-derived inhibitor known as tissue inhibitor of metalloproteinase (TIMP). Human follicular fluid and granulosa cells were obtained from preovulatory follicles of patients in an in vitro fertilization program. Chromatographic separation of follicular fluid on Sepharose 6B resulted in two peaks of inhibitory activity. The large molecular radius (Mr) inhibitor was similar in size to the serum-borne metalloproteinase inhibitor alpha 2-macroglobulin (i.e. Mr 700,000) whereas the small Mr inhibitor approximated the size of TIMP (i.e. Mr 29,000). Incubation of aliquots from either of the two peaks of inhibitor activity or an alpha 2-macroglobulin standard with an antibody to alpha 2-macroglobulin decreased the inhibitory activity in both the large Mr peak and the alpha 2-macroglobulin standard by 86.6 +/- 1.7% and 71.5 +/- 7.7% (n = 4, P less than 0.005), respectively, implying cross-reactivity with the alpha 2-macroglobulin antibody. The inhibitory activity in the small Mr peak, however, was unchanged. Northern analysis of total granulosa cell RNA demonstrated TIMP messenger RNA (mRNA) in all eight granulosa cell samples examined whereas alpha 2-macroglobulin mRNA was virtually undetectable. A positive correlation (r = 0.85, P less than 0.01) was observed between the levels of TIMP mRNA and the ratio of the follicular estradiol-progesterone concentration. However, inhibitor activity in the follicular fluid was not correlated with the levels of TIMP mRNA (r = 0.05). These findings confirm the presence of alpha 2-macroglobulin in follicular fluid and demonstrate that human preovulatory granulosa cells contain mRNA for TIMP, an inhibitor that regulates metalloproteinases such as collagenase, gelatinase, and proteoglycanase. Additionally, the expression of TIMP mRNA is steroid related and may be hormonally regulated. It is proposed that TIMP produced in the granulosa cell compartment in conjunction with alpha 2-macroglobulin from the serum may act to control the site and extent of ovarian connective tissue remodeling.

The modulation of ovarian steroidogenesis by epidermal growth factor (EGF) was investigated in cultured rat granulosa cells. Granulosa cells, obtained from ovaries of immature, hypophysectomized, estrogen-treated rats, were incubated for 2 days with EGF, follicle-stimulating hormone (FSH), or EGF plus FSH. Treatment with EGF did not affect estrogen production, but stimulated progestin (i.e. progesterone and 20 alpha-hydroxy-pregn-4-en-3-one) production in a dose-dependent manner. Stimulation of progestin production by EGF appears to be the result of an increase in pregnenolone biosynthesis as well as increases in the activities of 20 alpha-hydroxysteroid dehydrogenase and 3 beta-hydroxysteroid dehydrogenase/isomerase. Treatment with FSH increased both estrogen and progestin production by cultured granulosa cells. When cells were treated concomitantly with EGF, FSH-stimulated estrogen production was inhibited, while progestin production was further enhanced. The EGF enhancement of FSH-stimulated progestin production appears to be the result of synergistic increases in pregnenolone biosynthesis and 20 alpha-hydroxysteroid dehydrogenase activity, resulting in substantial increases in 20 alpha-hydroxypregn-4-en-3-one but not progesterone production. The effects of EGF were shown to be time-dependent. The concept of a direct action of EGF on rat granulosa cells is reinforced by the demonstration of high affinity (Kd approximately 3 X 10(-10) M), low capacity (approximately 5,000 sites/cell) EGF binding sites in these cells. Thus, EGF interacts with specific granulosa cell receptors to stimulate progestin but to inhibit estrogen biosynthesis.

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) may produce its effects by altering gene expression in susceptible cells. In mouse hepatoma cells, TCDD induces the transcription of the cytochrome P1-450 gene, whose product, aryl hydrocarbon hydroxylase, contributes both to the detoxification and to the metabolic activation of carcinogenic polycyclic aromatic hydrocarbons. A DNA fragment containing sequences flanking the 5' end of the cytochrome P1-450 gene was isolated and analyzed. This DNA fragment contains a cis-acting control element with at least three functional domains: a putative promoter, an inhibitory domain upstream from the promoter that blocks its function, and a TCDD-responsive domain still farther (1265 to 1535 base pairs) upstream of the promoter. These findings, together with results from earlier studies, imply that transcription of the cytochrome P1-450 gene is under both positive and negative control by at least two trans-acting regulatory factors.

