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PubMed1996

Regulation of cultured human chorion cell chemokine production by group B streptococci and purified bacterial products.

PROBLEM: To determine if different strains of group B streptococci (GBS) and purified bacterial products regulate chemokine production by cultured human chorion cells. METHOD OF STUDY: Primary cultures of human chorion cells were established from placentae isolated from normal women at term gestation having repeat cesarean section. Five different strains of heat-killed GBS were incubated with confluent chorion cells at 10(7) bacteria/ml for 16 hours at 37 degrees C. In separate experiments, lipoteichoic acid and sialic acid at various concentrations were incubated with chorion cells for 16 hours at 37 degrees C. Culture supernatants were collected and then assayed to determine concentrations of interleukin-8 (IL-8) and macrophage inflammatory protein-1 alpha (MIP-1 alpha) by ELISA. RESULTS: We found that GBS stimulated chorion cell production of MIP-1 alpha in a strain-specific fashion. We also found that both lipoteichoic acid and sialic acid stimulated concentration-dependent increases in chorion cell IL-8 production. Chorion cells, however, did not increase MIP-1 alpha production in response to either lipoteichoic acid or sialic acid. Two strains of GBS tested induced concentration-dependent increases in both IL-8 and MIP-1 alpha, but both stimulated IL-8 production to a greater extent. Similarly, IL-1 beta also caused chorion cells to produce more IL-8 than MIP-1 alpha. CONCLUSIONS: Our data are the first to show that GBS and purified bacterial products can stimulate chemokine production by fetal gestational tissues. We suggest that chorion cells may produce specific types of chemokines to attract different types of inflammatory cells and thus may participate in the pathophysiology of infection-mediated preterm labor by directing specific inflammatory responses.

Amniotic Fluid↗

PubMed1983

Large-scale production of mammalian cells and their products: engineering principles and barriers to scale-up.

Mammalian cell products have great medical and clinical importance, but to date, production methods employed to manufacture these products on a large scale are not as cost efficient as they could be. The implementation of process control would greatly improve the productivity of these products. Recently developed methods to produce cells on a large scale, such as microcarriers, artificial capillaries, tubular spiral film, and microencapsulation must be optimized, and the problem of oxygen transfer limitation must be solved. The accumulation of potentially toxic waste products can inhibit growth and reduce productivity. This effect can be reduced by either adjusting the environmental parameters of a fed-batch culture, so that the cell's metabolism is shifted away from producing these compounds, or by continually perfusing medium through the culture. If these technical barriers can be overcome, the cost of producing products derived from mammalian cells can be greatly reduced.

Animals↗

PubMed2000

Mechanism of the incidental production of a melanin-like pigment during 6-demethylchlortetracycline production in Streptomyces aureofaciens.

The secondary metabolite 6-demethylchlortetracycline (6-DCT), which is produced by Streptomyces aureofaciens, is used as a precursor of semisynthetic tetracyclines. Strains that produce 6-DCT also produce a melanin-like pigment (MP). The correlation between MP production and 6-DCT production was investigated by using S. aureofaciens NRRL 3203. Production of both MP and 6-DCT was repressed by phosphate or ammonium ions, suggesting that syntheses of these compounds are controlled by the same regulators. Ten chlortetracycline-producing recombinants were derived from 6-DCT-producing mutant NRRL 3203 by gene replacement. All of the recombinants produced chlortetracycline but not MP, indicating that MP production is the results of a defect in the 6-methylation step and suggesting that the polyketide nonaketideamide is a common intermediate leading to MP as well as 6-DCT. To further examine the possibility that MP might be synthesized via the 6-DCT-biosynthetic pathway, mutants defective in 6-DCT biosynthesis were derived from a 6-DCT producer. Some of these mutants were able to produce MP, while others, including mutants with mutations in the gene encoding anhydrotetracycline oxygenase, an enzyme catalyzing the penultimate step in the pathway, produced neither 6-DCT nor MP. Production of 6-DCT and production of MP were restored simultaneously by integrative transformation with the corresponding 6-DCT-biosynthetic genes, indicating that some of 6-DCT-biosynthetic enzymes are indispensable for MP production. These findings suggest that a defect in the 6-methylation step results in redirection of carbon flux from a certain intermediate in the 6-DCT-biosynthetic pathway to a shunt pathway and results in MP production.

