Effect of an altered hormonal environment by blood plasma collected after adrenocorticotropic administration on embryo development and gene expression in porcine embryos

Early embryonic development may be affected by adrenal hyperactivity in stressful situations which may lead to endocrine changes in the embryo environment. A sensitive period in porcine embryo development is the 4-cell stage when the embryo genome activation occurs. A mixed in vivo-in vitro system was implemented to test whether an altered milieu around this stage could affect embryo development and blastocyst quality in the porcine model. After in vitro maturation and fertilisation, presumptive zygotes were exposed for 24 h to plasma collected after ovulation from adrenocorticotropic hormone (ACTH) treated, non-ACTH-treated sows; and, medium without plasma, supplemented with bovine serum albumin. Subsequently, embryo development and differences in gene expression were tested among treatments. Cleavage and blastocyst rates did not differ between treatments. Blastocyst quality by morphology assessment was similar when all the resulting blastocysts were included in the analysis. However, when only expanded blastocysts (and onwards) were included in the analysis, the blastocysts from the non-ACTH plasma group showed better quality score. Blastocyst quality by morphological assessment was not mirrored by the transcription levels of various important genes for embryo development whose gene expression profile did not significantly differ among groups. It is likely that the effect of the altered environment provided by plasma from ACTH-treated sows was too short to affect embryo development. Therefore, a brief exposure to an altered endocrine environment may not have harmful consequences for the embryo once fertilisation occurs. (c) 2021 The Authors. Published by Elsevier Inc

The proximal promoter region of mTert is sufficient to regulate telomerase activity in ES cells and transgenic animals

BACKGROUND: The reverse transcriptase of telomerase (Tert) controls telomerase activity maintaining the end of linear chromosomes in eukaryotic cells. Telomerase function is highly active in undifferentiated multipotent stem cells, decreases with cell differentiation and is generally absent from most somatic cells in the adult. Its absence is responsible of telomeres shortening in such somatic cells. Using an in vivo transgenic model and an in vitro culture differentiation of adult stem cells, we examined the elements of the mouse Tert (mTert) promoter that control telomerase activity. RESULTS: Three constructs comprising 1, 2 or 5 kb of the mTert promoter sequence coupled to the coding sequence of the green fluorescent protein (EGFP) were electroporated into embryonic stem (ES) cells. Transformed ES cells were able to mimic the expected mTert expression, which was associated to green fluorescence. One and 5 kb promoter produced the higher expression of EGFP, on ES cells. When ES cells were allowed to differentiate to embryoid bodies and to other cell types, they lost gradually the expression of mTert-EGFP as consequence of differentiation. No differences were found among the three constructs analyzed. We then generated transgenic mice with the three constructs. Expression of the reporter gene was monitored by reverse transcription-PCR analysis and EGFP visualization. The mRNA expression of the three constructs was lower than the endogenous mTert, but mimicked the endogenous mTert transcription pattern; however, no fluorescent expression of EGFP was detected in adult tissues. EGFP expression of the three constructs was visualized at the blastocysts stage and in new ES cells generated from them; in the germinal ring of E13 dpc foetuses; in ES-like colonies and in germinal stem cells generated from neonatal and adult testis cells; and in neuroesferes generated from E14 dpc foetuses' brain cells. CONCLUSION: The 1 kb promoter upstream of the initiating ATG codon of mTert contains all the regulatory elements to control telomerase expression in ES cells during in vitro loss of pluripotency. The transgenic mouse lines generated represent an appropriate system to analyze the expression of mouse Tert gene under physiological condition and during establishment of stem cell lines generated from embryonic or adult tissues

An ancient testis-specific IQ motif-containing H gene regulates specific transcript isoform expression during spermatogenesis

