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Cancer therapies often cause serious side effects, affecting the quality of life for cancer survivors. The ovary can be affected by cancer therapies, causing premature ovarian insufficiency, leading to endocrine dysfunction and infertility. Thus, maintaining ovarian function against cancer treatment is an unmet need for female cancer patients. Cyclophosphamide (CPA), a common chemotherapeutic agent, is metabolized in vivo and forms DNA crosslinks, inducing apoptosis in rapidly proliferating tumor cells. However, the underlying mechanism of the CPA-induced oocyte death in ovarian reserve remains unclear. This study aims to investigate the mechanism of oocyte death in primordial follicles by generating oocyte-specific Abl1 and p63 knockout and Pik3ca* knockin mouse models using Gdf9-iCre+. Postnatal day 7 female mice were i.p. injected w/wo CPA. Notably, we found CPA drastically reduced the number of primordial follicles without increasing the number of growing follicles. The quantification of surviving follicles validated that 90% of the primordial follicles from oocyte-specific Abl1 knockout mice were lost following CPA treatment in vivo and in vitro. Concurrently, high expression of CHK2 was detected in the oocytes of the ovary cultured with CPA metabolite in vitro. This implied CHK2 rather than ABL is a possible tyrosine kinase for CPA-induced oocyte death. Most of all, p63 was a key player for CPA-induced oocyte death, supported by the result that p63 knockout oocytes were rescued after CPA treatment. To clarify whether the ovaries lose follicles via activation or apoptosis pathway, PIK3CA* mice were examined with CPA. As expected, primordial follicles and primordial follicles with activated oocytes in the ovary of PIK3CA* mice survived against CPA due to constitutive PI3K activity in them. This indicates activated follicles resist gonadotoxic reagents, although p63 is expressed inside their oocytes. Accordingly, the apoptosis markers, BAX and cleaved PARP were highly induced with CPA injection in a time-dependent manner in the ovaries from wild-type female mice but not from the p63 knockout. The double-strand break also occurred in the nucleus of oocytes post CPA administration as γH2AX was detected. Interestingly, OPA1, a protein required for mitochondrial fusion, was highly induced inside the oocyte cytoplasm of p63 knockout, while oocytes in wild-type mice time-dependently lost the OPA1 expression by CPA treatment. This indicates that oocytes without p63 in the nucleus survive and induce mitochondrial fusion to escape apoptosis by mitochondrial damages. In conclusion, CPA induces depletion of oocytes of primordial follicles through the CHK2-TAp63 apoptotic pathway.

ENDO 2022

Cyclophosphamide Depletes Primordial Follicles via the p63 Apoptotic Pathway

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ENDO’s comprehensive program provides attendees the opportunity to learn about the latest developments in hormone science and medicine from renowned investigators, expert clinicians, and educators from all over the world. 

technical paper

While androgens are traditionally associated with male physiology, recently it has become more apparent that balanced levels of androgens are crucial to maintaining female health and fertility as well. Specifically, androgens are necessary for granulosa cell proliferation and follicle survival and growth. Excess androgen levels in females are a hallmark in polycystic ovary syndrome (PCOS), whereas insufficient androgen signaling due to androgen receptor (AR) knockout leads to diminished ovarian reserve (DOR) – with both extremes resulting in anovulatory infertility. However, little is known about the factors regulating AR in granulosa cells. Our previous findings demonstrate that paxillin, a cytoplasmic adaptor protein, mediates AR signaling in prostate cancer cells. However, our current interest is whether this interaction is also found in granulosa cells. As such, our results show that paxillin indeed enhances AR protein expression in granulosa cells. We have also demonstrated through immunofluorescence and proximity ligation assays that paxillin colocalizes with AR, notably in membrane-bound complexes. Moreover, paxillin-null KGN and mouse granulosa cells express lower levels of AR, as demonstrated through Western blotting and immunofluorescence imaging. We then conducted mRNA and protein degradation experiments in paxillin-null KGN cells in order to determine the mechanism by which paxillin increases AR expression. These results suggest that paxillin acts by preventing AR mRNA and protein degradation, possibly by trafficking AR to focal adhesions, where it may be sequestered from degradation. Finally, to investigate the role of paxillin in female fertility, we developed a novel granulosa cell-specific paxillin knockout mouse model using an Amhr2-driven Cre/lox system. These knockout mice experience altered estrous cycles compared to controls, spending fewer days in estrus and more days in metestrus/diestrus. When bred continuously through 6 months of age, the paxillin KO mice produced significantly more litters compared to their littermate controls. Taken together, these results suggest that paxillin acts as a regulator of androgen-mediated female fertility in granulosa cells, and may serve as a potential target for therapeutics in fertility-related diseases such as PCOS and DOR.