Anti-Bacterial Agents↗

PubMed2004

Alginic acid oligosaccharide suppresses Th2 development and IgE production by inducing IL-12 production.

BACKGROUND: Since allergen-specific IgE is directly involved in the type I allergic reaction, development of a method for inhibiting Th2 responses which lead to the induction of IgE production would be a useful approach for preventing allergic disorders. The ability and mechanism of alginic acid oligosaccharide (ALGO), an oligosaccharide obtained from natural edible polysaccharide, for suppressing Th2 responses was examined in detail. METHODS: Lymph node cells obtained from beta-lactoglobulin (beta-LG)-primed BALB/c mice were cultured in vitro with an antigen for 3 days in the absence or presence of ALGO. The amount of cytokine in each culture supernatant was measured. The effect of ALGO on Th2 development was also examined by using ovalbumin specific T cell receptor transgenic mice. Antibody production in the serum of BALB/c mice that had been immunized with beta-LG or beta-LG plus ALGO was investigated. RESULTS: The production of IFN-gamma induced by antigen stimulation was upregulated by ALGO in a dose-dependent manner. IL-12 production was also enhanced by ALGO, and the addition of the anti-IL-12 antibody to the culture abrogated the effect of ALGO. On the other hand, IL-4 production by antigen-stimulated splenocytes of transgenic mice was suppressed in the presence of ALGO. Furthermore, IgE production by ALGO-treated mice was significantly inhibited compared with control mice. CONCLUSIONS: These results indicate that ALGO suppressed antigen-induced Th2 development by inducing IL-12 production. ALGO also inhibited in vivo IgE production. These findings suggest that ALGO is expected to be an edible anti-allergic agent.

Alginates↗

PubMed2005

Endothelin-1 in osteoarthritic chondrocytes triggers nitric oxide production and upregulates collagenase production.

The mechanism of endothelin-1 (ET-1)-induced nitric oxide (NO) production, MMP-1 production and MMP-13 production was investigated in human osteoarthritis chondrocytes. The cells were isolated from human articular cartilage obtained at surgery and were cultured in the absence or presence of ET-1 with or without inhibitors of protein kinase or LY83583 (an inhibitor of soluble guanylate cyclase and of cGMP). MMP-1, MMP-13 and NO levels were then measured by ELISA and Griess reaction, respectively. Additionally, inducible nitric oxide synthase (iNOS) and phosphorylated forms of p38 mitogen-activated protein kinase, p44/42, stress-activated protein kinase/Jun-N-terminal kinase and serine-threonine Akt kinase were determined by western blot. Results show that ET-1 greatly increased MMP-1 and MMP-13 production, iNOS expression and NO release. LY83583 decreased the production of both metalloproteases below basal levels, whereas the inhibitor of p38 kinase, SB202190, suppressed ET-1-stimulated production only. Similarly, the ET-1-induced NO production was partially suppressed by the p38 kinase inhibitor and was completely suppressed by the protein kinase A kinase inhibitor KT5720 and by LY83583, suggesting the involvement of these enzymes in relevant ET-1 signalling pathways. In human osteoarthritis chondrocytes, ET-1 controls the production of MMP-1 and MMP-13. ET-1 also induces NO release via iNOS induction. ET-1 and NO should thus become important target molecules for future therapies aimed at stopping cartilage destruction.

Aminoquinolines↗

PubMed2000

Applying environmental product design to biomedical products research.

