Endometriosis (EMs) is an estrogen (E2)-dependent inflammatory disorder. Although EMs is considered a benign disease, it presents with malignant characteristics, such as migration and invasion. An increasing number of studies have shown that aberrantly expressed circular RNAs (circRNAs) play an essential role in disease development and progression. However, the mechanisms by which circRNAs exert their pathological effects in EMs remain unclear. Hsa_circ_0001649, a novel cancer-associated circRNA, has been previously reported to be downregulated in several cancer types and related to cell migration and invasion. In the present study, real-time PCR (qRT-PCR) was carried out to measure hsa_circ_0001649 levels in human tissues, human primary endometrial stromal cells (ESCs) and a human endometrial stromal cell line (ThESCs). Matrix metalloproteinase 9 (MMP9) levels in ESCs and ThESCs were assessed by qRT-PCR and Western blotting, and the migration and invasion capacities of ThESCs were evaluated by transwell assay. As a result, hsa_circ_0001649 expression was significantly decreased in ectopic and eutopic endometrial samples compared with that in normal endometrial samples. E2 decreased hsa_circ_0001649 expression but increased MMP9 expression in ESCs and ThESCs. Furthermore, ThESCs were more invasive under E2 stimulation. However, these effects disappeared when ICI or hsa_circ_0001649 transfection was used. Collectively, our findings reveal that decreased hsa_circ_0001649 expression plays a role in E2-increased MMP9 expression through E2 receptors (ERs), which have critical functions in EMs.
Qi Li, Na Li, Hengwei Liu, Yu Du, Haitang He, Ling Zhang, and Yi Liu
Gonçalo Pereira, Ricardo Bexiga, João Chagas e Silva, Elisabete Silva, Christelle Ramé, Joëlle Dupont, Yongzhi Guo, Patrice Humblot, and Luís Lopes-da-Costa
Adipokines emerged as regulators of metabolism and inflammation in several scenarios. This study evaluated the relationship between adipokines (adiponectin, chemerin and visfatin) and cytological (subclinical) endometritis, by comparing healthy (without), transient (recovered by 45 days postpartum (DPP)) and persistent (until 45 DPP) endometritis cows (n = 49). Cows with persistent endometritis had higher adiponectin concentrations in plasma (at 21 DPP, P < 0.05 and at 45 DPP, P < 0.01) and in uterine fluid (at 45 DPP, P < 0.001), and higher chemerin concentrations in plasma (P < 0.05) and uterine fluid (P < 0.01) at 45 DPP than healthy cows. Cows with persistent endometritis had higher gene transcription in the cellular pellet of uterine fluid and protein expression in the endometrium of these adipokines and their receptors than healthy cows. Adiponectin plasma concentrations allowed to discriminate healthy from persistent endometritis cows, in 87% (21 DPP) and 98% (45 DPP) of cases, and adiponectin and chemerin uterine fluid concentrations at 45 DPP allowed for this discrimination in 100% of cases. Cows with concentrations above the cutoff were a minimum of 3.5 (plasma 21 DPP), 20.4 (plasma 45 DPP), and 33.3 (uterine fluid 45 DPP) times more at risk of evidencing persistent endometritis at 45 DPP than cows with concentrations below the cutoff. Overall, results indicate a relationship between adipokine signalling and the inflammatory status of the postpartum uterus of dairy cows, evidencing that adipokines represent suitable biomarkers of subclinical endometritis, able to predict the risk of persistence of inflammation.
