Antigenic variation of foot-and-mouth disease virus serotype A.

The current measures to control foot-and-mouth disease (FMD) include vaccination, movement control and slaughter of infected or susceptible animals. One of the difficulties in controlling FMD by vaccination arises due to the substantial diversity found among the seven serotypes of FMD virus (FMDV) and the strains within these serotypes. Therefore, vaccination using a single vaccine strain may not fully cross-protect against all strains within that serotype, and therefore selection of appropriate vaccines requires serological comparison of the field virus and potential vaccine viruses using relationship coefficients (r1 values). Limitations of this approach are that antigenic relationships among field viruses are not addressed, as comparisons are only with potential vaccine virus. Furthermore, inherent variation among vaccine sera may impair reproducibility of one-way relationship scores. Here, we used antigenic cartography to quantify and visualize the antigenic relationships among FMD serotype A viruses, aiming to improve the understanding of FMDV antigenic evolution and the scope and reliability of vaccine matching. Our results suggest that predicting antigenic difference using genetic sequence alone or by geographical location is not currently reliable. We found co-circulating lineages in one region that were genetically similar but antigenically distinct. Nevertheless, by comparing antigenic distances measured from the antigenic maps with the full capsid (P1) sequence, we identified a specific amino acid substitution associated with an antigenic mismatch among field viruses and a commonly used prototype vaccine strain, A22/IRQ/24/64.

Toward a global foot and mouth disease vaccine bank network.

A network of foot and mouth (FMD) vaccine banks has been initiated with the support of vaccine bank managers and technical advisors that participated in a workshop held at the Institute for Animal Health, Pirbright, in the United Kingdom in April 2006. Terms of Reference that provide guidance for coordinated activities are under consultation. Practical and economic benefits can be realised from collaboration, which will be achieved through mutually acceptable mechanisms for the exchange of information and materials relevant to vaccine banks and their management. If administrative and technical hurdles can be overcome, the network has the potential to contribute significantly to the improved control of FMD worldwide. A 'global' and interactive vaccine bank association could be created by agreeing a system of resource sharing that could orchestrate additional emergency cover with vaccine or antigen from the reserves of network members.

Cortisol induction of growth hormone synthesis in a clonal line of rat pituitary tumor cells in culture.

Growth hormone synthesis increased markedly after addition of glucocorticoids at physiological concentrations to cultures of the GH(1) line of rat pituitary tumor cells. Stimulation of hormone protein synthesis by corticosteroids was selective, since (i) the rate of hormone synthesis increased while total protein synthesis decreased, and (ii) cortisol analogs, biologically inactive metabolites, and sex steroids did not induce growth hormone synthesis.

Review of the Global Distribution of Foot-and-Mouth Disease Virus from 2007 to 2014.

Foot-and-mouth disease (FMD) virus affects livestock worldwide. There are seven different serotypes, each with a diversity of topotypes, genetic lineages and strains. Some lineages have different properties that may contribute to sporadic spread beyond their recognized endemic areas. The objective of this study was to review the most significant FMD epidemiological events that took place worldwide between 2007 and 2014. Severe epidemics were caused by FMD virus (FMDV) lineage O/Asia/Mya-98 in Japan and South Korea in 2010, both previously free of disease. In India, where FMD is endemic, the most important event was the re-emergence of lineage O/ME-SA/Ind-2001 in 2008. Notably, this lineage, normally restricted to India, Bangladesh, Nepal and Bhutan, was also found in Saudi Arabia and Libya in 2013 and has caused several outbreaks in Tunisia and Algeria in 2014-2015. In January 2011, FMDV-positive wild boars were found in Bulgaria, where the disease last occurred in 1996, followed by 12 outbreaks in livestock infected with FMDV O/ME-SA/PanAsia2. In 2012, FMDV SAT2 caused outbreaks in Egypt and the Palestinian Autonomous Territories. Another significant event was the emergence of FMDV Asia1 Sindh-08 in the Middle East. In South America, one outbreak of FMDV serotype O, topotype Euro-SA was reported in Paraguay in 2011, which was recognized as FMD-free with vaccination at the time. Lessons learned from past events, point out the need for an integrated strategy that comprises coordinated global and regional efforts for FMDV control and surveillance. Specific local characteristics related to host, environment and virus that condition FMD occurrence should be carefully considered and incorporated to adapt appropriate strategies into local plans. In this review, we compiled relevant epidemiological FMD events to provide a global overview of the current situation. We further discussed current challenges present in different FMD areas.

