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 biology.

Adenylate kinase 9 is essential for sperm function and male fertility in mammals.

Despite passing routine laboratory tests for semen quality, bulls used in artificial insemination exhibit significant variation in fertility. Routine analysis of fertility data identified a dairy bull with extreme subfertility (10% pregnancy rate). To characterize the subfertility phenotype, a range of in vitro, in vivo, and molecular assays were carried out. Sperm from the subfertile bull exhibited reduced motility and severely reduced caffeine-induced hyperactivation compared to controls. Ability to penetrate the zona pellucida, cleavage rate, cleavage kinetics, and blastocyst yield after IVF or AI were significantly lower than in control bulls. Whole-genome sequencing from semen and RNA sequencing of testis tissue revealed a critical mutation in adenylate kinase 9 (AK9) that impaired splicing, leading to a premature termination codon and a severely truncated protein. Mice deficient in AK9 were generated to further investigate the function of the gene; knockout males were phenotypically indistinguishable from their wild-type littermates but produced immotile sperm that were incapable of normal fertilization. These sperm exhibited numerous abnormalities, including a low ATP concentration and reduced motility. RNA-seq analysis of their testis revealed differential gene expression of components of the axoneme and sperm flagellum as well as steroid metabolic processes. Sperm ultrastructural analysis showed a high percentage of sperm with abnormal flagella. Combined bovine and murine data indicate the essential metabolic role of AK9 in sperm motility and/or hyperactivation, which in turn affects sperm binding and penetration of the zona pellucida. Thus, AK9 has been found to be directly implicated in impaired male fertility in mammals.

Preovulatory follicular fluid secretome added to in vitro maturation medium influences the metabolism of equine cumulus-oocyte complexes.

BACKGROUND: In vitro embryo production is a highly demanded reproductive technology in horses, which requires the recovery (in vivo or post-mortem) and in vitro maturation (IVM) of oocytes. Oocytes subjected to IVM exhibit poor developmental competence compared to their in vivo counterparts, being this related to a suboptimal composition of commercial maturation media. The objective of this work was to study the effect of different concentrations of secretome obtained from equine preovulatory follicular fluid (FF) on cumulus-oocyte complexes (COCs) during IVM. COCs retrieved in vivo by ovum pick up (OPU) or post-mortem from a slaughterhouse (SLA) were subjected to IVM in the presence or absence of secretome (Control: 0 µg/ml, S20: 20 µg/ml or S40: 40 µg/ml). After IVM, the metabolome of the medium used for oocyte maturation prior (Pre-IVM) and after IVM (Post-IVM), COCs mRNA expression, and oocyte meiotic competence were analysed. RESULTS: IVM leads to lactic acid production and an acetic acid consumption in COCs obtained from OPU and SLA. However, glucose consumption after IVM was higher in COCs from OPU when S40 was added (Control Pre-IVM vs. S40 Post-IVM: 117.24 ± 7.72 vs. 82.69 ± 4.24; Mean µM ± SEM; p < 0.05), while this was not observed in COCs from SLA. Likewise, secretome enhanced uptake of threonine (Control Pre-IVM vs. S20 Post-IVM vs. S40 Post-IVM: 4.93 ± 0.33 vs. 3.04 ± 0.25 vs. 2.84 ± 0.27; Mean µM ± SEM; p < 0.05) in COCs recovered by OPU. Regarding the relative mRNA expression of candidate genes related to metabolism, Lactate dehydrogenase A (LDHA) expression was significantly downregulated when secretome was added during IVM at 20-40 µg/ml in OPU-derived COCs (Control vs. S20 vs. S40: 1.77 ± 0.14 vs. 1 ± 0.25 vs. 1.23 ± 0.14; fold change ± SEM; p < 0.05), but not in SLA COCs. CONCLUSIONS: The addition of secretome during in vitro maturation (IVM) affects the gene expression of LDHA, glucose metabolism, and amino acid turnover in equine cumulus-oocyte complexes (COCs), with diverging outcomes observed between COCs retrieved using ovum pick up (OPU) and slaughterhouse-derived COCs (SLA).

