Browsing by Author "Moreno, Ricardo D."

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    A decrease of docosahexaenoic acid in testes of mice fed a high-fat diet is associated with impaired sperm acrosome reaction and fertility
    (2021) Bunay, Julio; Gallardo, Luz-Maria; Luis Torres-Fuentes, Jorge; Veronica Aguirre-Arias, M.; Orellana, Renan; Sepulveda, Nestor; Moreno, Ricardo D.
    Obesity is a major worldwide health problem that is related to most chronic diseases, including male infertility. Owing to its wide impact on health, mechanisms underlying obesity-related infertility remain unknown. In this study, we report that mice fed a high-fat diet (HFD) for over 2 months showed reduced fertility rates and increased germ cell apoptosis, seminiferous tubule degeneration, and decreased intratesticular estradiol (E2) and E2-to-testosterone ratio. Interestingly, we also detected a decrease in testicular fatty acid levels, behenic acid (C22:0), and docosahexaenoic acid (DHA, 22:6n-3), which may be related to the production of dysfunctional spermatozoa. Overall, we did not detect any changes in the frequency of seminiferous tubule stages, sperm count, or rate of in vitro capacitation. However, there was an increase in spontaneous and progesterone-induced acrosomal exocytosis (acrosome reaction) in spermatozoa from HFD-fed mice. These data suggest that a decrease in E2 and fatty acid levels influences spermatogenesis and some steps of acrosome biogenesis that will have consequences for fertilization. Thus, our results add new evidence about the adverse effect of obesity in male reproduction and suggest that the acrosomal reaction can also be affected under this condition.
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    Antigen retrieval by citrate solution improves western blot signal
    (2019) Patino-Garcia, Daniel; Rocha-Perez, Nadia; Moreno, Ricardo D.; Orellana, Renan
    In the present work, we describe and evaluate an additional step to the standard western blot protocol to increase signal strength after revealing. Weak or absence of signal is a common issue in western blot protocol leading to unexpected results. In our Antigen Retrieval for Western Blot Method (ARWB method), after transfer, the membrane was incubated in a citrate buffer following normal antigen retrieval procedure used for immunohistochemistry. Later, standard protocol was performed in order to reveal and compare with unexposed membranes to this antigen retrieval step. Signal in bands obtained by the modified protocol resulted significantly higher (in all 13 antibodies analyzed) compared to standard protocol. Some bands were only visible after citrate incubation. This method is a simple and economical way to improve results in western blot analysis.
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    Daily exposure to phthalates and alkylphenols alters miR biogenesis and expression in mice ovaries
    (2020) Patino-Garcia, Daniel; Cruz-Fernandes, Leonor; Bunay, Julio; Orellana, Renan; Moreno, Ricardo D.
    Reproductive hormone imbalance in infertile women is correlated to high levels of phthalates and alkylphenols, which are among endocrine-disrupting chemicals (EDCs). Previous studies have shown that they interfere with gene expression by deregulating levels of microRNAs (miRs), small non-coding RNAs targeting mRNAs encoding enzymes in the hormone biosynthesis pathway. However, this effect depends on the target organ, dose and whether or not they are alone or in mixtures. Our goal was to study whether the biosynthesis, and a specific group of miRs targeting mRNAs encoding enzymes in steroid hormone biosynthesis, are deregulated in the ovaries of female mice chronically exposed to a mixture of three phthalates (DEHP+DBP+BBP) and two alkylphenols (NP+OP) at a human environmentally relevant dose. We performed qPCR and Western blot assays along with a bioinformatics approach and found that this mixture modified the biogenesis machinery of miRs, inducing an increase in the mRNA levels of Drosha and Dicerl and DROSHA protein levels. In addition, we found changes in the precursor and mature forms of miR-96-5p, miR-200b-3p, miR-365-3p, miR-378a-3p and miR-503-5p which target steroidogenic pathway enzymes. Finally, using primary granulosa cell culture, we confirmed that miR-200b-3p targets Cyp19a1, transcript encoding CYP19A1, the enzyme that produces estradiol (E-2). These results indicate that chronic exposure to phthalates and alkylphenols mixture alters the biogenesis of ovary miRs and increases the expression of miRs implicated in the control of steroidal hormone synthesis in female mice, thus contributing to reproductive pathologies.
