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In-vitro spermatogenesis through testis modelling: Toward the generation of testicular organoids.

ABSTRACT: BACKGROUND:The testicular organoid concept has recently been introduced in tissue engineering to refer to testicular cell organizations modeling testicular architecture and function. The testicular organoid approach gives control over which and how cells reaggregate, which is not possible in organotypic cultures, thereby extending the applicability of in-vitro spermatogenesis (IVS) systems. However, it remains unclear which culture method and medium allow reassociation of testicular cells into a functional testicular surrogate in-vitro. OBJECTIVE:The aim of this paper is to review the different strategies that have been used in an attempt to create testicular organoids and generate spermatozoa. We want to provide an up-to-date list on culture methodologies and media compositions that have been used and determine their role in regulating tubulogenesis and differentiation of testicular cells. SEARCH METHOD:A literature search was conducted in PubMed, Web of Science, and Scopus to select studies reporting the reorganization of testicular cell suspensions in-vitro, using the keywords: three-dimensional culture, in-vitro spermatogenesis, testicular organoid, testicular scaffold, and tubulogenesis. Papers published before the August 1, 2019, were selected. OUTCOME:Only a limited number of studies have concentrated on recreating the testicular architecture in-vitro. While some advances have been made in the testicular organoid research in terms of cellular reorganization, none of the described culture systems is adequate for the reproduction of both the testicular architecture and IVS. CONCLUSION:Further improvements in culture methodology and medium composition have to be made before being able to provide both testicular tubulogenesis and spermatogenesis in-vitro.

SUBMITTER: Richer G

PROVIDER: S-EPMC7496450 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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In-vitro spermatogenesis through testis modelling: Toward the generation of testicular organoids.

Richer Guillaume G Baert Yoni Y Goossens Ellen E

Andrology 20200109 4

<h4>Background</h4>The testicular organoid concept has recently been introduced in tissue engineering to refer to testicular cell organizations modeling testicular architecture and function. The testicular organoid approach gives control over which and how cells reaggregate, which is not possible in organotypic cultures, thereby extending the applicability of in-vitro spermatogenesis (IVS) systems. However, it remains unclear which culture method and medium allow reassociation of testicular cell ...[more]

PMID: 31823507

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Project description:Spermatogenesis is a complex cell differentiation process that includes marked genetic, cellular, functional and structural changes. It requires tight regulation, because disturbances in any of the spermatogenic processes would lead to fertility deficiencies as well as disorders in offspring. To increase our knowledge of signal transduction during sperm development, we carried out a large-scale identification of the phosphorylation events that occur in the human male gonad. Metal oxide affinity chromatography using TiO2 combined with LC-MS/MS was conducted to profile the phosphoproteome of adult human testes with full spermatogenesis. A total of 8187 phosphopeptides derived from 2661 proteins were identified, resulting in the most complete report of human testicular phosphoproteins to date. Phosphorylation events were enriched in proteins functionally related to spermatogenesis, as well as to highly active processes in the male gonad, such as transcriptional and translational regulation, cytoskeleton organization, DNA packaging, cell cycle and apoptosis. Moreover, 174 phosphorylated kinases were identified. The most active human protein kinases in the testis were predicted both by the number of phosphopeptide spectra identified and the phosphorylation status of the kinase activation loop. The potential function of cyclin-dependent kinase 12 (CDK12) and p21-activated kinase 4 (PAK4) has been explored by in silico, protein-protein interaction analysis, immunodetection in testicular tissue, and a functional assay in a human embryonal carcinoma cell line. The colocalization of CDK12 with Golgi markers suggests a potential crucial role of this protein kinase during sperm formation. PAK4 has been found expressed in human spermatogonia, and a role in embryonal carcinoma cell response to apoptosis has been observed. Together, our protein discovery analysis confirms that phosphoregulation by protein kinases is highly active in sperm differentiation and opens a window to detailed characterization and validation of potential targets for the development of drugs modulating male fertility and tumor behavior.

