Single-cell roadmap of human gonadal development.

Gonadal development is a complex process that involves sex determination followed by divergent maturation into either testes or ovaries1. Historically, limited tissue accessibility, a lack of reliable in vitro models and critical differences between humans and mice have hampered our knowledge of human gonadogenesis, despite its importance in gonadal conditions and infertility. Here, we generated a comprehensive map of first- and second-trimester human gonads using a combination of single-cell and spatial transcriptomics, chromatin accessibility assays and fluorescent microscopy. We extracted human-specific regulatory programmes that control the development of germline and somatic cell lineages by profiling equivalent developmental stages in mice. In both species, we define the somatic cell states present at the time of sex specification, including the bipotent early supporting population that, in males, upregulates the testis-determining factor SRY and sPAX8s, a gonadal lineage located at the gonadal-mesonephric interface. In females, we resolve the cellular and molecular events that give rise to the first and second waves of granulosa cells that compartmentalize the developing ovary to modulate germ cell differentiation. In males, we identify human SIGLEC15+ and TREM2+ fetal testicular macrophages, which signal to somatic cells outside and inside the developing testis cords, respectively. This study provides a comprehensive spatiotemporal map of human and mouse gonadal differentiation, which can guide in vitro gonadogenesis.

The deubiquitinase USP8 targets ESCRT-III to promote incomplete cell division.

In many vertebrate and invertebrate organisms, gametes develop within groups of interconnected cells called germline cysts formed by several rounds of incomplete divisions. We found that loss of the deubiquitinase USP8 gene in Drosophila can transform incomplete divisions of germline cells into complete divisions. Conversely, overexpression of USP8 in germline stem cells is sufficient for the reverse transformation from complete to incomplete cytokinesis. The ESCRT-III proteins CHMP2B and Shrub/CHMP4 are targets of USP8 deubiquitinating activity. In Usp8 mutant sister cells, ectopic recruitment of ESCRT proteins at intercellular bridges causes cysts to break apart. A Shrub/CHMP4 variant that cannot be ubiquitinated does not localize at abscission bridges and cannot complete abscission. Our results uncover ubiquitination of ESCRT-III as a major switch between two types of cell division.

Aged mice ovaries harbor stem cells and germ cell nests but fail to form follicles.

BACKGROUND: We recently published evidence to suggest that two populations of stem cells including very small embryonic-like stem cells (VSELs) and ovarian stem cells (OSCs) in ovary surface epithelium (OSE) undergo proliferation/differentiation, germ cell nests (GCN) formation, meiosis and eventually differentiate into oocytes that assemble as primordial follicles on regular basis during estrus cycle. Despite presence of stem cells, follicles get exhausted with advancing age in mice and result in senescence equivalent to menopause in women. Stem cells in aged ovaries can differentiate into oocytes upon transplantation into young ovaries, however, it is still not well understood why follicles get depleted with advancing age despite the presence of stem cells. The aim of the present study was to study stem cells and GCN in aged ovaries. METHODS: OSE cells from aged mice (> 18 months equivalent to > 55 years old women) were enzymatically separated and used to study stem cells. Viable (7-AAD negative) VSELs in the size range of 2-6 µm with a surface phenotype of Lin-CD45-Sca-1+ were enumerated by flow cytometry. Immuno-fluorescence and RT-PCR analysis were done to study stem/progenitor cells (OCT-4, MVH, SCP3) and transcripts specific for VSELs (Oct-4A, Sox-2, Nanog), primordial germ cells (Stella), germ cells (Oct-4, Mvh), early meiosis (Mlh1, Scp1) and ring canals (Tex14). RESULTS: Putative VSELs and OSCs were detected as darkly stained, spherical cells with high nucleo-cytoplasmic ratio along with germ cells nests (GCN) in Hematoxylin & Eosin stained OSE cells smears. Germ cells in GCN with distinct cytoplasmic continuity expressed OCT-4, MVH and SCP3. Transcripts specific for stem cells, early meiosis and ring canals were detected by RT-PCR studies. CONCLUSION: Rather than resulting as a consequence of accelerated loss of primordial follicle and their subsequent depletion, ovarian senescence/menopause occurs as a result of stem cells dysfunction. VSELs and OSCs exist along with increased numbers of GCNs arrested in pre-meiotic or early meiotic stage in aged ovaries and primordial follicle assembly is blocked possibly due to age-related changes in their microenvironment.

brinker levels regulated by a promoter proximal element support germ cell homeostasis.

