Nuclear Progestin Receptor (Pgr) Knockouts in Zebrafish Demonstrate Role for Pgr in Ovulation but Not in Rapid Non-Genomic Steroid Mediated Meiosis Resumption

Progestins, progesterone derivatives, are the most critical signaling steroid for initiating final oocyte maturation (FOM) and ovulation, in order to advance fully-grown immature oocytes to become fertilizable eggs in basal vertebrates. It is well-established that progestin induces FOM at least partly through a membrane receptor and a non-genomic steroid signaling process, which precedes progestin triggered ovulation that is mediated through a nuclear progestin receptor (Pgr) and genomic signaling pathway. To determine whether Pgr plays a role in a non-genomic signaling mechanism during FOM, we knocked out Pgr in zebrafish using transcription activator-like effector nucleases (TALENs) and studied the oocyte maturation phenotypes of Pgr knockouts (Pgr-KOs). Three TALENs-induced mutant lines with different frame shift mutations were generated. Homozygous Pgr-KO female fish were all infertile while no fertility effects were evident in homozygous Pgr-KO males. Oocytes developed and underwent FOM normally in vivo in homozygous Pgr-KO female compared to the wild-type controls, but these mature oocytes were trapped within the follicular cells and failed to ovulate from the ovaries. These oocytes also underwent normal germinal vesicle breakdown (GVBD) and FOM in vitro, but failed to ovulate even after treatment with human chronic gonadotropin (HCG) or progestin (17α,20β-dihydroxyprogesterone or DHP), which typically induce FOM and ovulation in wild-type oocytes. The results indicate that anovulation and infertility in homozygous Pgr-KO female fish was, at least in part, due to a lack of functional Pgr-mediated genomic progestin signaling in the follicular cells adjacent to the oocytes. Our study of Pgr-KO supports previous results that demonstrate a role for Pgr in steroid-dependent genomic signaling pathways leading to ovulation, and the first convincing evidence that Pgr is not essential for initiating non-genomic progestin signaling and triggering of meiosis resumption

Embedding art in histology teaching: Visual thinking strategies (VTS) to enhance visual literacy

BACKGROUND/AIMS Histology is a visually challenging subject for novice students. Visual thinking strategies (VTS) uses the viewing of art to improve visual literacy in classroom settings (Housen, 2002), including medical programmes (Reilly, Ring, & Duke, 2005), but has not been evaluated in histology. This project assessed the impact of VTS on students' observational skills, perceptions of histological images, and practical report marks. METHODS Participants were third-year biomedical students (n=133) studying histology in 2021. Students were shown a novel histology image and wrote their observations (pre-VTS). An experienced VTS facilitator guided students through an approximately 20-minute session exploring a never-before-seen artwork. After the VTS-activity, students were shown a new histology image and wrote their observations (post-VTS). Pre- and post-VTS descriptions were scored for measures of observational richness and compared. Responses to open-ended reflective questions were analysed by inductive thematic analysis. Report marks were compared with those from a previous year. RESULTS/CONCLUSIONS While there was no significant effect of the VTS activity on the students’ pre-/post-VTS descriptions, nor on their practical report marks, 46% of students reported that VTS changed how they viewed histological images and improved their observational skills. This study suggests that a one-off VTS activity at the beginning of a histology class can benefit students’ experience of unfamiliar microscopic images and improve enjoyment of this challenging subject. REFERENCES Housen, A.C. (2002) Aesthetic thought, critical thinking and transfer. Arts and Learning Research Journal, 18, 99-132. Reilly, J. M., Ring, J., & Duke, L. (2005) Visual thinking strategies: a new role for art in medical education. Family Medicine, 37, 250-252

Periconceptional alcohol exposure causes female-specific perturbations to trophoblast differentiation and placental formation in the rat

The development of pathologies during pregnancy, including pre-eclampsia, hypertension and fetal growth restriction (FGR), often originates from poor functioning of the placenta. In vivo models of maternal stressors, such as nutrient deficiency, and placental insufficiency often focus on inadequate growth of the fetus and placenta in late gestation. These studies rarely investigate the origins of poor placental formation in early gestation, including those affecting the pre-implantation embryo and/or the uterine environment. The current study characterises the impact on blastocyst, uterine and placental outcomes in a rat model of periconceptional alcohol exposure, in which 12.5% ethanol is administered in a liquid diet from 4 days before until 4 days after conception. We show female-specific effects on trophoblast differentiation, embryo-uterine communication, and formation of the placental vasculature, resulting in markedly reduced placental volume at embryonic day 15. Both sexes exhibited reduced trophectoderm pluripotency and global hypermethylation, suggestive of inappropriate epigenetic reprogramming. Furthermore, evidence of reduced placental nutrient exchange and reduced pre-implantation maternal plasma choline levels offers significant mechanistic insight into the origins of FGR in this model

