A novel lysosome-targeted fluorescent probe for precise formaldehyde detection in water samples, living cells and breast cancer tumors.

This study investigated the potential ability of the fluorescent probe Ly-CHO to detect formaldehyde (FA) in living cells and tumor-bearing mice. Ly-CHO exhibited great selectivity, excellent sensitivity, and rapid response to FA, making it a valuable tool for tracking FA concentration changes. The probe was also found to target lysosomes specifically. Furthermore, Ly-CHO showed an obvious fluorescence increase in endogenous CHO detection after adding tetrahydrogen folic acid (THFA). This study validated Ly-CHO's possibility for FA imaging in vivo, with potential applications in understanding formaldehyde-related diseases.

Bisphenol P and bisphenol M promote triple-negative breast cancer metastasis through activation of AKT pathways.

Bisphenol P (BPP) and bisphenol M (BPM) are increasing in our living environment as analogues of bisphenol A (BPA), but little is known about their biological effect. In this study, we investigated the effects of low to medium dose exposure of BPP and BPM on triple negative breast cancer (TNBC). We found that BPP and BPM exposure didn't affect proliferation of TNBC cell lines MDA-MB-231 and 4 T1, but significantly promoted cells migration and invasion. The effect of BPP and BPM on promoting TNBC metastasis was further confirmed in mouse models. Low concentrations of BPP and BPM significantly increased the expression of epithelial-mesenchymal transition (EMT) marker and related proteins such as N-cadherin, MMP-9, MMP-2 and Snail, and also enhanced phosphorylation of AKT both in vitro and in vivo. When PI3K inhibitor wortmannin was applied to specifically inhibit phosphorylation of AKT, the expression of target genes markedly decreased, and the TNBC metastasis induced by low-concentration BPP and BPM were reversed. In conclusion, these results showed that PI3K/AKT signaling regulate BPP/BPM-induced metastasis of TNBC by triggering EMT. This study provides insights into the effects and the potential mechanisms of BPP and BPM on TNBC, raising concerns about the risk of using these two bisphenols as the alternative of BPA.

Modulation of tumour hypoxia by ultrasound-responsive microbubbles to enhance the sono-photodynamic therapy effect on triple-negative breast cancer.

Sono-photodynamic therapy (SPDT) is an oxidative stress-dependant antitumour treatment modality. Due to the hypoxic tumour microenvironment, the antitumour effect of SPDT is limited. In this study, we developed lipid vesicles to transport a photosensitizer (chlorin e6, Ce6) and oxygen into tumours to promote SPDT efficiency on triple-negative breast cancer in vitro and in vivo. The results showed that compared with the same concentration of free Ce6, Lipo-Ce6 produced a higher singlet oxygen level under light irradiation. Cellular Lipo-Ce6 accumulation was 4-fold higher than that of free Ce6. The cytotoxicity on 4T1 cells caused by Lipo-Ce6-SPDT was significantly stronger than that caused by free Ce6-SPDT, and oxygen microbubbles (O2MB) further enhanced the cytotoxicity of Lipo-Ce6-SPDT under hypoxic conditions. Cellular ROS production in the Lipo-Ce6-SPDT+O2MB group was approximately 2.5-fold higher than that in the Lipo-Ce6-SPDT+C3F8MB group. Furthermore, O2MB rapidly relieved 4T1 subcutaneous xenograft hypoxia conditions under ultrasound exposure and significantly improved the antitumour activity of SPDT in vivo. These results indicate that the combination of O2MB and a high-activity liposome photosensitizer can significantly enhance the antitumour efficiency of SPDT for hypoxic tumours.

Gut microbiota mediated the individualized efficacy of Temozolomide via immunomodulation in glioma.

