Retinoic Acid Induced Protein 14 (Rai14) is dispensable for mouse spermatogenesis

Background Retinoic Acid Induced Protein 14 (Rai14) is an evolutionarily conserved gene that is highly expressed in the testis. Previous experiments have reported that small interfering RNA (siRNA)-mediated gene knockdown (KD) of Rai14 in rat testis disrupted spermatid polarity and transport. Of note, a gene knockout (KO) model is considered the “gold standard” for in vivo assessment of crucial gene functions. Herein, we used CRISPR/Cas9-based gene editing to investigate the in vivo role of Rai14 in mouse testis. Methods Sperm concentration and motility were assayed using a computer-assisted sperm analysis (CASA) system. Histological and immunofluorescence (IF) staining and transmission electron microscopy (TEM) were used to visualize the effects of Rai14 KO in the testes and epididymides. Terminal deoxynucleotidyl transferase-dUTP nick-end labeling (TUNEL) was used to determine apoptotic cells. Gene transcript levels were calculated by real-time quantitative PCR. Results Rai14 KO in mice depicted normal fertility and complete spermatogenesis, which is in sharp contrast with the results reported previously in a Rai14 KD rat model. Sperm parameters and cellular apoptosis did not appear to differ between wild-type (WT) and KO group. Mechanistically, in contrast to the well-known role of Rai14 in modulating the dynamics of F-actin at the ectoplasmic specialization (ES) junction in the testis, morphological changes of ES junction exhibited no differences between Rai14 KO and WT testes. Moreover, the F-actin surrounded at the ES junction was also comparable between the two groups. Conclusion In summary, our study demonstrates that Rai14 is dispensable for mouse spermatogenesis and fertility. Although the results of this study were negative, the phenotypic information obtained herein provide an enhanced understanding of the role of Rai14 in the testis, and researchers may refer to these results to avoid conducting redundant experiments.

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Capacitation-Induced Mitochondrial Activity Is Required for Sperm Fertilizing Ability in Mice by Modulating Hyperactivation

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.767161 ◽

2021 ◽

Vol 9 ◽

Author(s):

María Milagros Giaccagli ◽

Matías Daniel Gómez-Elías ◽

Jael Dafne Herzfeld ◽

Clara Isabel Marín-Briggiler ◽

Patricia Sara Cuasnicú ◽

...

Keyword(s):

Mitochondrial Activity ◽

Computer Assisted ◽

Specific Staining ◽

Sperm Analysis ◽

Functional Changes ◽

Fertilizing Ability ◽

Sperm Fertilizing Ability

To become fully competent to fertilize an egg, mammalian sperm undergo a series of functional changes within the female tract, known as capacitation, that require an adequate supply and management of energy. However, the contribution of each ATP generating pathway to sustain the capacitation-associated changes remains unclear. Based on this, we investigated the role of mitochondrial activity in the acquisition of sperm fertilizing ability during capacitation in mice. For this purpose, the dynamics of the mitochondrial membrane potential (MMP) was studied by flow cytometry with the probe tetramethylrhodamine ethyl ester (TMRE). We observed a time-dependent increase in MMP only in capacitated sperm as well as a specific staining with the probe in the flagellar region where mitochondria are confined. The MMP rise was prevented when sperm were exposed to the mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazine (CCCP) or the protein kinase A (PKA) inhibitor H89 during capacitation, indicating that MMP increase is dependent on capacitation and H89-sensitive events. Results showed that whereas nearly all motile sperm were TMRE positive, immotile cells were mostly TMRE negative, supporting an association between high MMP and sperm motility. Furthermore, CCCP treatment during capacitation did not affect PKA substrate and tyrosine phosphorylations but produced a decrease in hyperactivation measured by computer assisted sperm analysis (CASA), similar to that observed after H89 exposure. In addition, CCCP inhibited the in vitro sperm fertilizing ability without affecting cumulus penetration and gamete fusion, indicating that the hyperactivation supported by mitochondrial function is needed mainly for zona pellucida penetration. Finally, complementary in vivo fertilization experiments further demonstrated the fundamental role of mitochondrial activity for sperm function. Altogether, our results show the physiological relevance of mitochondrial functionality for sperm fertilization competence.

