Evolutionary analysis of genes coding for Cysteine-RIch Secretory Proteins (CRISPs) in mammals

Cysteine-RIch Secretory Proteins (CRISP) are expressed in the reproductive tract of mammalian males and are involved in fertilization and related processes. Due to their important role in sperm performance and sperm-egg interaction, these genes are likely to be exposed to strong selective pressures, including postcopulatory sexual selection and/or male-female coevolution. We here perform a comparative evolutionary analysis of Crisp genes in mammals. Currently, the nomenclature of CRISP genes is confusing, as a consequence of discrepancies between assignments of orthologs, particularly due to numbering of CRISP genes. This may generate problems when performing comparative evolutionary analyses of mammalian clades and species. To avoid such problems, we first carried out a study of possible orthologous relationships and putative origins of the known CRISP gene sequences. Furthermore, and with the aim to facilitate analyses, we here propose a different nomenclature for CRISP genes (EVAC1-4, "EVolutionarily-analyzed CRISP") to be used in an evolutionary context.Fil: Arévalo, Lena. Consejo Superior de Investigaciones Científicas. Museo Nacional de Ciencias Naturales; España. Universitat Bonn; AlemaniaFil: Brukman, Nicolás Gastón. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Cuasnicu, Patricia Sara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Roldan, Eduardo R. S.. Consejo Superior de Investigaciones Científicas. Museo Nacional de Ciencias Naturales; Españ

Crisp1 and alopecia areata in C3H/HeJ mice

Alopecia areata (AA), a cell mediated autoimmune disease, is the second most common form of hair loss in humans. While the autoimmune disease is responsible for the underlying pathogenesis, the alopecia phenotype is ultimately due to hair shaft fragility and breakage associated with structural deficits. Quantitative trait genetic analyses using the C3H/HeJ mouse AA model identified cysteine-rich secretory protein 1 (Crisp1), a hair shaft structural protein, as a candidate gene within the major AA locus. Crisp1 transcripts in the skin at various times during disease development were barely detectable. In situ hybridization identified Crisp1 expression within the medulla of hair shafts from clinically normal strains of mice but not C3H/HeJ mice with AA. Follow-up work with 5-day-old C3H/HeJ mice with normal hair also had essentially no expression of Crisp1. Other non-inflammatory based follicular dystrophy mouse models with similar hair shaft abnormalities also have little or no Crisp1 expression. Shotgun proteomics, used to determine strain difference in hair proteins, confirmed that there was very little CRISP1 within normal C3H/HeJ mouse hair in comparison to 11 other strains. However, mutant mice with hair medulla defects also had undetectable levels of CRISP1 in their hair. Crisp1 null mice had normal skin, hair follicles, and hair shafts indicating that the lack of the CRISP1 protein does not translate directly into defects in the hair shaft or hair follicle. These results suggest that CRISP1 may be an important structural component of mouse hair and that its strain-specific dysregulation may indicate a predisposition to hair shaft disease such as AA.Fil: Sundberg, John P.. Vanderbilt University; Estados Unidos. The Jackson Laboratory; Estados UnidosFil: Awgulewitsch, Alejandro. Medical University of South Carolina; Estados UnidosFil: Pruett, Nathan D.. Medical University Of South Carolina; Estados UnidosFil: Potter, Cristhoper S.. The Jackson Laboratory; Estados UnidosFil: Silva, Kathleen A.. The Jackson Laboratory; Estados UnidosFil: Stearns, Timothy M.. The Jackson Laboratory; Estados UnidosFil: Sundberg, Beth A.. The Jackson Laboratory; Estados UnidosFil: Weigel Muñoz, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Cuasnicu, Patricia Sara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: King, Lloyd E. Jr. Vanderbilt University; Estados UnidosFil: Rice, Robert H.. University of California. Department of Nutrition and Department of Environmental Toxicology; Estados Unido