Prolactin (Prl), beta 2-adrenergic agents and human chorionic gonadotropin (hCG) are luteotropic in rats, whereas gonadotropin releasing hormone (GnRH) exerts direct inhibitory effects on ovarian steroidogenesis. The present study examined the modulation of the progestin biosynthetic pathway by the luteotropic agents, as well as the actions of GnRH. Rat granulosa cells were primed with follicle-stimulating hormone (FSH) to increase their responsiveness to the luteotropic agents. Subsequent treatment for 2 days with Prl, terbutaline (a beta 2-adrenergic agonist) or hCG stimulated the production of progesterone, 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P), pregnenolone and the activity of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD). In contrast, treatment with Prl or terbutaline, but not hCG, inhibited 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activity by decreasing the apparent maximal velocity of the enzyme with no change in its Km value. Concomitant treatment with GnRH inhibited progesterone, but increased 20 alpha-OH-P production stimulated by Prl or terbutaline. These effects were associated with a stimulation of 20 alpha-HSD activity, while neither 3 beta-HSD activity nor pregnenolone biosynthesis was decreased. In contrast, GnRH inhibited progesterone production in hCG-treated cells without affecting 20 alpha-OH-P production. This was associated with an inhibitory effect of GnRH on pregnenolone biosynthesis with no effect upon 3 beta-HSD activity. Thus, Prl and the beta 2-agonist stimulate progesterone production in granulosa cells by increasing pregnenolone production and 3 beta-HSD activity as well as by decreasing 20 alpha-HSD activity, while hCG stimulates progesterone production by increasing pregnenolone production and 3 beta-HSD activity. The inhibitory effect of GnRH on Prl- or terbutaline-stimulated progesterone production appears to result from a preferential increase in 20 alpha-HSD activity, while the GnRH inhibition of hCG-stimulated progesterone production appears to result from a preferential inhibition of pregnenolone production.

Harris (1) first proposed that the anterior pituitary gland is controlled by hypothalamic releasing factors. After the subsequent purification and sequencing of gonadotropin-releasing hormone (GnRH) (2, 3) and the synthesis of potent GnRH agonists and antagonists (4, 5), many laboratories have investigated the effects of these peptides on various reproductive functions. In both males and females, administration of GnRH or its potent agonists results in sustained increases in serum gonadotropins. In women, administration of GnRH causes preovulatory-like surges of gonadotropins (6, 7). In several animal species, a single injection of GnRH induces preovulatory- like surges of LH that trigger ovulation (8–11). Since pituitary gonadotropins are essential for normal gonadal functions and hypothalamic GnRH was believed to act solely on the pituitary gland, treatment with high doses of GnRH and its agonists had been predicted to be a potential means for enhancing fertility. Paradoxically, long term administration...

The direct effects of adrenocortical steroids on basal and FSH-stimulated production of progesterone by ovarian granulosa cells were investigated in vitro. Granulosa cells from immature, hypophysectomized, estrogen-treated rats were cultured for 2 days in the presence or absence of FSH and/or corticoids. Treatment with FSH resulted in substantial increases in the accumulation of progesterone, whereas dexamethasone by itself was only minimally effective. However, concomitant treatment with increasing concentrations of various native and synthetic corticoids led to dose-dependent increases (200-500%) in the FSH-stimulated accumulation of progesterone. The stimulatory potencies of the corticoids tested correlated with their glucocorticoid rather than their mineralocorticoid potencies (dexamethasone greater than cortisol much greater than aldosterone). Treatment with dexamethasone also led to significant increases in the accumulation of progesterone stimulated by prostaglandin E2, cholera toxin, or dibutyryl cAMP. In the presence or absence of FSH, treatment with dexamethasone, cortisol, or corticosterone led to significant decreases in the activity of 20 alpha-hydroxysteroid dehydrogenase. In addition, treatment with dexamethasone led to significant increases in basal and FSH-stimulated activities of 3 beta-hydroxysteroid dehydrogenase. Our findings indicate that glucocorticoids synergize with FSH and other trophic agents in the accumulation of progesterone in accordance with their glucocorticoid rather than mineralocorticoid potencies and that this direct effect may be accounted for, at least in part, by the stimulation of 3 beta-hydroxysteroid dehydrogenase and the inhibition of 20 alpha-hydroxysteroid dehydrogenase.