Paxillin and Androgen Receptor Interact to Modulate Female Fertility

Introduction: Estrogen is vital to human reproduction and acts primarily through two receptors: estrogen receptor alpha (ER𝛼, encoded by ESR1) and estrogen receptor beta (ERβ, encoded by ESR2). Surprisingly, very few human ESR1 pathogenic variants have been recognized, despite the existence of >1000 different androgen receptor pathogenic variants causing androgen insensitivity syndrome. Functional analysis of an ESR1 homozygous missense variant (p.Glu375His) in the ER𝛼 ligand-binding domain (LBD) showed a 240-fold decrease in estrogen response in a patient with complete estrogen insensitivity and abnormal pubertal development. We predict that less severe ESR1 variants will present with unexplained infertility. Previously, we performed whole exome sequencing on 200 females with unexplained infertility and identified 4 likely pathogenic ESR1 heterozygous missense variants confirmed by Sanger sequencing, including one variant (p.Thr313Met) in the LBD. In this study, we performed in vitro functional analysis to evaluate if the p.Thr313Met likely pathogenic variant found in a patient with unexplained infertility causes incomplete estrogen insensitivity.
Hypothesis: We hypothesize that the p.Thr313Met likely pathogenic variant in the ERa LBD will result in incomplete estrogen insensitivity.
Methods: The p.Thr313Met variant plasmid was constructed using site-directed mutagenesis (SDM), and confirmed by Sanger sequencing. An in vitro cell model was used to evaluate estrogenic activity. HepG2 cells were transiently transfected with WT ESR1, p.Thr313Met, or p.Glu375His plasmid, along with the Firefly estrogen response element (ERE) luciferase reporter (3X ERE TATA luc) and Renilla luciferase (pRL-TK luc) plasmids using the Effectene transfection protocol. Cells were treated with 17β-estradiol (0.01, 0.1, 1, 10, and 100 nM). A dual luciferase reporter assay was used to compare relative Firefly and Renilla luciferase activity between WT ESR1, p.Thr313Met, and p.Glu375His plasmids using a luminometer. Estrogen dose response curves were generated, and a two-way ANOVA with Tukey's test was performed to compare the relative luciferase activity across WT and variant plasmids using GraphPad Prism v.9. Differences were considered significant at p<0.05.
Results: Estrogen response curves were generated for WT, p.Thr313Met, and p.Glu375His plasmids. The p.Thr313Met variant showed lower overall activity with a right shifted dose response curve with a statistically significant decrease in relative luciferase activity in comparison to WT (p<0.05). The p.Thr313Met variant was more active at higher doses (1 and 10 nM) with a left shifted dose curve when compared to the p.Glu375His variant (p<0.05).
Conclusion: We have identified a human ESR1 variant that impairs estrogen signaling in vitro, suggesting incomplete estrogen insensitivity as a putative mechanism for unexplained infertility. The identification of ESR1 pathogenic variants and the evaluation of their functionality could provide improved diagnosis and genetic counseling for patients with unexplained infertility.

