scholarly journals Involvement of Cytokines and Hormones in the Development of Spermatogenesis In Vitro from Spermatogonial Cells of Cyclophosphamide-Treated Immature Mice

2021 ◽  
Vol 22 (4) ◽  
pp. 1672
Author(s):  
Ronnie Solomon ◽  
Ali AbuMadighem ◽  
Joseph Kapelushnik ◽  
Bat-Chen Amano ◽  
Eitan Lunenfeld ◽  
...  
Keyword(s):  
Culture Conditions ◽  
Seminiferous Tubules ◽  
Chemotherapy Treatment ◽  
Isolated Cells ◽  
Testicular Cells ◽  
Testicular Weight ◽  
Significant Impairment ◽  
Prepubertal Boys

Aggressive chemotherapy treatment may lead to male infertility. Prepubertal boys do not produce sperm at this age, however, they have spermatogonial stem cells in their testes. Here, we examined the effect of intraperitoneal injection of cyclophosphamide (CP) on the capacity of immature mice (IM) to develop spermatogenesis in vivo and in vitro [using methylcellulose culture system (MCS)]. Our results show a significant decrease in testicular weight, total number of testicular cells, and the number of Sertoli, peritubular, premeiotic, and meiotic/post-meiotic cells, but an increase in the percentages of damaged seminiferous tubules in CP-treated IM compared to control. The functionality of Sertoli cells was significantly affected. The addition of testosterone to isolated cells from seminiferous tubules of CP-treated IM significantly increased the percentages of premeiotic (CD9-positive cells) and meiotic/post-meiotic cells (ACROSIN-positive cells) developed in MCS compared to control. The addition of FSH did not affect developed cells in MCS compared to control, but in combination with testosterone, it significantly decreased the percentages of CD9-positive cells and ACROSIN-positive cells. The addition of IL-1 did not affect developed cells in MCS compared to control, but in combination with testosterone, it significantly increased the percentages of VASA-positive cells and BOULE-positive cells compared to IL-1 or testosterone. Addition of TNF significantly increased only CD9-positive cells in MCS compared to control, but in combination with testosterone, it significantly decreased ACROSIN-positive cells compared to testosterone. Our results show a significant impairment of spermatogenesis in the testes of CP-treated IM, and that spermatogonial cells from these mice proliferate and differentiate to meiotic/post-meiotic cells under in vitro culture conditions.

scholarly journals Microfluidic bioprinting for the in vitro generation of novel biomimetic human testicular tissues

2021 ◽  
Author(s):  
Meghan Alice Robinson ◽  
Erin Bedford ◽  
Luke Witherspoon ◽  
Stephanie Willerth ◽  
Ryan Flannigan
Keyword(s):  
Cancer Survival ◽  
Fertility Restoration ◽  
Survival Rates ◽  
Testicular Tissue ◽  
Seminiferous Tubules ◽  
Vitro Fertilization ◽  
Testicular Cells ◽  
Human Spermatogenesis

Advances in cancer treatments have greatly improved pediatric cancer survival rates, leading to quality of life considerations and in particular fertility restoration. Accordingly, pre-pubertal patients have the option to cryopreserve testicular tissue for experimental restorative therapies, including in vitro spermatogenesis, wherein testicular tissue is engineered in vitro and spermatozoa are collected for in vitro fertilization (IVF). Current in vitro systems have been unable to reliably support the generation of spermatozoa from human testicular tissues, likely due to the inability for the dissociated testicular cells to recreate the native architecture of testicular tissue found in vivo. Recent advances in 3-D bioprinting can place cells into geometries at fine resolutions comparable to microarchitectures found in native tissues, and therefore hold promise as a tool for the development of a biomimetic in vitro system for human spermatogenesis. This study assessed the utility of bioprinting technology to recreate the precise architecture of testicular tissue and corresponding spermatogenesis for the first time. We printed testicular cell-laden hollow microtubules at similar resolutions to seminiferous tubules, and compared the results to testicular organoids. We show that the human testicular cells retain their viability and functionality post-printing, and illustrate an intrinsic ability to reorganize into their native cytoarchitecture. This study provides a proof of concept for the use of 3-D bioprinting technology as a tool to create biomimetic human testicular tissues.

