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Key Event Title
Granulosa cell proliferation of gonadotropin-independent follicles, Reduced
|Level of Biological Organization|
Key Event Components
Key Event Overview
AOPs Including This Key Event
Key Event Description
Granulosa cell function
Granulosa cells of the ovary play an important structural and functional role during folliculogenesis. They form the ovarian follicle architecture and transmit molecular messages to the oocyte through gap junction channels, ensuring developmental competence (Kidder & Vanderhyden, 2010). Folliculogenesis can be roughly divided into two phases: gonadotropin independent and gonadotropin-dependent by the requirement for the gonadotropin follicle-stimulating hormone (FSH) to grow (Hsueh, Kawamura, Cheng, & Fauser, 2015). During the gonadotropin-independent growth phase, growth factors secreted by the follicle (e.g. GDF-9 by the oocyte and AMH by the granulosa cells) control the necessary morphological changes of granulosa cells and their proliferation (Hsueh et al., 2015). The growth can be histologically observed as proliferation of the granulosa cells as the flat granulosa cells of primordial follicles become cuboidal and increase in numbers (Gougeon, 2010). The connection between granulosa cell numbers and follicle growth during gonadotropin-independent growth is well described (Gougeon & Chainy, 1987).
Reduced granulosa cell proliferation as Key Event
Knock-out mouse models have demonstrated that granulosa cell proliferation is a prerequisite for normal follicle growth and fertility. For example, deletion of the oocyte-specific growth factor Gdf-9 that stimulates granulosa cells halt folliculogenesis at the primary follicle stage in mice: the granulosa cells fail to proliferate to generate secondary follicles, the oocytes degenerate, and the mice are sterile (Dong et al., 1996). Conversely, mice administered Gdf-9 have accelerated granulosa cell proliferation and higher numbers of primary and secondary follicles compared to non-treated ones (Vitt, McGee, Hayashi, & Hsueh, 2000). AMH is a growth factor secreted by granulosa cells during the gonadotropin-independent follicle growth stage, and it inhibits the activation of primordial follicles to keep the growing and dormant follicles in balance. In mice overexpressing AMH, follicle growth to antral stages is inhibited and the numbers of all developmental stages of follicles decline faster by age than in wildtype controls (Pankhurst et al., 2018). Exposure of human ovarian tissue to AMH in culture prevents follicle growth (Carlsson et al., 2006).
How It Is Measured or Detected
Decreased granulosa cell proliferation can be measured in cell culture. There are commercially available human granulosa cell tumor lines, for instance KGN (#RCB1154) “Granulosa cell tumor”, available from the Riken Cell Bank. This cell line is representative of undifferentiated granulosa cells at early stages of follicle development making it suitable to study interactions of preantral pathways independent from hormonal control from theca cells and hypothalamic-pituitary axis (Nishi et al., 2001). Well-established assays to detect proliferation include methods to assess DNA synthesis (e.g. BrdU), cellular metabolism (e.g. MTT, XTT, ATP detection assays), and proliferation proteins (e.g. PCNA, Ki67, MCM-2) (Adan, Kiraz, & Baran, 2016). The same methods can also be used in ovarian follicle or tissue culture.
Granulosa cell proliferation manifests as increased numbers of granulosa cells within ovarian follicles (Gougeon & Chainy, 1987). Analysis of follicle growth is based on the numbers of granulosa cell layers which is also reflected in the diameter of the follicle (Gougeon & Chainy, 1987). Granulosa cell proliferation is inseparably connected to folliculogenesis, and therefore numbers of follicles in different developmental stages reflect proliferation of granulosa cells. Granulosa cell proliferation can therefore be measured by counting follicles in different stages (primordial, primary, secondary) or by measuring the follicle diameters. Changes in the proliferation of granulosa cells during the preantral growth phase would lead to altered proportions of follicles in different stages. For example, inhibition of granulosa cell proliferation can lead to reduced numbers of secondary follicles (Dong et al., 1996; Pankhurst et al., 2018). Therefore, studying ratios between follicles in different developmental stages can reveal changes in the proliferation of granulosa cells. Follicle counts are already suggested endpoints in the Extended One-Generation Reproductive Toxicity Study; EOGRTS (OECD 443) (Test No. 443: Extended One-Generation Reproductive Toxicity Study, 2018).
