A genetically modified minipig model for Alzheimer's disease with SORL1 haploinsufficiency.

The established causal genes in Alzheimer's disease (AD), APP, PSEN1, and PSEN2, are functionally characterized using biomarkers, capturing an in vivo profile reflecting the disease's initial preclinical phase. Mutations in SORL1, encoding the endosome recycling receptor SORLA, are found in 2%-3% of individuals with early-onset AD, and SORL1 haploinsufficiency appears to be causal for AD. To test whether SORL1 can function as an AD causal gene, we use CRISPR-Cas9-based gene editing to develop a model of SORL1 haploinsufficiency in Göttingen minipigs, taking advantage of porcine models for biomarker investigations. SORL1 haploinsufficiency in young adult minipigs is found to phenocopy the preclinical in vivo profile of AD observed with APP, PSEN1, and PSEN2, resulting in elevated levels of ß-amyloid (Aß) and tau preceding amyloid plaque formation and neurodegeneration, as observed in humans. Our study provides functional support for the theory that SORL1 haploinsufficiency leads to endosome cytopathology with biofluid hallmarks of autosomal dominant AD.

Characterization of the endometrial transcriptome in early diestrus influencing pregnancy status in dairy cattle after transfer of in vitro-produced embryos.

Modifications of the endometrial transcriptome at day 7 of the estrus cycle are crucial to maintain gestation after transfer of in vitro-produced (IVP) embryos, although these changes are still largely unknown. The aim of this study was to identify genes, and their related biological mechanisms, important for pregnancy establishment based on the endometrial transcriptome of recipient lactating dairy cows that become pregnant in the subsequent estrus cycle, upon transfer of IVP embryos. Endometrial biopsies were taken from Holstein Friesian cows on day 6-8 of the estrus cycle followed by embryo transfer in the following cycle. Animals were classified retrospectively as pregnant (PR, n = 8) or nonpregnant (non-PR, n = 11) cows, according to pregnancy status at 26-47 days. Extracted mRNAs from endometrial samples were sequenced with an Illumina platform to determine differentially expressed genes (DEG) between the endometrial transcriptome from PR and non-PR cows. There were 111 DEG (false discovery rate < 0.05), which were mainly related to extracellular matrix interaction, histotroph metabolic composition, prostaglandin synthesis, transforming growth factor-ß signaling as well as inflammation and leukocyte activation. Comparison of these DEG with DEG identified in two public external data sets confirmed the more fertile endometrial molecular profile of PR cows. In conclusion, this study provides insights into the key early endometrial mechanisms for pregnancy establishment, after IVP embryo transfer in dairy cows.

Simulated physiological oocyte maturation has side effects on bovine oocytes and embryos.

PURPOSE: Oocyte maturation is a complex process involving nuclear and cytoplasmic modulations, during which oocytes acquire their ability to become fertilized and support embryonic development. The oocyte is apparently "primed" for maturation during its development in the dominant follicle. As bovine oocytes immediately resume meiosis when cultured, it was hypothesized that delaying resumption of meiosis with cyclic nucleotide modulators before in vitro maturation (IVM) would allow the oocytes to acquire improved developmental competence. METHODS: We tested the Simulated Physiological Oocyte Maturation (SPOM) system that uses forskolin and 3-isobutyl-1-methylxanthine for 2 h prior to IVM against two different systems of conventional IVM (Con-IVM). We evaluated the ultrastructure of matured oocytes and blastocysts and also assessed the expression of 96 genes related to embryo quality in the blastocysts. RESULTS: In summary, the SPOM system resulted in lower blastocyst rates than both Con-IVM systems (30 ± 9.1 vs. 35 ± 8.7; 29 ± 2.6 vs. 38 ± 2.8). Mature SPOM oocytes had significantly increased volume and number of vesicles, reduced volume and surface density of large smooth endoplasmic reticulum clusters, and lower number of mitochondria than Con-IVM oocytes. SPOM blastocysts showed only subtle differences with parallel undulations of adjacent trophectoderm plasma membranes and peripherally localized ribosomes in cells of the inner cell mass compared with Con-IVM blastocysts. SPOM blastocysts, however, displayed significant downregulation of genes related to embryonic developmental potential when compared to Con-IVM blastocysts. CONCLUSIONS: Our results show that the use of the current version of the SPOM system may have adverse effects on oocytes and blastocysts calling for optimized protocols for improving oocyte competence.

Identification of potential biomarkers in donor cows for in vitro embryo production by granulosa cell transcriptomics.

