A detailed protocol for a rapid analysis of testicular cell populations using flow cytometry

Rotgers, Emmi; Cisneros-Montalvo, Sheyla; Jahnukainen, Kirsi; Sandholm, Jouko; Toppari, Jorma; Nurmio, Mirja

doi:10.1111/andr.12066

Cited by 27 publications

(28 citation statements)

References 26 publications

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“…Mouse testes were dissociated according to the protocol described by Rotgers et al (16). Briefly, mouse testes were minced and incubated in DMEM/F12 containing 1 mg/ml collagenase (C9891; MilliporeSigma), 1 mg/ml hyaluronidase (H3506; MilliporeSigma), and 1 mg/ml DNase I (DN25; MilliporeSigma) at 37°C for 10 min and then filtered through 40 mm nylon mesh.…”

Section: Flow Cytometry To Analyze the Population Of Testicular Cellsmentioning

confidence: 99%

Sertoli cell–specific coxsackievirus and adenovirus receptor regulates cell adhesion and gene transcriptionviaβ‐catenin inactivation and Cdc42 activation

Huang

Zhang

et al. 2019

FASEB j.

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Blood‐testis barrier (BTB) and apical ectoplasmic specialization (ES) serve as structural supports for germ cell (GC) development. We demonstrated that the Sertoli cell (SC)‐specific coxsackievirus and adenovirus receptor (CXADR) knockout (SC‐CXADR−/−), but not the GC‐specific knockout, impaired spermatogenesis. An increase in GC apoptosis and premature loss of elongated spermatids were observed in SC‐CXADR−/− testes. The BTB function was compromised in SC‐CXADR−/− testes with dysregulation of oocludin and zonula occludens‐1 expression at the basal compartment of the seminiferous epithelium. An integrated omics analyses confirmed that altered gene ontology terms identified in SC‐CXADR−/− testes are highly associated with spermatid development and differentiation, spermatogenesis, and sperm motility and are considered as unique testicular function terms. Leptin, Nasp, Tektin3, Larp 7, and acrosin, which are highly associated with male fertility, were found to be down‐regulated in SC‐CXADR−/− testes. Based on the data from the omics analyses, we employed the CXADR‐deficient SC model to further investigate the molecular mechanisms involved. We unraveled that SC‐CXADRs are required for β‐catenin inactivation and cell division cycle protein 42 (Cdc42) activation, resulting in maintaining the integrity and function of the BTB and apical ES as well as inhibiting gene transcription, such as the Myc gene, in the testes. We demonstrated for the first time that CXADR is an important mediator governing β‐catenin and Cdc42 signaling that is essential for spermatogenesis. The molecular mechanisms identified herein may provide new insights to unravel the novel functions and signaling cascades of CXADR in other key CXADR‐expressing tissues.—Huang, K., Ru, B., Zhang, Y., Chan, W.‐L., Chow, S.‐C., Zhang, J., Lo, C., Lui, W.‐Y. Sertoli cell‐specific coxsackievirus and adenovirus receptor regulates cell adhesion and gene transcription via β‐catenin inactivation and Cdc42 activation. FASEB J. 33, 7588–7602 (2019). http://www.fasebj.org

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Section: Flow Cytometry To Analyze the Population Of Testicular Cellsmentioning

confidence: 99%

Sertoli cell–specific coxsackievirus and adenovirus receptor regulates cell adhesion and gene transcriptionviaβ‐catenin inactivation and Cdc42 activation

Huang

Zhang

et al. 2019

FASEB j.

