In vitro oogenesis from murine premeiotic germ cells using a new three-dimensional culture system.

A faithful reconstitution of the complete process of oogenesis in vitro is helpful for understanding the molecular mechanisms, genetics, and epigenetic changes related to gametogenesis; it can also be useful for clinical drug screening, disease research, and regenerative medicine. To this end, given the consensus that murine female germ cells initiate meiosis at E13.5, substantial works have reported the successful generation of fertile oocytes using E12.5 female gonads as starting materials. Nevertheless, our data demonstrated that murine germ cells at E12.5 have heterogeneously initiated a meiotic transcriptional program based on a measurement of pre-mRNAs (unspliced) and mature mRNAs (spliced) at a single-cell level. Therefore, to establish a platform that faithfully recapitulates the entire process in vitro (from premeiotic murine germ cells to fully developed oocytes), we here report a novel three-dimensional organoid culture (3-DOC) system, which successfully induced fully developed oocytes from E11.5 premeiotic female germ cells (oogonia). Compared with 2D culture and other 3D culture methods, this new culture system is more cost-effective and can create high-quality oocytes similar to in vivo oocytes. In summary, our new culture platform provides an experimental model for future research in regenerative medicine and reproductive biology.

H3K4me3 as a target of di(2-ethylhexyl) phthalate (DEHP) impairing primordial follicle assembly.

Di (2-ethylhexyl) phthalate (DEHP) is a widely used plastics additive that growing evidence indicates as endocrine disruptor able to negatively affect various reproductive processes both in female and male animals, including humans. However, the precise molecular mechanism of such actions is not completely understood. In the present study, scRNA-seq was performed on the ovaries of offspring from mothers exposed to DEHP from 16.5 days post coitum to 3 days post-partum, when the primordial follicle (PF) stockpile is established. While the histological observations of the offspring ovaries from DEHP exposed mothers confirmed previous data about a distinct reduction of oocytes enclosed in PFs. Focusing on oocytes, scRNA-seq analyses showed that the genes that mostly changed by DEHP were enriched GO terms related to histone H3-K4 methylation. Moreover, we observed H3K4me3 level, an epigenetics modification of H3 that is crucial for chromatin transcription, decreased by 40.28% (P < 0.01) in DEHP-treated group compared with control. When the newborn ovaries were cultured in vitro, the DEHP effects were abolished by tamoxifen (an estrogen receptor antagonist) or overexpression of Smyd3 (one specific methyltransferase of H3K4me3), in particular, the percentage of oocyte enclosed in PF was increased by 15.39% in DEHP plus Smyd3 overexpression group than of DEHP group (P < 0.01), which was accompanied by the upregulation of H3K4me3. Collectively, the present results discover Smyd3-H3K4me3 as a novel target of the deleterious ER-mediated effect of DEHP on PF formation during early folliculogenesis in the mouse and highlight epigenetics changes as prominent targets of endocrine disruptors like DEHP.

Oxidative stress as a plausible mechanism for zearalenone to induce genome toxicity.

Zearalenone (ZEN), a common non-steroidal estrogenic mycotoxin of the Fusarium genus, is one of the most frequent and powerful contaminant of grains and cereal products representing a serious threat for people and livestock health. In fact, ZEN causes cytotoxicity and genotoxicity in a variety of cell types at least in part through binding to estrogen receptors (ERs). The main pathways through which ZEN induces such effects remain, however, elusive. In particular, how the mycotoxin causes DNA damage, dysregulates DNA repair mechanisms, changes epigenome of targeted cells and, not least, affects chromatin conformation and non-coding RNA (ncRNA), is unclear. In the present paper, following extensive review of the literature about such ZEN effects and our own experience in studying the effects of this compound on reproductive processes, we propose that increased production of reactive oxygen species (ROS) and consequently oxidative stress (OS) are central in ZEN genotoxicity. Besides to shed light on the action mechanisms of the mycotoxin, this notion might help to develop effective strategies to counteract its deleterious biological effects.

The proliferation role of LH on porcine primordial germ cell-like cells (pPGCLCs) through ceRNA network construction.

BACKGROUND: The transdifferentiation of skin-derived stem cells (SDSCs) into primordial germ cell-like cells (PGCLCs) is one of the major breakthroughs in the field of stem cells research in recent years. This technology provides a new theoretical basis for the treatment of human infertility. However, the transdifferentiation efficiency of SDSCs to PGCLCs is very low, and scientists are still exploring ways to improve this efficiency or promote the proliferation of PGCLCs. This study aims to investigate the molecular mechanism of luteinising hormone (LH) to enhance porcine PGCLCs (pPGCLCs) proliferation. RESULTS: In this study, we dissected the proliferation regulatory network of pPGCLCs by whole transcriptome sequencing, and the results showed that the pituitary-secreted reproductive hormone LH significantly promoted the proliferation of pPGCLCs. We combined whole transcriptome sequencing and related validation experiments to explore the mechanism of LH on the proliferation of pPGCLCs, and found that LH could affect the expression of Hippo signalling pathway-related mRNAs, miRNAs and lncRNAs in pPGCLCs. CONCLUSIONS: For the first time, we found that LH promotes pPGCLCs proliferation through the competing endogenous RNA (ceRNA) regulatory networks and Hippo signalling pathway. This finding may help to elucidate the molecular mechanism by which LH promotes pPGCLCs proliferation.

