Comparative analysis of apoptotic pathways in rat, mouse, and hamster spermatozoa

Cisternas, Pablo; Moreno, Ricardo D.

doi:10.1002/mrd.20561

Cited by 21 publications

(13 citation statements)

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“…Protein was extracted by homogenizing isolated cauda spermatozoa as described previously (Cisternas and Moreno 2006). In brief, isolated cauda spermatozoa in Buffer A (1% Triton X-100, 1 M NaCl, 1 mM EDTA, 10 mg/ml phenylmethane sulfonyl-fluoride, 20 mM TRISHCl pH 7.0, plus protease inhibitors leupeptin, aprotinin, and E-64) were centrifuged for 10 min at 9300g at 4°C.…”

Section: Protein Extraction and Western Blot Analysismentioning

confidence: 99%

Differential localization of α’ and β subunits of protein kinase CK2 during rat spermatogenesis

et al. 2009

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Protein kinase CK2 is a serine/threonine kinase expressed in organisms from yeast to human and is composed of a catalytic subunit (alpha or alpha') and a regulatory subunit (beta) forming a holoenzyme with the possible subunit combinations alpha(2)beta(2), alpha'(2)beta(2), or alphaalpha'beta(2). This kinase has been shown to be involved in embryonic development and gametogenesis. We have studied the expression of the CK2alpha' and CK2beta subunits during the first wave of spermatogenesis and in adult testis in the rat. Western blot analyses have demonstrated that both CK2alpha' and CK2beta are expressed in testes from birth to adulthood. A more detailed study of the protein localization of CK2alpha' and CK2beta by immunohistochemistry suggests that CK2alpha' and CK2beta are localized in the nuclei of Sertoli cells in 5-day-old rats, whereas they appear to have a cytoplasmic localization in older animals. In adult testes, CK2alpha' and CK2beta subunits are present in spermatocytes. Both subunits exhibit a similar expression pattern with the highest level in spermatocytes at stages VIII-XIV. Interestingly, CK2beta is highly expressed in spermatogonia, whereas CK2alpha' is barely detectable. Mature epididymal spermatozoa express CK2alpha' in the acrosome and CK2beta in the flagellum. This new evidence therefore indicates that protein kinase CK2 has a possible role at various stages during mammalian spermatogenesis, a process that involves proliferation, meiosis, apoptosis, and differentiation. CK2 might thus emerge as a new pivotal control enzyme at various levels in mammalian spermatogenesis.

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Section: Protein Extraction and Western Blot Analysismentioning

confidence: 99%

Differential localization of α’ and β subunits of protein kinase CK2 during rat spermatogenesis

et al. 2009

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“…Protein extraction was done by homogenizing isolated seminiferous tubules in a buffer containing 1 M NaCl, 1 mM EDTA, 10 mg/ml PMSF, 1% Triton X-100, 20 mM Tris-HCl pH 7.4, and centrifuging for 10 min at 9,300g (Cisternas and Moreno, 2006). The samples were run on a 15% or 20% polyacrylamide gel (SDS-PAGE) under reducing and denaturing conditions, and then transferred to nitrocellulose at 350 mA for 1.5 hr.…”

Section: Protein Extraction and Western Blotmentioning

confidence: 99%

Relevance of caspase activity during apoptosis in pubertal rat spermatogenesis

Codelia

Cisternas

Moreno

2007

Molecular Reproduction Devel

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Caspases are a family of cysteine-proteases, activated upon several different stimuli, which execute apoptosis in many cell death models. Previous work of our group has shown rats have the highest rate of apoptosis during the first wave of spermatogenesis (between 20 and 25 days after birth), as evaluated by TUNEL and caspase activity. However, the hierarchical order of caspase activation and the relevance of each caspase during germ cell apoptosis are not clear. Thus, the goal of this work is to take a pharmacological approach to dissect the apoptosis pathway of caspase activation. Results showed that intratesticular injection of a caspase-8 inhibitor (z-IETD-fmk), or a pan-caspase inhibitor (z-VAD- fmk), significantly decreased the cleavage of p115 and PARP, two endogenous substrates of caspases, in 22-day-old rats. Additionally, these inhibitors promoted a significant reduction in the number of apoptotic germ cells. On the other hand, intratesticular injection of two different inhibitors of the intrinsic pathway (z-LEHD-fmk and minocycline) did not have any effect upon caspase substrates cleavage (p115 and PARP) or the number of apoptotic germ cells. Therefore, we conclude that the extrinsic pathway of apoptosis plays an important role in physiological germ cell apoptosis during the first round of spermatogenesis in the rat.

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“…The acrosome reaction was evaluated by the Coomassie blue dye technique (Bendahmane et al, ; Cisternas and Moreno, ). In brief, fixed spermatozoa were washed three times with 100 mM ammonium acetate by centrifugation at 3,000 rpm, layered into microscope glass slides and dried at 30°C.…”

Section: Methodsmentioning

confidence: 99%

Involvement of a P2X7 Receptor in the Acrosome Reaction Induced by ATP in Rat Spermatozoa

Torres-Fuentes

Ríos

Moreno

2015

Journal Cellular Physiology

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The acrosome reaction (AR) is the exocytosis of the acrosomal vesicle in response to different physiological and non-physiological stimuli. Particularly in mammals, the AR is needed for sperm to fuse with the oocyte plasma membrane, and it occurs only in capacitated sperm. Previous evidence in the literature indicates that extracellular ATP induces the AR in capacitated human and bovine spermatozoa, but its receptor has not yet been identified. The aim of this work was to define a putative ATP receptor in rat spermatozoa using pharmacological and biochemical approaches. We found that ATP induced the AR only in capacitated rat spermatozoa, which was inhibited in the presence of two general inhibitors of ATP receptors (P2 receptors), Suramin, and oxidized ATP (oATP), and one inhibitor of P2X receptor (pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid [PPADS]). In addition, the AR induced by ATP in capacitated rat spermatozoa was inhibited by brilliant blue-G (BB-G) and 17-β-oestradiol, two blockers of P2X7 receptors. Moreover, the ATP analog 2'(3')-O-(4-benzoylbenzoyl) ATP (BzATP) was almost 500 times more potent than ATP to induce the AR, which agrees with the pharmacology of a P2X7 receptor. Here, we show the presence of P2X7 receptor by Western blot and its localization in the tail and acrosome by indirect immunofluorescence. Finally, we quantify the presence of ATP in the rat oviduct during the estrous cycle. We found that the ATP concentration within the lumen of the oviduct is similar to those required to induce acrosome reaction, which agree with its role during in vivo fertilization. Therefore, our results strongly suggest that ATP induces the AR in capacitated rat spermatozoa through a P2X7 receptor, which may be functional during in vivo fertilization.

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Comparative analysis of apoptotic pathways in rat, mouse, and hamster spermatozoa

Cited by 21 publications

References 50 publications

Differential localization of α’ and β subunits of protein kinase CK2 during rat spermatogenesis

Differential localization of α’ and β subunits of protein kinase CK2 during rat spermatogenesis

Relevance of caspase activity during apoptosis in pubertal rat spermatogenesis

Involvement of a P2X7 Receptor in the Acrosome Reaction Induced by ATP in Rat Spermatozoa

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