View full text Add to dashboard Cite

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“…Individual germ cell populations were gated based on their DNA content according to Ref. as depicted in Fig E. Median Fluo4‐AM signals of each population were normalised to those observed for the respective wild‐type control.…”

Section: Methodsmentioning

confidence: 99%

“…Individual germ cell populations were gated based on their DNA content according to Ref. [36,37] as depicted in Fig 5E. Median Fluo4-AM signals of each population were normalised to those observed for the respective wild-type control. In addition, median forward scatter of the 1C-co population was compared between wild-type controls or SPPL2c-deficient animals to ensure that differences in Fluo4-AM staining were not caused by differences in cell size.…”

Section: Calcium Measurement In Testis Suspensionsmentioning

confidence: 99%

The intramembrane protease SPPL 2c promotes male germ cell development by cleaving phospholamban

et al. 2019

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Signal peptide peptidase (SPP) and the four homologous SPP‐like (SPPL) proteases constitute a family of intramembrane aspartyl proteases with selectivity for type II‐oriented transmembrane segments. Here, we analyse the physiological function of the orphan protease SPPL2c, previously considered to represent a non‐expressed pseudogene. We demonstrate proteolytic activity of SPPL2c towards selected tail‐anchored proteins. Despite shared ER localisation, SPPL2c and SPP exhibit distinct, though partially overlapping substrate spectra and inhibitory profiles, and are organised in different high molecular weight complexes. Interestingly, SPPL2c is specifically expressed in murine and human testis where it is primarily localised in spermatids. In mice, SPPL2c deficiency leads to a partial loss of elongated spermatids and reduced motility of mature spermatozoa, but preserved fertility. However, matings of male and female SPPL2c−/− mice exhibit reduced litter sizes. Using proteomics we identify the sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA2)‐regulating protein phospholamban (PLN) as a physiological SPPL2c substrate. Accumulation of PLN correlates with a decrease in intracellular Ca2+ levels in elongated spermatids that likely contribute to the compromised male germ cell differentiation and function of SPPL2c−/− mice.

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“…Flow cytometry analysis for the behavior of the testicular cell populations was performed as previously described (Rotgers et al ., 2015). Four E2F1 +/+ and E2F1 −/− animals at P20 and P40 were included in the assay.…”

Section: Methodsmentioning

confidence: 99%

E2F1 controls germ cell apoptosis during the first wave of spermatogenesis

et al. 2015

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SummaryCell cycle control during spermatogenesis is a highly complex process owing to the control of the mitotic expansion of the spermatogonial cell population and following meiosis, induction of DNA breaks during meiosis and the high levels of physiological germ‐cell apoptosis. We set out to study how E2F1, a key controller of cell cycle, apoptosis, and DNA damage responses, functions in the developing and adult testis. We first analyzed the expression pattern of E2f1 during post‐natal testis development using RNA in situ hybridization, which showed a differential expression pattern of E2f1 in the adult and juvenile mouse testes. To study the function of E2f1, we took advantage of the E2F1−/− mouse line, which was back‐crossed to C57Bl/6J genetic background. E2f1 loss led to a severe progressive testicular atrophy beginning at the age of 20 days. Spermatogonial apoptosis during the first wave of spermatogenesis was decreased. However, already in the first wave of spermatogenesis an extensive apoptosis of spermatocytes was observed. In the adult E2F1−/− testes, the atrophy due to loss of spermatocytes was further exacerbated by loss of spermatogonial stem cells. Surprisingly, only subtle changes in global gene expression array profiling were observed in E2F1−/− testis at PND20. To dissect the changes in each testicular cell type, an additional comparative analysis of the array data was performed making use of previously published data on transcriptomes of the individual testicular cell types. Taken together, our data indicate that E2F1 has a differential role during first wave of spermatogenesis and in the adult testis, which emphasizes the complex nature of cell cycle control in the developing testis.

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A detailed protocol for a rapid analysis of testicular cell populations using flow cytometry

Cited by 27 publications

References 26 publications

Sertoli cell–specific coxsackievirus and adenovirus receptor regulates cell adhesion and gene transcriptionviaβ‐catenin inactivation and Cdc42 activation

Sertoli cell–specific coxsackievirus and adenovirus receptor regulates cell adhesion and gene transcriptionviaβ‐catenin inactivation and Cdc42 activation

The intramembrane protease SPPL 2c promotes male germ cell development by cleaving phospholamban

E2F1 controls germ cell apoptosis during the first wave of spermatogenesis

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