A limited BMP signaling range in the stem cell niche of the ovary protects against germ cell tumors and promotes germ cell homeostasis. The canonical repressor of BMP signaling in both the Drosophila embryo and wing disc is the transcription factor Brinker (Brk), yet the expression and potential role of Brk in the germarium has not previously been described. Here, we find that brk expression requires a promoter-proximal element (PPE) to support long-distance enhancer action as well as to drive expression in the germarium. Furthermore, PPE subdomains have different activities; in particular, the proximal portion acts as a damper to regulate brk levels precisely. Using PPE mutants as well as tissue-specific RNA interference and overexpression, we show that altering brk expression within either the soma or the germline affects germ cell homeostasis. Remarkably, we find that Decapentaplegic (Dpp), the main BMP ligand and canonical antagonist of Brk, is upregulated by Brk in the escort cells of the germarium, demonstrating that Brk can positively regulate this pathway.

Drosophila p53 isoforms have overlapping and distinct functions in germline genome integrity and oocyte quality control.

p53 gene family members in humans and other organisms encode a large number of protein isoforms whose functions are largely undefined. Using Drosophila as a model, we find that a p53B isoform is expressed predominantly in the germline where it colocalizes with p53A into subnuclear bodies. It is only p53A, however, that mediates the apoptotic response to ionizing radiation in the germline and soma. In contrast, p53A and p53B are both required for the normal repair of meiotic DNA breaks, an activity that is more crucial when meiotic recombination is defective. We find that in oocytes with persistent DNA breaks p53A is also required to activate a meiotic pachytene checkpoint. Our findings indicate that Drosophila p53 isoforms have DNA lesion and cell type-specific functions, with parallels to the functions of mammalian p53 family members in the genotoxic stress response and oocyte quality control.

Effects of whole-life exposure to low-dose cadmium with post-weaning high-fat diet on offspring testes in a male mouse model.

Although several studies have reported testicular impairments caused by cadmium (Cd) or obesity alone, the combined effect of Cd and obesity on the testes and its underlying mechanism remains unclear. We examined the combined effect of whole-life exposure to low-dose Cd started at preconception and post-weaning high-fat diet (HFD) on the testes of offspring mice. At weaning, male offspring parented with and without exposure to low-dose Cd were continued on the same drinking water regimen as their parents and fed with either a normal diet (ND) or HFD for 10 or 24 weeks. Whole-life exposure to Cd resulted in its accumulation in testes, and HFD induced obesity and lipid metabolism disorder. Exposure to Cd or HFD alone significantly decreased Johnsen scores, disrupted testicular structure, and increased germ cell apoptosis at both 10 and 24 weeks. However, co-exposure to Cd and HFD did not induce the toxic effects that were induced by either alone, as revealed by preserved testicular structure and spermatogenesis, lack of significant apoptosis, and increased cell proliferation. Mechanistically, the combined effects of low-dose Cd and HFD consumption were associated with the activation of the JAK/STAT pathway. These findings suggest that co-exposure to low-dose Cd and HFD did not cause Cd- or HFD-induced testicular injury, probably because of the activation of the JAK/STAT pathway to prevent germ cell apoptosis.

Differential repression of Otx2 underlies the capacity of NANOG and ESRRB to induce germline entry.