Control of oocyte release by progesterone receptor-regulated gene expression

The progesterone receptor (PGR) is a nuclear receptor transcription factor that is essential for female fertility, in part due to its control of oocyte release from the ovary, or ovulation. In all mammals studied to date, ovarian expression of PGR is restricted primarily to granulosa cells of follicles destined to ovulate. Granulosa cell expression of PGR is induced by the pituitary Luteinizing Hormone (LH) surge via mechanisms that are not entirely understood, but which involve activation of Protein Kinase A and modification of Sp1/Sp3 transcription factors on the PGR promoter. Null mutations for PGR or treatment with PGR antagonists block ovulation in all species analyzed, including humans. The cellular mechanisms by which PGR regulates ovulation are currently under investigation, with several downstream pathways having been identified as PGR-regulated and potentially involved in follicular rupture. Interestingly, none of these PGR-regulated genes has been demonstrated to be a direct transcriptional target of PGR. Rather, in ovarian granulosa cells, PGR may act as an inducible coregulator for constitutively bound Sp1/Sp3 transcription factors, which are key regulators for a discrete cohort of ovulatory genes

Pgrmc1 Knockout Impairs Oocyte Maturation in Zebrafish

Recent investigations suggest progestin receptor membrane component 1 (PGRMC1) associates with and transports a wide range of molecules such as heme, cytochromes P450, steroids with 21 carbons, membrane progestin receptor alpha (mPRα/Paqr7), epidermal growth factor receptor (EGFR), and insulin receptor. It is difficult to discriminate the true functions of PGRMC1 from the functions of its associated molecules using biochemical and pharmacological approaches. To determine the physiological function(s) of PGRMC1, we generated global knockouts for pgrmc1 (pgrmc1−/−) in zebrafish. We found a reduction in both spawning frequency and the number of embryos produced by female mutants. We also observed reduced sensitivity of fully-grown immature oocytes to a progestin hormone and a reduced number of oocytes undergone meiotic maturation both in vivo and in vitro in pgrmc1−/−. This reduced sensitivity to progestin corresponds well with significant reduced expression of mPRα, the receptor mainly responsible for mediating oocyte maturation and meiosis resumption in fish. The results provide in vivo and in vitro evidence for the physiological functions of Pgrmc1 in oocyte maturation and fertility, as well as a plausible molecular mechanism via regulation of mPRα, which in turn directly regulates oocyte maturation and affects fertility in zebrafish

Perfil epidemiológico da violência contra mulheres no estado da Paraíba de 2009 a 2019

The objective of this study is to delineate the epidemiological profile of notifications of violence against women in the state of Paraíba, from 2009 to 2019. It is a cross-sectional, descriptive study conducted through online consultation of the TabNet Information System in the section on Notifiable Diseases and Injuries, using the field for interpersonal violence. As part of the results, we identified that physical violence is the most frequent type, accounting for 45.2% of cases. It was observed that the highest number of complaints involved black women, accounting for 60.11% of the cases; in the age group of 20 to 29 years, 22.71%; with incomplete primary education, 14.21%. Moreover, 59.2% of the situations of violence against women predominantly occur in residential areas. Regarding referrals to the health sector, the findings reveal that, in 54.15% of the records, it is defined as unknown. The study pointed out a significant weakness in the data records on violence, both due to lack of information and inadequate completion of the forms. Therefore, it is crucial to reinforce the importance of proper completion of compulsory notification forms and the training of professionals to provide accurate evidence on the problem and support management efforts.O objetivo desse estudo é traçar o perfil epidemiológico das notificações de violência contra mulher no estado da Paraíba, no período de 2009 a 2019. Trata-se de um estudo transversal, descritivo, realizado por meio de consulta online ao Sistema de Informação TabNet na seção de Doenças e Agravos de Notificação através do campo violência interpessoal. Como parte dos resultados, identificamos que a violência mais frequente é a física 45,2%. Foi observado que o maior número de queixas envolve mulheres negras, com 60,11% dos casos; na faixa etária de 20 a 29 anos, 22,71%; com ensino fundamental incompleto, 14,21%. Ademais, 59,2% das situações de violência contra a mulher ocorre, predominantemente, em locais residenciais. Quanto aos encaminhamentos ao setor de saúde, os achados revelam que, nesse item, em 54,15% dos registros é definido como ignorado. O estudo apontou uma grande fragilidade nos registros de dados sobre violência, tanto pela falta de informações quanto pelo preenchimento inadequado das fichas. Por isso, é crucial reforçar a importância do preenchimento adequado das fichas de notificação compulsória e a qualificação dos profissionais para fornecer evidências precisas sobre o problema e subsidiar a gestão para os enfrentamentos

Comparison of blood glucose concentration measured using a glucometer with plasma glucose concentration measured using a Cobas analyser on complementary rat samples.