Fertilization induces a transient exposure of phosphatidylserine in mouse eggs

Phosphatidylserine (PS) is normally localized to the inner leaflet of the plasma membrane and the requirement of PS translocation to the outer leaflet in cellular processes other than apoptosis has been demonstrated recently. In this work we investigated the occurrence of PS mobilization in mouse eggs, which express flippase Atp8a1 and scramblases Plscr1 and 3, as determined by RT-PCR; these enzyme are responsible for PS distribution in cell membranes. We find a dramatic increase in binding of flouresceinated-Annexin-V, which specifically binds to PS, following fertilization or parthenogenetic activation induced by SrCl2 treatment. This increase was not observed when eggs were first treated with BAPTA-AM, indicating that an increase in intracellular Ca2+ concentration was required for PS exposure. Fluorescence was observed over the entire egg surface with the exception of the regions overlying the meiotic spindle and sperm entry site. PS exposure was also observed in activated eggs obtained from CaMKIIγ null females, which are unable to exit metaphase II arrest despite displaying Ca2+ spikes. In contrast, PS exposure was not observed in TPEN-activated eggs, which exit metaphase II arrest in the absence of Ca2+ release. PS exposure was also observed when eggs were activated with ethanol but not with a Ca2+ ionophore, suggesting that the Ca2+ source and concentration are relevant for PS exposure. Last, treatment with cytochalasin D, which disrupts microfilaments, or jasplakinolide, which stabilizes microfilaments, prior to egg activation showed that PS externalization is an actin-dependent process. Thus, the Ca2+ rise during egg activation results in a transient exposure of PS in fertilized eggs that is not associated with apoptosis.Fil: Curia, Claudio Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Ernesto, Juan Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Stein, Paula. University of Pennsylvania; Estados UnidosFil: Busso, Dolores. Pontificia Universidad Católica de Chile; ChileFil: Schultz, Richard. University of Pennsylvania; Estados UnidosFil: Cuasnicu, Patricia Sara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Cohen, Debora Juana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentin

Standards in semen examination:publishing reproducible and reliable data based on high-quality methodology

Biomedical science is rapidly developing in terms of more transparency, openness and reproducibility of scientific publications. This is even more important for all studies that are based on results from basic semen examination. Recently two concordant documents have been published: the 6th edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen, and the International Standard ISO 23162:2021. With these tools, we propose that authors should be instructed to follow these laboratory methods in order to publish studies in peer-reviewed journals, preferable by using a checklist as suggested in an Appendix to this article.Peer reviewe

Introductory Overview of the Epididymis: Present Progress, Future Directions (Proceedings of the Fifth International Conference on the Epididymis)

Universidade Federal de São Paulo, Escola Paulista Med UNIFESP, Dept Pharmacol, Sect Expt Endocrinol, São Paulo, BrazilInst Biol & Med Expt IBYME CONICET, Buenos Aires, DF, ArgentinaUniversidade Federal de São Paulo, Escola Paulista Med UNIFESP, Dept Pharmacol, Sect Expt Endocrinol, São Paulo, BrazilWeb of Scienc

Cysteine-Rich Secretory Proteins (CRISP) and their role in mammalian fertilization

Epididymal protein CRISPI is a member of the CRISP (Cysteine-RIch Secretory proteins) family and is involved in sperm-egg fusion through its interaction with complementary sites on the egg surface. Results from our laboratory have shown that this binding ability resides in a 12-amino-acid region corresponding to a highly conserved motif of the CRISP family, named Signature 2 (S2). In addition to this, our results revealed that CRISP1 could also be involved in the previous step of sperm binding to the zona pellucida, identifying a novel role for this protein in fertilization. As another approach to elucidate the participation of CRISP1 in fertilization, a mouse line containing a targeted disruption of CRISP1 was generated. Although CRISP1-deficient mice exhibited normal fertility, CRISP1-defficient sperm presented a decreased level of protein tyrosine phosphorylation during capacitation, and an impaired ability to fertilize both zona-intact and zona-free eggs in vitro, confirming the proposed roles for the protein in fertilization. Evidence obtained in our laboratory indicated that testicular CRISP2 would also be involved in sperm-egg fusion. Competition assays between CRISP1 and CRISP2, as well as the comparison of their corresponding S2 regions, suggest that both proteins bind to common complementary sites in the egg. Together, these results suggest a functional cooperation between CRISP1 and CRISP2 to ensure the success of fertilization