Primordial germ cells (PGCs) arise from cells of the post-implantation epiblast in response to cytokine signaling. PGC development can be recapitulated in vitro by differentiating epiblast-like cells (EpiLCs) into PGC-like cells (PGCLCs) through cytokine exposure. Interestingly, the cytokine requirement for PGCLC induction can be bypassed by enforced expression of the transcription factor (TF) NANOG. However, the underlying mechanisms are not fully elucidated. Here, we show that NANOG mediates Otx2 downregulation in the absence of cytokines and that this is essential for PGCLC induction by NANOG. Moreover, the direct NANOG target gene Esrrb, which can substitute for several NANOG functions, does not downregulate Otx2 when overexpressed in EpiLCs and cannot promote PGCLC specification. However, expression of ESRRB in Otx2+/- EpiLCs rescues emergence of PGCLCs. This study illuminates the interplay of TFs occurring at the earliest stages of PGC specification.

Frazzled/Dcc acts independently of Netrin to promote germline survival during Drosophila oogenesis.

The Netrin receptor Frazzled/Dcc (Fra in Drosophila) functions in diverse tissue contexts to regulate cell migration, axon guidance and cell survival. Fra signals in response to Netrin to regulate the cytoskeleton and also acts independently of Netrin to directly regulate transcription during axon guidance in Drosophila. In other contexts, Dcc acts as a tumor suppressor by directly promoting apoptosis. In this study, we report that Fra is required in the Drosophila female germline for the progression of egg chambers through mid-oogenesis. Loss of Fra in the germline, but not the somatic cells of the ovary, results in the degeneration of egg chambers. Although a failure in nutrient sensing and disruptions in egg chamber polarity can result in degeneration at mid-oogenesis, these factors do not appear to be affected in fra germline mutants. However, similar to the degeneration that occurs in those contexts, the cell death effector Dcp-1 is activated in fra germline mutants. The function of Fra in the female germline is independent of Netrin and requires the transcriptional activation domain of Fra. In contrast to the role of Dcc in promoting cell death, our observations reveal a role for Fra in regulating germline survival by inhibiting apoptosis.

El consumo semicrónico moderado de alcohol altera la foliculogénesis y la calidad núcleo-citoplasmática oocitaria, en el ratón / Moderate semi-chronic alcohol consumption alters folliculogenesis and oocyte nuclear-cytoplasmic quality in mice

Resumen El consumo crónico de alcohol es un problema de salud mundial que afecta particularmente a la población femenina. Sin embargo, los efectos de la ingesta semicrónica en cantidades moderadas a bajas en el ovario y el oocito son poco conocidos. En un modelo murino, se administró etanol al 10% en agua de bebida (hembras tratadas) o agua (hembras control) por 15 días, y luego de la superovulación o no (ovulación espontánea), se analizó el ciclo estral y la calidad ovárico-gamética. En las hembras tratadas, la frecuencia y duración del diestro aumentó, y las frecuencias de folículos y cuerpos lúteos disminuyeron vs hembras controles, valores que se restauraron luego de la superovulación. Sin embargo, en las hembras tratadas, la tasa de proliferación celular folicular y el desbalance de la expresión ovárica de VEGF (factor de crecimiento endotelial) persistieron luego de la superovulación. El número de ovocitos ovulados con metafase II anormal, fragmentados y activados partenogenéticamente fue mayor en las hembras tratadas respecto las controles. En conclusión, el consumo semicrónico moderado de alcohol produce anestro, ciclo estral irregular, foliculogénesis deficiente y anomalías núcleo-citoplasmáticas en los oocitos ovulados. Estas alteraciones podrían constituirse en un factor etiológico de pérdida gestacional temprana y desarrollo embrionario anormal luego del consumo de alcohol.

