Animal models of enhanced fertility are rare, as most genetically modified mouse models with reproductive phenotype display subfertility or infertility. Here, we describe the ovarian phenotype of the Dummerstorf line 2 (FL2) mouse strain, which exhibits high fertility and has been selectively bred for increased fertility over more than 190 generations. This long-term selection, outbred mouse line almost doubled the litter size to 21.5 (FL2) compared with 11.3 (unselected control line, ctrl), without showing any signs of growth retardation in the offspring. Here, we show that FL2 females ovulate 25.0 oocytes per cycle compared with 13.2 in ctrl. FL2 mice remain in the estrus phase for a shorter period during a 12-day observation period. Follicle-stimulating hormone (FSH) levels are decreased, both in estrus and diestrus, compared with ctrl, whereas luteinizing hormone levels are unaffected. The mRNA expression levels in the pituitary gland correspond to the gonadotropin levels in the blood. Progesterone levels are decreased in estrus in FL2. Hypothalamic expression levels of gonadotropin-releasing hormone (GnRH) are decreased in diestrus. Holistic gene expression analysis indicates complex and differential regulation in estrus and diestrus in ovaries of FL2 compared with ctrl. In particular, genes of the TGF-β pathway (such as Bmp3, Bmp7, and Inhba) and the Wnt pathway (such as Sfrp4 and Mkrn1) are differentially expressed in ovaries of FL2 females. These data indicate that reduced activity of the hypothalamic–pituitary–gonadal axis (in particular, lower levels of GnRH, FSH, and progesterone), combined with altered gene transcription in the ovaries, leads to higher ovulation rates in order to achieve the breeding objective of improved fertility.

Selenoproteins are proteins containing the 21st proteinogenic amino acid selenocysteine in their peptide chain. Selenocysteine lends specific properties to selenoproteins, and selenoenzymes are often catalytically superior to enzymes that would contain cysteine instead. Degradation of peroxides, in particular by GPX4, the master regulator of ferroptosis, is currently receiving a lot of attention owing to its potential in cancer or degenerative disorders. In this context, selenium metabolism has recently become again an active field of research based on the importance to provide selenium to GPX4. Apart from redox control, deiodinases are being identified as critical modulators of an increasing number of specific developmental decisions in many organs where they are modulating local thyroid hormone levels. An entirely new field of selenium research is centered around the biosynthesis of ethanolamine phospholipids. Overall, skeletal muscle, the endocrine system, the immune system, and in particular the brain, are sensitive to selenoprotein deficiency. We summarize the basic concepts of selenium and selenoprotein metabolism and provide a framework where selenium or selenoproteins are important for human health. Then we review new work that significantly advanced our understanding of selenium biology in humans and summarize the substantial body of data describing patients with inborn errors of selenoprotein biosynthesis or selenoprotein genes. Transgenic mouse models are reported where they help define a biological concept. We deliberately did not strive to cover in this work all the biochemical mechanisms of selenoenzymes or their interactions with effector proteins.

Type 3 iodothyronine deiodinase (DIO3) is a membrane-associated enzyme that inactivates thyroid hormones and promotes high-grade serous ovarian cancer (HGSOC) progression. Although DIO3 undergoes continuous recycling between the plasma membrane and endosomal compartments, the mechanisms governing its intracellular routing and functional stability remain unknown. We used Human ovarian cancer cells (ES-2, OVCAR3, OVSAHO, KURAMOCHI, NCI/ADR (NAR)), ovarian cancer tumors and normal tissues from patients, immortalized fallopian tube cells (FT109, FT237 and FT282) and HEK293T cells. DIO3 was knocked-down (shRNA) or overexpressed (CRISPR activation system). Flow cytometry (cell counts and apoptosis assay), proteomics, co-immunoprecipitation, proximity ligation assay, confocal imaging in the presence of endosomal transport inhibitors, and AlphaFold-based structural modeling were performed. We identify sorting nexins SNX2 and SNX6 as components of the DIO3 trafficking machinery in ovarian cancer. Computer-based structural analysis provide a potential interface for SNXs association, requiring intact linker and catalytic domains of DIO3. Studies in ovarian cancer cells suggest that SNX6 is linked with DIO3 progression through endosomal and Golgi compartments, whereas SNX2 is associated with its recycling to the plasma membrane via RAB11-positive vesicles. Disruption of these pathways destabilizes DIO3-SNXs colocalization and selectively impairs proliferation and survival of DIO3-overexpressing cells. These findings establish intracellular trafficking as a fundamental determinant of DIO3 stability and function and uncover sorting nexin-associated routing as a previously unrecognized dependency and therapeutic vulnerability in ovarian cancer cells.

