Primordial follicle assembly in the mouse occurs during perinatal ages and largely determines the ovarian reserve that will be available to support the reproductive life span. The development of primordial follicles is controlled by a complex network of interactions between oocytes and ovarian somatic cells that remain poorly understood. In the present research, using single-cell RNA sequencing performed over a time series on murine ovaries, coupled with several bioinformatics analyses, the complete dynamic genetic programs of germ and granulosa cells from E16.5 to postnatal day (PD) 3 were reported. Along with confirming the previously reported expression of genes by germ cells and granulosa cells, our analyses identified 5 distinct cell clusters associated with germ cells and 6 with granulosa cells. Consequently, several new genes expressed at significant levels at each investigated stage were assigned. By building single-cell pseudotemporal trajectories, 3 states and 1 branch point of fate transition for the germ cells were revealed, as well as for the granulosa cells. Moreover, Gene Ontology (GO) term enrichment enabled identification of the biological process most represented in germ cells and granulosa cells or common to both cell types at each specific stage, and the interactions of germ cells and granulosa cells basing on known and novel pathway were presented. Finally, by using single-cell regulatory network inference and clustering (SCENIC) algorithm, we were able to establish a network of regulons that can be postulated as likely candidates for sustaining germ cell-specific transcription programs throughout the period of investigation. Above all, this study provides the whole transcriptome landscape of ovarian cells and unearths new insights during primordial follicle assembly in mice.
Di (2-ethylhexyl) phthalate (DEHP), is the most common member of the class of phthalates that are used as plasticizers and have become common environmental contaminants. A number of studies have shown that DEHP exposure impacts reproductive health in both male and female mammals by acting as an estrogen analog. Here, we investigated the effects of DEHP on meiotic progression of fetal mouse oocytes by using an in vitro model of ovarian tissue culture. The results showed that 10 or 100 μM DEHP exposure inhibited the progression of oocytes throughout meiotic prophase I, specifically from the pachytene to diplotene stages. DEHP possibly impairs the ability to repair DNA double-strand breaks induced by meiotic recombination and as a consequence activates a pachytene check point. At later stages, such defects led to an increased number of oocytes showing apoptotic markers (TUNEL staining, expression of pro-apoptotic genes), resulting in reduced oocyte survival, gap junctions, and follicle assembly in the ovarian tissues. Microarray analysis of ovarian tissues exposed to DEHP showed altered expression of several genes including some involved in apoptosis and gonad development. The expression changes of some genes clustered in cell-cell communication and signal transduction, along with plasma membrane, extracellular matrix and ion channel function classes, were dependent on the DEHP concentration. Together, these results bring new support to the notion that exposure to DEHP during gestation might exert deleterious effects on ovary development, perturbing germ cell meiosis and the expression of genes involved in a wide range of biological processes including ovary development.
Diethylhexyl phthalate (DEHP) is a widely used industrial additive for increasing plastic flexibility. It disrupts the physiological functions of endogenous hormones and induces abnormal development of mammals. The objectives of the present study were to evaluate the effects of DEHP exposure on ovarian development of pregnant mice and whether the effects are inheritable. We found that the synthesis of oestradiol in pregnant mice after DEHP exposure was significantly decreased, and that the first meiotic progression of female fetal germ cells was delayed. Furthermore, the DNA methylation level of Stra8 was increased and the expression levels of Stra8 were significantly decreased. An accelerated rate of follicle recruitment in F1 mice was responsible for the depletion of the primordial-follicle pool. Maternal DEHP exposure also significantly accelerated the recruitment of primordial follicles in F2 mice. In conclusion, our results indicated that maternal DEHP exposure induced ovarian development deficiency, which was transgenerational in mice.
The ability of metal oxide nanoparticles to penetrate the skin has aroused a great deal of interest during the past decade due to concerns over the safety of topically applied sunscreens that contain physical UV-resistant metal particles, such as nano-Zinc oxide (nZnO). Previous studies demonstrate that metal oxide nanoparticles accumulate in skin furrows and hair follicles following topical application while little is known about the consequence of these nanoparticles on skin homeostasis. The current investigation tested the effects of nZnO (0.5 mg/day mouse) on hair follicle physiology. Topical application of Vaseline containing nZnO, bulk ZnO (bZnO), or ionized Zn to newborn mice vibrissa pad over a period of 7 consecutive days revealed that nZnO accumulated within hair follicles, and this induced the apoptosis of hair follicle stem cells (HFSCs). In vitro studies also indicated that nZnO exposure caused obvious DNA damage and induced apoptosis in HFSCs. Furthermore, it was found that nZnO exposure perturbed genes associated with HFSC apoptosis, cell communication, and differentiation. HFSCs transplantation assay demonstrated that the potential of HFSCs to differentiate was reduced. This investigation indicates a potential risk of topically applied ZnO nanoparticles on skin homeostasis.
Di (2-ethylhexyl) phthalate (DEHP) is a plasticizer which is widely used in the manufacture of plastics. As a common environmental contaminant and recognized endocrine disrupting chemical, DEHP is able to deregulate the functions of a variety of tissues, including the reproductive system both in males and females. In order to investigate the possible effects of DEHP on the first wave of folliculogenesis, occurring in the mouse ovary postnatally, mice were administered 20 or 40 μg/kg DEHP through intraperitoneal injection at days 5, 10 and 15 post partum (dpp). Following DEHP treatment the gene expression profile of control and exposed ovaries was compared by microarray analyses at 20 dpp. We found that in the exposed ovaries DEHP significantly altered the transcript levels of several immune response and steroidogenesis associated genes. In particular, DEHP significantly decreased the expression of genes essential for androgen synthesis by theca cells including Lhcgr, Cyp17a1, Star and Ldlr. Immunohistochemistry and immune flow cytometry confirmed reduced expression of LHCGR and CYP17A1 proteins in the exposed theca cells. These effects were associated to a significant reduction in ovarian concentrations of progesterone, 17β-estradiol and androstenedione along with a reduction of LH in the serum. Although we did not find a significant reduction of the number of primary, secondary or antral follicles in the DEHP exposed ovaries when compared to controls, we did observe that theca cells showed an altered structure of the nuclear envelope, fewer mitochondria, and mitochondria with a reduced number of cristae. Collectively, these results demonstrate a deleterious effect of DEHP exposure on ovarian steroidogenesis during the first wave of folliculogenesis that could potentially affect the correct establishment of the hypothalamic-pituitary-ovarian axis and the onset of puberty.
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