scholarly journals Assessment of follicular development in cryopreserved primate ovarian tissue by xenografting: prepubertal tissues are less sensitive to the choice of cryoprotectant

Reproduction ◽  
2011 ◽  
Vol 141 (4) ◽  
pp. 481-490 ◽  
Author(s):  
V von Schönfeldt ◽  
R Chandolia ◽  
L Kiesel ◽  
E Nieschlag ◽  
S Schlatt ◽  
...  
Keyword(s):  
Cancer Survival ◽  
Ovarian Tissue ◽  
Survival Rates ◽  
Follicular Development ◽  
Nuclear Antigen ◽  
Primate Model ◽  
Primordial Follicles ◽  
Cellular Compartments ◽  
Proliferating Cell ◽  
Human Primate

Improvements in cancer survival rates have renewed interest in the cryopreservation of ovarian tissue for fertility preservation. We used the marmoset as a non-human primate model to assess the effect of different cryoprotectives on follicular viability of prepubertal compared to adult ovarian tissue following xenografting. Cryopreservation was performed with dimethylsulfoxide (DMSO), 1,2-propanediol (PrOH), or ethylene glycol (EG) using a slow freezing protocol. Subsequently, nude mice received eight grafts per animal from the DMSO and the PrOH groups for a 4-week grafting period. Fresh, cryopreserved–thawed, and xenografted tissues were serially sectioned and evaluated for the number and morphology of follicles. In adult tissue, the percentage of morphologically normal primordial follicles significantly decreased from 41.2±4.5% (fresh) to 13.6±1.8 (DMSO), 9.5±1.7 (PrOH), or 6.8±1.0 (EG) following cryopreservation. After xenografting, the percentage of morphologically normal primordial (26.2±2.5%) and primary follicles (28.1±5.4%) in the DMSO group was significantly higher than that in the PrOH group (12.2±3 and 5.4±2.1% respectively). Proliferating cell nuclear antigen (PCNA) staining suggests the resumption of proliferative activity in all cellular compartments. In prepubertal tissues, primordial but not primary follicles display a similar sensitivity to cryopreservation, and no significant differences between DMSO and PrOH following xenografting were observed. In conclusion, DMSO shows a superior protective effect on follicular morphology compared with PrOH and EG in cryopreserved tissues. Xenografting has confirmed better efficacy of DMSO versus PrOH in adult but not in prepubertal tissues, probably owing to a greater capacity of younger animals to compensate for cryoinjury.

scholarly journals 202.Estrogen actions on follicle formation and early follicle development

2004 ◽  
Vol 16 (9) ◽  
pp. 202
Author(s):  
K. L. Britt ◽  
P. K. Saunders ◽  
S. J. McPherson ◽  
M. L. Misso ◽  
E. R. Simpson ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Proliferating Cell Nuclear Antigen ◽  
Leydig Cells ◽  
Follicular Development ◽  
Primordial Follicle ◽  
Nuclear Antigen ◽  
Proliferative Index ◽  
Mouse Ovary ◽  
Primordial Follicles ◽  
Proliferating Cell

Estradiol 17 beta (E2) effects late follicular development whilst primordial follicle formation and early activation are thought to be independent of E2. To test this hypothesis we compared numbers of primordial and primary follicles in wildtype and E2 deficient ArKO mice, and the immunohistochemical staining or mRNA expression of Mullerian inhibiting substance (MIS), Wilms tumour 1 (WT-1), and growth differentiation factor (GDF9), known to effect early follicular differentiation. Proliferating cell nuclear antigen (PCNA) staining was a marker of proliferative index. The effects of E2 replacement for 3 wk in 7 wk old ArKO and wildtype mice on these parameters were also tested. We used unbiased, assumption-free stereological methods for quantification of early follicular numbers in the mouse ovary (1). ArKO mice had reduced numbers of primordial and primary follicles compared to wildtype (63%, p<0.001 and 60%, p=0.062 of Wt respectively). This reduction was not corrected by E2 treatment, suggesting that E2 effects the initial formation or activation of primordial follicles. There was a significant increase in the diameters of the oocytes in primordial follicles of ArKO mice compared to wildtype. There were no differences in the immunostaining of MIS, WT-1 and PCNA in primordial and primary follicles between wildtype and ArKO mice. The only difference was as a consequence of Sertoli and Leydig cells in ovaries of ArKO mice. GDF9 mRNA expression was markedly increased in ArKO ovaries. E2 treatment restored the ovarian follicular morphology, and consequently the immunostaining patterns, but had no effect on early follicle numbers. In conclusion, E2 has a role in controlling the size of the oocyte and primordial follicle pools in mice. Supported by NH&MRC RegKey #241000 and 198705. (1) Britt and Myers (2004) Reproduction 127,:569–580.

