Establishment, maintenance and functional integrity of the blood–testis barrier in organotypic cultures of fresh and frozen/thawed prepubertal mouse testes

Rondanino, Christine; Maouche, Ahmed; Dumont, Ludovic; Oblette, Antoine; Rives, Nathalie

doi:10.1093/molehr/gax017

Cited by 34 publications

(18 citation statements)

References 61 publications

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“…In tubules containing spermatocytes or round spermatids a heterogeneous staining pattern with immunopositive and negative reactions was observable in the basal part of the seminiferous epithelium, apparently independent from the containing GC population. Such a heterogeneous immunostaining pattern for claudin-3 was also reported by Rondanino et al [ 83 ] in fresh testicular tissues. A possible explanation for the seemingly heterogeneous immunostaining results in the present study could be that different GC (sub)populations were not definitely determined in the histological sections at hand.…”

Section: Discussionsupporting

confidence: 83%

Loss of connexin43 in murine Sertoli cells and its effect on blood-testis barrier formation and dynamics

Hollenbach¹,

Jung

Noelke³

et al. 2018

PLoS ONE

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Connexin43 (Cx43) is the predominant testicular gap junction protein and in cases of impaired spermatogenesis, Cx43 expression has been shown to be altered in several mammals. Amongst other functions, Cx43 is supposed to regulate junction formation of the blood-testis barrier (BTB). The aim of the present study was to investigate the expression pattern of different tight junction (TJ) proteins of the murine BTB using SC-specific Cx43 knockout mice (SCCx43KO). Adult homozygous male SCCx43KO mice (SCCx43KO-/-) predominantly show an arrest of spermatogenesis and SC-only tubules that might have been caused by an altered BTB assembly, composition or regulation. TJ molecules claudin-3, -5 and -11 were examined in adult wild type (WT) and SCCx43KO-/- mice using immunohistochemistry (IHC) and quantitative real-time PCR (qRT-PCR). In this context, investigation of single tubules with residual spermatogenesis in SCCx43KO-/- mice was particularly interesting to identify a potential Cx43-independent influence of germ cells (GC) on BTB composition and dynamics. In tubules without residual spermatogenesis, a diffuse cytoplasmic distribution pattern for claudin-11 protein could be demonstrated in mutant mice. Nevertheless, claudin-11 seems to form functional TJ. Claudin-3 and -5 could not be detected immunohistochemically in the seminiferous epithelium of those tubules. Correspondingly, claudin-3 and -5 mRNA expression was decreased, providing evidence of generally impaired BTB dynamics in adult KO mice. Observations of tubules with residual spermatogenesis suggested a Cx43-independent regulation of TJ proteins by GC populations. To determine initial BTB formation in peripubertal SCCx43KO-/- mice, immunohistochemical staining and qRT-PCR of claudin-11 were carried out in adolescent SCCx43KO-/- and WT mice. Additionally, BTB integrity was functionally analysed using a hypertonic glucose fixative. These analyses revealed that SCCx43KO-/- mice formed an intact BTB during puberty in the same time period as WT mice, which however seemed to be accelerated.

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Section: Discussionsupporting

confidence: 83%

Loss of connexin43 in murine Sertoli cells and its effect on blood-testis barrier formation and dynamics

Hollenbach¹,

Jung

Noelke³

et al. 2018

PLoS ONE

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“…Moreover, a study recently demonstrates that testicular function in cultured testis fragment model, as expressed in gene expression profiling and testosterone levels, is qualitatively similar to results observed in vivo 13 . However, the yield of spermatozoa produced in organotypic cultures is low compared with age‐matched in vivo control 14‐16 and our recent data showed that meiotic and post‐meiotic progression could be impaired in cultured testicular explants 16,17 …”