Inhibin is a gonadal protein hormone that suppresses the secretion of FSH from pituitary gonadotrophs. It has previously been characterized as a heterodimer of two dissimilar subunits (alpha, 18 kilodaltons and beta, 14 kilodaltons) the smaller of which exists in two forms (beta A and beta B) and can form dimers that stimulate the secretion of FSH. In the present work, cDNA clones encoding the inhibin alpha- and beta A-subunits have been isolated from rat ovary and characterized. The alpha-inhibin cDNA predicts a precursor protein of 366 amino acids containing the 133 amino acid mature alpha-subunit at its COOH-terminus. The beta A-inhibin cDNA predicts a precursor protein of 424 amino acids containing the 116 amino acid beta A-subunit at its COOH-terminus. Analysis of rat ovarian RNA indicates that alpha-inhibin mRNA levels are stimulated by PMSG treatment in vivo. In cultured granulosa cells, FSH also stimulates alpha-inhibin mRNA, and the FSH effect is suppressed by cotreatment with GnRH. Hybridization in situ to rat ovarian tissue demonstrates that both the alpha-inhibin and beta A-inhibin mRNAs are specifically expressed in granulosa cells of the developing follicles.

To evaluate the roles of plasminogen activator (PA) and PA inhibitor (PAI) in human ovulation, we obtained follicular fluid and granulosa cells from individual preovulatory follicles of patients undergoing gamete intrafallopian tube transfer. The follicular fluid samples (n = 25) were analyzed for total tissue-type PA antigen, PA enzyme activity by fibrin autography, PAI activity, PAI type 1 (PAI-1) antigen, and PAI-1 mRNA. The follicular fluid of preovulatory follicles contained low levels of total tissue-type PA antigen (less than 1 ng/mL). Fibrin autography experiments indicated little or no detectable PA activity associated with free or unbound PA. The results of the PAI activity assay and PAI-1 antigen determination support the concept of a relative abundance of PAI compared with PA. Hybridization analysis was used to measure the relative amounts of granulosa cell PAI-1 mRNA. The levels of PAI-1 mRNA correlate with follicular fluid PAI concentrations in individual follicles. Taken together, these results support the idea that there is very little free, or active, PA in follicular fluid of human preovulatory follicles, but there is an abundance of PAI. Furthermore, PAI-1 produced by the granulosa cells may represent a major form of PAI in follicular fluid.

Summary. The present review summarizes our studies on the hormonal control of the differentiation of granulosa cells in vitro. Studies on the hormonal regulation of granulosa cell oestrogen and progestagen biosynthesis by various regulatory agents facilitate our understanding of the control processes involved in follicular maturation and luteinization in vivo. This multiple hormonal control mechanism also provides an interesting model for future studies on the diverse mode of hormone action.

