spatula-montevideo-device-for-the-vitrification-of-mammalian-embryos

Spatula Montevideo Device for the Vitrification of Mammalian Embryos

This study describes an efficient and cost-effective vitrification method for mouse and sheep embryo cryopreservation known as the Spatula Montevideo ...

Institut Pasteur de Montevideo

To evaluate the effect of the nonsteroidal anti-inflammatory drugs tolfenamic acid and flunixin meglumine in pregnancy rate and embryo survival of recipient mice subjected to embryo transfer.

Administration of the nonsteroidal anti-inflammatory drug tolfenamic acid at embryo transfer improves maintenance of pregnancy and embryo survival in recipient mice.

This study was conducted to compare the effect of minimum volume Spatula MVD vitrification (VIT) versus traditional slow freezing (SLF) of mouse embryos. A total of 2,617 8-cell in vivo derived and 2-cell in vitro produced B6D2 mouse embryos were subjected to freezing/thawing or vitrification/warming, while fresh embryos were used as control group. Embryo recovery, survival and development rate, pregnancy rate and offspring production were analyzed. In Experiment 1, 8-cell in vivo derived embryos were subjected to in vitro culture, resulting in greater survival and development rates at 3.5 days post coitum stage in VIT than in SLF group (P < 0.05). Although both methods reached an acceptable hatching rate (41.0% and 49.7% for VIT and SLF, respectively; P=NS), it was significantly lower respect to the control group (67.8%, P < 0.01). In Experiment 2, 2-cell in vitro produced mouse embryos showed a similar recovery rate from the device after freezing/thawing or vitrification/warming (∼84%), however survival rate was significantly higher for vitrified/warmed (94.7%) than frozen/thawed embryos (85.1%; P < 0.01). Vitrified/warmed and control fresh embryos were transferred to surrogate mothers, revealing no differences both in pregnancy and offspring production rates. Our data demonstrate that minimum volume Spatula MVD method is a simple home-made useful technique for vitrification of 2-cell and 8-cell mouse embryos produced either in vitro or in vivo.

Minimum volume Spatula MVD vitrification method improves embryo survival compared to traditional slow freezing, both for in vivo and in vitro produced mice embryos.

Rodents used in biomedical research are maintained behind barriers to exclude microbial contaminants. Several check points have to be monitored to eliminate the potential of introducing adventitious agents into the facility. Microbiological monitoring of a mouse facility environment enables to evaluate the efficiency of sanitization and cleaning procedures, air quality, and technician good practices. At our SPF mouse facility, we implemented an environmental microbiological monitoring program based in sedimentation and swabbing, inexpensive and easy to use methods. The aim of this work was to evaluate the results and the efficiency of the monitoring program after seven years. The median for bacteria and fungi counts in the SPF sampled areas was ≤2 CFU/2 h for settle plates and <1 CFU per swabbing plate, satisfying the requirements for grade C of the EU-GMP, with some modifications. The environmental monitoring program was useful to detect early warning of problems and enabled us to define a safe range of microbiological counts. In addition, SPF status defined for our mice was maintained throughout this study, confirmed by our HM program. This work could encourage directors and technicians of other mouse facilities in Latin America and rest of the world to implement this kind of program.

Establishment of an environmental microbiological monitoring program in a mice barrier facility.

The aim of this study was to evaluate the effect of an environmental enrichment (EE) plan on the reproductive performance of Swiss Webster mice and their female offspring used as recipients for embryo transfer. A total of 54 breeder mice and 60 F1 females, used as foster mothers, were allocated in two experimental groups to receive or not receive EE for physical well-being. Reproductive outcomes of the Swiss trios such as birth rate and pup number, litter size, pups' weight at weaning, interlitter interval and time to first litter were analyzed. Environmental enrichment significantly increased pups weight from breeding trios compared to the control group (14.4 ± 0.1 vs. 13.8g ± 0.1, EE vs. control, respectively;  < 0.01). Other parameters did not differ between both groups. Reproductive parameters of female offspring used as recipients for embryo transfer did not differ among groups subjected or not to EE. These data demonstrate that the EE protocol applied in Swiss Webster breeder mice positively enhanced pups weight, and did not interfere with other reproductive outcomes. In conclusion, this study supports the implementation of EE plans usually applied for animal welfare in mouse facilities with slight improvement in reproductive performance.

Long-Term Effect of Environmental Enrichment on Reproductive Performance of Swiss Webster Mice and Their Female Offspring.

