Progestin augmentation of gonadotropin-stimulated progesterone production by cultured rat granulosa cells.

Ovarian progesterone production is stimulated by FSH and LH. Concomitant treatment with a synthetic progestin, R5020, (10(-6)M) increases the FSH-stimulated production of progesterone and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) in cultured rat granulosa cells. Likewise, R5020 augments the LH-stimulated progestin production in FSH-primed cells. Furthermore, the FSH stimulation of pregnenolone biosynthesis is enhanced by 10(-6) M of progesterone or R5020. These in vitro findings suggest that progestins may exert an autoregulatory positive feedback action to enhance gonadotropin-stimulated production of progesterone and 20 alpha-OH-P.

Induction of guanosine triphosphate-cyclohydrolase by follicle-stimulating hormone enhances interleukin-1 beta-stimulated nitric oxide synthase activity in granulosa cells.

In cultured granulosa cells, interleukin-1 beta (IL-1 beta) induced a time-dependent (16-72 h) and dose-related (0.3-30 ng/ml) stimulation of nitric oxide (NO) synthase (NOS) activity, as determined by the catalytic conversion of [3H]arginine to [3H]citrulline and NO2- accumulation in the culture medium. Although FSH alone failed to stimulate NOS activity, concomitant treatment with the gonadotropin (200 ng/ ml) or the cell-permeant cAMP analog (Bu)2cAMP (0.5 mM) markedly enhanced IL-1 beta-induced NO generation in cultured granulosa cells. The effect of IL-1 beta on citrulline biosynthesis and NO2- accumulation was abrogated by the NOS inhibitor NG-methyl-L-arginine or the IL-1-receptor antagonist protein. In contrast bacterial endotoxin (lipopolysaccharide), interferon-gamma, or tumor necrosis factor-alpha, which are well known inducers of inducible NOS (iNOS) in a variety of immunocompetent and nonimmunocompetent cell types, failed to increase [3H]citrulline formation or NO2- accumulation in untreated or FSH-stimulated cells. As demonstrated by reverse transcriptase-PCR analysis, IL-1 beta-stimulated NO generation was accompanied by a time-dependent increase in messenger RNA levels for iNOS and GTP-cyclohydrolase (GTPCH), the rate-limiting step for de novo tetrahydrobiopterin (BH4) biosynthesis. Treatment with FSH augmented only GTPCH messenger RNA expression, and a more than additive GTPCH signal was observed when cells were simultaneously challenged with IL-1 beta and FSH. Treatment with the GTPCH inhibitor 2,4-diamino-6-hydroxypyrimidine prevented IL-1 beta-induced NOS activity in untreated or FSH-stimulated cells, and this inhibition was completely reversed by sepiapterin, a substrate for BH4 biosynthesis, via an alternative pterin salvage pathway present in many cell types. As BH4 is an essential cofactor for NOS catalytic activity, these observations strongly suggest that FSH-induced biosynthesis of endogenous BH4 is essential for full iNOS biosynthetic capacity in IL-1 beta-stimulated granulosa cells.

Interleukin-1 beta stimulates sphingomyelin hydrolysis in cultured granulosa cells: evidence for a regulatory role of ceramide on progesterone and prostaglandin biosynthesis.

