Puberty marks the transition to reproductive competence and is driven by activation of the hypothalamo/pituitary/gonadal axis, culminating in first ovulation in females. In mice, puberty onset is commonly inferred from vaginal opening and estrous cyclicity, but the precise timing of first ovulation remains unclear. Here, we aimed to define reliable parameters of functional puberty in female C57BL/6J mice by integrating external, endocrine, and morphological measures. Vaginal opening and daily vaginal cytology were combined with daily serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) measurements and ovarian histology using the Pubertal Ovarian Maturation Score (Pub-Score), which estimates ovulation based on follicular development and corpus luteum morphology. Notably, we observed substantial inter-individual variability in puberty-related events, and no link between estrous cytology and first ovulation. Pub-Score analysis indicated that first ovulation occurred between 36 and 54 days postnatal, with a mean around 44 days, with no correlation with vaginal cytology. LH surges were detected in some of the females that had ovulated and showed limited temporal correspondence with vaginal cytology or Pub-Score estimates. Together, these findings demonstrate that puberty in female mice is a gradual, asynchronous process and that external or cytological markers alone are insufficient to define functional reproductive maturity.

STUDY QUESTION What is the effect of attenuating the physiological hypergonadotropic activity encountered at minipuberty on female reproductive function in a mouse model? SUMMARY ANSWER Decreasing the surge of gonadotropins at minipuberty extended reproductive lifespan, coinciding with alterations in neuroendocrine and ovarian aging. WHAT IS KNOWN ALREADY Minipuberty is characterized by the tremendous activation of the gonadotrope axis, as evidenced by elevated levels of gonadotropins regulating folliculogenesis and the synthesis of ovarian hormones, but its role in fertility remains unclear. STUDY DESIGN, SIZE, DURATION To determine the link between gonadotrope axis activity at minipuberty and reproductive parameters, we used a pharmacological approach to suppress gonadotropin levels in Swiss mice by injecting daily a GnRH receptor antagonist (GnRHR) (Ganirelix, 10 mu g/mouse) or its vehicle between 10 and 16 postnatal days, to cover the entire duration of minipuberty. We analyzed the onset of puberty and estrous cyclicity as well as fertility in young (3-5 months) and middle-aged (11 months) mice from control (CTR) and antagonist-treated groups (n = 17-20 mice/age and treatment group). Ovaries and brains were collected, fixed, and sectioned (for histology, follicle count, and immunohistochemistry) or frozen (for analysis of follicular markers, aging, and inflammation) from adult females, and blood was collected by cardiac puncture for hormonal assays (n = 3-8 mice/age and treatment group). PARTICIPANTS/MATERIALS, SETTING, METHODS To analyze the initiation of puberty, we monitored vaginal opening and performed vaginal smears in CTR and antagonist-treated mice. We studied estrous cyclicity on vaginal smears at the beginning of reproductive life. Mice were mated several times with males to assess fertility rates, delay of conception, and litter size. To evaluate ovarian function, we counted follicles at different stages and corpora lutea, and we determined the relative intra-ovarian abundance of key follicular markers by real-time RT-PCR, as well as the levels of circulating anti-M & uuml;llerian hormone (AMH) and progesterone by ELISA and GC-MS, respectively. We also analyzed features of ovarian aging and inflammation by histology and by measuring the relative intra-ovarian abundance of some markers using real-time RT-PCR. To determine the impact on neuroendocrine determinants related to the CTR of reproduction, we analyzed circulating gonadotropin levels using Luminex assays as well as kisspeptin and GnRH immunoreactivity in the hypothalamus by immunohistochemistry. MAIN RESULTS AND THE ROLE OF CHANCE Our results show that the treatment had no impact on the initiation of puberty, estrous cyclicity, or fertility at the beginning of reproductive life. However, it increased reproductive lifespan, as shown by the higher percentage of antagonist-treated females than CTRs still fertile at 11 months of age (33% versus 6%; P = 0.0471). There were no significant differences in the number of kisspeptin and GnRH neurons, nor in the density of kisspeptin- and GnRH-immunoreactive neurons in the hypothalamic areas involved in reproduction between the two groups of mice studied at either 4 or 11 months. In addition, basal levels of FSH were comparable between the two groups at 4 and 11 months, but not those of LH at 11 months which were much lower in females treated with antagonist than in their age-matched CTRs (237 +/- 59.6 pg/ml in antagonist-treated females versus 1027 +/- 226.3 pg/ml in CTRs, P = 0.0069). Importantly, at this age, antagonist-treated mice had basal LH levels comparable to young mice (e.g. in 4-month-old CTRs: 294 +/- 71.75 pg/ml, P > 0.05). Despite their prolonged reproductive lifespan and delayed neuroendocrine aging, antagonist-treated mice exhibited earlier depletion of their follicles, as shown by lower numbers of primordial, primary, and preantral follicles associated with lower circulating AMH levels and relative intra-ovarian abundance of Amh transcripts than CTR mice. However, they exhibited comparable completion of folliculogenesis, as suggested by the numbers of antral follicles and corpora lutea, relative intra-ovarian abundance of Cyp19a1, Inhba, and Inhbb transcripts, and circulating progesterone levels that all remained similar to those of the CTR group. These observed alterations in ovarian function were not associated with increased ovarian aging or inflammation. LARGE-SCALE DATA None. LIMITATIONS, REASONS FOR CAUTION This study was carried out on mice, which is a validated research model. However, human research is needed for further validation. WIDER IMPLICATIONS OF THE FINDINGS This study, which is the first to investigate the physiological role of minipuberty on reproductive parameters, supports the idea that suppressing the high postnatal levels of gonadotropins may have long-term effects on female fertility by extending the duration of reproductive life. Perturbations in gonadotropin levels during this period of life, such as those observed in infants born prematurely, may thus have profound consequences on late reproductive functions. STUDY FUNDING/COMPETING INTEREST(S) This research was conducted with the financial support of ANR AAPG2020 (ReproFUN), CNRS, Inserm, Universit & eacute; Paris Cit & eacute;, and Sorbonne Universit & eacute;. The authors declare that they have no conflicts of interest.

