Effects of ovarian disaggregation on adult murine follicle yield and viability

2017 ◽  
Vol 29 (12) ◽  
pp. 2400 ◽  
Author(s):  
Fiona Young ◽  
John Drummond ◽  
Emma Akers ◽  
Louise Bartle ◽  
David Kennedy ◽  
...  
Keyword(s):  
Molecular Markers ◽  
Granulosa Cells ◽  
Mitochondrial Function ◽  
Ovarian Tissue ◽  
Fluorescein Isothiocyanate ◽  
Rapid Screening ◽  
Animal Origin ◽  
Adult Mice ◽  
Cellular Cytoskeleton ◽  
Collagenase Iv

Follicles are isolated from ovaries for numerous reasons, including IVM, but adult murine yields are <2 follicles mg−1. The aim of the present study was to optimise ovarian disaggregation and develop methods applicable to the rapid screening of follicle viability. Ovaries from adult mice (n = 7) were halved and disaggregated mechanically, or by using collagenase IV (Col-IV; 590 U mL−1) or animal origin-free collagenase IV (AOF) at 590 or 1180 U mL−1. Isolated follicles were stained with 4′,6′-diamidino-2-phenylindole (DAPI; nuclei), chloromethyl-X-rosamine (CMXRos; mitochondria) or fluorescein isothiocyanate-conjugated anti-α-tubulin antibody. Follicle diameters and staining were measured and analysed using ImageJ, and data analysed using GraphPad Prism. Col-IV disaggregation yielded the highest number of follicles (17 ± 10 follicles mg−1 ovarian tissue). All disaggregation methods released more secondary follicles (86 ± 20 per ovary; P < 0.05) than any other size cohort. Mechanical and Col-IV disaggregation yielded similar numbers of morphologically intact follicles, whereas AOF disaggregation caused more damage (P < 0.01). As the morphological disruption increased, DAPI and CMXRos staining decreased (P < 0.05), and tubulin localisation became more heterogeneous. Col-IV disaggregation gave the best yield of morphologically intact follicles containing viable granulosa cells. In conclusion, we improved adult murine follicle yields and applied molecular markers to assess follicle morphology, cellular cytoskeleton and mitochondrial function.

scholarly journals Ovarian tissue freezing and activation after thawing: an update

2021 ◽  
Vol 26 (1) ◽  
Author(s):  
Li-fan Peng
Keyword(s):  
Granulosa Cells ◽  
Clinical Decision Making ◽  
Ovarian Function ◽  
Ovarian Tissue ◽  
Clinical Decision ◽  
The Body ◽  
Endocrine Function ◽  
Ovarian Tissue Cryopreservation ◽  
Main Body ◽  
Tissue Cryopreservation

Abstract Background With the growth of women’s age, ovarian failure can be caused by various factors. For the women who need chemotherapy because of cancer factors, the preservation of fertility is more urgent. The treatment of cancer is also a process in which all tissues and organs of the body are severely damaged, especially in the reproductive system. Main body As a new fertility preservation technology, autologous ovarian tissue cryopreservation and transplantation is developing rapidly and showing great potentiality in preserving ovarian endocrine function of young cervical cancer patients. Vitrification and slow freezing are two common techniques applied for ovarian tissue cryopreservation. Thus, cryopreserved/thawed ovarian tissue and transplantation act as an important method to preserve ovarian function during radiotherapy and chemotherapy, and ovarian cryopreservation by vitrification is a very effective and extensively used method to cryopreserve ovaries. The morphology of oocytes and granulosa cells and the structure of organelles were observed under the microscope of histology; the hormone content in the stratified culture medium of granulosa cells with the diameter of follicle was used to evaluate the development potential of ovarian tissue, and finally the ovarian tissue stimulation was determined by the technique of ovarian tissue transplantation. Conclusions Although there are some limitations, the team members still carry out this review to provide some references and suggestions for clinical decision-making and further clinical research.