Beta-2-Adrenergic stimulation of ornithine decarboxylase activity in porcine granulosa cells in vitro.

Epinephrine, norepinephrine, and isoproterenol produced dose-dependent stimulation of ornithine decarboxylase (EC 4.1.1.17) activity in isolated porcine granulosa cells maintained under defined conditions in vitro. beta- but not alpha-receptor-blocking agents prevented enzyme stimulation by catecholamines. Application of preferential beta-1 and beta-2-receptor antagonists and agonists localized the epinephrine effect to beta-2-adrenergic mediation. Epinephrine action was enhanced by the phosphodiesterase inhibitor, 1-methyl-3-isobutyl-xanthine, but not by saturating concentrations of the cyclic AMP analogue, 8-bromocyclic AMP, of follicle-stimulating hormone, or of prostaglandin E2. However, stimulation by epinephrine was additive to that of luteinizing hormone. Follicular fluid obtained from immature Graafian follicles contained concentrations of norepinephrine and epinephrine active in vitro. Thus, catecholamines may participate in the regulation of ornithine decarboxylase activity in the ovary. Catecholamine effects may be mediated by beta-2-receptors linked to the adenylate cyclase system.

Insulin degradation by isolated fat cells and their subcellular fractions.

Isolated frt cells and purified subcellular fractions of fat cells have been shown to degrade insulin to biologically inactive trichloroacetic-acid-soluble fragments. Further study of this activity has revealed the following characteristics: 1 Most of the insulin-degrading enzymes are intracellular, inaccessible to insulin or trypsin when fat cells are intact. More that 90 per cent of the recovered activity is found in the high-speed supernatant (cytosol) when cell fractionation studies are performed. 2. The plasma membrane contains significant insulin-degradative capacity, as shown by tryptic digestion of intact cells and cell fractionation. 3. The pH optimum of the cell-membrane insulin-degrading site is more acid than that of the cytosol activity, but the tow enzyme systems are similar with regard to substrate specificity, response to metabolic inhibitors, and elution volume of degradation products on gel filtration. 4. The plasma-membrane-degrading activity differs from the specific insulin-binding site with regard to saturation kinetics, optimum temperature, substrate specificity, sensitivity to sulfhydryl-blocking agents, and trypsin snesitivity.

Impact of zanamivir on antibiotic use for respiratory events following acute influenza in adolescents and adults.

BACKGROUND: Influenza infections commonly lead to respiratory tract complications that result in antibiotic treatment. OBJECTIVES: To determine frequency of respiratory events leading to antibiotic use following influenza illness in adolescents and adults, and to assess whether treatment with topical zanamivir prevents these complications. METHODS: Meta-analysis of 7 randomized, double-blind, placebo-controlled trials; 3815 mainly healthy adolescents and adults (mean age, 34 years) with an influenzalike illness of less than 2 days' duration were randomly assigned to receive combined inhaled and intranasal zanamivir, inhaled zanamivir, or corresponding placebos. Twelve percent of enrolled subjects were high-risk patients. The main outcome was the incidence of respiratory events leading to antibiotic prescriptions in patients with proven influenza. RESULTS: Influenza infections were laboratory confirmed in 2499 (66%) of 3815 patients (influenza A in 88% and B in 12%). Placebo recipients developed a respiratory event leading to antibiotic use in 17% of cases, mainly for acute bronchitis or acute sinusitis. Among zanamivir-treated patients (n = 1494) the incidence of respiratory events leading to the use of antimicrobials was 11% (relative risk [RR] compared with placebo, 0.69; 95% confidence interval [CI], 0.57-0.84). Intranasal and inhaled zanamivir seemed to reduce the number of upper (RR, 0.59; 95% CI, 0.36-0.97) and lower respiratory tract events (RR, 0.64; 95% CI, 0.38-1.08). Inhaled zanamivir reduced the number of lower respiratory tract events (RR, 0.60; 95% CI, 0.42-0.85), but the reduction in the number of upper respiratory tract events was not statistically significant (RR, 0.90; 95% CI, 0.63-1.27). CONCLUSIONS: Respiratory complications or worsening of symptoms leading to antibiotic use occurred in about 17% of adolescents or adults with influenza infection. Early treatment of influenza illness with zanamivir reduced the number of these antibiotic prescriptions. Arch Intern Med. 2000;160:3234-3240.