The objective was to evaluate the influence of colony aging in a Swiss Webster (SW) outbred stock used as recipients for embryo transfer. In the first study, a retrospective analysis was performed throughout several generations during a 38-month period in 2,398 embryos transferred to 108 SW recipients. A decrease in the percentage of live pups from transferred embryos was found at the end of the period. Impairment occurred due to the incidence of maternal cannibalism that increased from 0% to 67-100% (P<0.05), while pregnancy rate (pregnant/transferred recipients) and number of pups per delivered female were not affected throughout the period (P=NS). A following study was carried out to compare the reproductive performance of SW stock vs. B6D2F1 hybrid females in a 5-year interval. The study was conducted on a total of 893 embryos transferred to 40 females (20 SW and 20 B6D2F1) in Year #1, and 514 embryos transferred to 30 females (15 SW and 15 B6D2F1) in Year #5. No cases of maternal cannibalism were found on Year #1 in any of the strains (0/10 and 0/10). However, an incidence of 44,4% (4/9) was seen on Year #5 for SW, while for B6D2F1 the incidence was 0% (0/12) (P<0.05). Further examination of the uterus showed endometrial cysts and abnormal implantation sites in SW on Year #5 but not in B6D2F1 females. In conclusion, this study reports an impairment of the reproductive performance of an early aged SW outbred stock colony mainly due to the occurrence of maternal cannibalism. This finding has important implications for embryo transfer programs conducted in mouse facilities.

Colony aging affects the reproductive performance of Swiss Webster females used as recipients for embryo transfer.

SPATS1 (spermatogenesis-associated, serine-rich 1) is an evolutionarily conserved, testis-specific protein that is differentially expressed during rat male meiotic prophase. Some reports have suggested a link between SPATS1 underexpression/mutation and human pathologies such as male infertility and testicular cancer. Given the absence of functional studies, we generated a Spats1 loss-of-function mouse model using CRISPR/Cas9 technology. The phenotypic analysis showed no overt phenotype in Spats1-/- mice, with both males and females being fertile. Flow cytometry and histological analyses did not show differences in the testicular content and histology between WT and knockout mice. Moreover, no significant differences in sperm concentration, motility, and morphology, were observed between WT and KO mice. These results were obtained both for young adults and for aged animals. Besides, although an involvement of SPATS1 in the Wnt signaling pathway has been suggested, we did not detect changes in the expression levels of typical Wnt pathway-target genes in mutant individuals. Thus, albeit Spats1 alteration might be a risk factor for male testicular health, we hereby show that this gene is not individually essential for male fertility and spermatogenesis in mouse.

SPATS1 (spermatogenesis-associated, serine-rich 1) is not essential for spermatogenesis and fertility in mouse.

CCDC28B (coiled-coil domain-containing protein 28B) was identified as a modifier in the ciliopathy Bardet-Biedl syndrome (BBS). Our previous work in cells and zebrafish showed that CCDC28B plays a role regulating cilia length in a mechanism that is not completely understood. Here we report the generation of a Ccdc28b mutant mouse using CRISPR/Cas9 (Ccdc28b mut). Depletion of CCDC28B resulted in a mild phenotype. Ccdc28b mut animals i) do not present clear structural cilia affectation, although we did observe mild defects in cilia density and cilia length in some tissues, ii) reproduce normally, and iii) do not develop retinal degeneration or obesity, two hallmark features of reported BBS murine models. In contrast, Ccdc28b mut mice did show clear social interaction defects as well as stereotypical behaviors. This finding is indeed relevant regarding CCDC28B as a modifier of BBS since behavioral phenotypes have been documented in BBS. Overall, this work reports a novel mouse model that will be key to continue evaluating genetic interactions in BBS, deciphering the contribution of CCDC28B to modulate the presentation of BBS phenotypes. In addition, our data underscores a novel link between CCDC28B and behavioral defects, providing a novel opportunity to further our understanding of the genetic, cellular, and molecular basis of these complex phenotypes.

Generation and characterization of Ccdc28b mutant mice links the Bardet-Biedl associated gene with mild social behavioral phenotypes.

Cystic echinococcosis is caused by the larval stages (hydatids) of cestode parasites belonging to the species cluster Echinococcus granulosus , with E. granulosus  being the main infecting species. Hydatids are bladderlike structures that attain large sizes within various internal organs of livestock ungulates and humans. Hydatids are protected by the massive acellular laminated layer (LL), composed mainly of mucins. Parasite growth requires LL turnover, and abundant LL-derived particles are found at infection sites in infected humans, raising the question of how LL materials are dealt with by the hosts. In this article, we show that E. granulosus  LL mucins injected into mice are taken up by Kupffer cells, the liver macrophages exposed to the vascular space. This uptake is largely dependent on the intact mucin glycans and on Clec4F, a C-type lectin receptor which, in rodents, is selectively expressed in Kupffer cells. This uptake mechanism operates on mucins injected both in soluble form intravenously (i.v.) and in particulate form intraperitoneally (i.p.). In mice harboring intraperitoneal infections by the same species, LL mucins were found essentially only at the infection site and in the liver, where they were taken up by Kupffer cells via Clec4F. Therefore, shed LL materials circulate in the host, and Kupffer cells can act as a sink for these materials, even when the parasite grows in sites other than the liver.