Yizi Wang, Minghui Chen, Jian Xu, Xinyan Liu, Yuwei Duan, Canquan Zhou, and Yanwen Xu
Luteinization is the event of corpus luteum formation, a way of follicle cells transformation and a process of steroidogenesis alteration. As the core clock gene, Bmal1 was involved in regulation of ovulation process and luteal function afterwards. Till now, the underlying roles of luteinization played by Bmal1 remain unknown. To explore the unique role of Bmal1 in luteal steroidogenesis and its underlying pathway, we investigated luteal hormone synthesis profile in Bmal1 knockout female mice. We found that luteal hormone synthesis was notably impaired, and phosphorylation of PI3K/NfκB pathway was significantly activated. Then, the results were verified in in vitro cultured cells, including isolated Bmal1 interference granulosa cells (GCs) and theca cells (TCs) respectively. Hormones levels of supernatant culture media and mRNA expressions of steroidogenesis-associated genes (star, Hsd3β2, cyp19a1 in GCs, Lhcgr, star, Hsd3β2, cyp17a1 in TCs) were mutually decreased, while the phosphorylation of PI3K/NfκB was promoted during in vitro luteinization. After PI3K specific-inhibitor LY294002 intervention, mRNA expressions of Lhcgr and Hsd32 were partially-rescued in Bmal1 interference TCs, together with significantly increased androstenedione and T synthesis. Further exploration in TCs demonstrated BMAL1 interacted directly but negatively with NfκB p65 (RelA), a subunit which was supposed as a mediator in Bmal1-governed PI3K signaling regulation. Taken together, we verified the novel role of Bmal1 in luteal steroidogenesis, achieving by negative interplay with RelA mediated PI3K/NfκB pathway.
Roseanne Rosario, Hazel L Stewart, Emily Walshe, and Richard A Anderson
In female mammals, reproductive potential is determined during fetal life by the formation of a non-renewable pool of primordial follicles. Initiation of meiosis is one of the defining features of germ cell differentiation and is well established to commence in response to retinoic acid. WIN 18,446 inhibits the conversion of retinol to retinoic acid, and therefore it was used to explore the impact of reduced retinoic acid synthesis on meiotic progression and thus germ cell development and subsequent primordial follicle formation. e13.5 mouse fetal ovaries were cultured in vitro and treated with WIN 18,446 for the first 3 days of a total of up to 12 days. Doses as low as 0.01 µM reduced transcript levels of the retinoic acid response genes Stra8 and Rarβ without affecting germ cell number. Higher doses resulted in germ cell loss, rescued with the addition of retinoic acid. WIN 18,446 significantly accelerated the progression of prophase I; this was seen as early as 48 h post treatment using meiotic chromosome spreads and was still evident after 12 days of culture using Tra98/Msy2 immunostaining. Furthermore, ovaries treated with WIN 18,446 at e13.5 but not at P0 had a higher proportion of growing follicles compared to vehicle controls, thus showing evidence of increased follicle activation. These data therefore indicate that retinoic acid is not necessary for meiotic progression but may have a role in the regulation of its progression and germ cell survival at that time and provide evidence for a link between meiotic arrest and follicle growth initiation.
Hui Li, Qianhui Huang, Yu Liu, and Lana X. Garmire
Human placenta is a complex and heterogeneous organ interfacing between the mother and the fetus that supports fetal development. Alterations to placental structural components are associated with various pregnancy complications. To reveal the heterogeneity among various placenta cell types in normal and diseased placentas, as well as elucidate molecular interactions within a population of placental cells, a new genomics technology called single cell RNA-Seq (or scRNA-seq) has been employed in the last couple of years. Here we review the principles of scRNA-seq technology, and summarize the recent human placenta studies at scRNA-seq level across gestational ages as well as in pregnancy complications such as preterm birth and preeclampsia. We list the computational analysis platforms and resources available for the public use. Lastly, we discuss the future areas of interest for placenta single cell studies, as well as the data analytics needed to accomplish them.