PURPOSE:Protein-altering gene variants in SEPSECS disrupt the biosynthesis of selenoproteins, leading to a spectrum of neurological diseases. METHODS:We studied 27 individuals with biallelic SEPSECS variants, identifying 13 unreported gene variants. To better understand and diagnose the disorder, broad biochemical correlation of neurological symptoms, focused metabolomics, and structural and in vitro activity analyses were deployed. RESULTS:Our results suggest three general clinical courses: (i) severe early-onset with cerebellar or cerebral atrophy, (ii) milder early-onset with gradual deterioration, and (iii) late-onset, mild disease. SEPSECS variants primarily affect the brain. In only one individual out of eight, thyroid hormone measurements suggested a defect of T4 to T3 conversion. Accompanied increase in glutathione and sulfur metabolites in plasma indicates elevated oxidative stress. Variants mapping to conserved N- and C-termini and catalytic site elicit SEPSECS misfolding, aggregation, thermal instability, and loss of function, that could ultimately lead to ferroptosis of neurons and perhaps oligodendrocytes. For differential diagnosis and monitoring therapeutic attempts, we recommend measuring levels of plasma selenium, glutathione and sulfur metabolites, GPX activity, and SELENOP. Given the pontine involvement in less than half of the cases, we suggest renaming the syndrome from PCH2D to SEPSECS-related neurodevelopmental disorder. CONCLUSION:Our study expands the understanding of SEPSECS-related neurodevelopmental disorders, highlighting the need for updated diagnostic criteria and potential treatment strategies.

ABSTRACT Selenoproteins are a specialised group of proteins that incorporate selenium, an essential micronutrient, in the form of selenocysteine. The SECIS binding protein 2 (SBP2), encoded by SECISBP2 , is a crucial component of the selenocysteine incorporation machinery. SECISBP2 deficiency compromises selenoprotein synthesis, and its knockout causes embryonic lethality in mice. Selenium is critical to cardiac function, and nutritional deficiency causes Keshan disease, a progressive cardiomyopathy. Biallelic variants in SECISBP2 cause pleiotropic phenotypes including abnormal thyroid hormone metabolism, neurodevelopmental disorders and aortic aneurysms. No reported phenotypes to date include cardiomyopathy. We report a consanguineous South Asian family with a history of perinatal deaths due to progressive cardiomyopathy and intractable arrhythmias with a rare homozygous loss-of-function splice site variant in SECISBP2 . The SECISBP2 c.1303-2A>G variant was homozygous in four affected offspring and heterozygous in the parents. One child without cardiac disease did not carry this variant. RNA sequencing confirmed that almost all transcripts would undergo nonsense-mediated decay. Further, we observed a pronounced decrease in GPX1 and SELENOH selenoprotein mRNA, as well as a large decrease in SELENOH, GPX1, GPX3 and GPX4 cardiac protein abundance in homozygotes, a molecular hallmark of SECISBP2 deficiency. Notably, we observed a >12-fold decrease in cardiac GPX4, a key selenoprotein that suppresses lipid peroxidation and ferroptosis, suggesting a possible ferroptosis-mediated mechanism for heart failure. Our analysis suggests that c.1303-2A>G is likely the most damaging homozygous variant discovered to date. For the first time, we show that SECISBP2 is essential for human life, with almost complete loss-of-function causing a lethal perinatal cardiomyopathy characterised by pronounced cardiac selenoprotein loss.