Mucins Shed from the Laminated Layer in Cystic Echinococcosis Are Captured by Kupffer Cells via the Lectin Receptor Clec4F.

Emerging evidence suggests that immune receptors may participate in many aging-related processes such as energy metabolism, inflammation, and cognitive decline. CD300f, a TREM2-like lipid-sensing immune receptor, is an exceptional receptor as it integrates activating and inhibitory cell-signaling pathways that modulate inflammation, efferocytosis, and microglial metabolic fitness. We hypothesize that CD300f can regulate systemic aging-related processes and ultimately healthy lifespan. We closely followed several cohorts of two strains of CD300f and WT mice of both sexes for 30 months and observed an important reduction in lifespan and healthspan in knockout mice. This was associated with systemic inflammaging, increased cognitive decline, reduced brain glucose uptake observed by FDG PET scans, enrichment in microglial aging/neurodegeneration phenotypes, proteostasis alterations, senescence, increased frailty, and sex-dependent systemic metabolic changes. Moreover, the absence of CD300f altered macrophage immunometabolic phenotype. Taken together, we provide strong evidence suggesting that myeloid cell CD300f immune receptor contributes to healthy aging.

CD300f immune receptor contributes to healthy aging by regulating inflammaging, metabolism, and cognitive decline.

Microinjection of CRISPR/Cas9 requires the availability of zygotes that implies animal breeding, superovulation schemes, and embryo collection. Vitrification of zygotes may allow having ready-to-use embryos and to temporally dissociate the workload of embryo production from microinjection. In this study, fresh (F group) or vitrified (V group) zygotes were microinjected with CRISPR/Cas9 system to test the hypothesis that vitrified zygotes could be a suitable source of embryos for microinjection. In Experiment 1 (in vitro evaluation), B6D2F1/J zygotes were microinjected and cultured until blastocyst stage. Embryo survival and cleavage rates after microinjection were similar between groups (~50% and ~80% respectively; P = NS). Development rate was significantly higher for F than V group (55.0% vs. 32.6%, respectively; P<0.05). Mutation rate did not show statistical differences among groups (P = NS). In Experiment 2 (in vivo evaluation), C57BL/6J zygotes were microinjected and transferred to recipient females. Embryo survival was significantly lower in fresh than in vitrified zygotes (49.2% vs. 62.7%, respectively; P<0.05). Cleavage rate did not show statistical differences (~70%; P = NS). Pregnancy rate (70.0% vs. 58.3%) and birth rate (11.9% vs. 11.2%) were not different between groups (F vs. V group; P = NS). Offspring mutation rate was higher for F than V group, in both heterodimer analysis (73.7% vs. 33.3%, respectively; P = 0.015) and Sanger sequencing (89.5% vs. 41.7%, respectively; P = 0.006). In conclusion, vitrified-warmed zygotes present a viable alternative source for CRISPR/Cas9 microinjection when the production of fresh embryos is impeded by limited technical support. The possibility of zygote cryobanking to perform microinjection sessions on demand seems to be a suitable alternative to avoid the breeding and maintenance of animals all over the year, enhancing the implementation of CRISPR technology.

Zygote cryobanking applied to CRISPR/Cas9 microinjection in mice.

Infertility affects a considerable number of couples at reproductive age, with an incidence of 10-15%. Approximately 25% of cases are classified as idiopathic infertility. Often, errors during the meiotic stage appear to be related to idiopathic infertility. A crucial component during the first meiotic prophase is the synaptonemal complex (SC), which plays a fundamental role in homologous chromosome pairing and meiotic recombination. In many studies with infertile patients, mutations affecting SC-coding genes have been identified. The generation of humanized models has high physiological relevance, helping to clarify the molecular bases of pathology, which in turn is essential for the development of therapeutic procedures. Here, we report the generation and characterization of genetically modified mice carrying a mutation equivalent to SYCE1 c.197-2A>G, previously found in male infertile patients, aiming to determine the actual effects of this mutation on reproductive capacity and to study the underlying molecular mechanisms. Homozygous mutants were infertile. SYCE1 protein was not detected and Syce1 transcript presented minimal levels, suggesting transcript degradation underlying the infertility mechanism. Additionally, homozygous mutants showed impaired homologous chromosome synapsis, meiotic arrest before the pachytene stage, and increased apoptosis of meiotic cells. This study validates the variant as pathogenic and causative of infertility, since the observed dramatic phenotype was attributable to this single homozygous point mutation, when compared to WT and heterozygous littermates. Moreover, although this homozygous point mutation has been only found in infertile men thus far, we anticipate that if it were present in women, it would cause infertility as well, as homozygous female mice also exhibited an infertility phenotype.