ESR1 Pathogenic Variant With Incomplete Estrogen Insensitivity

Although obesity is closely associated with reproductive dysfunction, many women with obesity remain eumenorrheic. The degree to which obesity affects menstrual cycle characteristics in this population is limited to endocrine assessments with few data available on how endocrine aberrations align with defects in antral follicle development.We have previously shown alterations in the distribution of recruitable (2-5mm) versus selectable (6-9mm) follicles in those with obesity that were associated with decreased gonadotropin and progesterone production despite normal menstrual cycle length. The objectives of the present study were to further contrast follicular growth and endocrine dynamics between obese and non-obese eumenorrheic women with the goal of determining any alterations in distinct events in folliculogenesis.
21 non-obese (total percent body fat <35%) and 21 obese (total percent body fat ≥35%) reproductive age females with regular menstrual cycles (21-35d) were evaluated by serial ovarian transvaginal ultrasonography and venipuncture every-other-day for one complete inter-ovulatory interval (IOI). The number and diameters of all follicles ≥2mm at each visit were documented offline using the Grid Method. Growth profiles of individual follicles which grew to ≥7mm were assessed using the Identity Method to characterize antral follicle recruitment, selection, and ovulation. Clinical, morphologic, and endocrine features were compared across groups using t-tests and mixed models.
Women with obesity exhibited fewer recruitment events versus non-obese women (1±0.6 vs. 2±0.9; p=0.010). They also had fewer selectable follicles (4±3 vs. 8±6 follicles/participant, p=0.022) which grew faster but remained static longer and regressed slower (all growth rates, p<0.05) compared to their non-obese counterparts. Anti-Mullerian hormone levels were lower in the obese versus non-obese group (4.40±3.01 vs. 5.94±2.49ng/mL, p=0.023). Of the selectable follicles, fewer progressed to ≥10mm in the obese versus non-obese group (1±0.7 vs. 2±0.8 follicles, p=0.030) with ultimately fewer anovulatory follicles achieving dominance (0±0.7 vs. 1±0.9 follicle, p=0.029). Ovulatory follicles in the obese group were selected at smaller diameters (7.5±1.6 vs. 9.5±1.9mm, p=0.001) versus their non-obese counterparts. However, the kinetics of dominant follicle growth were similar between groups. Post-ovulation, progesterone concentrations across the luteal phase were lower in women with obesity (4.95±3.34 vs. 7.85±5.74ng/mL, p=0.001) and the prevalence of luteal phase defects (LPDs) higher versus non-obese women, defined by either integrated (76 vs. 29%, p=0.002) or maximum (71 vs. 24%, p=0.002) progesterone levels.
Eumenorrheic women with obesity display evidence of suppressed follicle dynamics as evidenced by fewer recruitment events, selectable follicles, and anovulatory dominant follicles.  LPDs are more prevalent in women with obesity during natural ovulatory cycles which may underlie subfertility in this population. A smaller size at selection suggests premature responsiveness to luteinizing hormone which may result in suboptimal luteinization. Further studies are needed to elucidate the degree to which these defects impact fertility and fecundity in our increasingly obese demographic.

Women with obesity display defects in follicular recruitment, selection, and dominance alongside luteal phase dysfunction during natural ovulatory cycles