Regulation of deoxyribonucleic acid synthesis in cultured porcine granulosa cells by growth factors and hormones.

To assess the potential role of hormones and growth factors in ovarian follicular growth, we have developed a simple method for the evaluation of DNA synthesis in cultured porcine granulosa cells. Optimal conditions were found to entail newly established cultures that were growth arrested by serum deprivation and then treated with growth-promoting agents. Such treatment resulted in an easily quantitated 3- to 30-fold increase in [3H]thymidine incorporation into trichloroacetic acid-precipitable macromolecules. The radioactivity incorporated was localized to the DNA band on cesium chloride gradients and showed excellent correlation with labeling indices. Insulin, multiplication-stimulating activity, epidermal growth factor (EGF), platelet-derived growth factor, and ovarian follicular fluid were potent stimulators of DNA synthesis in this assay. While EGF and insulin effects were additive, indicating discrete modes of action, the effects of insulin and multiplication-stimulating activity were not additive at maximally effective concentrations. In contrast to the effects of these stimulatory substances, BSA and porcine relaxin were devoid of mitogenic activity under these circumstances. The major trophic hormones implicated in follicular growth, FSH, LH, and estradiol, as well as the cAMP analog 8-bromo-cAMP were without mitogenic effects. Instead, treatment with LH, 8-bromo-cAMP, and the combination of estradiol and FSH resulted in a distinct decrease in DNA synthesis. These data confirm and extend previous evidence of the mitogenic action of insulin-like peptides and EGF, and suggest that the hormones generally believed to regulate granulosa cell replication in vivo lack direct mitogenic effects in vitro.

Effects of 2-hydroxyestradiol on the number of granulosa cell beta-adrenergic receptors.

Recent studies have shown that 2-hydroxyestradiol (2-OH-E2) synthesized locally from estradiol (E2) stimulates progesterone production by granulosa cells (GC) and enhances the action of other trophic hormones. A particularly strong synergistic interaction between 2-OH-E2 and beta-adrenergic agonists was observed. Therefore, the present studies were undertaken to determine if this synergism was due to a 2-OH-E2-stimulated increase in the number of beta-adrenergic binding sites in GC. Binding of the 125I-labeled beta-adrenergic ligand iodocyanopindolol ([125I]iodo-CYP) to GC was evaluated and was found to be saturable with time and ligand concentration, and of high affinity (Kd = 29 +/- 8 pM; maximum binding = 0.55 +/- 0.02 fmol/10(6) cells). Rank order potency for agonist/antagonist binding was consistent with a beta-adrenergic receptor: propranolol greater than isoproterenol greater than norepinephrine. 2-OH-E2 did not compete for the [125I]iodo-CYP-binding sites. Incubation of cultured porcine GC with 2-OH-E2 caused a time- and dose-dependent increase in the number of specific [125I]iodo-CYP-binding sites. Averaged over eight separate experiments, 4-day treatment with 4 micrograms/ml 2-OH-E2 increased the number of [125I]iodo-CYP-binding sites 3.1 +/- 0.9-fold above the control value (P less than 0.05). A similar 4-day treatment with 2 micrograms/ml E2 or 200 ng/ml LH or FSH was without effect on [125I]iodo-CYP binding (P greater than 0.05) despite stimulation of progesterone secretion to a degree similar to that seen with 2-OH-E2. These results support the hypothesis that 2-OH-E2 produced by GC may enhance progesterone production stimulated by catecholamines, in part by increasing the numbers of beta-adrenergic binding sites on GC.