325 VIABILITY AND GROWTH OF CATTLE PREANTRAL FOLLICLES AFTER IN VITRO CULTURE OF OVARIAN FRAGMENTS IN α-TOCOPHEROL

2010 ◽  
Vol 22 (1) ◽  
pp. 318 ◽  
Author(s):  
L. A. Lisboa ◽  
E. R. Andrade ◽  
M. F. Hertel ◽  
F. A. Melo-Sterza ◽  
K. Moreno ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Ovarian Tissue ◽  
Nuclear Antigen ◽  
Cortical Tissue ◽  
Chi Square ◽  
Primordial Follicles ◽  
Preantral Follicles ◽  
Chi Square Test ◽  
Proliferating Cell

The development of culture systems to support the initiation of growth of primordial follicles is important to the study of the factors that control the earliest stages of folliculogenesis. The aims of the present study were to investigate the effects of α-tocopherol on survival, activation, and growth of cattle preantral follicles using histological and immunohistochemistry proliferating cell nuclear antigen (PCNA) studies. The ovarian cortex was divided into small fragments; one fragment was immediately fixed in Bouin (control). The other fragments were cultured for 2, 4, 6, or 8 d in culture plates with Minimum Essential Medium supplemented with insulin-transferrin-selenium (ITS), pyruvate, glutamine, hypoxanthine, BSA, and antibiotics (MEM+); and MEM+ plus α-tocopherol (5, 25, 50, 100, or 200 ng mL-1). Preantral follicles were classified according to their developmental stage (primordial, intermediate, primary, or secondary) and on the basis of morphological features (normal or degenerated). Pair-wise comparisons were done using Tukey’s procedure. Chi-square test was used to compare the percentage of follicles with PCNA-positive granulosa cells. All analyses were done with the SAS software (SAS Institute, Cary, NC, USA), and P < 0.05 was considered significant. The results showed that, compared with non-cultured cortical tissue (Day 0), the culture of ovarian tissue significantly reduced (P < 0.05) the percentage of normal follicles in all media tested, except for tissue cultured in the presence of 200 ng mL-1 of a-tocopherol. Furthermore, in all media tested, the percentage of primordial follicles was significantly reduced (P < 0.05), with a concomitant increase in the percentage of developing follicles. The highest percentage of secondary follicles was observed after 6 days of culture in MEM plus 200 ng mL-1 of a-tocopherol. The PCNA analysis confirmed the viability of follicles cultured with 200 ng mL-1 of a-tocopherol after 6 d. After 8 days of in vitro culture, we observed severe follicular degeneration in all media tested, suggesting that other supplements are recommended for longer periods of culture. In conclusion, the results of the present study indicate that 200 ng mL-1 of a-tocopherol maintains the survival of cattle preantral follicles and promotes activation of primordial follicles after 6 days of in vitro culture. Financial support: L. A. Lisboa is a recipient of CAPES support; E. R. Andrade and A. A. Alfieri are recipients of PRODOC/CAPES fellowships.

scholarly journals The Effects of High-Dose Qinggan Huoxue Recipe on Acute Liver Failure Induced by D-Galactosamine in Rats

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Hong Zhu ◽  
Yang Zhang ◽  
Xiaoyu Hu ◽  
Cheng Yi ◽  
Sen Zhong ◽  
...  
Keyword(s):  
Liver Function ◽  
Liver Failure ◽  
Acute Liver Failure ◽  
Survival Curve ◽  
Survival Rates ◽  
High Mobility ◽  
Nuclear Antigen ◽  
High Dose ◽  
Improve Liver Function ◽  
Proliferating Cell

Qinggan Huoxue Recipe is a traditional Chinese medicine, which has been usually used to improve liver function in hepatitis. In order to investigate the effects of high-dose Qinggan Huoxue Recipe on acute liver failure and explore the potential mechanism, we had built acute liver failure models in rats by intraperitoneal injection of D-galactosamine (D-GalN). High-dose Qinggan Huoxue Recipe was delivered by gavage. After treatment, the blood alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), albumin (ALB), cholinesterase (CHE), and prothrombin time (PT) were determined. The pathological score of liver tissue was recorded. Proliferating cell nuclear antigen (PCNA) immunohistochemistry staining and fluorescence quantitative reverse transcription polymerase chain reaction (qRT-PCR) of high mobility group box 1 (HMGB1), toll-like receptor 4 (TLR4), nuclear factor-kappa B (NF-κB), and Caspase-3 were performed. The survival curve was also depicted. Our results demonstrated that high-dose Qinggan Huoxue Recipe could significantly improve liver function and increase survival rates in rats with acute liver failure. These effects were supposed to be mediated by suppressing inflammatory reaction and apoptosis.

