scholarly journals Significant Benefits of Nanoparticles Containing a Necrosis Inhibitor on Mice Testicular Tissue Autografts Outcomes

2019 ◽  
Vol 20 (23) ◽  
pp. 5833 ◽  
Author(s):  
Federico Del Vento ◽  
Maxime Vermeulen ◽  
Bernard Ucakar ◽  
Jonathan Poels ◽  
Anne des Rieux ◽  
...  
Keyword(s):  
Germ Cells ◽  
Reproductive Potential ◽  
Testicular Tissue ◽  
Mice Model ◽  
Post Transplantation ◽  
Tissue Integrity ◽  
Prepubertal Boys ◽  
Hematoxylin Eosin ◽  
Alginate Matrix ◽  
Active Caspase

Fertility preservation for prepubertal boys relies exclusively on cryopreservation of immature testicular tissue (ITT) containing spermatogonia as the only cells with reproductive potential. Preclinical studies that used a nude mice model to evaluate the development of human transplanted ITT were characterized by important spermatogonial loss. We hypothesized that the encapsulation of testicular tissue in an alginate matrix supplemented with nanoparticles containing a necrosis inhibitor (NECINH-NPS) would improve tissue integrity and germ cells’ survival in grafts. We performed orthotopic autotransplantation of 1 mm³ testicular tissue fragments recovered form mice (aged 4–5 weeks). Fragments were either non-encapsulated, encapsulated in an alginate matrix, or encapsulated in an alginate matrix containing NECINH-NPs. Grafts were recovered 5- and 21-days post-transplantation. We evaluated tissue integrity (hematoxylin-eosin staining), germ cells survival (immunohistochemistry for promyelocytic leukemia zinc-finger, VASA, and protein-boule-like), apoptosis (immunohistochemistry for active-caspase 3), and lipid peroxidation (immunohistochemistry for malondialdehyde). NECINH-NPs significantly improved testicular tissue integrity and germ cells’ survival after 21 days. Oxidative stress was reduced after 5 days, regardless of nanoparticle incorporation. No effect on caspase-dependent apoptosis was observed. In conclusion, NECINH-NPs in an alginate matrix significantly improved tissue integrity and germ cells’ survival in grafts with the perspective of higher reproductive outcomes.

scholarly journals Haploid Germ Cells Generated in Organotypic Culture of Testicular Tissue From Prepubertal Boys

2018 ◽  
Vol 9 ◽  
Author(s):  
Francesca de Michele ◽  
Jonathan Poels ◽  
Maxime Vermeulen ◽  
Jérôme Ambroise ◽  
Damien Gruson ◽  
...  
Keyword(s):  
Germ Cells ◽  
Organotypic Culture ◽  
Testicular Tissue ◽  
Prepubertal Boys

scholarly journals Accelerated and Improved Vascular Maturity after Transplantation of Testicular Tissue in Hydrogels Supplemented with VEGF- and PDGF-Loaded Nanoparticles

2021 ◽  
Vol 22 (11) ◽  
pp. 5779
Author(s):  
Federico Del Vento ◽  
Jonathan Poels ◽  
Maxime Vermeulen ◽  
Bernard Ucakar ◽  
Maria Grazia Giudice ◽  
...  
Keyword(s):  
Fertility Restoration ◽  
Smooth Muscle Actin ◽  
Testicular Tissue ◽  
Significant Loss ◽  
Short Term ◽  
Tissue Integrity ◽  
Vascular Structures ◽  
Tissue Grafts ◽  
Old Mice ◽  
Hematoxylin Eosin

