Thyroid disorders are clinically associated with impaired fertility in women, and these abnormalities can be improved by restoring the euthyroid state. The exact mechanisms of thyroid effect on female fertility are not well known; however, it is conceivable that thyroid hormones (THs) might act on ovarian physiology via receptors in granulosa cells. This work is aimed at evaluating the effects of THs on non-tumoral granulosa cells and follicles.
Freshly isolated rat ovarian follicles and granulosa cells were exposed to T3 or T4 (THs). Cell growth and viability were evaluated by cell counting and the MTT assay, respectively, follicle growth was evaluated by volume measurements. Apoptosis was evaluated by the TUNEL assay and active Caspase 3 staining. rGROV cells were exposed to T3, and apoptosis was induced by serum deprivation. Bcl2, Bcl-2-associated X protein (BAX), Akt and pAkt expression were evaluated by western blot.
T3 induced a 40% increase in follicle volume (after 7 days). This increase was presumably due to the observed decrease (33%) in the apoptotic rate of the granulosa cell population. Both T3 and T4 caused a dose-dependent increase in rat granulosa cell number and viability. In addition, THs decreased the cell apoptotic rate in a dose-dependent manner. In both conditions, T3 appeared to be more efficient. In rGROV cells, 100 nM T3 induced cell growth and, in the absence of growth factors, reduced cell apoptosis by 40%, downregulating Caspase 3 and BAX. This effect was associated with an increase in pAkt levels. The involvement of the PI3 K pathway was confirmed by the ability of the PI3 K specific inhibitor (LY-294,002) to abolish T3 pro-survival action.
THs influence cell survival of ovarian granulosa cells. This effect likely contributes to the TH-induced follicle volume increase.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Tax calculation will be finalised during checkout.
Thyroid Hormone Receptors
Dittrich R, Beckmann MW, Oppelt PG, Hoffmann I, Lotz L, Kuwert T, Mueller A (2011) Thyroid hormone receptors and reproduction. J Reprod Immunol 90:58–66. https://doi.org/10.1016/j.jri.2011.02.009
Poppe K, Velkeniers B, Glinoer D (2007) Thyroid disease and female reproduction. Clin Endocrinol (Oxf) 66:309–321. https://doi.org/10.1111/j.1365-2265.2007.02752.x
Vissenberg R, Manders VD, Mastenbroek S, Fliers E, Afink GB, Ris-Stalpers C, Goddijn M, Bisschop PH (2015) Pathophysiological aspects of thyroid hormone disorders/thyroid peroxidase autoantibodies and reproduction. Hum Reprod Update 21:378–387. https://doi.org/10.1093/humupd/dmv004
Duarte-Guterman P, Navarro-Martin L, Trudeau VL (2014) Mechanisms of crosstalk between endocrine systems: regulation of sex steroid hormone synthesis and action by thyroid hormones. Gen Comp Endocrinol 203:69–85. https://doi.org/10.1016/j.ygcen.2014.03.015
Saran S, Gupta BS, Philip R, Singh KS, Bende SA, Agroiya P, Agrawal P (2016) Effect of hypothyroidism on female reproductive hormones. Indian J Endocrinol Metab 20:108–113. https://doi.org/10.4103/2230-8210.172245
Krassas GE, Poppe K, Glinoer D (2010) Thyroid function and human reproductive health. Endocr Rev 31:702–755. https://doi.org/10.1210/er.2009-0041
Hapon MB, Gamarra-Luques C, Jahn GA (2010) Short term hypothyroidism affects ovarian function in the cycling rat. Reprod Biol Endocrinol 8:14. https://doi.org/10.1186/1477-7827-8-14
Fedail JS, Zheng K, Wei Q, Kong L, Shi F (2014) Roles of thyroid hormones in follicular development in the ovary of neonatal and immature rats. Endocrine 46:594–604. https://doi.org/10.1007/s12020-013-0092-y
Zhang C, Guo L, Zhu B, Feng Y, Yu S, An N, Wang X (2013) Effects of 3, 5, 3′-triiodothyronine (t3) and follicle stimulating hormone on apoptosis and proliferation of rat ovarian granulosa cells. Chin J Physiol 56:298–305. https://doi.org/10.4077/CJP.2013.BAB186
