Reproduction, fertility, and development最新文献

Context: In diabetes, abnormalities of granulosa cells (GCs) and steroidogenesis are associated with hyperglycaemia-induced oxidative stress. Betaine has beneficial effect in experimental model of diabetes by reducing oxidative stress, inflammation, and apoptosis.

Aims: In this study we investigate the effects of betaine to prevent oxidative stress in GCs induced by high glucose and improve steroidogenesis.

Methods: Primary GCs, isolated from ovarian follicles of C57BL/6 mice were cultured in 5mM (control) and 30mM (hyperglycaemia) of glucose and in presence of 5mM of betaine for 24h. Then antioxidant enzymes, malondialdehyde, oestradiol and progesterone were measured. In addition, the expression of Nrf2 and NF-κB , antioxidant enzymes (Sod1 , Gpx and Cat ) were analysed by qRT-PCR assay.

Key results: We observed significant (P NF-κB and down-regulation of Nrf2 due to high concentration of glucose. Also significant (P Cat , Sod1 and GPx ) and activity reduction of these enzymes as well as significant (P NF-κB and up-regulating the expression of Nrf2 , Cat , Sod1 and GPx . It was also shown that betaine in the presence of FSH significantly (P Conclusion: Betaine compensated the antioxidant stress in mouse GCs under hyperglycaemic condition via regulation of Nrf2/NF-κB at transcription level.

Implications: As betaine is a natural product and no side effect has been reported to today, we suggest more research needs to be carried out especially on patients whom suffer from diabetes to find the probability of using betaine as a therapeutic agent.

Context: Infertility is a common disease among women of childbearing age and seriously endangers the reproductive health of human beings.

Aims: We aimed to study the active effect and mechanism of betulonic acid (BTA) on tubal inflammatory infertility.

Methods: An inflammatory model was established in isolated rat oviduct epithelial cells. Immunofluorescence of cytokeratin 18 was performed in cells. The therapeutic effect of BTA on cells was observed. Subsequently, we added JAK/STAT inhibitor AG490 and MAPK inhibitor U0126 and measured the levels of inflammatory factors via enzyme-linked immunosorbent assay and qRT-PCR. CCK-8 assay was applied to test cell proliferation, whereas flow cytometry was used to measure apoptosis. The levels of TLR4, IκBα, JAK1, JAK2, JAK3, Tyk2, STAT3, p38, ERK and the phosphorylation of p65 were determined by Western blotting.

Key results: Betulonic acid inhibited the activation of TLR4 and NF-κB signalling pathways, and significantly downregulated IL-1β, IL-6, and TNF-α, with high doses being the most effective. Furthermore, high-dose BTA promoted the proliferation of oviduct epithelial cells and inhibited apoptosis. In addition, BTA inhibited the activation of JAK/STAT signalling pathway to perform effectively in oviduct epithelial cells inflammation. The addition of AG490 led to the inhibition of the JAK/STAT signalling pathway. BTA also inhibited the activation of MAPK signalling pathway in oviduct epithelial cells inflammation. Under U0126 treatment, the inhibition of proteins in MAPK pathway by BTA was weakened.

Conclusions: Therefore, BTA inhibited the TLR, JAK/STAT and MAPK signalling pathways.

Implications: Our study provided a new therapeutic strategy for infertility caused by oviduct inflammation.

The European Union (EU) has developed cultural policy initiatives that seek to promote cultural Europeanization with the purpose of constructing European identity narratives and facilitating citizens' sense of belonging to Europe and the EU. The article focuses on the citizens' perspective to cultural Europeanization through ethnographic research on one central action in the EU cultural policy, European Heritage Label (EHL). We analyse the interviews conducted in selected EHL sites with Central and East European (CEE) citizens who were visiting the sites as well as with cultural heritage practitioners working at three EHL sites located in CEE countries. We ask how the practitioners and the visitors engage with European identity narratives and elaborate their European belonging. We especially scrutinize how everyday encounters and experiences, such as mobility, shape identifications with 'Europe' and perceptions of what is 'European'. The interviews are interpreted in the theoretical framework of 'being' and 'becoming' European. This framework indicates a centuries-long liminal position of the Central and Eastern Europe. It enables us to scrutinize CEE citizens' sense of belonging to Europe in an intersection of dual Europeanization, i.e. cultural Europeanization and 'Europeanization' of the CEE countries to overcome this liminal position and become 'true' Europeans.

Context and aims: Melatonin is a powerful antioxidant regulating various biological functions, including alleviating male reproductive damage under pathological conditions. Here, we aim to analyse the effect of melatonin on normal male reproduction in mice.

Methods: Male mice received an intraperitoneal injection of melatonin (10mg/kg body weight) for 35 consecutive days. The testis and epididymis morphology, and epididymal sperm parameters were examined. PCNA, HSPA2, SYCP3, ZO-1 and CYP11A1 expressions in epididymis or testis were detected by immunohistochemistry or Western blotting. Male fertility was determined by in vivo and in vitro fertilisation (IVF) experiments. The differentially expressed sperm proteins were identified by proteomics.