The principal themes for the Biomedical Research and the Environment Conference Committee on Environmental Economics in Biomedical Research include the following: healthcare delivery companies and biomedical research organizations, both nonprofit and for-profit, need to improve their environmental performance; suppliers of healthcare products will be called upon to support this need; and improving the environmental profile of healthcare products begins in research and development (R&D). The committee report begins with requirements from regulatory authorities (e.g., U.S. Environmental Protection Agency [EPA], the U.S. Food and Drug Administration), and the healthcare delivery sector). The 1998 American Hospital Association and EPA Memorandum of Understanding to reduce solid waste and mercury from healthcare facilities is emblematic of these requirements. The dominant message from the requirements discussion is to ensure that R&D organizations do not ignore customer, environmental, and regulatory requirements in the early stages of product development. Several representatives from healthcare products manufacturers presented their companies' approaches to meeting these requirements. They reported on efforts to ensure that their R&D processes are sensitive to the environmental consequences from manufacturing, distributing, using, and disposing of healthcare products. These reports describe representatives' awareness of requirements and the unique approaches their R&D organizations have taken to meet these requirements. All representatives reported that their R&D organizations have embraced environmental product design because it avoids the potential of returning products to R&D to improve the environmental profile. Additionally, several reports detailed cost savings, sustainability benefits, and improvements in environmental manufacturing or redesign, and increased customer satisfaction. Many companies in healthcare delivery are working to improve environmental performance. Fundamental to these efforts is the necessity of motivating suppliers to improve the environmental profile of new products used in the healthcare delivery sector.

Biomedical Technology↗

PubMed2003

Lipoxygenase products regulate nitric oxide and inducible nitric oxide synthase production in interleukin-1beta stimulated vascular smooth muscle cells.

In cultured vascular smooth muscle cells (VSMCs), interleukin-1beta (IL-1beta) stimulates inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production. IL-1beta also activates phospholipase A2 (PLA2), and induces lipoxygenase (LOX) and cyclooxygenase-2 (COX-2). The present study investigated whether these metabolites are involved in the regulation of IL-1beta-induced NO production and iNOS expression. Pretreatment with ONO-RS-082, the secretory PLA2 (sPLA2) inhibitor, at 1 to 10 micromol/l reduced IL-1beta-stimulated nitrite production and iNOS expression. Nordihydroguaiaretic acid (NDGA, 1 to 10 micromol/l), the LOX inhibitor, also reduced IL-1beta (10 ng/ml)-stimulated nitrite production and iNOS expression in a dose-dependent manner. Exogenous 12(S)-hydroxyeicosatetraenoic acids (HETE) enhanced the IL-1beta-stimulated nitrite production and iNOS expression. On the other hand, the COX inhibitors, indomethacin and NS-398, had little effect on nitrite production or iNOS expression. These results suggest that LOX products play important roles in the regulation of stimulus-induced NO production in VSMCs.

Aminobenzoates↗

PubMed1999

Effect of fibrous by-products on production and ruminal fermentation in lactating dairy cows.

Lactating dairy cows were used in experiments to determine the effects of feeding a combination of fibrous by-products to replace a portion of alfalfa hay or grain. Cows were fed a control diet, consisting of alfalfa hay, corn, soybean meal, and corn silage or one of four treatment diets. In these diets, a combination of soy hulls, corn gluten feed, and wheat midds replaced approximately 30 or 60% of alfalfa hay or 25 or 50% of corn and soybean meal. A 56-d production study used 50 midlactation dairy cows in a randomized complete block design. No differences in milk production or composition among treatments were measured, except for the diet in which 60% of the alfalfa hay was replaced with fibrous by-products. Cows fed this diet had a significantly lower percentage of milk fat compared with other treatments. A fermentation study used five fistulated, multiparous lactating dairy cows in a 5 x 5 Latin square design. Cows were fed one of the five experimental diets used in the production study during five consecutive 14-d periods. Rumen acetate to propionate ratio was highest for the control and 50% concentrate replacement diets (3.27) and lowest for the 60% hay replacement diet (2.78). This shift in ruminal volatile fatty acid profile corresponded to the change in milk fat percentage, measured during the production study. A mixture of fibrous by-products fed as an alternative to hay or grain ingredients could potentially decrease feed costs without a resultant decrease in milk production by mid-lactation dairy cows.