Vitamin K 2,3-epoxide reductase complex subunit 1-like 1 (VKORC1L1) catalyzes the reduction of vitamin K within the endoplasmic reticulum (ER) and has recently been shown to suppress ferroptosis. However, its physiological role remains largely unknown. Here, we show that Vkorc1l1-/- mice spontaneously developed progressive stages of steatotic liver disease (SLD) on a chow diet, independent of metabolic dysfunction. Mechanistically, despite the loss of VKORC1L1-mediated ferroptosis suppression, Vkorc1l1-/- mice activated compensatory NRF2-driven antioxidant responses and enhanced hepatic triacylglycerol (TAG) synthesis. These adaptations promoted the sequestration of polyunsaturated fatty acids into lipid droplets (LDs), thereby limiting ferroptosis-induced hepatic injury. Furthermore, metabolism of Vkorc1l1-/- mice was reprogrammed to a chronic fasting-like phenotype characterized by with enhanced mitochondrial function. Notably, the radical-trapping antioxidant menaquinone-4, but not phylloquinone, both of which are substrates of VKORC1L1, decreased lipid droplet number, whereas inhibition of VKORC1L1 through warfarin enhanced LD biogenesis in HepG2 cells. In humans, a common VKORC1L1 risk haplotype was associated with reduced hepatic VKORC1L1 expression and with increased risk of metabolic dysfunction-associated steatotic liver disease in the UK Biobank and alcohol-associated liver cirrhosis in case–control cohorts. This work identifies VKORC1L1 as a key regulator of SLD pathogenesis by orchestrating metabolic rewiring to maintain redox homeostasis and suppress ferroptosis in vivo.

Selenocysteine (Sec) metabolism is crucial for cellular function and ferroptosis prevention and begins with the uptake of the Sec carrier, selenoprotein P (SELENOP). Following uptake, Sec released from SELENOP is metabolized via selenocysteine lyase (SCLY), producing selenide, a substrate for selenophosphate synthetase 2 (SEPHS2), which provides the essential selenium donor, selenophosphate (H2SePO3-), for the biosynthesis of the Sec-tRNA. Here, we discovered an alternative pathway in Sec metabolism mediated by peroxiredoxin 6 (PRDX6), independent of SCLY. Mechanistically, we demonstrate that PRDX6 can readily react with selenide and interact with SEPHS2, potentially acting as a selenium delivery system. Moreover, we demonstrate the functional significance of this alternative route in human cancer cells, revealing a notable association between elevated expression of PRDX6 and human MYCN-amplified neuroblastoma subtype. Our study sheds light on a previously unrecognized aspect of Sec metabolism and its implications in ferroptosis, offering further possibilities for therapeutic exploitation.

Inborn errors of selenoprotein expression arise from deleterious variants in genes encoding selenoproteins or selenoprotein biosynthetic factors, some of which are associated with neurodegenerative disorders. This study shows that bi-allelic selenocysteine tRNA-specific eukaryotic elongation factor (EEFSEC) variants cause selenoprotein deficiency, leading to progressive neurodegeneration. EEFSEC deficiency, an autosomal recessive disorder, manifests with global developmental delay, progressive spasticity, ataxia, and seizures. Cerebral MRI primarily demonstrated a cerebellar pathology, including hypoplasia and progressive atrophy. Exome or genome sequencing identified six different bi-allelic EEFSEC variants in nine individuals from eight unrelated families. These variants showed reduced EEFSEC function in vitro, leading to lower levels of selenoproteins in fibroblasts. In line with the clinical phenotype, an eEFSec-RNAi Drosophila model displays progressive impairment of motor function, which is reflected in the synaptic defects in this model organisms. This study identifies EEFSEC deficiency as an inborn error of selenocysteine metabolism. It reveals the pathophysiological mechanisms of neurodegeneration linked to selenoprotein metabolism, suggesting potential targeted therapies.

