scholarly journals Spata19 Inactivation as a Cause of Oligospermia

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Mahsa Zargar ◽  
◽  
Abbas Jamshidizad ◽  
Aidin Rahim-Tayefeh ◽  
Ehsan Hashemi ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Cell Apoptosis ◽  
Leydig Cells ◽  
Sperm Count ◽  
Specific Gene ◽  
Rt Pcr ◽  
Histological Studies ◽  
Adhesive Molecule ◽  
Exon 4 ◽  
The Brain

Background: Spermatogenesis associated 19 (Spata19) was introduced as a testis-specific gene that was probably involved in spermatogenesis cell apoptosis. Therefore, this study aimed to investigate the effect of Spata19 inactivation on sperm count. Materials and Methods: We generated global Spata19 knockout mice by CRISPR/Cas9 nickase technology. Disability was validated in three levels of DNA, RNA, and protein using PCR, RT-PCR, and immunohistochemistry. Histological studies were performed for testis. Sperm characteristics were also assessed with CASA software. Results: Spata19 knockout mice had a 43 nucleotides deletion in exon 4 of this gene. The presence and absence of Spata19 were confirmed in normal and knockout mice, respectively. The presence of Spata19 in normal NMRI mice was detected in the brain, heart, and thymus by semi-nested RT-PCR and in Leydig cells by immunohistochemistry. Histological studies revealed a decrease in sperm count in knockout mice. Also, CASA parameters were significantly reduced (P<0.05). Conclusion: These data indicate that Spata19 inactivation is a cause of oligospermia, and its role could be beyond an adhesive molecule.

Cigarette smoke exposure aggravates air space enlargement and alveolar cell apoptosis in Smad3 knockout mice

2011 ◽  
Vol 301 (4) ◽  
pp. L391-L401 ◽  
Author(s):  
Laszlo Farkas ◽  
Daniela Farkas ◽  
David Warburton ◽  
Jack Gauldie ◽  
Wei Shi ◽  
...  
Keyword(s):  
Cigarette Smoke ◽  
Knockout Mice ◽  
Cell Apoptosis ◽  
Transforming Growth Factor ◽  
Smoke Exposure ◽  
Cigarette Smoke Exposure ◽  
Chronic Obstructive ◽  
Specific Gene ◽  
Alveolar Cell ◽  
Air Space

The concept of genetic susceptibility factors predisposing cigarette smokers to develop emphysema stems from the clinical observation that only a fraction of smokers develop clinically significant chronic obstructive pulmonary disease. We investigated whether Smad3 knockout mice, which develop spontaneous air space enlargement after birth because of a defect in transforming growth factor-β (TGF-β) signaling, develop enhanced alveolar cell apoptosis and air space enlargement following cigarette smoke exposure. We investigated Smad3−/− and Smad3+/+ mice at different adult ages and determined air space enlargement, alveolar cell proliferation, and apoptosis. Furthermore, laser-capture microdissection and real-time PCR were used to measure compartment-specific gene expression. We then compared the effects of cigarette smoke exposure on Smad3−/− and littermate controls. Smad3 knockout resulted in the development of air space enlargement in the adult mouse and was associated with decreased alveolar VEGF levels and activity and increased alveolar cell apoptosis. Cigarette smoke exposure aggravated air space enlargement and alveolar cell apoptosis. We also found increased Smad2 protein expression and phosphorylation, which was enhanced following cigarette smoke exposure, in Smad3-knockout animals. Double immunofluorescence analysis revealed that endothelial apoptosis started before epithelial apoptosis. Our data indicate that balanced TGF-β signaling is not only important for regulation of extracellular matrix turnover, but also for alveolar cell homeostasis. Impaired signaling via the Smad3 pathway results in alveolar cell apoptosis and alveolar destruction, likely via increased Smad2 and reduced VEGF expression and might represent a predisposition for accelerated development of emphysema due to cigarette smoke exposure.

scholarly journals Hexose-6-Phosphate Dehydrogenase and 11β-Hydroxysteroid Dehydrogenase-1 Tissue Distribution in the Rat

Endocrinology ◽  
2007 ◽  
Vol 149 (2) ◽  
pp. 525-533 ◽  
Author(s):  
Elise P. Gomez-Sanchez ◽  
Damian G. Romero ◽  
Angela F. de Rodriguez ◽  
Mary P. Warden ◽  
Zygmunt Krozowski ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Dehydrogenase Activity ◽  
Leydig Cells ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Hydroxysteroid Dehydrogenase ◽  
Interstitial Cells ◽  
Rat Tissues ◽  
Rt Pcr ◽  
Reduced Nicotinamide Adenine Dinucleotide ◽  
The Brain

