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Tiantian Zhang - One of the best experts on this subject based on the ideXlab platform.
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The use of fetal bovine serum for cryopreservation of stage III zebrafish (Danio rerio) Ovarian Follicles.
Cryo letters, 2012Co-Authors: Tiziana Zampolla, David M Rawson, Tiantian ZhangAbstract:: Although several studies on fish Ovarian Follicles cryopreservation have been carried out, their cryopreservation still remains unsuccessful. In this study, for the first time the effect of Fetal Bovine Serum (FBS) in combination with several concentrations of methanol (1, 2 or 4M) as cryoprotectant on stage III Ovarian Follicles viability was investigated and cryopreservation using controlled slow cooling was undertaken. Membrane integrity assessed by trypan blue (TB) staining and ATP level of stage III Ovarian Follicles were evaluated following cryoprotectant treatment and cryopreservation. The results showed that the survival of Ovarian Follicles after cryopreservation, assessed by TB staining, was significantly decreased using all three concentrations of CPAs solutions when compared to room temperature controls. The results showed a further decrease of viability when the TB test was performed 2h post-thaw. 2M methanol+10 percent FBS resulted in higher survival rate compared with the other methanol concentrations in combination with 10 percent FBS, viability results obtained 10 min and 2 hours post-thaw were 70.2 +/- 4.2 percent and 46.2 +/- 10.4 percent respectively. Whilst results obtained with 1M methanol supplemented with 10 percent FBS 10min and 2 hours post-thaw were 51.8 +/- 2.4 and 3.4 +/- 1.5 and those obtained with 4M supplemented with 10 percent FBS were 36.0 +/- 5.3 and 6.4 +/- P content was observed with all three cryoprotectant solutions indicating a compromised metabolic status. The addition of 10 percent fetal bovine serum to media used for cryopreservation zebrafish Ovarian Follicles did not allow the achievement of successful cryopreservation.
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18. Vitrification of zebrafish (Danio rerio) Ovarian Follicles
Cryobiology, 2012Co-Authors: Leandro Godoy, Tiziana Zampolla, Danilo Pedro Streit, Adriana Bos-mikich, Tiantian ZhangAbstract:Overfishing and environmental factors such as pollution have contributed to the significant decline of fish populations worldwide. Successful cryopreservation of fish gametes and embryos would have important applications in conservation and aquaculture. Although cryopreservation of fish semen has already been achieved successfully, cryopreservation of fish embryos and oocytes remains elusive. Despite the widespread use of vitrification for oocyte cryopreservation in humans and domestic mammals, vitrification of fish oocytes has not been studied to date. This study aimed at developing a cryopreservation protocol for stage III zebrafish Ovarian Follicles within fragment using vitrification. Vitrification ability of methanol, ethanol, Me 2 SO, propylene glycol and ethylene glycol were tested using a range of concentrations made up in 90% Leibovitz L-15 medium. Solutions were tested for vitrification ability using 0.25 ml plastic straw and a fibreplug™ (CryoLogic Ltd.) loaded by a pipette. The transparent glassy appearance during cooling and warming was used to identify vitrified solution. Based on results obtained from this stage, the vitrification ability of 24 combinations using the mentioned cryoprotectants was studied. Three vitrification solutions (VS) (V6B: 1.5 M methanol + 6.0 M ethylene glycol + 0.5 M sucrose; V1B: 1.5 M methanol + 4.5 M propylene glycol; and V2: 1.5 M methanol + 5.5 M Me 2 SO) were selected for subsequent experiments based on their vitrifying ability. For VS toxicity tests, Ovarian Follicles were equilibrated in L-15 medium containing 1.5 M methanol for 30 min at room temperature. After equilibration Follicles were exposed to VS for 3 min in a stepwise manner. Afterwards the cryoprotectants were gradually removed in three steps and viability was assessed by trypan blue staining. For vitrification procedures, immediately after exposure to VS Ovarian Follicles were loaded in either plastic straw or fibreplug™ and plunged into liquid nitrogen, being stored for at least 20 min. The warming was performed at 28 °C and cryoprotectants were gradually removed in three steps. Ovarian follicle viability was assessed by three means: trypan blue staining, measurement of ATP level in the Follicles and assessment of mitochondrial membrane potential and distribution by using JC-1 molecular probe. Results obtained from toxicity tests showed Follicles viability of 74% for V1B; 30% for V2 and 78% for V6B. After vitrification, Ovarian Follicles presented a viability of 60% when fibreplug and V1B were used, V2 showed viability of 42% (using fibreplug), and using plastic straws V6B solution showed only 2% of viability. [ATP] soon after warming dropped down to 22% (V2) and 7% (V1B) and two hr later the concentration in V2 was 15% and for V1B a very low concentration of 3.5%. The viable Follicles in V1B and V2 seemed to be opaque and morphologically intact, when assessed immediately after warming. These results are different to the results described so far in the literature, where zebrafish Follicles were observed to become swollen and translucent even during the warming process after being exposed to controlled slow cooling protocols. However, 30 min after warming most of the Follicles started to become semi-translucent and lightly swollen. A complete loss of internal structure pattern and also loss of fluorescence indicated Follicles with no mitochondrial membrane potential after vitrification.