Study question: What is the role of the physiological hypergonadotropic activity encountered at minipuberty on the implementation of female reproductive function, in a mouse model with manipulated minipubertal gonadotropin levels? Summary answer: Elevated minipubertal levels of gonadotropins may have long-term effects on fertility by mediating neuroendocrine aging and ovarian follicle depletion. What is known already: Minipuberty is characterized by the tremendous activation of the gonadotropin axis, as evidenced by elevated levels of gonadotropins regulating folliculogenesis as well as the synthesis of ovarian hormones including estradiol, testosterone, and AMH. Study design, size, duration: To determine whether hypergonadotropic activity of the gonadotropin axis at mini-puberty could impact reproductive parameters and female fertility, we used a pharmacological approach to suppress gonadotropin levels in Swiss mice by injecting daily a GnRH receptor antagonist (GnRHR) (Ganirelix, 10 ug/mouse) or its vehicle between 10 and 16 postnatal days, to cover the entire duration of minipuberty. We analyzed the onset of puberty and estrous cyclicity as well as fertility in young (3 to 5 months) and middle-aged (11 months) mice from control (CTR) and antagonist-treated groups (n = 17 to 20 mice/age and treatment group). Ovaries and brains were collected, fixed and sectioned (for histology, follicle count and immunohistochemistry) or frozen (for analysis of follicular markers, aging and inflammation) from adult females, and blood was collected by cardiac puncture for hormonal assays (n = 3 to 8 mice/age and treatment group). Participants/materials, setting, methods: To analyze the initiation of puberty, we monitored vaginal opening and performed vaginal smears to detect first estrus and diestrus 2 in control and antagonist-treated mice. We studied estrous cyclicity on vaginal smears to detect the occurrence of the different stages of the cycle at the beginning of reproductive life. Young and middle-aged mice of the two groups were mated several times with males to assess fertility rates, delay of conception and litter size. To evaluate ovarian function, we counted follicles at the primordial, primary, secondary and tertiary stages and corpora lutea by morphometric analyses, and we determined the relative intra-ovarian abundance of follicular markers (Amh, Inhba, Inhbb, Cyp19a1, Lhcgr, Fshr) by real-time RT-PCR, as well as the levels of circulating AMH and progesterone by ELISA and GC/MS, respectively. We also analyzed features of ovarian aging and inflammation (presence of oocyte-depleted follicles and multinucleated giant cells) by histology and by measuring the relative intra-ovarian abundance of Sirt1, Sod2, Tnfa and Il1b using real-time RT-PCR. To determine the impact on neuroendocrine determinants related to the control of reproduction, we analyzed circulating gonadotropin levels using Luminex assays as well as kisspeptin and GnRH immunoreactivity by immunohistochemistry in the hypothalamus, in both young and middle-aged mice. Main results and the role of chance: Our results show that the treatment had no impact on the initiation of puberty, estrous cyclicity, or fertility at the beginning of reproductive life. However, it increased reproductive lifespan, as shown by the higher percentage of antagonist-treated females than controls (33% versus 6%) still fertile at 11 months of age (P=0.0471). There were no significant differences in the number of kisspeptin and GnRH neurons, nor in the density of kisspeptin- and GnRH-immunoreactivity in the hypothalamic areas involved in reproduction between the two groups of mice studied at either 4 or 11 months. In addition, basal levels of LH and FSH were comparable between the two groups at 4 months, but not those of LH at 11 months which were much lower in females treated with antagonist than in their age-matched controls (237 +/- 59.60 pg/mL in antagonist-treated females versus 1027 +/- 226.3 pg/mL in controls, P=0.0069). Importantly, at this age, antagonist-treated mice had basal LH levels comparable to young mice (e.g., in 4-month-old controls: 294 +/- 71.75 pg/mL, P > 0.05), while those of control females were higher (P= 0.0091). Despite their prolonged reproductive lifespan and delayed neuroendocrine aging, antagonist-treated mice exhibited earlier depletion of their follicles, as shown by lower numbers of primordial, primary, and secondary follicles associated with lower circulating AMH levels and relative intra-ovarian abundance of Amh transcripts than control mice. However, they exhibited comparable completion of folliculogenesis, as suggested by the numbers of tertiary follicles and corpora lutea, relative intra-ovarian abundance of Cyp19a1, Inha and Inhb transcripts, and circulating progesterone levels that all remained similar to those of the control group. These observed alterations in ovarian function were not associated with increased ovarian aging or inflammation. Large scale data: none Limitations, reasons for caution: This study was carried out on mice, which is a validated research model. However, human research is needed for further validation. Wider implications of the findings: This study, which is the first to investigate the physiological role of minipuberty on reproductive parameters, supports the idea that high postnatal levels of gonadotropins may have long-term effects on female fertility by regulating the duration of reproductive life. Changes in gonadotropin levels during this period of life, such as those observed in infants born prematurely, may thus have profound consequences on late reproductive functions. ### Competing Interest Statement The authors have declared no competing interest.