Postnatal Catch-Up Growth Programs Telomere Dynamics and Glucose Intolerance in Low Birth Weight Mice.

Low birth weight and rapid postnatal weight gain are independent predictors of obesity and diabetes in adult life, yet the molecular events involved in this process remain unknown. In inbred and outbred mice, this study examines natural intrauterine growth restriction (IUGR) in relation to body weight, telomere length (TL), glucose tolerance, and growth factor gene (Igf1, Igf2, Insr, Igf1r, and Igf2r) mRNA expression levels in the brain, liver, and muscle at 2- and 10 days of age and then at 3- and 9 months of age. At birth, ~15% of the animals showed IUGR, but by 3 and 9 months, half of these animals had regained the same weight as controls without IUGR (recuperated group). At 10 days, there was no difference in TL between animals undergoing IUGR and controls. However, by 3 and 9 months of age, the recuperated animals had shorter TL than the control and IUGR-non recuperated animals and also showed glucose intolerance. Further, compared to controls, Igf1 and Igf2 growth factor mRNA expression was lower in Day 2-IUGR mice, while Igf2r and Insr mRNA expression was higher in D10-IUGR animals. Moreover, at 3 months of age, only in the recuperated group were brain and liver Igf1, Igf2, Insr, and Igf2r expression levels higher than in the control and IUGR-non-recuperated groups. These data indicate that catch-up growth but not IUGR per se affects TL and glucose tolerance, and suggest a role in this latter process of insulin/insulin-like growth signaling pathway gene expression during early development.

Minor Splicing Factors Zrsr1 and Zrsr2 Are Essential for Early Embryo Development and 2-Cell-Like Conversion.

Minor splicing plays an important role in vertebrate development. Zrsr1 and Zrsr2 paralog genes have essential roles in alternative splicing, mainly participating in the recognition of minor (U12) introns. To further explore their roles during early embryo development, we produced Zrsr1mu and Zrsr2mu mutant mice, containing truncating mutations within the second zinc finger domain. Both homozygous mutant mice were viable with a normal lifespan. When we crossed a homozygous Zrsr2mu/mu female with Zrsr1mu/mu male, the double heterozygotes were non-viable, giving rise to embryos that stopped developing mainly between the 2- and 4-cell stages, just after zygotic gene activation. RNA-seq analysis of Zrsr1/2mu 2-cell embryos showed altered gene and isoform expression of thousands of genes enriched in gene ontology terms and biological pathways related to ribosome, RNA transport, spliceosome, and essential zygotic gene activation steps. Alternative splicing was analyzed, showing a significant increase in intron retention in both U2 and U12 intron-containing genes related to cell cycle and mitotic nuclear division. Remarkably, both Zrsr1 and Zrsr2 were required for the conversion of mouse-induced pluripotent stem cells into 2C-like cells. According to our results, Zrsr1 or Zrsr2 are necessary for ZGA and both are indispensable for the conversion of induced pluripotent stem cells into 2C-like cells.

D-Chiro-Inositol Treatment Affects Oocyte and Embryo Quality and Improves Glucose Intolerance in Both Aged Mice and Mouse Models of Polycystic Ovarian Syndrome.

Polycystic ovarian syndrome (PCOS) is the main cause of female infertility. It is a multifactorial disorder with varying clinical manifestations including metabolic/endocrine abnormalities, hyperandrogenism, and ovarian cysts, among other conditions. D-Chiro-inositol (DCI) is the main treatment available for PCOS in humans. To address some of the mechanisms of this complex disorder and its treatment, this study examines the effect of DCI on reproduction during the development of different PCOS-associated phenotypes in aged females and two mouse models of PCOS. Aged females (8 months old) were treated or not (control) with DCI for 2 months. PCOS models were generated by treatment with dihydrotestosterone (DHT) on Days 16, 17, and 18 of gestation, or by testosterone propionate (TP) treatment on the first day of life. At two months of age, PCOS mice were treated with DCI for 2 months and their reproductive parameters analyzed. No effects of DCI treatment were produced on body weight or ovary/body weight ratio. However, treatment reduced the number of follicles with an atretic cyst-like appearance and improved embryo development in the PCOS models, and also increased implantation rates in both aged and PCOS mice. DCI modified the expression of genes related to oocyte quality, oxidative stress, and luteal sufficiency in cumulus-oocyte complexes (COCs) obtained from the aged and PCOS models. Further, the phosphorylation of AKT, a main metabolic sensor activated by insulin in the liver, was enhanced only in the DHT group, which was the only PCOS model showing glucose intolerance and AKT dephosphorylation. The effect of DCI in the TP model seemed mediated by its influence on oxidative stress and follicle insufficiency. Our results indicate that DCI works in preclinical models of PCOS and offer insight into its mechanism of action when used to treat this infertility-associated syndrome.

