Effect of recombinant human vascular endothelial growth factor on testis tissue xenotransplants from prepubertal boys: a three-case study

Research question Does recombinant human vascular endothelial growth factor (VEGF-165) improve the efficiency of human immature testis tissue (ITT) xenotransplantation? Design ITT fragments from three prepubertal boys were cultured for 5 days with VEGF-165 or without (control) before xenotransplantation into the testes of immunodeficient mice. Xenotransplants were recovered at 4 and 9 months after transplantation, and vascularization, seminiferous tubule integrity, number of spermatogonia and germ cell differentiation were evaluated by histology and immunohistochemistry. Results Transplants from donor 1 and donor 2 treated with VEGF demonstrated higher vascular surface (P = 0.004) and vessel density (P = 0.011) overall and contained more intact seminiferous tubules (P = 0.039) with time, compared with controls. The number of spermatogonia was increased over time (P < 0.001) irrespective of treatment and donor, whereas, for the VEGF-treated transplants, the increase was even higher over time (P = 0.020). At 9 months, spermatocytes were present in the xenotransplants, irrespective of treatment. No transplants could be recovered from donor 3, who had already received treatment with cyclosporine for aplastic anaemia before biopsy. Conclusions In-vitro pre-treatment of human prepubertal testis tissue with VEGF improved transplant vascularization in two out of three cases, resulting in improved seminiferous tubule integrity and spermatogonial survival during xenotransplantation. Although further studies are warranted, we suggest VEGF be considered as a factor for improving the efficiency of immature testis tissue transplantation in the future

What is the best protocol to cryopreserve immature mouse testicular cell suspensions?

Research question From a clinical perspective, which parameters grant optimal cryopreservation of mouse testicular cell suspensions? Design We studied the effect of different cryopreservation rates, the addition of sugars, different vessels and the addition of an apoptotic inhibitor on the efficiency of testicular cell suspension cryopreservation. After thawing and warming, testicular cell suspensions were transplanted to recipient mice for further functional assay. After selecting the optimal cryopreservation procedure, a second experiment compared the transplantation efficiency between the selected freezing protocol and fresh testicular cell suspensions. Results Multiple- and single-step freezing did not differ significantly in terms of recovered viable cells (RVC) (33 ± 28% and 38 ± 25%). The addition of sucrose did not result in a higher RVC (33 ± 20%). Cells frozen in vials recovered better than those frozen in straws (52 ± 20% versus 33 ± 20%; P = 0.0049). The inclusion of an apoptosis inhibitor (z-VAD[Oe]-FMK) significantly increased the RVC after thawing (61 ± 18% versus 50 ± 17%; P = 0.0480). When comparing the optimal cryopreservation procedure with fresh testicular cell suspensions, a lower RVC (63 ± 11% versus 92 ± 4%; P < 0.0001) and number of donor-derived spermatogonial stem cell colonies per testis (34.04 ± 2.34 versus 16.78 ± 7.76; P = 0.0051) were observed. Conclusion Upon freeze–thawing or vitrification–warming, and assessment of donor-derived spermatogenesis after transplantation, Dulbecco's modified Eagle's medium supplemented with 1.5M dimethyl-sulphoxide, 10% fetal calf serum and 60 µM of Z-VAD-(OMe)-FMK in vials at a freezing rate of −1°C/min was optimal

Of mice and human embryos:is there an ethically preferred order of preclinical research on new assisted reproductive technologies?

It is widely acknowledged that the responsible introduction of new assisted reproductive technologies (ARTs) requires preclinical safety research, including the use of animal models and human embryos. However, the moral sensitivity of human embryo research has led to regulations and guidance stating that human embryos may only be used for research that cannot also be conducted with animals. We call this the 'use animals first' (UAF) rule. In the field of ART research, this translates into the notion of an ideal chain of consecutive preclinical research steps, where research using human embryos may only be considered as a further step after promising results have been obtained in animals first. This may lead to research ethics committees requiring animal studies that are in fact a waste of time and money, while exposing animals to an infringement of their wellbeing for no good purpose. In this paper, we explore the possible moral arguments behind the UAF-rule and test their validity. We conclude that there are no convincing grounds for upholding this rule and recommend replacing it.</p

Establishment of CRISPR/Cas9 genome editing in witloof (Cichorium intybus var. foliosum)

Cichorium intybus var. foliosum (witloof) is an economically important crop with a high nutritional value thanks to many specialized metabolites, such as polyphenols and terpenoids. However, witloof plants are rich in sesquiterpene lactones (SL) which are important for plant defense but also impart a bitter taste, thus limiting industrial applications. Inactivating specific genes in the SL biosynthesis pathway could lead to changes in the SL metabolite content and result in altered bitterness. In this study, a CRISPR/Cas9 genome editing workflow was implemented for witloof, starting with polyethylene glycol (PEG) mediated protoplast transfection for CRISPR/Cas9 vector delivery, followed by whole plant regeneration and mutation analysis. Protoplast transfection efficiencies ranged from 20 to 26 %. A CRISPR/Cas9 vector targeting the first exon of the phytoene desaturase (CiPDS) gene was transfected into witloof protoplasts and resulted in the knockout of CiPDS, giving rise to an albino phenotype in 23% of the regenerated plants. Further implementing our protocol, the SL biosynthesis pathway genes germacrene A synthase (GAS), germacrene A oxidase (GAO), and costunolide synthase (COS) were targeted in independent experiments. Highly multiplex (HiPlex) amplicon sequencing of the genomic target loci revealed plant mutation frequencies of 27.3, 42.7, and 98.3% in regenerated plants transfected with a CRISPR/Cas9 vector targeting CiGAS, CiGAO, and CiCOS, respectively. We observed different mutation spectra across the loci, ranging from consistently the same +1 nucleotide insertion in CiCOS across independent mutated lines, to a complex set of 20 mutation types in CiGAO across independent mutated lines. These results demonstrate a straightforward workflow for genome editing based on transfection and regeneration of witloof protoplasts and subsequent HiPlex amplicon sequencing. Our CRISPR/Cas9 workflow can enable gene functional research and faster incorporation of novel traits in elite witloof lines in the future, thus facilitating the development of novel industrial applications for witloof