Hypothalamic gonadotropin releasing hormone (GnRH) and its analogs have been shown to regulate progestin production by ovarian granulosa cells treated with FSH and prolactin. The present study examines the direct modulation of an ovarian progesterone metabolizing enzyme by GnRH and its analogs in vitro and in vivo . Granulosa cells from hypophyseclomized, diethylstilbestrol-treated rats were cultured with GnRH or its analogs. After 2 days, cells were homogenized and 20α-hydroxysteroid dehydrogenase (20α-OH-SDH) activity was measured using a direct enzyme assay. GnRH stimulated 20α-OH-SDH activity in a dose-dependent manner with an ED 50 of 8.95 × 10 −9 M and a GnRH aganist ([des-Gly 10 , d -Lcu 6 , (N 2 Me)Leu 7 ,Pro 9 NHEt]GnRH) was shown to be 5.6 fold more potent than GnRH. The stimulatory effect of GnRH was inhibited by concomitant treatment with a GnRH antagonist ([ d -pGlu 1 , d -Phe 2 d -Trp 3,6 ]GnRH) with a half maximal inhibitory dose ratio of 3 1 ([antagonist]/[GnRH]). Time course studies indicated that 12 h of GnRH treatment were required to stimulate a measurable increase in enzyme activity. Cycloheximide treatment inhibited the GnRH action, suggesting that GnRH induces the synthesis of 20α-OH-SDH. Furthermore, treatment with GnRH and its analogs in hypophysectomizcd rats in vivo stimulated enzyme activity without affecting the K m of the enzyme, whereas the stimulatory effect of GnRH was blocked by concomitant treatment with a potent GnRH antagonist. These studies demonstrated that GnRH and its analogs may directly regulate the synthesis of ovarian 20α-OH-SDH in vitro and in vivo .

1. Cell Culture Approach to the Study of Granulosa Cells In 1672, Regnier de Graaf first described the differentiation of ovarian follicles into the corpus luteum. He stated that “age and coitus cause very great changes in eggs (follicles). In young animals, they are very small and in more developed ones they are larger. After coitus, they (the follicles) so alter as to resemble the globules (the corpora lutea) …, being one or more according as the animal will produce one or more fetuses.” (1) Follicles as the functional unit: Ovarian follicles have since been shown to be the basic functional unit of the ovary and consist of an outer layer of theca interna cells which encircle inner layers of granulosa cells. Granulosa cells, in turn, surround the innermost oocyte-cumulus cell complex. The maturation of ovarian follicles and their transformation into corpora lutea is regulated by a specific set of hormones and has been a major research area for ovarian physiologists for several hundred years.

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) dioxin, produces a diverse set of biological responses which, in some cases, reflects the altered expression of specific genes. An intracellular receptor protein binds TCDD saturably and with high affinity and mediates several of TCDD's biological effects. In mouse hepatoma cells, TCDD induces aryl hydrocarbon hydroxylase activity by activating the transcription of the cytochrome P1-450 gene. Studies of receptor-defective variant cells indicate that the activation of cytochrome P1-450 gene transcription requires functional TCDD receptors. Analysis of the DNA that flanks the 5′-end of the mouse cytochrome P1-450 gene reveals at least three control regions: a promoter, an inhibitory element, and a dioxin-responsive element (DRE). Therefore, expression of the cytochrome P1-450 gene represents a balance between negative and positive control. The DRE contains two discrete, non-overlapping DNA domains that respond to TCDD. Each TCDD-responsive domain acts independently of the other, each requires TCDD receptors for function, and each has the properties of a transcriptional enhancer. For example, the function of the DREs is relatively independent of both their location and their orientation with respect to the promoter. Together, the DREs and the TCDD-receptor complex constitute a dioxin-responsive enhancer system. Exposure of cells to TCDD results in the protection of a specific DNA domain from exonuclease digestion. This protection requires TCDD receptors. The protected domain maps to a DRE. This observation implies that the TCDD-receptor complex interacts with the DRE to activate the transcription of the cytochrome P1-450 gene.

