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  • Author: B K Tsang x
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Sodium-dependent intracellular pH regulation in granulosa cells of the domestic hen (Gallus domesticus)

E. K. Asem and B. K. Tsang

ABSTRACT

The existence and importance of the Na+/H+ exchanger in intracellular pH (pHi) regulation in ovarian cells was studied in acid-loaded avian granulosa cells by monitoring the recovery of normal pHi using a trapped fluorescein derivative as an indicator. The resting pHi of freshly isolated granulosa cells from preovulatory follicles was 680 ± 0·08 when the extracellular pH (pHo) and sodium concentration (Nao +) were 7·3 and 144 mmol/l respectively. While exposure of granulosa cells to high pHo (pHo > 745) medium shifted the pHi upward with time, incubation of the cells in low pHo (pH < 6·80) buffer resulted in a significant decrease in pHi. In contrast, pHi remained constant when pHo was varied between the broad range of 6·8–7·4. When the cytoplasm was acidified by treatment with nigericin in choline+ buffer, both the magnitude and rate of recovery of normal pHi was suppressed significantly with decreasing pHo, but increased in high pHo medium. The recovery of pHi was dependent upon the concentration of extracellular sodium, in that the recovery rate and magnitude increased concomitantly with increases in Nao + concentrations, while the recovery was abolished when Nao + was completely replaced with choline+. In addition, the sodium ionophore monensin enhanced the recovery rate of normal pHi in a concentration-dependent manner. This action of monensin was observed only when sodium was present in the incubation medium, indicating that Nao + entry is important for the recovery of normal pHi. Monensin also evoked further cytoplasmic alkalinization in fully recovered cells, with a relative net effect dependent upon the level of Nao + present. The recovery of pHi by acid-loaded cells was attenuated in a concentration-dependent manner by the Na+/H+ exchange inhibitor amiloride. These results clearly demonstrate in granulosa cells the presence of a pHi-regulating system that requires extracellular Na+ and is sensitive to amiloride.

DOI:
https://doi.org/10.1677/jme.0.0010095
Volume 1: Issue 2,
Pages:
95–103 (9 total)
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Influence of growth factors on the plasminogen activator activity of avian granulosa cells from follicles at different maturational stages of preovulatory development

M Lafrance, F Croze, and B K Tsang

ABSTRACT

Granulosa cells from the first (Fl), third (F3) and fifth and sixth (F5–6) preovulatory follicles and the small yellow follicles (SYFs; diameter 6–8 mm) were cultured for 21 h in the absence and presence of murine and human epidermal growth factors, fibroblast growth factor, transforming growth factors α and β-I (TGFα, TGFβ), platelet-derived growth factor and insulin-like growth factor-I at concentrations of 0·1–100 ng/ml. Plasminogen activator (PA) activities in the cell (PAc) and in the medium (PAm) were measured by fibrinolysis and fibrin overlay methods. Basal PAc and PAm activities were highest in cell cultures from the less mature follicles (F5–6 and SYF) and decreased as the follicles matured (F3>F1). PAc activity was greater than PAm activity, irrespective of the stage of follicular development. All growth factors examined at the 100 ng/ml level were effective in increasing PAc and PAm activities in cultures of granulosa cells from Fl follicles. However, only TGFα was able to increase PA activities at lower concentrations. The stimulation of the PA activities of granulosa cells from F3 follicles was inconsistent. None of the growth factors significantly increased PA activities in granulosa cells from F5–6 follicles and SYFs, as determined by fibrinolysis. The major PAc and PAm species (characterized by fibrin overlay) had a molecular mass of about 35 kDa, which is characteristic of the urokinase type. Both assay methods detected a stimulatory effect of the growth factors on PA activities in the granulosa cells from Fl follicles. However, an increase in PA activities in cells from F3 and F5–6 follicles and SYFs was indicated only after fibrin overlay analysis. Tritiated thymidine was incorporated into the DNA of granulosa cells at all stages of follicular development and was enhanced by all growth factors, although TGFα and TGFβ were the most effective and had a ranked order of activity: F3, F5–6>F1, SYF. The present findings show that, of the growth factors examined, TGFα may be an effective regulator of PA activity in avian granulosa cells during follicular development, in addition to its observed mitogenic action.

DOI:
https://doi.org/10.1677/jme.0.0110291
Volume 11: Issue 3,
Pages:
291–304 (14 total)
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Calcium ionophores increase intracellular pH in chicken granulosa cells

