Selective depletion of AMH-expressing granulosa cells in vivo impairs follicular development and fertility in female mice

Endo, T.; Tamemasa, M.; Hayakawa, K.; Okada, F.; Oyama, N.; Watanabe, K.; Lai, T.; Nakano, Y.; Fujioka, Y.; Goto, M.; Takahashi, R.; Tomita, A.; Sugiura, K.; Hirate, Y.; Mizuno, N.; Kanai, Y.; Kanai-Azuma, M.

doi:10.64898/2026.08.04.742350

bioRxiv · 10.64898/2026.08.04.742350

Selective depletion of AMH-expressing granulosa cells in vivo impairs follicular development and fertility in female mice

Endo, T.·Tamemasa, M.·Hayakawa, K.·Okada, F.·Oyama, N.·Watanabe, K.·Lai, T.·Nakano, Y.·Fujioka, Y.·Goto, M.·Takahashi, R.·Tomita, A.·Sugiura, K.·Hirate, Y.·Mizuno, N.·Kanai, Y.·Kanai-Azuma, M.

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Abstract

In mammals, ovarian follicle development is a highly coordinated process that underlies female fertility. Granulosa cells expressing anti-Mullerian hormone (AMH) are widely used as a marker of growing follicles. However, the in vivo roles of granulosa cells in follicular development and female fertility remain unclear. Here, we analyzed AMH-toxin receptor-mediated cell knockout (AMH-TRECK) transgenic (Tg) mice on a NOG background, in which AMH-expressing granulosa cells are specifically depleted by diphtheria toxin (DT). We first found that, after a single DT injection into postnatal AMH-TRECK Tg females, AMH-expressing granulosa cells in primary and secondary follicles exhibited cleaved caspase-3 signals 1 day later and were depleted 4 days later. Second, after repeated DT injections weekly from 1 to 7 weeks of age in AMH-TRECK Tg females, antral follicles and corpora lutea were rarely observed, and the numbers of primordial, primary, and secondary follicles were decreased. Following PMSG and hCG stimulation, repeated DT-injected Tg females exhibited a reduced number of ovulated oocytes with a low proportion of mature oocytes, resulting in reduced IVF rates and fertility. Further, after a cessation of repeated DT treatment, ovarian weight and follicular development recovered: the numbers of primary, secondary, and antral follicles were recovered, whereas the primordial follicle pool remains reduced. We conclude that selective depletion of AMH-expressing granulosa cells in vivo impairs follicular development and fertility. Our model enables assessment of the in vivo effects of granulosa cell depletion and may provide a useful platform for future transplantation-based studies to understand complex follicular dynamics.

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Endo, T., Tamemasa, M., Hayakawa, K., Okada, F., Oyama, N., Watanabe, K., Lai, T., Nakano, Y., Fujioka, Y., Goto, M., Takahashi, R., Tomita, A., Sugiura, K., Hirate, Y., Mizuno, N., Kanai, Y., Kanai-Azuma, M.. 2026-08-05. Selective depletion of AMH-expressing granulosa cells in vivo impairs follicular development and fertility in female mice. https://doi.org/10.64898/2026.08.04.742350

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