Dual-analysis was conducted on n = 8-9 per sex per group. (A) Bland-Altman plot to visualise the difference in the glucometer and Cobas readings versus the average of the readings. This indicates that the glucometer tended to underestimate circulating glucose levels. (B) Comparison of glucose levels across each Treat/Sex group by method. White bars indicate control and black bars indicate EtOH-exposed animals within each sex. Data analysed using a two-way ANOVA, with treatment/sex (Control Male, EtOH Male, Control Female, EtOH Female) and method (glucometer/Cobas) as factors. Although this showed an effect of the method, this was consistent across all treatment/sex combinations (i.e. Treat/Sex was not significantly different). Therefore, although the glucometer underestimates circulating glucose levels, there is no bias for a particular Treat/Sex. Therefore, blood glucose can be used in place of plasma glucose if required, as long as all treatment groups use this substitution.</p

The role of nuclear progesterone receptor (PGR) in regulating gene expression, morphology and function in the ovary and oviduct during the periovulatory period.

Ovulation requires sequential molecular events and structural remodelling in the ovarian follicle for the successful release of a mature oocyte into the oviduct. Critical to this process is progesterone receptor (PGR), a transcription factor highly yet transiently expressed in granulosa cells (GC) of preovulatory follicles and abundant in the oviduct. Progesterone receptor knockout (PRKO) mice are anovulatory, with a specific and complete defect in follicle rupture. Therefore, this model was used to examine the critical molecular and biochemical mechanisms necessary for successful ovulation. Progesterone is known to affect oviductal cells in vitro, but how PGR regulates oviductal structure and function is poorly understood. A systematic evaluation of ovarian and oviductal morphology during the periovulatory period revealed no structural defects in PRKO mice relative to heterozygous (PR+/-) littermates. However, in response to the LH surge/hCG treatment, ovulation only occurred in PR+/- ovaries, with numerous corpora lutea observed and cumulus oocyte complexes (COCs) in oviducts, while PRKO ovaries did not ovulate and showed entrapped COCs within large, luteinising follicles. Transplantation of PRKO ovaries into wild-type mice (PRWT) did not rescue the infertility phenotype. Therefore, although PGR is expressed in other tissues, ovarian PGR is essential for ovulation. Further experiments identified PGR-regulated processes at multiple levels. In whole ovaries 10 h post-hCG, inflammatory genes were disrupted in PRKO mice, including cytokines, endothelial adhesion factors, vasoconstrictors, T-cell antigens, and the prostaglandin synthase, Ptgs2. In GCs and COCs isolated 8 h post-hCG, microarray analyses identified 296 and 44 differentially expressed genes respectively between PRKO and PR+/- samples. Gene ontology analysis revealed associations with the processes of proteolysis, vascular remodelling/angiogenesis, inflammatory responses, cell adhesion, migration and invasion. The latter three processes were characterised in periovulatory COCs using in vitro assays and were shown to be transiently activated, peaking at ovulation then declining dramatically in COCs collected from the oviduct immediately post-ovulation. However, periovulatory PRKO and PR+/- COCs showed similar rates of adhesion, migration and invasion in the presence of collagen I. Upregulation of the chemokine receptor, Cxcr4, by LH/hCG via PGR in both GCs and COCs was validated by RT-PCR and immunohistochemistry. Mitochondrial membrane potential was altered in PRKO oocytes compared to PR+/- and therefore their developmental potential may be reduced. Further, a bioassay measuring retention of prostaglandin (PGE2) within the matrix of expanded COCs suggested that the matrix integrity of PRKO COCs may be compromised. Therefore, PGR in granulosa cells appears to have down-stream impacts on COCs. In oviducts, microarray analysis comparing gene expression in PRKO and PR+/- mice 8 h post-hCG, when P4 levels are high, identified 1003 PGR-regulated genes. Gene ontology analysis identified significant associations with the functions of cell adhesion, migration, invasion, chemotaxis, muscle contraction and vasoconstriction. Several genes were confirmed to be PGR-regulated by RT-PCR (Adamts1, Itga8 and Edn3) and were induced by LH/hCG. Therefore, the identification of novel gene targets for PGR regulation in the ovary and oviduct exposes several new, down-stream influences of PGR on inflammation, the COC and oviductal function, highlighting the essential role of PGR as master regulator in the ovary and oviduct during the periovulatory period.Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 201

Supplemental Table for Manuscript Submitted to the International Journal of Molecular Sciences

&lt;p&gt;Contains details of primers used for qPCR examining expression of factors that regulate the maintenance of the ovarian reserve in neonatal rat ovaries.&lt;/p&gt