Gonadotropins and estradiol stimulate immunoreactive insulin-like growth factor-I production by porcine granulosa cells in vitro.

Previous studies have established the ovarian granulosa cell as a site of insulin-like growth factor-I (IGF-I) secretion and action, suggesting an autocrine function for this peptide in the ovary. To better understand how this putative autocrine system is regulated and its interface with the classic ovarian trophic hormones FSH, LH, and estradiol (E2), we have studied the effects of these hormones on the secretion of immunoreactive IGF-I (iIGF-I) by cultured porcine granulosa cells. Immature granulosa cells were cultured under serum-free conditions which were optimized to allow maximal iIGF-I production and hormonal responsivity. Measurements of iIGF-I were made after minimizing the influence of IGF-binding proteins by either acid gel filtration or reverse phase chromatography. Since the two preparative procedures gave roughly comparable results, the more expeditious reverse phase procedure was chosen for most samples. Cycloheximide virtually eliminated measurable iIGF-I in culture, suggesting that the peptide measured was newly synthesized, and degradation of IGF-I by cultured granulosa cells was negligible. Consequently, the medium levels provided an accurate indication of cellular secretion over the collection period. Under optimal culture conditions, iIGF-I was readily measurable and responsive to treatment with ovarian trophic hormones. The iIGF-I levels in several experiments with these hormones were as follows: FSH treatment, 1.58 +/- 0.21 times the control value (n = 5 experiments); E2 treatment, 1.26 +/- 0.12 times the control value (n = 5); E2 plus FSH, 3.12 X 0.31 times the control value (n = 8); LH, 1.33 +/- 0.12 times the control value (n = 3); LH plus FSH, 1.78 +/- 0.2 times the control value (n = 1). To assess the role of cAMP in the mediation of gonadotropin effects in this system, granulosa cells were treated with a phosphodiesterase inhibitor (methylisobutylxanthine), which resulted in iIGF-I levels 1.61 +/- 0.7 times the control level. In the presence of FSH, a further stimulatory effect was demonstrated (3.76 +/- 0.29 times control). In addition, the cAMP analog 8-bromo-cAMP dramatically increased iIGF-I levels (6.3 +/- 0.72 times control). These data provide the first demonstration that gonadal iIGF-I secretion can be stimulated by the principal hormones involved in trophic regulation of the ovary. As with other gonadotropin-dependent functions of granulosa cells, this effect appears to be mediated by cAMP and enhanced by E2. This interface between circulating hormones and autocrine systems could provide an important mechanism to amplify the effects of gonadotropic hormones on a local level.

Insulin and insulin-like growth factors (IGFs) stimulate production of IGF-binding proteins by ovarian granulosa cells.