Morphology and morphometry of preantral follicles, and immunolocalization of angiogenic factors in ovarian tissue from the neotropical primateSapajus apella

Zygote ◽  
2018 ◽  
Vol 26 (5) ◽  
pp. 424-429
Author(s):  
AB Brito ◽  
DCC Brito ◽  
W B Silva ◽  
APR Rodrigues ◽  
JR Figueiredo ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Developmental Stages ◽  
Ovarian Tissue ◽  
Angiogenic Factors ◽  
Oocyte Nucleus ◽  
Oocyte Diameter ◽  
Preantral Follicles ◽  
Cell Counts ◽  
Secondary Stage ◽  
Morphology And Morphometry

SummaryOvarian biopsies from five health adult monkeys were collected by exploratory laparotomy. Preantral follicles (primordial, primary, and secondary) were classified as normal or degenerated and submitted to morphometric analysis in which granulosa cell counts and the areas of follicles, oocytes, and oocyte nuclei were measured. Ovarian fragments were also immunolabelled for the quantitative analysis of VEGFA and CD31 protein expression in the ovarian tissue and in the preantral follicles. In total, 213 preantral follicles was examined for morphometry and morphological classification. From this total, 20 (9.4%) were follicles enclosing two or more oocytes, i.e. multi-oocyte follicles (MOFs). From the 193 follicles enclosing only one oocyte, 46.3% were classified as primordial, 24,1% as transition, 23.3% as primary, and 6.3% as secondary follicles. The mean number of granulosa cells surrounding primordial, transition, primary, and secondary follicles was 9.2, 12.1, 18.7, and 45.3, respectively. Increase in oocyte diameter was observed from primary to secondary follicles, while the oocyte nucleus increased only when follicles reached the secondary stage. The expression of CD31 was strong in vessels, corpus luteum, and in normal oocytes and granulosa cells from preantral follicles at all developmental stages. Likewise, VEGFA expression was observed in vessels and preantral follicles (granulosa cells, the oocyte and the oocyte nucleus). We characterized the morphology, and morphometry and expression of angiogenic factors in normal and atretic preantral follicles fromSapajus apella. This description can support the analysis of follicular quality and survival after procedures such as transplantation and cryopreservation.

scholarly journals Ovarian Tumor Mitochondria Exhibit Abnormal Phenotypes and Blunted Associations with Biobehavioral Factors

2021 ◽  
Author(s):  
Snehal Bindra ◽  
Marlon McGill ◽  
Marina Triplett ◽  
Anisha Tyagi ◽  
Premal Thaker ◽  
...  
Keyword(s):  
Molecular Markers ◽  
Respiratory Chain ◽  
Ovarian Tissue ◽  
Mitochondrial Respiratory Chain ◽  
Ovarian Tumors ◽  
Mitochondrial Content ◽  
Benign Tissue ◽  
Mitochondrial Respiratory Chain Activity ◽  
Respiratory Chain Activity ◽  
And Function

Abstract Tumor cells exhibit mitochondrial alterations and are also influenced by biobehavioral processes, but the intersection of biobehavioral factors and tumor mitochondria remains unexplored. Here we examined multiple biochemical and molecular markers of mitochondrial content and function in benign and cancerous ovarian tissue in parallel with exploratory analyses of biobehavioral factors. First, analysis of a publicly-available database (n=1,435) showed that gene expression of specific mitochondrial proteins in ovarian tumors is associated with survival. Quantifying multiple biochemical and molecular markers of mitochondrial content and function in 51 benign and 128 high-grade epithelial ovarian tumors revealed that compared to benign tissue, tumors exhibit 3.3-8.4-fold higher mitochondrial content and respiratory chain enzymatic activities (P<0.001) but similar mitochondrial DNA levels (-3.1%), documenting abnormal mitochondrial phenotypes in tumors. Mitochondrial respiratory chain activity was also associated with interleukin-6 (IL-6) levels in ascites. In benign tissue, negative biobehavioral factors were inversely correlated with mitochondrial content and respiratory chain activities, whereas positive biobehavioral factors tended to be positively correlated with mitochondrial measures, although effect sizes were small to medium (r=-0.43 to 0.47). In contrast, serous tumors showed less pronounced biobehavioral-mitochondrial correlations. These results document abnormal mitochondrial functional phenotypes in ovarian tumors and warrant further research on the link between biobehavioral factors and mitochondria in cancer.