Abstract Disclosure: M. Chester: None. M. Devillers: None. L. Naule: None. F. Souaré: None. R. Corre: None. D. Quintas: None. C. Petrovic: None. J. Cohen-Tannoudji: None. S. Mhaouty-Kodja: None. C.J. Guigon: None. After birth, there is a transient activation of the hypothalamic-pituitary-ovary (HPO) axis, with high secretion of LH and FSH acting on the ovary to promote the secretion of sex steroids and in particular that of estradiol (E2). This period, called mini-puberty, occurs between 10-17 days postnatal (dpn) in mice. During reproductive life, E2 plays an important role on the HPO axis by regulating GnRH pulsatility and, thus, the cyclic fluctuations of gonadotropin hormone levels required for follicular development and ovulation. E2 acts on hypothalamic kisspeptin neurons of the arcuate (ARC) and the anteroventral periventricular nucleus (AVPV), which project to GnRH neurons and regulate their activity by negative and positive feedbacks, respectively. The possibility that E2 contributes, during mini-puberty, to the maturation of hypothalamic structures involved in puberty onset and reproductive function, has never been investigated. The aim of this study was to elucidate the putative roles of mini-puberty on reproduction. We developed a mouse model injected with a GnRH receptor antagonist (GonadoSTOP mice) suppressing LH and FSH surges to cause a 50% decrease in E2 levels during mini-puberty. Our results show that GonadoSTOP mice exhibited later first diestrus 2 (D2) (31.0 dpn versus 29.9 dpn in CTR; n=55-56 mice/group; P=0.0285), suggesting a possible delay in the timing of puberty. During reproductive life, GonadoSTOP mice spent a longer % of time in D2 than control mice, with no alteration in total estrous cycle time (64.4% versus 56% in CTR; n=18-19 mice/group; P=0.0350). Immunohistochemical analysis of kisspeptin neurons in ARC and AVPV of ovariectomized and hormonally-primed mice showed that their numbers were significantly lower in AVPV from GonadoSTOP mice than in control mice (n=5-6 brains/group; P=0.038). In fertility studies, we observed no difference in the time to conception or % of pregnant mice between the two groups at early reproductive life (3, 4 and 5 months). Contrary to these results, at the time of reproductive decline in control mice (11 months), a substantial % of GonadoSTOP mice could still be pregnant (47% versus 18% of CTR mice; P=0.0017). At this age, GonadoSTOP mice had lower LH levels than control mice (P=0.0074), but similar levels to young control mice at 4 months (P>0.05). RT-qPCR analyses of markers reflecting ovarian activity (Cyp19a1, Inhbb, Amh, Gdf9) at 4 and 11 months revealed that ovarian aging occurred similarly in both groups, suggesting that reproductive longevity in GonadoSTOP mice does not have an ovarian origin. Taken together these data suggest that transient and sustained activation of the HPO axis at mini-puberty may regulate key aspects of reproductive function with long-term impact on fertility, possibly by acting on the hypothalamus. Presentation: Friday, June 16, 2023