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    Differential localization of α' and β subunits of protein kinase CK2 during rat spermatogenesis
    (2009) Alvarado-Diaz, Carlos P.; Tapia, Julio C.; Antonelli, Marcelo; Moreno, Ricardo D.
    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 alpha alpha'beta(2). This kinase has been shown to be involved in embryonic development and gametogenesis. We have studied the expression of the CK2 alpha' and CK2 beta subunits during the first wave of spermatogenesis and in adult testis in the rat. Western blot analyses have demonstrated that both CK2 alpha' and CK2 beta are expressed in testes from birth to adulthood. A more detailed study of the protein localization of CK2 alpha' and CK2 beta by immunohistochemistry suggests that CK2 alpha' and CK2 beta 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, CK2 alpha' and CK2 beta subunits are present in spermatocytes. Both subunits exhibit a similar expression pattern with the highest level in spermatocytes at stages VIII-XIV. Interestingly, CK2 beta is highly expressed in spermatogonia, whereas CK2 alpha' is barely detectable. Mature epididymal spermatozoa express CK2 alpha' in the acrosome and CK2 beta 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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    Embryo Buoyancy and Cell Death Gene Expression During Embryogenesis of Yellow-Tail Kingfish Seriola lalandi
    (2021) Palomino, Jaime; Gomez, Camila; Otarola, Maria Teresa; Dettleff, Phillip; Patino-Garcia, Daniel; Orellana, Renan; Moreno, Ricardo D.
    In pelagic fish, embryo buoyancy is a noteworthy aspect of the reproductive strategy, and is associated with overall quality, survival, and further developmental success. In captivity, the loss of buoyancy of early embryos correlates with high mortality that might be related to massive cell death. Therefore, the aim of this study was to evaluate under captivity conditions the expression of genes related to the apoptosis process during the early embryonic development of the pelagic fish Seriola lalandi, and its relationship to the buoyancy of embryos. The relative expression of bcl2, bax-like, casp9, casp8, and casp3 was evaluated by RT-qPCR and FasL/Fas protein levels by western blot in five development stages of embryos sorted as floating or low-floating. All genes examined were expressed in both floating and low-floating embryos up to 24 h of development. Expression of the pro-apoptotic factors bax, casp9, casp8, and casp3 was higher in low-floating as compared with floating embryos in a developmental stage-specific manner. In contrast, there was no difference in expression of bcl2 between floating and low-floating embryos. Fas protein was detected as a single band in floating embryos without changes in expression throughout development; however, in low-floating embryos, three higher intensity reactive bands were detected in the 24-h embryos. Interestingly, FasL was only detected at 24-h in floating embryos, whereas in low-floating samples this ligand was present at all stages, with a sharp increase as development progressed. Cell death, as evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, was highly increased in low-floating embryos as compared to floating embryos throughout all developmental stages, with the highest levels observed during the gastrula stage and at 24 h. The results of this study suggest that an increase in cell death, probably associated with the intrinsic and extrinsic apoptosis pathways, is present in low-floating embryos that might explain their lower developmental potential under captivity conditions.
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    Etoposide induces apoptosis and upregulation of TACE/ADAM17 and ADAM10 in an in vitro male germ cell line model
    (2011) Lizama, Carlos; Ludwig, Andreas; Moreno, Ricardo D.