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Estrogen modulates the recruitment of myelopoietic cell progenitors in rat through a stromal cell-independent mechanism involving apoptosis

Blood ◽

10.1182/blood.v87.7.2683.bloodjournal8772683 ◽

1996 ◽

Vol 87 (7) ◽

pp. 2683-2692 ◽

Cited By ~ 33

Author(s):

NK Shevde ◽

JW Pike

Keyword(s):

Estrogen Receptor ◽

Ovarian Function ◽

Morphological Changes ◽

Estrogen Treatment ◽

Ovariectomized Rats ◽

Terminal Deoxynucleotidyl Transferase ◽

Protective Effects ◽

Hematopoietic Stem

Loss of ovarian function leads to a significant increase in the number of bone-resorbing osteoclasts. Estrogen replacement is known to manifest bone protective effects in the treatment of postmenopausal osteoporosis. In the present study, we used ovariectomized rats to examine the effects of estrogen loss at the osteoclast progenitor colony forming unit-granulocyte macrophage (CFU-GM) level. A significant increase in CFU-GM number was observed as early as 7 days following ovariectomy, and correlated directly with an increase in the number of osteoclast-like cells generated in marrow cultures. The increase in CFU-GM following ovariectomy was abrogated in animals that received estrogen treatment in vivo. A similar suppressive effect was observed on CFU-GM number when ovariectomized rat marrow was treated with estrogen in vitro. This effect was blocked in the presence of the estrogen antihormone ICI 164,384. Thus, the data suggest the possibility that estrogen exerts a direct effect on osteoclast progenitors, and does so through the estrogen receptor-mediated mechanism. Ovariectomy also led to an increase in the early hematopoietic stem/progenitor cell population (Thy 1.1+ cells) as determined by FLOW cytometry methods. Morphological changes as well as terminal deoxynucleotidyl transferase assays revealed that estrogen treatment negated growth factor-induced proliferation of these early progenitors by promoting apoptosis. The cellular effects of estrogen in vitro together with the immunocytochemical detection of the estrogen receptor in these cells, strongly support the contention that in addition to osteoclast progenitors such as CFU-GM, earlier hematopoietic progenitors are also unique cellular targets for estrogen action.

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Validation of the Turkey Semen Cryopreservation by Evaluating the Effect of Two Diluents and the Inseminating Doses

Animals ◽

10.3390/ani10081329 ◽

2020 ◽

Vol 10 (8) ◽

pp. 1329

Author(s):

Michele Di Iorio ◽

Giusy Rusco ◽

Roberta Iampietro ◽

Lucia Maiuro ◽

Achille Schiavone ◽

...

Keyword(s):

Sperm Quality ◽

Sperm Concentration ◽

Membrane Integrity ◽

In Vivo Test ◽

Sperm Analysis ◽

Fertilizing Ability ◽

Vitro Analysis ◽

Hatching Rates

This study was designed to test the fertilizing ability of cryopreserved turkey semen, and here, two experiments were performed: an in vitro analysis to assess the effects of Tselutin and Lake diluents and an in vivo test to determine the fertility and hatching rates by also studying the feat of three insemination doses (250, 400 and 600 × 106 sperm/hen). Pooled semen samples were diluted with Tselutin or Lake extender which contained 20% of dimethylsulfoxide and 1 mM of Ficoll at final sperm concentration of 3 × 109 sperm/mL. Thereafter, semen was packaged into straws and frozen on liquid nitrogen. The post-thaw sperm quality was evaluated considering motility (computer-aided sperm analysis—CASA system) and membrane integrity (flow cytometry). Significantly higher values of progressive motility and some kinetic parameters in semen frozen with Lake were found. When we compared the extenders in vivo, no significant effects were detected, whilst sperm concentration significantly affected both fertility and hatching rates, with the best results obtained with the sperm concentration of 400 × 106 sperm/hen. From the results obtained, it emerged that the extender type only affected sperm motility characteristics, not the fertilizing ability of frozen-thawed semen, while inseminating dose markedly affected fertility and hatching rates.

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Downregulation of CRABP2 Inhibit the Tumorigenesis of Hepatocellular Carcinoma In Vivo and In Vitro

BioMed Research International ◽

10.1155/2020/3098327 ◽

2020 ◽

Vol 2020 ◽

pp. 1-12

Author(s):

Qingmin Chen ◽

Ludong Tan ◽

Zhe Jin ◽

Yahui Liu ◽

Ze Zhang

Keyword(s):

