scholarly journals Development of Spermatogenesis In Vitro in Three-Dimensional Culture from Spermatogonial Cells of Busulfan-Treated Immature Mice

2018 ◽  
Vol 19 (12) ◽  
pp. 3804 ◽  
Author(s):  
Ali AbuMadighem ◽  
Ronnie Solomon ◽  
Alina Stepanovsky ◽  
Joseph Kapelushnik ◽  
QingHua Shi ◽  
...  
Keyword(s):  
Male Infertility ◽  
In Vitro Culture ◽  
Seminiferous Tubule ◽  
Three Dimensional ◽  
Testicular Tissue ◽  
Biologically Active ◽  
Isolated Cells ◽  
Dead Box ◽  
Testicular Weight

Aggressive chemotherapy may lead to permanent male infertility. Prepubertal males do not generate sperm, but their testes do contain spermatogonial cells (SPGCs) that could be used for fertility preservation. In the present study, we examined the effect of busulfan (BU) on the SPGCs of immature mice, and the possible induction of the survivor SPGCs to develop spermatogenesis in 3D in-vitro culture. Immature mice were injected with BU, and after 0.5–12 weeks, their testes were weighed and evaluated histologically compared to the control mice. The spermatogonial cells [Sal-like protein 4 (SALL4) and VASA (a member of the DEAD box protein family) in the testicular tissue were counted/seminiferous tubule (ST). The cells from the STs were enzymatically isolated and cultured in vitro. Our results showed a significant decrease in the testicular weight of the BU-treated mice compared to the control. This was in parallel to a significant increase in the number of severely damaged STs, and a decrease in the number of SALL4 and VASA/STs compared to the control. The cultures of the isolated cells from the STs of the BU-treated mice showed a development of colonies and meiotic and post-meiotic cells after four weeks of culture. The addition of homogenates from adult GFP mice to those cultures induced the development of sperm-like cells after four weeks of culture. This is the first study demonstrating the presence of biologically active spermatogonial cells in the testicular tissue of BU-treated immature mice, and their capacity to develop sperm-like cells in vitro.

scholarly journals The Expression Levels and Cellular Localization of Pigment Epithelium Derived Factor (PEDF) in Mouse Testis: Its Possible Involvement in the Differentiation of Spermatogonial Cells

2021 ◽  
Vol 22 (3) ◽  
pp. 1147
Author(s):  
Noy Bagdadi ◽  
Alaa Sawaied ◽  
Ali AbuMadighem ◽  
Eitan Lunenfeld ◽  
Mahmoud Huleihel
Keyword(s):  
Cellular Localization ◽  
Pigment Epithelium ◽  
Testicular Tissue ◽  
Seminiferous Tubules ◽  
Target Cells ◽  
Isolated Cells ◽  
Expression Levels ◽  
Cellular Origin ◽  
Pigment Epithelium Derived Factor

Pigment epithelium derived factor (PEDF) is a multifunctional secretory soluble glycoprotein that belongs to the serine protease inhibitor (serpin) family. It was reported to have neurotrophic, anti-angiogenic and anti-tumorigenic activity. Recently, PEDF was found in testicular peritubular cells and it was assumed to be involved in the avascular nature of seminiferous tubules. The aim of this study was to determine the cellular origin, expression levels and target cells of PEDF in testicular tissue of immature and adult mice under physiological conditions, and to explore its possible role in the process of spermatogenesis in vitro. Using immunofluorescence staining, we showed that PEDF was localized in spermatogenic cells at different stages of development as well as in the somatic cells of the testis. Its protein levels in testicular homogenates and Sertoli cells supernatant showed a significant decrease with age. PEDF receptor (PEDF-R) was localized within the seminiferous tubule cells and in the interstitial cells compartment. Its RNA expression levels showed an increase with age until 8 weeks followed by a decrease. RNA levels of PEDF-R showed the opposite trend of the protein. Addition of PEDF to cultures of isolated cells from the seminiferous tubules did not changed their proliferation rate, however, a significant increase was observed in number of meiotic/post meiotic cells at 1000 ng/mL of PEDF; indicating an in vitro differentiation effect. This study may suggest a role for PEDF in the process of spermatogenesis.

scholarly journals Beyond 3D culture models of cancer

2015 ◽  
Vol 7 (283) ◽  
pp. 283ps9-283ps9 ◽  
Author(s):  
Kandice Tanner ◽  
Michael M. Gottesman
Keyword(s):  
In Vitro Culture ◽  
Three Dimensional ◽  
3D Culture ◽  
Drug Efficacy ◽  
Spatiotemporal Evolution ◽  
Culture Models ◽  
Dynamic Interplay