scholarly journals Sphingosine 1-phosphate protects against radiation-induced ovarian injury in female rats—impact on mitochondrial-related genes

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Jiahui Zhao ◽  
Shuyun Zhang ◽  
Liesong Chen ◽  
Xiaolong Liu ◽  
Haihong Su ◽  
...  
Keyword(s):  
Ionizing Radiation ◽  
Protective Effect ◽  
Mitochondrial Function ◽  
Target Genes ◽  
Ovarian Function ◽  
Ovarian Tissue ◽  
Differentially Expressed ◽  
Female Rats ◽  
Sphingosine 1 Phosphate ◽  
Radiation Induced

Abstract The toxic effects of ionizing radiation on the gonads have been widely recognized. Sphingosine 1-phosphate (S1P) has a protective effect on ovarian injury, and although it is known that mitochondria are involved in this process, the specific mechanism is not fully understood. The present study analysed the changes in the serum AMH and ovarian histology in Sprague-Dawley female rats exposed to X-ray radiation only or co-administered with S1P. The mRNA expression profile of ovarian tissue was further analysed via next-generation sequencing and bioinformatics approaches to screen out candidate mitochondria-related genes. Finally, differentially expressed target genes were verified by real-time PCR. The results showed that ionizing radiation could reduce the serum AMH level, destroy ovarian structure and decrease the number of follicles in rats, while S1P administration significantly attenuated the impairment of ovarian function. Gene ontology (GO) and KEGG pathway analysis revealed that a variety of genes related to mitochondrial function were differentially expressed, and the protective effect of S1P on mitochondria was more obvious in the acute phase 24 h after radiation. The differentially expressed mitochondrial function-related genes associated with the protective effect of S1P were UQCRH, MICU2 and GPX4, which were subsequently verified by RT-PCR. Therefore, ionizing radiation has a significant effect on ovarian function, and S1P has a protective effect on radiation-induced ovarian injury, in which mitochondria may play an important role. This study sheds new light on the mechanism of radiation-induced ovarian injury and helps develop a novel potential strategy to control it.

scholarly journals Gene Expression in Granulosa Cells From Small Antral Follicles From Women With or Without Polycystic Ovaries

2019 ◽  
Vol 104 (12) ◽  
pp. 6182-6192 ◽  
Author(s):  
Lisa Ann Owens ◽  
Stine Gry Kristensen ◽  
Avi Lerner ◽  
Georgios Christopoulos ◽  
Stuart Lavery ◽  
...  
Keyword(s):  
Gene Expression ◽  
Granulosa Cells ◽  
Polycystic Ovary ◽  
Ovarian Tissue ◽  
Polycystic Ovaries ◽  
Vitro Fertilization ◽  
Antral Follicles ◽  
Oocyte Aspiration ◽  
And Function