METABOLIC CHANGES IN TESTICULAR CELLS FROM RATS AFTER LONG-TERM EXPOSURE TO 37 °C IN VIVO OR IN VITRO

1980 ◽  
Vol 85 (3) ◽  
pp. 471-479 ◽  
Author(s):  
F. F. G. ROMMERTS ◽  
F. H. DE JONG ◽  
J. A. GROOTEGOED ◽  
H. J. VAN DER MOLEN
Keyword(s):  
Sertoli Cells ◽  
Leydig Cells ◽  
Biochemical Properties ◽  
Effect Of Temperature ◽  
Seminiferous Tubules ◽  
Experimental Conditions ◽  
Testicular Cells ◽  
Old Rats

Biochemical properties of isolated Leydig cells, Sertoli cells and spermatocytes from rat testes have been investigated after in-vivo or in-vitro exposure of these cells to abdominal temperature (37 °C). The rate of production of testosterone and pregnenolone by isolated Leydig cells from cryptorchid and normal testes from mature rats was not different. Production of pregnenolone by mitochondria prepared from cryptorchid testes was 6·7 times higher than production by mitochondria from normal testes. Sertoli cells prepared from immature rats and incubated in vitro at 32 or 37 °C showed, on day 1 of the culture period, an initial twofold increase in the secretion of androgen-binding protein which was absent after 6 days in culture. In contrast, incorporation of [3H]leucine into secreted proteins was stimulated twofold on day 1 as well as by day 6 of culture. Secretion of oestradiol was increased 30-fold by day 6 when compared with the level found on day 1 when cells had been cultured at 37 °C and the increased secretion of oestradiol was maintained for approximately 2 days when the temperature of incubation was decreased to 32 °C Spermatocytes isolated from seminiferous tubules incubated for 20 h at 37 °C were active in the synthesis of RNA. No degeneration of these cells was observed in testes of 25-day-old rats 5 days after experimental cryptorchidism, whereas under similar conditions massive degeneration of spermatocytes was shown in the testes of mature rats. These results suggest that the effects of temperature on the different testicular cells greatly depend on the experimental conditions used to study the effect of temperature.

scholarly journals Selective ablation of adult GFAP-expressing tanycytes leads to hypogonadotropic hypogonadism in males

2021 ◽  
Author(s):  
Lucile BUTRUILLE ◽  
Martine BATAILLER ◽  
Marie-Line CATEAU ◽  
Ariane SHARIF ◽  
Valerie LEYSEN ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Radial Glia ◽  
Third Ventricle ◽  
Seminiferous Tubules ◽  
Male Reproductive Function ◽  
Intracerebroventricular Infusion ◽  
Testicular Weight ◽  
Selective Ablation

In adult mammals, neural stem cells emerge in three neurogenic regions, the subventricular zone of the lateral ventricle (SVZ), the subgranular zone of the dentate gyrus of the hippocampus (SGZ) and the hypothalamus. In the SVZ and the SGZ, neural stem/progenitor cells (NSPCs) express the glial fibrillary acidic protein (GFAP) and selective ablation of these NSPCs drastically decreases cell proliferation in vitro and in vivo. In the hypothalamus, GFAP is expressed by α-tanycytes, which are specialized radial glia-like cells in the wall of the third ventricle. To explore the role of these hypothalamic GFAP-positive tanycytes, we used transgenic mice expressing herpes simplex virus thymidine kinase (HSV-Tk) under the control of the mouse Gfap promoter and 4-week intracerebroventricular infusion of the antiviral agent ganciclovir (GCV) that kills dividing cells expressing Tk. While GCV drastically reduced the number and growth of hypothalamus-derived neurospheres from adult transgenic mice in vitro, it caused hypogonadism in vivo. The selective death of dividing tanycytes expressing GFAP indeed caused a marked decrease in testosterone levels and testicular weight, as well as vacuolization of the seminiferous tubules and loss of spermatogenesis. In addition, GCV-treated GFAP-Tk mice showed impaired sexual behavior, but no alteration in food intake or body weight. Our results also show that the selective ablation of GFAP-expressing tanycytes leads to a sharp decrease in the number of gonadotropin-releasing hormone (GnRH)-immunoreactive neurons and blunted LH secretion. Altogether, our data show that GFAP-expressing tanycytes play a central role in the regulation of male reproductive function.