To assess the role of inhibitors of proteolytic enzymes, such as plasminogen activator (PA) and collagenase in the ovulatory process, inhibitor activity and mRNA levels were examined in periovulatory rat and human ovaries. In the rat, immature animals received 20 IU of pregnant mare serum gonadotropin (PMSG) followed 52 h later by 10 IU of hCG. Ovaries were removed at intervals from 0 to 20 h after human chorionic gonadotropin (hCG) administration. Inhibitor activity for metalloproteinases, such as collagenase, increased from 60.5 +/- 4.1 inhibitor units/ovary at 0 h (i.e., time of hCG treatment) to a maximum of 218.2 +/- 11.4 units/ovary at 8 h after hCG before decreasing at 12 h (time of ovulation) and 20 h (122.2 +/- 7.9 and 71.6 +/- 8.1 units/ovary, respectively). Human follicular fluid and granulosa cells were obtained from preovulatory follicles of patients in our in vitro fertilization program. Metalloproteinase inhibitor activity was evaluated in follicular fluid as well as the levels of PA and PA inhibitor (PAI) mRNA by Northern analysis. Increasing metalloproteinase inhibitor activity was positively correlated with follicular levels of estradiol (p less than 0.001) and progesterone (p less than 0.02, N = 26). Chromatographic separation of follicular fluid resulted in two peaks of metalloproteinase inhibitor activity. The large molecular weight (MW) inhibitor had an approximate size of 700 kilodaltons (kDa) and may represent alpha 2-macroglobulin, a serum-derived inhibitor. The small MW inhibitor shared many of the characteristics of tissue-derived inhibitors of metalloproteinases. Partial purification of the small MW inhibitor by Concanavalin A-Sepharose and Heparin-Sepharose chromatography demonstrated the inhibitor to be a glycoprotein with an approximate MW = 28-29 K. Northern analysis of human granulosa cell total RNA from preovulatory follicles showed little or no detectable tissue-type PA or urokinase-type PA mRNA. In contrast, two species of PA inhibitor type-1 mRNA were detected in relative abundance. The present findings demonstrate the presence of proteolytic inhibitors in periovulatory ovaries of the rat and human. These ovarian inhibitors may play a role in regulating connective tissue remodeling during follicular rupture.

The direct modulation of 20α-hydroxysteroid dehydrogenase (20α-OH-SDH) by regulatory agents of luteal functions was examined using in vitro cultures of ovarian granulose cells from immature, hypophysectomized, estrogen-treated rats. Without FSH priming, treatment with isoproterenol, PRL, or prostaglandin F2α (PGF2α) resulted in minimal modulation of the 20α-OH-SDH activity of granulosa cells. In contrast, after in vitro FSH priming, these hormones or agents caused marked changes in 20α-OH-SDH activity. Isoproterenol treatment inhibited 20α-OH-SDH activity in a dose-dependent manner (ED50, 3.4 × 10-10 M; maximal inhibition, 62% decrease). Similar inhibition of 20α-OH-SDH activity was observed after treatment with epinephrine, norepinephrine, and a β2-adrenergic agonist (terbutalino sulfate), whereas a β1 -adrenergic agonist (dobutamine) was less effective. Furthermore, the inhibitory effect of epinephrine was blocked by concomitant treatment with a β2- antagonist (IPS 339) but not by a β1-antagonist (pract...

Studies with rat ovarian cells indicate that proteolytic enzymes, such as plasminogen activator (PA), play a role in the tissue remodeling that occurs before ovulation. In the rat, gonadotropins appear to increase granulosa cell tissue-type plasminogen activator (t-PA) content by increasing the cellular concentration of t-PA mRNA as well as by modulating the activity of a specific PA inhibitor (PAI). We obtained granulosa cells from preovulatory follicles of women undergoing either in vitro fertilization or gamete intra-fallopian tube transfer in order to evaluate the roles of PA and PAI in human ovulation. Samples of granulosa cell total RNA were hybridized with probes for t-PA, urokinase-type PA, PAI type 1 (PAI-1), or inhibin A-chain (as a control). Northern analyses revealed that the RNA of granulosa cells from preovulatory follicles contained little or no detectable PA mRNA. In contrast, two species of PAI mRNA were detected in relative abundance. The signal intensity produced by the PAI-1 probe varied by about 8-fold among patient samples, suggesting that PAI-1 may be useful as a marker of follicular maturation and differentiation. These results demonstrate that human granulosa cells collected immediately before ovulation contain PA inhibitor mRNA, yet have little or no PA mRNA.