Maternal Zearalenone exposure impacted ovarian follicle formation and development of suckled offspring.

Zearalenone (ZEN) is a secondary metabolite, which is mainly produced by Fusarium fungi and exists in various feeds and agricultural products. Recently, an increasing amount of data has shown that ZEN, as an estrogen-like hormone, can have harmful effects on the female reproductive system, especially on oogenesis and folliculogenesis. Breast milk is considered to be the ideal form of nutrition for infants; however, there are some records of contaminants in food, such as mycotoxins, which may be transferred from maternal blood to milk. In this study, we investigated the toxic effects of breast milk on folliculogenesis in offspring following maternal ZEN exposure. Our results showed that maternal ZEN exposure significantly inhibited the process of primordial follicle (PF) assembly and reduced the number of PFs in suckled offspring's ovaries. In addition, RNA-seq analysis showed that RIG-I-like receptor (RLRs) signaling pathways were activated after exposed to ZEN, which increased the expression levels of DNA damage (γ-H2AX, RAD51, and PARP1) and apoptosis related protein (BAX/BCL2 and Caspase-3). Finally, ZEN exposure interfered with follicular development, as evidenced by the reduced percentages of oocyte maturation and embryonic development when the offspring grew to adolescence. It is worth noting that maternal ZEN exposure disrupted the tri-methylation levels of H3K4, H3K9, and H3K27 in the offspring's oocytes. Our results indicated that maternal ZEN exposure affected ovarian development in offspring through the breast milk, which may be detrimental to their reproductive capability in adult life.

A hypothetical role for autophagy during the day/night rhythm-regulated melatonin synthesis in the rat pineal gland.

Melatonin is a highly conserved molecule that regulates day/night rhythms; it is associated with sleep improvement, reactive oxygen species (ROS) scavenging, anti-aging effects, and seasonal and circadian rhythms and has been a hot topic of research for decades. Using single-cell RNA sequencing, a recent study describes a single-cell transcriptome atlas for the rat pineal gland. Based on a more comprehensive analysis of the retrieved data (Mays et al., PLoS One, 2018, 13, e0205883), results from the current study unveiled the underappreciated gene regulatory network behind different cell populations in the pineal gland. More importantly, our study here characterized, for the first time, the day/night activation of autophagy flux in the rat pineal gland, indicating a potential role of autophagy in regulating melatonin synthesis in the rat pineal gland. These findings emphasized a hypothetical role of day/night autophagy in linking the biological clock with melatonin synthesis. Furthermore, ultrastructure analysis of pinealocytes provided fascinating insights into differences in their intracellular structure between daytime and nighttime. In addition, we also provide a preliminary description of cell-cell communication in the rat pineal gland. In summary, the current study unveils the day/night regulation of autophagy in the rat pineal gland, raising a potential role of autophagy in day/night-regulated melatonin synthesis.

Inflammatory cytokines as key players of apoptosis induced by environmental estrogens in the ovary.

Natural and synthetic environmental estrogens (EEs), interfering with the physiological functions of the body's estrogens, are widespread and are rising much concern for their possible deleterious effects on human and animal health, in particular on reproduction. In fact, increasing evidence indicate that EEs can be responsible for a variety of disfunctions of the reproductive system especially in females such as premature ovarian insufficiency (POI). Because of their great structural diversity, the modes of action of EEs are controversial. One important way through which EEs exert their effects on reproduction is the induction of apoptosis in the ovary. In general, EEs can exert pro-and anti-apoptotic effects by agonizing or antagonizing numerous estrogen-dependent signaling pathways. In the present work, results concerning apoptotic pathways and diseases induced by representative EEs (such as zearalenone, bisphenol A and di-2-ethylhexyl phthalate), in ovaries throughout development are presented into an integrated network. By reviewing and elaborating these studies, we propose inflammatory factors, centered on the production of tumor necrosis factor (TNF), as a major cause of the induction of apoptosis by EEs in the mammalian ovary. As a consequence, potential strategies to prevent such EE effect are suggested.

Di (2-ethylhexyl) phthalate impairs primordial follicle assembly by increasing PDE3A expression in oocytes.

