The Continued Absence of Functional Germline Stem Cells in Adult Ovaries

Ovaries are central to development, fertility, and reproduction of women. A particularly interesting feature of ovaries is their accelerated aging compared to other tissues, leading to loss of function far before other organs senesce. The limited pool of ovarian follicles is generated before birth and once exhausted, menopause will inevitably commence around the age of 50 years marking the end of fertility. Yet, there are reports suggesting the presence of germline stem cells and neo-oogenesis in adult human ovaries. These observations have fueled a long debate, created experimental fertility treatments, and opened business opportunities. Our recent analysis of cell types in the ovarian cortex of women of fertile age could not find evidence of germline stem cells. Like before, our work has been met with critique suggesting methodological shortcomings. We agree that excellence starts with methods and welcome discussion on the pros and cons of different protocols. In this commentary, we discuss the recent re-interpretation of our work.Peer reviewe

Reply : Revisiting Claims of the Continued Absence of Functional Germline Stem Cells in Adult Ovaries

Non peer reviewe

The E-cadherin/AmotL2 complex organizes actin filaments required for epithelial hexagonal packing and blastocyst hatching

Epithelial cells connect via cell-cell junctions to form sheets of cells with separate cellular compartments. These cellular connections are essential for the generation of cellular forms and shapes consistent with organ function. Tissue modulation is dependent on the fine-tuning of mechanical forces that are transmitted in part through the actin connection to E-cadherin as well as other components in the adherens junctions. In this report we show that p100 amotL2 forms a complex with E-cadherin that associates with radial actin filaments connecting cells over multiple layers. Genetic inactivation or depletion of amotL2 in epithelial cells in vitro or zebrafish and mouse in vivo, resulted in the loss of contractile actin filaments and perturbed epithelial packing geometry. We further showed that AMOTL2 mRNA and protein was expressed in the trophectoderm of human and mouse blastocysts. Genetic inactivation of amotL2 did not affect cellular differentiation but blocked hatching of the blastocysts from the zona pellucida. These results were mimicked by treatment with the myosin II inhibitor blebbistatin. We propose that the tension generated by the E-cadherin/AmotL2/actin filaments plays a crucial role in developmental processes such as epithelial geometrical packing as well as generation of forces required for blastocyst hatching.Peer reviewe

Characterization and target genes of nine human PRD-like homeobox domain genes expressed exclusively in early embryos

PAIRED (PRD)-like homeobox genes belong to a class of predicted transcription factor genes. Several of these PRD-like homeobox genes have been predicted in silico from genomic sequence but until recently had no evidence of transcript expression. We found recently that nine PRD-like homeobox genes, ARGFX, CPHX1, CPHX2, DPRX, DUXA, DUXB, NOBOX, TPRX1 and TPRX2, were expressed in human preimplantation embryos. In the current study we characterized these PRD-like homeobox genes in depth and studied their functions as transcription factors. We cloned multiple transcript variants from human embryos and showed that the expression of these genes is specific to embryos and pluripotent stem cells. Overexpression of the genes in human embryonic stem cells confirmed their roles as transcription factors as either activators (CPHX1, CPHX2, ARGFX) or repressors (DPRX, DUXA, TPRX2) with distinct targets that could be explained by the amino acid sequence in homeodomain. Some PRD-like homeodomain transcription factors had high concordance of target genes and showed enrichment for both developmentally important gene sets and a 36 bp DNA recognition motif implicated in Embryo Genome Activation (EGA). Our data implicate a role for these previously uncharacterized PRD-like homeodomain proteins in the regulation of human embryo genome activation and preimplantation embryo development.Peer reviewe

Multiphoton microscopy and ultrafast spectroscopy: Imaging meets quantum (MUSIQ) roadmap

In April 2019 the EU Marie Skłodowska-Curie Actions (MSCA) Innovative Training Networks (ITN) MUSIQ officially started. The network brought together a unique team of world-leading academics and industrial partners at the forefront of optical micro-spectroscopy and ultrafast laser technology developments merged with fundamental studies of coherent light-matter interaction phenomena, development of quantitative image analysis tools beyond state-of-the-art, and biomedical/pharmaceutical real-world applications. The unique vision of MUSIQ has been to develop and apply the next-generation optical microscopy technologies exploiting quantum coherent nonlinear phenomena. This Roadmap has been written collectively by the MUSIQ early-stage researchers and their supervisors. It provides a summary of the achievements within MUSIQ to date, with an outlook towards future directions