Differential isoform expression and alternative splicing in sex determination in mice.

BACKGROUND: Alternative splicing (AS) may play an important role in gonadal sex determination (GSD) in mammals. The present study was designed to identify differentially expressed isoforms and AS modifications accompanying GSD in mice. RESULTS: Using deep RNA-sequencing, we performed a transcriptional analysis of XX and XY gonads during sex determination on embryonic days 11 (E11) and 12 (E12). Analysis of differentially expressed genes (DEG) identified hundreds of genes related to GSD and early sex differentiation that may represent good candidates for sex reversal. Expression at time point E11 in males was significantly enriched in RNA splicing and mRNA processing Gene Ontology terms. Differentially expressed isoform analysis identified hundreds of specific isoforms related to GSD, many of which showed no differences in the DEG analysis. Hundreds of AS events were identified as modified at E11 and E12. Female E11 gonads featured sex-biased upregulation of intron retention (in genes related to regulation of transcription, protein phosphorylation, protein transport and mRNA splicing) and exon skipping (in genes related to chromatin repression) suggesting AS as a post-transcription mechanism that controls sex determination of the bipotential fetal gonad. CONCLUSION: Our data suggests an important role of splicing regulatory mechanisms for sex determination in mice.

Extracellular vesicles derived from endometrial human mesenchymal stem cells enhance embryo yield and quality in an aged murine model†.

Advanced age is a risk factor undermining women's fertility. Hence, the optimization of assisted reproduction techniques is an interdisciplinary challenge that requires the improvement of in vitro culture systems. Here, we hypothesize that supplementation of embryo culture medium with extracellular vesicles from endometrial-derived mesenchymal stem cells (EV-endMSCs) may have a positive impact on the embryo competence of aged oocytes. In this work, 24 weeks old B6D2 female mice were used as egg donors and in vitro fertilization assays were performed using males from the same strain (8-12 weeks); the presumptive zygotes were incubated in the presence of 0, 10, 20, 40, or 80 µg/ml of EV-endMSCs. The results from the proteomic analysis of EV-endMSCs and the classification by Reactome pathways allowed us to identify proteins closely related with the fertilization process. Moreover, in our aged murine model, the supplementation of the embryo culture medium with EV-endMSCs improved the developmental competence of the embryos as well as the total blastomere count. Finally, gene expression analysis of murine blastocysts showed significant changes on core genes related to cellular response to oxidative stress, metabolism, placentation, and trophectoderm/inner cell mass formation. In summary, we demonstrate that EV-endMSCs increase the quality of the embryos, and according to proteomic and genomic analysis, presumably by modulating the expression of antioxidant enzymes and promoting pluripotent activity. Therefore, EV-endMSCs could be a valuable tool in human assisted reproduction improving the developmental competence of aged oocytes and increasing the odds of implantation and subsequent delivery.

Longitudinal analysis of somatic and germ-cell telomere dynamics in outbred mice.

Although telomere length (TL) shortens with age in most tissues, an age-related increase in length has been described in sperm through a mechanism that is not yet fully understood. Changes in TL with age in the same individual have not been explored. This longitudinal study examines TL dynamics in somatic tissue and gametes during an entire lifespan in an outbred mouse population (from 8 to up to 114 weeks of age). Our findings indicate a reduced life expectancy in males compared to females (84.75 ± 9.23 vs. 113.16 ± 0.20 weeks) and significant variability in TL dynamics between individuals. While with aging, a clear reduction in TL was produced in somatic cells and oocytes, telomeres in sperm cells significantly lengthened. Finally, we found evidence indicating that telomere elongation in sperm during aging may be dependent on different mechanisms, such as the survival of spermatogonia with longer telomeres and the alternative lengthening of telomeres mechanism in meiotic and postmeiotic spermatogenic cells.