Ligand blot analysis of granulosa cell (GC)-conditioned culture medium revealed several easily measurable insulin-like growth factor (IGF)-binding proteins (IGFBPs), including IGFBP-3 [40-44 kilodaltons (kDa)] and IGFBP-2 (34 kDa). In the present study, IGF-I, in a dose-dependent manner, significantly stimulated the production of these IGFBPs. Insulin, but not IGF-II, mimicked IGF-I's action on IGFBP-3 and -2 production, but was less potent. The synthetic IGF, long R3-IGF-I, which has very low affinity for IGFBPs and only slightly reduced affinity for the IGF-I (type I) receptor, had significantly greater potency in stimulating IGFBP-3 and -2 production compared to IGF-I. Des-(1-3)-IGF-I had similar effects. IGF-I, IGF-II, and the IGF-I analogs, but not insulin, also induced production of an unidentified 30-kDa IGFBP not normally detectable in these cultures. However, in the presence of epidermal growth factor (which was without independent effect on the 30-kDa IGFBP), insulin also induced this 30-kDa IGFBP. By Northern analysis the expression of IGFBP-3 mRNA was found to be significantly stimulated by IGF-I. In summary, insulin stimulated IGFBP-3 and -2 production in a manner that mimics that of IGF-I and the more potent long R3-IGF-I. However, its low potency suggested that IGFBP production is regulated via the IGF-I (type I) receptor. The much higher potency of long R3-IGF-I compared to that of IGF-I suggests that the IGFBPs themselves modulate the action of IGFs by sequestering exogenous IGFs. Thus, one cellular response to IGF stimulation is the production of IGFBPs, which, in turn, reduce or negate the biological activity of the IGFs. The effects of insulin-like peptides are exerted at least in part by increasing levels of mRNA for specific BPs.

Growth factors regulate immunoreactive insulin-like growth factor-I production by cultured porcine granulosa cells.

The effects of various growth factors on the production of immunoreactive insulin-like growth factor I (iIGF-I) in short term (3-day) cultures of porcine granulosa cells was investigated. Epidermal growth factor (EGF) was shown to be a potent dose-dependent stimulator of iIGF-I production, achieving a 3.6-fold stimulation at a dose of 10 ng/ml. Transforming growth factor-alpha (10 ng EGF equivalents/ml) was also stimulatory. Platelet-derived growth factor (10 ng/ml) had no effect of its own, but enhanced EGF-stimulated iIGF-I production. The acidic and basic fibroblast growth factors (100 ng/ml) had no effect alone or in combination with EGF. Transforming growth factor-beta (10 ng/ml) had no effect of its own, but inhibited EGF-stimulated iIGF-I production. The interactive effects of EGF and FSH (200 ng/ml) on iIGF-I production were investigated in short term and longer term (7-day) cultures. In short term cultures under conditions optimized for EGF-dependent iIGF-I production, FSH had no effect of its own and inhibited EGF action. Conversely, in longer term cultures optimized for FSH-dependent iIGF-I production, EGF had no effect of its own and inhibited FSH action. Thus IGF production by cultured porcine granulosa cells is regulated in a complex manner and is highly dependent on the culture conditions. Our results suggest that IGF production in the ovary may also be regulated in a complex manner which is dependent on the developmental state of the follicle.

2-Hydroxyestradiol modulates a facilitative action of catecholamines on porcine granulosa cells.

Recent studies have disclosed a novel intraovarian paracrine system whereby catecholestrogens [e.g. 2-hydroxyestradiol (2-OH-E2)], synthesized locally from estradiol (E2) can stimulate progesterone secretion by granulosa cells (GC). Since these studies suggested that effects of 2-OH-E2 were discrete from those of E2, the present studies were undertaken to determine if the effects of 2-OH-E2 could be mediated through or interact with the catecholamine response system of GC. First, the effects of 2-OH-E2 were compared with those of E2 and epinephrine (EPI) using undifferentiated porcine GC. After 4 days of treatment, saturating concentrations of EPI (1 microgram/ml), 2-OH-E2 (4 micrograms/ml), and E2 (1 microgram/ml) stimulated progesterone production per cell 2-, 9-, and 10-fold, respectively. Saturating doses of EPI plus 2-OH-E2 or EPI plus E2 caused further significant increases in progesterone production above the effects of any single treatment, and the effects of EPI plus 2-OH-E2 were significantly greater than that of EPI plus E2. Isoproterenol mimicked the effect of EPI. Neither propranolol (a beta-antagonist) nor phentolamine (an alpha-antagonist) blocked the effects of 2-OH-E2, suggesting that effects of 2-OH-E2 were not mediated through alpha- or beta-adrenergic receptors. Collectively, these data suggest that 2-OH-E2, beta-adrenergic agonists, and E2 use separate response systems. To further evaluate the interaction between catecholamines and 2-OH-E2, GC were treated with increasing doses of 2-OH-E2 with or without EPI or isoproterenol. EPI and isoproterenol caused a synergistic increase in progesterone production above that induced by all doses of 2-OH-E2 along (average 3.8 +/- 0.7- and 3.2 +/- 0.4-fold enhancement for EPI and isoproterenol, respectively). This synergistic effect was blocked with addition of propranolol, indicating a beta-adrenergic mediation for the catecholamine portion of this effect. Studies using the catechol-O-methyltransferase inhibitor, U-0521, and the O-methyl derivative of EPI, metanephrine, suggested that catechol-O-methyltransferase present in GC dramatically reduces the potency of EPI, but is not involved in the synergism between EPI and 2-OH-E2. In conclusion, 2-OH-E2 is a more efficacious stimulator of progesterone secretion than catecholamines and synergizes with beta-adrenergic agonists to further stimulate progesterone production by GC. The mechanism by which catecholamines and 2-OH-E2 interact within GC is unknown.4+owever, this catecholamine/2-OH-E2 interaction