Objective:The monocarboxylate transporter (MCT) 8 is a specific transporter for thyroid hormones. Pathogenic variants in MCT8 lead to a severe psychomotor disorder called MCT8 deficiency. A recently published patient carries a MCT8V235 to leucine substitution that was incapable of T3 transport. Analyses of our MCT8 homology model predicted steric clashes between Leu235 and Phe285 as well as Gln288, possibly affecting another transport-sensitive phenylalanine at position 287. Methods:We analyzed the occurrence of potential van der Waals (VDW) interactions between Leu235 and Phe285 as well as Gln288 in the homology model. We overexpressed MCT8V235 and MCT8F287 mutants with altered side-chain properties in cells to assess their role in T3 transport function. In addition, we created an MCT8V235L,F285A double mutant. Results:Mutations of MCT8V235 to alanine, threonine or isoleucine, as well as the analysis of potential VDW interactions, helped us to identify Phe285, but not Gln288, as the amino acid responsible for the inactivity of MCT8V235L. The hypothesis was supported by activity measurements of an MCT8V235L,F285A double mutant that showed rescued T3 transport with KM values similar to wild-type MCT8. The analyses of MCT8F287 mutated to tyrosine, tryptophan and valine revealed that the size and/or the aromatic properties of the amino acid side chain are crucial for proper membrane expression and T3 transport. Conclusion:We were able to restore transport activity of MCT8V235L by introducing a second mutation (MCT8V235L,F285A). We speculate that the additional mutation prevents a shift of Phe287 into the potential transport cavity, eventually restoring T3 transport.

CONTEXT:Monocarboxylate transporter (MCT) 8 facilitates thyroid hormone (TH) transport across the blood-brain barrier. Pathogenic variants in SLC16A2 cause MCT8 deficiency (Allan-Herndon-Dudley syndrome), characterized by intellectual and motor disability and abnormal thyroid function tests. MCT8 deficiency typically affects males due to its X-linked inheritance. OBJECTIVE:Here, we report 8 female patients with heterozygous pathogenic variants in SLC16A2 who presented with variable neurocognitive impairment, behavioral problems, and TH function abnormalities. METHODS:We performed X-chromosome inactivation studies in female patients in whom heterozygous pathogenic variants in SLC16A2 were identified. The effect of SLC16A2 variants on TH transport was assessed in transfected cells and patient-derived fibroblasts. RESULTS:In all patients (mean age 8.6 years; range, 2.3-25 years) routine care genetic analyses identified heterozygous variants in SLC16A2 (p.(R445C), p.(N193I), p.(G276R), t(X;20), resulting in a breakpoint in intron 1, t(X;19), resulting in a breakpoint in SLC16A2, p.(I562Sfs566*), p.(G221R)). All missense variants showed substantially reduced MCT8-mediated TH uptake in transiently transfected cells. X-chromosome inactivation studies in patient cells showed skewed X-inactivation in all 7 evaluated individuals. In 5 out of 7 evaluated cases, MCT8-mediated 3,5,3'-triiodothyronine (T3) uptake in patient-derived fibroblasts was impaired to a similar degree as in fibroblasts derived from male patients with MCT8 deficiency. CONCLUSION:Female patients with heterozygous pathogenic variants in SLC16A2 and skewed X-chromosome inactivation may present with variable neuro(psycho)logical, behavioral, and thyroid function test abnormalities. Female patients presenting with neurocognitive impairment and abnormal TH function tests (low free thyroxine and/or high total T3 concentrations) should be tested for genetic variants in SLC16A2.