P–125 Evaluation of N-acetylcysteine (NAC) effect on in vitro culture of immature mouse testis following vitrification

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
P Nikoosokhan ◽  
B Ebrahimi ◽  
A Alizadeh ◽  
S Hajiaghalou
Keyword(s):  
Stem Cells ◽  
Cell Survival ◽  
Culture Medium ◽  
Caspase 3 ◽  
Testicular Tissue ◽  
Culture Conditions ◽  
Seminiferous Tubules ◽  
Immature Mouse ◽  
Prepubertal Boys

Abstract Study question Can the Culture of cryopreserved immature mouse testicular tissue in the presence of NAC improves the developmental process and prevent apoptosis induction during the culture? Summary answer An appropriate dosage of NAC in the culture medium of immature mouse testicular tissue was associated with increased cell survival and spermatogonia stem cell regeneration. What is known already Spermatogonial stem cells (SSCs) are the most advanced type of stem cells in the testes of prepubertal boys which are the main targets of oncological treatments. Therefore, the only possible alternative to maintain fertility in prepubertal boys is to preserve SSCs before their depletion by cryopreserving the testicular tissue. Despite the possibility of obtaining viable spermatozoa using cryopreserved testicular tissue cultivated in vitro,cryopreservation methods and damages caused by the culture procedure would be obstacles for maintaining the testicular tissueand it seems that optimizing the culture condition is vital. Study design, size, duration Testis tissues were harvested from 6-days-old immature NMRI male mice (n = 100) after cervical dislocation and vitrified. After 3 days testicular biopsies were warmed and distributed into control, culture Ӏ (not supplemented with NAC) and culture ӀӀ (supplemented with NAC) groups. To determine the appropriate NAC concentration 8 different dosages of NAC were evaluated in terms of cell survival and the best dose, a culture medium containing 125mmol/L NAC was selected to continue the study. Participants/materials, setting, methods Vitrified-warmed fragments (2mm3) obtaining from immature NMRI mice were cultured in vitro for 7 days on agar gel. The effects of culture conditions were assessed by Morphological evaluation of seminiferous tubules (using Hematoxylin-eosin staining). Cell viability, protein expression (caspase–3), and gene expression (Bax, Bcl2, Caspase–3, plzf) were evaluated by flow cytometry, immunofluorescence staining, and real time polymerase chain reaction respectively. Additionally, Malondialdehyde (MDA) concentration in the culture medium was measured by MAD Assay Kit. Main results and the role of chance Significant (p < 0.01) increase in cell viability was observed in the culture ӀӀ group after 7 days of culture compared to the culture Ӏ. Bax/Bcl2 ratio was significantly (p < 0.01) lower in the culture ӀӀ group compared to the control and culture Ӏ group. The expression of caspase–3 showed a significant (p < 0.001) increase in the culture ӀӀ group while immunofluorescence analysis showed low expression of it in all groups. These results were consistent with the high level of Bcl2expression that inhibited Caspase–3 expression and consequently the inhibition of apoptosis, and on the other hand, the presence of NAC showed that plzf expressions significantly (p < 0.001) increased in culture ӀӀ group compared to the control and culture Ӏ group. Although the presence of NAC did not inhibit all the deleterious effects of culture medium on tissue morphology, NAC was able to maintain better integrity of tissue and seminiferous tubules within central regions compared to the group without NAC. The decrease in MDA level in the presence of NAC (culture ӀӀ) was also a good indicator to confirm the desired results obtained from the presence of NAC in the culture medium. Limitations, reasons for caution Although the findings of the study were satisfactory in mice tissue after 1 week of culture, it is essential to replicate the experiments using human tissue and evaluate the quality and reproductive potential of surviving spermatogonia after long-term storage to become clinically applicable. Wider implications of the findings: This study highlights the necessity for further experiments to improve the testicular tissue culture conditions for better spermatogonial survival and differentiation to sperm, as the prepubertal fertility restoration methods are promising to be implemented in the clinic in the near future. Trial registration number Not applicable

scholarly journals Multiscale modelling of drug transport and metabolism in liver spheroids