The mechanisms underlying the spatiotemporal evolution of tumor ecosystems present a challenge in evaluating drug efficacy. In this Perspective, we address the use of three-dimensional in vitro culture models to delineate the dynamic interplay between the tumor and the host microenvironment in an effort to attain realistic platforms for assessing pharmaceutical efficacy in patients.

scholarly journals In vitro culture of hFOB1.19 osteoblast cells on TGF-β1-SF-CS three-dimensional scaffolds

2015 ◽  
Vol 13 (1) ◽  
pp. 181-187 ◽  
Author(s):  
SHUANG TONG ◽  
LEI XUE ◽  
DA-PENG XU ◽  
ZI-MEI LIU ◽  
YANG DU ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Three Dimensional ◽  
Osteoblast Cells ◽  
Tgf Β1

scholarly journals Cell migration and organization in three-dimensional in vitro culture driven by stiffness gradient

2016 ◽  
Vol 113 (11) ◽  
pp. 2496-2506 ◽  
Author(s):  
Danielle Joaquin ◽  
Michael Grigola ◽  
Gubeum Kwon ◽  
Christopher Blasius ◽  
Yutao Han ◽  
...  
Keyword(s):  
Cell Migration ◽  
In Vitro Culture ◽  
Three Dimensional

111 EQUINE AMNIOTIC EPITHELIAL OR BONE MARROW MESENCHYMAL STEM CELLS DIFFERENTLY SUPPORT IN VITRO EMBRYO DEVELOPMENT IN A BOVINE IN VITRO CULTURE MODEL

2009 ◽  
Vol 21 (1) ◽  
pp. 156 ◽  
Author(s):  
F. Cremonesi ◽  
V. Maggio ◽  
A. Lange Consiglio
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
In Vitro Culture ◽  
Calf Serum ◽  
Biologically Active ◽  
Bovine Embryo ◽  
Epithelial Stem Cells ◽  
Marrow Mesenchymal Stem Cells

There are indications that the culture system and the medium composition can affect embryo quality. In fact, various studies have been shown that the in vitro culture environment is one of the key determinants of the blastocyst output. In light of this, recently, some studies used co-culture with mouse embryonic fibroblasts in the effort to improve the development of bovine and ovine in vitro-derived embryos. Despite the progress in equine IVM and ICSI technologies and the different culture conditions reported for preimplantation development of ICSI fertilized horse oocytes, the yield of blastocysts remained low. In the present study we investigated the benefits of co-culturing bovine embryos with equine bone marrow mesenchymal stem cells (BM-MSC) or equine amniotic epithelial stem cells (AE-SC) on blastocyst development. This study employed the bovine embryo as a model and represents the initial step towards standardization of a protocol for the culture of equine embryos in our laboratory. BM specimens were obtained aseptically from sternal aspirates of horses under local anaesthesia and layered over Hystopaque™ 1.080, then centrifuged for 20 min at 400g and 4°C. Cell pellets were resuspended in 10 mL Dulbecco Modified Earle’s Medium supplemented with 10% fetal calf serum, 1% non-essential amino acids, penicillin (100 U mL–1) and streptomycin (100 μg mL–1) and seeded in 24-well plates. Amniotic membranes were obtained from fresh placentas and, to release the AE cells, amniotic fractions were incubated at 37°C with 0.05% trypsin for 45 min. Separated AE cells were plated on 25 cm2 flask in standard culture media containing 10 ng mL–1 epidermal growth factor. Seven hundred fifty cumulus–oocyte complexes with a homogeneous cytoplasm and two or more layers of cumulus cells were used. After IVM and IVF cumulus-free presumptive zygotes were randomly transferred into one of three co-culture systems in which they were cultured for up to Day 7: 1) co-culture with granulosa cells (control); 2) co-culture with BM-MSC; 3) co-culture with AE-SC. The culture medium was TCM 199 + 10% fetal bovine serum, pyruvate and gentamicin at 38.5°C in 5% CO2. Statistical analyses was performed by chi square test. Blastocysts developmental rates were similar among control, AE-SC and BM-MSC (35%, 41% and 30%, respectively), but the co-culture with AE-SC gave a significantly greater percentage of blastocysts compared to BM-MSC (P < 0.05). In conclusion, despite the absence of a significant increment in blastocysts attainment using stem cells as feeders for embryo culture, the AE-SC monolayer create a more suitable microenvironment necessary for inducing local cell activation and proliferation of the growing embryos in comparison with BM-MSC. It can be suggested that these cells secrete biologically active substances including signaling molecules and growth factors of epithelial nature different from those of the BM cells of mesenchymal origin. Regione Lombardia is acknowledged for the “Dote Ricercatori” fellowship to V.M.