Abstract Context Polycystic ovary syndrome (PCOS) is the most common cause of anovulation. A key feature of PCOS is arrest of follicles at the small- to medium-sized antral stage. Objective and Design To provide further insight into the mechanism of follicle arrest in PCOS, we profiled (i) gonadotropin receptors; (ii) characteristics of aberrant steroidogenesis; and (iii) expression of anti-Müllerian hormone (AMH) and its receptor in granulosa cells (GCs) from unstimulated, human small antral follicles (hSAFs) and from granulosa lutein cells (GLCs). Setting GCs from hSAFs were collected at the time of cryopreservation of ovarian tissue for fertility preservation and GLCs collected during oocyte aspiration before in vitro fertilization/intracytoplasmic sperm injection. Participants We collected hSAF GCs from 31 women (98 follicles): 10 with polycystic ovaries (PCO) and 21 without. GLCs were collected from 6 women with PCOS and 6 controls undergoing IVF. Main Outcome Measures Expression of the following genes: LHCGR, FSHR, AR, INSR, HSD3B2, CYP11A1, CYP19, STAR, AMH, AMHR2, FST, INHBA, INHBB in GCs and GLCs were compared between women with PCO and controls. Results GCs in hSAFs from women with PCO showed higher expression of LHCGR in a subset (20%) of follicles. Expression of FSHR (P < 0.05), AR (P < 0.05), and CYP11A1 (P < 0.05) was lower, and expression of CYP19A1 (P < 0.05), STAR (P < 0.05), HSD3B2 (P = NS), and INHBA (P < 0.05) was higher in PCO GCs. Gene expression in GL cells differed between women with and without PCOS but also differed from that in GCs. Conclusions Follicle arrest in PCO is characterized in GCs by differential regulation of key genes involved in follicle growth and function.

Expression and immunolocalisation of follicle-stimulating hormone receptors in gonads of newborn and adult female horses

2016 ◽  
Vol 28 (9) ◽  
pp. 1340 ◽  
Author(s):  
Dragos Scarlet ◽  
Ingrid Walter ◽  
Juraj Hlavaty ◽  
Christine Aurich
Keyword(s):  
Western Blot ◽  
Granulosa Cells ◽  
Hormone Receptors ◽  
Ovarian Function ◽  
Ovarian Tissue ◽  
Cumulus Cells ◽  
Protein Levels ◽  
Fshr Gene ◽  
Luteal Tissue ◽  
Immunofluorescence Labelling

In mares, FSH and its receptor (FSHR) are essential for ovarian function. The objective of the present study was to analyse FSHR gene expression at the mRNA and protein levels in ovarian tissue from newborn and adult horses. Expression of mRNA was analysed by reverse transcription polymerase chain reaction, whereas FSHR protein was visualised by immunohistochemistry (IHC), immunofluorescence labelling (IF) and western blot. FSHR mRNA was detected in ovarian follicles and luteal tissue from adult mares, as well as in the ovaries of neonates. Follicular growth up to 4 mm in diameter was already present in neonates. Using IHC and IF, FSHR protein was detected in granulosa cells, cumulus cells and inconsistently in oocytes, independent of the animal’s age or the stage of folliculogenesis. A lower FSHR expression was observed in theca cells in comparison to granulosa cells. FSHR was abundant in the ovarian stroma cells of neonates but not of adults. Luteal cells stained positive for FSHR independent of the stage of corpus luteum development. The presence of FSHR protein in various cell populations of the ovary was confirmed by western blot. In conclusion, FSHR is present in horse ovaries consistently from birth onwards and expression remains constant during the oestrous cycle.

Ultrastructural localisation of calcium deposits in the mouse ovary

2003 ◽  
Vol 15 (8) ◽  
pp. 415 ◽  
Author(s):  
M. Sedmíková ◽  
R. Rajmon ◽  
J. Petr ◽  
M. Vaňková ◽  
J. Rozinek ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Follicular Development ◽  
Mouse Ovary ◽  
Antral Follicles ◽  
Mouse Oocytes ◽  
Adult Mice ◽  
Calcium Deposits ◽  
Cellular Compartments ◽  
Meiotic Competence