2020 ◽  
Vol 10 (2) ◽  
pp. 20190041 ◽  
Author(s):  
Joseph A. Leedale ◽  
Jonathan A. Kyffin ◽  
Amy L. Harding ◽  
Helen E. Colley ◽  
Craig Murdoch ◽  
...  
Keyword(s):  
Drug Transport ◽  
Temporal Dynamics ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Isolated Cells ◽  
Modelling Framework ◽  
The Impact ◽  
Cells Cultured

In early preclinical drug development, potential candidates are tested in the laboratory using isolated cells. These in vitro experiments traditionally involve cells cultured in a two-dimensional monolayer environment. However, cells cultured in three-dimensional spheroid systems have been shown to more closely resemble the functionality and morphology of cells in vivo . While the increasing usage of hepatic spheroid cultures allows for more relevant experimentation in a more realistic biological environment, the underlying physical processes of drug transport, uptake and metabolism contributing to the spatial distribution of drugs in these spheroids remain poorly understood. The development of a multiscale mathematical modelling framework describing the spatio-temporal dynamics of drugs in multicellular environments enables mechanistic insight into the behaviour of these systems. Here, our analysis of cell membrane permeation and porosity throughout the spheroid reveals the impact of these properties on drug penetration, with maximal disparity between zonal metabolism rates occurring for drugs of intermediate lipophilicity. Our research shows how mathematical models can be used to simulate the activity and transport of drugs in hepatic spheroids and in principle any organoid, with the ultimate aim of better informing experimentalists on how to regulate dosing and culture conditions to more effectively optimize drug delivery.

scholarly journals The insulin sensitiser metformin regulates chicken Sertoli and germ cell populations

Reproduction ◽  
2016 ◽  
Vol 151 (5) ◽  
pp. 527-538 ◽  
Author(s):  
M Faure ◽  
E Guibert ◽  
S Alves ◽  
B Pain ◽  
C Ramé ◽  
...  
Keyword(s):  
Germ Cells ◽  
Sertoli Cells ◽  
Energy Content ◽  
Morphological Changes ◽  
Seminiferous Tubules ◽  
Reduce Food Intake ◽  
Testicular Weight

Abstract Metformin, an insulin sensitiser from the biguanide family of molecules, is used for the treatment of insulin resistance in type 2 diabetes individuals. It increases peripheral glucose uptake and may reduce food intake. Based on the tight link between metabolism and fertility, we investigated the role of metformin on testicular function using in vitro culture of Sertoli cells and seminiferous tubules, complemented by in vivo data obtained following metformin administration to prepubertal chickens. In vitro, metformin treatment reduced Sertoli cell proliferation without inducing apoptosis and morphological changes. The metabolism of Sertoli cells was affected because lactate secretion by Sertoli cells increased approximately twofold and intracellular free ATP was negatively impacted. Two important pathways regulating proliferation and metabolism in Sertoli cells were assayed. Metformin exposure was not associated with an increased phosphorylation of AKT or ERK. There was a 90% reduction in the proportion of proliferating germ cells after a 96-h exposure of seminiferous tubule cultures to metformin. In vivo, 6-week-old chickens treated with metformin for 3 weeks exhibited reduced testicular weight and a 50% decrease in testosterone levels. The expression of a marker of undifferentiated germ cells was unchanged in contrast to the decrease in expression of ‘protamine’, a marker of differentiated germ cells. In conclusion, these results suggest that metformin affects the testicular energy content and the proliferative ability of Sertoli and germ cells. Reproduction (2016) 151 527–538

scholarly journals Identification of Premeiotic, Meiotic, and Postmeiotic Cells in Testicular Biopsies Without Sperm from Sertoli Cell-Only Syndrome Patients

