Separation of mouse spermatogenic cells by sedimentation velocity

Romrell, Lynn J.; Bellvé, Anthony R.; Fawcett, Don W.

doi:10.1016/0012-1606(76)90262-1

Cited by 495 publications

(293 citation statements)

References 13 publications

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“…To profile small RNA expression during spermatogenesis in postnatal mice, three types of germ cells were isolated using gravity density gradient sedimentation (Romrell et al 1976;Bellve et al 1977). Type A spermatogonia (A), pachytene spermatocytes (PS), and round spermatids (RS) were isolated from 8-d-old mice, 17-d-old mice, and adult mice, respectively.…”

Section: Profiles Of Small Rnas In Mouse Spermatogenesismentioning

confidence: 99%

piRNA profiling during specific stages of mouse spermatogenesis

Gan

Lin²,

Zhang³

et al. 2011

RNA

111

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PIWI-interacting RNAs (piRNAs) are a class of small RNAs abundantly expressed in animal gonads. piRNAs that map to retrotransposons are generated by a ''ping-pong'' amplification loop to suppress the activity of retrotransposons. However, the biogenesis and function of other categories of piRNAs have yet to be investigated. In this study, we first profiled the expression of small RNAs in type A spermatogonia, pachytene spermatocytes, and round spermatids by deep sequencing. We then focused on the computational analysis of the potential piRNAs generated in the present study as well as other published sets. piRNAs mapping to retrotransposons, mRNAs, and intergenic regions had different length distributions and were differentially regulated in spermatogenesis. piRNA-generating mRNAs (PRMRs), whose expression positively correlated with their piRNA products, constituted one-third of the protein-coding genes and were evolutionarily conserved and enriched with splicing isoforms and antisense transcripts. PRMRs with piRNAs preferentially mapped to CDSs and 39 UTRs partitioned into three clusters differentially expressed during spermatogenesis and enriched with unique sets of functional annotation terms related to housekeeping activities as well as spermatogenesis-specific processes. Intergenic piRNAs were divided into 2992 clusters probably representing novel transcriptional units that have not been reported. The transcripts of a large number of genes involved in spermatogenesis are the precursors of piRNAs, and these genes are intricately regulated by alternative splicing and antisense transcripts. piRNAs, whose regulatory role in gene expression awaits to be identified, are clearly products of a novel regulatory process that needs to be defined.

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Section: Profiles Of Small Rnas In Mouse Spermatogenesismentioning

confidence: 99%

piRNA profiling during specific stages of mouse spermatogenesis

Gan

Lin²,

Zhang³

et al. 2011

RNA

111

View full text Add to dashboard Cite

show abstract

“…Mixed spermatogenic cells, pachytene spermatocytes (91.5%), and round spermatids (92%) were isolated from adult CD-1 mice as described previously (Romrell et al, 1976;Bellve et al, 1977;O'Brien et al, 1989). Total RNA was isolated using Trizol reagent (Life Technologies, Grand Island, NY).…”

Section: Northern Analysismentioning

confidence: 99%

Brca1 and Brca2 expression patterns in mitotic and meiotic cells of mice

et al. 1998

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The mouse homologues of the breast cancer susceptibility genes, Brca1 and Brca2, are expressed in a cell cycle-dependent fashion in vitro and appear to be regulated by similar or overlapping pathways. Therefore, we compared the non isotopic in situ hybridization expression patterns of Brca1 and Brca2 mRNA in vivo in mitotic and meiotic cells during mouse embryogenesis, mammary gland development, and in adult tissues including testes, ovaries, and hormonally altered ovaries. Brca1 and Brca2 are expressed concordantly in proliferating cells of embryos, and the mammary gland undergoing morphogenesis and in most adult tissues. The expression pattern of Brca1 and Brca2 correlates with the localization of proliferating cell nuclear antigen, an indicator of proliferative activity. In the ovary, Brca1 and Brca2 exhibited a comparable hormone-independent pattern of expression in oocytes, granulosa cells and thecal cells of developing follicles. In the testes, Brca1 and Brca2 were expressed in mitotic spermatogonia and early meiotic prophase spermatocytes. Northern analyses of prepubertal mouse testes revealed that the time course of Brca2 expression was delayed in spermatogonia relative to Brca1. Thus, while Brca1 and Brca2 share concordant cell-speci®c patterns of expression in most proliferating tissues, these observations suggest that they may have distinct roles during meiosis.