Zhang C, Wang X, Wang Z, Niu W, Zhu B, Xia G (2013) Effect of different culture systems and 3, 5, 3′-triiodothyronine/follicle-stimulating hormone on preantral follicle development in mice. PLoS ONE 8:e61947. https://doi.org/10.1371/journal.pone.0061947
Wakim AN, Polizotto SL, Buffo MJ, Marrero MA, Burholt DR (1993) Thyroid hormones in human follicular fluid and thyroid hormone receptors in human granulosa cells. Fertil Steril 59:1187–1190. https://doi.org/10.1016/S0015-0282(16)55974-3
Kinne A, Schulein R, Krause G (2011) Primary and secondary thyroid hormone transporters. Thyroid Res 4(Suppl 1):S7. https://doi.org/10.1186/1756-6614-4-S1-S7
Kersseboom S, Visser TJ (2011) Tissue-specific effects of mutations in the thyroid hormone transporter MCT8. Arq Bras Endocrinol Metabol 55:1–5. https://doi.org/10.1210/jcem.82.6.3997
Aghajanova L, Lindeberg M, Carlsson IB, Stavreus-Evers A, Zhang P, Scott JE, Hovatta O, Skjoldebrand-Sparre L (2009) Receptors for thyroid-stimulating hormone and thyroid hormones in human ovarian tissue. Reprod Biomed Online 18:337–347. https://doi.org/10.1016/S1472-6483(10)60091-0
Zhang SS, Carrillo AJ, Darling DS (1997) Expression of multiple thyroid hormone receptor mRNAs in human oocytes, cumulus cells, and granulosa cells. Mol Hum Reprod 3:555–562. https://doi.org/10.1093/molehr/3.7.555
Maruo T, Hayashi M, Matsuo H, Yamamoto T, Okada H, Mochizuki M (1987) The role of thyroid hormone as a biological amplifier of the actions of follicle-stimulating hormone in the functional differentiation of cultured porcine granulosa cells. Endocrinology 121:1233–1241. https://doi.org/10.1210/endo-121-4-1233
Matsuda F, Inoue N, Manabe N, Ohkura S (2012) Follicular growth and atresia in mammalian ovaries: regulation by survival and death of granulosa cells. J Reprod Dev 58:44–50. https://doi.org/10.1262/jrd.2011-012
Markstrom E, Svensson EC, Shao R, Svanberg B, Billig H (2002) Survival factors regulating ovarian apoptosis – dependence on follicle differentiation. Reproduction 123:23–30. https://doi.org/10.1530/rep.0.1230023
Gougeon A (1986) Dynamics of follicular growth in the human: a model from preliminary results. Hum Reprod 1:81–87. https://doi.org/10.1093/oxfordjournals.humrep.a136365
McGee EA, Hsueh AJ (2000) Initial and cyclic recruitment of ovarian follicles. Endocr Rev 21:200–214. https://doi.org/10.1210/edrv.21.2.0394
Jiang JY, Cheung CK, Wang Y, Tsang BK (2003) Regulation of cell death and cell survival gene expression during ovarian follicular development and atresia. Front Biosci 8:d222–d237
Palumbo A, Yeh J (1995) Apoptosis as a basic mechanism in the ovarian cycle: follicular atresia and luteal regression. J Soc Gynecol Investig 2:565–573. https://doi.org/10.1177/107155769500200310
Boone DL, Carnegie JA, Rippstein PU, Tsang BK (1997) Induction of apoptosis in equine chorionic gonadotropin (eCG)-primed rat ovaries by anti-eCG antibody. Biol Reprod 57:420–427
Tilly JL, Kowalski KI, Johnson AL, Hsueh AJ (1991) Involvement of apoptosis in ovarian follicular atresia and postovulatory regression. Endocrinology 129:2799–2801. https://doi.org/10.1210/endo-129-5-2799
Verga-Falzacappa C, Patriarca V, Bucci B, Mangialardo C, Michienzi S, Moriggi G, Stigliano A, Brunetti E, Toscano V, Misiti S (2009) The TRbeta1 is essential in mediating T3 action on Akt pathway in human pancreatic insulinoma cells. J Cell Biochem 106:835–848. https://doi.org/10.1002/jbc.22045
Verga-Falzacappa C, Timperi E, Bucci B, Amendola D, Piergrossi P, D’Amico D, Santaguida MG, Centanni M, Misiti S (2012) T(3) preserves ovarian granulosa cells from chemotherapy-induced apoptosis. J Endocrinol 215:281–289. https://doi.org/10.1530/JOE-12-0153
Cecconi S, Gualtieri G, Di BA, Troiani G, Cifone MG, Canipari R (2000) Evaluation of the effects of extremely low frequency electromagnetic fields on mammalian follicle development. Hum Reprod 15:2319–2325. https://doi.org/10.1093/humrep/15.11.2319
Verga-Falzacappa C, Mangialardo C, Patriarca V, Bucci B, Amendola D, Raffa S, Torrisi MR, Silvestrini G, Ballanti P, Moriggi G, Stigliano A, Brunetti E, Toscano V, Misiti S (2009) Thyroid hormones induce cell proliferation and survival in ovarian granulosa cells COV434. J Cell Physiol 221:242–253. https://doi.org/10.1002/jcp.21849