Acetates↗

PubMed1987

Production performance of White Leghorn layers fed lactobacillus fermentation products.

A series of trials was conducted to determine if adding a Lactobacillus fermentation product (LAC) to the feed of laying hens would improve their production performance. Feeding a liquid, nonviable LAC product to either cage or floor housed laying hens did not improve hen-day egg production, feed efficiency, nor egg size during a 48 week experimental period. Laying hens fed a dried, nonviable LAC product did not show any improvement in hen-day egg production nor feed efficiency compared with laying hens fed no LAC or zinc bacitracin. Addition of a viable LAC product to ratios of differing protein levels did not improve hen-day egg production, livability, or egg size of laying hens.

Animal Feed↗

PubMed2001

Prostaglandin E2 up-regulates macrophage-derived chemokine production but suppresses IFN-inducible protein-10 production by APC.

PGE(2) has been known to suppress Th1 responses. We studied the role of PGE(2) in two representative chemokines, macrophage-derived chemokine (MDC) and IFN-inducible protein-10, production by LPS- or CD40-stimulated spleen cells. The production of MDC, one of the ligands for CCR4 preferentially expressed on Th2, was enhanced in nonstimulated, LPS-, CD40-, or CD3-stimulated spleen cells by the pretreatment with PGE(2), while the production of IFN-inducible protein-10, a representative ligand for CXC chemokine receptor 3 expressed on Th1, was suppressed. MDC production was also enhanced by IL-4, IL-5, and intracellular cAMP-elevating agents such as dibutyryl cAMP and 3-isobutyl-1-methylxanthine, and the effect of IL-4, IL-5, and PGE(2) was additive. However, the pretreatment with IL-6, IL-10, or TGF-beta, or the neutralization of IFN-gamma or IL-12 had no effect on MDC production. B cells, macrophages, and dendritic cells were main producers of MDC, while T cells produced only a small amount of MDC. MDC production by B cells was equally stimulated by LPS and anti-CD40 Ab, while that by macrophages and dendritic cells was more markedly stimulated by anti-CD40 Ab, and PGE(2) further enhanced MDC production by these stimulated cells. These results indicate that PGE(2) regulates Th1/Th2-related chemokine production by B cells, macrophages, and dendritic cells, and that this is a new function of PGE(2) for the regulation of Th2 immune responses at the induction and activation stages.

Adjuvants, Immunologic↗

PubMedDate not supplied

The effect of organic and hydraulic shock loads on the production of soluble microbial products in anaerobic digesters.

Anaerobic chemostats were used to investigate the effects of organic and hydraulic shock loads on the production of soluble microbial products (SMP). Production of SMP was found to increase during glucose spikes, reaching up 15% of the influent chemical oxygen demand. These SMP appear to be utilization-associated products produced as a result of the temporarily high organic load, and chemical analysis and ultrafiltration experiments revealed that most of these compounds are difficult to identify and that the majority of them are present in the low molecular weight (MW) range. Production of SMP also increased when the hydraulic retention time was reduced from 15 to 3 days, and an increase in DNA concentration in the bulk solution suggested enhanced cell lysis. Although the cause of lysis was not clear, it is believed that most of the SMP produced under such conditions were biomass-associated products following cell death. While the majority of these compounds lay in the low MW range, as much as 35% were found to have MWs greater than 1 kDa. During the period when the anaerobic chemostat was fed no alkalinity and the pH remained lower than 6.5 for more than a week, a slightly higher production of SMP and a shift in the MW distribution towards the production of higher MW SMP was observed.