Background: SKYLIGHT 2 (NCT04003142) investigated safety and efficacy of fezolinetant (a neurokinin-3 receptor antagonist) on frequency and severity of moderate-to-severe vasomotor symptoms (VMS) and sleep disturbance. In this analysis, persistence of fezolinetant efficacy was investigated over 52 weeks.
Methods: This double-blind Phase 3 study randomized women aged ≥40–65 years with moderate-to-severe VMS associated with menopause (average of ≥7 hot flashes/day) to once-daily placebo or fezolinetant 30mg or 45mg for 12 weeks. Women completing 12 weeks entered an extension period, with those on placebo re-randomized to fezolinetant 30mg or 45mg (placebo/fezolinetant), and those originally on fezolinetant remaining on their dose for an additional 40-weeks. Fezolinetant efficacy was evaluated vs placebo for 12-weeks through change in VMS frequency, VMS severity, and Patient-reported Outcomes Measurement Information System Sleep Disturbance–Short Form 8b (PROMIS) Total Score. Persistence of efficacy for fezolinetant was evaluated descriptively (without statistical comparisons) over the extension period. Results: The analysis comprised 484 women (fezolinetant 30mg n=166, fezolinetant 45mg n=167, placebo/fezolinetant 30mg n=76, placebo/fezolinetant 45mg n=75). Improvement in VMS frequency and severity observed through week 12 (statistically significant differences vs placebo) was maintained throughout the 52-week total study period for those receiving fezolinetant. Fezolinetant demonstrated further reductions in VMS frequency and severity from baseline to beyond week 12. For VMS frequency, there was a least squares (LS) mean (SE) baseline-to-week 12 reduction of –6.83 (0.39) VMS/day for fezolinetant 30mg and –7.50 (0.39) for 45mg, and a mean (SD) baseline-to-week 52 reduction of –8.03 (4.53) for fezolinetant 30mg and –8.48 (3.98) for 45mg. For VMS severity, LS mean (SE) baseline-to-week 12 reduction was –0.64 (0.06) for 30mg and –0.77 (0.06) for 45mg, and mean (SD) baseline-to-week 52 reduction was –0.83 (0.82) for fezolinetant 30mg, and –0.95 (0.78) for 45mg. Women re-randomized from placebo to fezolinetant experienced a reduction in frequency and severity of VMS consistent with that in women receiving fezolinetant throughout the study. Fezolinetant also reduced PROMIS-assessed sleep disturbance, with a LS mean (SE) baseline-to-week 12 reduction of –4.1 (0.5) for fezolinetant 30mg and –5.5 (0.5) for 45mg (statistically significant difference vs placebo for fezolinetant 45mg) and a mean (SD) baseline-to-week 52 reduction of – 6.3 (7.3) for fezolinetant 30mg and –5.7 (7.9) for 45mg. The safety profile observed for the 40-week extension period was consistent with that of the 12-week placebo-controlled period. Conclusion: Fezolinetant 30mg and 45mg once daily were efficacious for treatment of moderate-to-severe VMS associated with menopause. Efficacy was persistent and reductions in VMS frequency were maintained during the extension period, at levels consistent with weeks 1 through 12. Fezolinetant 45mg improved sleep at week 12 and improvement was maintained through the active treatment extension period. No safety signals of concern were apparent for either fezolinetant dose.

Fezolinetant for Treatment of Moderate-to-severe Vasomotor Symptoms Associated with Menopause: Results from a 52-week Study (Skylight 2)

Approximately 70% of breast cancers express estrogen receptor alpha (ERα), a hormone-regulated transcription factor.  Upon binding to endogenous and exogenous ligands, ERα dimerizes and binds to regulatory regions across the genome known as enhancers.  Enhancers act as nucleation sites for the recruitment of coregulatory proteins and ultimately regulate target gene transcription.  We have previously used global run-on sequencing (GRO-seq) to detect transcription at active enhancers that generates enhancer RNAs (eRNAs).  eRNAs are poorly characterized noncoding RNAs and are thought to regulate enhancer function and gene expression.  To better understand the functions of these eRNAs, we annotated the estrogen-regulated eRNA transcriptome in ERα-positive breast cancer cells using precision nuclear run-on of capped RNA (PRO-cap) and polyA-depleted RNA-seq.  We then cloned selected eRNAs, fused them to single guide RNAs, and targeted them to their ERα enhancers of origin using CRISPR/dCas9.  eRNAs promoted expression of cognate, but not heterologous, target genes by increasing ERα recruitment and simulating p300-catalyzed H3K27 acetylation at the enhancer.  We identified a ~40 nucleotide functional eRNA regulatory motif (FERM) present in many estrogen-regulated eRNAs that is necessary and sufficient to promote gene expression by increasing ERα recruitment and stimulating p300-catalyzed H3K27 acetylation.  The FERM interacted with BCAS2, an RNA-binding protein amplified in breast cancers, to exert its effect on enhancer assembly and dynamics.  Overexpression of a targeted eRNA controlling expression of an oncogene increased cell proliferation, implicating eRNAs in regulating breast cancer cell growth.  Together, we provide a genome-wide annotation of estrogen-regulated eRNAs, demonstrate the mechanisms by which targeted eRNAs promote target gene expression in vivo, identify the FERM element within eRNAs that is critical for regulating target gene expression, and show the biological implications of oncogenic eRNAs in breast cancer growth.  Collectively, our results demonstrate the biological and mechanistic roles of eRNAs in breast cancer.
  This work is supported by grants from the NIH/NIDDK (DK058110) and CPRIT (RP160319) to W.L.K.