Abstract Chronic alcohol consumption is a global health problem that particularly affects the female population. However, the ef-fects of semi-chronic ethanol intake in low-moderate amounts on the ovary and oocyte are poorly understood. In a mouse model, 10% ethanol was administered in drinking water (treated females) or water (control females) for 15 days, and after superovulation or not (spontaneous ovulation), the estrous cycle and ovarian-gametic quality were analyzed. In treated females, the frequency and duration of the diestrus increased, and the frequencies of follicles and corpus luteum decreased vs control females, values that restored after superovulation. However, in treated females, the follicular cell proliferation rate and the imbalance in ovarian expression of VEGF (endothelial growth factor) persisted after superovulation. The number of ovulated oocytes with abnormal metaphase II, fragmented and parthenogenetically activated was higher in treated females than in control ones. In conclusion, moderate semi-chronic alcohol consumption produces anestrum, irregular estrous cycle, poor folliculogenesis, and nuclear-cytoplasmic abnormalities in ovulated oocytes. These alterations could constitute an etiological factor of early gestational loss and abnormal embryonic development after alcohol consumption.

Neonatal Porcine Germ Cells Dedifferentiate and Display Osteogenic and Pluripotency Properties.

Gonocytes are progenitors of spermatogonial stem cells in the neonatal testis. We have previously shown that upon culturing, neonatal porcine gonocytes and their colonies express germ cell and pluripotency markers. The objectives of present study were to investigate in vitro trans-differentiation potential of porcine gonocytes and their colonies into cells from three germinal layers, and to assess pluripotency of cultured gonocytes/colonies in vivo. For osteogenic and tri-lineage differentiation, cells were incubated in regular culture media for 14 and 28 days, respectively. Cells were cultured for an additional 14 days for osteogenic differentiation or 7 days for differentiation into derivates of the three germinal layers. Osteogenic differentiation of cells and colonies was verified by Alizarin Red S staining and tri-lineage differentiation was confirmed using immunofluorescence and gene expression analyses. Furthermore, upon implantation into recipient mice, the cultured cells/colonies developed teratomas expressing markers of all three germinal layers. Successful osteogenic differentiation from porcine germ cells has important implications for bone regeneration and matrix formation studies. Hence, gonocytes emerge as a promising source of adult pluripotent stem cells due to the ability to differentiate into all germinal layers without typical biosafety risks associated with viral vectors or ethical implications.

Disparities between Uptake of Germline BRCA1/2 Gene Tests and Implementation of Post-test Management Strategies in Epithelial Ovarian, Fallopian Tube, or Primary Peritoneal Cancer Patients.

BACKGROUND: To assess the rate of germline BRCA gene tests in epithelial ovarian cancer (EOC) patients and uptake of post-test risk management strategies in BRCA1/2-mutated patients. METHODS: Institutional databases were searched to identify patients who were diagnosed with epithelial ovarian, fallopian tube, or primary peritoneal cancer (EOC) between 2009 and 2019 in two academic hospitals. Retrospective review on medical records was performed to collect clinico-pathologic variables, including performance of germline BRCA gene test and its results, as well as conduct of breast cancer screening tests and cascade testing. If annual mammography +/- breast ultrasonography was performed, it was considered that regular breast cancer surveillance was done. RESULTS: A total of 840 women with EOC were identified during the study period. Of these, 454 patients (54.0%) received BRCA gene testing and 106 patients (106/454, 23.3%) were positive for BRCA1/2 mutations. The rate of BRCA tests has markedly increased from 25.8% in 2009-2012 to 62.7% in 2017-2019. Among the 93 patients with BRCA1/2 mutation without previous personal breast cancer history, 20 patients (21.5%) received annual mammography with or without breast ultrasonography for regular surveillance. Among the 106 BRCA1/2-mutated EOC patients, cascade testing on family members was performed only in 13 patients (12.3%). CONCLUSION: Although BRCA1/2 gene tests have been substantially expanded, the uptake of post-test risk management strategies, including breast cancer screening for BRCA1/2-mutated patients and cascade testing for family members, has remained low. Strategies to increase its uptake and education about the importance of post-test risk managements are needed.

Heading towards a dead end: The role of DND1 in germ line differentiation of human iPSCs.