Avascular transplantation of frozen–thawed testicular tissue fragments represents a potential future technique for fertility restoration in boys with cancer. A significant loss of spermatogonia was observed in xeno-transplants of human tissue most likely due to the hypoxic period before revascularization. To reduce the effect of hypoxia–reoxygenation injuries, several options have already been explored, like encapsulation in alginate hydrogel and supplementation with nanoparticles delivering a necrosis inhibitor (NECINH) or VEGF. While these approaches improved short-term (5 days) vascular surfaces in grafts, neovessels were not maintained up to 21 days; i.e., the time needed for achieving vessel stabilization. To better support tissue grafts, nanoparticles loaded with VEGF, PDGF and NECINH were developed. Testicular tissue fragments from 4–5-week-old mice were encapsulated in calcium-alginate hydrogels, either non-supplemented (control) or supplemented with drug-loaded nanoparticles (VEGF-nanoparticles; VEGF-nanoparticles + PDGF-nanoparticles; NECINH-nanoparticles; VEGF-nanoparticles + NECINH-nanoparticles; and VEGF-nanoparticles + PDGF-nanoparticles + NECINH-nanoparticles) before auto-transplantation. Grafts were recovered after 5 or 21 days for analyses of tissue integrity (hematoxylin–eosin staining), spermatogonial survival (immuno-histo-chemistry for promyelocytic leukemia zinc finger) and vascularization (immuno-histo-chemistry for α-smooth muscle actin and CD-31). Our results showed that a combination of VEGF and PDGF nanoparticles increased vascular maturity and induced a faster maturation of vascular structures in grafts.

Fertility preservation for prepubertal boys: lessons learned from the past and update on remaining challenges towards clinical translation

2020 ◽  
Author(s):  
Christine Wyns ◽  
Marc Kanbar ◽  
Maria Grazia Giudice ◽  
Jonathan Poels
Keyword(s):  
Stem Cells ◽  
Fertility Preservation ◽  
Clinical Application ◽  
Germ Cells ◽  
Testicular Tissue ◽  
Reproductive Age ◽  
Reproductive Capacity ◽  
Patient Counselling ◽  
Final Maturation ◽  
Prepubertal Boys

Abstract BACKGROUND Childhood cancer incidence and survivorship are both on the rise. However, many lifesaving treatments threaten the prepubertal testis. Cryopreservation of immature testicular tissue (ITT), containing spermatogonial stem cells (SSCs), as a fertility preservation (FP) option for this population is increasingly proposed worldwide. Recent achievements notably the birth of non-human primate (NHP) progeny using sperm developed in frozen-thawed ITT autografts has given proof of principle of the reproductive potential of banked ITT. Outlining the current state of the art on FP for prepubertal boys is crucial as some of the boys who have cryopreserved ITT since the early 2000s are now in their reproductive age and are already seeking answers with regards to their fertility. OBJECTIVE AND RATIONALE In the light of past decade achievements and observations, this review aims to provide insight into relevant questions for clinicians involved in FP programmes. Have the indications for FP for prepubertal boys changed over time? What is key for patient counselling and ITT sampling based on the latest achievements in animals and research performed with human ITT? How far are we from clinical application of methods to restore reproductive capacity with cryostored ITT? SEARCH METHODS An extensive search for articles published in English or French since January 2010 to June 2020 using keywords relevant to the topic of FP for prepubertal boys was made in the MEDLINE database through PubMed. Original articles on fertility preservation with emphasis on those involving prepubertal testicular tissue, as well as comprehensive and systematic reviews were included. Papers with redundancy of information or with an absence of a relevant link for future clinical application were excluded. Papers on alternative sources of stem cells besides SSCs were excluded. OUTCOMES Preliminary follow-up data indicate that around 27% of boys who have undergone testicular sampling as an FP measure have proved azoospermic and must therefore solely rely on their cryostored ITT to ensure biologic parenthood. Auto-transplantation of ITT appears to be the first technique that could enter pilot clinical trials but should be restricted to tissue free of malignant cells. While in vitro spermatogenesis circumvents the risk linked to cancer cell contamination and has led to offspring in mice, complete spermatogenesis has not been achieved with human ITT. However, generation of haploid germ cells paves the way to further studies aimed at completing the final maturation of germ cells and increasing the efficiency of the processes. WIDER IMPLICATIONS Despite all the research done to date, FP for prepubertal boys remains a relatively young field and is often challenging to healthcare providers, patients and parents. As cryopreservation of ITT is now likely to expand further, it is important not only to acknowledge some of the research questions raised on the topic, e.g. the epigenetic and genetic integrity of gametes derived from strategies to restore fertility with banked ITT but also to provide healthcare professionals worldwide with updated knowledge to launch proper multicollaborative care pathways in the field and address clinical issues that will come-up when aiming for the child’s best interest.