    Etoposide is a widely used anticancer drug in the treatment of different tumors. Etoposide is known to activate a wide range of intracellular signals, which may in turn induce cellular responses other than apoptosis. ADAM10 and TACE/ADAM17 belong to a family of transmembrane extracellular metalloproteinases involved in paracrine/juxtacrine regulation of many signaling pathways. The aim of this work was to evaluate if etoposide induces upregulation of ADAM10 or TACE/ADAM17 in two cell lines (GC-1 and GC-2) derived from male germ cells. Results showed that etoposide induced apoptosis in a dose-response manner in both GC-1 and GC-2 cells. Apoptosis started to increase 6 h after etoposide addition in GC-2 cells, whereas the same was observed 18 h after addition to the GC-1 cells. Protein and mRNA levels of ADAM10 and TACE/ADAM17 increased 18.h after etoposide was removed from the GC-1 cells. In GC-2 cells, the protein levels of both proteins increased 12 h after etoposide was removed. ADAM] 0 mRNA increased after 3 h and then steadily decreased up to 12 h after removal, whereas TACE/ADAM17 mRNA decreased after etoposide removal. Finally, apoptosis was prevented in GC-1 and GC-2 cells by the addition of pharmacological inhibitors of ADAM10 and TACE/ADAM17 to the culture medium of etoposide-treated cells. Our results show for the first time that etoposide upregulates ADAM10 and TACE/ADAM17 mRNA and protein levels. In addition, we also show that ADAM] 0 and TACE/ADAM17 have a role in etoposide-induced apoptosis. (c) 2010 Elsevier B.V. All rights reserved.
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    Histomorphological changes between short and long days in ovary guanacos (Lama guanicoe)
    (2023) Correa E, Lina Maria; Moreno, Ricardo D.; Luis Riveros, Jose
    This is the first morpho-histological comparison of guanaco ovaries between reproductive (long-days) and non-reproductive (short-days) seasons, and oestrogen receptor-alpha (ER alpha) and beta (ER beta) detection. Different stages of follicle development were found in the cortical area, but no corpus luteum was detected. The size and frequency of antral follicles and large atretic follicles were higher in long-day ovaries than short-days, consistent with ovarian activity in this season. Differential expression of ER alpha and ER beta was observed in follicles at different stages of development between short and long days. These data reveal histological and molecular differences between reproductive and non-reproductive seasons of guanaco ovaries.
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    Human globozoospermia-related genes and their role in acrosome biogenesis
    (2023) Moreno, Ricardo D.
    The mammalian acrosome is a secretory vesicle attached to the sperm nucleus whose fusion with the overlying plasma membrane is required to achieve fertilization. Acrosome biogenesis starts during meiosis, but it lasts through the entire process of haploid cell differentiation (spermiogenesis). Acrosome biogenesis is a stepwise process that involves membrane traffic from the Golgi apparatus, but it also seems that the lysosome/endosome system participates in this process. Defective sperm head morphology is accompanied by defective acrosome shape and function, and patients with these characteristics are infertile or subfertile. The most extreme case of acrosome biogenesis failure is globozoospermia syndrome, which is primarily characterized by the presence of round-headed spermatozoa without acrosomes with cytoskeleton defects around the nucleus and infertility. Several genes participating in acrosome biogenesis have been uncovered using genetic deletions in mice, but only a few of them have been found to be deleted or modified in patients with globozoospermia. Understanding acrosome biogenesis is crucial to uncovering the molecular basis of male infertility and developing new diagnostic tools and assisted reproductive technologies that may help infertile patients through more effective treatment techniques.This article is categorized under:Reproductive System Diseases > Environmental FactorsInfectious Diseases > Stem Cells and DevelopmentReproductive System Diseases > Molecular and Cellular Physiology
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    Hypothalamic-pituitary-gonadal axis response to photoperiod changes in female guanacos (Lama guanicoe)
    (2024) Correa, Lina Maria; Moreno, Ricardo D.; Riveros, Jos e Luis
    The guanaco, a wild South American camelid, is renowned for its remarkable resilience to extreme conditions. Despite this, little is known about how reproductive hormones in female camelids are influenced during their seasonal breeding period, which occurs during long photoperiod. To explore this, the study investigated the response of the hypothalamic-pituitary-gonadal axis in female guanacos during short days (10L:14D; July) and long days (16L:8D; December) in the Mediterranean ecosystem (33(degrees)38 ' 28 '' S, 70(degrees)34 ' 27 '' W). Blood samples from 14 adult animals were collected, and measurements of melatonin, 178-estradiol, FSH, and LH concentrations were taken. The results showed that melatonin concentration was lower (P < 0.05) during long days than short days, whereas 178-estradiol, FSH, and LH concentrations were higher (P < 0.05) during long days compared to short days. Furthermore, the study detected the expression of the melatonin receptor 1A and kisspeptin in the hypothalamus and pituitary, suggesting that the pineal gland of female guanacos is sensitive to seasonal changes in day length. These findings also indicate a seasonal variation in the concentration of reproductive hormones, likely linked to the distinct modulation of the hypothalamic-pituitary-gonadal axis of female guanacos during short and long days.