BACKGROUND: Temozolomide (TMZ) is the preferred chemotherapy strategy for glioma therapy. As a second-generation alkylating agent, TMZ provides superior oral bio-availability. However, limited response rate (less than 50%) and high incidence of drug resistance seriously restricts TMZ's application, there still lack of strategies to increase the chemotherapy sensitivity. METHODS: Luci-GL261 glioma orthotopic xenograft model combined bioluminescence imaging was utilized to evaluate the anti-tumor effect of TMZ and differentiate TMZ sensitive (S)/non-sensitive (NS) individuals. Integrated microbiomics and metabolomics analysis was applied to disentangle the involvement of gut bacteria in TMZ sensitivity. Spearman's correlation analysis was applied to test the association between fecal bacteria levels and pharmacodynamics indices. Antibiotics treatment combined TMZ treatment was used to confirm the involvement of gut microbiota in TMZ response. Flow cytometry analysis, ELISA and histopathology were used to explore the potential role of immunoregulation in gut microbiota mediated TMZ response. RESULTS: Firstly, gut bacteria composition was significantly altered during glioma development and TMZ treatment. Meanwhile, in vivo anti-cancer evaluation suggested a remarkable difference in chemotherapy efficacy after TMZ administration. Moreover, 16s rRNA gene sequencing and non-targeted metabolomics analysis revealed distinct different gut microbiota and immune infiltrating state between TMZ sensitive and non-sensitive mice, while abundance of differential gut bacteria and related metabolites was significantly correlated with TMZ pharmacodynamics indices. Further verification suggested that gut microbiota deletion by antibiotics treatment could accelerate glioma development, attenuate TMZ efficacy and inhibit immune cells (macrophage and CD8α+ T cell) recruitment. CONCLUSIONS: The current study confirmed the involvement of gut microbiota in glioma development and individualized TMZ efficacy via immunomodulation, hence gut bacteria may serve as a predictive biomarker as well as a therapeutic target for clinical TMZ application.

Active fractions of golden-flowered tea (Camellia nitidissima Chi) inhibit epidermal growth factor receptor mutated non-small cell lung cancer via multiple pathways and targets in vitro and in vivo.

As a medicine-food homology (MFH) plant, golden-flowered tea (Camellia nitidissima Chi, CNC) has many different pharmacologic activities and is known as "the queen of the tea family" and "the Panda of the Plant world". Several studies have revealed the pharmacologic effects of CNC crude extract, including anti-tumor, anti-oxidative and hepatoprotective activity. However, there are few studies on the anti-tumor active fractions and components of CNC, yet the underlying mechanism has not been investigated. Thus, we sought to verify the anti-non-small cell lung cancer (NSCLC) effects of four active fractions of CNC. Firstly, we determined the pharmacodynamic material basis of the four active fractions of CNC (Camellia. leave. saponins, Camellia. leave. polyphenols, Camellia. flower. saponins, Camellia. flower. polyphenols) by UPLC-Q-TOF-MS/MS and confirmed the differences in their specific compound contents. Then, MTT, colony formation assay and EdU incorporation assay confirmed that all fractions of CNC exhibit significant inhibitory on NSCLC, especially the Camellia. leave. saponins (CLS) fraction on EGFR mutated NSCLC cell lines. Moreover, transcriptome analysis revealed that the inhibition of NSCLC cell growth by CLS may be via three pathways, including "Cytokine-cytokine receptor interaction," "PI3K-Akt signaling pathway" and "MAPK signaling pathway." Subsequently, quantitative real-time PCR (RT-qPCR) and Western blot (WB) revealed TGFB2, INHBB, PIK3R3, ITGB8, TrkB and CACNA1D as the critical targets for the anti-tumor effects of CLS in vitro. Finally, the xenograft models confirmed that CLS treatment effectively suppressed tumor growth, and the key targets were also verified in vivo. These observations suggest that golden-flowered tea could be developed as a functional tea drink with anti-cancer ability, providing an essential molecular mechanism foundation for MFH medicine treating NSCLC.

Opportunities and challenges of patient-derived models in cancer research: patient-derived xenografts, patient-derived organoid and patient-derived cells.

BACKGROUND: As reported, preclinical animal models differ greatly from the human body. The evaluation model may be the colossal obstacle for scientific research and anticancer drug development. Therefore, it is essential to propose efficient evaluation systems similar to clinical practice for cancer research. MAIN BODY: While it has emerged for decades, the development of patient-derived xenografts, patient-derived organoid and patient-derived cell used to be limited. As the requirements for anticancer drug evaluation increases, patient-derived models developed rapidly recently, which is widely applied in basic research, drug development, and clinical application and achieved remarkable progress. However, there still lack systematic comparison and summarize reports for patient-derived models. In the current review, the development, applications, strengths, and challenges of patient-derived models in cancer research were characterized. CONCLUSION: Patient-derived models are an indispensable approach for cancer research and human health.