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Development of in vitro culture method for early stage zebrafish (Danio rerio) Ovarian Follicles for use in cryopreservation studies
Theriogenology, 2010Co-Authors: S. Tsai, David M Rawson, Tiantian ZhangAbstract:There have been no reported methods for in vitro growth of early stage Ovarian Follicles for fish and their cryopreservation is still under investigation. If cryopreservation of early stage Ovarian Follicles can be achieved, in vitro procedures for Ovarian follicle culture, development, ovulation and fertilisation after cryopreservation would be needed. The aim of the present study was to develop an in vitro culture method for early stage zebrafish Ovarian Follicles for use after their cryopreservation. Procedures for in vitro culture of stage I (primary growth) and stage II (cortical alveolus) Ovarian Follicles were developed. The effects of concentration of L-15 medium, pH and the concentration of human chorionic gonadotropin (hCG) and activin A were studied. The results demonstrated that early stage zebrafish Ovarian Follicles can be cultured in vitro for 24 h, stage I and II Ovarian Follicles can grow to the sizes of early stage II and stage III Ovarian Follicles after hCG treatment. The method developed here is effective for assessing early stage zebrafish Ovarian Follicles growth competence in vitro. The results from the present study indicated that in vitro culture is the most reliable method for assessing Ovarian follicle viability when compared with vital dye staining methods.
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Development of cryopreservation protocols for early stage zebrafish (Danio rerio) Ovarian Follicles using controlled slow cooling.
Theriogenology, 2009Co-Authors: S. Tsai, David M Rawson, Tiantian ZhangAbstract:Abstract Cryopreservation of germplasm of aquatic species offers many benefits to the fields of aquaculture, conservation and biomedicine. Although successful fish sperm cryopreservation has been achieved with many species, there has been no report of successful cryopreservation of fish embryos and late stage oocytes which are large, chilling sensitive and have low membrane permeability. In the present study, cryopreservation of early stage zebrafish Ovarian Follicles was studied for the first time using controlled slow freezing. The effect of cryoprotectant, freezing medium, cooling rate, method for cryoprotectant removal, post-thaw incubation time and Ovarian follicle developmental stage were investigated. Stages I and II Ovarian Follicles were frozen in 4 M methanol and 3 M DMSO in either L-15 medium or KCl buffer. Ovarian follicle viability was assessed using trypan blue, FDA + PI staining and ADP/ATP assay. The results showed that KCl buffer was more beneficial than L-15 medium, methanol was more effective than DMSO, optimum cooling rates were 2–4 °C/min, stepwise removal of cryoprotectant improved Ovarian follicle viability significantly and stage I Ovarian Follicles were more sensitive to freezing. The results also showed that FDA + PI staining and ADP/ATP assay were more sensitive than TB staining. The highest follicle viabilities after post-thaw incubation for 2 h obtained with FDA + PI staining were 50.7 ± 4.0% although ADP/ATP ratios of the cryopreserved Follicles were significantly increased indicating increased cell damage. Studies are currently being carried out on in vitro maturation of these cryopreserved Ovarian Follicles.