The DND microRNA-mediated repression inhibitor 1 (DND1) is a conserved RNA binding protein (RBP) that plays important roles in survival and fate maintenance of primordial germ cells (PGCs) and in the development of the male germline in zebrafish and mice. Dead end was shown to be expressed in human pluripotent stem cells (PSCs), PGCs and spermatogonia, but little is known about its specific role concerning pluripotency and human germline development. Here we use CRISPR/Cas mediated knockout and PGC-like cell (PGCLC) differentiation in human iPSCs to determine if DND1 (1) plays a role in maintaining pluripotency and (2) in specification of PGCLCs. We generated several clonal lines carrying biallelic loss of function mutations and analysed their differentiation potential towards PGCLCs and their gene expression on RNA and protein levels via RNA sequencing and mass spectrometry. The generated knockout iPSCs showed no differences in pluripotency gene expression, proliferation, or trilineage differentiation potential, but yielded reduced numbers of PGCLCs as compared with their parental iPSCs. RNAseq analysis of mutated PGCLCs revealed that the overall gene expression remains like non-mutated PGCLCs. However, reduced expression of genes associated with PGC differentiation and maintenance (e.g., NANOS3, PRDM1) was observed. Together, we show that DND1 iPSCs maintain their pluripotency but exhibit a reduced differentiation to PGCLCs. This versatile model will allow further analysis of the specific mechanisms by which DND1 influences PGC differentiation and maintenance.

Multifaceted roles of centrosomes in development, health, and disease.

The centrosome is a membrane-less organelle consisting of a pair of barrel-shaped centrioles and pericentriolar material and functions as the major microtubule-organizing center and signaling hub in animal cells. The past decades have witnessed the functional complexity and importance of centrosomes in various cellular processes such as cell shaping, division, and migration. In addition, centrosome abnormalities are linked to a wide range of human diseases and pathological states, such as cancer, reproductive disorder, brain disease, and ciliopathies. Herein, we discuss various functions of centrosomes in development and health, with an emphasis on their roles in germ cells, stem cells, and immune responses. We also discuss how centrosome dysfunctions are involved in diseases. A better understanding of the mechanisms regulating centrosome functions may lead the way to potential therapeutic targeting of this organelle in disease treatment.

Efficient gene transfer into zebra finch germline-competent stem cells using an adenoviral vector system.

Zebra finch is a representative animal model for studying the molecular basis of human disorders of vocal development and communication. Accordingly, various functional studies of zebra finch have knocked down or introduced foreign genes in vivo; however, their germline transmission efficiency is remarkably low. The primordial germ cell (PGC)-mediated method is preferred for avian transgenic studies; however, use of this method is restricted in zebra finch due to the lack of an efficient gene transfer method for the germline. To target primary germ cells that are difficult to transfect and manipulate, an adenovirus-mediated gene transfer system with high efficiency in a wide range of cell types may be useful. Here, we isolated and characterized two types of primary germline-competent stem cells, PGCs and spermatogonial stem cells (SSCs), from embryonic and adult reproductive tissues of zebra finch and demonstrated that genes were most efficiently transferred into these cells using an adenovirus-mediated system. This system was successfully used to generate gene-edited PGCs in vitro. These results are expected to improve transgenic zebra finch production.