Twins labeling derivatization-based LC-MS/MS strategy for absolute quantification of paired prototypes and modified metabolites.

Modified metabolites play significant roles in disease occurrence, progression and diagnosis. Sensitive and accurate analytical methods for the quantification of these metabolites are therefore of great importance. In this study, a liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous measurement of 13 pairs of prototypes and their modified forms covering nucleobases, nucleosides and amino acids. In order to improve the quantification sensitivity and accuracy, two structure analogs named N-dimethyl-amino naphthalene-1-sulfonyl chloride (Dns-Cl) and N-diethyl-amino naphthalene-1-sulfonyl chloride (Dens-Cl) were introduced for twins labeling derivatization. Dns-labeling was utilized to react with target analytes while the Dens-labeling of standard compounds provided one-to-one internal standards. With the introduce of naphthalene and easily ionizable moiety tertiary ammonium, chromatography retention and separation of these polar metabolites were notably improved on C18 columns and the detection sensitivity was increased up to 400 folds. The method is sensitive with the lower limit of quantification (LLOQ) values of 0.002-0.5 µg/mL. Comparisons of the performance of twins labeling derivatization and traditional chemical isotope labeling (CIL) derivatization verified the ability of our method in the absolute quantification. The established method was applied to human lung adenocarcinoma cell line A549 and its cisplatin resistant derivative A549/DDP. Significant shifts in 12 metabolites as well as 9 modified-to-prototypical ratios in A549/DDP were observed, demonstrating the utility of our method and the potential role of modified metabolites in mediating anticancer drug resistance. The method can be easily extended to determine other types of modified metabolites in various biological matrices, which will greatly expand our knowledge on these metabolites.

Akkermansia Muciniphila Potentiates the Antitumor Efficacy of FOLFOX in Colon Cancer.

FOLFOX (oxaliplatin, fluorouracil and calcium folinate) is the first-line chemotherapy regimen for colon cancer therapy in the clinic. It provides superior efficacy than oxaliplatin alone, but the underlying mechanism remains unclear. In the present study, pharmacomicrobiomics integrated with metabolomics was conducted to uncover the role of the gut microbiome behind this. First, in vivo study demonstrated that FOLFOX exhibited better efficacy than oxaliplatin alone in colon cancer animal models. Second, 16S rDNA gene sequencing analysis showed that the abundance of Akkermansia muciniphila (A. muciniphila) remarkably increased in the FOLFOX treated individuals and positively correlated with the therapeutic effect. Third, further exploration confirmed A. muciniphila colonization significantly enhanced the anti-cancer efficacy of FOLFOX. Last, metabolomics analysis suggested dipeptides containing branched-chain amino acid (BCAA) might be responsible for gut bacteria mediated FOLFOX efficacy. In conclusion, our study revealed the key role of A. muciniphila in mediating FOLFOX efficacy, and manipulating A. muciniphila might serve as a novel strategy for colon cancer therapy.

Oleic Acid and Insulin as Key Characteristics of T2D Promote Colorectal Cancer Deterioration in Xenograft Mice Revealed by Functional Metabolomics.

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers with high mortality worldwide. Type 2 diabetes mellitus (T2D), known as a risk factor of CRC, can promote the deterioration of CRC, but the underlying mechanism is elusive. In this study, we aimed to reveal the relationship between CRC and T2D from the perspective of small-molecule metabolism. First, a list of common dysregulated metabolites in CRC and T2D was obtained by retrieving existing metabolomics publications. Among these metabolites, oleic acid (OA) was found to be able to promote the proliferation and migration of colon carcinoma cell HCT116. Further experiments proved that insulin could significantly strengthen this promotion and showed a synergistic effect with OA. Mechanism study found that OA and insulin acted synergistically through the extracellular signal-regulated kinase (ERK)1/2/c-Myc/cyclin D1 pathway. In addition, the combination of ERK1/2 inhibitor SCH772984 and cyclin-dependent kinase (CDK)4/6 inhibitor palbociclib showed a remarkable inhibitory effect on tumor growth in vivo. Taken together, the current study found that OA plays an important role in CRC development by using a functional metabolomics approach. More importantly, insulin and OA were confirmed to synergistically promote the deterioration of CRC in vitro and in vivo via ERK1/2/c-Myc/cyclin D1 pathway. Our findings may shed light on CRC treatment among the T2D population.