Pancreatic islet transplantation under the kidney capsule of mice: model of refinement for molecular and ex-vivo graft analysis

2021 ◽  
pp. 002367722110040
Author(s):  
Julien Thévenet ◽  
Valery Gmyr ◽  
Nathalie Delalleau ◽  
François Pattou ◽  
Julie Kerr-Conte
Keyword(s):  
Islet Transplantation ◽  
Pain Treatment ◽  
Ex Vivo ◽  
Well Being ◽  
Diabetic Patients ◽  
Mice Model ◽  
Pancreatic Islet Transplantation ◽  
Post Transplantation ◽  
Kidney Capsule

Diabetes cell therapy by human islet transplantation can restore an endogenous insulin secretion and normal glycaemic control in type 1 diabetic patients for as long as 10 years post transplantation. Before transplantation, each clinical islet preparation undergoes extensive in-vitro and in-vivo quality controls. The in-vivo quality control assay consists of transplanting human islets under the kidney capsule of immunocompromised mice. Currently, it is considered the best predictive factor to qualify clinical transplant efficiency. This chimeric model offers a wide area of study since it combines the possibility of producing not only quantitative but also a maximum of qualitative data. Today’s technological advances allow us to obtain more accurate and stronger data from the animals used in research while ensuring their comfort and well-being throughout the protocol, including cage enrichment and pain treatment during and after surgery. As demonstrated in this valuable model, we are able to generate more usable results (Refine), while reducing the number of animals used (Reduce), by focusing on the development of ex-vivo analysis techniques (Replace), which clearly highlights the Burch and Russell 3Rs concept.

scholarly journals Effects of microgravity or simulated launch on testicular function in rats

1992 ◽  
Vol 73 (2) ◽  
pp. S174-S185 ◽  
Author(s):  
R. P. Amann ◽  
D. R. Deaver ◽  
B. R. Zirkin ◽  
G. S. Grills ◽  
W. J. Sapp ◽  
...  
Keyword(s):  
Germ Cells ◽  
Quantitative Methods ◽  
Smooth Endoplasmic Reticulum ◽  
Testicular Tissue ◽  
Seminiferous Tubules ◽  
Specific Gene ◽  
Fixed Tissue ◽  
Treatment Groups ◽  
Essentially Normal ◽  
Peripheral Blood Plasma

Testes from flight rats on COSMOS 2044 and simulated-launch, vivarium, or caudal-elevation control rats (5/group) were analyzed by subjective and quantitative methods. On the basis of observations of fixed tissue, it was evident that some rats had testicular abnormalities unassociated with treatment and probably existing when they were assigned randomly to the four treatment groups. Considering rats without preexisting abnormalities, diameter of seminiferous tubules and numbers of germ cells per tubule cross section were lower (P less than 0.05) in flight than in simulated-launch or vivarium rats. However, ratios of germ cells to each other or to Sertoli cells and number of homogenization-resistant spermatids did not differ from values for simulated-launch or vivarium controls. Expression of testis-specific gene products was not greatly altered by flight. Furthermore, there was no evidence for production of stress-inducible transcripts of the hsp70 or hsp90 genes. Concentration of receptors for rat luteinizing hormone in testicular tissue and surface density of smooth endoplasmic reticulum in Leydig cells were similar in flight and simulated-launch rats. However, concentrations of testosterone in testicular tissue or peripheral blood plasma were reduced (P less than 0.05) in flight rats to less than 20% of values for simulated-launch or vivarium controls. Thus spermatogenesis was essentially normal in flight rats, but production of testosterone was severely depressed. Exposure to microgravity for greater than 2 wk might result in additional changes. Sequelae of reduced androgen production associated with microgravity on turnover of muscle and bone should be considered.