202 EFFECTS OF ASCORBIC ACID ON IN VITRO CULTURE OF CATTLE PREANTRAL FOLLICLES

2010 ◽  
Vol 22 (1) ◽  
pp. 259
Author(s):  
E. R. Andrade ◽  
R. van den Hurk ◽  
L. A. Lisboa ◽  
M. F. Hertel ◽  
F. A. Melo-Sterza ◽  
...  
Keyword(s):  
Ascorbic Acid ◽  
In Vitro Culture ◽  
Ovarian Tissue ◽  
Immunohistochemical Analysis ◽  
Development Stage ◽  
Nuclear Antigen ◽  
Primordial Follicles ◽  
Preantral Follicles ◽  
Ovarian Cortex

The mechanisms that regulate the gradual exit of ovarian follicles from the nongrowing, primordial pool are poorly understood. The objective of this study was to evaluate the effects of adding ascorbic acid to the media for in vitro culture of cattle ovarian fragments and to determine the effects of this addition on the growth activation and viability of preantral follicles. The ovarian cortex was divided into small fragments; 1 fragment was immediately fixed in Bouin’s solution (control). The other fragments were cultured for 2, 4, 6, or 8 days on culture plates in minimum essential medium (MEM) supplemented with insulin-transferrin-selenium (ITS), pyruvate, glutamine, hypoxantine, BSA, and antibiotics (MEM+) or in MEM+ plus ascorbic acid (5, 25, 50, 100, or 200 μg mL-1). Ovarian tissue was processed for classical histology, TEM, and immunohistochemical demonstration of proliferating cell nuclear antigen (PCNA). Preantral follicles were classified according to their development stage (primordial, intermediate, primary, and secondary) and on the basis of morphological features (normal or degenerated). Pair-wise comparisons were done using Tukey’s procedure. Chi-square test was used to compare percentages of follicles with PCNA-positive granulosa cells. All analyses were done with Statistical Analysis System (SAS Institute, Cary, NC, USA); P ≤ 0.05 was considered significant. Compared with control fragments, the percentage of primordial follicles was reduced (P ≤ 0.05) and the percentage of growing follicles was increased (P ≤ 0.05) in cultured cortical fragments, independent of the tested medium or incubation time. Furthermore, compared with control tissue, culture of ovarian cortex for 8 days reduced the percentages of healthy, viable follicles (P ≤0.05), but not when cultures were supplemented with 25, 50, and 100 μg mL-1 of ascorbic acid. Ultrastructural and immunohistochemical analysis of ovarian cortical fragments cultured for 8 days, however, showed the integrity and viability of follicles only when fragments were cultured in the presence of 50 μg mL-1 of ascorbic acid. In conclusion, this study demonstrated that addition of ascorbic acid to MEM at a concentration of 50 μg mL-1 not only stimulates the activation and subsequent growth of cattle primordial follicles that are cultured in vitro for 8 days but also safeguards the viability of these preantral follicles. E. R. Andrade and A. A. Alfieri are recipients of the PRODOC/CAPES fellowship.

Effects of jacalin and follicle-stimulating hormone on in vitro goat primordial follicle activation, survival and gene expression

Zygote ◽  
2014 ◽  
Vol 23 (4) ◽  
pp. 537-549 ◽  
Author(s):  
Regislane P. Ribeiro ◽  
Antonia M.L.R. Portela ◽  
Anderson W.B. Silva ◽  
José J.N. Costa ◽  
José R.S. Passos ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ovarian Tissue ◽  
Follicle Stimulating Hormone ◽  
Primordial Follicle ◽  
Nuclear Antigen ◽  
Primordial Follicles ◽  
Primordial Follicle Activation ◽  
Follicle Activation ◽  
Stimulating Hormone