Estradiol (E2) is a major hormone-controlling folliculogenesis whose dysfunction may participate in polycystic ovary syndrome (PCOS) infertility. To determine whether both the concentration and action of E2 could be impaired in non-hyperandrogenic overweight PCOS women, we isolated granulosa cells (GCs) and follicular fluid (FF) from follicles of women undergoing ovarian stimulation (27 with PCOS, and 54 without PCOS). An analysis of the transcript abundance of 16 genes in GCs showed that androgen and progesterone receptor expressions were significantly increased in GCs of PCOS (by 2.7-fold and 1.5-fold, respectively), while those of the steroidogenic enzymes CYP11A1 and HSD3B2 were down-regulated (by 56% and 38%, respectively). Remarkably, treatment of GC cultures with E2 revealed its ineffectiveness in regulating the expression of several key endocrine genes (e.g., GREB1 or BCL2) in PCOS. Additionally, a comparison of the steroid concentrations (measured by GC/MS) in GCs with those in FF of matched follicles demonstrated that the significant decline in the E2 concentration (by 23%) in PCOS FF was not the result of the E2 biosynthesis reduction. Overall, our study provides novel hallmarks of PCOS by highlighting the ineffective E2 signaling in GCs as well as the dysregulation in the expression of genes involved in follicular growth, which may contribute to aberrant folliculogenesis in non-hyperandrogenic women with PCOS.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

In females, androgens contribute to ovarian diseases such as polycystic ovarian syndrome (PCOS), but their action is also crucial for ovarian physiology, i.e., follicular growth and estradiol (E2) synthesis during reproductive life, in interaction with the gonadotropins LH and FSH. However, it is unclear whether androgens already play a role in the ovary at mini-puberty, a phase of postnatal development with active follicular growth and high E2 levels. Therefore, we analyzed the potential actions of androgens on the ovary and their possible interaction with gonadotropins during this period in mice. We used molecular-based studies and pharmacological approaches in vivo and on cultured ovaries. We found that mini-pubertal ovaries produce significant amounts of testosterone and display androgen receptor (AR) expression in growing follicles, both under the control of LH. By blocking AR signaling either in vivo or in ovarian cultures, we found that this pathway may participate in the regulation of prepubertal E2 synthesis and follicular growth, possibly by regulating the expression of a number of key intra-ovarian regulators, including FSH receptor (Fshr), the aromatase enzyme converting androgens into estrogens (Cyp19a1) and the cell cycle inhibitor p27KIP1 (Cdkn1b). We further showed that AR may stimulate FSH-mediated regulation of Cyp19a1 through its action on Fshr mRNA abundance. Overall, this work supports the idea that AR signaling is already activated in mini-pubertal ovaries to regulate E2 synthesis and follicular growth, at the interplay with LH and FSH signaling. Its early action may, thus, contribute to the implementation of early ovarian function with possible impacts on reproductive function.