Hepatocellular Carcinoma ◽

Retinoic Acid ◽

Cell Lines ◽

Molecular Mechanisms ◽

Tumor Stage ◽

Migration And Invasion ◽

Hcc Cells

Cellular retinoic acid-binding protein 2 (CRABP2) binds retinoic acid (RA) in the cytoplasm and transports it into the nucleus, allowing for the regulation of specific downstream signal pathway. Abnormal expression of CRABP2 has been detected in the development of several tumors. However, the role of CRABP2 in hepatocellular carcinoma (HCC) has never been revealed. The current study aimed to investigate the role of CRABP2 in HCC and illuminate the potential molecular mechanisms. The expression of CRABP2 in HCC tissues and cell lines was detected by western blotting and immunohistochemistry assays. Our results demonstrated that the expression levels of CRABP2 in HCC tissues were elevated with the tumor stage development, and it was also elevated in HCC cell lines. To evaluate the function of CRABP2, shRNA-knockdown strategy was used in HCC cells. Cell proliferation, metastasis, and apoptosis were analyzed by CCK-8, EdU staining, transwell, and flow cytometry assays, respectively. Based on our results, knockdown of CRABP2 by shRNA resulted in the inhibition of tumor proliferation, migration, and invasion in vitro, followed by increased tumor apoptosis-related protein expression and decreased ERK/VEGF pathway-related proteins expression. CRABP2 silencing in HCC cells also resulted in the failure to develop tumors in vivo. These results provide important insights into the role of CRABP2 in the development and development of HCC. Based on our findings, CRABP2 may be used as a novel diagnostic biomarker, and regulation of CRABP2 in HCC may provide a potential molecular target for the therapy of HCC.

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In vivo analysis of the role of aberrant histone deacetylase recruitment and RARα blockade in the pathogenesis of acute promyelocytic leukemia

Journal of Experimental Medicine ◽

10.1084/jem.20050616 ◽

2006 ◽

Vol 203 (4) ◽

pp. 821-828 ◽

Cited By ~ 24

Author(s):

Hiromichi Matsushita ◽

Pier Paolo Scaglioni ◽

Mantu Bhaumik ◽

Eduardo M. Rego ◽

Lu Fan Cai ◽

...

Keyword(s):

Retinoic Acid ◽

Fusion Protein ◽

Acute Promyelocytic Leukemia ◽

Histone Deacetylases ◽

Retinoic Acid Receptor ◽

Promyelocytic Leukemia ◽

Dominant Negative

The promyelocytic leukemia–retinoic acid receptor α (PML-RARα) protein of acute promyelocytic leukemia (APL) is oncogenic in vivo. It has been hypothesized that the ability of PML-RARα to inhibit RARα function through PML-dependent aberrant recruitment of histone deacetylases (HDACs) and chromatin remodeling is the key initiating event for leukemogenesis. To elucidate the role of HDAC in this process, we have generated HDAC1–RARα fusion proteins and tested their activity and oncogenicity in vitro and in vivo in transgenic mice (TM). In parallel, we studied the in vivo leukemogenic potential of dominant negative (DN) and truncated RARα mutants, as well as that of PML-RARα mutants that are insensitive to retinoic acid. Surprisingly, although HDAC1-RARα did act as a bona fide DN RARα mutant in cellular in vitro and in cell culture, this fusion protein, as well as other DN RARα mutants, did not cause a block in myeloid differentiation in vivo in TM and were not leukemogenic. Comparative analysis of these TM and of TM/PML−/− and p53−/− compound mutants lends support to a model by which the RARα and PML blockade is necessary, but not sufficient, for leukemogenesis and the PML domain of the fusion protein provides unique functions that are required for leukemia initiation.

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Molecular targeting of retinoic acid metabolism in neuroblastoma: the role of the CYP26 inhibitor R116010 in vitro and in vivo

British Journal of Cancer ◽

10.1038/sj.bjc.6603779 ◽

2007 ◽

Vol 96 (11) ◽

pp. 1675-1683 ◽

Cited By ~ 24

Author(s):

J L Armstrong ◽

G A Taylor ◽

H D Thomas ◽

A V Boddy ◽

C P F Redfern ◽

...

Keyword(s):

Retinoic Acid ◽

Acid Metabolism ◽

Molecular Targeting

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Artificial fertilisation in a terrestrial toadlet (Pseudophryne guentheri): effect of medium osmolality, sperm concentration and gamete storage

Reproduction Fertility and Development ◽

10.1071/rd12223 ◽

2013 ◽

Vol 25 (8) ◽

pp. 1134 ◽

Cited By ~ 17

Author(s):

Aimee J. Silla

Keyword(s):

