Effects of dbcAMP on progesterone synthesis by cultured goat luteal cell subpopulations isolated from early and late luteal stage corpora lutea

This research aimed to investigate the effects of dbcAMP on steroid accumulation by culturing two distinct luteal cell subpopulations isolated from early and late luteal stage corpora lutea. Cells were isolated from corpora lutea collected from eight Angora goats on either the 5th or 15th days of their estrous cycles. Cell isolation was performed by enzymatic digestion using collagenase and DNase. Isolated cells were separated into two distinct subpopulations enriched with small and large luteal cells by percoll density-gradient centrifugation. Isolated cells were stained in order to detect 3β-hydroxysteroid dehydrogenase (3β-HSD). Cells stained positively for 3β-HSD activity (5 x 104 cell⁄well) were incubated with dbcAMP in the absence or presence of 22(R)-hydroxycholesterol (22R-HC) for periods of up to 7 days. Large luteal cell enriched subpopulations produced more basal progesterone (P < 0.05) than did the small luteal cell enriched subpopulations. Treatment of cells with 22R-HC alone induced 4.00 to 11.60 times increase in steroid synthesis depending on type of cells incubated, luteal age and days of incubation. Incubation of cells with 1 mM dbcAMP in the absence or presence of 22R-HC induced in a significant increase (P < 0.01) in steroid accumulation in all treated groups. In contrast, when cells are treated with low dose dbcAMP (0.1 mM), treatment induced stimulation failed to reach significant level in most treated groups. In conclusion, although treatment of goat luteal cells with dbcAMP induces an increase in steroid accumulation, a high dose is necessary to reach significant levels. Stimulatory effect of dbcAMP on steroidogenesis maintains during long life culturing.

Effects of dbcAMP on progesterone synthesis by cultured goat luteal cell subpopulations isolated from early and late luteal stage corpora lutea

This research aimed to investigate the effects of dbcAMP on steroid accumulation by culturing two distinct luteal cell subpopulations isolated from early and late luteal stage corpora lutea. Cells were isolated from corpora lutea collected from eight Angora goats on either the 5th or 15th days of their estrous cycles. Cell isolation was performed by enzymatic digestion using collagenase and DNase. Isolated cells were separated into two distinct subpopulations enriched with small and large luteal cells by percoll density-gradient centrifugation. Isolated cells were stained in order to detect 3β-hydroxysteroid dehydrogenase (3β-HSD). Cells stained positively for 3β-HSD activity (5 x 104 cell⁄well) were incubated with dbcAMP in the absence or presence of 22(R)-hydroxycholesterol (22R-HC) for periods of up to 7 days. Large luteal cell enriched subpopulations produced more basal progesterone (P < 0.05) than did the small luteal cell enriched subpopulations. Treatment of cells with 22R-HC alone induced 4.00 to 11.60 times increase in steroid synthesis depending on type of cells incubated, luteal age and days of incubation. Incubation of cells with 1 mM dbcAMP in the absence or presence of 22R-HC induced in a significant increase (P < 0.01) in steroid accumulation in all treated groups. In contrast, when cells are treated with low dose dbcAMP (0.1 mM), treatment induced stimulation failed to reach significant level in most treated groups. In conclusion, although treatment of goat luteal cells with dbcAMP induces an increase in steroid accumulation, a high dose is necessary to reach significant levels. Stimulatory effect of dbcAMP on steroidogenesis maintains during long life culturing.(AU)

Effects of cholesterol, FSH and LH on steroidogenic activity of cat granulosa cells cultured in vitro

The aim of this study was to examine the effects of 22R-hydroxycholesterol (22R-HC), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) on estradiol and progesterone production by cat granulosa cells. Granulosa cells from follicles were collected and cultured for up to 5 days in 24 well plates containing Dulbecco’s Modified Eagle’s Medium (DMEM)/HAM F-12 supplemented with 10-7 M androstenedione, 0.1% ITS premix and 0.1% bovine serum albumin, in the presence or absence of 22R-HC (10 μg/ml), FSH or LH (10, 100 ng/ml each) on first and third day. Additionally, 5% fetal calf serum was added into the culture medium for the first 24 h. Treatment of cells with 22R-HC resulted in an increase (P < 0.05) in progesterone and estradiol production on days 3 and 5 of the culture. Incubation of cells with FSH (10 and 100 ng/ml) resulted in significant stimulations of progesterone (P < 0.001) whilst incubation had no effect on estradiol production. None of the LH doses (10 and 100 ng/ml) had any effect on progesterone production by granulosa cells during the culture time. With the inclusion of 22R-HC into the culture system, progesterone synthesis was enhanced (P < 0.001) in the presence of all FSH doses.