The Ovum Pick Up-In vitro Production (OPU-IVP) of embryos is an advanced reproductive technology used in cattle production but the complex biological mechanisms behind IVP outcomes are not fully understood. In this study we sequenced RNA of granulosa cells collected from Holstein cows at oocyte aspiration prior to IVP, to identify candidate genes and biological mechanisms for favourable IVP-related traits in donor cows where IVP was performed separately for each animal. We identified 56 genes significantly associated with IVP scores (BL rate, kinetic and morphology). Among these, BEX2, HEY2, RGN, TNFAIP6 and TXNDC11 were negatively associated while Mx1 and STC1 were positively associated with all IVP scores. Functional analysis highlighted a wide range of biological mechanisms including apoptosis, cell development and proliferation and four key upstream regulators (COX2, IL1, PRL, TRIM24) involved in these mechanisms. We found a range of evidence that good IVP outcome is positively correlated with early follicular atresia. Furthermore we showed that high genetic index bulls can be used in breeding without reducing the IVP performances. These findings can contribute to the development of biomarkers from follicular fluid content and to improving Genomic Selection (GS) methods that utilize functional information in cattle breeding, allowing a widespread large scale application of GS-IVP.

Effects of post-mortem storage conditions of bovine epididymides on sperm characteristics: investigating a tool for preservation of sperm from endangered species.

The aim of this study was to establish and validate a reliable and efficient protocol for the recovery and cryopreservation of epididymal spermatozoa used for in vitro fertilization, using bulls of two different age classes. Testicles from 26 (37-51 weeks old, group 1) and 19 (52-115 weeks old, group 2) Danish Holstein bulls were collected after slaughter and stored at 5°C. After 0, 24 or 48 h, epididymides were isolated and spermatozoa collected. Assessments included spermatozoal motility, viability and morphology before and after cryopreservation and in vitro embryo production. Results showed that live spermatozoa can be collected from epididymides of bulls after their death. Storage of the testicles at 5°C for 24 h followed by cryopreservation of recovered epididymal spermatozoa resulted in 21% (group 1) and 31% (group 2) blastocysts produced in vitro. These results illustrate that epididymal spermatozoa recovered from testicles kept in specific conditions can be used to preserve genetic material from endangered and threatened species or populations in nature as well as in domestic and zoo animals.

Analysis of nucleolar morphology and protein localization as an indicator of nuclear reprogramming.

When a cell is reprogrammed to a new phenotype, the nucleolus undergoes more or less dramatic modulations, which can be used as a marker for the occurrence of the reprogramming. This phenomenon is most pronounced when differentiated cells are reprogrammed to totipotency when they are submitted to cloning by somatic cell nuclear transfer. However, when cells are reprogrammed by less fundamental means, as for example treatment by Xenopus extract or expression of pluripotency genes, more subtle nucleolar modulations can also be noted. The monitoring and understanding of the reprogramming-related nucleolar modulations are based upon detailed knowledge about the nucleolar changes that occur during normal development from the developing oocyte over oocyte maturation and fertilization to the activation of the embryonic genome in the early embryo. Below, the ultrastructural and molecular modulations of the nucleolus are summarized in this developmental context, but also as they occur in assisted reproductive technologies such as in vitro fertilization and somatic cell nuclear transfer. Moreover, detailed protocols for monitoring the nucleolar changes by transmission electron microscopy and immunocytochemistry are presented.

Increasing efficiency in production of cloned piglets.

The low efficiency in obtaining piglets after production of cloned embryos was challenged in two steps-first by performing in vitro culture for 5-6 days after cloning to obtain later-stage embryos for more precise selection for transfer, and second by reducing the number of embryos transferred per recipient sow. The data set consisted of combined results from a 4-year period where cloning was performed to produce piglets that were transgenic for important human diseases. For this, different transgenes and cell types were used, and the cloning work was performed by several persons using oocytes from different pig breeds, but following a standardized and optimized protocol. Results showed that in vitro culture is possible with a relatively stable rate of transferable embryos around 41% and a pregnancy rate around 90%. Furthermore, a reduction from around 80 embryos to 40 embryos transferred per recipient was possible without changing the efficiency of around 14% (piglets born out of embryos transferred). It was concluded that this approach can increase the efficiency in obtaining piglets by means of in vitro culture and selection of high-quality embryos with subsequent transfer into more recipients. Such changes can also reduce the need for personnel, time, and material when working with this technology.