Spermatogenic cells express more alternatively spliced RNAs than most whole tissues; however, the regulation of these events remains unclear. Here, we have characterized the function of a testis-specific IQ motif-containing H gene (Iqch) using a mutant mouse model. We found that Iqch is essential for the specific expression of RNA isoforms during spermatogenesis. Using immunohistochemistry of the testis, we noted that Iqch was expressed mainly in the nucleus of spermatocyte and spermatid, where IQCH appeared juxtaposed with SRRM2 and ERSP1 in the nuclear speckles, suggesting that interactions among these proteins regulate alternative splicing (AS). Using RNA-seq, we found that mutant Iqch produces alterations in gene expression, including the clear downregulation of testis-specific lncRNAs and protein-coding genes at the spermatid stage, and AS modifications – principally increased intron retention – resulting in complete male infertility. Interestingly, we identified previously unreported spliced transcripts in the wild-type testis, while mutant Iqch modified the expression and use of hundreds of RNA isoforms, favouring the expression of the canonical form. This suggests that Iqch is part of a splicing control mechanism, which is essential in germ cell biologyThis study was funded by the Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación (PID2021-122507OB-I00 and PID2020-117491GB-I00) and the European Union NextGenerationEU/PRTR. P.N.-L. was supported by a pre-doctoral fellowship from the Ministerio de Ciencia e Innovación (PRE2019-088813) and M.L. was supported by a Juan de la Cierva postdoctoral contract (FJC2019-040385-I) from the Ministerio de Ciencia e Innovación. Open access funding provided by Consejo Superior de Investigaciones Cientıficas. Deposited in PMC for immediate releas

Sex-Dimorphic Behavioral Alterations and Altered Neurogenesis in U12 Intron Splicing-Defective Zrsr1 Mutant Mice

Mutant mice with respect to the splicing factor Zrsr1 present altered spermatogenesis and infertility. To investigate whether Zrsr1 is involved in the homeostatic control that the hypothalamus exerts over reproductive functions, we first analyzed both differential gene and isoform expression and alternative splicing alterations in Zrsr1 mutant (Zrsr1mu) hypothalamus; second, we analyzed the spontaneous and social behavior of Zrsr1mu mice; and third, we analyzed adult cell proliferation and survival in the Zrsr1mu hypothalamus. The Zrsr1mu hypothalamus showed altered expression of genes and isoforms related to the glutathione metabolic process, synaptonemal complex assembly, mRNA transport, and altered splicing events involving the enrichment of U12-type intron retention (IR). Furthermore, increased IR in U12-containing genes related with the prolactin, progesterone, and gonadotropin-releasing hormone (GnRH) reproductive signaling pathway was observed. This was associated with a hyperactive phenotype in both males and females, with an anxious phenotype in females, and with increased social interaction in males, instead of the classical aggressive behavior. In addition, Zrsr1mu females but not males exhibited reduced cell proliferation in both the hypothalamus and the subventricular zone. Overall, these results suggest that Zrsr1 expression and function are relevant to organization of the hypothalamic cell network controlling behavior

Non-coding RNAs derived from the foot-and-mouth disease virus genome trigger broad antiviral activity against coronaviruses

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of a potentially severe respiratory disease, the coronavirus disease 2019 (COVID-19), an ongoing pandemic with limited therapeutic options. Here, we assessed the anti-coronavirus activity of synthetic RNAs mimicking specific domains in the non-coding regions of the foot-and-mouth disease virus (FMDV) genome (ncRNAs). These molecules are known to exert broad-spectrum antiviral activity in cell culture, mice and pigs effectively triggering the host innate immune response. The ncRNAs showed potent antiviral activity against SARS-CoV-2 after transfection in human intestinal Caco-2 and lung epithelium Calu-3 2B4 cells. When the in vivo efficacy of the FMDV ncRNAs was assessed in K18-hACE2 mice, administration of naked ncRNA before intranasal SARS-CoV-2 infection significantly decreased the viral load and the levels of pro-inflammatory cytokines in the lungs compared with untreated infected mice. The ncRNAs were also highly efficacious when assayed against common human HCoV-229E and porcine transmissible gastroenteritis virus (TGEV) in hepatocyte-derived Huh-7 and swine testis ST cells, respectively. These results are a proof of concept of the pan-coronavirus antiviral activity of the FMDV ncRNAs including human and animal divergent coronaviruses and potentially enhance our ability to fight future emerging variants

Zrsr2 and functional U12-dependent spliceosome are necessary for follicular development

