Germ Cell Maintenance and Sustained Testosterone and Precursor Hormone Production in Human Prepubertal Testis Organ Culture with Tissues from Boys 7 Years+ under Conditions from Adult Testicular Tissue

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Abstract

Human prepubertal testicular tissues are rare, but organ culture conditions to develop a system for human in vitro-spermatogenesis are an essential option for fertility preservation in prepubertal boys subjected to gonadotoxic therapy. To avoid animal testing in line with the 3Rs principle, organ culture conditions initially tested on human adult testis tissue were applied to prepubertal samples (n = 3; patient ages 7, 9, and 12 years). Tissues were investigated by immunostaining and transmission electron microscopy (TEM), and the collected culture medium was profiled for steroid hormones by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Culture conditions proved suitable for prepubertal organ culture since SSCs and germ cell proliferation could be maintained until the end of the 3-week-culture. Leydig cells (LCs) were shown to be competent for steroid hormone production. Three additional testis tissues from boys of the same age were examined for the number of germ cells and undifferentiated spermatogonia (SPG). Using TEM micrographs, eight tissues from patients aged 1.5 to 13 years were examined, with respect to the sizes of mitochondria (MT) in undifferentiated SPG and compared with those from two adult testicular tissues. Mitochondrial sizes were shown to be comparable between adults and prepubertal boys from approximately 7 years of age, which suggests the transition of SSCs from normoxic to hypoxic metabolism at about or before this time period.

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Proteomics. Tissue-based map of the human proteome.

Mathias Uhlén, Linn Fagerberg, Björn Hallström … (2015)

Resolving the molecular details of proteome variation in the different tissues and organs of the human body will greatly increase our knowledge of human biology and disease. Here, we present a map of the human tissue proteome based on an integrated omics approach that involves quantitative transcriptomics at the tissue and organ level, combined with tissue microarray-based immunohistochemistry, to achieve spatial localization of proteins down to the single-cell level. Our tissue-based analysis detected more than 90% of the putative protein-coding genes. We used this approach to explore the human secretome, the membrane proteome, the druggable proteome, the cancer proteome, and the metabolic functions in 32 different tissues and organs. All the data are integrated in an interactive Web-based database that allows exploration of individual proteins, as well as navigation of global expression patterns, in all major tissues and organs in the human body. Copyright © 2015, American Association for the Advancement of Science.

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The adult human testis transcriptional cell atlas

Jingtao Guo, Edward J. Grow, Hana Mlcochova … (2018)

Human adult spermatogenesis balances spermatogonial stem cell (SSC) self-renewal and differentiation, alongside complex germ cell-niche interactions, to ensure long-term fertility and faithful genome propagation. Here, we performed single-cell RNA sequencing of ~6500 testicular cells from young adults. We found five niche/somatic cell types (Leydig, myoid, Sertoli, endothelial, macrophage), and observed germline-niche interactions and key human-mouse differences. Spermatogenesis, including meiosis, was reconstructed computationally, revealing sequential coding, non-coding, and repeat-element transcriptional signatures. Interestingly, we identified five discrete transcriptional/developmental spermatogonial states, including a novel early SSC state, termed State 0. Epigenetic features and nascent transcription analyses suggested developmental plasticity within spermatogonial States. To understand the origin of State 0, we profiled testicular cells from infants, and identified distinct similarities between adult State 0 and infant SSCs. Overall, our datasets describe key transcriptional and epigenetic signatures of the normal adult human testis, and provide new insights into germ cell developmental transitions and plasticity.

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In vitro production of functional sperm in cultured neonatal mouse testes.

Takuya Sato, Kumiko Katagiri, Ayako Gohbara … (2011)

Spermatogenesis is one of the most complex and longest processes of sequential cell proliferation and differentiation in the body, taking more than a month from spermatogonial stem cells, through meiosis, to sperm formation. The whole process, therefore, has never been reproduced in vitro in mammals, nor in any other species with a very few exceptions in some particular types of fish. Here we show that neonatal mouse testes which contain only gonocytes or primitive spermatogonia as germ cells can produce spermatids and sperm in vitro with serum-free culture media. Spermatogenesis was maintained over 2 months in tissue fragments positioned at the gas-liquid interphase. The obtained spermatids and sperm resulted in healthy and reproductively competent offspring through microinsemination. In addition, neonatal testis tissues were cryopreserved and, after thawing, showed complete spermatogenesis in vitro. Our organ culture method could be applicable through further refinements to a variety of mammalian species, which will serve as a platform for future clinical application as well as mechanistic understanding of spermatogenesis.