Estimation of viability of bovine granulosa cells

Metcalf, Mary G.

doi:10.1530/jrf.0.0650425

Cited by 13 publications

(6 citation statements)

References 10 publications

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“…Granulosa and theca cells were cultured in the absence of serum for 48 h and then treated with TGFa (10 ng/ml) for an additional 24 h. Cultures were incubated in the presence of [ 3 H]thymidine for 4 h, followed by determination of the amount of [ 3 H]thymidine incorporated into DNA as previously described (18,24). TGFa significantly stimulated DNA synthesis in both granulosa and theca cells (Fig.…”

Section: Resultsmentioning

confidence: 96%

Regulation of Ovarian Cell Growth through the Local Production of Transforming Growth Factor-α by Theca Cells*

1988

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The rapid proliferation of a tissue often requires the local production of a specific growth factor. The ovarian follicle is a rapidly growing tissue which contains two primary somatic cell types, granulosa cells and theca cells. Theca cells and granulosa cells were isolated from bovine ovaries and cultured to assess the possible local production of a growth factor within the ovarian follicle. Serum-free conditioned medium from theca cells, but not from granulosa cells, was found to contain a component that specifically bound to the epidermal growth factor (EGF) receptor. Therefore, theca cells appear to produce an EGF-like substance as a potential regulator of follicle cell growth. This result provides physiological significance to the previous observation that granulosa cells contain EGF receptors and respond to EGF to increase cell proliferation. Transforming growth factor-alpha (TGF alpha) is a protein that is structurally and functionally related to EGF and binds to the EGF receptor. Using a molecular probe to TGF alpha, theca cells were found to express the TGF alpha gene, which is consistent with the presence of an EGF-like substance in conditioned medium, but granulosa cells had no detectable TGF alpha gene expression. Similar analysis with a molecular probe to EGF demonstrated the apparent lack of EGF gene expression in theca cells or granulosa cells. As previously demonstrated with granulosa cells, the data presented indicate that theca cells also contain high affinity EGF receptors. TGF alpha was found to stimulate the growth of both granulosa and theca cells. These observations imply that within the ovarian follicle TGF alpha is produced by theca cells, which can subsequently have both a paracrine and an autocrine role in regulating follicle cell proliferation. Results presented demonstrate production of TGF alpha by a normal adult mesenchymal tissue and provide an example of a growth factor-mediated mesenchymal-epithelial cell interaction between theca cells and granulosa cells.

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Section: Resultsmentioning

confidence: 96%

Regulation of Ovarian Cell Growth through the Local Production of Transforming Growth Factor-α by Theca Cells*

1988

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“…Internucleosomal DNA fragmentation was also detected in GC isolated from healthy follicles, indicating that apoptosis in the bovine species may occur very early in the atretic process before other morphological and biochemical signs of degeneration or dysfunction are evident. Therefore, the morphological criteria used previously [22] for classifying healthy and atretic follicles are not very accurate. Our findings are consistent with one study in cows in which follicles were collected during the luteal phase [9] and a study in ewes [24].…”

Section: Discussionmentioning

confidence: 99%

“…From around 8 mm, follicles express LH receptors on GC and require pulsatile LH stimulation to continue growing [21]. Small, medium, and large follicles were then classified as healthy or atretic according to previously established morphological criteria [22]. Healthy follicles had vascularized (pink or red) theca interna and clear amber follicular fluid (FF) with no debris, and they contained Ͼ 25% of the maximum number of GC that could be present for a given follicle size (Ͼ 25% G max ).…”

Section: Collection Of Ovaries and Classification Of Folliclesmentioning

confidence: 99%

Morphological and Biochemical Identification of Apoptosis in Small, Medium, and Large Bovine Follicles and the Effects of Follicle-Stimulating Hormone and Insulin-Like Growth Factor-I on Spontaneous Apoptosis in Cultured Bovine Granulosa Cells1

Yang

Rajamahendran

2000

Biology of Reproduction

132

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The first objective of this study was to determine whether the death of bovine granulosa cells (GC) isolated from small ( 8 mm) follicles during follicular atresia occurs by apoptosis. The second objective was to establish an in vitro model system to elucidate the developmental (GC from follicles of different sizes) and hormonal (FSH and insulin-like growth factor-I [IGF-I]) regulation of bovine GC apoptosis during follicular atresia. Bovine ovaries were obtained from a nearby slaughterhouse. Follicles were classified by morphometric criteria as healthy or atretic. Apoptosis in GC from follicles of different sizes was analyzed by both morphological and biochemical methods. Bovine GC were cultured for 48 h at a density of 5 x 10(6) cells/ml in serum-free media at 39 degrees C to determine the effects of FSH and IGF-I on apoptosis. The results showed that apoptosis occurred in GC from all sizes of follicles. Apoptosis in GC was also detected in some healthy follicles. Degenerate GC displayed the morphological characteristics of apoptosis, including nuclei with marginated chromatin, a single condensed nucleus, multiple nuclear fragments, and/or membrane-bound structures containing variable amounts of chromatin and/or cytoplasm (apoptotic bodies). All GC classified as apoptotic on the basis of their morphology contained fragmented DNA measured by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) technique. Cells that had undergone apoptosis were observed mainly in GC and in scattered theca cells. Throughout the GC layer, apoptotic cell death was more prevalent among antral GC than among mural GC. Interestingly, morphological results showed that no apoptosis occurred in cumulus cells. A time-dependent, spontaneous onset of apoptosis occurred in GC from small, medium, and large follicles during in vitro serum-free culture. The rate of DNA fragmentation in the culture of GC from small follicles was higher than that from medium and large follicles. FSH attenuated apoptotic cell death in GC from medium follicles more effectively than in those from small follicles. IGF-I also suppressed apoptosis in cultured GC from small follicles. In conclusion, this study showed that 1) GC death during bovine follicular development and atresia occurs by apoptosis; 2) apoptosis occurs in GC and theca cells; however, apoptosis does not occur in cumulus cells even in atretic antral follicles; 3) GC from all small, medium, and large follicles undergo spontaneous onset of apoptosis when cultured under serum-free conditions; and 4) FSH and IGF-I can attenuate apoptosis in cultured bovine GC.