38 Vitrification of prepubertal lamb spermatogonia using a novel vitrification system

2019 ◽  
Vol 31 (1) ◽  
pp. 145 ◽  
Author(s):  
S. Ledda ◽  
S. Pinna ◽  
S. Nieddu ◽  
D. Natan ◽  
A. Arav ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
In Vitro Culture ◽  
Dimethyl Sulfoxide ◽  
Fertility Preservation ◽  
Testicular Tissue ◽  
Gonadal Tissue ◽  
New Device ◽  
Vitrification Solution ◽  
Tissue Cryopreservation

Vitrification is a method extensively used for preserving oocytes and embryos and is also gaining acceptance for preserving gonadal tissue. Cryopreservation of spermatogonial stem cells is an applicable method for young males seeking fertility preservation before starting a treatment or can be a tool for genetic preservation of rare or high-value animals. The aim of this work was to evaluate the cryopreservation of testicular tissue from young lambs by vitrification using a new device named E.Vit (FertileSafe, Ness Ziona, Israel) that permits all cryopreservation procedures to be performed in straw. The new device consists of a 0.3-mL straw (Cryo Bio System, IMV, L’Aigle, France) with a capsule containing 50-µm pores inserted at one end. Testicular tissue extracts were prepared from testes of slaughtered lambs (n=10, 40 days old), opened by sagittal sectioning with a microblade and collecting small pieces of testicular tissue (1mm3) from the middle part of the rete testis. Three pieces of gonadal tissue were inserted into each E.Vit device. Each straw was sequentially loaded vertically in two 1.5-mL microtubes, which contained the following solutions: first, the equilibrating solution (7.5% dimethyl sulfoxide+7.5% ethylene glycol+20% FCS in TCM-199) for 6min, followed by 90min in the vitrification solution (18% dimethyl sulfoxide+18% ethylene glycol+0.5M Trehalose+BSA in TCM-199). After exposure to the equilibrating solution and vitrification solution, the solutions were removed and the straws were directly loaded into LN2. The warming procedure consisted of placing the straws directly into 5-mL tubes containing 100, 50, and 25% warming solution (1M sucrose in TCM-199+20% FCS) at 38.6°C for 5min each before arrival into the holding medium. Samples were recovered from the straws incubated at 38.6°C in 5% CO2 in air in TCM 199+5% FCS and evaluated at 0 and 2h post-warming for viability using trypan blue staining. Expression of a panel of specific genes (SOD2, HSP90b, BAX, POUF5/OCT4, TERT, CIRBP, KIF11, AR, FSHR) was analysed by real-time PCR in cryopreserved tissue in vitro cultured for 2h post-warming (2hV), in fresh controls immediately after tissue dissection (0hF), and after 2h of in vitro culture (2hF). The majority of cells survived after vitrification, although viability immediately after warming (0hV: 56%±1.45) or after 2h of in vitro culture (IVC) (2hV: 54±7%) was significantly lower compared with non-cryopreserved fresh controls (0hF: 89%±1.45; ANOVA P&lt;0.05). Expression analysis showed specific patterns for the different genes. Notably, BAX transcript abundance was not affected by vitrification or IVC, indicating an acceptable level of stress for the cells. The genes HSP90b and CIRBP were down-regulated in 2hF but increased in 2hV, as expected. Expression of SOD1 and OCT4 was altered by vitrification but not by IVC. Conversely, expression of TERT, KIF11, and AR was affected by both IVC and cryopreservation (ANOVA P&lt;0.05). This novel protocol for testicular tissue cryopreservation of prepubertal animals may be a promising strategy for fertility preservation and can contribute as a new approach in the development of large-scale biodiversity programs.

In vitro culture of precision-cut testicular tissue as a novel tool for the study of responses to LH

2009 ◽  
Vol 46 (1) ◽  
pp. 45-53 ◽  
Author(s):  
Andy Michael Laughlin ◽  
Thomas H. Welsh ◽  
Charles C. Love ◽  
Dickson D. Varner ◽  
Alan R. Parrish ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Testicular Tissue
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