Effects of carbachol on rat Sertoli cell proliferation and muscarinic acetylcholine receptors regulation: an in vitro study

Lucas, Thaís F.G.; Avellar, Maria Christina W.; Porto, Catarina S.

doi:10.1016/j.lfs.2004.05.006

Cited by 20 publications

(18 citation statements)

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“…The testes from 15-day-old rats were removed and decapsulated, and Sertoli cells were isolated (Lucas et al 2004(Lucas et al , 2008. Aliquots of 1 ml of the cell suspension were plated in 12-well plates at a density of w4!10 4 cells/well (about 10 000 cells/ cm 2 ) in phenol red-free F12/DMEM containing 0.02 g/l gentamicin (Sigma Chemical Co.), pH 7.2-7.4, supplemented with 10 mg/ml insulin, 10 mg/ml transferrin, 10 ng/ml sodium selenite, and 10 ng/ml epidermal growth factor (Sigma).…”

Section: Primary Cultures Of Sertoli Cellsmentioning

confidence: 99%

“…Previous studies in our laboratory indicated that incorporation of [methyl-3 H] thymidine (2 mCi/ml, specific activity 79.0 Ci/ mmol, Amersham Biosciences) in cultured Sertoli cells increased linearly between 2 and 10 h of incubation. All studies were performed using 6 h of [methyl-3 H] thymidine incubation (Lucas et al 2004).…”

Section: [Methyl-3 H] Thymidine Incorporation Assaysmentioning

confidence: 99%

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Locally produced relaxin may affect testis and vas deferens function in rats

Cardoso¹,

Nascimento²,

Royer³

et al. 2010

Reproduction

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We have previously shown that the rat testis and vas deferens contain high levels of the relaxin receptor, RXFP1. The present study was undertaken to determine the expression of relaxin in these tissues, and the effect of exogenous relaxin on Sertoli cell proliferation and on the mRNA levels of some proteins that may contribute to epithelial secretion and tissue reorganization in the vas deferens. Relaxin mRNA levels in testis and vas deferens were much lower than in the prostate. Sertoli cells seem to be an important source of relaxin mRNA in testis. Relaxin immunoreactivity was detected in the seminiferous epithelium but not in the interstitial compartment. The relaxin precursor was expressed in the vas deferens, and relaxin immunoreactivity was detected in apical cells of the vas deferens. Castration, but not treatment with the anti-estrogen ICI 182,780, dramatically reduced relaxin mRNA levels in the prostate and vas deferens, and this effect was prevented by testosterone. Rxfp1 mRNA levels in the vas deferens and prostate were not affected by castration or treatment with ICI 182,780. Exogenous relaxin increased the incorporation of 3 H-thymidine in cultured Sertoli cells, and treatment of the vas deferens with 100 ng/ml relaxin increased the mRNA levels for the cystic fibrosis chloride channel (cystic fibrosis transmembrane regulator) about three times, and doubled mRNA levels for the inducible form of nitric oxide synthase and metalloproteinase 7. These results suggest that locally produced relaxin acts as an autocrine or paracrine agent in the testis and vas deferens to affect spermatogenesis and seminal fluid composition.

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Section: Primary Cultures Of Sertoli Cellsmentioning

confidence: 99%

Section: [Methyl-3 H] Thymidine Incorporation Assaysmentioning

confidence: 99%

Locally produced relaxin may affect testis and vas deferens function in rats

Cardoso¹,

Nascimento²,

Royer³

et al. 2010

Reproduction

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“…The experimental procedures were conducted according to guidelines for the care and use of laboratory animals as approved by the Research Ethical Committee from UNIFESP-EPM (protocol 121/08). The testes were removed and decapsulated and Sertoli cells were prepared as described previously (Lucas et al 2004(Lucas et al , 2008a(Lucas et al , 2008b(Lucas et al , 2010. The cells (4.5!10 5 cells/ml) were plated at a density of 4!10 6 cells/ 9 ml in 100 mm dish (about 5!10 4 cells/cm 2 ) to allow the formation of monolayer in phenol red-free Ham F12/DMEM (1:1, Gibco, Invitrogen) containing 0.02 g/l gentamicin (Sigma Chemical Co.), pH 7.2-7.4, and supplemented with 10 mg/ml insulin, 10 mg/ml transferrin, 10 ng/ml sodium selenite, and 10 ng/ml epidermal growth factor (Sigma).…”