In granulosa cells labeled to isotopic steady-state with [3H]serine, addition of interleukin-1 beta (IL1 beta) or bacterial sphingomyelinase (SMase) induced a rapid decrease (approximately 60% by 10 min) in cellular [3H]Sphingomyelin content and a prolonged generation (up to 60 min) of [3H]ceramide, the immediate lipid-moiety generated in response to sphingomyelin hydrolysis. In FSH-treated cells, IL1 beta (0.3-30 ng/ml) inhibited progesterone biosynthesis in a dose-dependent manner, an effect that was also observed in cells exposed to increasing concentrations of bacterial SMase (0.003-0.3 U/ml) or the membrane-permeable ceramide analogue N-hexanoylsphingosine (C6-cer:0.1-10 microM). Abrogation of progesterone biosynthesis was not a sole consequence of inadequate cAMP biosynthesis because cyclic nucleotide levels remained elevated (3- to 4-fold over untreated cultures) after addition of IL1 beta, SMase, or two different cell permeable ceramide analogues (C2-cer and C6-cer) to gonadotropin-stimulated granulosa cells. Moreover, taken into account that exogenous SMase or C6-cer partially abolished progesterone biosynthesis induced by But2cAMP (0.5 mM) or cholera toxin (CTX: 1 microgram/ml), the above mentioned results support the notion that activation of the sphingomyelin pathway exerts its inhibitory effects on granulosa cell steroidogenic activity at site(s) of action both proximal and distal to cAMP generation. As determined by RT-PCR analysis, the inhibitory effect of IL1 beta, SMase, or C6-cer on gonadotropin-stimulated steroidogenesis was accompanied by arrested transcription of the mitochondrial cholesterol side chain cleavage enzyme (P450scc) and 3 beta-hydroxysteroid dehydrogenase/delta 5-4isomerase, the two FSH-inducible steps involved in progesterone biosynthesis. Although bacterial SMase or the ceramide analogue C6-cer alone did not exactly reproduce the effect of IL1 beta on granulosa cell prostaglandin E2 (PGE2) biosynthesis, both agents augmented net PGE2 production and messenger RNA levels of the inducible prostaglandin endoperoxide synthase/cyclooxygenase (PGHS-2) in cytokine-treated cells. Although the effect on PGHS-2 messenger RNA may account for the facilitatory role of ceramide on IL1 beta-induced PGE2 biosynthesis, neither SMase nor the membrane-permeant ceramide analogue were able to augment prostaglandin accumulation in the presence of exogenously added arachidonate precursor. Collectively, whereas these results show that ceramide triggers a negative-effector pathway that is both necessary and sufficient to reproduce the inhibitory effect of IL1 beta on FSH-stimulated granulosa cell steroidogenesis, they also support the notion that sphingomyelin hydrolysis may be important for cytokine-induced PGHS-2 expression but not sufficient to reproduce IL1 beta-stimulated PGE2 biosynthesis.

Testicular 3 beta-hydroxysteroid dehydrogenase/delta 5-4 isomerase in the hypophysectomized rat: effect of treatment with 5 alpha-dihydrotestosterone.

The in-vivo regulatory effect of androgens on steroidogenesis was investigated. Adult (2 to 3 months old) hypophysectomized rats were treated intratesticularly with increasing doses of 5 alpha-dihydrotestosterone (DHT; 10-200 micrograms/100 g body weight) or vehicle (50 microliters dimethyl sulphoxide; DMSO) in the contralateral testis. Intratesticular testosterone concentrations were extremely low in hypophysectomized rats 15-20 days after surgery. Treatment with DHT caused a dose-dependent inhibition of testicular 3 beta-hydroxysteroid dehydrogenase/delta 5-4 isomerase (3 beta-HSD) 2 h later, and this effect was apparent at the dose of 20 micrograms/100 g body weight (P less than 0.01). The inhibitory effect of 3 beta-HSD was not due to a possible interference of DHT in the enzyme assay, since various concentrations of the androgen (0.1-100 mumol/l) were ineffective as inhibitors of 3 beta-HSD. The highest dose of DHT used in this study (200 micrograms/100 g body weight) resulted in a rapid (1-2 h) and transient (4-6 h) inhibition (approximately 80%) of 3 beta-HSD activity. Pretreatment of rats with the antiandrogen cyproterone acetate (5 mg/rat) or the protein synthesis inhibitor cycloheximide (10 mg/rat) did not affect the enzyme activity of testes injected with DMSO, but counteracted the inhibitory effect of DHT on 3 beta-HSD activity in the contralateral testis. The results presented suggest that the inhibitory effect of the non-aromatizable androgen DHT is receptor-mediated and involves the synthesis of a factor(s) that modulates 3 beta-HSD activity.