Project description:Study questionCan transplanted primate testicular cells form seminiferous tubules de novo, supporting complete spermatogenesis?Summary answerCryopreserved testicular cells from a prepubertal monkey can reorganize in an adult monkey recipient testis forming de novo seminiferous tubular cords supporting complete spermatogenesis.What is known alreadyDe novo morphogenesis of testicular tissue using aggregated cells from non-primate species grafted either subcutaneously or in the testis can support spermatogenesis.Study design, size, durationTwo postpubertal rhesus monkeys (Macaca mulatta) were given testicular irradiation. One monkey was given GnRH-antagonist treatment from 8 to 16 weeks after irradiation, while the other received sham injections. At 16 weeks, cryopreserved testicular cells from two different prepubertal monkeys [43 × 106 viable (Trypan-blue excluding) cells in 260 μl, and 80 × 106 viable cells in 400 μl] were transplanted via ultrasound-guided injections to one of the rete testis in each recipient, and immune suppression was given. The contralateral testis was sham transplanted. Testes were analyzed 9 months after transplantation.Participants/materials, setting, methodsSpermatogenic recovery was assessed by testicular volume, weight, histology and immunofluorescence. Microsatellite genotyping of regions of testicular sections obtained by LCM determined whether the cells were derived from the host or transplanted cells.Main results and the role of chanceTransplanted testis of the GnRH-antagonist-treated recipient, but not the sham-treated recipient, contained numerous irregularly shaped seminiferous tubular cords, 89% of which had differentiating germ cells, including sperm in a few of them. The percentages of donor genotype in different regions of this testis were as follows: normal tubule, 0%; inflammatory, 0%; abnormal tubule region, 67%; whole interior of abnormal tubules, >99%; adluminal region of the abnormal tubules, 92%. Thus, these abnormal tubules, including the enclosed germ cells, were derived de novo from the donor testicular cells.Large scale dataNot applicable.Limitations, reasons for cautionThe de novo tubules were observed in only one out of the two monkeys transplanted with prepubertal donor testicular cells.Wider implications of the findingsThese findings may represent a promising strategy for restoration of fertility in male childhood cancer survivors. The approach could be particularly useful in those exposed to therapeutic agents that are detrimental to the normal development of the tubule somatic cells affecting the ability of the endogenous tubules to support spermatogenesis, even from transplanted spermatogonial stem cells.Study funding/competing interest(s)This work was supported by research grants P01 HD075795 from Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD/NIH) to K.E.O and Cancer Center Support Grant P30 CA016672 from NCI/NIH to The University of Texas MD Anderson Cancer Center. The authors declare that they have no competing interests.

Project description:Spermatogenesis is a complex cell differentiation process that includes marked genetic, cellular functional and structural changes. It requires tight regulation, since disturbances in any of the spermatogenic stages would lead to fertility deficiencies. In order to increase our knowledge of signal transduction during sperm development, we carried out a large-scale identification of the phosphorylation events that occur in the human gonad. Metal oxide affinity chromatography using TiOx combined with LC-MS/MS was conducted to profile the phosphoproteome of human testes with full spermatogenesis. A total of 8187 phosphopeptides derived from 2661 proteins were identified, resulting in the most complete report of human testicular phosphoproteins to date. Phosphorylation events were enriched in proteins functionally related to spermatogenesis, as well as to highly active processes in the male gonad, such as transcriptional and translational regulation, cytoskeleton organization, DNA packaging, cell cycle and apoptosis. Moreover, 174 phosphorylated kinases were identified. The most active and abundant human protein kinases in the testis were predicted both by the phosphorylation status of the kinase activation loop and the number of phosphopeptide spectra identified. The potential function of two of those kinases, cyclin-dependent kinase 12 (CDK12) and p21-activated kinase 4(PAK4), has been explored by protein-protein interaction analysis, immunodetection in human and mouse testicular tissue, and functional assay in a human embryonal carcinoma cell line. The co-localization of CDK12 with Golgi markers and probably pro-acrosomal vesicles suggests a potential crucial role of this protein kinase in sperm formation. PAK4 expression has been found limited to human spermatogonia, and a role in embryonal carcinoma cell response to apoptosis has been observed. Together, our data confirm that phosphoregulation by protein kinases is highly active in sperm differentiation, and open a window to detailed characterization and validation of potential targets for the development of drugs modulating male fertility, and tumor behavior.

Dataset Information

In-vitro spermatogenesis through testis modelling: Toward the generation of testicular organoids.

Publications

In-vitro spermatogenesis through testis modelling: Toward the generation of testicular organoids.

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