SUMMARY ZRSR2 is a splicing factor involved in recognition of 30 -intron splice sites that is frequently mutated in myeloid malignancies and several tumors; however, the role of mutations of Zrsr2 in other tissues has not been analyzed. To explore the bio logical role of ZRSR2,we generated threeZrsr2 mutantmouse lines. AllZrsr2 mutant lines exhibited blood cell anomalies, and in two lines, oogenesis was blocked at the secondary follicle stage. RNA-seq of Zrsr2mu secondary follicles showed aberrations in gene expression and showed altered alternative splicing (AS) events involving enrichment of U12-type intron retention (IR), supporting the functional Zrsr2 action in minor spliceosomes. IR events were preferentially associated with centriole repli cation, protein phosphorylation, and DNA damage checkpoint. Notably, we found alterations in AS events of 50 meiotic genes. These results indicate that ZRSR2 mu tations alter splicing mainly in U12-type introns, which may affect peripheral blood cells, and impede oogenesis and female fertility

Characterization of preovulatory follicular fluid secretome and its effects on equine oocytes during in vitro maturation

12 Pág.In vitro maturation (IVM) of oocytes is clinically used in horses to produce blastocysts but current conditions used for horses are suboptimal. We analyzed the composition of equine preovulatory follicular fluid (FF) secretome and tested its effects on meiotic competence and gene expression in oocytes subjected to IVM. Preovulatory FF was obtained, concentrated using ultrafiltration with cut-off of 10 kDa, and stored at -80 °C. The metabolic and proteomic composition was analyzed, and its ultrastructural composition was assessed by cryo-transmission microscopy. Oocytes obtained post-mortem or by ovum pick up (OPU) were subjected to IVM in the absence (control) or presence of 20 or 40 μg/ml (S20 or S40) of secretome. Oocytes were then analyzed for chromatin configuration or snap frozen for gene expression analysis. Proteomic analysis detected 255 proteins in the Equus caballus database, mostly related to the complement cascade and cholesterol metabolism. Metabolomic analysis yielded 14 metabolites and cryo-transmission electron microscopy analysis revealed the presence of extracellular vesicles (EVs). No significant differences were detected in maturation rates among treatments. However, the expression of GDF9 and BMP15 significantly increased in OPU-derived oocytes compared to post-mortem oocytes (fold increase ± SEM: 9.4 ± 0.1 vs. 1 ± 0.5 for BMP15 and 9.9 ± 0.3 vs. 1 ± 0.5 for GDF9, respectively; p < 0.05). Secretome addition increased the expression of TNFAIP6 in S40 regardless of the oocyte source. Further research is necessary to fully understand whether secretome addition influences the developmental competence of equine oocytes.Projects PID2020-112723RB-I00 and PID2021-122507OB-I00 funded by MCIN1/AEI/10.13039/501100011033 and by “ERDF A way of making Europe”. José Manuel Ortiz Rodríguez was funded by PNR - DIMIVET- University of Bologna - CUP J45F21002000001, Ministry of University and Research (D.M. 737/2021). This work was supported by ‘Junta de Extremadura’ (Spain) and ‘Fondo Europeo de Desarrollo Regional’; Reference: IB20005. Marcos Luis Calero was supported by a grant “Plan Propio de Iniciación a la Investigación, Desarrollo Tecnológico e Innovación. Acción II” from the University of Extremadura (Ref. Beca RC1). Grant FJC2021-047675-I funded by MCIN2/AEI /10.13039/501100011033 and European Union NextGenerationEU/PRTR to Federica Marinaro. We appreciate the kind help of Paula Navarrete López for proteomic figures handling. The help of the veterinary staff of the INCARSA abbatoir is greatly acknowledged. This paper is dedicated to de memory of Dr. Juan Florencio Macías Núñez who seeded the curiosity and love for research in B. M-G.Peer reviewe

Aislamiento, caracterización y reprogramación de células multipotentes murinas en ratones transgénicos "mTert-EGFP"

