Evidence for biological rhythm in spermatogenesis in the pubertal hamster (Mesocricetus auratus): a flow cytometric study

Vigodner, Margarita; Lewy, Hadas; Lewin, L.; Shochat, L.; Golan, Rachel

doi:10.1016/j.lfs.2003.07.028

Cited by 3 publications

(5 citation statements)

References 15 publications

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“…S1). Based on previous studies detailing the DNA content of various testicular germ cell populations we hypothesized that these two populations might represent round and elongated spermatids, respectively, since both are haploid but differ from each other in terms of DNA compaction. Indeed, when we sorted both populations (termed “sub1N” and “1 N” based on PI signal) and loaded the obtained cell fractions onto glass slides via cytospin, we morphologically observed a high percentage of cells in the sub1N‐sorted fraction that displayed phenotypical characteristics of elongated spermatids (oval‐shaped, highly condensed nucleus) and, vice versa, a high percentage of cells in the 1 N‐sorted cell fraction that displayed characteristics of round spermatids (round nucleus).…”

Section: Resultsmentioning

confidence: 99%

Simultaneous Purification of Round and Elongated Spermatids from Testis Tissue Using a FACS‐Based DNA Ploidy Assay

et al. 2018

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Spermiogenesis is the final phase of spermatogenesis during which post‐meiotic haploid round spermatids (rSpt) differentiate into elongated spermatozoa and includes several critical cell‐specific processes like DNA condensation, formation of the acrosome, and production of the flagellum. Disturbances in this process will lead to complications in sperm development and subsequently cause infertility. As such, studying spermiogenesis has clinical relevance in investigating the etiology of male infertility and will improve our scientific understanding of male germ cell formation. Here, we were able to purify round spermatid and elongated spermatid fractions from a single cryopreserved human testicular tissues sample with an efficiency of 85.4% ± 4.9% and 97.6% ± 0.6%, respectively. We confirmed the cell types by morphology and immunohistochemistry for histone H4 and PNA protein expression. The purity was measured by manual counting of histone H4 positive (round) and negative (elongated) spermatids in both sorted 1 N cell fractions. This method can be applied to both human and rodent studies. Especially in studies with limited access to testicular tissue, this method provides a reliable means to simultaneously isolate these cell types with high purity. Our method allows for further investigation of germ cell development and the process of spermiogenesis in particular, as well as provides a tool to study the etiology of male infertility, including morphological and biochemical assessment of round and elongating spermatids from subfertile men. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

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Section: Resultsmentioning

confidence: 99%

Simultaneous Purification of Round and Elongated Spermatids from Testis Tissue Using a FACS‐Based DNA Ploidy Assay

et al. 2018

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“…Multi-parametric FCM analyses have been historically employed to study postnatal development of the male gonad in different species. Many such reports exist for diverse mammalian species, including mouse [53,54], rat [20,55], guinea pig [21], hamster [56,57], cat [58], pig [59], and several primates [60,61] including man [62,63], among others.…”

Section: Fcm As An Analytical Tool Of Spermatogenesis 41 Testis Posmentioning

confidence: 99%

Contributions of Flow Cytometry to the Molecular Study of Spermatogenesis in Mammals

Rodríguez-Casuriaga

Geisinger

2021

IJMS

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Mammalian testes are very heterogeneous organs, with a high number of different cell types. Testicular heterogeneity, together with the lack of reliable in vitro culture systems of spermatogenic cells, have been an obstacle for the characterization of the molecular bases of the unique events that take place along the different spermatogenic stages. In this context, flow cytometry has become an invaluable tool for the analysis of testicular heterogeneity, and for the purification of stage-specific spermatogenic cell populations, both for basic research and for clinical applications. In this review, we highlight the importance of flow cytometry for the advances on the knowledge of the molecular groundwork of spermatogenesis in mammals. Moreover, we provide examples of different approaches to the study of spermatogenesis that have benefited from flow cytometry, including the characterization of mutant phenotypes, transcriptomics, epigenetic and genome-wide chromatin studies, and the attempts to establish cell culture systems for research and/or clinical aims such as infertility treatment.

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“…Testis developmental schedule and cell composition for different mammalian species such as mouse [Meistrich et al, 1978;Janca et al, 1986;Petit et al, 1995], rat [Clausen et al, 1982;Van Kroonenburgh et al, 1985;Suter et al, 1997;Malkov et al, 1998], hamster [Vigodner et al, 2003[Vigodner et al, , 2004, pig [Oskam et al, 2008], cat [Neubauer et al, 2004], several primates [Aravindan et al, 1990;Aslam et al, 2002;Wistuba et al, 2003] including man [Blanchard et al, 1991;Hittmair et al, 1992], and others, have been assessed by FC (see fig. 1 A as an example).…”

Section: Flow Cytometry As a Way To Analyze And Sort Testicular Cell mentioning

confidence: 99%

“…Propidium iodide (PI), which is the most commonly used dye to quantitatively assess DNA content, has been extensively used to identify testicular cell populations [Weiss et al, 1997;Malkov et al, 1998;Lee et al, 2001;Vigodner et al, 2004]. It is an intercalating agent that binds DNA in a stoichiometric manner with little or no sequence preference, can be excited with a UV (351 nm) or blue (488 nm) laser and exhibits its fluorescence emission maximum at 619 nm.…”

Section: Dna Stains For Spermatogenesis Assessmentmentioning

confidence: 99%

“…PI staining generally allows distinguishing among the 4 previously mentioned cell populations from mature testes according to their relative ploidy (levels of fluorescence): haploid mature cells, haploid immature, diploid, and tetraploid ( fig. 1 B, a-c) [Vigodner et al, 2004]. When cell size and granularity were also considered, 7 distinct subpopulations could be distinguished in the dot plots obtained from rodent testicular cell suspensions after PI staining [Malkov et al, 1998].…”

Section: Dna Stains For Spermatogenesis Assessmentmentioning

confidence: 99%

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Flow Cytometry for Gene Expression Studies in Mammalian Spermatogenesis

Geisinger

Rodríguez-Casuriaga²

2010

Cytogenet Genome Res

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Mammalian spermatogenesis is nowadays still poorly understood at the molecular level, mainly due to the heterogeneous nature of testes, which contain a high number of different cell types, and to the lack of spermatogenic cell culture systems for in vitro studies. As a consequence, the development and/or application of methodological approaches aiming at the enrichment or purification of specific testicular cell types are of great interest and have been addressed by scientists for at least 4 decades. Among the many applications that flow cytometry (FC) has gained since its invention, analysis and sorting of spermatogenic cell populations represent a promising strategy to efficiently overcome testis heterogeneity drawback. Surprisingly, FC has been only rarely used as a preparative method for downstream gene expression studies in specific spermatogenic stages. This work aims to provide an overview of FC for spermatogenic studies including preparation of testicular single cell suspensions, dyes for DNA staining, and our own experience with rodent testis material.

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Evidence for biological rhythm in spermatogenesis in the pubertal hamster (Mesocricetus auratus): a flow cytometric study

Cited by 3 publications

References 15 publications

Simultaneous Purification of Round and Elongated Spermatids from Testis Tissue Using a FACS‐Based DNA Ploidy Assay

Simultaneous Purification of Round and Elongated Spermatids from Testis Tissue Using a FACS‐Based DNA Ploidy Assay

Contributions of Flow Cytometry to the Molecular Study of Spermatogenesis in Mammals

Flow Cytometry for Gene Expression Studies in Mammalian Spermatogenesis

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