Intracellular concentrations of the glucocorticoids cortisol and corticosterone are modulated by the enzymes 11β-hydroxysteroid dehydrogenase (11β-HSD) 1 and 2. 11β-HSD1 is a reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent microsomal reductase that converts the inactive glucocorticoids cortisone and 11-dehydrocorticosterone to their active forms, cortisol and corticosterone. Hexose-6-phosphate dehydrogenase (H6PDH) is an enzyme that generates NADPH from oxidized NADP (NADP+) within the endoplasmic reticulum. In the absence of NADPH or H6PDH to regenerate NADPH, 11β-HSD1 acts as a dehydrogenase and inactivates glucocorticoids, as does 11β-HSD2. A monoclonal antibody against H6PDH was produced to study the possibility that 11β-HSD1 in the absence of H6PDH may be responsible for hydroxysteroid dehydrogenase activity in tissues that do not express significant amounts of 11β-HSD2. H6PDH and 11β-HSD1 expression was surveyed in a variety of rat tissues by real-time RT-PCR, Western blot analysis, and immunohistochemistry. H6PDH was found in a wide variety of tissues, with the greatest concentrations in the liver, kidney, and Leydig cells. Although the brain as a whole did not express significant amounts of H6PDH, some neurons were clearly immunoreactive by immunohistochemistry. H6PDH was amply expressed in most tissues examined in which 11β-HSD1 was also expressed, with the notable exception of the renal interstitial cells, in which dehydrogenase activity by 11β-HSD1 probably moderates activation of the glucocorticoid receptor because rat renal interstitial cells do not have significant amounts of mineralocorticoid receptors. This antibody against the H6PDH should prove useful for further studies of enzyme activity requiring NADPH generation within the endoplasmic reticulum.

The Protective Effect of Metformin against Oxandrolone-Induced Infertility in Male Rats

2020 ◽  
Vol 26 ◽  
Author(s):  
Abdulqader Fadhil Abed ◽  
Yazun Bashir Jarrar ◽  
Hamzeh J Al-Ameer ◽  
Wajdy Al-Awaida ◽  
Su-Jun Lee
Keyword(s):  
Gene Expression ◽  
Male Infertility ◽  
Protective Effect ◽  
Histological Examination ◽  
Sperm Count ◽  
Serum Testosterone ◽  
Male Rats ◽  
P Value ◽  
Protective Effects ◽  
Rt Pcr

Background: Oxandrolone is a synthetic testosterone analogue that is widely used among bodybuilders and athletes. However, oxandrolone causes male infertility. Recently, it was found that metformin reduces the risk of infertility associated with diabetes mellitus. Aim: This study aimed to investigate the protective effects of metformin against oxandrolone-induced infertility in male rats. Methods: Rats continuously received one of four treatments (n=7) over 14 days: control DMSO administration, oxandrolone administration, metformin administration, or co-administration of oxandrolone and metformin. Doses were equivalent to those used for human treatment. Subsequently, testicular and blood samples were collected for morphological, biochemical, and histological examination. In addition, gene expression of the testosterone synthesizing enzyme CYP11A1 was analyzed in the testes using RT-PCR. Results: Oxandrolone administration induced male infertility by significantly reducing relative weights of testes by 48%, sperm count by 82%, and serum testosterone levels by 96% (ANOVA, P value < 0.05). In addition, histological examination determined that oxandrolone caused spermatogenic arrest which was associated with 2-fold downregulation of testicular CYP11A1 gene expression. However, co-administration of metformin with oxandrolone significantly ameliorated toxicological alterations induced by oxandrolone exposure (ANOVA, P value < 0.05). Conclusion: Metformin administration protected against oxandrolone-induced infertility in male rats. Further clinical studies are needed to confirm the protective effect of metformin against oxandrolone-induced infertility among athletes.

scholarly journals Subarachnoid diverticulum associated with feline infectious peritonitis in a Siberian cat

2020 ◽  
Vol 6 (2) ◽  
pp. 205511692094147
Author(s):  
Christopher Hoey ◽  
George Nye ◽  
Angela Fadda ◽  
Janet Bradshaw ◽  
Emi N Barker
Keyword(s):  
Cervical Spinal Cord ◽  
Serum Biochemistry ◽  
Acute Onset ◽  
Neurological Deficits ◽  
Rt Pcr ◽  
Feline Infectious Peritonitis ◽  
Case Summary ◽  
Choroid Plexitis ◽  
The Brain ◽  
Feline Coronavirus