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Studies on chilling sensitivity of early stage zebrafish (Danio rerio) Ovarian Follicles
Cryobiology, 2009Co-Authors: S. Tsai, David M Rawson, Tiantian ZhangAbstract:Cryopreservation of fish gametes is of great importance in aquaculture, conservation and human genomic research. The creation of gamete cryobanks allows the storage of genetic material of targeted species for almost unlimited time periods. Cryopreservation has been successfully applied to fish sperm of many species, but there has been no success with fish embryos and oocytes. One of the obstacles to fish oocyte cryopreservation is their high chilling sensitivity and especially at subzero temperatures. Although studies on late stage oocyte cryopreservation has been carried out, there have been no reported studies on cryopreservation of early stage Ovarian Follicles. The aim of this study is to investigate the chilling sensitivity of early stage zebrafish Ovarian Follicles before developing protocols for their cryopreservation. Experiments were conducted with stage I (primary growth), stage II (cortical alveolus) and stage III (vetillogenesis) Ovarian Follicles, which were chilled in KCl buffer and L-15 medium for up to 144h at -1 degrees C in a low temperature bath. Ovarian Follicles were also exposed to 2M methanol or 2M DMSO in L-15 medium for up to 168h at -1 and -5 degrees C, respectively. Control Follicles were kept at 28 degrees C. Ovarian follicle viability was assessed using trypan blue staining. The results showed that stage I and II Ovarian Follicles are less sensitive to chilling than stage III Follicles. These results were also confirmed following in vitro maturation of the chilled Ovarian Follicles. The results also showed that L-15 medium is more beneficial than KCl buffer for Ovarian Follicles at all stages. The presence of both methanol and DMSO reduced chilling sensitivity of Ovarian Follicles at all stages with methanol being the most effective. The study indicated that stage I and II Follicles are less sensitive to chilling than stage III Follicles, and that early stage zebrafish Ovarian Follicles may be better candidates for cryopreservation.
Ariella Shikanov - One of the best experts on this subject based on the ideXlab platform.
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Synthetic PEG Hydrogel for Engineering the Environment of Ovarian Follicles.
Methods in molecular biology (Clifton N.J.), 2018Co-Authors: Uziel Mendez, Hong Zhou, Ariella ShikanovAbstract:The functional unit within the ovary is the Ovarian follicle, which is also a morphological unit composed of three basic cell types: the oocyte, granulosa, and theca cells. Similar to human Ovarian Follicles, mouse Follicles can be isolated from their Ovarian environment and cultured in vitro to study folliculogenesis, or follicle development for days or weeks. Over the course of the last decade, follicle culture in a three-dimensional (3D) environment exponentially improved the outcomes of in vitro folliculogenesis. Follicle culture in 3D environments preserves follicle architecture and promotes the cross talk between cells in the follicle. Hydrogels, such as polyethylene glycol (PEG), have been used for various physiological systems for regenerative purposes because they provide a 3D environment similar to soft tissues, allow diffusion of nutrients, and can be readily modified to present biological signals, including cell adhesion ligands and proteolytic degradation facilitated by enzymes secreted by the encapsulated cells. This chapter outlines the application of PEG hydrogels to the follicle culture, including the procedures to isolate, encapsulate, and culture mouse Ovarian Follicles. The tunable properties of PEG hydrogels support co-encapsulation of Ovarian Follicles with somatic cells, which further promote follicle survival and growth in vitro through paracrine and juxtacrine interactions.
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Three-Dimensional Hydrogel-Based Culture to Study the Effects of Toxicants on Ovarian Follicles.
Methods in molecular biology (Clifton N.J.), 2018Co-Authors: Hong Zhou, Ariella ShikanovAbstract:Various toxicants, such as drugs and their metabolites, can cause potential Ovarian toxicity. As the functional units of the ovary, Ovarian Follicles are susceptible to this type of damage at all developmental stages. Studying the effects of toxicants on Ovarian Follicles is an important task. Three-dimensional (3D) hydrogels, such as fibrin alginate interpenetrating networks (FA-IPNs), can support Ovarian follicle culture in vitro for extended periods of time and serve as a suitable tool for studying ovotoxicity. Growing Follicles encapsulated in the FA-IPN can proteolytically degrade the fibrin component in the FA-IPN. The degradation of fibrin mirrors the follicle growth and serves as a surrogate reporter for follicle health. The speed of fibrin degradation can be further controlled by aprotinin, a small molecule that inhibits plasmin-driven proteolytic degradation, which further expands the application of the described system. In this chapter, we describe methods to (1) isolate and encapsulate mouse Ovarian Follicles in FA-IPN, (2) follow follicle growth and development in vitro, and (3) evaluate the effects of toxicants on folliculogenesis using fibrin degradation.