Estrogen-regulated Enhancer RNAs Contain a Functional Motif that Controls Enhancer Assembly and Function in Breast Cancer Cells

Invasive lobular carcinoma of the breast (ILC) affects ~40,000 US women annually and is a top ten most diagnosed cancer in women. ~95% of ILC express estrogen receptor alpha (ER), but ILC is associated with poor long-term outcomes and anti-estrogen resistance, suggesting ER function is distinct in ILC cells. We reported that MDC1 (mediator of DNA damage checkpoint 1) is an ILC-specific ER co-regulator required for ER-driven proliferation, and that MDC1 knockdown dysregulates the ER transcriptome in ILC. Mechanisms of MDC1 co-regulator activity are unknown, but must be defined to understand endocrine response and resistance in ILC.
To understand MDC1 co-regulator functions, we profiled ER/MDC1 genomic binding (ie. "cistrome") using CUT&RUN in ER+ ILC cell line MDA MB 134VI (MM134). We identified ~2500 binding sites each for ER and MDC1, but only ~8% of sites were bound by both factors. However, using BETA to integrate cistrome and transcriptome data predicted that both ER and MDC1 activate estrogen-regulated genes with over half of direct ER target genes also direct MDC1 targets (p=9.8x10-375). Among shared ER: MDC1 direct target genes (n=760), 58% had MDC1 binding in the promoter region with ER bound at nearby enhancers, while only 15% were linked to shared ER: MDC1 binding sites, suggesting MDC1 primarily facilitates promoter access for ER-bound enhancers. Further, MDC1 knockdown reduced ER binding at enhancer sites at ER: MDC1 target genes, implicating MDC1 in regulating chromatin access.
Importantly, MDC1 does not have known enzymatic activity to directly regulate chromatin structure. We profiled MDC1-interacting proteins in ILC cells by RIME (Rapid immunoprecipitation mass spectrometry of endogenous proteins), and determined whether knockdown of putative ER: MDC1 partners disrupted ER function. This identified the SWI/SNF complex protein BRG1 (SMARCA4) as a critical ER: MDC1 partner. BRG1 was required for ILC cell growth, and BRG1 knockdown specifically suppressed ER regulation of MDC1-dependent ER target genes.
Taken together, our data suggest that in ILC cells, MDC1 facilitates ER access to and activity at target gene promoters. The BRG1 SWI/SNF complex is a putative ER: MDC1 partner and mediator of MDC1 co-regulator functions. Ongoing research is focused on understanding how MDC1 regulates ILC-specific ER activity by licensing access to ILC-specific target genes, and the role of the SWI/SNF complex in mediating genomic ER: MDC1 activity.

Endocrine Response And Resistance Is Driven By Novel Transcriptional Co-Regulator Activity In Invasive Lobular Carcinoma Of The Breast