The inducible isoform of CREM (inducible cAMP early repressor, ICER) is a repressor of CYP19 rat ovarian promoter.

The synthesis of estradiol by the granulosa cells is a prominent event in ovarian physiology and depends on the expression of P450(AROM). FSH induces the expression of P450(AROM) in granulosa cells as a result of the presence in the ovarian promoter of a CRE (cAMP response element)-like sequence (CLS). In rodents, LH downregulates aromatase expression during luteinization by an as yet undescribed mechanism. In granulosa cells, LH increases the expression of the inducible cAMP early repressor (ICER), an isoform of CREM (cAMP-responsive element modulator) that represses cAMP-induced transcription. The possibility that ICER represses the activity of the aromatase ovarian promoter, thus being part of the mechanism underlying the effects of LH was investigated. We have found that: (1) nuclear proteins from forskolin-stimulated granulosa cells were specifically bound to an oligonucleotide containing the CLS sequence of the CYP19 ovarian promoter and one out of the two protein-DNA complexes formed was supershifted by an anti-CREM antibody; (2) in granulosa cells, forskolin-induced increases in P450(AROM) promoter luciferase reporter gene activity were prevented by the transient overexpression of ICER; (3) similar results were obtained in 8-Br-cAMP-stimulated R2C cells, a Leydig tumor cell line routinely used for the study of P450(AROM) promoter activity; (4) both ICER mRNA levels and P450(AROM) promoter-driven luciferase activity were elevated 6 and 12 h after stimulation of R2C cells with 8-Br-cAMP and were decreased 24 and 48 h later; (5) in an R2C polyclonal line overexpressing ICER, the promoter activity at early stages of stimulation was completely attenuated, while 24 and 48 h downregulation was prevented in another R2C line stably transfected with an antisense ICER construct. These results suggest that ICER represses CYP19 ovarian promoter and that LH-induced expression of ICER may serve to downregulate P450(AROM) transcription in granulosa cells during luteinization.

Effect of the protein phosphatase inhibitor okadaic acid on FSH-induced granulosa cell steroidogenesis.

To address a possible role of type 1 and 2A serine/threonine protein phosphatases (PP1 and PP2A) in regulating granulosa cell hormonal responses, we investigated the effects of okadaic acid (OA) on FSH- and cAMP-induced steroidogenesis in these cells. When added alone (0.01-1 nmol/l), the cell-permeant phosphatase inhibitor did not affect progesterone and 3 beta-hydroxysteroid dehydrogenase/delta 5-4 isomerase (3 beta-HSD) enzyme activity, whereas when added with FSH it dose-dependently augmented (minimal effective dose, 0.1 nmol/l) gonadotropin-stimulated steroidogenesis in cultured granulosa cells. A similar stimulatory effect of the toxin was observed in cells cultured for 48 h with the cell-permeant analogue dibutyryl cAMP (1 mmol/l), or when granulosa cells were stimulated with the cAMP-inducing agents cholera toxin (1 microgram/ml), forskolin (15 mumol/l) or 1-methyl-3-isobutyl-xanthine (0.1 mmol/l). The observed effect of OA on FSH-supported granulosa cell steroidogenesis was not a consequence of increased cAMP generation, and time course experiments also revealed that a minimal time period of 12 h was necessary for OA (0.1 and 1 nmol/l) to significantly enhance FSH-induced progesterone and 3 beta-HSD enzyme activity. Since OA also inhibits the dephosphorylation of protein kinase C (PKC) substrates, we also compared the effect of OA and the PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) on FSH-induced granulosa cell steroidogenic activity. While activation of the PKC pathway with the tumor promoter TPA (10 nmol/l) inhibited progesterone and cAMP accumulation in FSH-stimulated granulosa cells, treatment with OA augmented steroidogenesis and did not affect gonadotropin-induced cAMP generation. Collectively these results suggest that PP1 and PP2A may be important in regulating the phosphorylation state of proteins implicated in the cAMP-protein kinase A-stimulated steroidogenic activity of these cells.