In vivo and in vitro protective effects of the Wuzi Yanzong pill against experimental spermatogenesis disorder by promoting germ cell proliferation and suppressing apoptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Wuzi Yanzong pill (WZYZP) is a classical traditional Chinese medicine (TCM) formula originated from the Tang dynasty. WZYZP has a long history of use for reinforcing kidney and alleviating male infertility in China. AIM OF THE STUDY: The effect of WZYZP on male infertility and the mechanism underlying this effect was not clarified clearly. Therefore, this study aimed to investigate the protective effect of WZYZP in experimental spermatogenesis disorder via in vivo and in vitro studies, to promote the use of this formula for the treatment of spermatogenesis disorder. MATERIAL AND METHODS: Male SD rats were exposed to tripterygium glycosides to induce experimental spermatogenesis disorder, and WZYZP was subsequently administrated at different dosages for treatment. Sperm counts, sperm motility, and serum hormone levels were detected. HE staining and TUNEL staining were performed to evaluate the pathological lesions and apoptosis of testes, respectively. Next, germ cells were isolated from spermatogenesis disorder-model rats and treated with WZYZP- containing serum at different concentrations. CCK-8 assay and flow cytometry assay were performed to detect cell proliferation and apoptosis. Immunofluorescence assay, qRT-PCR and Western blotting analyses were performed to detect the expression of Beclin 1, LC3 and TGF-ß-PI3k/AKT-mTOR pathway - related factors, including TGF-ß, PI3K, AKT, mTOR, 4 EBP-1 and p70S6K. RESULTS: In vivo experiments showed that WZYZP protected against spermatogenesis disorder in model rats by improving sperm count and motility, as well as restoring serum hormone levels. HE and TUNEL staining demonstrated that the pathological injuries and cell apoptosis in testes of the model rats were alleviated by WZYZP treatment. Moreover, in vitro experiments of germ cells isolated from spermatogenesis disorder-model rats showed that WZYZP treatment increased the cell proliferation, inhibited cell apoptosis and autophagy. qRT-PCR and Western blotting assay results showed that this protective effect was associated with the regulation of the TGF-ß/PI3K/AKT/mTOR signaling pathway. The expression levels of p-PI3K/PI3K, p-AKT/AKT, p-mTOR/mTOR, 4 EBP-1 and p70S6K were increased, while TGF-ß was inhibited in the WZYZP treated groups. CONCLUSION: The results showed that WZYZP could protect against experimental spermatogenesis disorder by increasing the germ cell proliferation and inhibiting their apoptosis. Our support the clinical use of this formula for the management of spermatogenesis disorder.

A chromodomain protein mediates heterochromatin-directed piRNA expression.

PIWI-interacting RNAs (piRNAs) play significant roles in suppressing transposons, maintaining genome integrity, and defending against viral infections. How piRNA source loci are efficiently transcribed is poorly understood. Here, we show that in Caenorhabditis elegans, transcription of piRNA clusters depends on the chromatin microenvironment and a chromodomain-containing protein, UAD-2. piRNA clusters form distinct focus in germline nuclei. We conducted a forward genetic screening and identified UAD-2 that is required for piRNA focus formation. In the absence of histone 3 lysine 27 methylation or proper chromatin-remodeling status, UAD-2 is depleted from the piRNA focus. UAD-2 recruits the upstream sequence transcription complex (USTC), which binds the Ruby motif to piRNA promoters and promotes piRNA generation. Vice versa, the USTC complex is required for UAD-2 to associate with the piRNA focus. Thus, transcription of heterochromatic small RNA source loci relies on coordinated recruitment of both the readers of histone marks and the core transcriptional machinery to DNA.

Altered germline cyst formation and oogenesis in Tex14 mutant mice.

During oocyte differentiation in mouse fetal ovaries, sister germ cells are connected by intercellular bridges, forming germline cysts. Within the cyst, primary oocytes form via gaining cytoplasm and organelles from sister germ cells through germ cell connectivity. To uncover the role of intercellular bridges in oocyte differentiation, we analyzed mutant female mice lacking testis-expressed 14 (TEX14), a protein involved in intercellular bridge formation and stabilization. In Tex14 homozygous mutant fetal ovaries, germ cells divide to form a reduced number of cysts in which germ cells remained connected via syncytia or fragmented cell membranes, rather than normal intercellular bridges. Compared with wild-type cysts, homozygous mutant cysts fragmented at a higher frequency and produced a greatly reduced number of primary oocytes with precocious cytoplasmic enrichment and enlarged volume. By contrast, Tex14 heterozygous mutant germline cysts were less fragmented and generate primary oocytes at a reduced size. Moreover, enlarged primary oocytes in homozygous mutants were used more efficiently to sustain folliculogenesis than undersized heterozygous mutant primary oocytes. Our observations directly link the nature of fetal germline cysts to oocyte differentiation and development.

Response to stress in biological disorders: Implications of stress granule assembly and function.