Selenocysteine (Sec), the 21st proteogenic amino acid, is a key component of selenoproteins, where it performs critical roles in redox reactions. Sec incorporation during translation is unique and highly sensitive to selenium levels. Encoded by the UGA codon, typically a termination signal, its insertion necessitates the presence of a selenocysteine insertion sequence (SECIS) within the 3' untranslated region (UTR) of selenoprotein mRNAs. This SECIS element orchestrates the recruitment of specialized molecular factors, including SECISBP2, the unique tRNA[Ser]Sec, and its dedicated elongation factor, EEFSEC. The extended variable arm of tRNA[Ser]Sec permits its specific recognition by EEFSEC. While the structure of the ribosome-bound complex is known, the precise mechanism by which EEFSEC-tRNA[Ser]Sec recodes UGA in the presence of SECIS and SECISBP2 remains unclear. tRNA[Ser]Sec has relatively few epitranscriptomic modifications, but those at the anticodon loop are crucial. Key modifications include N6-isopentenyladenosine (i6A) at position 37 and two forms of 5-methoxycarbonylmethyluridine (mcm5U and mcm5Um) at position 34. The ratio of these isoforms varies with tissue type and selenium levels, influencing mRNA-specific Sec recoding. A C65G mutation in the acceptor stem, identified in patients, disrupts these modifications at position 34, impairing selenoprotein synthesis. This highlights the essential role of wobble position modifications in anticodon function. tRNA[Ser]Sec exemplifies the complex regulation of UGA codon recoding and underscores the interplay of structural and epitranscriptomic factors in selenoprotein translation.

Presenilin proteins (PS1 and PS2) represent the catalytic subunit of γ-secretase and play a critical role in the generation of the amyloid β (Aβ) peptide and the pathogenesis of Alzheimer disease (AD). However, PS proteins also exert multiple functions beyond Aβ generation. In this study, we examine the individual roles of PS1 and PS2 in cellular cholesterol metabolism. Deletion of PS1 or PS2 in mouse models led to cholesterol accumulation in cerebral neurons. Cholesterol accumulation was also observed in the lysosomes of embryonic fibroblasts from Psen1-knockout (PS1-KO) and Psen2-KO (PS2-KO) mice and was associated with decreased expression of the Niemann-Pick type C1 (NPC1) protein involved in intracellular cholesterol transport in late endosomal/lysosomal compartments. Mass spectrometry and complementary biochemical analyses also revealed abnormal N-glycosylation of NPC1 and several other membrane proteins in PS1-KO and PS2-KO cells. Interestingly, pharmacological inhibition of N-glycosylation resulted in intracellular cholesterol accumulation prominently in lysosomes and decreased NPC1, thereby resembling the changes in PS1-KO and PS2-KO cells. In turn, treatment of PS1-KO and PS2-KO mouse embryonic fibroblasts (MEFs) with the chaperone inducer arimoclomol partially normalized NPC1 expression and rescued lysosomal cholesterol accumulation. Additionally, the intracellular cholesterol accumulation in PS1-KO and PS2-KO MEFs was prevented by overexpression of NPC1. Collectively, these data indicate that a loss of PS function results in impaired protein N-glycosylation, which eventually causes decreased expression of NPC1 and intracellular cholesterol accumulation. This mechanism could contribute to the neurodegeneration observed in PS KO mice and potentially to the pathogenesis of AD.

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

CONTEXT:Monocarboxylate transporter 8 (MCT8) deficiency is a rare X-chromosomal inherited disease leading to severe cognitive impairment, muscular hypotonia, and symptoms of peripheral thyrotoxicosis. Experimental approaches aiming to functionally rescue mutant MCT8 activity by the chemical chaperone phenylbutyrate (PB) demonstrated promising effects in vitro for several MCT8 missense mutations. OBJECTIVE:The objective was to evaluate biochemical and clinical effects of PB in doses equivalent to those approved for the treatment of urea cycle disorders in a boy with MCT8 deficiency due to a novel MCT8 missense mutation c.703G>T (p.V235L). RESULTS:During a treatment period of 13 months, PB led to a significant decrease of elevated thyrotropin and triiodothyronine (T3) serum concentrations, while free thyroxine (fT4) increased. The weight z-score of the toddler remained remarkably stable during the treatment period. Neurodevelopmental assessments (BSID-III) revealed a slight increase of gross motor skills from developmental age 4 to 6 months. However, increasing liver enzyme serum activities and accumulation of phenylacetate in urine led to treatment interruptions and dose alterations. In vitro analyses in MDCK1 cells confirmed the pathogenicity of MCT8 p.V235L. However, while PB increased expression of the mutant protein, it did not rescue T3 transport, suggesting a PB effect on thyroid function tests independent of restoring MCT8 activity. CONCLUSION:In a clinical attempt of PB treatment in MCT8 deficiency we observed a significant improvement of thyroid hormone function tests, tendencies toward body weight stabilization and slight neurodevelopmental improvement. Hepatotoxicity of PB may be a limiting factor in MCT8 deficiency and requires further investigation.