Concomitant effects of growth hormone on secretion of insulin-like growth factor I and progesterone by cultured porcine granulosa cells.

The observation that GH deficiency delays the onset of puberty has raised the question of the effect of GH on gonadal development. In addition, recent studies in the rat have indicated that GH is able to elevate ovarian levels of immunoreactive insulin-like growth factor I (iIGF-I) in vivo and enhance FSH-induced granulosa cell differentiation in vitro. To evaluate further the possibility of direct effects of GH on ovarian function, we have studied the action of GH on the secretion of iIGF-I and progesterone by cultured porcine granulosa cells from immature follicles. The effects of GH were compared with those of estradiol (E2) and FSH, hormones previously shown to stimulate steroidogenesis and iIGF-I production in this system. GH-stimulated cultures secreted 7.8 times as much iIGF-I per cell as control cultures, while cultures treated with E2 plus FSH secreted 4.5 times as much, and the combination of all three hormones produced an additional increment. The GH-dependent immunoreactivity was localized to two peaks on gel filtration which coeluted with authentic IGF-I and with an IGF-binding protein. In contrast to the results with iIGF-I secretion, GH was a relatively ineffective stimulator of progesterone secretion, resulting in levels 2.6 times the control value, compared to levels 7.4-fold the control value in cultures treated with E2 plus FSH. However, when the three agonists were combined, a synergistic interaction was observed which resulted in progesterone values 33.3 times the control value. In parallel studies, PRL was unable to mimic the effects of GH on iIGF-I or progesterone secretion. In summary, GH has direct stimulatory actions on porcine granulosa cells. Compared to E2 and FSH, established stimulators of these cells, GH is at least comparable in effectiveness with regard to iIGF-I secretion, but less effective as a stimulator of steroidogenesis. However, GH dramatically enhances the effects of E2 and FSH on progesterone secretion. These effects of GH could be important during the onset of puberty, when GH levels in plasma are elevated.

Proteolytic degradation of insulin-like growth factor (IGF)-binding protein-3 by porcine ovarian granulosa cells in culture: regulation by IGF-I.