Embryo responses to stress induced by assisted reproductive technologies.

Assisted reproductive technology (ART) has led to the birth of millions of babies. In cattle, thousands of embryos are produced annually. However, since the introduction and widespread use of ART, negative effects on embryos and offspring are starting to emerge. Knowledge so far, mostly provided by animal models, indicates that suboptimal conditions during ART can affect embryo viability and quality, and may induce embryonic stress responses. These stress responses take the form of severe gene expression alterations or modifications in critical epigenetic marks established during early developmental stages that can persist after birth. Unfortunately, while developmental plasticity allows the embryo to survive these stressful conditions, such insult may lead to adult health problems and to long-term effects on offspring that could be transmitted to subsequent generations. In this review, we describe how in mice, livestock, and humans, besides affecting the development of the embryo itself, ART stressors may also have significant repercussions on offspring health and physiology. Finally, we argue the case that better control of stressors during ART will help improve embryo quality and offspring health.

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.

CB1 cannabinoid receptor drives oocyte maturation and embryo development via PI3K/Akt and MAPK pathways.

Endocannabinoids have been recognized as mediators of practically all reproductive events in mammals. However, little is known about the role of this system in oocyte maturation. In a mouse model, we observed that activation of cannabinoid receptor 1 (CB1) during in vitro oocyte maturation modulated the phosphorylation status of Akt and ERK1/2 and enhanced the subsequent embryo production. In the absence of CB1, in vivo oocyte maturation was impaired and embryo development delayed. Cannabinoid receptor 2 (CB2) was unable to rescue these effects. Finally, we confirmed abnormal oocyte maturation rather than impaired embryonic transport through the oviduct in CB1 knockouts. Our data suggest that cannabinoid agonists may be useful in vitro maturation supplements. For in vitro fertilization patients intolerant to gonadotropins, this could be a promising and only option.-López-Cardona, A. P., Pérez-Cerezales, S., Fernández-González, R., Laguna-Barraza, R., Pericuesta, E., Agirregoitia, N., Gutiérrez-Adán, A., Agirregoitia, E. CB1 cannabinoid receptor drives oocyte maturation and embryo development via PI3K/Akt and MAPK pathways.

Extracellular vesicles derived from endometrial human mesenchymal stem cells improve IVF outcome in an aged murine model.

Advanced age reduces the success of in vitro fertilization (IVF) being this effect partly mediated by an overproduction of reactive oxygen species (ROS) that trigger apoptosis. It has been demonstrated that extracellular vesicles derived from endometrial mesenchymal stem cells (EV-endMSCs) exert an antioxidant effect and can be used as IVF coadjutants. In this work, endMSCs were isolated from human menstrual blood (n = 4) and characterized according to multipotentiality and surface marker expression prior EV-endMSCs isolation. Oocytes were obtained from 21 B6D2 mice (24 weeks) and coincubated with sperm from young males (8-12 weeks). Presumptive zygotes were incubated in the presence of 0, 10, 20, 40 or 80 µg/ml of EV-endMSCs in KSOM medium. Blastocyst yield was evaluated, and 25 blastocysts per group were used for qPCR. Blastocyst rate was 29.4% in control; 45.2% for 10 µg/ml, 62.9% for 20 µg/ml, 55.5% for 40 µg/ml and 53.8% in the 80 µg/ml (n = 124-130 oocytes) being all the increases significantly different when compared against control (p < 0.05). The 20-80 µg/ml treatments decreased the expression of glutathione peroxidase (Gpx1), and the 10-40 µg/ml treatments reduced the expression of superoxide dismutase (Sod1; p < 0.05) compared to control; Bax mRNA expression did not vary. Our results suggest that the increased developmental competence of the embryos could be partly mediated by the EV-endMSCs' ROS scavenger activity.

Elimination of methylation marks at lysines 4 and 9 of histone 3 (H3K4 and H3K9) of spermatozoa alters offspring phenotype.