Partial characterization of a thyrotropin releasing hormone-sensitive glycosyl-phosphatidylinositol in pituitary lactotrophes.

Metabolic labelling experiments performed with cultured pituitary lactotrophes revealed the presence of a glycosyl-phosphatidylinositol (GPtdIns) structurally related to GPtdIns lipids isolated from other cell types as demonstrated by: (i) metabolic incorporation of [3H]galactose, [3H]glucosamine and [3H]inositol into the polar inositolphosphoglycan moiety (InsPG) and [3H]myristate and [3H]palmitate into the diacylglycerol (DAG) backbone of GPtdIns; (ii) sensitivity of the [3H]labelled GPtdIns to nitrous acid deamination and; (iii) sensitivity of GPtdIns to phosphatidylinositol (PtdIns)-specific phospholipase C (PLC) hydrolysis. In cultured pituitary cells labelled to isotopic steady state with 10 microCi/ml of [3H]glucosamine, treatment with hypothalamic TRH (10(-6) M) induced a rapid and transient hydrolysis (ca. 50%) of the labelled GPtdIns. Moreover, as demonstrated in [3H]inositol labelled cells, treatment with thyrotropin releasing hormone (TRH) elicited the cleavage of [3H]GPtdIns in a similar manner, and this effect was followed by the phosphoinositide (PtdIns, PtdInsP and PtdInsP2) hydrolysis 30 s later. These results suggest that the phosphodiesterase cleavage of GPtdIns could be an early event implicated in TRH action in pituitary lactotrophes.

Inhibition of steroidogenesis in neonatal Leydig cells by unknown factor(s) present in spent media of androgen-treated cultured testicular cells from adult rats.

Treatment of cultured testicular cells from adult rats with 5 alpha-dihydrotestosterone (DHT; 10(-6) M) or the synthetic androgen methyltrienolone (R1881; 10(-6) M) inhibited Leydig cell 3 beta-hydroxysteroid dehydrogenase/delta 5-4 isomerase (3 beta-HSD) enzyme activity, whereas no effect of both androgens on cultured cells derived from neonatal animals could be observed. The inhibitory effect of DHT or R1881 on Leydig cell 3 beta-HSD enzyme activity, however, was abolished when adult cells were cultured in the presence of the anti-androgen cyproterone acetate (CPA; 10(-6) M) or the protein synthesis inhibitor cycloheximide (CX; 1 microgram/ml). Testicular cells from adult animals were also cultured in the presence of the different treatments described above, and the spent media was collected and thereafter used as conditioned culture medium (CCM) in subsequent experiments performed with neonatal cells. Dispersed testicular cells from neonatal rats were cultured for 12 days in McCoy's 5a medium of in CCM derived from R1881-treated adult cells, and fresh culture medium or CCM was replaced every 2 days. The human chorionic gonadotropin (hCG)-stimulated testosterone production of neonatal cells was abolished in the presence of CCM derived from R1881-treated adult cells. Nevertheless, the steroidogenic response to hCG recovered when neonatal cells were cultured for two additional days in McCoy's 5a medium. Treatment of neonatal cells with increasing concentrations of hCG (0.1-10 ng/ml) resulted in a dose-dependent augmentation in Leydig cell 3 beta-HSD enzyme activity and testosterone production.(ABSTRACT TRUNCATED AT 250 WORDS)

Progestin regulation of progesterone biosynthetic enzymes in cultured rat granulosa cells.