SummaryThis study aims to investigate the effects of jacalin and follicle-stimulating hormone (FSH) on activation and survival of goat primordial follicles, as well as on gene expression in cultured ovarian tissue. Ovarian fragments were cultured for 6 days in minimum essential medium (MEM) supplemented with jacalin (10, 25, 50 or 100 μg/ml – Experiment 1) or in MEM supplemented with jacalin (50 μg/ml), FSH (50 ng/ml) or both (Experiment 2). Non-cultured and cultured tissues were processed for histological and ultrastructural analysis. Cultured tissues from Experiment 2 were also stored to evaluate the expression of BMP-15, KL (Kit ligand), c-kit, GDF-9 and proliferating cell nuclear antigen (PCNA) by real-time polymerase chain reaction (PCR). The results of Experiment 1 showed that, compared with tissue that was cultured in control medium, the presence of 50 μg/ml of jacalin increased both the percentages of developing follicles and viability. In Experiment 2, after 6 days, higher percentages of normal follicles were observed in tissue cultured in presence of FSH, jacalin or both, but no synergistic interaction between FSH and jacalin was observed. These substances had no significant effect on the levels of mRNA for BMP-15 and KL, but FSH increased significantly the levels of mRNA for PCNA and c-kit. On the other hand, jacalin reduced the levels of mRNA for GDF-9. In conclusion, jacalin and FSH are able to improve primordial follicle activation and survival after 6 days of culture. Furthermore, presence of FSH increases the expression of mRNA for PCNA and c-kit, but jacalin resulted in lower GDF-9 mRNA expression.

scholarly journals Herbal Compound “Jiedu Huayu” Reduces Liver Injury in Rats via Regulation of IL-2, TLR4, and PCNA Expression Levels

2017 ◽  
Vol 2017 ◽  
pp. 1-9 ◽  
Author(s):  
Minggang Wang ◽  
Qinglan Shi ◽  
Rongzhen Zhang ◽  
Hua Qiu ◽  
Dewen Mao ◽  
...  
Keyword(s):  
Western Blot ◽  
Therapeutic Potential ◽  
Survival Rates ◽  
Interleukin 2 ◽  
Nuclear Antigen ◽  
Traditional Use ◽  
Qrt Pcr ◽  
Hepatocyte Regeneration ◽  
Herbal Compound ◽  
Proliferating Cell

Aim of the Study. To investigate the preventative effects of Jiedu Huayu (JDHY) on D-galactosamine (D-GalN) and lipopolysaccharide-induced acute liver failure (ALF) and to evaluate the possible mechanisms of action. Materials and Methods. ALF was induced in Wistar rats by administrating D-GalN (900 mg/kg) and lipopolysaccharide (10 μg/kg). After treatment with JDHY granules, the levels of blood alanine aminotransferase, aspartate aminotransferase, total bilirubin, and prothrombin time were determined. Proliferating cell nuclear antigen was detected by immunohistochemistry staining. The expression of interleukin-2 (IL-2) and toll-like receptor 4 (TLR4) was examined by fluorescence quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot. Results. JDHY treatment dramatically improved liver function and increased survival rates in an ALF model in rats. We observed a decrease in IL-2 and TLR4 expression following treatment with JDHY in liver cells from ALF rats using qRT-PCR and Western blot analysis. Conclusion. We hypothesize that the therapeutic potential of JDHY for treating ALF is due to its modulatory effect on the suppression of inflammation and by promoting hepatocyte regeneration. Our results contribute towards validation of the traditional use of JDHY in the treatment of liver disease.

scholarly journals NMN does not protect the ovarian reserve from cancer treatments

Reproduction ◽  
2020 ◽  
Vol 159 (2) ◽  
pp. 105-113 ◽  
Author(s):  
Jessica Stringer ◽  
Ella Groenewegen ◽  
Seng H Liew ◽  
Karla Hutt
Keyword(s):  
Ovarian Reserve ◽  
Cancer Survival ◽  
Survival Rates ◽  
Primordial Follicle ◽  
Cancer Therapies ◽  
Γ Irradiation ◽  
Cancer Treatments ◽  
Primordial Follicles ◽  
Nicotinamide Mononucleotide ◽  
Cyclophosphamide Treatment