Marta Almada, Lia Costa, Bruno Fonseca, Patrícia Alves, Jorge Braga, Daniela Gonçalves, Natércia Teixeira, and Georgina Correia-da-Silva
Proliferation, differentiation and apoptosis of trophoblast cells are required for normal placental development. Impairment of those processes may lead to pregnancy-related diseases. Disruption of endoplasmic reticulum (ER) homeostasis has been associated with several reproductive pathologies including recurrent pregnancy loss and preeclampsia. In the unfolded protein response (UPR), specific ER-stress signalling pathways are activated to restore ER homeostasis, but if the adaptive response fails, apoptosis is triggered. Protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1 (IRE1) and Activating transcription factor 6 (ATF6) are central players in UPR and in ER-stress-induced apoptosis, as well as downstream transcription factors, as C/EBP homologous protein (CHOP). Our previous studies have shown that the endocannabinoid 2-arachidonoylglycerol (2-AG) modulates trophoblast cell turnover. Nevertheless, the role of ER-stress on 2-AG induced apoptosis and cannabinoid signalling in trophoblast has never been addressed. In this work, we used BeWo cells and human primary cytotrophoblasts isolated from term-placenta. The expression of ER-stress markers was analysed by qRT-PCR and Western blotting. ROS generation was assessed by fluorometric methods, while apoptosis was detected by the evaluation of caspase -3/-7 activities and Poly (ADP-ribose) polymerase (PARP) cleavage. Our findings indicate that 2-AG is able to induce ER-stress and apoptosis. Moreover, the eukaryotic initiation factor 2 (eIF2α)/CHOP pathway involved in ER-stress-induced apoptosis is triggered through a mechanism dependent on cannabinoid receptor CB2 activation. The results bring novel insights on the importance of ER-stress and cannabinoid signalling on 2-AG mechanisms of action in placenta.
Hang Qi, Huiyu Zhang, Xiaoya Zhao, Ya Qin, Guiling Liang, Xiaoqing He, and Jian Zhang
Tubal endometriosis (tubal EM) is a subtype of endometriosis (EM) associated with fallopian tube impairments and infertility. Since the molecular mechanism underlying tubal EM is not clear, we assume that an aberrant transcriptome of fallopian tube epithelium and microenvironment changes caused by cytokines in tubal fluid are possible causes. The aim of this study was to identify potential hub mRNAs/proteins of tubal EM through integrated transcriptomic and proteomic analyses and to elucidate significant pathways, cellular functions, and interaction networks during the initiation and progression of tubal EM. We obtained human fallopian tube epithelium and tubal fluid samples from patients with and without tubal EM. Tubal epithelia were analyzed using microarray, and tubal fluid was analyzed using quantitative label-free LC-MS/MS. We identified differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) and determined common mRNAs/protein. We observed 35 commonly deregulated mRNAs/proteins, and IPA indicated that cellular movement, inflammatory response, and immune cell trafficking were significantly activated during the pathogenesis of tubal EM. We also identified acute phase response signaling pathway activation as a unique pathogenesis signature of tubal EM. Our results demonstrate that an integrated analysis of the transcriptome and proteome has the potential to reveal novel disease mechanisms at a molecular level.
Hannah L Morgan, Isaac Ampong, Nader Eid, Charlène Rouillon, Helen R Griffiths, and Adam J Watkins
The link between male diet and sperm quality has received significant investigation. However, the impact diet and dietary supplements have on the testicular environment has been examined to a lesser extent. Here, we establish the impact of a sub-optimal low protein diet (LPD) on testicular morphology, apoptosis and serum fatty acid profiles. Furthermore, we define whether supplementing a LPD with specific methyl donors abrogates any detrimental effects of the LPD. Male C57BL6 mice were fed either a control normal protein diet (NPD; 18% protein; n = 8), an isocaloric LPD (LPD; 9% protein; n = 8) or an LPD supplemented with methyl donors (MD-LPD; choline chloride, betaine, methionine, folic acid, vitamin B12; n = 8) for a minimum of 7 weeks. Analysis of male serum fatty acid profiles by gas chromatography revealed elevated levels of saturated fatty acids and lower levels of mono- and polyunsaturated fatty acids in MD-LPD males when compared to NPD and/or LPD males. Testes of LPD males displayed larger seminiferous tubule cross section area when compared to NPD and MD-LPD males, while MD-LPD tubules displayed a larger luminal area. Furthermore, TUNNEL staining revealed LPD males possessed a reduced number of tubules positive for apoptosis, while gene expression analysis showed MD-LPD testes displayed decreased expression of the pro-apoptotic genes Bax, Csap1 and Fas when compared to NPD males. Finally, testes from MD-LPD males displayed a reduced telomere length but increased telomerase activity. These data reveal the significance of sub-optimal nutrition for paternal metabolic and reproductive physiology.