Progestins have recently been shown to augment gonadotropin-stimulated progesterone and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) biosynthesis in cultured rat granulosa cells. The mechanism by which progestins autoregulate ovarian progestin biosynthesis was investigated by studying the modulation of pregnenolone biosynthesis as well as the activities of the enzymes 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD). Granulosa cells obtained from immature hypophysectomized, estrogen-treated rats were cultured with FSH and/or progestins. Pregnenolone production was measured in the presence of cyanoketone (10(-6) M) to inhibit 3 beta-HSD activity. Enzymatic activities of 3 beta-HSD and 20 alpha-HSD were determined in cell homogenates by direct enzyme assays. FSH stimulated pregnenolone production, while treatment with progesterone or R5020 alone was ineffective. Concomitant treatment with the progestins further enhanced FSH-stimulated pregnenolone production in a dose-dependent manner with minimal effective doses of 10(-8) and 10(-7) M for R5020 and progesterone, respectively. In FSH-primed cells, LH increased pregnenolone accumulation, and concomitant treatment with R5020 also enhanced the LH action. Furthermore, the gonadotropins stimulated the activity of 3 beta-HSD, and this effect was further enhanced by concomitant treatment with either R5020 or progesterone in a dose-dependent manner. In addition, the 20 alpha-HSD activities were enhanced by progestins in cells treated with FSH but not with LH. Thus, both natural and synthetic progestins stimulate the gonadotropin-induced progesterone production in cultured granulosa cells via enhancing the 3 beta-HSD enzyme as well as pregnenolone biosynthesis.

Androgen regulation of progestin biosynthetic enzymes in FSH-treated rat granulosa cells in vitro.

The influence of androgens on the FSH modulation of progestin biosynthetic enzymes was studied in vitro. Granulosa cells obtained from immature, hypophysectomized, estrogen-treated rats were cultured for 3 days in a serum-free medium containing FSH (20 ng/ml) with or without increasing concentrations (10(-9)-10(-6)M) or 17 beta-hydroxy-5 alpha-androstan-3-one (dihydrotestosterone; DHT), 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-diol), or the synthetic androgen 17 beta-hydroxy-17-methyl-4,9,11-estratrien-3-one (methyltrienolone; R1881). FSH treatment increased progesterone and 20 alpha-hydroxy-4-pregnen-3-one (20 alpha-OH-P) production by 10.2- and 11-fold, respectively. Concurrent androgen treatment augmented FSH-stimulated progesterone and 20 alpha-OH-P production in a dose-related manner (R1881 greater than 3 alpha-diol greater than DHT). In the presence of an inhibitor of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), the FSH-stimulated pregnenolone (3 beta-hydroxy-5-pregnen-20-one) production (a 20-fold increase) was further enhanced by co-treatment with R1881, 3 alpha-diol or DHT. Furthermore, FSH treatment increased 4.4-fold the activity of 3 beta-HSD, which converts pregnenolone to progesterone. This stimulatory action of FSH was further augmented by concurrent androgen treatment. In contrast, androgen treatment did not affect FSH-stimulated activity of a progesterone breakdown enzyme, 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD). These results demonstrate that the augmenting effect of androgens upon FSH-stimulated progesterone biosynthesis is not due to changes in the conversion of progesterone to 20 alpha-OH-P, but involves an enhancing action upon 3 beta-HSD/delta 5,delta 4-isomerase complexes and additional enzymes prior to pregnenolone biosynthesis.

Use of flow cytometry for enumeration of Mycoplasma mycoides subsp. mycoides large-colony type in broth medium.

AIMS: The potential of using flow cytometry (FC) in combination with a fluorescent dye (SYBR green-I) for rapidly estimating Mycoplasma mycoides subSPS. mycoides large-colony type (MmmLC) in broth culture was examined. METHODS AND RESULTS: The FC analysis was performed by staining the MmmLC cells with a fluorescent dye, SYBR green-I (SYBR), and the results were compared with plate count method (colony forming units, - CFUs). There was a good correlation (linear regression, r(2) = 0.93) between mycoplasma counts determined by FC (cells ml(-1)) and by traditional plate count method (CFU ml(-1)). The lowest bacterial concentration detected by FC and traditional plate count was of the order of 10(4) cells ml(-1) and 10(3) CFU ml(-1), respectively. FC method allowed results in 20-30 min, whereas at least 24 h were necessary to obtain results with the traditional plate count method (CFU). CONCLUSION: Growth rates of MmmLC in broth medium determined by FC were highly reproducible and correlated well with mycoplasma counts assessed by the plate count method. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings suggest that FC could be a good alternative to replace other time-consuming techniques that are currently used to enumerate mycoplasma in broth medium, such as plate count method (CFU).