Bacteria, Anaerobic↗

PubMed1988

Interleukin 1 alpha production by peripheral blood monocytes from patients with chronic liver disease and effect of sera on interleukin 1 alpha production.

We investigated the role of interleukin 1 alpha (IL 1 alpha) in the pathogenesis of chronic liver disease. IL 1 alpha production by peripheral blood monocytes was measured with a specific, sensitive double-antibody radioimmunoassay. When monocytes were cultured for two days with bacterial lipopolysaccharide (LPS), IL 1 alpha production in asymptomatic hepatitis B virus carrier (ASC) and patients with chronic active hepatitis (CAH) was equivalent to that of controls (168 +/- 31 U/ml, mena +/- SD), while IL 1 alpha levels generated by monocytes from liver cirrhosis (LC) (117 +/- 45 U/ml, p less than 0.01) were significantly lower than controls. When normal monocytes were cultured together with LPS and IFN gamma, mena IL 1 alpha production was 297 +/- 56 U/ml. IL 1 alpha production in ASC did not differ from controls. On the other hand, IL 1 alpha production in patients with CAH (241 +/- 58 U/ml, p less than 0.05) and LC (189 +/- 70 U/ml, p less than 0.01) were significantly diminished in comparison with controls although there was considerable overlap. Serial study demonstrated that IL 1 alpha production rose significantly during acute deterioration of illness with marked rise in serum alanine aminotransferase. The addition of sera to normal monocytes cultures resulted in significantly enhanced suppression (p less than 0.05) for IL 1 alpha production in comparison with that of control sera. These findings indicate that decreased monocyte function and serum inhibitor(s) for IL 1 alpha production could contribute to the pathogenesis of chronic liver disease.

Carrier State↗

PubMed2005

Advanced glycation end-product-induced mitogenesis and collagen production are dependent on angiotensin II and connective tissue growth factor in NRK-49F cells.

Diabetic nephropathy (DN) is characterized by glomerulopathy and tubulointerstitial expansion followed by renal fibrosis. Angiotensin II (Ang II) and connective tissue growth factor (CTGF) are involved in the pathogenesis of DN, while Janus kinase 2 (JAK2) is important in advanced glycation end-product (AGE)-induced effects in renal interstitial (NRK-49F) fibroblasts. Thus, we studied the role of Ang II, CTGF, and JAK2 in AGE-induced effects in NRK-49F cells. We found that AGE (150 microg/ml) increased mitogenesis and type I collagen production at 7 days while Ang II (10(-7)M) increased mitogenesis and type I collagen production at 3 days. We also found that AGE (150 microg/ml) increased angiotensinogen protein at 2 days, which was attenuated by AG-490 (a JAK2 inhibitor). AGE (150 microg/ml) increased CTGF mRNA and protein expression at 3 and 5 days, respectively. Ang II (10(-7)M) increased CTGF mRNA and protein expression at 1 and 2 days, respectively, which were attenuated by AG-490. Moreover, losartan (a type I angiotensin receptor blocker) and captopril (an angiotensin converting enzyme inhibitor) attenuated AGE-induced CTGF mRNA/protein expression while attenuating AGE-induced mitogenesis and type I collagen production. AG-490 and CTGF antisense (but not sense) oligodeoxynucleotide (ODN) attenuated Ang II (10(-7)M) and AGE-induced mitogenesis and type I collagen production at 3 and 7 days, respectively. We concluded that AGE (150 microg/ml)-induced mitogenesis and type I collagen production are dependent on the Ang II-JAK2-CTGF pathway in NRK-49F cells. Moreover, Ang II-induced mitogenesis and type I collagen production are dependent on the JAK2-CTGF pathway.

Angiotensin II↗

PubMed2006

Vaginal douches and other feminine hygiene products: women's practices and perceptions of product safety.