192 IDENTIFICATION AND CHARACTERIZATION OF Oct4-EGFP EXPRESSING CELLS IN TRANSGENIC PIG TESTIS

2014 ◽  
Vol 26 (1) ◽  
pp. 210
Author(s):  
M. Nowak-Imialek ◽  
N. Lachmann ◽  
D. Herrmann ◽  
F. Jacob ◽  
H. Niemann
Keyword(s):  
Stem Cells ◽  
Cell Population ◽  
Germ Cells ◽  
Germ Line ◽  
Cell Mass ◽  
Testicular Tissue ◽  
Egfp Expression ◽  
Cell Populations ◽  
Marker Genes ◽  
Transgenic Pigs

We have produced germ line transgenic pigs carrying the entire 18-kb genomic sequence of the murine Oct4 gene fused to the enhanced green fluorescent protein (EGFP) cDNA (OG2 construct; Nowak-Imialek et al., 2011 Stem Cells Dev.). Expression of the EGFP reporter construct is confined to germ line cells, the inner cell mass, and trophectoderm of blastocysts, and testicular germ cells, including putative spermatogonial stem cells (SSC). SSC are unique among stem cells because they can both self-renew and differentiate into spermatozoa. In-depth knowledge on porcine SSC has been hampered by the inability to isolate these cells from the complex cell population of the testis. In the Oct4-EGFP transgenic mouse, SSC are the only adult stem cells that express Oct4. Fluorescence microscopy of testicular tissue isolated from transgenic piglets revealed minimum numbers of EGFP-positive cells, whereas testicular tissue isolated from adult transgenic boars contained a high amount of EGFP fluorescent cells. Northern blot analysis confirmed stronger EGFP expression in the testis of adult transgenic pigs than in the testis from transgenic piglets. Time course and the signal intensity of EGFP expression in Oct4-EGFP testis paralleled mRNA expression of the endogenous Oct4 gene. Here, we used adult Oct4-EGFP transgenic pigs as a model for fluorescence-activated cell sorting (FACS)-based isolation of EGFP-expressing cells from testes. To obtain a single-cell suspension, the testes were enzymatically dissociated using two digestion steps. Thereafter, FACS based on EGFP expression was successfully used to purify specific testicular cell populations. Two cell populations, i.e. EGFP+ (14%) and EGFP– (45%) could be isolated. Subsequently, qualitative PCR analyses were performed on EGFP+, EGFP–, and unsorted cell populations using marker genes specific for pluripotency and undifferentiated germ cells (OCT4, FGFR3, UTF1, PGP9.5, GFRα1, CD90, SALL4), differentiating germ cells (c-KIT), meiosis (BOLL), spermatids (PRM2), and somatic cells (VIM, LHCGR). All of the genes, including OCT4, UTF1, FGFR3, PGP9.5, CD90, SALL4, and GFRα1 were expressed at least 3-fold and up to 12-fold greater in the EGFP-positive population. Vimentin, which is mainly expressed in Sertoli cells and LHCGR, which is mainly expressed in Leydig cells, were expressed in unsorted and EGFP– cell populations and at very low level in EGFP+ cells. Moreover, expression of the c-KIT and PRM2 markers were detected also in EGFP+ cell population, indicating that these cells contain also differentiating spermatogonia. To explore the characteristics of the Oct4-EGFP expressing cells in greater detail, localization in the porcine testis sections and analysis of co-expression with germ cell markers using immunohistochemistry is currently underway.