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“…A second strategy is the obtainment of highly enriched stage-specific cell populations from adult animals, separated by gravimetric decantation (Staput) (26)(27)(28)(29) or centrifugal elutriation (e.g., 18,[30][31][32]. However, because of the relative cell-type abundance and size difference, this approach only allows the successful separation of highly enriched fractions containing P spermatocytes (i.e., medium meiotic prophase), or round spermatids (RS; i.e.…”

mentioning

confidence: 99%

High‐purity flow sorting of early meiocytes based on DNA analysis of guinea pig spermatogenic cells

et al. 2011

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Mammalian spermatogenesis is still nowadays poorly understood at the molecular level. Testis cellular heterogeneity is a major drawback for spermatogenic gene expression studies, especially when research is focused on stages that are usually very short and poorly represented at the cellular level such as initial meiotic prophase I (i.e., leptotene [L] and zygotene [Z]). Presumably, genes whose products are involved in critical meiotic events such as alignment, pairing and recombination of homologous chromosomes are expressed during the short stages of early meiotic prophase. Aiming to characterize mammalian early meiotic gene expression, we have found the guinea pig (Cavia porcellus) as an especially attractive model. A detailed analysis of its first spermatogenic wave by flow cytometry (FCM) and optical microscopy showed that guinea pig testes exhibit a higher representation of early meiotic stages compared to other studied rodents, partly because of their longer span, and also as a result of the increased number of cells entering meiosis. Moreover, we have found that adult guinea pig testes exhibit a peculiar 4C DNA content profile, with a bimodal peak for L/Z and P spermatocytes that is absent in other rodents. Besides, we show that this unusual 4C peak allows the separation by FCM of highly pure L/Z spermatocyte populations aside from pachytene ones, even from adult individuals. To our knowledge, this is the first report on an accurate and suitable method for highly pure early meiotic prophase cell isolation from adult mammals, and thus sets an interesting approach for gene expression studies aiming at a deeper understanding of the molecular groundwork underlying male gamete production. ' International Society for Advancement of CytometryKey terms meiosis; spermatogenesis; flow cytometry; cell sorting; guinea pig; meiotic prophase; gene expression SPERMATOGENESIS can be viewed as the simultaneous and coordinated execution of three individual programs of gene expression: somatic proliferation of spermatogonia, meiosis, and spermiogenesis (1). In the seminiferous tubules of adult mammals, germ cells in different maturation steps coexist, with C (round spermatids, elongating and elongated spermatids, spermatozoa), 2C (several types of G1 spermatogonia, secondary spermatocytes) and 4C (different stages of primary spermatocytes, G2 spermatogonia) DNA content. Moreover, somatic Sertoli cells (2C) coexist with germ cells inside the tubules, which are surrounded by peritubular myoid cells and immersed in a stroma containing fibroblasts, lymphocytes, mastocytes, macrophages, and Leydig cells (all of them 2C).Beside the obvious importance of spermatogenesis as a source of male reproductive cells, along the lengthy first meiotic prophase significant events such as the recognition, alignment, pairing (synapsis), and recombination (crossing over) of homologous chromosomes take place (2-5). Particularly, meiotic homologous recombination has vital importance for genetic variability (6). Furthermore, mutations of

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Separation of mouse spermatogenic cells by sedimentation velocity

Cited by 495 publications

References 13 publications

piRNA profiling during specific stages of mouse spermatogenesis

piRNA profiling during specific stages of mouse spermatogenesis

Brca1 and Brca2 expression patterns in mitotic and meiotic cells of mice

High‐purity flow sorting of early meiocytes based on DNA analysis of guinea pig spermatogenic cells

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