Primordial follicle oocytes are extremely vulnerable to DNA damage caused by exogenous agents, such as those commonly used to treat cancer. Consequently, female cancer patients often have diminished ovarian reserve, which if severe enough, can cause premature ovarian failure and early menopause. Advances in cancer therapies have resulted in significantly improved cancer survival rates; therefore, it is becoming increasingly important to devise strategies to protect the ovarian reserve from cancer treatments, to avoid loss of fertility and endocrine dysfunction. In this study, we aimed to determine whether supplementation with nicotinamide mononucleotide (NMN) could preserve the ovarian reserve following exposure to DNA-damaging cancer treatments. Adult female mice (n = 5–6/group) received saline or NMN (500 mg/kg/day) for 8 days. Mice were left untreated or exposed to γ-irradiation (0.1 Gy) or cyclophosphamide (150 mg/kg) on day 7 and ovaries and serum collected for analysis on day 12. We report that γ-irradiation treatment significantly reduced the number of primordial follicles, but supplementation with NMN did not prevent the observed follicle loss. Similarly, cyclophosphamide treatment significantly reduced primordial follicle numbers, but these losses were not prevented by NMN supplementation. In conclusion, depletion of the ovarian reserve following γ-irradiation or cyclophosphamide was not protected by NMN supplementation under the conditions employed in this study.

Proliferating cell nuclear antigen in laryngeal cancer

1998 ◽  
Vol 112 (3) ◽  
pp. 310-313 ◽  
Author(s):  
Tomasz Kręcicki ◽  
Michal Jeleń
Keyword(s):  
Proliferating Cell Nuclear Antigen ◽  
Univariate Analysis ◽  
Survival Rates ◽  
Paraffin Section ◽  
Nuclear Antigen ◽  
Control Group ◽  
Satisfactory Outcome ◽  
Significant Difference ◽  
N Stage ◽  
Proliferating Cell

AbstractThe expression of proliferating cell nuclear antigen in paraffin sections from 154 cases of laryngeal squamous cell carcinoma were examined. There was significant difference in PCNA expression between the control group and cancer patients (p<0.001). The mean score of PCNA was higher in patients with poor prognosis than in patients with satisfactory outcome after treatment (p<0.05). There was no significant correlation between the PCNA count and the patient's age and sex, T and N stage and site of the tumour.Univariate analysis revealed that the PCNA score correlated with the patients' survival rates. In multivariate analysis the prognostic value of PCNA was on the statistical borderline (p = 0.049). In our study clinical features like N and T status had a more important influence on survival rate. Nevertheless it appears that the immunohistological examination of PCNA in paraffin section could be a complementary prognostic tool for laryngeal carcinoma. PCNA expression may also be a valuable tool for differentiating malignant from benign laryngeal epithelium.

scholarly journals microRNA 376a regulates follicle assembly by targeting Pcna in fetal and neonatal mouse ovaries

Reproduction ◽  
2014 ◽  
Vol 148 (1) ◽  
pp. 43-54 ◽  
Author(s):  
Huan Zhang ◽  
Xiaohua Jiang ◽  
Yuanwei Zhang ◽  
Bo Xu ◽  
Juan Hua ◽  
...  
Keyword(s):  
Proliferating Cell Nuclear Antigen ◽  
Primordial Follicle ◽  
Neonatal Mouse ◽  
Nuclear Antigen ◽  
Mrna Levels ◽  
Microrna Target ◽  
Ovarian Insufficiency ◽  
Primordial Follicles ◽  
Reproductive Life ◽  
Proliferating Cell

In mammals, the primordial follicle pool, providing all oocytes available to a female throughout her reproductive life, is established perinatally. Dysregulation of primordial follicle assembly results in female reproductive diseases, such as premature ovarian insufficiency and infertility. Female mice lackingDicer1(Dicer), a gene required for biogenesis of microRNAs, show abnormal morphology of follicles and infertility. However, the contribution of individual microRNAs to primordial follicle assembly remains largely unknown. Here, we report that microRNA 376a (miR-376a) regulates primordial follicle assembly by modulating the expression of proliferating cell nuclear antigen (Pcna), a gene we previously reported to regulate primordial follicle assembly by regulating oocyte apoptosis in mouse ovaries. miR-376a was shown to be negatively correlated withPcnamRNA expression in fetal and neonatal mouse ovaries and to directly bind toPcnamRNA 3′ untranslated region. Cultured 18.5 days postcoitum mouse ovaries transfected with miR-376a exhibited decreasedPcnaexpression both in protein and mRNA levels. Moreover, miR-376a overexpression significantly increased primordial follicles and reduced apoptosis of oocytes, which was very similar to those in ovaries co-transfected with miR-376a and siRNAs targetingPcna. Taken together, our results demonstrate that miR-376a regulates primordial follicle assembly by modulating the expression ofPcna. To our knowledge, this is the first microRNA–target mRNA pair that has been reported to regulate mammalian primordial follicle assembly and further our understanding of the regulation of primordial follicle assembly.

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