Influence of follicular health on the steroidogenic and morphological characteristics of bovine granulosa cells in vitro

Henderson, Kathryn; McNatty, Kenneth P.; Smith, Paul A.; Gibb, M.; O’Keeffe, L. E.; Lun, S.; Heath, D. A.; Prisk, M. D.

doi:10.1530/jrf.0.0790185

Cited by 39 publications

(20 citation statements)

References 15 publications

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“…This corresponded also with the results received form the annexin V-FITC test and TUNEL method revealing apoptotic cells. This is in agreement with Henderson et al [61], who found that granulosa cells isolated from non-atretic cow follicles cultured under the influence of testosterone secreted diminishing amounts of progesterone. This confirms that follicles used in our cultures, both granulosal and follicular, were healthy and non-atretic.…”

Section: Discussionsupporting

confidence: 93%

Does 2-hydroxyflutamide Inhibit Apoptosis in Porcine Granulosa Cells? — An <i>In Vitro</i> Study

Duda

Durlej

Knet

et al. 2012

Journal of Reproduction and Development

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Abstract. In mammalian ovaries, the majority of follicles are lost before ovulation by atresia. This degenerative process is initiated or caused by granulosa cell apoptosis. To reveal the androgen-dependent mechanism of selective follicular atresia, the culture model system for agonism and antagonism of the androgen receptor has been established. We examined the influence of an androgen receptor antagonist, 2-hydroxyflutamide (2-Hf), on the incidence of apoptosis in cultured porcine granulosa cells. They were incubated (6 and 12-h) in the presence of testosterone (T, 10 -7 M), 2-Hf (1.7×10 -4 M) or both T and 2-Hf (T+2-Hf), and then analyzed by flow cytometry with fluorescein labelled annexin V. To better imitate in vivo conditions, the intact porcine follicles (6-8 mm in diameter) have been incubated in an organ culture system with the addition of the same factors. Sections obtained from follicles fixed after culture were stained with hematoxylin and eosin, and the presence of apoptosis-related DNA strand breaks was evaluated by the TUNEL method. Estradiol and progesterone concentrations in the culture media were measured by radioimmunoassays. The addition of T or 2-Hf to the culture media caused an increase in the number of apoptotic granulosa cells, while treatment with T+2-Hf decreased it in both in vitro and organotypic models. Follicles cultured with the addition of T or 2-Hf exhibited morphological changes indicating follicular atresia. Granulosal estradiol secretion was considerably stimulated by T+2-Hf. The highest increase in follicular estradiol secretion was observed after the anti-androgen addition. In both granulosal and follicular cultures, the production of progesterone declined in the presence of T or 2-Hf but increased after their simultaneous addition. In conclusion, androgen receptor antagonist 2-Hf attenuates induction of granulosa cell apoptosis in the presence of a high T level. The nature of this protective mechanism as yet is unknown and requires further research. Key words: Apoptosis, Granulosa cells, 2-hydroxyflutamide, Ovarian follicles, Pig (J. Reprod. Dev. 58: [438][439][440][441][442][443][444] 2012) T he ovary plays two distinct functions in female reproduction: it produces germ cells (oocytes) and provides a proper hormonal environment for ovarian follicles undergoing cycles of growth and development induced by gonadotropins [1][2][3]. Although gonadotropins are predominant regulators of follicular development, it is now clear that there is a wide range of additional regulators of follicular maturation produced within the ovarian tissue such as steroids and peptide hormones, as well as ovarian growth factors. They act by paracrine mechanisms to modulate, either to amplify or attenuate, the functions of gonadotropins [4]. Amongst the several hundred thousand primordial follicles present in the mammalian ovary, only 1% of them develop to the preovulatory stage and finally ovulate [5]. The remaining ones will be eliminated via a degenerative process called atresia [6,7]. Atresia ...

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Section: Discussionsupporting

confidence: 93%

Does 2-hydroxyflutamide Inhibit Apoptosis in Porcine Granulosa Cells? — An <i>In Vitro</i> Study

Duda

Durlej

Knet

et al. 2012

Journal of Reproduction and Development

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“…Atresia was associated with a gradual decrease in follistatin expression by granulosa cells, as during atresia of the ovine follicle (Braw-Tal 1994). Theca interna cells remained morphologically and functionally healthy during the early stages of atresia, expressing all three of the mRNAs studied here, and continued to produce steroids (McNatty et al 1984a, Henderson et al 1987, Jolly et al 1994. As atresia progressed, there was a decrease in all three mRNAs expression in thecal cells, and apoptotic cells were occasionally detected.…”