During fasting, hepatocytes produce glucose in response to hormonal signals. Glucagon and glucocorticoids are principal hormones secreted during fasting that cooperate in regulating glucose production via increasing hepatic gluconeogenesis. However, how these hormone signals are integrated in hepatocytes and translated to a biological output is unknown. We used genome-wide profiling of gene expression, enhancer dynamics and transcription factor binding in primary mouse hepatocytes to uncover the mode of cooperation between glucagon and corticosterone (the major glucocorticoid in mice). We found that compared to a single treatment with either hormone alone, a dual treatment initiates a pro-gluconeogenic gene program in hepatocytes by synergistically inducing gluconeogenic genes. We show that the mechanism driving synergistic gene expression is based on glucagon-mediated enhancer activation, leading to increased binding of the glucocorticoid receptor (GR) upon corticosterone stimulation. This was shown by glucagon-dependent increases in the active enhancer mark H3K27ac at GR binding sites (both signals were measured genome-wide via chromatin immunoprecipitation sequencing). Thus, glucagon-dependent enhancer activation, mediated by the glucagon-activated transcription factor CREB, assists GR loading to specific enhancers located near synergistic gluconeogenic genes. This 'assisted loading' mechanism is enhancer-specific as GR was able to efficiently bind other enhancers without assistance by CREB. Interestingly, we found that the glucagon-CREB axis does not only activate single enhancers but is also able to activate entire enhancer clusters. Indeed, CREB binding in one enhancer unit within a cluster, led to cluster-wide enhancer activation, thereby assisting the loading of GR in other enhancer units within the cluster that are unbound by CREB directly. These chromatin and gene expression changes collectively lead to synergistic glucose production from hepatocytes in the presence of glucagon and corticosterone. In summary, we show that hepatocytes integrate extracellular signals by an enhancer-specific mechanism: glucagon activates enhancers, thereby assisting the loading of GR upon stimulation by corticosterone. This chromatin-bound integration of endocrine signals leads to synergistic gene induction and a tailored response to fasting.

Hormone-controlled cooperative binding of transcription factors within enhancer clusters drives synergistic induction of fasting-regulated genes

The mineralocorticoid receptor (MR) is a ligand activated transcription factor that plays an important role in cardiovascular physiology and disease. MR transactivation is regulated by ligand-dependent conformational change in the MR and recruitment of coregulator binding partners to form a unique DNA-binding complex at the hormone response element in target gene promoters. Differences in the recruitment of coregulatory proteins can promote tissue-, ligand- or gene-specific transcriptional outputs. The goal of this study was to evaluate a candidate MR coregulator, the circadian gene timeless for ligand and cell-selective regulation of MR transactivation. While timeless is not essential for mammalian clock function and does not bind DNA, timeless RNA and protein levels oscillate over 24hr. Co-expression of timeless dose-dependently down-regulated MR transactivation of an MR-responsive reporter in HEK293 cells, yet enhanced transactivation mediated by other steroid receptors. Timeless also inhibited MR transactivation of synthetic and native gene promoters, and expression of endogenous MR target genes, in H9c2 cardiac myoblasts. Immunofluorescence staining of timeless, and MR showed colocalization to the nucleus in the presence of aldosterone and co-immunoprecipitation which suggested an interaction, although a direct interaction was not confirmed. Taken together, these data suggest timeless is a novel MR coregulatory protein that may contribute to time-of-day-dependent MR transcriptional outputs and suggest new opportunities for the development of MR antagonists with selective actions.

Regulation of mineralocorticoid receptor transactivation by circadian protein timeless.