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    Nonylphenol releases arachidonic acid in rat Sertoli cells via activation of PKA and PLA2
    (2024) Valtierra, Florencia X. Santiago; Urriola-Munoz, Paulina; Godoy-Sepulveda, Rodrigo; Moreno, Ricardo D.; Reyes, Juan G.; Valles, Ana S.; Oresti, Gerardo M.
    Nonylphenol (NP), an endocrine-disrupting chemical, is an environmental contaminant, and many notorious effects on male fertility have been reported in animal models and wild-type species. Here, we evaluated the effects of NP in follicle-stimulating hormone (FSH) signal transduction pathways and lipid metabolism using an in vitro model of rat Sertoli cell (SC) primary culture. Results show that an acute (1 h) SC exposure to NP (10 mu M) increased the intra- and extra-cellular concentrations of free fatty acids (FFAs), mainly arachidonic acid (20:4n-6). Phosphatidylinositol seemed to be the major phospholipid source of this 20:4n-6 release by activation of the protein kinase A (PKA)/cytoplasmic phospholipase A2 (cPLA2) pathway. NP also increased diacylglycerols (DAG) levels and the expression (mRNA) of cyclooxygenase 2 (Cox2) and prostaglandin E2 (PGE2) levels. It is noteworthy that accumulation of lipid droplets took place after 24 h NP exposition, which was prevented by both a PKA inhibitor and a PLA2 inhibitor. Like FSH, NP triggers the release of 20:4n-6, which is a substrate for PGE2 synthesis via PKA/PLA2 activation. In addition, NP induces the formation of DAG, which could be required as a cofactor of the PKC-mediated activation of the COX2 inflammatory pathway. Our findings suggest that NP alters lipid homeostasis in SCs by inducing the activation of pro-inflammatory pathways that may trigger adverse effects in testis physiology over time. Concomitantly, the SC enhances the acylation of surplus FFAs (including 20:4n-6) in neutral lipids as a protective mechanism to shield itself from lipotoxicity and pro-inflammatory signals.
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    Proacrosin/acrosin quantification as an indicator of acrosomal integrity in fresh and frozen dog spermatozoa
    (ELSEVIER, 2006) Cortes, Constanza J.; Codelia, Veronica A.; Manosalva, Iris; de Lange, Johanna; De los Reyes, Monica; Moreno, Ricardo D.
    The scope of the present study was to evaluate the presence and activation of proacrosin/acrosin as a tool to determine the acrosomal status of fresh and frozen/thawed dog spermatozoa. Monoclonal antibody C5F11, directed against human acrosin, cross-reacted with dog spermatozoa and labeled the acrosome of both fresh and frozen/thawed dog spermatozoa. Frozen/thawed spermatozoa had a lesser proportion of labeled spermatozoa than fresh spermatozoa (P < 0.05). When live spermatozoa were labeled with soybean trypsin inhibitor conjugated with Alexa 488 (SBTI-Alexa 488), the proportion of acrosome-labeled fresh spermatozoa was less than frozen/thawed spermatozoa (P < 0.05). By using Western blots and enzymatic activity, frozen/thawed spermatozoa had a greater proportion of active acrosin than fresh spermatozoa. In addition, beta 1,4-galactosyl-transferase (GaIT), a plasma membrane bound protein, remained attached to frozen/thawed spermatozoa. Proacrosin is activated during freezing/thawing of dog spermatozoa, and that proacrosin/acrosin may be a good indicator of acrosomal integrity of frozen/thawed spermatozoa. (c) 2005 Elsevier B.V. All rights reserved.