La presente tesis está compuesta por tres artículos científicos publicados (Pericuesta et al., 2006; Ramirez et al., 2006; Ramírez et al., 2007) y un último trabajo realizado para completar el análisis de las células troncales en tejidos adultos. La importancia de las células troncales ha crecido enormemente en las últimas décadas debido al gran potencial que presentan para tratar enfermedades hasta hoy incurables como el Parkinson, la diabetes tipo I, la enfermedad de Alzheimer, esclerosis múltiple, daños en la espina dorsal e incluso el cáncer, así como para realizar ensayos específicos de drogas y toxinas. La presente tesis pretende analizar distintos aspectos relacionados con la pluripotencia de las células troncales murinas, en particular aquellos aspectos relacionados con los procesos de aislamiento, regulación y reprogramación. Para abordar este estudio se ha diseñado un modelo transgénico murino en el que se utiliza como marcador de célula indiferenciada, la actividad del promotor de la transcriptasa inversa de la enzima telomerasa asociado con la proteína de fluorescencia verde (GFP)

Prion protein expression regulates embryonic stem cell pluripotency and differentiation.

Cellular prion protein (PRNP) is a glycoprotein involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs). Although the physiological function of PRNP is largely unknown, its key role in prion infection has been extensively documented. This study examines the functionality of PRNP during the course of embryoid body (EB) differentiation in mouse Prnp-null (KO) and WT embryonic stem cell (ESC) lines. The first feature observed was a new population of EBs that only appeared in the KO line after 5 days of differentiation. These EBs were characterized by their expression of several primordial germ cell (PGC) markers until Day 13. In a comparative mRNA expression analysis of genes playing an important developmental role during ESC differentiation to EBs, Prnp was found to participate in the transcription of a key pluripotency marker such as Nanog. A clear switching off of this gene on Day 5 was observed in the KO line as opposed to the WT line, in which maximum Prnp and Nanog mRNA levels appeared at this time. Using a specific antibody against PRNP to block PRNP pathways, reduced Nanog expression was confirmed in the WT line. In addition, antibody-mediated inhibition of ITGB5 (integrin αvβ5) in the KO line rescued the low expression of Nanog on Day 5, suggesting the regulation of Nanog transcription by Prnp via this Itgb5. mRNA expression analysis of the PRNP-related proteins PRND (Doppel) and SPRN (Shadoo), whose PRNP function is known to be redundant, revealed their incapacity to compensate for the absence of PRNP during early ESC differentiation. Our findings provide strong evidence for a relationship between Prnp and several key pluripotency genes and attribute Prnp a crucial role in regulating self-renewal/differentiation status of ESC, confirming the participation of PRNP during early embryogenesis

Mitochondrial and metabolic adjustments during the final phase of follicular development prior to IVM of bovine oocytes

In vitro maturation (IVM) leads to reduced developmental rates compared to the use of in vivo matured oocytes. This reduction can be attributed to the suboptimal environment experienced during IVM, but the use of incompetent oocytes also plays a significant role. The objective of this study has been to characterize the mitochondrial and metabolic differences between competent and incompetent bovine oocytes selected prior to IVM based on Brilliant Cresyl Blue (BCB) staining. BCB selection allowed to sort two populations of cumulus-oocyte complexes (COCs) exhibiting diverse developmental competence despite showing a similar size and thereby being morphologically undistinguishable otherwise. Nuclear maturation rates were similar in both populations, but cleavage and blastocysts rates were significantly higher in BCB+ compared with BCB-. Mitochondrial distribution was similar between both groups, but mtDNA content experienced a 1.9-fold increase between BCB- and BCB+ oocytes, suggesting that a significant mtDNA synthesis must occur at the last stages of follicular development to achieve full competence prior to IVM. Consistently, transcriptional analysis in cumulus cells revealed an upregulation of the mitochondrial transcription factor TFAM in BCB-. Transcriptional analysis also suggested a decrease in both anaerobic glycolysis and pentose phosphate pathway (PPP) in BCB+ COCs, as the anaerobic glycolysis enzymes GAPDH and LDHA and the positive regulator of G6PD activity SIRT2 were upregulated in BCB- cumulus cells. These results suggest that during the final stages of follicular development a significant mtDNA replication must occur to achieve full oocyte developmental competence, and that this replication may be linked to anaerobic glycolysis and PPP activities