Hypo-glycosylated hFSH drives ovarian follicular development more efficiently than fully-glycosylated hFSH: enhanced transcription and PI3K and MAPK signaling.

STUDY QUESTION: Does hypo-glycosylated human recombinant FSH (hFSH18/21) have greater in vivo bioactivity that drives follicle development in vivo compared to fully-glycosylated human recombinant FSH (hFSH24)? SUMMARY ANSWER: Compared with fully-glycosylated hFSH, hypo-glycosylated hFSH has greater bioactivity, enabling greater follicular health and growth in vivo, with enhanced transcriptional activity, greater activation of receptor tyrosine kinases (RTKs) and elevated phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and Mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling. WHAT IS KNOWN ALREADY: Glycosylation of FSH is necessary for FSH to effectively activate the FSH receptor (FSHR) and promote preantral follicular growth and formation of antral follicles. In vitro studies demonstrate that compared to fully-glycosylated recombinant human FSH, hypo-glycosylated FSH has greater activity in receptor binding studies, and more effectively stimulates the PKA pathway and steroidogenesis in human granulosa cells. STUDY DESIGN, SIZE, DURATION: This is a cross-sectional study evaluating the actions of purified recombinant human FSH glycoforms on parameters of follicular development, gene expression and cell signaling in immature postnatal day (PND) 17 female CD-1 mice. To stimulate follicle development in vivo, PND 17 female CD-1 mice (n = 8-10/group) were treated with PBS (150 µl), hFSH18/21 (1 µg/150 µl PBS) or hFSH24 (1 µg/150 µl PBS) by intraperitoneal injection (i.p.) twice daily (8:00 a.m. and 6:00 p.m.) for 2 days. Follicle numbers, serum anti-Müllerian hormone (AMH) and estradiol levels, and follicle health were quantified. PND 17 female CD-1 mice were also treated acutely (2 h) in vivo with PBS, hFSH18/21 (1 µg) or hFSH24 (1 µg) (n = 3-4/group). One ovary from each mouse was processed for RNA sequencing analysis and the other ovary processed for signal transduction analysis. An in vitro ovary culture system was used to confirm the relative signaling pathways. PARTICIPANTS/MATERIALS, SETTING, METHODS: The purity of different recombinant hFSH glycoforms was analyzed using an automated western blot system. Follicle numbers were determined by counting serial sections of the mouse ovary. Real-time quantitative RT-PCR, western blot and immunofluorescence staining were used to determine growth and apoptosis markers related with follicle health. RNA sequencing and bioinformatics were used to identify pathways and processes associated with gene expression profiles induced by acute FSH glycoform treatment. Analysis of RTKs was used to determine potential FSH downstream signaling pathways in vivo. Western blot and in vitro ovarian culture system were used to validate the relative signaling pathways. MAIN RESULTS AND THE ROLE OF CHANCE: Our present study shows that both hypo- and fully-glycosylated recombinant human FSH can drive follicular growth in vivo. However, hFSH18/21 promoted development of significantly more large antral follicles compared to hFSH24 (P < 0.01). In addition, compared with hFSH24, hFSH18/21 also promoted greater indices of follicular health, as defined by lower BAX/BCL2 ratios and reduced cleaved Caspase 3. Following acute in vivo treatment with FSH glycoforms RNA-sequencing data revealed that both FSH glycoforms rapidly induced ovarian transcription in vivo, but hypo-glycosylated FSH more robustly stimulated Gαs and cAMP-mediated signaling and members of the AP-1 transcription factor complex. Moreover, hFSH18/21 treatment induced significantly greater activation of RTKs, PI3K/AKT and MAPK/ERK signaling compared to hFSH24. FSH-induced indices of follicle growth in vitro were blocked by inhibition of PI3K and MAPK. LARGE SCALE DATA: RNA sequencing of mouse ovaries. Data will be shared upon reasonable request to the corresponding author. LIMITATIONS, REASONS FOR CAUTION: The observations that hFSH glycoforms have different bioactivities in the present study employing a mouse model of follicle development should be verified in nonhuman primates. The gene expression studies reflect transcriptomes of whole ovaries. WIDER IMPLICATIONS OF THE FINDINGS: Commercially prepared recombinant human FSH used for ovarian stimulation in human ART is fully-glycosylated FSH. Our findings that hypo-glycosylated hFSH has greater bioactivity enabling greater follicular health and growth without exaggerated estradiol production in vivo, demonstrate the potential for its development for application in human ART. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by NIH 1P01 AG029531, NIH 1R01 HD 092263, VA I01 BX004272, and the Olson Center for Women's Health. JSD is the recipient of a VA Senior Research Career Scientist Award (1IK6 BX005797). This work was also partially supported by National Natural Science Foundation of China (No. 31872352). The authors declared there are no conflicts of interest.