Abstract Disclosure: M. Devillers: None. C.M. François: None. M. Chester: None. R. Corre: None. V. Cluzet: None. F. Giton: None. J. Cohen-Tannoudji: None. C.J. Guigon: None. Introduction: In females, androgens contribute to ovarian diseases such as polycystic ovary syndrome (PCOS), but their action is also crucial for ovarian physiology, i.e., follicular growth and estradiol (E2) synthesis during reproductive life, in interaction with the gonadotropins LH and FSH. However, it is unclear whether androgens already play a role in the ovary at mini-puberty, a phase of postnatal development with active follicular growth and high E2 levels. Hypothesis/question: Given their role in the adult ovary, we hypothesized that androgens may regulate early ovarian function at mini-puberty. Therefore, we analyzed their potential actions on the ovary and their possible interaction with gonadotropins during this period. Methods: We used the mouse as an experimental model to measure the intra-ovarian content and serum levels of testosterone by gas chromatography-mass spectrometry (GC-MS) at different prepubertal ages, including mini-puberty. We performed molecular-based studies (immunohistochemistry, RT-qPCR), morphometric studies and pharmacological approaches in vivo and in cultured ovaries. Main results: We found that mini-pubertal ovaries produce significant amounts of testosterone and display androgen receptor (AR) expression in growing follicles, both under the control of LH. By blocking AR signaling with flutamide either in vivo or in ovarian cultures, we found that this pathway may participate in the regulation of prepubertal E2 synthesis and follicular growth, possibly by regulating the expression of a number of key intra-ovarian regulators, including FSH receptor (Fshr), the aromatase enzyme converting androgens into estrogens (Cyp19a1) and the cell cycle inhibitor p27KIP1 (Cdkn1b). We further showed that AR may stimulate FSH-mediated regulation of Cyp19a1 through its action on Fshr mRNA abundance. Conclusion: This work supports the idea that AR signaling is already activated in mini-pubertal ovaries to regulate E2 synthesis and follicular growth, in interaction with LH and FSH signaling. This early physiological action of androgens may contribute to the implementation of early ovarian function with possible impacts on reproductive function. Presentation: Friday, June 16, 2023

The DNA polymerase zeta (Polζ) plays a critical role in bypassing DNA damage. REV3L, the catalytic subunit of Polζ, is also essential in mouse embryonic development and cell proliferation for reasons that remain incompletely understood. In this study, we reveal that REV3L protein interacts with heterochromatin components including repressive histone marks and localizes in pericentromeric regions through direct interaction with HP1 dimer. We demonstrate that Polζ/REV3L ensures progression of replication forks through difficult‐to‐replicate pericentromeric heterochromatin, thereby preventing spontaneous chromosome break formation. We also find that Rev3l‐deficient cells are compromised in the repair of heterochromatin‐associated double‐stranded breaks, eliciting deletions in late‐replicating regions. Lack of REV3L leads to further consequences that may be ascribed to heterochromatin replication and repair‐associated functions of Polζ, with a disruption of the temporal replication program at specific loci. This is correlated with changes in epigenetic landscape and transcriptional control of developmentally regulated genes. These results reveal a new function of Polζ in preventing chromosome instability during replication of heterochromatic regions. Translesion synthesis DNA polymerase ζ (Polζ), with its catalytic subunit REV3L, is known to also limit DNA breaks in proliferating mammalian genomes, but specific consequences for replication and repair have remained unknown. This work demonstrates specific REV3L functions in limiting breaks and structural variations in heterochromatin. HP1‐mediated targeting of REVL3, the catalytic subunit of translesion synthesis Polζ, influences replication fork progression and epigenetic and transcriptional landscapes of developmentally‐regulated genes.