Influence of vaccination on the nitric oxide response of gilthead seabream following infection with Photobacterium damselae subsp. piscicida.

The nitric oxide (NO) response of vaccinated and non-vaccinated juvenile gilthead seabream was studied in vivo and the NO response of isolated kidney macrophages of fish was studied in vitro. Fish were vaccinated with formalin-killed Photobacterium damselae subsp. piscicida (Pdp) with or without Freund's incomplete adjuvant (FIA) and control fish received phosphate buffered saline (PBS). Thirty days later, fish were injected with a sublethal dose of Pdp and 3 fish/group were bled at time periods thereafter and serum nitrite and citrulline levels were determined as a measure of the NO response. All infected groups showed an increase in NO metabolites from 6h to 27 days, with peak levels at 24 h. However, the response in bacterin-vaccinated fish was significantly higher than in the non-vaccinated group and the bacterin plus FIA resulted in a further significant enhancement. Similarly enhanced NO responses were produced in vitro by isolated macrophages obtained from vaccinated compared with non-vaccinated fish 30 days after vaccination following infection, with the response in macrophages from fish vaccinated with the bacterin plus FIA being significantly higher than those from fish vaccinated with the bacterin alone. Thus, vaccination resulted in an enhanced NO response to infection with Pdp in vivo and in vitro. Furthermore, the level of protection of fish to experimental challenge with virulent Pdp correlated with the level of the NO responses in the different groups.

Effects of starvation in rats on serum levels of testosterone, dihydrotestosterone and testicular 3 beta-hydroxysteroid dehydrogenase activity.

Adult male rats were maintained on the following dietary regimes: full-fed for 28 days (controls); full-fed 21 days followed by no feed for seven days (acutely starved); no feed for seven days followed by 1/4 feed for 14 days (chronically starved); no feed for seven days, 1/4 of the normal diet for 1/4 days, and completes return of feed for seven days (refed). Serum testosterone (T) and dihydrotestosterone (DHT) were measured by radioimmunoassay and testicular delta 5-3 beta-hydroxysteroid-dehydrogenase (3 beta-HSD) activity was measured by a pregnenolone depletion method. Prostate, seminal vesicles and seminal fluid weight, as well as number of epididymal spermatozoa were reduced in all starved groups. Serum androgens and testicular delta 5-3 beta-HSD activity were significantly reduced in acutely and chronically starved rats. After seven days of refeeding, serum levels of T, DHT, as well as testicular 3 beta-HSD activity rose to control values. In addition, the accessory organ weights and number of epididymal spermatozoa followed a similar pattern. These data provide further evidence that starvation results in impaired testicular function which is reversed by refeeding.

The effect of starvation, restricted feed intake and refeeding on acid phosphatase activity of the hypothalamus and frontal cerebral cortex of the rat.

Acid phosphatase (EC: 3.1.3.2) activity at the hypothalamus and frontal cerebral cortex has been studied in male rats maintained on different dietary regimes. The enzyme activity in the cerebral cortex did not undergo any observable changes in any of the experimental groups. Enzyme activity in the hypothalamus, rose after food deprivation in both acutely and chronically starved rats, while the enzyme activity returned to physiological levels in refed animals. The hypothalamus neuroendocrine role and a possible interregional heterogeneity for acid phosphatase in rat brain might explain the two enzymic activity patterns observed in the present experiment.

The effect of starvation, diabetes or hypophysectomy on the testicular function in rat.

Serum levels of testosterone, in vitro production of testosterone and the activity of testicular delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) were measured in normal, starved, streptozotocin diabetic and hypophysectomized rats seven days after initiation of the experiment. Similar impaired testicular functions were found in the starved and diabetic animals. The greatest alteration corresponded to the hypophysectomized group. The 3 beta-HSD activity correlates in all groups with hormone levels or hormone production in vitro. The impaired testicular function in the starved and diabetic groups is discussed.