A four-component combination derived from Huang-Qin Decoction significantly enhances anticancer activity of irinotecan.

Huang-Qin Decoction (HQD) is a classic prescription for diarrhea in Chinese medicine treatment. Recent studies have demonstrated that HQD and its modified formulation PHY906 could ameliorate irinotecan (CPT-11) induced gastrointestinal (GI) toxicity and enhance its anticancer therapeutic efficacy. Nevertheless, which constituents in HQD are effective is still unclear so far. The study aims to screen out the key bioactive components combination from HQD that could enhance the anticancer effect of CPT-11. First, the potential bioactive constituents were obtained through system pharmacology strategy. Then the bioactivity of each constituent was investigated synthetically from the aspects of NCM460 cell migration, TNF-α release of THP-1-derived macrophage and MTT assay in HCT116 cell. The contribution of each constituent in HQD was evaluated using the bioactive index Ei, which taken the content and bioactivity into comprehensive consideration. And then, the most contributing constituents were selected out to form a key-component combination. At last, the bioefficacy of the key-component combination was validated in vitro and in vivo. As a result, a key-component combination (HB4) consisting of four compounds baicalin, baicalein, glycyrrhizic acid and wogonin was screened out. In vitro assessment indicated that HB4 could enhance the effect of CPT-11 on inhibiting cell proliferation and inducing apoptosis in HCT116. Furthermore, the in vivo study confirmed that HB4 and HQD have similar pharmacological activity and could both enhance the antitumor effect of CPT-11 in HCT116 xenograft model. Meanwhile, HB4 could also reduce the CPT-11 induced GI toxicity.

MicroRNA-130a Increases and Predicts Cardiotoxicity during Adjuvant Chemotherapy in Human Epidermal Growth Factor Receptor-2-Positive Breast Cancer.

PURPOSE: This study aimed to investigate the changes in microRNA-130a (miR-130a) and its correlation with cardiotoxicity during epirubicin/cyclophosphamide followed by docetaxel plus trastuzumab (EC-D+T) adjuvant chemotherapy in human epidermal growth factor receptor-2-positive (HER2⁺) breast cancer patients. METHODS: A total of 72 HER2⁺ breast cancer patients who underwent resection and were scheduled to receive EC-D+T adjuvant therapy were consecutively enrolled. The expression of miR-130a and cardiotoxicity (defined as any of the following situations: 1) absolute decline of left ventricular ejection fraction (LVEF) ≥ 10% and LVEF < 53%; 2) heart failure; 3) acute coronary artery syndromes; and 4) fatal arrhythmia) were assessed every 3 months throughout the 15-month EC-D+T treatment. RESULTS: The accumulating cardiotoxicity rate was 12 (16.7%), of which the incidence of heart failure, acute coronary syndrome, life-threatening arrhythmias, ΔLVEF ≥ 10%, and LVEF < 53% was 0 (0.0%), 1 (1.4%), 0 (0.0%), and 12 (16.7%), respectively. Baseline miR-130a expression was negatively correlated with LVEF (%) and positively correlated with cardiac troponin I. The expression of miR-130a gradually increased in both cardiotoxicity and non-cardiotoxicity patients during EC-D+T treatment, while the increment of miR-130a was more obvious in cardiotoxicity patients compared with non-cardiotoxicity patients. Further logistic regression and receiver operating characteristic curve analysis indicated that miR-130a was an independent predictive factor for increased cardiotoxicity risk. CONCLUSION: MiR-130a increases constantly and predicts high cardiotoxicity risk during EC-D+T adjuvant chemotherapy in HER2⁺ breast cancer patients.

PKA and AMPK Signaling Pathways Differentially Regulate Luteal Steroidogenesis.