Section: Discussionmentioning

confidence: 73%

Gene expression for LH receptor, 17α-hydroxylase and StAR in the theca interna of preantral and early antral follicles in the bovine ovary

Braw–Tal

Roth²

2005

Reproduction

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The onset of gene expression for three proteins that play pivotal roles in theca interna function, namely the LH receptor (LH-R), cytochrome P450 17a-hydroxylase (17aOH) and the steroidogenic acute regulatory protein (StAR), was determined. Ovaries were obtained on day 9 of the oestrus cycle from mature synchronized dairy cows (n 5 5) and gene expression in preantral and antral follicles up to 4 mm in diameter was evaluated by in situ hybridization. LH-R and 17aOH mRNAs were observed first, in the theca interna of large preantral follicles (type 4), concurrent with its morphological differentiation. StAR mRNA appeared later during follicular growth, in follicles >1 mm in diameter (type 6). LH-R and 17aOH mRNAs were found exclusively in the thecal cells, whereas StAR mRNA appeared in thecal cells, granulosa cells of late atretic follicles and oocytes. In early atresia, thecal cells expressed all three mRNAs, and their expression decreased gradually as atresia progressed. Atresia in granulosa cells was characterized by massive apoptosis of periantral, but not peribasal cells, that differentiated into luteallike cells expressing StAR. In summary, our study suggests that in spite of the presence of 17aOH, a key enzyme in steroidogenesis, the ability to produce steroids by bovine follicles smaller than 1 mm in diameter must be very limited due to the absence of StAR protein.During the early stages of atresia, thecal cells remain morphologically and functionally healthy, and continue to express all three studied mRNAs.

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“…These studies also found either a lower level of E 2 (Irving- Rodgers et al 2003b) or no significant reduction (McNatty et al 1984) respectively at these sizes of follicles. In a latter study, however, significant differences in E 2 production by cultured granulosa cells from follicles classified as healthy and atretic were observed (Henderson et al 1987), indicating that there is a difference in granulosa cells in Figure 6 A cartoon of antral (A) and basal (B) atresia illustrating the changes over three time points as cell death progresses either from the apical regions or from the basal region. In basal atresia, the known changes in follicular fluid hormone, expression of steroidogenic enzymes in the apical cells, breaching of the follicular basal lamina and migration of cells from the theca including macrophages, and death of steroidogenic cells and endothelial cells in the theca and changes in insulin-like factor 3 in the thecal interna are indicated.…”

Section: Atresia Of Antral Follicles !5 MM In Diametermentioning

confidence: 78%

Morphological classification of bovine ovarian follicles

Rodgers¹,

Irving-Rodgers²

2010

Reproduction

128

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Follicle classification is an important aid to the understanding of follicular development and atresia. Some bovine primordial follicles have the classical primordial shape, but ellipsoidal shaped follicles with some cuboidal granulosa cells at the poles are far more common. Preantral follicles have one of two basal lamina phenotypes, either a single aligned layer or one with additional layers. In antral follicles !5 mm diameter, half of the healthy follicles have columnar shaped basal granulosa cells and additional layers of basal lamina, which appear as loops in cross section ('loopy'). The remainder have aligned single-layered follicular basal laminas with rounded basal cells, and contain better quality oocytes than the loopy/columnar follicles. In sizes O5 mm, only aligned/rounded phenotypes are present. Dominant and subordinate follicles can be identified by ultrasound and/or histological examination of pairs of ovaries. Atretic follicles !5 mm are either basal atretic or antral atretic, named on the basis of the location in the membrana granulosa where cells die first. Basal atretic follicles have considerable biological differences to antral atretic follicles. In follicles O5 mm, only antral atresia is observed. The concentrations of follicular fluid steroid hormones can be used to classify atresia and distinguish some of the different types of atresia; however, this method is unlikely to identify follicles early in atresia, and hence misclassify them as healthy. Other biochemical and histological methods can be used, but since cell death is a part of normal homoeostatis, deciding when a follicle has entered atresia remains somewhat subjective.

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Influence of follicular health on the steroidogenic and morphological characteristics of bovine granulosa cells in vitro

Cited by 39 publications

References 15 publications

Does 2-hydroxyflutamide Inhibit Apoptosis in Porcine Granulosa Cells? — An <i>In Vitro</i> Study

Does 2-hydroxyflutamide Inhibit Apoptosis in Porcine Granulosa Cells? — An <i>In Vitro</i> Study

Gene expression for LH receptor, 17α-hydroxylase and StAR in the theca interna of preantral and early antral follicles in the bovine ovary

Morphological classification of bovine ovarian follicles

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