Interstitial lung fibroblast activation coupled with extracellular matrix production is a pathological signature of pulmonary fibrosis, and is governed by transforming growth factor (TGF)-β1/Smad signalling. TGF-β1 and oxidative stress cooperate to drive fibrosis. Cells can produce reactive oxygen species through activation and/or induction of NADPH oxidases, such as dual oxidase (DUOX1/2). Since DUOX enzymes, as extracellular hydrogen peroxide (H2O2--)-generating systems, are involved in extracellular matrix formation and in wound healing in different experimental models, we hypothesised that DUOX-based NADPH oxidase plays a role in the pathophysiology of pulmonary fibrosis.Our in vivo data (idiopathic pulmonary fibrosis patients and mouse models of lung fibrosis) showed that the NADPH oxidase DUOX1 is induced in response to lung injury. DUOX1-deficient mice (DUOX1+/- and DUOX1-/-) had an attenuated fibrotic phenotype. In addition to being highly expressed at the epithelial surface of airways, DUOX1 appears to be well expressed in the fibroblastic foci of remodelled lungs. By using primary human and mouse lung fibroblasts, we showed that TGF-β1 upregulates DUOX1 and its maturation factor DUOXA1 and that DUOX1-derived H2O2 promoted the duration of TGF-β1-activated Smad3 phosphorylation by preventing phospho-Smad3 degradation. Analysis of the mechanism revealed that DUOX1 inhibited the interaction between phospho-Smad3 and the ubiquitin ligase NEDD4L, preventing NEDD4L-mediated ubiquitination of phospho-Smad3 and its targeting for degradation.These findings highlight a role for DUOX1-derived H2O2 in a positive feedback that amplifies the signalling output of the TGF-β1 pathway and identify DUOX1 as a new therapeutic target in pulmonary fibrosis.

Toxic effects of nanoparticles on female reproductive health have been documented but the underlying mechanisms still need to be clarified. Here, we investigated the effect of carbon black nanoparticles (CB NPs) on the pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which are key regulators of gonadal gametogenesis and steroidogenesis. To that purpose, we subjected adult female mice to a weekly non-surgical intratracheal administration of CB NPs at an occupationally relevant dose over 4 weeks. We also analyzed the effects of CB NPs in vitro, using both primary cultures of pituitary cells and the LβT2 gonadotrope cell line. We report here that exposure to CB NPs does not disrupt estrous cyclicity but increases both circulating FSH levels and pituitary FSH β-subunit gene (Fshb) expression in female mice without altering circulating LH levels. Similarly, treatment of anterior pituitary or gonadotrope LβT2 cells with increasing concentrations of CB NPs dose-dependently up-regulates FSH but not LH gene expression or release. Moreover, CB NPs enhance the stimulatory effect of GnRH on Fshb expression in LβT2 cells without interfering with LH regulation. We provide evidence that CB NPs are internalized by LβT2 cells and rapidly activate the cAMP/PKA pathway. We further show that pharmacological inhibition of PKA significantly attenuates the stimulatory effect of CB NPs on Fshb expression. Altogether, our study demonstrates that exposure to CB NPs alters FSH but not LH expression and may thus lead to gonadotropin imbalance.

Background Macrophages play pivotal roles in tumor progression and the response to anticancer therapies, including radiotherapy (RT). Dual oxidase (DUOX) 1 is a transmembrane enzyme that plays a critical role in oxidant generation. Methods Since we found DUOX1 expression in macrophages from human lung samples exposed to ionizing radiation, we aimed to assess the involvement of DUOX1 in macrophage activation and the role of these macrophages in tumor development. Results Using Duox1 −/− mice, we demonstrated that the lack of DUOX1 in proinflammatory macrophages improved the antitumor effect of these cells. Furthermore, intratumoral injection of Duox1 −/− proinflammatory macrophages significantly enhanced the antitumor effect of RT. Mechanistically, DUOX1 deficiency increased the production of proinflammatory cytokines (IFNγ, CXCL9, CCL3 and TNFα) by activated macrophages in vitro and the expression of major histocompatibility complex class II in the membranes of macrophages. We also demonstrated that DUOX1 was involved in the phagocytotic function of macrophages in vitro and in vivo . The antitumor effect of Duox1 −/− macrophages was associated with a significant increase in IFNγ production by both lymphoid and myeloid immune cells. Conclusions Our data indicate that DUOX1 is a new target for macrophage reprogramming and suggest that DUOX1 inhibition in macrophages combined with RT is a new therapeutic strategy for the management of cancers.