OBJECTIVE: Use of vaginal douche products has been linked with a variety of reproductive health problems; nonetheless, the practice of douching persists. The goals of this study were to 1) determine the use of vaginal douches and other feminine hygiene products, 2) ascertain how safe women think vaginal douche products are, and 3) evaluate women's readiness to stop douching. METHODS: A random-digit-dial computer-assisted telephone survey was conducted among US women between the ages of 18 and 44. RESULTS: Of the 2,602 women interviewed, 11.8% (n = 307) engaged in regular douching (White: 9.1%; African American: 27.7%; Hispanic: 15.0%). Women who douched, compared to women who did not douche, used other feminine hygiene products significantly more often (vaginal sprays [ p < .0001], wipes/towelettes [ p < 0.01], vaginal powder [ p < 0.0001] and bubble bath for feminine cleansing [ p < 0.001]). Women who douched also were more likely than nondouchers to agree with the statement, "Douche products are safe to use; otherwise they wouldn't be on the market" (70.3% vs. 33.4%, respectively; p < 0.0001). Nearly all women (90.0%) who douched had no intention to discontinue the practice. CONCLUSION: Compared with women who do not douche, women who douche use other feminine hygiene products at a much higher rate and also believe that douche products are safe. Women who douche will remain resistant to stopping the practice without innovative interventions. Given that most women start douching in adolescence, teens should be targeted for prevention efforts.

Adolescent↗

PubMed2000

Shelf life of modified atmosphere packed cooked meat products: addition of Na-lactate as a fourth shelf life determinative factor in a model and product validation.

Cooked meat products are often post-contaminated because of a packaging and/or slicing step after the pasteurisation process. The shelf life is therefore limited and can be extended by adding Na-lactate. A previously developed model for the spoilage of gas packed cooked meat products, including temperature, water activity and dissolved CO2 as independent variables, was extended with a fourth factor: the Na-lactate concentration in the aqueous phase of the meat product. Models were developed for the maximum specific growth rate mu(max) and the lag phase lambda of the specific spoilage organism Lactobacillus sake subsp. carnosum. Quadratic response surface equations were compared with extended Ratkowsky models. In general, response surface equations fitted the experimental data best but in the case of mu(max) the response surface model predicted illogical growth behaviour at low water activities and high Na-lactate concentrations. A extensive product validation of the mathematical models was performed by means of inoculated as well as naturally contaminated industrially prepared cooked meat products. The deviations of the experimentally determined versus predicted growth parameters in inoculated cooked meat products were in general small. Both types of models were also able to predict the shelf life of naturally contaminated cooked meat products, except for pâté where an under-estimation of the shelf life was predicted by the response surface equations. The validation studies revealed higher accuracy of the extended Ratkowsky models in comparison to the response surface equations. A significant shelf life extending effect of Na-lactate was predicted, which was more pronounced at low refrigerated temperatures. A synergistic effect has also been noticed between Na-lactate and carbon dioxide which, at least partly, could be explained by the pH-decreasing effect of CO2.

Animals↗

PubMed1998

Presence of dopa and amino acid hydroperoxides in proteins modified with advanced glycation end products (AGEs): amino acid oxidation products as a possible source of oxidative stress induced by AGE proteins.

Glycation and subsequent Maillard or browning reactions of glycated proteins, leading to the formation of advanced glycation end products (AGEs), are involved in the chemical modification of proteins during normal aging and have been implicated in the pathogenesis of diabetic complications. Oxidative conditions accelerate the browning of proteins by glucose, and AGE proteins also induce oxidative stress responses in cells bearing AGE receptors. These observations have led to the hypothesis that glycation-induced pathology results from a cycle of oxidative stress, increased chemical modification of proteins via the Maillard reaction, and further AGE-dependent oxidative stress. Here we show that the preparation of AGE-collagen by incubation with glucose under oxidative conditions in vitro leads not only to glycation and formation of the glycoxidation product Nepsilon-(carboxymethyl)lysine (CML), but also to the formation of amino acid oxidation products on protein, including m-tyrosine, dityrosine, dopa, and valine and leucine hydroperoxides. The formation of both CML and amino acid oxidation products was prevented by anaerobic, anti-oxidative conditions. Amino acid oxidation products were also formed when glycated collagen, prepared under anti-oxidative conditions, was allowed to incubate under aerobic conditions that led to the formation of CML. These experiments demonstrate that amino acid oxidation products are formed in proteins during glycoxidation reactions and suggest that reactive oxygen species formed by redox cycling of dopa or by the metal-catalysed decomposition of amino acid hydroperoxides, rather than by redox activity or reactive oxygen production by AGEs on protein, might contribute to the induction of oxidative stress by AGE proteins.