ESHRE good practice recommendations for add- ons in reproductive medicine

The draft of the paper “ESHRE Good practice recommendations for add-ons in reproductive medicine” was published for public review for 4 weeks, between 1 November and 1 December 2022. This report summarizes all reviewers, their comments and the reply of the working group and is published on the ESHRE website as supporting documentation to the paper. During the stakeholder review, a total of 274 comments (including 24 duplicates) were received from 46 reviewers. Reviewers included professionals and representatives of donor-conceived offspring organisations. The comments were focussed on the content of the guideline (209 comments), language and style (31 comments), or were remarks that did not require a reply (10 comments). All comments to the language and format were checked and corrected where relevant. The comments to the content of the paper (n=209) were assessed by the working group and where relevant, adaptations were made in the paper (n=94; 45%). Adaptations included revisions and/or clarifications of the text, and amendments to the recommendations. For a number of comments, the working group considered them outside the scope of the paper or not appropriate/relevant (n=115; 55%).peer-reviewe

Developmental Expression, Function, and Regulation of Multidrug Resistance in the Mouse Placenta and Fetal Brain

During pregnancy, 64-96% of women take at least one prescription drug. The placenta is the primary barrier between substrates in maternal and fetal circulation. The blood-brain barrier (BBB) acts as an additional barrier for the fetal brain, which is particularly susceptible to the effects of xenobiotics. Multidrug resistance phosphoglycoprotein (P-gp; encoded by Abcb1 mRNA) and breast cancer resistance protein (Bcrp1; encoded by Abcg2 mRNA) are efflux transporters localized on placental syncytiotrophoblast and capillary endothelial cells of the BBB. Placental Abcb1/P-gp and Abcg2/Bcrp1 limit maternal-fetal transfer of endogenous and exogenous substrates. Similarly, the neuroprotective roles of Abcb1/P-gp and Abcg2/Bcrp1 in the adult BBB have been demonstrated. However, developmental changes in expression and function and regulation of Abcb1/P-gp and Abcg2/Bcrp1 in these tissues are poorly understood. This thesis investigates gestational changes in expression and function of Abcb1/P-gp and Abcg2/Bcrp1 in the placenta and fetal brain, in addition to regulation by steroids, progesterone and glucocorticoids. The effects of glucocorticoids on Abcb1/P-gp and Abcg2/Bcrp1 in the placenta and fetal brain are of importance given that 10% of pregnant women are treated with synthetic glucocorticoids during the management of threatened preterm labour. These studies demonstrate that the decrease in placental Abcb1/P-gp mediated fetal protection near term is compensated by an increase in Abcb1/P-gp and Abcg2/Bcrp1 mediated neuroprotection in the fetal brain; likely in preparation for life ex-utero. The lack of effects of progesterone and the dose-, age- and sex- dependent regulatory effects of synthetic glucocorticoid have highlighted the complexity associated with regulation of these transporters. Further, these studies are the first to report sexually dimorphic glucocorticoid effects on Abcb1/P-gp and Abcg2/Bcrp1 expression and function, with the female fetus being particularly susceptible to glucocorticoid these effects. In this regard, Abcb1/P-gp and Abcg2/Bcrp1 transport capacity may be altered when synthetic glucocorticoid is administered as a co-therapy, and as such, recipient sex should be considered during pharmacotherapy. Understanding the regulation of Abcb1/P-gp and Abcg2/Bcrp1 expression and function in the placenta and fetal brain during normal development and under pathological conditions is critical for fetal health and development, particularly when therapeutic strategies are utilized in pregnancy.Ph

Effects of sertraline and fluoxetine on p-glycoprotein at barrier sites: in vivo and in vitro approaches.

Retention of substances from systemic circulation in the brain and testes are limited due to high levels of P-glycoprotein (P-gp) in the luminal membranes of brain and testes capillary endothelial cells. From a clinical perspective, P-gp rapidly extrudes lipophilic therapeutic agents, which then fail to reach efficacious levels. Recent studies have demonstrated that acute administration of selective serotonin reuptake inhibitors (SSRI) can affect P-gp function, in vitro and in vivo. However, little is known concerning the time-course of these effects or the effects of different SSRI in vivo.The P-gp substrate, tritiated digoxin ([(3)H] digoxin), was co-administered with fluoxetine or sertraline to determine if either compound increased drug accumulation within the brains and testes of mice due to inhibition of P-gp activity. We undertook parallel studies in endothelial cells derived from brain microvessels to determine the dose-response and time-course of effects.In vitro, sertraline resulted in rapid and potent inhibition of P-gp function in brain endothelial cells, as determined by cellular calcein accumulation. In vivo, a biphasic effect was demonstrated. Brain accumulation of [(3)H] digoxin was increased 5 minutes after treatment with sertraline, but by 60 minutes after sertraline treatment, brain accumulation of digoxin was reduced compared to control. By 240 minutes after sertraline treatment brain digoxin accumulation was elevated compared to control. A similar pattern of results was obtained in the testes. There was no significant effect of fluoxetine on P-gp function, in vitro or in vivo.Acute sertraline administration can modulate P-gp activity in the blood-brain barrier and blood-testes barrier. This clearly has implications for the ability of therapeutic agents that are P-gp substrates, to enter the brain when co-administered with SSRI