Section: Introductionsupporting

confidence: 70%

Activation of the cannabinoid receptor type 2 by the agonist JWH133 promotes the first wave of in vitro spermatogenesis

et al. 2020

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Background Oncological procedures have irreversible side effects on germ cells for childhood cancer survival boys. In vitro culture of prepubertal testicular tissue has been proposed to restore fertility; however, recent data on animal models showed that meiotic and post‐meiotic progression was impaired. Objectives As potential key inducers of the mitosis‐meiosis switch, type 2 cannabinoid receptor (CB2) has been proposed to play a central role in the meiotic entry of male germ cells. Herein, the in vitro first spermatogenesis wave in mice was used to understand the impact of CB2 activation on the differentiation of spermatogonia until elongated spermatids. Materials and methods A first set of cultured testicular explants of 6.5 days post‐partum (dpp) mice was performed to assess the impact of a range of JWH133 supplementation (10 nm, 100 nm, 1 µm, 10 µm). Then, the progressive development of germ cells at key timepoints of spermatogenesis was evaluated throughout (i) in vitro culture (day 2 [D2], D3, D6, D10, D18, and D30) coupled with (ii) in vivo counterparts (8.5, 9.5, 12.5, 16.5, 24.5, and 36.5 dpp). Results CB2 was detected at the plasma membrane of cells, and a successful completion of spermatogenesis was obtained in vitro. One day after the activation of CB2 by 1 μm of the agonist JWH133, percentage of zygotene spermatocyte I increased. Conclusion After 30 days of culture, (i) an enrichment of haploid germ cells detected by flow cytometry, (ii) a reduced necrotic area, and (iii) an increase in the density of post‐meiotic germ cells were observed. We showed that the activation of CB2 improves in vitro entry into meiosis and differentiation of spermatogonia, mimicking physiological meiotic transition.

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“…Due to the unique architecture and constant turnover of stem cells, prepubertal testes may be more prone to impairments in functionality due to the way they are handled, transported, and maintained. Although attempts to achieve in vitro spermatogenesis using mouse and human ITT have been reported [7,17,18,[28][29][30][31][32][33], to our knowledge, this is the first report comparing the functionality of ITT handled at different temperature and time periods before the organ culture is initiated. We believe that this is an important prerequisite for the successful in vitro generation of spermatozoa.…”

Section: Discussionmentioning

confidence: 97%

Impact of Temperature and Time Interval Prior to Immature Testicular-Tissue Organotypic Culture on Cellular Niche

et al. 2020

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Cryopreservation of immature-testicular-tissue (ITT) prior to gonadotoxic treatment, while experimental, is the only recommended option for fertility preservation in prepubertal boys. The handling and manipulation of ITT before cryopreservation could influence the functionality of cells during fertility restoration, which this study explored by evaluating cellular niche and quality of mouse ITT subjected to various temperatures and time durations in vitro. ITT from 6-day-old mice were handled at ultraprofound-hypothermic, profound-hypothermic, and mild-warm-ischemic temperatures for varying time periods prior to 14-day organotypic culture. Viability, functionality, synaptonemal complex and chromatin remodeling markers were assessed. Results have shown that cell viability, testosterone level, and in vitro proliferation ability did not change when ITT were held at ultraprofound-hypothermic-temperature up to 24 h, whereas cell viability was significantly reduced (P < 0.01), when held at profound-hypothermic-temperature for 24 h before culture. Further, cell viability and testosterone levels in cultured cells from profound-hypothermic group were comparable to corresponding ultraprofound-hypothermic group but with moderate reduction in postmeiotic cells (P < 0.01). In conclusion, holding ITT at ultraprofound-hypothermic-temperature is most suitable for organotypic culture, whereas short-term exposure at profound-hypothermic-temperature may compromise postmeiotic germ cell yield post in vitro culture. This data, albeit in mouse model, will have immense value in human prepubertal fertility restoration research.

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Establishment, maintenance and functional integrity of the blood–testis barrier in organotypic cultures of fresh and frozen/thawed prepubertal mouse testes

Cited by 34 publications

References 61 publications

Loss of connexin43 in murine Sertoli cells and its effect on blood-testis barrier formation and dynamics

Loss of connexin43 in murine Sertoli cells and its effect on blood-testis barrier formation and dynamics

Activation of the cannabinoid receptor type 2 by the agonist JWH133 promotes the first wave of in vitro spermatogenesis

Impact of Temperature and Time Interval Prior to Immature Testicular-Tissue Organotypic Culture on Cellular Niche

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