Amino Acids↗

PubMed2006

[Effect of advanced oxidation protein products on nitric oxide production in mouse peritoneal macrophages].

OBJECTIVE: To investigate the effect of advanced oxidation protein products (AOPP) on nitric oxide (NO) production in mouse peritoneal macrophages (MPMs). METHODS: MPMs were incubated in the absence or presence of lipopolysaccharide (LPS) with AOPP-modified bovine serum albumin (BSA) prepared by exposure of BSA to hypoclorous acid or pre-treated with AOPP-BSA and subsequent stimulation with LPS. NO production in the supernatants of the culture media was determined spectrophotometrically using Griess method. The cell viability was measured by MTT assay. RESULTS: BSA induced significant NO production in MPMs. AOPP modification of BSA significantly inhibited NO production, and AOPP-BSA exhibited time- and dose-dependent inhibition of NO production induced by LPS in MPMs incubated together with LPS or pre-treated before LPS stimulation. CONCLUSION: AOPP-BSA is capable of inhibiting inducible NO production in MPMs.

Animals↗

PubMed1998

Steroid production, cell proliferation, and apoptosis in cultured bovine antral and mural granulosa cells: development of an in vitro model to study estradiol production.

This study was undertaken to characterize the relationship between changes in steroid production, cell cycle activity (ie, cell proliferation) and apoptosis in antral and mural bovine granulosa cells cultured in vitro. This was done to select conditions promoting optimal estradiol production by bovine granulosa cells cultured in completely defined conditions. In the first experiment, antral granulosa cells were cultured over the entire 4 days of the culture period in the presence of either 0, 2, or 10 ng/ml of FSH (chronic conditions) or were maintained under minimal FSH support (0.5 ng/ml FSH) for the first 3 days of culture and then were challenged over the fourth day of culture with either 0, 2, or 10 ng/ml FSH (challenged conditions). Compared with cells exposed to constant FSH levels (chronic conditions), the FSH-induced production of estradiol was higher (P < 0.006) and that of progesterone was lower (P < 0.02) over the last 24 h of culture, when antral granulosa cells were maintained under minimal FSH support during the first 3 days of culture (challenged conditions). In the second experiment, dynamics of estradiol and progesterone productions, conversion of [14C]androstenedione into subsequent steroid metabolites, DNA content, cell cycle activity, and apoptosis (as assessed by flow cytometry) of antral and mural granulosa cells over the first 3 days of culture under minimal FSH support and in response to a challenge with FSH during the last 24 h of culture were evaluated. Estradiol production as well as the conversion of androstenedione into testosterone and estradiol were greater (P < 0.01) in antral than in mural granulosa cells cultured under challenged conditions. A higher proportion of mural than antral granulosa cells were in the proliferative state at the end of culture (P < 0.03). This may be related to the decreased ability of mural cells to produce estradiol. FSH suppressed (P < 0.05) the spontaneous onset of apoptosis in both cell types. These results suggest that functional differences between these two cell compartments need to be considered in studying bovine granulosa cells in vitro. Because of their large (400 to 600%) FSH-induced estradiol production, antral granulosa cells cultured under challenged conditions provide a model that can be used to examine substances for their ability to alter estradiol production and apoptosis in bovine granulosa cells.

Animals↗