Iodothyronine deiodinases (Dio) are selenocysteine-containing membrane enzymes that activate and inactivate the thyroid hormones (TH) through reductive iodide eliminations. The three deiodinase isoforms are homodimers sharing highly conserved amino acid sequences, but they differ in their regioselectivities for the deiodination reaction and regulatory features. We have now solved a crystal structure of the mouse deiodinase 2 (Dio2) catalytic domain. It reveals a high overall similarity to the deiodinase 3 structure, supporting the proposed common mechanism, but also Dio2-specific features, likely mediating its unique properties. Activity studies with an artificially enforced Dio dimer further confirm that dimerization is required for activity and requires both the catalytic core and the enzyme’s N-terminus. Cross-linking studies reveal the catalytic core’s dimerization interface, providing insights into the architecture of the complete, active Dio homodimer.

Background: 3,5,3'-Triiodothyroacetic acid (TRIAC) is a T3-receptor agonist pharmacologically used in patients to mitigate T3 resistance. It is additionally explored to treat some symptoms of patients with inactivating mutations in the thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8, SLC16A2). MCT8 is expressed along the blood-brain barrier, on neurons, astrocytes, and oligodendrocytes. Hence, pathogenic variants in MCT8 limit the access of TH into and their functions within the brain. TRIAC was shown to enter the brain independently of MCT8 and to modulate expression of TH-dependent genes. The aim of the study was to identify transporters that facilitate TRIAC uptake into cells. Methods: We performed a whole-genome RNAi screen in HepG2 cells stably expressing a T3-receptor-dependent luciferase reporter gene. Validation of hits from the primary and confirmatory secondary screen involved a counter screen with siRNAs and compared the cellular response to TRIAC to the effect of T3, in order to exclude siRNAs targeting the gene expression machinery. MDCK1 cells were stably transfected with cDNA encoding C-terminally myc-tagged versions of the identified TRIAC-preferring transporters. Several individual clones were selected after immunocytochemical characterization for biochemical characterization of their 125I-TRIAC transport activities. Results: We identified SLC22A9 and SLC29A2 as transporters mediating cellular uptake of TRIAC. SLC22A9 encodes the organic anion transporter 7 (OAT7), a sodium-independent organic anion transporter expressed in the plasma membrane in brain, pituitary, liver, and other organs. Competition with the SLC22A9/OAT7 substrate estrone-3-sulfate reduced 125I-TRIAC uptake. SLC29A2 encodes the equilibrative nucleoside transporter 2 (ENT2), which is ubiquitously expressed, including pituitary and brain. Coincubation with the SLC29A2/ENT2 inhibitor nitrobenzyl-6-thioinosine reduced 125I-TRIAC uptake. Moreover, ABCD1, an ATP-dependent peroxisomal pump, was identified as a 125I-TRIAC exporter in transfected MDCK1 cells. Conclusions: Knowledge of TRIAC transporter expression patterns, also during brain development, may thus in the future help to interpret observations on TRIAC effects, as well as understand why TRIAC may not show a desirable effect on cells or organs not expressing appropriate transporters. The identification of ABCD1 highlights the sensitivity of our established screening assay, but it may not hold significant relevance for patients undergoing TRIAC treatment.