Case summary A 7-month-old Siberian cat was presented for investigation of acute onset multifocal neurological deficits. Neurological examination documented dull mental status and an ambulatory left hemiparesis. Serum biochemistry documented marked hyperglobulinaemia. MRI of the brain identified marked leptomeningeal contrast enhancement extending along the brainstem caudally to involve the cranial cervical spinal cord. MRI of the cervical spine further identified a subarachnoid diverticulum that extended from the level of the obex to the C2–C3 vertebrae. Cerebrospinal fluid quantitative RT-PCR was positive for the presence of feline coronavirus. Histopathology revealed pyogranulomatous meningitis and choroid plexitis, uveitis and nephritis. Relevance and novel information This article describes the first reported case of a subarachnoid diverticulum associated with feline infectious peritonitis.

scholarly journals The Impact of Bone on the Biology of Aging

2020 ◽  
Vol 4 (Supplement_1) ◽  
pp. 740-740
Author(s):  
Gerard Karsenty
Keyword(s):  
Muscle Function ◽  
Male Fertility ◽  
Leydig Cells ◽  
Memory Loss ◽  
Age Related ◽  
Systematic Exploration ◽  
Biology Of Aging ◽  
The Impact ◽  
The Brain ◽  
Regulate Energy Metabolism

Abstract We hypothesized that bone may secrete hormones that regulate energy metabolism and reproduction. Testing this hypothesis revealed that the osteoblast-specific secreted protein osteocalcin is a hormone regulating glucose homeostasis and male fertility by signaling through a GPCR, Gprc6a, expressed in pancreatic β bells and Leydig cells of the testes. The systematic exploration of osteocalcin biology, revealed that it regulates an unexpectedly large spectrum of physiological functions in the brain and peripheral organs and that it has most features of an antigeromic molecule. As will be presented at the meeting, this body of work suggests that harnessing osteocalcin for therapeutic purposes may be beneficial in the treatment of age-related diseases such as depression, age-related memory loss and the decline in muscle function seen in sarcopenia.

scholarly journals Silencing of MEG3 attenuated the role of lipopolysaccharides by modulating the miR-93-5p/PTEN pathway in Leydig cells

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Xu Zhou ◽  
Jingliang He ◽  
Jinbo Chen ◽  
Yu Cui ◽  
Zhenyu Ou ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Apoptosis ◽  
Leydig Cells ◽  
Treatment Strategies ◽  
Pten Expression ◽  
Luciferase Reporter ◽  
Western Blots ◽  
New Treatment ◽  
Lps Treatment

Abstract Background Leydig cells reflect the activation of inflammation, decrease of androgen production, inhibition of cell growth and promotion of cell apoptosis under orchitis. Maternally expressed gene 3 (MEG3) exerts a crucial role in various human diseases, but under orchitis, the role and underlying molecular mechanism of MEG3 in Leydig cells remain unclear. Methods Lipofectamine 2000 was used for the cell transfections. qPCR and western blots assay were applied to assess the gene expression. ELISA assay was used to measure the TNFα, IL6 and testosterone secretion. CCK8 and EdU assay was employ to test the cell viability and proliferation respectively. Luciferase reporter and RIP assay were introduced to detect the binding of miR-93-5p with MEG3 and PTEN. Results Lipopolysaccharides (LPS) induced TNFα and IL6 secretion, lowered testosterone production, inhibited cell viability and proliferation, and induced cell apoptosis in Leydig cells. MEG3 was upregulated in Leydig cells treated with LPS and that knockdown of MEG3 inhibited the role of LPS in Leydig cells. MEG3 absorbed miR-93-5p and that suppression of miR-93-5p restored the role of silenced MEG3 in Leydig cells under LPS treatment. miR-93-5p inhibited PTEN expression and that over-expressed PTEN alleviated the effect of miR-93-5p in Leydig cells treated with LPS. LPS activated the MEG3/miR-93-5p/PTEN signalling pathway in Leydig cells. Conclusions This study revealed that MEG3 serves as a molecular sponge to absorb miR-93-5p, thus leading to elevation of PTEN expression in Leydig cells under LPS treatment, offering a theoretical basis on which to establish potential new treatment strategies for orchitis.