Section: Sertoli Cell Culturementioning

confidence: 99%

“…Incorporation of [methyl-3 H]thymidine into cell DNA was estimated as described by Guizzetti et al (1996) with slight modifications (Lucas et al 2004(Lucas et al , 2008a. In previous studies, we determined that incorporation of [methyl-3 H]thymidine (2 mCi/ml, specific activity 79.0 Ci/mmol, GE Healthcare Life Science, Pittsburgh, PA, USA) in cultured Sertoli cells was time dependent and linear from 2 to 10 h of incubation.…”

Section: [Methyl-3 H]thymidine Incorporation Assaysmentioning

confidence: 99%

“…In previous studies, we determined that incorporation of [methyl-3 H]thymidine (2 mCi/ml, specific activity 79.0 Ci/mmol, GE Healthcare Life Science, Pittsburgh, PA, USA) in cultured Sertoli cells was time dependent and linear from 2 to 10 h of incubation. All studies were performed after incubation with [methyl-3 H]thymidine for 6 h (Lucas et al 2004(Lucas et al , 2008a. On day 4 of culture, primary Sertoli cells were incubated with 2 mCi/ml [methyl-…”

Section: [Methyl-3 H]thymidine Incorporation Assaysmentioning

confidence: 99%

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Na+/K+-ATPase α1 isoform mediates ouabain-induced expression of cyclin D1 and proliferation of rat Sertoli cells

et al. 2012

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Novel roles for the interaction of cardiotonic steroids to Na H]thymidine, both of which were dependent on MAPK3/1 but not AKT intracellular cascade, as shown by pretreatment with MEK (MAP2K1/2) inhibitor U0126 and PI3K inhibitor wortmannin respectively. Moreover, the effect of ouabain on these proliferation parameters was completely prevented by phospho-cAMP response element-binding protein (CREB)/CREB-binding protein complex inhibitor KG501 and only partially by nuclear factor kB nuclear translocation inhibitor SN50. Pretreatment with estrogen receptor antagonist ICI 182 780 showed that MAPK3/1 activation by ouabain does not involve this receptor. The Na C /K C -ATPase a1 isoform, but not a4, was detected in Sertoli cells, suggesting that ouabain effects in Sertoli cells are mediated via a1. Taken together, these results show a rapid ouabain action in the Sertoli cells, which in turn can modulate nuclear transcriptional events essential for Sertoli cell proliferation in a critical period of testicular development. Our findings are important to understand the role of ouabain in the testis and its possible implications in male infertility. Reproduction (2012) 144 737-745

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Epididymal protease inhibitor (EPPIN) is differentially expressed in the male rat reproductive tract and immunolocalized in maturing spermatozoa

Silva

Patrão

Tsuruta

et al. 2012

Molecular Reproduction Devel

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EPPIN (epididymal protease inhibitor; SPINLW1), an antimicrobial cysteine-rich protein containing both Kunitz and whey acidic protein (WAP)-type four disulfide core protease inhibitor consensus sequences, is a target for male contraception because of its critical role in sperm motility. Here, we characterized EPPIN's expression and cellular distribution in rat tissues and its in vivo regulation by androgens in the epididymis. EPPIN (mRNA and protein) was abundantly expressed in the rat testis and epididymis; we also found that the vas deferens, seminal vesicles, and brain were novel sites of EPPIN expression. PCR studies demonstrated that in addition to Sertoli cells, spermatogenic cells expressed Eppin mRNA. EPPIN was immunolocalized in Sertoli cells and spermatogenic cells (pachytene spermatocytes and round and elongated spermatids) and in epithelial cells and spermatozoa from efferent ductules and epididymis. EPPIN staining was observed on the middle and principal pieces of the flagellum of testicular spermatozoa. Epididymal spermatozoa had more intense EPPIN staining on the flagellum, and the EPPIN staining became apparent on the head and neck regions. This suggested that the EPPIN found on maturing spermatozoa was secreted primarily by the epithelial cells of the epididymis. Surgical castration down-regulated EPPIN expression levels (mRNA and protein) in the caput and cauda epididymis, an effect reversed by testosterone replacement. Altogether, our data suggested that EPPIN expression in rats is more widespread than in humans and mice, and is androgen-dependent in the epididymis. This species could be used as an experimental model to further study EPPIN's role in male fertility.

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Effects of carbachol on rat Sertoli cell proliferation and muscarinic acetylcholine receptors regulation: an in vitro study

Cited by 20 publications

References 73 publications

Locally produced relaxin may affect testis and vas deferens function in rats

Locally produced relaxin may affect testis and vas deferens function in rats

Na+/K+-ATPase α1 isoform mediates ouabain-induced expression of cyclin D1 and proliferation of rat Sertoli cells

Epididymal protease inhibitor (EPPIN) is differentially expressed in the male rat reproductive tract and immunolocalized in maturing spermatozoa

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