It is indispensable for cells to adapt and respond to environmental stresses, in order for organisms to survive. Stress granules (SGs) are condensed membrane-less organelles dynamically formed in the cytoplasm of eukaryotes cells to cope with diverse intracellular or extracellular stress factors, with features of liquid-liquid phase separation. They are composed of multiple constituents, including translationally stalled mRNAs, translation initiation factors, RNA-binding proteins and also non-RNA-binding proteins. SG formation is triggered by stress stimuli, viral infection and signal transduction, while aberrant assembly of SGs may contribute to tissue degenerative diseases. Recently, a growing body of evidence has emerged on SG response mechanisms for cells facing high temperatures, oxidative stress and osmotic stress. In this review, we aim to summarize factors affecting SGs assembly, present the impact of SGs on germ cell development and other biological processes. We particularly emphasize the significance of recently reported RNA modifications in SG stress responses. In parallel, we also review all current perspectives on the roles of SGs in male germ cells, with a particular focus on the dynamics of SG assembly.

Detection of Caenorhabditis elegans Germ Cell Apoptosis Following Exposure to Environmental Contaminant Mixtures: A Crude Oil-Dispersant Mixture Example.

Crude oil disasters, such as the Deepwater Horizon accident, have caused severe environmental contamination and damage, affecting the health of marine and terrestrial organisms. Some previous studies have demonstrated cleanup efforts using chemical dispersant induced more potent toxicities than oil alone due to an increase in bioavailability of crude oil components, such as PAHs. However, there still lacks a systematic procedure that provides methods to determine genotypic and phenotypic changes following exposure to environmental toxicants or toxicant mixture, such as dispersed crude oil. Here, we describe methods for identifying a mechanism of dispersed crude oil-induced reproductive toxicity in the model organisms, Caenorhabditis elegans (C. elegans). Due to the genetic malleability of C. elegans, two mutant strains outlined in this chapter were used to identify a pathway responsible for inducing apoptosis: MD701 bcIs39 [lim-7p::ced-1::GFP + lin-15(+)], a mutant strain that allows visualization of apoptotic bodies via a green fluorescent protein fused to CED-1; and TJ1 (cep-1(gk138) I.), a p53/CEP-1 defective strain that is unable to activate apoptosis via the p53/CEP-1 pathway. In addition, qRT-PCR was utilized to demonstrate the aberrant expression of apoptosis (ced-13, ced-3, ced-4, ced-9, cep-1, dpl-1, efl-1, efl-2, egl-1, egl-38, lin-35, pax-2, and sir-2.1) and cytochrome P450 (cyp14a3, cyp35a1, cyp35a2, cyp35a5, and cyp35c1) protein-coding genes following exposure to dispersed crude oil. The procedure outlined here can be applicable to determine whether environmental contaminants, most of time contaminant mixture, cause reproductive toxicity by activation of the proapoptotic, p53/CEP-1 pathway.

Direct repression of Nanog and Oct4 by OTX2 modulates the contribution of epiblast-derived cells to germline and somatic lineage.

In mammals, the pre-gastrula proximal epiblast gives rise to primordial germ cells (PGCs) or somatic precursors in response to BMP4 and WNT signaling. Entry into the germline requires activation of a naïve-like pluripotency gene regulatory network (GRN). Recent work has shown that suppression of OTX2 expression in the epiblast by BMP4 allows cells to develop a PGC fate in a precise temporal window. However, the mechanisms by which OTX2 suppresses PGC fate are unknown. Here, we show that, in mice, OTX2 prevents epiblast cells from activating the pluripotency GRN by direct repression of Oct4 and Nanog. Loss of this control during PGC differentiation in vitro causes widespread activation of the pluripotency GRN and a deregulated response to LIF, BMP4 and WNT signaling. These abnormalities, in specific cell culture conditions, result in massive germline entry at the expense of somatic mesoderm differentiation. Increased generation of PGCs also occurs in mutant embryos. We propose that the OTX2-mediated repressive control of Oct4 and Nanog is the basis of the mechanism that determines epiblast contribution to germline and somatic lineage.