Sperm Concentration ◽

Sperm Storage ◽

Computer Assisted ◽

Sperm Viability ◽

Short Term ◽

Terrestrial Environment ◽

Sperm Analysis ◽

Fertilisation Success ◽

Computer Assisted Sperm Analysis ◽

Analysis System

Anurans exhibit a greater reproductive diversity than any other vertebrate order. However, studies investigating the effects of the external fertilisation environment on fertilisation success are limited to aquatic-breeding species. This study investigated the effects of fertilisation medium osmolality, sperm concentration and short-term oocyte storage on fertilisation success in a terrestrial-breeding anuran, Pseudophryne guentheri. Split-clutch experimental designs were used to determine optimal fertilisation conditions. To determine the effect of short-term sperm storage, sperm viability was assessed using fluorescence microscopy and percentage sperm motility and velocity quantified with a computer-assisted sperm analysis system. Fertilisation success was highest in media ranging in osmolality from 25 mOsm kg–1 to 100 mOsm kg–1, representing a broader range and higher optimal osmolality than previously reported for aquatic breeders. High rates of fertilisation (>75%) were achieved in relatively low sperm concentrations (2.5 × 104 mL–1). Oocytes stored in isotonic solutions (200 mOsm kg–1) retained fertilisation capacity (32%) after 8 h of storage, while sperm suspensions maintained motility (≥26%) for 13 days. Additional studies on terrestrial-breeding anurans will be required to ascertain whether the optimal fertilisation conditions reported reflect adaptations to achieve fertilisation in a terrestrial environment.

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Rapid Magneto-Sonoporation of Adipose-Derived Cells

Materials ◽

10.3390/ma14174877 ◽

2021 ◽

Vol 14 (17) ◽

pp. 4877

Author(s):

Miriam Filippi ◽

Boris Dasen ◽

Arnaud Scherberich

Keyword(s):

Stem Cells ◽

Tissue Repair ◽

Morphological Changes ◽

Adipose Derived Stem Cells ◽

Resonance Imaging ◽

Membrane Resealing ◽

First Time ◽

Stimulation Of

By permeabilizing the cell membrane with ultrasound and facilitating the uptake of iron oxide nanoparticles, the magneto-sonoporation (MSP) technique can be used to instantaneously label transplantable cells (like stem cells) to be visualized via magnetic resonance imaging in vivo. However, the effects of MSP on cells are still largely unexplored. Here, we applied MSP to the widely applicable adipose-derived stem cells (ASCs) for the first time and investigated its effects on the biology of those cells. Upon optimization, MSP allowed us to achieve a consistent nanoparticle uptake (in the range of 10 pg/cell) and a complete membrane resealing in few minutes. Surprisingly, this treatment altered the metabolic activity of cells and induced their differentiation towards an osteoblastic profile, as demonstrated by an increased expression of osteogenic genes and morphological changes. Histological evidence of osteogenic tissue development was collected also in 3D hydrogel constructs. These results point to a novel role of MSP in remote biophysical stimulation of cells with focus application in bone tissue repair.

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Erastin induces ferroptosis via ferroportin-mediated iron accumulation in endometriosis

Human Reproduction ◽

10.1093/humrep/deaa363 ◽

2020 ◽

Author(s):

Yajie Li ◽

Xinliu Zeng ◽

Dingheng Lu ◽

Minuo Yin ◽

Meirong Shan ◽

...

Keyword(s):