Context Monocarboxylate transporter 8 (MCT8) deficiency is a rare genetic disease that leads to severe global developmental delay. MCT8 facilitates thyroid hormone (TH) transport across the cell membrane, and the serum TH profile is characterized by high T3 and low T4 levels. Recent studies have shown that the chemical chaperone sodium phenylbutyrate (NaPB) restored mutant MCT8 function and increased TH content in patient-derived induced pluripotent stem cells, making it a potential treatment for MCT8 deficiency.Objective We aimed to assess the efficacy and safety of glycerol phenylbutyrate (GPB) in MCT8 deficiency.Methods We treated 2 monozygotic twins aged 14.5 years with MCT8 deficiency due to P321L mutation with escalating doses of GPB over 13 months. We recorded TH, vital signs, anthropometric measurements, and neurocognitive functions. Resting metabolic rate (RMR) was measured by indirect calorimetry. Serum metabolites of GPB were monitored as a safety measure. In vitro effects of NaPB were evaluated in MDCK1 cells stably expressing the MCT8P321L mutation. The effects of GPB were compared to the effects of DITPA and TRIAC, thyromimetic medications that the patients had received in the past.Results NaPB restored mutant MCT8 expression in MDCK1 cells and increased T3 transport into cells carrying the P321L mutation. GPB treatment reduced high T3 and increased low T4 levels. The patients showed a significant weight gain simultaneously with a reduction in RMR. Only minor neurocognitive improvement was observed, in hyperreflexia score and in cognitive functions. Serum metabolites did not exceed the toxic range, but elevated liver transaminases were observed.Conclusion In the first report of GPB treatment in MCT8 deficiency we found an improvement in TH profile and body mass index, with minor neurodevelopmental changes.

Brief summary: This translational study highlights the importance of 2 novel endocytic receptors that are involved in cellular androgen uptake and in the pathogenesis of androgen insensitivity syndrome (AIS) type II.

The development and maturation of ovarian follicles is a complex and highly regulated process, which is essential for successful ovulation. During recent decades, several mouse models provided insights into the regulation of folliculogenesis. In contrast to the commonly used transgenic or knockout mouse models, the Dummerstorf high-fertility mouse line 1 (FL1) is a worldwide unique selection experiment for increased female reproductive performance and extraordinary high fertility. Interactions of cycle-related alterations of parameters of the hypothalamic pituitary gonadal axis and molecular factors in the ovary lead to improved follicular development and therefore increased ovulation rates in FL1 mice. FL1 females almost doubled the number of ovulated oocytes compared to the unselected control mouse line. To gain insights into the cellular mechanisms leading to the high fertility phenotype we used granulosa cells isolated from antral follicles for mRNA sequencing. Based on the results of the transcriptome analysis we additionally measured hormones and growth factors associated with follicular development to complement the picture of how the signaling pathways are regulated. While IGF1 levels are decreased in FL1 mice in estrus, we found no differences in insulin, prolactin and oxytocin levels in FL1 mice compared to the control line. The results of the mRNA sequencing approach revealed that the actions of insulin, prolactin and oxytocin are restricted local to the granulosa cells, since hormonal receptor expression is differentially regulated in FL1 mice. Additionally, numerous genes, which are involved in important gonadotropin, apoptotic and metabolic signaling pathways in granulosa cells, are differentially regulated in granulosa cells of FL1 mice.We showed that an overlap of different signaling pathways reflects the crosstalk between gonadotropin and growth factor signaling pathways, follicular atresia in FL1 mice is decreased due to improved granulosa cell survival and by improving the efficiency of intracellular signaling, glucose metabolism and signal transduction, FL1 mice have several advantages in reproductive performance and therefore increased the ovulation rate. Therefore, this worldwide unique high fertility model can provide new insights into different factors leading to improved follicular development and has the potential to improve our understanding of high fertility.