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hydrogel based 3 dimensional 3d system for toxicity and high throughput htp analysis for cultured murine Ovarian Follicles
PLOS ONE, 2015Co-Authors: Hong Zhou, Malika Amattullah Malik, Aarthi Arab, Matthew Thomas Hill, Ariella ShikanovAbstract:Various toxicants, drugs and their metabolites carry potential Ovarian toxicity. Ovarian Follicles, the functional unit of the ovary, are susceptible to this type of damage at all stages of their development. However, despite of the large scale of potential negative impacts, assays that study Ovarian toxicity are limited. Exposure of cultured Ovarian Follicles to toxicants of interest served as an important tool for evaluation of toxic effects for decades. Mouse Follicles cultured on the bottom of a culture dish continue to serve an important approach for mechanistic studies. In this paper, we demonstrated the usefulness of a hydrogel based 3-dimensional (3D) mouse Ovarian follicle culture as a tool to study Ovarian toxicity in a different setup. The 3D in vitro culture, based on fibrin alginate interpenetrating network (FA-IPN), preserves the architecture of the Ovarian follicle and physiological structure-function relationship. We applied the novel 3D high-throughput (HTP) in vitro Ovarian follicle culture system to study the ovotoxic effects of an anti-cancer drug, Doxorobucin (DXR). The fibrin component in the system is degraded by plasmin and appears as a clear circle around the encapsulated follicle. The degradation area of the follicle is strongly correlated with follicle survival and growth. To analyze fibrin degradation in a high throughput manner, we created a custom MATLAB® code that converts brightfield micrographs of Follicles encapsulated in FA-IPN to binary images, followed by image analysis. We did not observe any significant difference between manually processed images to the automated MATLAB® method, thereby confirming that the automated program is suitable to measure fibrin degradation to evaluate follicle health. The cultured Follicles were treated with DXR at concentrations ranging from 0.005 nM to 200 nM, corresponding to the therapeutic plasma levels of DXR in patients. Follicles treated with DXR demonstrated decreased survival rate in greater DXR concentrations. We observed partial follicle survival of 35% ± 3% (n = 80) in 0.01nM treatment and 48% ± 2% (n = 92) in 0.005nM, which we identified as the IC50 for secondary Follicles. In summary, we established a 3D in vitro Ovarian follicle culture system that could be used in an HTP approach to measure toxic effects on Ovarian Follicles.
Antonella Cecchettini - One of the best experts on this subject based on the ideXlab platform.
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a fat body derived protein is selectively sulfated during transit to Ovarian Follicles in the stick insect carausius morosus
Developmental Biology, 1995Co-Authors: Franco Giorgi, Antonella Cecchettini, Massimo Masetti, M. T. Locci, Jt BradleyAbstract:A monoclonal antibody raised against Ovarian Follicles of the stick insect Carausius morosus reacted with two related polypeptides of 157 and 85 kDa in both the ovary and the hemolymph. In vitro cultured fat body proved capable of releasing the 157-kDa polypeptide into the culture medium and processing it to the lower-molecular-weight polypeptide of 85 kDa. This was further demonstrated by in vitro exposure to [35S]methionine. Under the same culturing conditions, Ovarian Follicles proved incapable of synthesizing and/or secreting the 85-kDa polypeptide. However, in vivo [35S]methionine-labeled Ovarian Follicles released both polypeptides when cultured in vitro for up to 24 hr. Vitellogenin polypeptides were labeled in vivo following exposure to [3H]glucosamine, while 157- and 85-kDa polypeptides were labeled only in Ovarian Follicles exposed in vivo to sodium [35S]sulfate. Under in vitro conditions, the 157-kDa polypeptide could be labeled with sodium [35S]sulfate only if Ovarian Follicles were cocultured with fat body. No sulfation occurred in fat body or Ovarian Follicles cultured separately. These experiments suggest that the 157-kDa polypeptide is a fat body-derived polypeptide that is sulfated upon transfer to the Ovarian follicle.