Detection of specific mRNAs in single nephron segments by use of the polymerase chain reaction

1990 ◽  
Vol 258 (5) ◽  
pp. F1470-F1474 ◽  
Author(s):  
T. Moriyama ◽  
H. R. Murphy ◽  
B. M. Martin ◽  
A. Garcia-Perez
Keyword(s):  
Polymerase Chain Reaction ◽  
Aldose Reductase ◽  
Collecting Duct ◽  
Specific Gene ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Nephron Segments ◽  
Single Nephron ◽  
Specific Mrnas ◽  
Polymerase Chain

We have developed a procedure to detect specific mRNAs in single renal nephron segments. This approach combines microdissection, reverse transcription (RT) of the target mRNA, and amplification of the resulting cDNA using the polymerase chain reaction (PCR). After microdissection, the sample is placed in a tube where it is permeabilized and where all reactions are performed directly without the need for isolation of the RNA. Our model target was the mRNA for aldose reductase. This enzyme catalyzes the conversion of glucose to sorbitol. Its expression is modulated by changes in extracellular osmolality in the renal medulla. RT-PCR of inner medullary collecting duct (1 mm) and glomeruli (6-10) yielded a product of the predicted length (670 base pairs) defined by the PCR primers. Its identity was confirmed by a specific oligonucleotide probe that differed from the primers. RT-PCR of proximal tubules (1 mm) resulted in no aldose reductase-specific amplification product. RT-PCR is generally applicable for measuring specific gene expression in single nephron segments or small numbers of cultured cells. Utility, limitations, and refinements of this approach are discussed.

scholarly journals Elevated Expression of the Testis-specific Gene WBP2NL in Breast Cancer

2015 ◽  
Vol 7 ◽  
pp. BIC.S19079 ◽  
Author(s):  
Seyedmehdi Nourashrafeddin ◽  
Mehdi Dianatpour ◽  
Mahmoud Aarabi ◽  
Maryam Beigom Mobasheri ◽  
Golnesa Kazemi-oula ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Line ◽  
Cell Lines ◽  
Specific Gene ◽  
Rt Pcr ◽  
Noncancerous Tissue ◽  
Ww Domain ◽  
Group I ◽  
Elevated Expression ◽  
Cancer Tissues

Breast cancer is one of the most common causes of cancer death in women; therefore, the study of molecular aspects of breast cancer for finding new biomarkers is important. Recent studies have shown that WW domain-binding protein 2 (WBP2) is important for the oncogenic property of breast cancer. WWP2 N-terminal-like ( WBP2NL) is a testis-specific signaling protein that induces meiotic resumption and oocyte activation events. Our previous study revealed that WBP2NL gene expression is elevated in actively dividing cells and it might be associated with cellular proliferation and tumorigenic process. However, the clinical relevance and importance of WBP2NL gene in cancer has not been understood yet. Therefore, we were interested in analyzing the expression of WBP2NL gene in human breast cancer tissues and breast cancer cell lines, for the first time. We used reverse transcription-polymerase chain reaction (RT-PCR) and semi-nested RT-PCR to evaluate the expression of WBP2NL in malignant breast cancer and adjacent noncancerous tissue (ANCT) samples, as well as MCF-7 and MDA-MB-231 cell lines. The WBP2NL gene was expressed in 45 out of 50 (90%) breast cancer tissues and overexpressed in the MDA-MB-231 cell line. We suggest that WBP2NL may play roles in breast cancer activation maybe through binding to a group I WW domain protein. The elevated expression of WBP2NL gene in breast cancer and MDA-MB-231 cell line leads us to suggest that WBP2NL might be considered as a novel prognostic factor for early diagnosis of breast cancer.

scholarly journals Alternative RNA splicing of the human endothelin-A receptor generates multiple transcripts

1996 ◽  
Vol 313 (3) ◽  
pp. 795-801 ◽  
Author(s):  
Yoshihiro MIYAMOTO ◽  
Takaaki YOSHIMASA ◽  
Hiroshi ARAI ◽  
Kazuhiko TAKAYA ◽  
Yoshihiro OGAWA ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Rna Splicing ◽  
Cdna Clones ◽  
Human Tissues ◽  
Rt Pcr ◽  
Endothelin A Receptor ◽  
Lower Molecular Mass ◽  
Novel Transcripts ◽  
Exon 4 ◽  
Endothelin A