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“…Granulosa cells were harvested only from healthy follicles. Healthy, developing follicles were assessed using criteria previously described (Metcalf 1982), including a vascularized pink theca externa and clear amber FF with no debris. Follicles were punctured with an 18-gauge needle and FF was aspirated.…”

Section: Preparation Of Gcmentioning

confidence: 99%

Effects of gonadotropins and insulin-like growth factors-I and -II on in vitro steroid production by bovine granulosa cells

Yang¹,

Rajamahendran²

1998

Can. J. Anim. Sci.

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Yang, M. Y. and Rajamahendran, R. 1998. Effects of gonadotropins and insulin-like growth factors-I and -II on in vitro steroid production by bovine granulosa cells. Can. J. Anim. Sci. 78: 587-597. The objectives of this study were: 1) to develop a bovine granulosa cell (GC) culture system; and 2) to use this system to evaluate the effects of gonadotropins (FSH and LH) and insulin-like growth factors-I and -II (IGF-I and IGF-II) on steroidogenesis of bovine GC derived from small, medium, and large antral follicles (diameters ≤4, 5-8 and >8 mm, respectively). Granulosa cells were cultured (concentration, 5 × 10 5 cells per well) in serum-free medium for 48 h with variable doses of hormones and growth factors. Concentrations of progesterone (P 4 ) and estradiol-17β (E 2 ) in the media were determined by radioimmunoassay. Basal E 2 production by GC from follicles of all sizes decreased with time of culture (P < 0.01) while basal P 4 production increased (P < 0.01). Basal E 2 and P 4 production increased with increasing size of follicles (P < 0.01). Only very low concentrations of FSH stimulated E 2 production from medium and large follicles. Follicle-stimulating hormone stimulated P 4 production by GC of follicles of all sizes (P < 0.05). Luteinizing hormone inhibited E 2 production by GC in medium and large follicles (P < 0.05), suggesting that LH is responsible for the rise in plasma E 2 through effects on both theca cells and GC. A dose of 100 ng mL -1 of IGF-I increased E 2 production by GC from medium and large follicles (P < 0.05). Progesterone production by GC from all categories of follicles was also stimulated by IGF-I (P < 0.05). Estradiol-17β production by GC from large follicles decreased in response to IGF-II (P < 0.05). The physiological role of IGF-II on steroidogenesis in the bovine ovary remains to be elucidated. In summary, these results demonstrate the development of a serum-free culture system for bovine GC, and that FSH, LH, IGF-I and IGF-II have different effects on steroidogenesis by bovine GC from different size follicles. de mettre au point un système de culture des cellules de granulosa bovine (CG) et 2) d'utiliser ce système pour évaluer les effets des hormones gonadotropes FSH et LH et des somatomédines I et II sur la stéroïdogénèse des CG dérivées des petits, moyens et grands follicules antraux (diamètres respectifs ≤4, 5-8 et >8 mm). Les cellules de granulosa étaient mises en culture pendant 48 h, à raison de 5 × 10 5 cellules par puit, dans un milieu sans sérum en présence de doses variables d'hormones et de facteurs de croissance. Les concentrations de progestérone (P 4 ) et d'oestradiol-17β (E 2 ) dans les milieux de culture étaient contrôlées par dosage immunoradiologique. La production basale de E 2 par les CG des follicules de toute tailles diminuait au fur et à mesure de la durée de culture (P < 0,01), tandis que celle de P 4 augmentait (P < 0,01). La production basale des deux hormones augmentait avec la taille des follicules (P < 0,01). Seules les très basses concentrations de ...

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Estimation of viability of bovine granulosa cells

Cited by 13 publications

References 10 publications

Regulation of Ovarian Cell Growth through the Local Production of Transforming Growth Factor-α by Theca Cells*

Regulation of Ovarian Cell Growth through the Local Production of Transforming Growth Factor-α by Theca Cells*

Morphological and Biochemical Identification of Apoptosis in Small, Medium, and Large Bovine Follicles and the Effects of Follicle-Stimulating Hormone and Insulin-Like Growth Factor-I on Spontaneous Apoptosis in Cultured Bovine Granulosa Cells1

Effects of gonadotropins and insulin-like growth factors-I and -II on in vitro steroid production by bovine granulosa cells

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