Luteinizing hormone (LH) via protein kinase A (PKA) triggers ovulation and formation of the corpus luteum, which arises from the differentiation of follicular granulosa and theca cells into large and small luteal cells, respectively. The small and large luteal cells produce progesterone, a steroid hormone required for establishment and maintenance of pregnancy. We recently reported on the importance of hormone-sensitive lipase (HSL, also known as LIPE) and lipid droplets for appropriate secretory function of the corpus luteum. These lipid-rich intracellular organelles store cholesteryl esters, which can be hydrolyzed by HSL to provide cholesterol, the main substrate necessary for progesterone synthesis. In the present study, we analyzed dynamic posttranslational modifications of HSL mediated by PKA and AMP-activated protein kinase (AMPK) as well as their effects on steroidogenesis in luteal cells. Our results revealed that AMPK acutely inhibits the stimulatory effects of LH/PKA on progesterone production without reducing levels of STAR, CYP11A1, and HSD3B proteins. Exogenous cholesterol reversed the negative effects of AMPK on LH-stimulated steroidogenesis, suggesting that AMPK regulates cholesterol availability in luteal cells. AMPK evoked inhibitory phosphorylation of HSL (Ser565). In contrast, LH/PKA decreased phosphorylation of AMPK at Thr172, a residue required for its activation. Additionally, LH/PKA increased phosphorylation of HSL at Ser563, which is crucial for enzyme activation, and decreased inhibitory phosphorylation of HSL at Ser565. The findings indicate that LH and AMPK exert opposite posttranslational modifications of HSL, presumptively regulating cholesterol availability for steroidogenesis.

[Impact of a Sewage Treatment Plant on the Accumulation of Microplastics in Freshwater Organisms in the Lijiang River of the Guilin Urban Section].

Microplastics (MPs, particle size<5 mm), as a new pollutant, have attracted wide attention in recent years. The distributions of MPs in effluent of a sewage treatment plant (STP) were examined. Surface water, sediment, and freshwater organism samples were taken from the STP discharge outlet in the Lijiang River tributary (S1), the confluence of tributaries and main streams in the Lijiang River (S2), and downstream locations in the Lijiang River (S3). The impact of STP discharge effluent on the characteristics and spatial distribution of MPs pollution in freshwater organisms was studied. The results showed that the freshwater organisms had a probability of uptake of MPs by 94.2%. The mean abundance of MPs in S1 (2.7 n·ind-1) was significantly higher than that of S3 (1.9 n·ind-1, P<0.05). The MPs found in S1 and S3 were mainly <0.10 mm, accounting for 46.0% and 30.5%, respectively. The fiber type of MPs was observed in the body of freshwater organisms. Polyethylene terephthalate was the major polymer form in S1 organisms, while polypropylene was the major polymer form in S3. The effluent discharged from the STP led to the accumulation of MPs in freshwater organisms.

Formation and characterization of lipid droplets of the bovine corpus luteum.

Establishment and maintenance of pregnancy depends on progesterone synthesized by luteal tissue in the ovary. Our objective was to identify the characteristics of lipid droplets (LDs) in ovarian steroidogenic cells. We hypothesized that LDs are a major feature of steroidogenic luteal cells and store cholesteryl esters. Whole bovine tissues, isolated ovarian steroidogenic cells (granulosa, theca, small luteal, and large luteal), and isolated luteal LDs were assessed for LD content, LD-associated proteins and lipid analyses. Bovine luteal tissue contained abundant lipid droplets, LD-associated perilipins 2/3/5, hormone-sensitive lipase, and 1-acylglycerol-3-phosphate O-acyltransferase ABHD5. Luteal tissue was enriched in triglycerides (TGs) compared to other tissues, except for adipose tissue. Luteal cells were distinguishable from follicular cells by the presence of LDs, LD-associated proteins, and increased TGs. Furthermore, LDs from large luteal cells were numerous and small; whereas, LDs from small luteal cells were large and less numerous. Isolated LDs contained nearly all of the TGs and cholesteryl esters present in luteal tissue. Isolated luteal LDs were composed primarily of TG, with lesser amounts of cholesteryl esters, diglyceride and other phospholipids. Bovine luteal LDs are distinct from LDs in other bovine tissues, including follicular steroidogenic cells.

Bioactivity of recombinant hFSH glycosylation variants in primary cultures of porcine granulosa cells.