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On the occurrence of proteolytic activities in Ovarian Follicles of the stick insect Carausius morosus (Br.)
Italian Journal of Zoology, 1994Co-Authors: Antonella CecchettiniAbstract:Abstract Two distinct proteolytic activities were demonstrated in developing Ovarian Follicles of the stick insect Carausius morosus using aldolase as a substrate. One has an optimum activity around pH 5 and it is strongly inhibited by pepstatin A. Because of these properties, it is classifiable as an aspartic proteinase. The second one has an alkaline pH optimum and it is inhibited by aprotinin. Based on these properties it is classifiable as a serine, trypsin‐like, proteinase. This latter activity has also been visualized as a negatively stained protein fraction of about 26 kD on polyacrylamide gels containing casein as substrate. While the aspartic proteinase is predominantly associated with Follicles approaching chorionogenesis, the serine proteinase is restricted to immature Follicles and disappears prior to completion of Ovarian development.
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postendocytic vitellin processing in Ovarian Follicles of the stick insect carausius morosus br
Archives of Insect Biochemistry and Physiology, 1993Co-Authors: Franco Giorgi, Vincenzo Ignacchiti, Massimo Masetti, Antonella Cecchettini, James T. BradleyAbstract:Newly laid eggs of the stick insect Carausius morosus contain two native vitellins (Vit A and Vit B). Under denaturing conditions, these vitellins resolved into 3 (A1, A2, and A3) and 2 (B1 and B2) polypeptides. All of these polypeptides had counterparts in the female hemolymph from which they were shown to be derived by in vivo labelling. During Ovarian development, the 2 vitellins changed both in charge and polypeptide composition. In EV and LV Follicles, Vit A resolved into 4 distinct vitellin polypeptides (A0, A1, A2 and A3). Using a panel of monoclonal antibodies, polypeptide A0 proved to be immunologically related to polypeptide A2. In Follicles about to begin choriongenesis, polypeptide A3 was gradually replaced by a lower Mr polypeptide. Over the same time period, polypeptide B1 changed in charge, but not in Mr. To confirm the existence of a polypeptide processing in C. morosus, Ovarian Follicles of different developmental stages were exposed in vivo to [35S]-methionine from 6 to 72 h. Data showed that A0 and B1 were the polypeptides most heavily labelled after short time exposures to the radioisotope. Polypeptides B2 and A3 were also labelled to some extent. With progressively longer exposures, polypeptides A1 and A2 also became labelled. In vivo exposure to [3H]-GlcNAc caused all vitellin polypeptides to become heavily labelled. Autoradiographic analysis of Ovarian Follicles labelled this way showed that, during development, radioactivity was gradually transferred from newly formed yolk spheres in the cortical ooplasm to the central ooplasm. Data were interpreted as suggesting a causal relationship between polypeptide processing and progressive yolk sphere fusion to yield the central ooplasm. © 1993 Wiley-Liss, Inc.
Jt Bradley - One of the best experts on this subject based on the ideXlab platform.
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a fat body derived protein is selectively sulfated during transit to Ovarian Follicles in the stick insect carausius morosus
Developmental Biology, 1995Co-Authors: Franco Giorgi, Antonella Cecchettini, Massimo Masetti, M. T. Locci, Jt BradleyAbstract:A monoclonal antibody raised against Ovarian Follicles of the stick insect Carausius morosus reacted with two related polypeptides of 157 and 85 kDa in both the ovary and the hemolymph. In vitro cultured fat body proved capable of releasing the 157-kDa polypeptide into the culture medium and processing it to the lower-molecular-weight polypeptide of 85 kDa. This was further demonstrated by in vitro exposure to [35S]methionine. Under the same culturing conditions, Ovarian Follicles proved incapable of synthesizing and/or secreting the 85-kDa polypeptide. However, in vivo [35S]methionine-labeled Ovarian Follicles released both polypeptides when cultured in vitro for up to 24 hr. Vitellogenin polypeptides were labeled in vivo following exposure to [3H]glucosamine, while 157- and 85-kDa polypeptides were labeled only in Ovarian Follicles exposed in vivo to sodium [35S]sulfate. Under in vitro conditions, the 157-kDa polypeptide could be labeled with sodium [35S]sulfate only if Ovarian Follicles were cocultured with fat body. No sulfation occurred in fat body or Ovarian Follicles cultured separately. These experiments suggest that the 157-kDa polypeptide is a fat body-derived polypeptide that is sulfated upon transfer to the Ovarian follicle.