A single post-ovulatory dose of ulipristal acetate impairs post-fertilization events in mice

Ulipristal acetate (UPA) is a selective progesterone receptor modulator used for emergency contraception that has proven to be highly effective in preventing pregnancy when taken up to 120 h after unprotected sexual intercourse. Even though it may act mainly by delaying or inhibiting ovulation, additional effects of UPA on post-fertilization events cannot be excluded. Therefore, the aim of this study was to determine whether a single post-ovulatory dose of UPA could prevent pregnancy using the mouse as a pre-clinical model. Mated females received a single dose of UPA (40 mg/kg) on Day E1.5 or E2.5 (E0.5: copulatory plug detection) and post-fertilization events were evaluated. Our studies revealed that UPA administration produced a significant decrease in the number of conceptuses compared to control. Moreover, UPA-treated females exhibited a lower number of early implantation sites on Day E5.5, despite normal in vivo embryo development and transport to the uterus at E3.5. Administration of UPA produced histological and functional alterations in the uterine horns, i.e., a dyssynchronous growth between endometrial glands and stroma, with non-physiological combination of both fractions compared to controls, and a completely impaired ability to respond to an artificial decidualization stimulus. Altogether, our results show that the administration of a single post-ovulatory dose of UPA impairs mouse pregnancy probably due to an effect on embryo-uterine interaction, supporting additional effects of the drug on post-fertilization events. Although these studies cannot be performed with human samples, our results with the mouse model provide new insights into the mechanism of action of UPA as an emergency contraception method254257264FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2015/20504-9This study was partially supported by grants BIO 486 (1480/2016) from the University of Rosario to M.J.M., 2015/20504-9 from the São Paulo Research Foundation (FAPESP) to L.B., PICT 2015-0471 to P.S.C. and PICT 2016-1057 to D.J.C. from the National Agency of Scientific and Technological Promotion (ANPCyT), Argentin

Impaired male fertility and abnormal epididymal epithelium differentiation in mice lacking CRISP1 and CRISP4

Abstract Epididymal Cysteine Rich Secretory Proteins 1 and 4 (CRISP1 and CRISP4) associate with sperm during maturation and play different roles in fertilization. However, males lacking each of these molecules individually are fertile, suggesting compensatory mechanisms between these homologous proteins. Based on this, in the present work, we generated double CRISP1/CRISP4 knockout (DKO) mice and examined their reproductive phenotype. Our data showed that the simultaneous lack of the two epididymal proteins results in clear fertility defects. Interestingly, whereas most of the animals exhibited specific sperm fertilizing ability defects supportive of the role of CRISP proteins in fertilization, one third of the males showed an unexpected epididymo-orchitis phenotype with altered levels of inflammatory molecules and non-viable sperm in the epididymis. Further analysis showed that DKO mice exhibited an immature epididymal epithelium and abnormal luminal pH, supporting these defects as likely responsible for the different phenotypes observed. These observations reveal that CRISP proteins are relevant for epididymal epithelium differentiation and male fertility, contributing to a better understanding of the fine-tuning mechanisms underlying sperm maturation and immunotolerance in the epididymis with clear implications for human epididymal physiology and pathology

RT-PCR analysis of flippases and scramblases expression in mouse eggs.

<p>Total mouse egg RNA was subjected to RT-PCR using specific primers for each tested enzyme. Products were separated on 2% agarose gels and stained with ethidium bromide (lane <b>C</b>). As positive controls, total RNA from different tissues were used for each tested enzyme (lane <b>A</b>): epididymis for <i>Atp8a1</i>, testis for <i>ATP8a2</i>, liver for <i>Plscr1</i>, kidney for <i>Plscr2</i>, lung for <i>Plscr3</i> and <i>Plscr4</i>, and cumulus cells for <i>Cd52</i>. As negative controls, water was used as template for the reverse transcription (lane <b>B</b>) and during the amplification reaction (lane <b>D</b>). The expression of <i>Cd52</i> was evaluated to control contamination with cumulus cells. Results are representative of 3 independent egg RNA isolations.</p