Background: NAD+-dependent PARP1 (Poly-(ADP-ribosyl)polymerase 1) generates the post-translational modification ADP-Ribosylation (ADPR). Given PARP1-ADPR determines cellular NAD+ availability, which in turn impacts a range of hormonally governed actions, we sought to identify the molecular roles of PARP1 in relation to glucocorticoids in the endocrine sensitive tissue skeletal muscle.
Importance: Activity of the glucocorticoid receptor (GR) is dictated by molecular partners whose actions skew the frequency with which GR produces meaningful transcriptional outcomes. As a result, our molecular understanding of this steroid hormone receptor remains incomplete.
Hypothesis: PARP1 through ADPR actions impacts skeletal muscle phenotype and co-regulates glucocorticoid-mediated transcriptional responses.
Experimental Design: Murine muscle myoblasts (C2C12) or primary murine muscle cultures were treated with PARP inhibitors (PJ34(10uM) or BYK204165(1uM)) or transfected with silencing RNA targeting PARP1. Cells were treated with ± dexamethasone (1uM).
Major Results: Analysis of differentiating muscle revealed PARP1 and ADPR are dynamic at days 0-5 (measured by Western immunoblot. p<0.001; n=6). Chemical inhibition of PARP1 significantly decreased ADPR (Western immunoblot. p<0.001; n=9) but did not prevent myotube formation. To identify if initial ADPR dynamics held consequence for fully differentiated myotubes, cells were treated with a single dose of the PARP1 inhibitor BYK204165 at induction of differentiation. Myotube lysates were collected 5 days later and were subjected to unlabelled SWATH-MS. This measured the abundance of 180 proteins as significantly different in PARP inhibitor-treated cells (n=7). Analyses of these proteins revealed over-representation of pathways governing muscle development (FDR 8.99-08), contraction (FDR 9.47-08, myofiber assembly (FDR 8.06-05), and energy metabolism (FDR 2.93-06). Indicating PARP1-ADPR regulates myogenesis and plays a role in the striated muscle contractile response.
ChIP assays for PARP1 binding in cells showed a (30-fold enrichment ±3.2SEM) at GR response elements and detectable ADPR was reduced when exposed to dexamethasone (Western immunoblot n=5). These data support ADPR as responsive to glucocorticoid exposure and PARP1 influences GR recruitment to the genome.
RNAseq of siPARP1 (n=4) versus scrambled myoblasts (n=5) identified differential expression of 165 genes. Gene ontology revealed over-representation in siPARP1 cells of those pathways regulating muscle differentiation, including actin-binding, cytoskeletal structure, and NAD+ binding genes. SiRNA of PARP1 had no influence over the ability of glucocorticoids to activate archetype target genes (DUSP1, MURF1, ATROGIN1 & GILZ), but differentially impacted the expression of 434 other genes.
Conclusions: PARP1 mediated ADPR is critical to the 'early phase' events of muscle cell differentiation. PARP1 influences short-term transcriptional behaviors of the glucocorticoid receptor; in turn, glucocorticoids themselves impact PARP1-genome interaction events. These findings are important for the understanding of general GR mechanisms and have relevance for the widespread functional decline observed during tissue glucocorticoid excess.

PARP1 Contributes to the Glucocorticoid Receptor Transcriptional Response And Influences the Skeletal Muscle Phenotype

The androgen receptor (AR) plays a crucial role in the development and maintenance of the male phenotype, as shown in patients with androgen insensitivity syndrome (AIS). The AR is a nuclear receptor that needs to homodimerize to execute its role as transcription factor. Dimerization can occur through three different modes: via the DNA-binding domain (DBD), via the ligand-binding domain and via an interaction between the LBD and the aminoterminal domain. Dimerization via the DBD is very well known and occurs through the D-box located in the second zinc finger. DBD dimerization is generally accepted for all steroid receptors, but some of them show that D-box mutations do not inactivate them completely. However, for the AR, no monomeric transactivation capacity was described yet. To study the role of D-box independent activity of the AR, we introduced a double point mutation in the D-box that is predicted to disrupt DBD dimerization (ARDmon). These two point mutations were found in separate patients suffering from partial AIS. In vitro studies with this mutant suggest that the remaining activity depends on the androgen response element under investigation. We show that ligand binding was not affected by the D-box mutation. Furthermore, BioID assays indicated no changes in coregulatory interactions. When introducing the corresponding mutation in mice, this led to a phenotype that is intermediate between global ARKO and wild type (WT) males. Based on their anogenital distance (AGD) and the absence of nipples at early age, the ARDmon/Y mice were identified as males. Follow-up of their AGD and body weight, however, revealed clear differences with both WT males and females. Furthermore, hypospadias was observed in the ARDmon/Y mice. Testes were smaller in size compared to the WT males and first analysis showed almost no expression of well-known AR regulated genes. Reminiscent structures of the epididymis were found.
In summary, the ARDmon mouse model shows an intermediate AIS phenotype. Via orchidectomy in combination with androgen replacement therapy, we will now investigate remaining androgen responses in the ARDmon/Y mice and elucidate response elements on which the AR can act in a D-box independent way. Additionally, we generated the first rodent PAIS model with a genetic cause of hypospadias.

Cyclophosphamide Depletes Primordial Follicles via the p63 Apoptotic Pathway

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