It is known that Di (2-ethylhexyl) phthalate (DEHP) may impact mammalian reproduction and that in females one target of the drug's action is follicle assembly. Here we revisited the phthalate's action on the ovary and from bioinformatics analyses of the transcriptome performed on newborn mouse ovaries exposed in vitro to DEHP, up-regulation of PDE3A, as one of the most important alterations caused by DEHP on early folliculogenesis, was identified. We obtained some evidence suggesting that the decrease of cAMP level in oocytes and the parallel decrease of PKA expression, consequent on the PDE3A increase, were a major cause of the reduction of follicle assembly in the DEHP-exposed ovaries. In fact, Pde3a RNAi on cultured ovaries reducing cAMP and PKA decrease counteracted the primordial follicle assembly impairment caused by the compound. Moreover, RNAi normalized the level of Kit, Nobox, Figla mRNA and GDF9, BMP15, CX37, γH2AX proteins in oocytes, and KitL transcripts in granulosa cells as well as their proliferation rate altered by DEHP exposure. Taken together, these results identify PDE3A as a new critical target of the deleterious effects of DEHP on early oogenesis in mammals and highlight cAMP-dependent pathways as major regulators of oocyte and granulosa cell activities crucial for follicle assembly. Moreover, we suggest that the level of intracellular cAMP in the oocytes may be an important determinant for their capability to repair DNA lesions caused by DNA damaging compounds including DEHP.

Maternal Bisphenol S exposure affects the reproductive capacity of F1 and F2 offspring in mice.

Bisphenol S (BPS) is an endocrine disruptor which is widely used in commercial plastic products. Previous studies have shown that exposure to BPS has toxic effects on various aspects of mammalian, but there are few reports about reproductive toxicity. In order to investigate the effects of maternal BPS exposure on the reproductive of F1 and F2 female mice, the pregnant mice were orally administered with different dosages of BPS only once every day from 12.5 to 15.5 days post-coitus (dpc). The results showed that maternal BPS exposure to 2 µg per kg of body weight per day (2 µg/kg) and 10 µg/kg accelerated the meiotic prophase I (MPI) of F1 female mice and the expression of the genes related to meiotic were increased. Further studies showed that maternal BPS exposure resulted in a significant increase in the percentage of oocytes enclosed in primordial follicles in the 3 days post-partum (3 dpp) ovaries of F1 female mice. And at the time of 21 days post-partum (21 dpp) in F1 female mice, the number of antral follicles were significantly lower compare to controls. In the study of five-week female mice of F1, we found that BPS disturbed the folliculogenesis, and the maturation rates and fertilization rates of oocytes were significantly decreased. Of note, maternal BPS exposure disrupted H3K4 and H3K9 tri-methylation levels in F1 ovaries. Maternal BPS exposure only affected the cyst breakdown in F2 female mice. Taken together, our results suggest that, maternal BPS exposure impaired the process of meiosis and oogenesis of F1 and F2 offspring, resulting in abnormal follicular development and serious damage to the reproduction.

Effects of Ochratoxin A exposure on DNA damage in porcine granulosa cells in vitro.

Ochratoxin A (OTA), a feed mycotoxin, tends to impair the reproductive performance of animals. Our previous studies have demonstrated that OTA exposure inhibits porcine ovarian granulosa cell (GC) proliferation and induces their apoptosis, but the underlying toxic mechanism is still uncertain. In this study, we explored the OTA exposure on porcine GCs in vitro and found that OTA exposure inhibited the proliferation of porcine GCs and arrested cell cycle of GCs in the G2/M phase. The results based on RNA-Seq revealed that 20 µM and 40 µM OTA exposure increase DNA damage of porcine GCs in vitro. The differentially expressed genes (DEGs) of 40 µM OTA exposure were enriched in the pathways of mismatch repair, nucleotide excision repair and homologous recombination in DNA replication compared with control group and 20 µM OTA exposure group. Meanwhile, OTA exposure increased the expression levels of DNA double-strand breaks (DSBs) gene γ-H2AX, and DNA repair related genes, such as BRCA1, XRCC1, PARP1, and RAD51. Above all, our research revealed that OTA might exert deleterious effects on porcine ovarian GCs, influencing DNA repair-related biological processes and causing DNA damage response.

[A review of mixed gas detection system based on infrared spectroscopic technique].

In order to provide the experiences and references to the researchers who are working on infrared (IR) mixed gas detection field. The proposed manuscript reviews two sections of the aforementioned field, including optical multiplexing structure and detection method. At present, the coherent light sources whose representative are quantum cascade laser (QCL) and inter-band cascade laser(ICL) become the mainstream light source in IR mixed gas detection, which replace the traditional non-coherent light source, such as IR radiation source and IR light emitting diode. In addition, the photon detector which has a super high detectivity and very short response time is gradually beyond thermal infrared detector, dominant in the field of infrared detector. The optical multiplexing structure is the key factor of IR mixed gas detection system, which consists of single light source multi-plexing detection structure and multi light source multiplexing detection structure. Particularly, single light source multiplexing detection structure is advantages of small volume and high integration, which make it a plausible candidate for the portable mixed gas detection system; Meanwhile, multi light source multiplexing detection structure is embodiment of time division multiplex, frequency division multiplexing and wavelength division multiplexing, and become the leading structure of the mixed gas detection system because of its wider spectral range, higher spectral resolution, etc. The detection method applied to IR mixed gas detection includes non-dispersive infrared (NDIR) spectroscopy, wavelength and frequency-modulation spectroscopy, cavity-enhanced spectroscopy and photoacoustic spectroscopy, etc. The IR mixed gas detection system designed by researchers after recognizing the whole sections of the proposed system, which play a significant role in industrial and agricultural production, environmental monitoring, and life science, etc.