Endocrine Abstracts

Objectives: Hypothyroidism is successfully treated by T4 replacement. Nevertheless, T4 treatment is not always well-tolerated. Regenerative medicine using thyroid follicular cells (TFCs) constitutes a very attractive alternative therapy; however, the minimal number of TFCs that have to be fixed remains unknown.

Methods: The severe hypothyroid phenotype of DUOXA^-/- mice will be rescued using targeted expression of the H₂O₂; enzymatic complex DUOX2/DUOXA2. The proportion of functional thyroid follicles necessary to recover a healthy thyroid function will be evaluated in triple transgenic animals (3TA: DUOXA^-/-/Tet:D2DA2^+/-/ TG:CreER^+/-) following doxycycline (DOX) induction and intraperitoneal injection with reduced doses of tamoxifen (TAM) to reactivate a restricted number of TFCs. Moreover, the proportion of active TFCs required in a follicle for TH synthesis will be evaluated by combining TH staining with RNAscope detection of the DUOX2 transcript as a marker of CreER^T2-mediated recombination.

Results: To evaluate how quickly TH synthesis resumes in 10 mg TAM-injected 3TA, thyroids have been collected 0 to 7 days after DOX induction. Four days after T4 withdrawal, reduction of the colloidal lumen was clearly observed in DUOXA^-/-/ Tet:D2DA2^+/- animals (2TA) and further enhanced at Day-7. In contrast, a preserved follicular structure was present in 3TA. Despite maximum induction of transgenes 2 days after DOX initiation, 3TA as well as 2TA showed elevation of TSH-dependent genes at Day-3. Meanwhile, iodinated thyroglobulin (TGI) starts to be detected in 3TA in a limited number of small follicles mainly localized in the center of the thyroid tissue. 24h later, staining of positive TGI increased to 89% of thyroid follicles together with a significant reduction of Nis, Tpo and TSHr expression. At Day-7, 95% of thyroid follicles were TGI positive. Next, animals were injected with various doses of TAM (10 to 0.1 mg) before DOX supplementation during 1 week. The vast majority of thyroid follicles were functional (TGI⁺) in 3TA injected with TAM from 10 to 0.5 mg. In contrast, injection with 0.25 or 0.1 mg of TAM resulted in hypothyroid phenotype with Nis, Tpo and TSHr overexpression and altered thyroid follicular structures. Concomitantly, these mice presented a clear reduction of TGI⁺ follicles below 50%. Finally, detection of the DUOX2 transcript confirmed the reduced number of recombined TFCs upon restricted TAM injection, but revealed that a very few number of active TFC would be sufficient to reconstitute a functional thyroid follicle.

Conclusions: These preliminary experiments clearly demonstrated that iodination of TG resumes very rapidly, as fast as 24h, after the restoration of the TFC function. Moreover, these data suggested that more than 80% of active follicles are required to support healthy thyroid function, but this could be achieved via a more limited proportion of active TFC suitable for TH synthesis.