Katerina Menelaou, Malwina Prater, Simon J Tunster, Georgina E T Blake, Colleen Geary Joo, James C Cross, Russell S Hamilton, and Erica D Watson
Assisted reproduction technologies (ARTs) are becoming increasingly common. Therefore, how these procedures influence gene regulation and foeto-placental development are important to explore. Here, we assess the effects of blastocyst transfer on mouse placental growth and transcriptome. C57Bl/6 blastocysts were transferred into uteri of B6D2F1 pseudopregnant females and dissected at embryonic day 10.5 for analysis. Compared to non-transferred controls, placentas from transferred conceptuses weighed less even though the embryos were larger on average. This suggested a compensatory increase in placental efficiency. RNA sequencing of whole male placentas revealed 543 differentially expressed genes (DEGs) after blastocyst transfer: 188 and 355 genes were downregulated and upregulated, respectively. DEGs were independently validated in male and female placentas. Bioinformatic analyses revealed that DEGs represented expression in all major placental cell types and included genes that are critical for placenta development and/or function. Furthermore, the direction of transcriptional change in response to blastocyst transfer implied an adaptive response to improve placental function to maintain foetal growth. Our analysis revealed that CpG methylation at regulatory regions of two DEGs was unchanged in female transferred placentas and that DEGs had fewer gene-associated CpG islands (within ~20 kb region) compared to the larger genome. These data suggested that altered methylation at proximal promoter regions might not lead to transcriptional disruption in transferred placentas. Genomic clustering of some DEGs warrants further investigation of long-range, cis-acting epigenetic mechanisms including histone modifications together with DNA methylation. We conclude that embryo transfer, a protocol required for ART, significantly impacts the placental transcriptome and growth.
Federica Lopes, Jin Liu, Stephanie Morgan, Rebecca Matthews, Lucy Nevin, Richard A Anderson, and Norah Spears
Chemotherapy drugs are administered to patients using combination regimens, and as such the possibility of multiplicative effects between drugs need to be investigated. This study examines the individual and combined effects of the chemotherapy drugs cisplatin and doxorubicin on the human ovary. Although cisplatin and doxorubicin are known to affect female fertility, there is limited information about their direct effects on the human ovary, and none examining the possibility of combined, multiplicative effects of co-exposure to these drugs. Here, human ovarian biopsies were obtained from 14 women at the time of caesarean section, with 38 mouse ovaries also obtained from neonatal C57Bl/6J mice. Tissue was cultured for 6 days prior to analyses, with chemotherapy drugs added to culture medium on the second day of culture only. Treatment groups of a single (5 μg/mL human; 0.5 μg/mL mouse) or double (10 μg/mL human; 1.0 μg/mL mouse) dose of cisplatin, a single (1 μg/mL human; 0.05 μg/mL mouse) or double (2 μg/mL human; 0.01 μg/mL mouse) dose of doxorubicin or a combination of a single dose of both drugs together were compared to controls without drug exposure. Exposure to cisplatin or doxorubicin significantly decreased follicle health in human and mouse, supporting the suitability of the mouse as a model for the human ovary. There was also a significant reduction of mouse follicle number. Human ovarian stromal tissue exhibited increased apoptosis and decreased cell proliferation. Crucially, there was no evidence indicating the occurrence of multiplicative effects between cisplatin and doxorubicin.