Porcine ovarian granulosa cells in culture secrete glycosylated insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3), which inhibits gonadotropin and IGF action in the ovary. Synthesis of IGFBP-3 is stimulated by IGF-I and attenuated by gonadotropin. The purpose of the present study was to determine whether IGFBP-3 levels were also regulated via proteolysis. Exogenously added nonglycosylated recombinant human IGFBP-3 (rhIGFBP-3) was significantly degraded over time by a soluble serine-specific protease, similar to plasmin, in control cultures and those treated with FSH, insulin, or several other classes of hormones. In contrast, degradation was greatly attenuated by the IGFs. Degraded rhIGFBP-3 exhibited much reduced affinity for [125I]IGF-II, suggesting that degradation could make available IGFs for cellular interaction. The mechanism of IGFBP-3 protease inhibition by IGFs is unclear. Mediation by IGF receptors is unlikely, as insulin at a dose that activated both insulin and type I IGF receptors did not alter intrinsic degradation of IGFBP-3 (as does IGF). Additionally, IGF-I attenuation of IGFBP-3 degradation was not inhibited by antagonism of receptor action with a tyrosine kinase inhibitor. Further, IGF-I inhibited degradation in cell-free conditioned medium. Direct stabilization of IGFBP-3 via binding of IGFs was suggested from these results. However, long R3 IGF-I attenuated IGFBP-3 degradation even though it has low affinity for IGFBPs. Inhibition of the protease by IGFs is also possible. We conclude that IGFs inhibit the degradation of exogenous nonglycosylated rhIGFBP-3. If active in vivo, this may serve to increase endogenous IGFBP-3 levels in follicular fluid.

FSH and LH stimulation of ornithine decarboxylase activity: studies with porcine granulosa cells in vitro.

Under defined conditions in vitro FSH and LH caused a dose-dependent stimulation of ornithine decarboxylase (ODC) activity in granulosa cells isolated from porcine ovarian follicles. In cells from small follicles, FSH was at least twice as potent an inducer of the enzyme activity as LH. The cells from medium-sized follicles were more responsive to both hormones than cells from small follicles. In addition, the cells from medium-sized follicles were approximately equally responsive to maximal FSH and LH stimulation. Incubation of cells from either small follicles or medium-sized follicles with maximum effective doses of LH plus FSH caused no additive effects.

Serum-free medium enhances growth and differentiation of cultured pig granulosa cells.

We have developed new serum-free culture techniques for swine granulosa cells from immature (1-3 mm) follicles. These methods have allowed more detailed examination of factors regulating both replication and cytodifferentiation of these cells. For optimal replication, collagen-coated culture dishes and a highly supplemented nutrient medium (a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-10), containing 5 micrograms/ml transferrin, 300 mU/ml insulin, 40 ng/ml hydrocortisone, 4 mg/ml BSA, and 2.5% (vol/vol) of a platelet extract (PE) was found to be essential. Cultures maintained in this serum-free complete medium (SFCM) grew to confluence and contained as many or more cells than replicate cultures maintained in 10% fetal calf serum (10% FCS) (e.g. SFCM: 1.89 +/- 0.17; 10% FCS: 1.12 +/- 0.02 cells per well X 10(-5) on day 6). In the absence of albumin, PE, or without collagen coating, the cell numbers were, respectively 4.5%, 9.8%, and 5.0% of that observed with complete SFCM. The mitogenic effect of the PE was due to heat-labile as well as heat-stable components and could not be replaced by platelet-derived growth factor. To evaluate cytodifferentiation, cells grown in SFCM were compared with those grown in 10% FCS with regard to progesterone secretion and FSH responsiveness. Basal progesterone levels were higher in SFCM at all stages in culture. FSH stimulated progesterone secretion in both 10% FCS and SFCM. However, FSH responsiveness was diminished after 4 days with 10% FCS, whereas cells in SFCM remained responsive for 10 days. Thus, this system seems to be highly suitable for the study of the regulation of growth and differentiation of granulosa cells.

Gonadotropin stimulation of porcine ovarian ornithine decarboxylase in vitro: the role of 3',5'-adenosine monophosphate.