The contribution of the contents of spermatozoa to the development of the embryo is currently being considered wider than was previously thought. Recent findings point to the participation of epigenetic marks present in the retained histones of mature spermatozoa on embryo and fetal development. Here we created a novel conditional transgenic mouse that expresses lysine (K) demethylase 1a (Kdm1a) during spermatogenesis when the testicles are subjected to heat stress. Using these animals under these conditions we were able to reduce the methylation level of histone 3 at lysines 4 and 9 (H3K4 and H3K9, respectively) in mature spermatozoa. The offspring of these transgenic mice were followed for correct development and growth after birth. We found that the offspring of males expressing Kdm1a suffered 20% of reabsorptions at Day 15 after implantation (vs 0.3% in the control). In addition, 35% of the offspring sired by these males showed some kind of abnormality (suckling defects, lack of movement coordination, dropping forelimbs, abnormal body curvature, absence of eyes, gigantisms and neuromuscular defects) and 25% died before postnatal Day 21. Some abnormalities were maintained to adulthood. These results show that alteration of epigenetic marks present in the retained histones of mature spermatozoa affect fetal development and have phenotypic consequences in the newborn.

Effects of vitrification on the expression of pluripotency, apoptotic and stress genes in in vitro-produced porcine blastocysts.

The aims of the present study were to: (1) evaluate the effect of vitrification and warming on quality parameters and expression levels of pluripotency, apoptotic and stress genes in in vitro-produced (IVP) porcine blastocysts; and (ii) determine the correlation between these parameters. To this end, total cell number, DNA fragmentation, peroxide levels and the relative transcript abundance of BCL-2 associated X protein (BAX), BCL2-like 1 (BCL2L1), heat shock protein 70 (HSPA1A), POU class 5 homeobox 1 (POU5F1), superoxide dismutase 1 (SOD1) and superoxide dismutase 2 (SOD2) were analysed in fresh and vitrified IVP blastocysts. The results suggest that vitrification procedures have no effect on total cell number and gene expression of BAX, BCL2L1, SOD1 and SOD2 or the BAX:BCL2L1 ratio. Nevertheless, a significant increase in DNA fragmentation (2.9±0.4% vs 11.9±2.0%) and peroxide levels (80.4±2.6 vs 97.2±3.1) were seen in vitrified compared with Day 7 fresh blastocysts. In addition, after blastocyst vitrification, relative transcript abundance was downregulated for POU5F1 and upregulated for HSPA1A. Finally, there was a significant correlation of POU5F1 and HSPA1A with DNA fragmentation (POU5F1, r=-0.561; HSPA1A, r=0.604) and peroxide levels (POU5F1, r=-0.590; HSPA1A, r=0.621). In conclusion, under the conditions of the present study, vitrification and warming of IVP porcine blastocysts resulted in altered expression of POU5F1 and HSPA1A, but had no effect on BAX, BCL2L1, SOD1 and SOD2 expression.

Characterisation of the deleted in azoospermia like (Dazl)-green fluorescent protein mouse model generated by a two-step embryonic stem cell-based strategy to identify pluripotent and germ cells.

The deleted in azoospermia like (Dazl) gene is preferentially expressed in germ cells; however, recent studies indicate that it may have pluripotency-related functions. We generated Dazl-green fluorescent protein (GFP) transgenic mice and assayed the ability of Dazl-driven GFP to mark preimplantation embryo development, fetal, neonatal and adult tissues, and in vitro differentiation from embryonic stem cells (ESCs) to embryoid bodies (EBs) and to primordial germ cell (PGC)-like cells. The Dazl-GFP mice were generated by a two-step ESC-based strategy, which enabled primary and secondary screening of stably transfected clones before embryo injection. During preimplantation embryo stages, GFP was detected from the zygote to blastocyst stage. At Embryonic Day (E) 12.5, GFP was expressed in gonadal ridges and in neonatal gonads of both sexes. In adult mice, GFP expression was found during spermatogenesis from spermatogonia to elongating spermatids and in the cytoplasm of oocytes. However, GFP mRNA was also detected in other tissues harbouring multipotent cells, such as the intestine and bone marrow. Fluorescence was maintained along in vitro Dazl-GFP ESC differentiation to EBs, and in PGC-like cells. In addition to its largely known function in germ cell development, Dazl could have an additional role in pluripotency, supporting these transgenic mice as a valuable tool for the prospective identification of stem cells from several tissues.