Vaccari S, Latini S, Barberi M, Teti A, Stefanini M, Canipari R (2006) Characterization and expression of different pituitary adenylate cyclase-activating polypeptide/vasoactive intestinal polypeptide receptors in rat ovarian follicles. J Endocrinol 191:287–299. https://doi.org/10.1677/joe.1.06470
Innocenti F, Cerquetti L, Pezzilli S, Bucci B, Toscano V, Canipari R, Stigliano A (2017) Effect of mitotane on mouse ovarian follicle development and fertility. J Endocrinol 234:29–39. https://doi.org/10.1530/JOE-17-0203
Abdul-Ghani R, Serra V, Gyorffy B, Jurchott K, Solf A, Dietel M, Schafer R (2006) The PI3 K inhibitor LY294002 blocks drug export from resistant colon carcinoma cells overexpressing MRP1. Oncogene 25:1743–1752. https://doi.org/10.1038/sj.onc.1209201
Pascual A, Aranda A (2013) Thyroid hormone receptors, cell growth and differentiation. Biochim Biophys Acta 1830:3908–3916. https://doi.org/10.1016/j.bbagen.2012.03.012
Zheng K, Sulieman FJ, Li J, Wei Q, Xu M, Shi F (2015) Nitric oxide and thyroid hormone receptor alpha 1 contribute to ovarian follicular development in immature hyper- and hypo-thyroid rats. Reprod Biol 15:27–33. https://doi.org/10.1016/j.repbio.2014.11.002
Dijkstra G, de Rooij DG, de Jong FH, Van Den HR (1996) Effect of hypothyroidism on ovarian follicular development, granulosa cell proliferation and peripheral hormone levels in the prepubertal rat. Eur J Endocrinol 134:649–654. https://doi.org/10.1530/eje.0.1340649
Chan WY, Ng TB (1995) Effect of hypothyroidism induced by propylthiouracil and thiourea on male and female reproductive systems of neonatal mice. J Exp Zool 273:160–169. https://doi.org/10.1002/jez.1402730209
Jiang JY, Umezu M, Sato E (2000) Improvement of follicular development rather than gonadotrophin secretion by thyroxine treatment in infertile immature hypothyroid rdw rats. J Reprod Fertil 119:193–199. https://doi.org/10.1530/jrf.0.1190193
Kobayashi N, Orisaka M, Cao M, Kotsuji F, Leader A, Sakuragi N, Tsang BK (2009) Growth differentiation factor-9 mediates follicle-stimulating hormone-thyroid hormone interaction in the regulation of rat preantral follicular development. Endocrinology 150:5566–5574. https://doi.org/10.1210/en.2009-0262
Zhang CP, Yang JL, Zhang J, Li L, Huang L, Ji SY, Hu ZY, Gao F, Liu YX (2011) Notch signaling is involved in ovarian follicle development by regulating granulosa cell proliferation. Endocrinology 152:2437–2447. https://doi.org/10.1210/en.2010-1182
Kim WG, Zhu X, Kim DW, Zhang L, Kebebew E, Cheng SY (2013) Reactivation of the silenced thyroid hormone receptor beta gene expression delays thyroid tumor progression. Endocrinology 154:25–35. https://doi.org/10.1210/en.2012
Zhang C, Xia G, Tsang BK (2011) Interactions of thyroid hormone and FSH in the regulation of rat granulosa cell apoptosis. Front Biosci (Elite Ed) 3:1401–1413. https://doi.org/10.2741/342
Goldman S, Dirnfeld M, Abramovici H, Kraiem Z (1993) Triiodothyronine (T3) modulates hCG-regulated progesterone secretion, cAMP accumulation and DNA content in cultured human luteinized granulosa cells. Mol Cell Endocrinol 96:125–131. https://doi.org/10.1210/jcem.82.6.3997
Dubuis JM, Dayer JM, Siegrist-Kaiser CA, Burger AG (1988) Human recombinant interleukin-1 beta decreases plasma thyroid hormone and thyroid stimulating hormone levels in rats. Endocrinology 123:2175–2181
Samuels HH, Stanley F, Casanova J (1979) Relationship of receptor affinity to the modulation of thyroid hormone nuclear receptor levels and growth hormone synthesis by L-triiodothyronine and iodothyronine analogues in cultured GH1 cells. J Clin Invest 63:1229–1240. https://doi.org/10.1172/JCI109418
Canipari R, Strickland S (1985) Plasminogen activator in the rat ovary. Production and gonadotropin regulation of the enzyme in granulosa and thecal cells. J Biol Chem 260:5121–5125