Franco Giorgi - One of the best experts on this subject based on the ideXlab platform.
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a fat body derived protein is selectively sulfated during transit to Ovarian Follicles in the stick insect carausius morosus
Developmental Biology, 1995Co-Authors: Franco Giorgi, Antonella Cecchettini, Massimo Masetti, M. T. Locci, Jt BradleyAbstract:A monoclonal antibody raised against Ovarian Follicles of the stick insect Carausius morosus reacted with two related polypeptides of 157 and 85 kDa in both the ovary and the hemolymph. In vitro cultured fat body proved capable of releasing the 157-kDa polypeptide into the culture medium and processing it to the lower-molecular-weight polypeptide of 85 kDa. This was further demonstrated by in vitro exposure to [35S]methionine. Under the same culturing conditions, Ovarian Follicles proved incapable of synthesizing and/or secreting the 85-kDa polypeptide. However, in vivo [35S]methionine-labeled Ovarian Follicles released both polypeptides when cultured in vitro for up to 24 hr. Vitellogenin polypeptides were labeled in vivo following exposure to [3H]glucosamine, while 157- and 85-kDa polypeptides were labeled only in Ovarian Follicles exposed in vivo to sodium [35S]sulfate. Under in vitro conditions, the 157-kDa polypeptide could be labeled with sodium [35S]sulfate only if Ovarian Follicles were cocultured with fat body. No sulfation occurred in fat body or Ovarian Follicles cultured separately. These experiments suggest that the 157-kDa polypeptide is a fat body-derived polypeptide that is sulfated upon transfer to the Ovarian follicle.
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postendocytic vitellin processing in Ovarian Follicles of the stick insect carausius morosus br
Archives of Insect Biochemistry and Physiology, 1993Co-Authors: Franco Giorgi, Vincenzo Ignacchiti, Massimo Masetti, Antonella Cecchettini, James T. BradleyAbstract:Newly laid eggs of the stick insect Carausius morosus contain two native vitellins (Vit A and Vit B). Under denaturing conditions, these vitellins resolved into 3 (A1, A2, and A3) and 2 (B1 and B2) polypeptides. All of these polypeptides had counterparts in the female hemolymph from which they were shown to be derived by in vivo labelling. During Ovarian development, the 2 vitellins changed both in charge and polypeptide composition. In EV and LV Follicles, Vit A resolved into 4 distinct vitellin polypeptides (A0, A1, A2 and A3). Using a panel of monoclonal antibodies, polypeptide A0 proved to be immunologically related to polypeptide A2. In Follicles about to begin choriongenesis, polypeptide A3 was gradually replaced by a lower Mr polypeptide. Over the same time period, polypeptide B1 changed in charge, but not in Mr. To confirm the existence of a polypeptide processing in C. morosus, Ovarian Follicles of different developmental stages were exposed in vivo to [35S]-methionine from 6 to 72 h. Data showed that A0 and B1 were the polypeptides most heavily labelled after short time exposures to the radioisotope. Polypeptides B2 and A3 were also labelled to some extent. With progressively longer exposures, polypeptides A1 and A2 also became labelled. In vivo exposure to [3H]-GlcNAc caused all vitellin polypeptides to become heavily labelled. Autoradiographic analysis of Ovarian Follicles labelled this way showed that, during development, radioactivity was gradually transferred from newly formed yolk spheres in the cortical ooplasm to the central ooplasm. Data were interpreted as suggesting a causal relationship between polypeptide processing and progressive yolk sphere fusion to yield the central ooplasm. © 1993 Wiley-Liss, Inc.