Since the purification and sequencing of gonadotropin releasing hormone (GnRH) and the subsequent synthesis of potent GnRH agonists and antagonists, many laboratories have been involved in investigating the effects of these peptides on reproductive functions. In women, administration of GnRH at a restricted time in the cycle results in preovulatory-like surges of gonadotropin (Yen et al., 1975; Grimes et al., 1975). Paradoxically, long-term administration of potent GnRH agonists is always associated with the inhibition of a variety of female reproductive functions such as ovarian steroidogenesis, ovulation, ovum transport, ovum implantation, pregnancy, uterine growth and ovarian-dependent mammary tumorigenesis.

The influence of triphenylethylene antiestrogens on FSH-stimulated production of estrogen, progesterone, and 20 alpha-hydroxy-4-pregnen-3-one (20 alpha-OH-P) was examined in cultured rat granulosa cells. The cells were cultured with increasing concentrations of FSH, with or without diethylstilbestrol (DES) or various antiestrogens. After 3 days, medium progesterone and 20 alpha-OH-P contents were determined. Cells were reincubated for an additional 8 h with delta 4-androstene-3,17-dione, and estrogen formation was measured. Steroid production was negligible by cultures treated with DES or antiestrogens alone. FSH treatment increased estrogen and progestin production, while the addition of DES (10(-7) M) further enhanced FSH-stimulated steroidogenesis. Likewise, treatment with 10(-6) M of the antiestrogens tamoxifen (Tam), nafoxidine (Naf), or clomiphene citrate (CC) also enhanced FSH-stimulated aromatase activity. In contrast, the antiestrogens each inhibited FSH stimulation of progesterone and 20 alpha-OH-P production (greater than 73% inhibition at 300 ng/ml FSH). Increasing concentrations (3 X 10(-8)-10(-6) M) of the antiestrogens augmented the stimulatory effect of FSH (10 ng/ml) on estrogen production in a dose-related manner (CC greater than Tam greater than Naf). Similar doses of these antiestrogens inhibited the stimulatory effect of FSH (300 ng/ml) on progesterone and 20 alpha-OH-P production (Tam greater than CC greater than Naf). The observed inhibition of progestin production is associated with decreases in FSH-stimulated pregnenolone biosynthesis in antiestrogen-treated cells incubated with 10(-6) M cyanoketone. Furthermore, the antiestrogens inhibited the binding of [3H]estradiol to ovarian estrogen receptors with binding affinity constants of 364, 437, and 2144 nM for CC, Tam, and Naf, respectively. Thus, antiestrogens exert disparate actions on granulosa cell estrogen and progestin biosyntheses. Like estrogens, CC, Tam, and Naf enhance FSH-stimulated aromatase activity with potencies comparable with their abilities to interact with ovarian estrogen receptors. However, unlike estrogens, the antiestrogens inhibit FSH-stimulated progestin biosynthesis, partially via suppression of pregnenolone biosynthesis. The present granulosa cell culture system provides a valuable model for elucidating the disparate actions of estrogens and antiestrogens on ovarian steroidogenesis.

Understanding the complex movement patterns of animals in natural environments is a key objective of ‘movement ecology’. Complexity results from behavioural responses to external stimuli but can also arise spontaneously in their absence. Drawing on theoretical arguments about decision-making circuitry, we predict that the spontaneous patterns will be scale-free and universal, being independent of taxon and mode of locomotion. To test this hypothesis, we examined the activity patterns of the European honeybee, and multiple species of noctuid moth, tethered to flight mills and exposed to minimal external cues. We also reanalysed pre-existing data forDrosophilaflies walking in featureless environments. Across these species, we found evidence of common scale-invariant properties in their movement patterns; pause and movement durations were typically power law distributed over a range of scales and characterized by exponents close to 3/2. Our analyses are suggestive of the presence of a pervasive scale-invariant template for locomotion which, when acted on by environmental cues, produces the movements with characteristic scales observed in nature. Our results indicate that scale-finite complexity as embodied, for instance, in correlated random walk models, may be the result of environmental cues overriding innate behaviour, and that scale-free movements may be intrinsic and not limited to ‘blind’ foragers as previously thought.