2019 ◽  
Vol 20 (3) ◽  
pp. 470 ◽  
Author(s):  
Maram Abofoul-Azab ◽  
Eitan Lunenfeld ◽  
Eliahu Levitas ◽  
Atif Zeadna ◽  
Johnny Younis ◽  
...  
Keyword(s):  
Sertoli Cell ◽  
Culture Conditions ◽  
Seminiferous Tubules ◽  
Testicular Sperm Extraction ◽  
Isolated Cells ◽  
Extraction Technology ◽  
Culture Systems ◽  
Proliferation And Differentiation ◽  
Before And After

Sertoli cell-only syndrome (SCOS) affects about 26.3–57.8% of azoospermic men, with their seminiferous tubules containing only Sertoli cells. Recently, it was reported that testicular biopsies from nonobstructive azoospermic (NOA) patients contained germ cells, and that sperm could be found in the tubules of 20% of SCOS patients using testicular sperm extraction technology. Since the patients without sperm in their testicular biopsies do not have therapy to help them to father a biological child, in vitro maturation of spermatogonial stem cells (SSCs) isolated from their testis is a new approach for possible future infertility treatment. Recently, the induction of human and mice SSCs proliferation and differentiation was demonstrated using different culture systems. Our group reported the induction of spermatogonial cell proliferation and differentiation to meiotic and postmeiotic stages in mice, rhesus monkeys, and prepubertal boys with cancer using 3D agar and methylcellulose (MCS) culture systems. The aim of the study was to identify the type of spermatogenic cells present in biopsies without sperm from SCOS patients, and to examine the possibility of inducing spermatogenesis from isolated spermatogonial cells of these biopsies in vitro using 3D MCS. We used nine biopsies without sperm from SCOS patients, and the presence of spermatogenic markers was evaluated by PCR and specific immunofluorescence staining analyses. Isolated testicular cells were cultured in MCS in the presence of StemPro enriched media with different growth factors and the development of colonies/clusters was examined microscopically. We examined the presence of cells from the different stages of spermatogenesis before and after culture in MCS for 3–7 weeks. Our results indicated that these biopsies showed the presence of premeiotic markers (two to seven markers/biopsy), meiotic markers (of nine biopsies, cAMP responsive element modulator-1 (CREM-1) was detected in five, lactate dehydrogenase (LDH) in five, and BOULE in three) and postmeiotic markers (protamine was detected in six biopsies and acrosin in three). In addition, we were able to induce the development of meiotic and/or postmeiotic stages from spermatogonial cells isolated from three biopsies. Thus, our study shows for the first time the presence of meiotic and/or postmeiotic cells in biopsies without the sperm of SCOS patients. Isolated cells from some of these biopsies could be induced to meiotic and/or postmeiotic stages under in vitro culture conditions.

scholarly journals Transcriptome comparisons of in vitro intestinal epithelia grown under static and microfluidic gut-on-chip conditions with in vivo human epithelia

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kornphimol Kulthong ◽  
Guido J. E. J. Hooiveld ◽  
Loes Duivenvoorde ◽  
Ignacio Miro Estruch ◽  
Victor Marin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Culture Conditions ◽  
Gene Set Enrichment Analysis ◽  
Shear Stresses ◽  
Static Culture ◽  
Dynamic Culture ◽  
Intestinal Segments ◽  
On Chip

AbstractGut-on-chip devices enable exposure of cells to a continuous flow of culture medium, inducing shear stresses and could thus better recapitulate the in vivo human intestinal environment in an in vitro epithelial model compared to static culture methods. We aimed to study if dynamic culture conditions affect the gene expression of Caco-2 cells cultured statically or dynamically in a gut-on-chip device and how these gene expression patterns compared to that of intestinal segments in vivo. For this we applied whole genome transcriptomics. Dynamic culture conditions led to a total of 5927 differentially expressed genes (3280 upregulated and 2647 downregulated genes) compared to static culture conditions. Gene set enrichment analysis revealed upregulated pathways associated with the immune system, signal transduction and cell growth and death, and downregulated pathways associated with drug metabolism, compound digestion and absorption under dynamic culture conditions. Comparison of the in vitro gene expression data with transcriptome profiles of human in vivo duodenum, jejunum, ileum and colon tissue samples showed similarities in gene expression profiles with intestinal segments. It is concluded that both the static and the dynamic gut-on-chip model are suitable to study human intestinal epithelial responses as an alternative for animal models.