A 59-year-old woman with locally invasive poorly differentiated thyroid cancer with synchronous lung, mediastinal, and bone metastases and a somatic BRAFK601E mutation with contraindication for antiangiogenic drugs was treated with dabrafenib and trametinib. During treatment, serum levels of thyroglobulin increased as early as day 7 up to 10-fold over baseline at week 4. Concurrently, clinical hyperthyroidism occurred, with free triiodothyronine and free thyroxine levels increasing to 6.6 and 4.4 times their upper reference limit. Fludeoxyglucose positron emission tomography/computed tomography at one and two months after treatment initiation showed a PERCIST metabolic response with a 82% decrease in fludeoxyglucose uptake, whereas disease remained morphologically stable according to RECIST criteria. A diagnostic radioactive iodine whole-body scan performed when the patient was thyrotoxic with an undetectable serum thyrotropin level, in the absence of any exogenous thyrotropin stimulation, showed high radioactive iodine uptake in the lung, mediastinum, and skull metastases. A biopsy performed two months after treatment initiation showed a more differentiated growth pattern and a decrease in the mitotic activity compared to baseline. An increase of thyroglobulin and thyroid peroxidase was observed at both the protein and mRNA levels. Sodium-iodide symporter mRNA expression increased by >750 times over its initial level, and sodium-iodide symporter protein expression became detectable under treatment. A decrease in general status due to thyrotoxicosis led to treatment discontinuation. Thyrotoxicosis resolved rapidly and radioactive iodine uptake decreased by >90%. This clinical case shows that redifferentiation itself is not necessarily associated with an antitumor effect.

We have developed new benign palladium nanoparticles able to catalyze the Suzuki-Miyaura cross-coupling reaction on human thyroglobulin (Tg), a naturally iodinated protein produced by the thyroid gland, in homogenates from patients' tissues. This represents the first example of a chemoselective native protein modification using transition metal nanoobjects in near-organ medium.

Background: Dual oxidases (DUOX1 and DUOX2) were initially identified as H2O2 sources involved in thyroid hormone synthesis. Congenital hypothyroidism (CH) resulting from inactivating mutations in the DUOX2 gene highlighted that DUOX2 is the major H2O2 provider to thyroperoxidase. The role of DUOX1 in the thyroid remains unknown. A recent study suggests that it could compensate for DUOX2 deficiency in CH. Both DUOX enzymes and their respective maturation factors DUOXA1 and DUOXA2 form a stable complex at the cell surface, which is fundamental for their enzymatic activity. Recently, intra-and intermolecular disulfide bridges were identified that are essential for the structure and the function of the DUOX2-DUOXA2 complex. This study investigated the involvement of cysteine residues conserved in DUOX1 toward the formation of disulfide bridges, which could be important for the function of the DUOX1-DUOXA1 complex. Methods: To analyze the role of these cysteine residues in both the targeting and function of dual oxidase, different human DUOX1 mutants were constructed, where the cysteine residues were replaced with glycine. The effect of these mutations on cell surface expression and H2O2-generating activity of the DUOX1-DUOXA1 complex was analyzed. Results: Mutations of two cysteine residues (C118 and C1165), involved in the formation of the intramolecular disulfide bridge between the N-terminal ectodomain and one of the extracellular loops, mildly altered the function and the targeting of DUOX1, while this bridge is crucial for DUOX2 function. Unlike DUOXA2, with respect to DUOX2, the stability of the maturation factor DUOXA1 is not dependent on the oxidative folding of DUOX1. Only mutation of C579 induced a strong alteration of both targeting and function of the oxidase by preventing the covalent interaction between DUOX1 and DUOXA1. Conclusion: An intermolecular disulfide bridge rather than an intramolecular disulfide bridge is important for both the trafficking and H(2)O(2)(-)generating activity of the DUOX1-DUOXA1 complex.

Fibrillarin (Fbl) is a highly conserved protein that plays an essential role in ribosome biogenesis and more particularly in the methylation of ribosomal RNAs and rDNA histones. In cellular models, FBL was shown to play an important role in tumorigenesis and stem cell differentiation. We used the zebrafish as an in vivo model to study Fbl function during embryonic development. We show here that the optic tectum and the eye are severely affected by Fbl depletion whereas ventral regions of the brain are less impacted. The morphogenesis defects are associated with impaired neural differentiation and massive apoptosis. Polysome gradient experiments show that fbl mutant larvae display defects in ribosome biogenesis and activity. Strikingly, flow cytometry analyses revealed different S-phase profiles between wild-type and mutant cells, suggesting a defect in S-phase progression.