Domain of Applicability
Mechanisms controlling folliculogenesis are well conserved between mammalian species, including mice, farm animals and humans (Adhikari & Liu, 2009; McGee & Hsueh, 2000).
Adan, A., Kiraz, Y., & Baran, Y. (2016). Cell Proliferation and Cytotoxicity Assays. Current Pharmaceutical Biotechnology, 17(14), 1213–1221. https://doi.org/10.2174/1389201017666160808160513
Adhikari, D., & Liu, K. (2009, August). Molecular mechanisms underlying the activation of mammalian primordial follicles. Endocrine Reviews, Vol. 30, pp. 438–464. https://doi.org/10.1210/er.2008-0048
Carlsson, I. B., Scott, J. E., Visser, J. A., Ritvos, O., Themmen, A. P. N., & Hovatta, O. (2006). Anti-Müllerian hormone inhibits initiation of growth of human primordial ovarian follicles in vitro. Human Reproduction, 21(9), 2223–2227. https://doi.org/10.1093/humrep/del165
Dong, J., Albertini, D. F., Nishimori, K., Kumar, T. R., Lu, N., & Matzuk, M. M. (1996). Growth differentiation factor-9 is required during early ovarian folliculogenesis. Nature, 383(6600), 531–535. https://doi.org/10.1038/383531a0
Gougeon, A. (2010). Croissance folliculaire dans l’ovaire humain: de l’entrée en croissance du follicule primordial jusqu’à la maturation préovulatoire. Annales d’Endocrinologie, 71(3), 132–143. https://doi.org/10.1016/j.ando.2010.02.021
Gougeon, A., & Chainy, G. B. N. (1987). Morphometric studies of small follicles in ovaries of women at different ages. Journal of Reproduction and Fertility, 81(2), 433–442. https://doi.org/10.1530/jrf.0.0810433
Hsueh, A. J. W., Kawamura, K., Cheng, Y., & Fauser, B. C. J. M. (2015). Intraovarian control of early folliculogenesis. Endocrine Reviews, Vol. 36, pp. 1–24. https://doi.org/10.1210/er.2014-1020
Kidder, G. M., & Vanderhyden, B. C. (2010). Bidirectional communication between oocytes and follicle cells: Ensuring oocyte developmental competence. Canadian Journal of Physiology and Pharmacology, Vol. 88, pp. 399–413. https://doi.org/10.1139/Y10-009
McGee, E. A., & Hsueh, A. J. W. (2000). Initial and Cyclic Recruitment of Ovarian Follicles*. Endocrine Reviews, 21(2), 200–214. https://doi.org/10.1210/edrv.21.2.0394
Nishi, Y., Yanase, T., Mu, Y.-M., Oba, K., Ichino, I., Saito, M., … Nawata, H. (2001). Establishment and Characterization of a Steroidogenic Human Granulosa-Like Tumor Cell Line, KGN, That Expresses Functional Follicle-Stimulating Hormone Receptor. Endocrinology, 142(1), 437–445. https://doi.org/10.1210/endo.142.1.7862
Pankhurst, M. W., Kelley, R. L., Sanders, R. L., Woodcock, S. R., Oorschot, D. E., & Batchelor, N. J. (2018). Anti-Müllerian hormone overexpression restricts preantral ovarian follicle survival. Journal of Endocrinology, 237(2), 153–163. https://doi.org/10.1530/JOE-18- 0005
Test No. 443: Extended One-Generation Reproductive Toxicity Study. (2018). https://doi.org/10.1787/9789264185371-en
Vitt, U. A., McGee, E. A., Hayashi, M., & Hsueh, A. J. W. (2000). In vivo treatment with GDF-9 stimulates primordial and primary follicle progression and theca cell marker CYP17 in ovaries of immature rats. Endocrinology, 141(10), 3814–3820. https://doi.org/10.1210/endo.141.10.7732