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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
|During development and at adulthood||High|
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 and 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 et al., 2015). During the gonadotropin-independent growth phase, growth factors secreted by the follicle, e.g. growth differentiation factor-9 (GDF9) by the oocyte and anti-Müllerian hormone (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 and Chainy, 1987).
Reduced granulosa cell proliferation as Key Event
Genetically modified 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 GDF9 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 GDF9 have accelerated granulosa cell proliferation and higher numbers of primary and secondary follicles compared to non-treated ones(Vitt et al., 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 inhibits 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 primordial to early antral 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 et al., 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 and 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 and Chainy, 1987). Granulosa cell proliferation is inseparably connected to folliculogenesis, and therefore numbers of follicles in different developmental stages reflect the 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 early follicle 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)(2018).
Domain of Applicability
Mechanisms controlling folliculogenesis are well conserved between mammalian species, including mice, farm animals and humans(Adhikari and Liu, 2009; McGee and Hsueh, 2000).
Adan, A., Kiraz, Y., and Baran, Y. (2016). Cell Proliferation and Cytotoxicity Assays. Current Pharmaceutical Biotechnology 17, 1213–1221. https://doi.org/10.2174/1389201017666160808160513.
Adhikari, D., and Liu, K. (2009). Molecular mechanisms underlying the activation of mammalian primordial follicles. Endocrine Reviews 30, 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., and Hovatta, O. (2006). Anti-Müllerian hormone inhibits initiation of growth of human primordial ovarian follicles in vitro. Human Reproduction 21, 2223–2227. https://doi.org/10.1093/humrep/del165.
Dong, J., Albertini, D.F., Nishimori, K., Kumar, T.R., Lu, N., and Matzuk, M.M. (1996). Growth differentiation factor-9 is required during early ovarian folliculogenesis. Nature 383, 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, 132–143. https://doi.org/10.1016/j.ando.2010.02.021.
Gougeon, A., and Chainy, G.B.N. (1987). Morphometric studies of small follicles in ovaries of women at different ages. Journal of Reproduction and Fertility 81, 433–442. https://doi.org/10.1530/jrf.0.0810433.
Hsueh, A.J.W., Kawamura, K., Cheng, Y., and Fauser, B.C.J.M. (2015). Intraovarian control of early folliculogenesis. Endocrine Reviews 36, 1–24. https://doi.org/10.1210/er.2014-1020.
Kidder, G.M., and Vanderhyden, B.C. (2010). Bidirectional communication between oocytes and follicle cells: Ensuring oocyte developmental competence. Canadian Journal of Physiology and Pharmacology 88, 399–413. https://doi.org/10.1139/Y10-009.
McGee, E.A., and Hsueh, A.J.W. (2000). Initial and Cyclic Recruitment of Ovarian Follicles*. Endocrine Reviews 21, 200–214. https://doi.org/10.1210/edrv.21.2.0394.
Nishi, Y., Yanase, T., Mu, Y.-M., Oba, K., Ichino, I., Saito, M., Nomura, M., Mukasa, C., Okabe, T., Goto, K., et al. (2001). Establishment and Characterization of a Steroidogenic Human Granulosa-Like Tumor Cell Line, KGN, That Expresses Functional Follicle-Stimulating Hormone Receptor. Endocrinology 142, 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., and Batchelor, N.J. (2018). Anti-Müllerian hormone overexpression restricts preantral ovarian follicle survival. Journal of Endocrinology 237, 153–163. https://doi.org/10.1530/JOE-18-0005.
Vitt, U.A., McGee, E.A., Hayashi, M., and 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, 3814–3820. https://doi.org/10.1210/endo.141.10.7732.
(2018). Test No. 443: Extended One-Generation Reproductive Toxicity Study (OECD).