Nicotinamide, a Recognized Inhibitor of SIRT1, Promotes Expansion in Ex Vivo Cultures of Short and Long-Term Repopulating Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2431-2431 ◽  
Author(s):  
Tony Peled ◽  
Hadas Shoham ◽  
Dorit Aschengrau ◽  
Dima Yackoubov ◽  
Gabi Frei ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Donor Cell ◽  
Mice Model ◽  
Short Term ◽  
Post Transplantation ◽  
Short And Long Term ◽  
Cells Cultured

Abstract Abstract 2431 Poster Board II-408 Nicotinamide (NAM), is a form of VitB3 that recognized and inhibits SIRT1, the human ortholog of the yeast Sir2 class III NAD+-dependent histone deacetylase. We have previously demonstrated that NAM inhibits in vitro differentiation and enhances expansion, migration, homing and NOD/SCID engraftment efficacy of cord blood (CB)-derived CD34+ cells cultured with cytokines. In the current study, the in vivo function of ex vivo cultured cells with NAM was tested in a congenic mice model (BALB/C, CD45.1/CD45.2) for BM transplantation. Purified CD117+ BM cells from BALB/C CD45.1 mice were cultured with a combination of 4 cytokines (FLT3, SCF, TPO, IL-6, 50 ng/ml each), with and without 0.5mM NAM for three weeks. Numbers of CFUc, CD117+ and CD117+Lin- cells were significantly (p < 0.05) higher in cultures treated with NAM as compared with cultured treated with cytokines alone. Non-cultured, freshly purified CD117+ cells (1000 and 2500 cells/mice) and their total progeny following expansion with or without NAM were transplanted into ablated (1000 Rad) CD45.2 mice (n = 10/cohort), 24h post irradiation (Fig 1). Three months post transplantation, all the mice in the control group (non-transplanted) died. The percent survival of mice transplanted with cells cultured with cytokines and NAM was remarkably higher over the survival of mice in the cohort transplanted with cells cultured with cytokines alone or non-cultured cells (Fig 1). FACS analysis (CD45.1-donor / CD45.2-host) of peripheral blood from mice transplanted with NAM cultured cells show 80% donor cell chimerism (CD45.1), 3 and 6 months post transplantation. Percent of donor derived Gr-1+ and CD3+ cells were similar in mice transplanted with non-cultured or NAM cultured cells. Percentages of donor cell chimerism (CD45.1) in secondary mice transplanted with total BM cells derived from primary recipients originally transplanted with non-cultured and NAM cultured cells were 47 and 73, respectively, 6 weeks after the secondary transplantation. In a different experiment, to follow time to engraftment during the first month post transplantation, mice transplanted with non-cultured cells or cells cultured with cytokines and NAM (n = 10/cohort) were bled at weekly intervals and peripheral blood samples were counted for WBC and analyzed by the FACS to determine donor cell chimerism and lineage engraftment. The results show accelerated engraftment (Fig 2) and higher levels of donor cell chimerism (Fig 3) in the cohort transplanted with NAM cultured cells relative to the cohort transplanted with non-cultured cells. Number of granulocytes, T, NK and B cells during the first month post transplant were also significantly (p<0.05) higher in mice transplanted with cells cultured with cytokines and NAM relative to their levels in mice transplanted with non-cultured cells. The results obtained in the congenic mice model for BMT suggest that NAM promotes expansion in ex vivo cultures of short and long-term repopulating cells, as demonstrated by accelerated donor derived engraftment during the first month post transplantation, higher survival of mice, sustained donor cell chimerism 6 month post transplantation and successful reconstitution of secondary recipients. NAM is thus a novel molecule that may be used to stimulate and expand hematopoietic repopulating cells, fasten post transplant engraftment and hopefully improve transplantation outcome. Current studies are designed to elucidate NAM mode of action. Fig 1: Three month survival Fig 1:. Three month survival Fig 2: Short-term Engrafoment Fig: 3 Percentage of Donor Chimerism Fig 2:. Short-term Engrafoment Fig: 3 Percentage of Donor Chimerism Disclosures: Peled: Gamida-Cell: Employment, Equity Ownership. Shoham:Gamida Cell: Employment. Aschengrau:Gamida Cell: Employment. Yackoubov:Gamida Cell: Employment. Frei:Gamida Cell: Employment. Nagler:Gamida Cell: Arnon Nagler, Consultancy. Peled:Gamida Cell: Consultancy.

Current State of Fertility Preservation in Cancer Patients—What is Established and What is Still Controversial?