In order to elucidate the regulatory mechanisms of expression of the human endothelin-A receptor (hET-AR) gene, we characterized hET-AR transcripts using reverse transcriptase (RT)-PCR analysis in a variety of human tissues. RT-PCR of lung mRNA using a set of primers from exons 2 and 5 showed two lower-molecular-mass transcripts in addition to the expected fragment. When RT-PCR with primers from exons 4 and 8 was performed, no transcripts other than the expected one were detected. PCR cloning utilizing a set of primers from exons 2 and 8 which covered the entire coding sequence revealed that the cDNA clones corresponding to the two novel transcripts contained deletions of 199 bp and 327 bp respectively compared with the previously described hET-AR cDNA. Comparison of their sequences with that of the hET-AR gene showed that the deleted sequences correspond exactly to exon 4 and exons 3 and 4 respectively, indicating that these lower-molecular-mass ET-AR transcripts result from alternative RNA splicing (designated ET-AR∆4 and ET-AR∆3,4 respectively). Alternative splicing of exon 4 results in a transcript which would be translated into a C-terminal truncated protein containing the first, second and third transmembrane domains, while the splicing out of exons 3 and 4 would produce a protein with five membrane-spanning domains but lacking the third and fourth domains present in the ET-AR protein. An RNase protection assay revealed that ET-AR∆4 and ET-AR∆3,4, as well as ET-AR, transcripts were observed in various human tissues, including the lung, aorta, atrium, kidney and placenta, which are known to express ET-AR abundantly. Thus we have isolated the cDNAs of novel transcripts of hET-AR which are generated by alternative RNA splicing, and these results suggest that this alternative RNA splicing might contribute to the regulation of ET-AR gene expression.

Application of Perfluorooctylbromide Nanoparticles with Ulinastatin for Early Brain Injury Caused by Carbon Monoxide Poisoning

2021 ◽  
Vol 13 (7) ◽  
pp. 1383-1390
Author(s):  
Guangcong Li ◽  
Dan Li
Keyword(s):  
Carbon Monoxide ◽  
Particle Size ◽  
Treatment Group ◽  
Cell Apoptosis ◽  
High Speed ◽  
Carbon Monoxide Poisoning ◽  
Neuronal Cell ◽  
Early Brain Injury ◽  
Group A ◽  
The Brain

ABSTRACTThis study aimed to explore the mechanism of perfluorooctylbromide (PFOB) nanoparticles (NPs) combined with ulinastatin (UTI) on early brain injury (EBI) caused by carbon monoxide poisoning (CMP). Firstly, PFOB NPs were prepared by high-speed dispersion and high-speed homogenization. The physicochemical characteristics of the particle size distribution and Zeta potential distribution of the NPs were analyzed using a laser particle size analyzer. The thermal and photoinduced phase transition characteristics of the NPs were analyzed under heating and laser irradiation conditions. Then, 50 Sprague Dawley (SD) rats were deemed as the research objects to establish the CMP rat models using hyperbaric oxygen chambers. According to different treatment methods, they were rolled into a healthy control group, a carbon monoxide (CO) model group, a PTOB treatment group, an UTI treatment group, and a PTOB + UTI treatment group. The brain tissues of each group of rats were collected 3 days after treatment. The neuronal cell apoptosis, expression of Caspase-3, messenger ribonucleic acid (mRNA) of inflammatory factors interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in rat brain tissue were detected through immunohistochemical staining, in situ cell apoptosis detection, Reverse transcription-polymerase chain reaction (RT-PCR), and Western blotting, so did the relative expression of target proteins B-cell lymphoma-2 (Bcl-2), Bcl2-Associated X (Bax) and myelin basic protein (MBP). As a result, the average particle size and the average Zeta potential of the prepared PFOB NPs was 103±31 nm and −23 ± 15 mV, respectively. When the PFOB NPs were heated to 80 °C, the particle size increased greatly and cracks appeared. The particle size of PFOB NPs also increased obviously after laser irradiation, and the PFOB inside the particles changed into gas phase. Compared to CO group, expression of Caspase-3, neuronal cell apoptosis rate, mRNA expression of IL-1β and TNF-α, and protein expression of Bax and Bcl-2 in the brain tissue of PTOB group, UTI group, and PFOB + UTI group were notably decreased (P < 0.05), while the MBP protein expression increased considerably (P < 0.05). Changes in PFOB + UTI group were more obvious than those in PTOB group and UTI group, and those indicators weren’t considerably different from the controls. In summary, PFOB NPs were successfully prepared with favorable phase transition characteristics. Moreover, PFOB NPs combined with UTI could reduce the apoptosis of brain neurons after CMP, improve the inflammatory response, and play a protective effect on EBI of CMP.

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