Follicle-enclosed mouse oocytes contain numerous calcium deposits. The ultrastructural distribution of calcium deposits in the nuclei, mitochondria and cytoplasm of mouse oocytes and granulosa cells of primary, secondary and antral follicles was examined using the combined oxalate–pyroantimonate method. The mitochondria of oocytes from all types of follicles had the highest levels of calcium deposits of all oocyte compartments, with the exception of primary follicles, in which oocyte nuclei contained the same level of calcium deposits as the mitochondria. Calcium deposits in the cytoplasm of oocytes from primary follicles were significantly lower than those in the cytoplasm of oocytes from secondary and antral follicles. Calcium deposits in the cytoplasm of granulosa cells were significantly lower than calcium deposits in the mitochondria of granulosa cells and this difference persisted throughout all categories of follicles. Calcium deposits in the nuclei of granulosa cells did not differ from levels in the mitochondria in primary and secondary follicles. In contrast, the nuclei of granulosa cells from antral follicles had lower levels of calcium deposits than the mitochondria. The differences observed in calcium deposits in various cellular compartments in oocytes and granulosa cells in the follicles of ovaries of adult mice can be attributed to their acquisition of meiotic competence and follicular development.

scholarly journals Identification of Molecular Markers Specific to the Bovine Species Used in the Production of Food Products of Animal Origin

2020 ◽  
Vol 77 (1) ◽  
pp. 62
Author(s):  
Lavinia Maria CHIŞ ◽  
Dan Cristian VODNAR
Keyword(s):  
Molecular Markers ◽  
Genomic Dna ◽  
Reference Range ◽  
Dna Amplification ◽  
Food Products ◽  
Animal Origin ◽  
Helpful Tool ◽  
Target Dna ◽  
Extraction Stage ◽  
Bovine Species

Molecular markers are a helpful tool for species detection of meat origin, in order to determine with accuracy the possible fraud of animal-based food products. In this study, blood, milk and cheese samples were taken from the Romanian “Bălțata” bovine species. In the blood extraction stage, the commercial Wizard Genomic DNA Extraction Kit was used, and for the milk and cheese extraction, the SureFood®PREP Advanced kit was used. Target DNA amplification in all the three matrices was performed with the StockMarks for Cattle kit. It was found that the analyzed loci had the size of the fragments within the reference range given by the kit used, which concludes that the blood, milk and the cheese have the same origin, from the same animal of the bovine species. Therefore, through the genotyping technique, traceability of food products can be achieved and the species of origin can be identified.

scholarly journals Detection of Residues of Antimicrobial Compounds in Eggs by the Rapid Screening Methods

2018 ◽  
Vol 62 (3) ◽  
pp. 48-55
Author(s):  
M. Krišová ◽  
I. Kožárová
Keyword(s):  
Potable Water ◽  
Rapid Screening ◽  
Animal Origin ◽  
Effective Control ◽  
Human Consumption ◽  
Screening Methods ◽  
Antimicrobial Compounds ◽  
Disease Treatment ◽  
The Public ◽  
Antimicrobial Residues

Abstract Eggs belong to the most frequently consumed products of animal origin worldwide, and therefore the safety of eggs is a substantiated issue. Conventional poultry rearing involves the use of antimicrobials added to their feed or potable water particularly for disease treatment, however, in some countries also for the prevention of diseases, promotion of growth and better utilisation of the feed. Thus, effective control of residues of such substances in eggs is very important for the protection of the public health. The aim of this study was to detect the potential presence of antimicrobial residues in fresh hen eggs using commercially available rapid screening methods (Premi®Test and EXP Ampulle test) and compare the results of both of these tests. We examined 22 samples randomly selected from among 66 samples purchased in 11 European countries. We respected the procedures as supplied by the manufacturers of the tests together with their respective test kits. The examination of eggs by the Premi®Test did not detect the presence of antimicrobial residues in the samples, while the EXP Ampulle test provided 8 positive and 6 dubious results. Our results allowed us to conclude that the EXP Ampulle appears to be more sensitive and allows one to carry out more effective control of the presence of antimicrobial residues in hen eggs intended for human consumption.

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