An efficient system to establish biopsy-derived trophoblastic cell lines from bovine embryos.

Trophoblastic cells play a crucial role in implantation and placentogenesis and can be used as a model to provide substantial information on the peri-implantation period. Unfortunately, there are few cell lines for this purpose in cattle because of the difficulty of raising successive cell stocks in the long-term. Our results show that the combination of a monolayer culture system in microdrops on a surface treated with gelatin and the employment of conditioned media from mouse embryonic fibroblasts support the growth of bovine trophoblastic cells lines from an embryo biopsy. Expression profiles of mononucleate- and binucleate-specific genes in established trophoblastic cells lines represented various stages of gestation. Moreover, the ability to expand trophoblastic cell lines for more than 2 yr together with pluripotency-related gene expression patterns revealed certain self-renewal capacity. In summary, we have developed a system to expand in vitro trophoblastic cells from an embryo biopsy that solves the limitations of using amplified DNA from a small number of cells for bovine embryo genotyping and epigenotyping and, on the other hand, facilitates the establishment of trophoblastic cell lines that can be useful as peri-implantation in vitro models.

Germ-cell culture conditions facilitate the production of mouse embryonic stem cells.

The derivation of embryonic stem-cell (ESC) lines from blastocysts is a very inefficient process. Murine ESCs are thought to arise from epiblast cells that are already predisposed to a primordial-germ-cell fate. During the process of ESC derivation from B6D2 F1 hybrid mice, if we first culture the embryo from the two-cell stage in medium supplemented with LIF, we improve the quality of the blastocyst. When the blastocyst is then cultured in a germ-line stem-cell culture medium (GSCm), we are able to more efficiently (28.3%) obtain quality ESC lines that have a normal karyotype, proper degree of chimerism, and exhibit germ-line transmission when microinjected into blastocysts. Although germ-cell-specific genes were expressed in all culture medium conditions, GSCm did not shift the transcriptome towards germ-cell specification. A correlation was further observed between ESC derivation efficiency and the expression of some imprinted genes and retrotransposable elements. In conclusion, the combination of LIF supplementation followed by culture in GSCm establishes a higher efficiency method for ESC derivation.

Susceptibility and transmissibility of SARS-CoV-2 variants in transgenic mice expressing the cat angiotensin-converting enzyme 2 (ACE-2) receptor.

The emergence of SARS-CoV-2 in 2019 and its rapid spread throughout the world has caused the largest pandemic of our modern era. The zoonotic origin of this pathogen highlights the importance of the One Health concept and the need for a coordinated response to this kind of threats. Since its emergence, the virus has caused >7 million deaths worldwide. However, the animal source for human outbreaks remains unknown. The ability of the virus to jump between hosts is facilitated by the presence of the virus receptor, the highly conserved angiotensin-converting enzyme 2 (ACE2), found in various mammals. Positivity for SARS-CoV-2 has been reported in various species, including domestic animals and livestock, but their potential role in bridging viral transmission to humans is still unknown. Additionally, the virus has evolved over the pandemic, resulting in variants with different impacts on human health. Therefore, suitable animal models are crucial to evaluate the susceptibility of different mammalian species to this pathogen and the adaptability of different variants. In this work, we established a transgenic mouse model that expresses the feline ACE2 protein receptor (cACE2) under the human cytokeratin 18 (K18) gene promoter's control, enabling high expression in epithelial cells, which the virus targets. Using this model, we assessed the susceptibility, pathogenicity, and transmission of SARS-CoV-2 variants. Our results show that the sole expression of the cACE2 receptor in these mice makes them susceptible to SARS-CoV-2 variants from the initial pandemic wave but does not enhance susceptibility to omicron variants. Furthermore, we demonstrated efficient contact transmission of SARS-CoV-2 between transgenic mice that express either the feline or the human ACE2 receptor.

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

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.