A rapid method of Sertoli cell isolation by DSA lectin, allowing mitotic analyses

Scarpino, Stefania; Morena, Anna Rita; Petersen, Cecilia; Fröysa, Berit; Söder, Olle; Boitani, Carla

doi:10.1016/s0303-7207(98)00190-7

Cited by 61 publications

(43 citation statements)

References 28 publications

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“…Purity of the pachytene spermatocyte sample was estimated at ;70% based on fluorescence analysis using anti-SYCP3 (a marker of the synaptonemal complex) and anti-phospho-H2AX (a marker of double-strand breaks and the sex body). Sertoli cells were isolated from 3-wk-old animals using Datura Stramonium agglutinin (DSA) coated dishes as previously described (Scarpino et al 1998), with a purity of ;95%.…”

Section: Methods Samplesmentioning

confidence: 99%

Birth and expression evolution of mammalian microRNA genes

et al. 2012

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MicroRNAs (miRNAs) are major post-transcriptional regulators of gene expression, yet their origins and functional evolution in mammals remain little understood due to the lack of appropriate comparative data. Using RNA sequencing, we have generated extensive and comparable miRNA data for five organs in six species that represent all main mammalian lineages and birds (the evolutionary outgroup) with the aim to unravel the evolution of mammalian miRNAs. Our analyses reveal an overall expansion of miRNA repertoires in mammals, with threefold accelerated birth rates of miRNA families in placentals and marsupials, facilitated by the de novo emergence of miRNAs in host gene introns. Generally, our analyses suggest a high rate of miRNA family turnover in mammals with many newly emerged miRNA families being lost soon after their formation. Selectively preserved mammalian miRNA families gradually evolved higher expression levels, as well as altered mature sequences and target gene repertoires, and were apparently mainly recruited to exert regulatory functions in nervous tissues. However, miRNAs that originated on the X chromosome evolved high expression levels and potentially diverse functions during spermatogenesis, including meiosis, through selectively driven duplicationdivergence processes. Overall, our study thus provides detailed insights into the birth and evolution of mammalian miRNA genes and the associated selective forces.

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Section: Methods Samplesmentioning

confidence: 99%

Birth and expression evolution of mammalian microRNA genes

et al. 2012

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“…Sertoli cell contamination in germ cell fractions was monitored by RPA analysis of FSH receptor expression. Sertoli cells were isolated from 7-and 14-day-old mice as described by Scarpino et al (28). Sertoli cell cultures appeared to contain less than 5% myoid cells, as determined by alkaline phosphatase staining, and less than 1% germ cells.…”

Section: Cell Isolationmentioning

confidence: 99%

Regulatory role of BMP2 and BMP7 in spermatogonia and Sertoli cell proliferation in the immature mouse

Puglisi

Montanari

Chiarella

et al. 2004

European Journal of Endocrinology

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Aim: The aim of this study was to determine the action of bone morphogenetic proteins (BMPs) on testicular cell proliferation during early postnatal life, a definite developmental time at which crucial changes in germ cell and Sertoli cell maturation occur. Methods: We investigated the effect of BMP2 and BMP7, two factors which belong to the relatively distant decapentaplegic (DPP) and 60A classes of the large BMP family, upon spermatogonial and Sertoli cell proliferation, and we examined the expression of activin/BMP type II and type I receptors. We used in vitro cultured testis fragments from 7-day-old mice, highly purified populations of somatic and germ cells and total testes from mice of different ages. Cell proliferation was assessed by BrdU labelling and [ 3 H]-thimidine incorporation. Ribonuclease protection assays and Northern blotting were performed to analyse receptor expression. Results and conclusions:We have demonstrated a stimulatory action of BMP2 and BMP7 in spermatogonia and Sertoli cell proliferation respectively. ActRIIB is the type II receptor expressed most in spermatogonia, whereas Sertoli cells specifically expressed BMPRIIB, in addition to ActRIIB. By contrast, the presence of ActRIIA was undetectable in either germ or somatic cells. The type I receptors ActRIA, ActRIB and BMPRIA were all found in both cell types, indicating that the observed effect of BMP2 and BMP7 on testicular cell proliferation may be mediated by a number of combinatorial interactions in the receptor complexes. These findings suggest that BMPs are involved in physiological paracrine signalling during the first wave of spermatogenesis.European Journal of Endocrinology 151 511-520

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“…Sertoli cells were isolated from 7-day-old mice as described previously (Scarpino et al, 1998). After three days in culture at 32°C, Sertoli cells were washed with medium and stored at -80°C or treated with ovine follicle stimulating hormone (FSH) (20 ng/ml) to evaluate cAMP production into the medium, as measured by enzymeimmunoassay (Amersham, Bucks, UK).…”

Section: Cell Preparationsmentioning

confidence: 99%

Ryanodine receptors are expressed and functionally active in mouse spermatogenic cells and their inhibition interferes with spermatogonial differentiation

Chiarella

Puglisi

Sorrentino

et al. 2004

Journal of Cell Science

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Ryanodine receptors (RyRs) are intracellular calcium release channels that are highly expressed in striated muscle and neurons but are also detected in several non-excitable cells. We have studied the expression of the three RyR isoforms in male germ cells at different stages of maturation by western blot and RT-PCR. RyR1 was expressed in spermatogonia, pachytene spermatocytes and round spermatids whereas RyR2 was found only in 5- to 10-day-old testis but not in germ cells. RyR3 was not revealed at the protein level, although its mRNA was detected in mixed populations of germ cells. Caffeine, a known agonist of RyRs, was able to induce release of Ca 2+ from intracellular stores in spermatogonia, pachytene spermatocytes and round spermatids, but not spermatozoa. Treatment with high doses of ryanodine, which are known to block RyR channel activity, reduced spermatogonial proliferation and induced meiosis in in vitro organ cultures of testis from 7-day-old mice. In conclusion, the results presented here indicate that RyRs are present in germ cells and that calcium mobilization through RyR channels could participate to the regulation of male germ maturation

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A rapid method of Sertoli cell isolation by DSA lectin, allowing mitotic analyses

Cited by 61 publications

References 28 publications

Birth and expression evolution of mammalian microRNA genes

Birth and expression evolution of mammalian microRNA genes

Regulatory role of BMP2 and BMP7 in spermatogonia and Sertoli cell proliferation in the immature mouse

Ryanodine receptors are expressed and functionally active in mouse spermatogenic cells and their inhibition interferes with spermatogonial differentiation

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