E. K. Asem, M. Li, and B. K. Tsang

ABSTRACT

Several hormone agonists exert their physiological actions by triggering an inositol phospholipid—Ca2+ signalling cascade and cytosolic alkalinization. Although calcium ionophores have been used extensively to probe the role of Ca2+ in the regulation of steroidogenesis in granulosa cells, the precise relationship between changes in intracellular Ca2+ (Ca2+ i) and pH (pHi) is unclear. In the present study we have used a fluorescent pH indicator, 2′7′-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein, to examine the influence of two Ca2+ ionophores, ionomycin and 4-Bromo-A23187 (4-Br-A23187), on pHi in chicken granulosa cells. Chicken granulosa cells from the largest preovulatory follicle were incubated with Ca2+ ionophores (0–2 μm) and/or inhibitors of Na+/H+ antiport (amiloride, dimethylamiloride and ethylisopropyl amiloride; 0·5, 5 and 50 μm respectively) in the presence of Na+ (or choline+; 0–144 mm) and/or Ca2+ (0–10 mm). Ionomycin or 4-Br-A23187 elicited a rapid and sustained cytosolic alkalinization. The magnitude of increase in pHi was dependent on the concentration of the Ca2+ ionophore and the presence of extracellular Ca2+ but independent of extracellular Na+. Pretreatment of the cells with amiloride or its analogues failed to affect the increase in pHi induced by the Ca2+ ionophores significantly. These findings demonstrate that, in addition to their widely reported effects on Ca2+ i redistribution in granulosa cells, 4-Br-A23187 and ionomycin cause Ca2+-dependent cytosolic alkalinization. This action of the Ca2+ ionophores is independent of the Na+/H+ antiport. Caution must be exercised in using Ca2+ ionophores as probes to define the role of Ca2+ in the regulation of granulosa cell function.

DOI:
https://doi.org/10.1677/jme.0.0090001
Volume 9: Issue 1,
Pages:
1–6 (6 total)
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Relaxin gene expression in the porcine follicle during preovulatory development induced by gonadotrophins

C. A. Bagnell, W. Tsark, L. Tashima, B. R. Downey, B. K. Tsang, and L. Ainsworth

ABSTRACT

Northern analysis was used to identify relaxin gene expression in ovaries of prepubertal pigs primed with pregnant mare's serum gonadotrophin (PMSG) and human chorionic gonadotrophin (hCG). The cellular distribution of relaxin transcript in the developing follicle was localized by in-situ hybridization histochemistry. Three probes complementary to non-overlapping regions of the porcine prorelaxin molecule were used to identify relaxin gene expression in ovarian follicular tissue collected 0, 48, 60, 72 and 84 h after treatment with PMSG/hCG. A 1 kb transcript was detected in ovarian extracts of prepubertal gilts from 48 to 84 h after PMSG stimulation. This corresponds to the molecular size of the relaxin transcript reported in the pregnant sow ovary. Relaxin mRNA levels increased in ovaries from animals 48 through 84 h after PMSG. In-situ hybridization showed that the site of relaxin synthesis was the theca interna layer of the developing follicle. Relaxin mRNA was not observed in other follicular cell types, in small or atretic follicles or in follicles from unstimulated animals. The distribution and relative concentration of relaxin mRNA showed a good correlation with in-vitro production and immunohistochemical localization of relaxin previously reported in the developing pig follicle. The presence of both protein and mRNA for relaxin in the growing follicle supports a role for relaxin as a local regulator of ovarian function.

DOI:
https://doi.org/10.1677/jme.0.0050211
Volume 5: Issue 3,
Pages:
211–219 (9 total)
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Developmental expression of the relaxin gene in the porcine corpus luteum

C A Bagnell, Q Zhang, K Ohleth, M L Connor, B R Downey, B K Tsang, and L Ainsworth

ABSTRACT

Northern analysis and in-situ hybridization were used to follow the development of relaxin gene expression in the newly forming corpus luteum (CL) after ovulation and throughout luteal development. Alkaline phosphatase (AP) was used as a marker of theca-derived lutein cells and the relationship between AP-positive and relaxin mRNA-containing cells was assessed. Ovaries from prepubertal pigs treated with pregnant mares serum gonadotrophin (PMSG)/human chorionic gonadotrophin (hCG) were collected during the periovulatory period and at various times during 19 days after ovulation. In addition, CL from cyclic pigs on days 10 and 16 were used to monitor relaxin gene expression in small and large luteal cells. Northern analysis revealed that relaxin gene expression increased with CL development in the PMSG/hCG-treated pig, reaching maximal levels at around day 14 post-ovulation. Thereafter, as the CL regressed, the level of relaxin mRNA declined. In CL from cyclic pigs at day 10 of the cycle, only small luteal cells expressed relaxin mRNA. However, by day 16 of the cycle, large luteal cells were the source of relaxin gene expression. In-situ hybridization studies revealed that in the early CL (up to 30 h post-ovulation), the relaxin gene transcript was observed in cells along the margins of the CL and in the core of the infolding follicle wall corresponding to the AP-positive, luteinized theca cell layer. As luteinization progressed, the theca and granulosa cell layers could no longer be distinguished morphologically (from 54 h after ovulation until day 9). However, the pattern of relaxin hybridization persisted along the periphery in bands of cells penetrating the CL, and coincided with areas of AP staining, indicating that the theca lutein cells were the site of relaxin gene expression. At day 14, relaxin hybridization and AP staining were distributed throughout the luteal tissue. With CL regression both AP staining and relaxin hybridization declined. This pattern of relaxin hybridization in the CL of the gonadotrophin-primed pig was identical to that observed in cyclic pigs on days 10 and 16 of the cycle. These findings indicate that theca interna cells retain their ability to express the relaxin gene following ovulation and luteinization. In the early CL, the small theca-derived lutein cells are the source of relaxin transcript. However, as the CL becomes fully differentiated, the large granulosa-derived lutein cells acquire the capacity to express the relaxin message.

DOI:
https://doi.org/10.1677/jme.0.0100087
Volume 10: Issue 1,
Pages:
87–97 (11 total)