Studies of Metabolism Mediated Mutagenicity In Vitro

1990 ◽  
Vol 18 (1_part_1) ◽  
pp. 243-250
Author(s):  
Dag Jenssen ◽  
Lennart Romert
Keyword(s):  
Cell Lines ◽  
Biological Effects ◽  
Animal Experiments ◽  
Culture Technique ◽  
Organ Perfusion ◽  
Isolated Cells ◽  
Toxicological Effect ◽  
In Vitro Methods

To understand the cause of the biological effects of xenobiotic metabolism in mammals, investigators have traditionally performed animal experiments by comparing the results of biochemical methods, such as measurement of enzyme activity analysis of the metabolites produced, with the observed toxicological effect. This article deals with in vitro methods for genotoxicity combined with drug metabolising preparations at the organelle, cell or organ levels, as exemplified by microsome preparations, isolated cells/cell lines and organ perfusion systems, respectively. The advantage of some of these methods for studying metabolism-mediated mutagenicity is that the measured endpoint reflects not only the bioactivating phase I reactions, but also the detoxifying phase II reactions, and the transfer of the non-conjugated reactive metabolites to other cells and their ability to cause mutations in these cells. In vivo, all these events are important factors in the initiation of cancer. A mechanistic advantage of the methods for metabolism-mediated mutagenicity in vitro is that the relevance of the different steps in metabolism for the mutational events can seldom be investigated in an in vivo assay. Furthermore, human studies can easily be performed using the co-culture technique with isolated human cells or cell lines.

scholarly journals TMOD-15. EFFICACY OF THE MDM2 INHIBITOR KRT-232 IN GLIOBLASTOMA PATIENT-DERIVED XENOGRAFT MODELS

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii231-ii231
Author(s):  
Rachael Vaubel ◽  
Ann Mladek ◽  
Yu Zhao ◽  
Shiv K Gupta ◽  
Minjee Kim ◽  
...  
Keyword(s):  
Target Genes ◽  
Xenograft Model ◽  
Culture Conditions ◽  
Patient Derived Xenograft ◽  
Fold Induction ◽  
Extended Survival ◽  
Mdm2 Amplification ◽  
Mdm2 Inhibitor

Abstract Non-genotoxic reactivation of p53 by MDM2 inhibitors represents a promising therapeutic strategy for tumors with wild-type TP53, particularly tumors harboring MDM2 amplification. MDM2 controls p53 levels by targeting it for degradation, while disruption of the MDM2-p53 interaction causes rapid accumulation of p53 and activation of the p53 pathway. We examined the efficacy of the small molecule MDM2 inhibitor KRT-232, alone and in combination with radiation therapy (RT), in MDM2-amplified and/or p53 wildtype patient-derived xenograft (PDX) models of glioblastoma in vitro and in vivo. In vitro, glioblastoma PDX explant cultures showed sensitivity to KRT-232, both tumors with MDM2 amplification (GBM108 and G148) and non-amplified but TP53-wildtype lines (GBM10, GBM14, and GBM39), with IC50s ranging from 300-800 nM in FBS culture conditions. A TP53 p.F270C mutant PDX (GBM43) was inherently resistant, with IC50 >3000 nM. In the MDM2-amplified GBM108 line, KRT-232 led to a robust (5-6 fold) induction of p53-target genes p21, PUMA, and NOXA, with initiation of both apoptosis and senescence. Expression of p21 and PUMA was greater with KRT-232 in combination with RT (25-35 fold induction), while stable knock-down of p53 in GBM108 led to complete resistance to KRT-232. In contrast, GBM10 showed lower induction of p21 and PUMA (2-3 fold) and was more resistant to KRT-232. In an orthotopic GBM108 xenograft model, treatment with KRT-232 +/- RT for one week extended survival from 22 days (placebo) to 46 days (KRT-232 alone); combination KRT-232 + RT further extended survival (77 days) over RT alone (31 days). KRT-232 is an effective treatment in a subset of glioblastoma pre-clinical models alone and in combination with RT. Further studies are underway to understand the mechanisms conferring innate sensitivity or resistance to KRT-232.

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