Previous studies have reported hypo-glycosylated FSH and fully-glycosylated FSH to be naturally occurring in humans, and these glycoforms exist in changing ratios over a woman's lifespan. The precise cellular and molecular effects of recombinant human FSH (hFSH) glycoforms, FSH21 and FSH24, have not been documented in primary granulosa cells. Herein, biological responses to FSH21 and FSH24 were compared in primary porcine granulosa cells. Hypo-glycosylated hFSH21 was significantly more effective than fully-glycosylated hFSH24 at stimulating cAMP accumulation and protein kinase A (PKA) activity, leading to the higher phosphorylation of CREB and ß-Catenin. Compared to fully-glycosylated hFSH24, hypo-glycosylated hFSH21 also induced greater levels of transcripts for HSD3B, STAR and INHA, and higher progesterone production. Our results demonstrate that hypo-glycosylated hFSH21 exerts more robust activation of intracellular signals associated with steroidogenesis than fully-glycosylated hFSH24 in primary porcine granulosa cells, and furthers our understanding of the differing bioactivities of FSH glycoforms in the ovary.

Luteinizing hormone regulates the phosphorylation and localization of the mitochondrial effector dynamin-related protein-1 (DRP1) and steroidogenesis in the bovine corpus luteum.

The corpus luteum is an endocrine gland that synthesizes and secretes progesterone. Luteinizing hormone (LH) activates protein kinase A (PKA) signaling in luteal cells, increasing delivery of substrate to mitochondria for progesterone production. Mitochondria maintain a highly regulated equilibrium between fusion and fission in order to sustain biological function. Dynamin-related protein 1 (DRP1), is a key mediator of mitochondrial fission. The mechanism by which DRP1 is regulated in the ovary is largely unknown. We hypothesize that LH via PKA differentially regulates the phosphorylation of DRP1 on Ser616 and Ser637 in bovine luteal cells. In primary cultures of steroidogenic small luteal cells (SLCs), LH, and forskolin stimulated phosphorylation of DRP1 (Ser 637), and inhibited phosphorylation of DRP1 (Ser 616). Overexpression of a PKA inhibitor blocked the effects of LH and forskolin on DRP1 phosphorylation. In addition, LH decreased the association of DRP1 with the mitochondria. Genetic knockdown of the DRP1 mitochondria receptor, and a small molecule inhibitor of DRP1 increased basal and LH-induced progesterone production. Studies with a general Dynamin inhibitor and siRNA knockdown of DRP1 showed that DRP1 is required for optimal LH-induced progesterone biosynthesis. Taken together, the findings place DRP1 as an important target downstream of PKA in steroidogenic luteal cells.

Yes-associated protein 1 is required for proliferation and function of bovine granulosa cells in vitro†.

Yes-associated protein 1 (YAP1) is a major component of the Hippo signaling pathway. Although the exact extracellular signals that control the Hippo pathway are currently unknown, increasing evidence supports a critical role for the Hippo pathway in embryonic development, regulation of organ size, and carcinogenesis. Granulosa cells (GCs) within the ovarian follicle proliferate and produce steroids and growth factors, which facilitate the growth of follicle and maturation of the oocyte. We hypothesize that YAP1 plays a role in proliferation and estrogen secretion of GCs. In the current study, we examined the expression of the Hippo signaling pathway in bovine ovaries and determined whether it was important for GC proliferation and estrogen production. Mammalian STE20-like protein kinase 1 (MST1) and large tumor suppressor kinase 2 (LATS2) were identified as prominent upstream components of the Hippo pathway expressed in granulosa and theca cells of the follicle and large and small cells of the corpus luteum. Immunohistochemistry revealed that YAP1 was localized to the nucleus of growing follicles. In vitro, nuclear localization of the downstream Hippo signaling effector proteins YAP1 and transcriptional co-activator with PDZ-binding motif (TAZ) was inversely correlated with GC density, with greater nuclear localization under conditions of low cell density. Treatment with verteporfin and siRNA targeting YAP1 or TAZ revealed a critical role for these transcriptional co-activators in GC proliferation. Furthermore, knockdown of YAP1 in GCs inhibited follicle-stimulating hormone (FSH)-induced estradiol biosynthesis. The data indicate that Hippo pathway transcription co-activators YAP1/TAZ play an important role in GC proliferation and estradiol synthesis, two processes necessary for maintaining normal follicle development.