Matikainen T, Perez GI, Zheng TS, Kluzak TR, Rueda BR, Flavell RA, Tilly JL (2001) Caspase-3 gene knockout defines cell lineage specificity for programmed cell death signaling in the ovary. Endocrinology 142:2468–2480. https://doi.org/10.1210/endo.142.6.8078
Laoag-Fernandez JB, Matsuo H, Murakoshi H, Hamada AL, Tsang BK, Maruo T (2004) 3,5,3′-Triiodothyronine down-regulates Fas and Fas ligand expression and suppresses caspase-3 and poly (adenosine 5′-diphosphate-ribose) polymerase cleavage and apoptosis in early placental extravillous trophoblasts in vitro. J Clin Endocrinol Metab 89:4069–4077. https://doi.org/10.1210/jc.2003-032208
Sukocheva OA, Carpenter DO (2006) Anti-apoptotic effects of 3,5,3′-tri-iodothyronine in mouse hepatocytes. J Endocrinol 191:447–458. https://doi.org/10.1677/joe.1.07061
Misiti S, Anastasi E, Sciacchitano S, Verga FC, Panacchia L, Bucci B, Khouri D, D’Acquarica I, Brunetti E, Di MU, Toscano V, Perfetti R (2005) 3,5,3′-Triiodo-L-thyronine enhances the differentiation of a human pancreatic duct cell line (hPANC-1) towards a beta-cell-Like phenotype. J Cell Physiol 204:286–296. https://doi.org/10.1002/jcp.20293
Davis PJ, Leonard JL, Davis FB (2008) Mechanisms of nongenomic actions of thyroid hormone. Front Neuroendocrinol 29:211–218. https://doi.org/10.1016/j.yfrne.2007.09.003
Minlikeeva AN, Freudenheim JL, Cannioto RA, Eng KH, Szender JB, Mayor P, Etter JL, Cramer DW, Diergaarde B, Doherty JA, Dork T, Edwards R, deFazio A, Friel G, Goodman MT, Hillemanns P, Hogdall E, Jensen A, Jordan SJ, Karlan BY, Kjaer SK, Klapdor R, Matsuo K, Mizuno M, Nagle CM, Odunsi K, Paddock L, Rossing MA, Schildkraut JM, Schmalfeldt B, Segal BH, Starbuck K, Terry KL, Webb PM, Zsiros E, Ness RB, Modugno F, Bandera EV, Chang-Claude J, Moysich KB (2017) History of thyroid disease and survival of ovarian cancer patients: results from the Ovarian Cancer Association Consortium, a brief report. Br J Cancer 117:1063–1069. https://doi.org/10.1038/bjc.2017.267
Journy NMY, Bernier MO, Doody MM, Alexander BH, Linet MS, Kitahara CM (2017) Hyperthyroidism, Hypothyroidism, and Cause-Specific Mortality in a Large Cohort of Women. Thyroid 27:1001–1010. https://doi.org/10.1089/thy.2017.0063
Ness RB, Grisso JA, Cottreau C, Klapper J, Vergona R, Wheeler JE, Morgan M, Schlesselman JJ (2000) Factors related to inflammation of the ovarian epithelium and risk of ovarian cancer. Epidemiology 11:111–117. https://doi.org/10.1097/00001648-200003000-00006
Rae MT, Gubbay O, Kostogiannou A, Price D, Critchley HO, Hillier SG (2007) Thyroid hormone signaling in human ovarian surface epithelial cells. J Clin Endocrinol Metab 92:322–327. https://doi.org/10.1210/jc.2006-1522
This study was supported by IBSA Institute Biochimique SA (Lugano, CH) (to M.C.) and by grants from Sapienza University of Rome, Ateneo Federato 2014–2015 (to R. C.).
Conflict of interest
On behalf of all of the authors, the corresponding author states that there is no conflict of interest.
Animals were maintained in accordance with the Italian Department of Health Guide for Care and Use of Laboratory Animals. Experimental protocols were approved by the ‘La Sapienza’ University Committee for Animal Care and Use. All of the authors certify that: (1) this material has not been published in whole or in part elsewhere; (2) the manuscript is not currently being considered for publication in another journal; (3) they were personally and actively involved in substantive work leading to the manuscript and will hold themselves jointly and individually responsible for its content; (4) this article does not contain any studies with human participants performed by any of the authors.
No informed consent.
About this article
Cite this article
Canipari, R., Mangialardo, C., Di Paolo, V. et al. Thyroid hormones act as mitogenic and pro survival factors in rat ovarian follicles. J Endocrinol Invest 42, 271–282 (2019). https://doi.org/10.1007/s40618-018-0912-2
- Thyroid hormones
- Granulosa cells