2016 ◽  
Vol 12 (01) ◽  
pp. 33
Author(s):  
Kutluk Oktay ◽  
Giuliano Bedoschi ◽  
◽  
◽  
◽  
...  
Keyword(s):  
Cancer Patients ◽  
Fertility Preservation ◽  
Cancer Care ◽  
Testicular Tissue ◽  
Semen Cryopreservation ◽  
Young Females ◽  
Current State ◽  
Preservation Method ◽  
Prepubertal Boys ◽  
Straightforward Approach

Fertility Preservation is an essential part of cancer care when treating young females and men. While semen cryopreservation is a straightforward approach for postpubertal men and there is the option of experimental testicular tissue freezing for prepubertal boys, the options for females are more tumultuous. The last 17 years brought us established approaches such the embryo and oocyte cryopreservation and the ovarian cryopreservation is ready to join the list. However, there still is no proven medical fertility preservation method and the controversy around the utility of GnRHa continues.

scholarly journals Cryopreservation of testicular tissue: an alternative to maintain the reproductive capacity in different animal species

2017 ◽  
Vol 47 (11) ◽  
Author(s):  
David Baruc Cruvinel Lima ◽  
Lúcia Daniel Machado da Silva
Keyword(s):  
Clinical Practice ◽  
Germ Cells ◽  
Animal Species ◽  
Testicular Tissue ◽  
Reproductive Capacity ◽  
Significant Progress ◽  
Breeding Programs ◽  
Artificial Breeding ◽  
Made In

ABSTRACT: Cryopreservation of testicular tissue enables the maintenance of reproductive capacity in different animal species, and contributes to the formation of gene banks for endangered species. The spermatogonia present in the testes can be grown in vitro and the sperm obtained can be used in artificial breeding programs. This review aimed to describe the main techniques of testicular cryopreservation, the main cryoprotectants used, as well as the progress made in different animal species thus far. In the last decade, significant progress has been made in obtaining viable and functional germ cells from testicular tissue. However, more research is needed to better establish protocols that can be used in clinical practice with various species.

scholarly journals Germ cell development in equine testis tissue xenografted into mice

Reproduction ◽  
2006 ◽  
Vol 131 (6) ◽  
pp. 1091-1098 ◽  
Author(s):  
R Rathi ◽  
A Honaramooz ◽  
W Zeng ◽  
R Turner ◽  
I Dobrinski
Keyword(s):  
Germ Cell ◽  
Germ Cells ◽  
Meiotic Division ◽  
Seminal Vesicles ◽  
Post Transplantation ◽  
Germ Cell Development ◽  
Immunodeficient Mice ◽  
Low Efficiency ◽  
Underlying Mechanisms ◽  
Testis Tissue

Grafting of testis tissue from immature animals to immunodeficient mice results in complete spermatogenesis, albeit with varying efficiency in different species. The objectives of this study were to investigate if grafting of horse testis tissue would result in spermatogenesis, and to assess the effect of exogenous gonadotropins on xenograft development. Small fragments of testis tissue from 7 colts (2 week to 4 years of age) were grafted under the back skin of castrated male immunodeficient mice. For 2 donor animals, half of the mice were treated with gonadotropins. Xenografts were analyzed at 4 and 8 months post-transplantation. Spermatogenic differentiation following grafting ranged from no differentiation to progression through meiosis with appearance of haploid cells. Administration of exogenous gonadotropins appeared to support post-meiotic differentiation. For more mature donor testis samples where spermatogenesis had progressed into or through meiosis, after grafting an initial loss of differentiated germ cells was observed followed by a resurgence of spermatogenesis. However, if haploid cells had been present prior to grafting, spermatogenesis did not progress beyond meiotic division. In all host mice with spermatogenic differentiation in grafts, increased weight of the seminal vesicles compared to castrated mice showed that xenografts were releasing testosterone. These results indicate that horse spermatogenesis occurs in a mouse host albeit with low efficiency. In most cases, spermatogenesis arrested at meiosis. The underlying mechanisms of this spermatogenic arrest require further investigation.

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