Mouse modeling of familial human SYCE1 c.197-2A>G splice site mutation leads to meiotic recombination failure and non-obstructive azoospermia.

The protein Deleted in Breast Cancer 1 (Dbc1) is an important regulator of various transcription factors and epigenetic modulators, significantly influencing metabolism, obesity, and aging-related processes. Knockout mice lacking Dbc1 exhibit severe obesity but remain protected from liver steatosis, insulin resistance, and atherosclerosis. We hypothesized that this phenotype of "healthy obesity" results from adipose tissue expansion, which prevents free fatty acid spillover and subsequent metabolic damage to peripheral tissues. To further investigate the putative role of Dbc1 in adipose cells during obesity and its effects on metabolic dysregulation, we generated conditional Dbc1 knockout (KO) mice by backcrossing with AdipoQ-CRE transgenic mice to selectively abrogate Dbc1 expression in all mature adipocytes (Dbc1LoxP/LoxP;CRE). These mice demonstrated effective deletion of Dbc1 in mature adipocytes across various fat depots. We assessed the impact of Dbc1 deletion on metabolic regulation in male and female mice fed standard chow and high-fat diets. Our findings revealed that Dbc1 knockout in mature adipocytes did not influence weight gain, glucose tolerance, or other metabolic dysregulation markers, irrespective of sex. However, Dbc1 KO adipocytes exhibited an mRNA expression profile indicative of heightened inflammation during obesity. These results suggest that the protective phenotype observed in whole-body Dbc1 KO obese mice is not attributable to Dbc1's function within mature adipocytes but likely involves other cell types in adipose tissue. Moreover, the specific deletion of Dbc1 in mature adipocytes unveils a novel role of Dbc1 in inflammation signaling during obesity.

Article	Year
Administration of the nonsteroidal anti-inflammatory drug tolfenamic acid at embryo transfer improves maintenance of pregnancy and embryo survival in recipient mice. Journal of assisted reproduction and genetics\| PubMed ID: 25561155	2015
Minimum volume Spatula MVD vitrification method improves embryo survival compared to traditional slow freezing, both for in vivo and in vitro produced mice embryos. Cryobiology\| PubMed ID: 30040919	2018
Establishment of an environmental microbiological monitoring program in a mice barrier facility. Anais da Academia Brasileira de Ciencias\| PubMed ID: 30304242	2018
Long-Term Effect of Environmental Enrichment on Reproductive Performance of Swiss Webster Mice and Their Female Offspring. Animals : an open access journal from MDPI\| PubMed ID: 32824668	2020
Colony aging affects the reproductive performance of Swiss Webster females used as recipients for embryo transfer. Animal reproduction\| PubMed ID: 33791029	2020
SPATS1 (spermatogenesis-associated, serine-rich 1) is not essential for spermatogenesis and fertility in mouse. PloS one\| PubMed ID: 33945571	2021
Generation and characterization of Ccdc28b mutant mice links the Bardet-Biedl associated gene with mild social behavioral phenotypes. PLoS genetics\| PubMed ID: 35653384	2022
Mucins Shed from the Laminated Layer in Cystic Echinococcosis Are Captured by Kupffer Cells via the Lectin Receptor Clec4F. Infection and immunity\| PubMed ID: 37162364	2023
CD300f immune receptor contributes to healthy aging by regulating inflammaging, metabolism, and cognitive decline. Cell reports\| PubMed ID: 37864797	2023
Zygote cryobanking applied to CRISPR/Cas9 microinjection in mice. PloS one\| PubMed ID: 38980864	2024
Mouse modeling of familial human SYCE1 c.197-2A>G splice site mutation leads to meiotic recombination failure and non-obstructive azoospermia. Molecular human reproduction\| PubMed ID: 39909494	2025
Adipocyte-specific deletion of Dbc1 does not recapitulate healthy obesity phenotype but suggests regulation of inflammation signaling. PloS one\| PubMed ID: 40315412	2025

Geraldine Schlapp

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