The role of cAMP as a mediator of gonadotropin stimulation of ovarian ornithine decarboxylase (ODC) activity was studied in granulosa cells isolated from small (1--2 mm) porcine ovarian follicles. These cells responded to both FSH and LH with significant increases in intracellular concentration of cAMP. At concentrations of gonadotropins which were saturating for the induction of ODC activity, FSH was a more potent stimulator of both cAMP production and ODC activity than LH. N,O'-Dibutyryl cAMP (1.0--10.0 mM) caused a dose-dependent stimulation of ODC activity which equaled the maximal effect of LH but was significantly less effective than the saturating dose of FSH. 8-Bromo-cAMP was more potent than N,O'-dibutyryl cAMP and as effective as FSH as an inducer of ODC activity. Addition of theophylline, a phosphodiesterase inhibitor, to the incubation medium resulted in a dose-dependent inhibition of ODC activity in both control and gonadotropin-stimulated cells. In contrast, 1-methyl,3-isobutyl xanthine, another phosphodiesterase inhibitor, potentiated effects of both submaximal and maximal effective doses of gonadotropins while producing no effect on basal ODC activity of these cells. The results of this study are consistent with the concept that cAMP can mediate gonadotropin stimulation of ODC in porcine granulosa cells. In addition, this study shows the importance of proper selection of cAMP analogs and phosphodiesterase inhibitors, and their concentration in studying such effects.

Demonstration of specific binding of prolactin by porcine corpora lutea.

Subcellular fractions from porcine corpora lutea of the reproductive cycle and pregnancy were shown to have specific binding sites for ovine prolactin (oPRL). Aside from oPRL, only ovine and bovine growth hormone preparations competed with [125I]iodo-oPRL for its binding site. These cross reactions were at a level consistent with the prolactin contamination of these preparations. Rat growth hormone, FSH, LH, TSH, insulin, and ACTH exhibited negligible cross-reactivity. Both corpora hemorrhagica and albicantia had lower specific binding of [125I]iodoPRL than did active corpora lutea of the reproductive cycle, while corpora lutea of pregnancy demonstrated a nearly 5-fold increase in specific binding compared with that of the cycle. Corpora lutea from animals with larger fetuses (greater gestational age) bound the most prolactin. Analysis of data from cold competition studies employing weighted non-linear least-square fitting to a three-parameter model, showed high-affinity binding of oPRL with an association constant (Ka, 23 C) of 2.0 X 10(9)M-1 for the binding site of the corpus luteum of the cycle. The Ka shows no appreciable change with pregnancy. In contrast, the binding site concentration (N) increases markedly from less than 10 fmol/mg protein in corpora lutea from non-pregnant animals to approximately 40 fmol/mg protein for animals at a gestational stage of 40-46 days. The observed Ka's are similar to values obtained for the prolactin binding site in porcine granulosa cells harvested from unruptured follicles and to the prolactin-binding site in the mammary gland.

Multihormone regulation of steroidogenesis in cultured porcine granulosa cells: studies in serum-free medium.

FSH, LH, and estradiol are known to modulate ovarian follicular differentiation. However, the cellular site of action and relative importance of the three hormones have remained uncertain. The recent development of a serum-free system for the culture of immature porcine granulosa cells has enabled us to reinvestigate these issues with better control of pituitary peptides and gonadal steroids. Progesterone production in response to FSH was higher in cells cultured in serum-free complete medium than in those grown in the presence of 10% fetal calf serum [10-fold vs. 1.5-2 fold (control)]. Ovine LH alone was also able to stimulate progesterone production in serum-free free complete medium (6-fold); this effect could not be accounted for by FSH contamination. The LH stimulation, however, was enhanced by FSH. Insulin was required for both FSH and LH stimulation of progesterone production. Estradiol stimulated progesterone production per se (2- to 3-fold) and also enhanced FSH and LH actions. The estimated ED50 for estradiol in FSH-treated cells was 20 ng/ml. Maximal levels of progesterone after 6 days were observed when the combination of FSH, LH, and estradiol was present from the onset of the culture. Incubations carried out in the presence of 5-cholesten-3 beta-25-diol indicated that the hormonal interactions take place, at least in part, at the level of the side-chain cleavage enzyme. These results indicate that FSH is the most important hormonal stimulus for progesterone synthesis in immature granulosa cells. However, LH, estradiol, and insulin (or insulin-like growth factors) exert direct actions on the granulosa cell that may be required for the development of optimal steroidogenic potential.