Key results: No visible structural changes and oxidative damage in the testis and epididymis, and no significant side effects on testis weight, testosterone levels, sperm motility, and sperm morphology were observed in the melatonin-treatment group compared with the control group. Spermatogenesis-related molecules of PCNA, SYCP3, ZO-1, and CYP11A1 showed no significant differences in melatonin-treated testis. However, PCNA and HSPA2 increased their expressions in the epididymal initial segments in the melatonin-treatment group. Normal sperm fertilisation, two-cell and blastocyst development were observed in the melatonin-treated group, but melatonin significantly enhanced the sperm binding ability characterised as more sperm binding to one oocyte (control 7.2±1.3 versus melatonin 11.8±1.5). Sperm proteomics demonstrated that melatonin treatment enhanced the biological process of cell adhesion in sperm.

Conclusions and implications: This study suggests that melatonin can promote sperm maturation and sperm function, providing important information for further research on the physiological function and protective effect of melatonin in male reproduction.

Context: Testicular torsion-detorsion results in loss of germ cells and infertility. Pentoxifylline has been shown to prevent tissue damage.

Aims: To determine the effect of pentoxifylline on germ cell survival in torsion-detorsion induced apoptosis Methods: Twenty male mice were divided into four groups of five animals each: Control, T1 (Torsion-detorsion+single dose 100mg/kg Pentoxifylline/IP), T2 (Torsion-detorsion+daily 20mg/kg pentoxifylline/IP for 2weeks, and T/D (Torsion-detorsion only). 35thday after torsion-detorsion, the left testes of all the animals were harvested for histological and biochemical analysis.

Key results: Histomorpholoical analysis showed significant increase (P <0.05) in seminiferous tubule diameter, Johnsen's score and germ cells of Control and T1 compared to T2 and T/D, with no significant difference (P >0.05) in testis weight, sertoli, leydig and myoid cells. Tunnel assay showed significant increase (P <0.05) in apoptotic cells of T/D and T2 animals compared to Control and T1. RT-PCR analysis showed significant high (P <0.01) mRNA expression of Bax gene in T/D compared to T1 and T2 and significant increase (P <0.05) of Bcl2 in Control, T1, T2 compared to T/D. Nrf2-ARE transcripts revealed significant increase (P <0.05) in Control and T1 compared to T2 and T/D. Western blot showed significantly increased (P <0.05) caspase-3 in T/D compared to Control, T1 and T2.

Conclusion: Pentoxifylline promotes spermatogenesis and suppressed apoptosis induced by testicular torsion-detorsion.

Implication: Pentoxifylline could serve as adjunct therapy to surgery in the treatment of torsion-detorsion induced germ cell apoptosis.

Context: Ovarian quiescence can be due to hormonal deficiency usually caused by apoptosis of granulosa cells responsible for oestrogen synthesis.

Aim: This study evaluated the regenerative effect of platelet rich plasma (PRP) on bovine in vitro models to understand its effect on granulosa cells.

Methods: Quiescent and healthy ovarian sections were cultured in the presence/absence of PRP for 72h and, at different times (0, 24, 48 and 72h), hematoxylin-eosin and immunohistochemical detection of Ki-67 were performed. Additionally, granulosa cells collected from healthy bovine ovaries were stressed with 100ng/mL of lipopolysaccharide (LPS) in presence/absence of PRP and evaluated at 0, 4, 8 and 24h for apoptosis by acridine orange and propidium iodide staining. Enzyme-linked immunosorbent assay tests were performed to evaluate oestrogen (E2) and anti-Müllerian hormone (AMH) concentrations on cultures of ovarian slices and granulosa cells.

Key results: In slides of quiescent ovaries treated with PRP, a marked and widespread positivity to Ki-67 was expressed by 40-60% of the follicular wall cells at 48h of culture. Levels of E2 and AMH were significantly higher compared to untreated quiescent samples reaching the levels of healthy control samples. PRP counteracted the LPS effect and apoptosis (at 24h, there were 93.44±3.51% live cells with LPS+PRP compared to 37±1.32% with LPS) and significantly increased concentrations of E2 and AMH.

Conclusions: PRP can stimulate granulosa cell proliferation and counteract inflammatory processes in vitro .

Implications: This treatment could improve the reproductive ability of quiescent females.

Context: Sperm storage is a complex and highly coordinated process that is regulated by a variety of factors. The BCL 2 protein family plays a key role in regulating apoptosis, and determines sperm survival.

Aims: The objective of this study was to explore the correlation between sperm storage and the BCL 2 protein family in the oviduct of Mauremys reevesii .

Methods: Hematoxylin-eosin (HE) staining, immunohistochemistry (IHC) and real-time quantitative polymerase chain reaction (RT-qPCR) techniques were used to investigate three parts of the reproductive tract (isthmus, uterus and vagina) of mated and unmated female M. reevesii .