Electron Microscopy ◽

Lipid Peroxidation ◽

Cell Viability ◽

Stromal Cells ◽

Morphological Changes ◽

Iron Accumulation ◽

Blue Staining ◽

Endometrial Stromal Cells

Abstract STUDY QUESTION Could erastin activate ferroptosis to regress endometriotic lesions? SUMMARY ANSWER Erastin could induce ferroptosis to regress endometriotic lesions in endometriosis. WHAT IS KNOWN ALREADY Ectopic endometrial stromal cells (EESCs) are in an iron overloading microenvironment and tend to be more sensitive to oxidative damage. The feature of erastin-induced ferroptosis is iron-dependent accumulation of lethal lipid reactive oxygen species (ROS). STUDY DESIGN, SIZE, DURATION Eleven patients without endometriosis and 21 patients with endometriosis were recruited in this study. Primary normal and ectopic endometrial stromal cells were isolated, cultured and subjected to various treatments. The in vivo study involved 10 C57BL/6 female mice to establish the model of endometriosis. PARTICIPANTS/MATERIALS, SETTING, METHODS The markers of ferroptosis were assessed by cell viability, lipid peroxidation level and morphological changes. The cell viability was measured by colorimetric method, lipid peroxidation levels were measured by flow cytometry, and morphological changes were observed by transmission electron microscopy. Immunohistochemistry and western blot were used to detect ferroportin (FPN) expression. Prussian blue staining and immunofluorescent microscopy of catalytic ferrous iron were semi-quantified the levels of iron. Adenovirus-mediated overexpression and siRNA-mediated knockdown were used to investigate the role of FPN on erastin-induced ferroptosis in EESCs. MAIN RESULTS AND THE ROLE OF CHANCE EESCs were more susceptible to erastin treatment, compared to normal endometrial stromal cells (NESCs) (P<0.05). Treatment of cultured EESCs with erastin dramatically increased the total ROS level (P<0.05, versus control), lipid ROS level (P<0.05, versus NESCs) and intracellular iron level (P<0.05, versus NESCs). The cytotoxicity of erastin could be attenuated by iron chelator, deferoxamine (DFO), and ferroptosis inhibitors, ferrostatin-1 and liproxstatin-1, (P<0.05, versus erastin) in EESCs. In EESCs with erastin treatment, shorter and condensed mitochondria were observed by electron microscopy. These findings together suggest that erastin is capable to induce EESC death by ferroptosis. However, the influence of erastin on NESCs was slight. The process of erastin-induced ferroptosis in EESCs accompanied iron accumulation and decreased FPN expression. The overexpression of FPN ablated erastin-induced ferroptosis in EESCs. In addition, knockdown of FPN accelerated erastin-induced ferroptosis in EESCs. In a mouse model of endometriosis, we found ectopic lesions were regressed after erastin administration. LARGE SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION This study was mainly conducted in primary human endometrial stromal cells. Therefore, the function of FPN in vivo need to be further investigated. WIDER IMPLICATIONS OF THE FINDINGS Our findings reveal that erastin may serve as a potential therapeutic treatment for endometriosis. STUDY FUNDING/COMPETING INTEREST(S) This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. The authors declare no conflict of interest.

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All-trans Retinoic Acid Counteracts Diarrhea and Inhibition of Downregulated in Adenoma Expression in Gut Inflammation

Inflammatory Bowel Diseases ◽

10.1093/ibd/izz249 ◽

2019 ◽

Vol 26 (4) ◽

pp. 534-545 ◽

Cited By ~ 1

Author(s):

Shubha Priyamvada ◽

Arivarasu N Anbazhagan ◽

Anoop Kumar ◽

Ishita Chatterjee ◽

Alip Borthakur ◽

...

Keyword(s):

Retinoic Acid ◽

Intestinal Epithelial ◽

Gut Inflammation ◽

Trans Retinoic Acid ◽

All Trans Retinoic Acid ◽

Function Expression ◽

Ifn Γ

Abstract Background Intestinal epithelial apical membrane Cl-/HCO3- exchanger DRA (downregulated in adenoma, SLC26A3) has emerged as an important therapeutic target for diarrhea, emphasizing the potential therapeutic role of agents that upregulate DRA. All-trans retinoic acid (ATRA), a key vitamin A metabolite, was earlier shown by us to stimulate DRA expression in intestinal epithelial cells. However, its role in modulating DRA in gut inflammation has not been investigated. Aims Our aim was to analyze the efficacy of ATRA in counteracting inflammation-induced decrease in DRA in vitro and in vivo. Methods Interferon-γ (IFN-γ)-treated Caco-2 cells and dextran sulfate sodium (DSS)-treated C57BL/6J mice served as in vitro and in vivo models of gut inflammation, respectively. The effect of ATRA on IFN-γ-mediated inhibition of DRA function, expression, and promoter activity were elucidated. In the DSS colitis model, diarrheal phenotype, cytokine response, in vivo imaging, myeloperoxidase activity, and DRA expression were measured in the distal colon. Results All-trans retinoic acid (10 μM, 24 h) abrogated IFN-γ (30 ng/mL, 24 h)-induced decrease in DRA function, expression, and promoter activity in Caco-2 cells. All-trans retinoic acid altered IFN-γ signaling via blocking IFN-γ-induced tyrosine phosphorylation of STAT-1. All-trans retinoic acid cotreatment (1 mg/kg BW, i.p. daily) of DSS-treated mice (3% in drinking water for 7 days) alleviated colitis-associated weight loss, diarrheal phenotype, and induction of IL-1β and CXCL1 and a decrease in DRA mRNA and protein levels in the colon. Conclusion Our data showing upregulation of DRA under normal and inflammatory conditions by ATRA demonstrate a novel role of this micronutrient in alleviating IBD-associated diarrhea.

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