Effects of cholesterol, FSH and LH on steroidogenic activity of cat granulosa cells cultured in vitro

The aim of this study was to examine the effects of 22R-hydroxycholesterol (22R-HC), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) on estradiol and progesterone production by cat granulosa cells. Granulosa cells from follicles were collected and cultured for up to 5 days in 24 well plates containing Dulbeccos Modified Eagles Medium (DMEM)/HAM F-12 supplemented with 10-7 M androstenedione, 0.1% ITS premix and 0.1% bovine serum albumin, in the presence or absence of 22R-HC (10 μg/ml), FSH or LH (10, 100 ng/ml each) on first and third day. Additionally, 5% fetal calf serum was added into the culture medium for the first 24 h. Treatment of cells with 22R-HC resulted in an increase (P < 0.05) in progesterone and estradiol production on days 3 and 5 of the culture. Incubation of cells with FSH (10 and 100 ng/ml) resulted in significant stimulations of progesterone (P < 0.001) whilst incubation had no effect on estradiol production. None of the LH doses (10 and 100 ng/ml) had any effect on progesterone production by granulosa cells during the culture time. With the inclusion of 22R-HC into the culture system, progesterone synthesis was enhanced (P < 0.001) in the presence of all FSH doses.(AU)

Genetic diversity among melon accessions (Cucumis melo) from Turkey based on SSR markers.

Melon (Cucumis melo) is an important vegetable crop in Turkey, where it is grown in many regions; the most widely planted lines are local winter types belonging to the var. inodorous. We examined 81 melon genotypes collected from different provinces of Turkey, compared with 15 reference melon genotypes obtained from INRA/France, to determine genetic diversity among Turkish melons. Twenty polymorphic primers were used to generate the SSR markers. PCR amplification was performed and electrophoresis was conducted. SSR data were used to generate a binary matrix. For cluster analysis, UPGMA was employed to construct a clustering dendrogram based on the genetic distance matrix. The cophenetic correlation was compared with the similarity matrix using the Mantel matrix correspondence test to evaluate the representativeness of the dendrogram. A total of 123 alleles were amplified using the 20 SSR primer sets. The number of alleles detected by a single primer set ranged from 2 to 12, with an average of 6.15. The similarity ranged from 0.22 to 1.00 in the dendrogram developed from microsatellite analysis. Based on this molecular data, we concluded that genetic diversity among these Turkish accessions is relatively high.

Genetic characterization of heat tolerant tomato (Solanum lycopersicon) genotypes by SRAP and RAPD markers.

We employed RAPD and sequence-related amplified polymorphism (SRAP) markers to evaluate polymorphisms in 15 tomato (Solanum lycopersicon) genotypes that were obtained from a tomato breeding program. Four local tomato genotypes selected from the Sanliurfa province (Southeastern Anatolia Region of Turkey), 10 heat-tolerant tomato genotypes, received from the Asian Vegetable Research and Development Center, and a sample of S. pimpinellifolium were genotyped with RAPD and SRAP markers. Eleven SRAP primer combinations were used and 66 bands were scored. The number of bands scored per primer combination ranged from three to 12, with a mean of six alleles per primer combination. All fragments scored for each primer combination were polymorphic. The percentage of polymorphic products ranged from 25 to 80%. The 15 tomato genotypes were screened for RAPD markers using 50 primers in a PCR-based DNA amplification procedure; 46 primers produced clear and good amplification. Ten of these 46 primers amplified monomorphic fragments in the tomato genotypes. A dendrogram was constructed by combining data from the RAPD and SRAP analyses. Similarity ratios of genotypes ranged from 0.87 to 0.99. The dendrogram was divided into two branches; the first main branch included only genotype CL 5915, and the second main branch included all the other genotypes.