Key results: Hematoxylin-eosin staining revealed many sperm stored in the oviduct. IHC showed positive immunostaining for the BCL 2 and BAX proteins in epithelial ciliated and glandular cells. RT-qPCR indicated that the mRNA expressions of anti-apoptotic genes (BCL 2 , MCL 1 , BCL- W , BCL-XL ) and the androgen receptor (AR) were significantly higher in mated turtles than unmated turtles. However, the expression of pro-apoptotic genes (BAX , BAD , BID and CASPASE 3 ) showed the opposite relationship.

Conclusions: These results suggest that sperm entering the oviduct can promote the synthesis of anti-apoptotic genes to protect themselves from various degradation factors.

Implications: These findings will help researchers understand the mechanisms of sperm storage.

Context: One of the main problems of porcine in vitro maturation (IVM) is incomplete cytoplasmatic maturation. Nuclear and cytoplasmic maturation will determine the future success of fertilisation and embryo development. Insulin-transferrin-selenium (ITS) has insulin-like and antioxidant effects, and metformin (M) is an insulin-sensitiser and antioxidant drug.

Aims: To assess the effects of adding ITS and/or M in porcine IVM media on cytoplasmic maturation and early embryo development.

Methods: Cumulus -oocyte complexes (COC) were IVM with M (10-4 M), ITS (0.1% v/v), M+ITS or no adding (Control).

Key results: ITS increased glucose consumption compared to Control and M (P <0.01), and M+ITS did not differ from ITS or Control. Redox balance: M, ITS and M+ITS increased glutathione (P <0.01) and decreased lipid peroxidation (P <0.005). The viability of cumulus cells by flow cytometry increased with M (P <0.005) and decreased with ITS (P <0.001); M+ITS did not differ from Control. After IVF, M increased penetration and decreased male pronucleus (P <0.05). Embryo development: cleavage increased with M (P <0.05), and blastocysts increased with ITS and M+ITS (P <0.05). The number of blastocyst cells increased with ITS (P <0.05).

Conclusions: Adding ITS and M+ITS to porcine IVM media benefits embryo development to blastocysts, but ITS alone has better effects than M+ITS.

Implications: ITS is an excellent tool to improve IVM and embryo development after IVF in pigs.

Context: 46,XY, disorders of sexual development (46,XY, DSD) is a congenital genetic disease whose pathogenesis is complex and clinical manifestations are diverse. The existing molecular research has often focused on single-centre sequencing data, instead of prediction based on big data.

Aims: This work aimed to fully understand the pathogenesis of 46,XY, DSD, and summarise the key pathogenic genes.

Methods: Firstly, the potential pathogenic genes were identified from public data. Secondly, bioinformatics was used to predict pathogenic genes, including hub gene analysis, protein-protein interaction (PPI) and function enrichment analysis. Lastly, the genomic DNA from two unrelated families were recruited, next-generation sequencing and Sanger sequencing were performed to verify the hub genes.

Key results: A total of 161 potential pathogenic genes were selected from MGI and PubMed gene sets. The PPI network was built which included 144 nodes and 194 edges. MCODE 4 was selected from PPI which scored the most significant P -value. The top 15 hub genes were ranked and identified by Cytoscape. Furthermore, three variants were found on SRD5A2 gene by genome sequencing, which belonged to the prediction hub genes.

Conclusions: Our results indicate that occurrence of 46,XY, DSD is attributed to a variety of genes. Bioinformatics analysis can help us predict the hub genes and find the most core network MCODE model.

Implications: Bioinformatic predictions may provide a novel perspective on better understanding the pathogenesis of 46,XY, DSD.

Context: The exact mechanisms regulating the initiation of porcine conceptus elongation are not known due to the complexity of the uterine environment.

Aims: To identify contributing factors for initiation of conceptus elongation in vitro , this study evaluated differential metabolite abundance within media following culture of blastocysts within unmodified alginate (ALG) or Arg-Gly-Asp (RGD)-modified alginate hydrogel culture systems.

Methods: Blastocysts were harvested from pregnant gilts, encapsulated within ALG or RGD or as non-encapsulated control blastocysts (CONT), and cultured. At the termination of 96h culture, media were separated into blastocyst media groups: non-encapsulated control blastocysts (CONT); ALG and RGD blastocysts with no morphological change (ALG- and RGD-); ALG and RGD blastocysts with morphological changes (ALG+ and RGD+) and evaluated for non-targeted metabolomic profiling by liquid chromatography (LC)-mass spectrometry (MS) techniques and gas chromatography-(GC-MS).

Key results: Analysis of variance identified 280 (LC-MS) and 1 (GC-MS) compounds that differed (P <0.05), of which 134 (LC-MS) and 1 (GC-MS) were annotated. Metabolites abundance between ALG+ vs ALG-, RGD+ vs RGD-, and RGD+ vs ALG+ were further investigated to identify potential differences in metabolic processes during the initiation of elongation.

Conclusions: This study identified changes in phospholipid, glycosphingolipid, lipid signalling, and amino acid metabolic processes as potential RGD-independent mechanisms of elongation and identified changes in lysophosphatidylcholine and sphingolipid secretions during RGD-mediated elongation.

Implications: These results illustrate changes in phospholipid and sphingolipid metabolic processes and secretions may act as mediators of the RGD-integrin adhesion that promotes porcine conceptus elongation.