Genetic engineering a large animal model of human hypophosphatasia in sheep.

The availability of tools to accurately replicate the clinical phenotype of rare human diseases is a key step toward improved understanding of disease progression and the development of more effective therapeutics. We successfully generated the first large animal model of a rare human bone disease, hypophosphatasia (HPP) using CRISPR/Cas9 to introduce a single point mutation in the tissue nonspecific alkaline phosphatase (TNSALP) gene (ALPL) (1077 C > G) in sheep. HPP is a rare inherited disorder of mineral metabolism that affects bone and tooth development, and is associated with muscle weakness. Compared to wild-type (WT) controls, HPP sheep have reduced serum alkaline phosphatase activity, decreased tail vertebral bone size, and metaphyseal flaring, consistent with the mineralization deficits observed in human HPP patients. Computed tomography revealed short roots and thin dentin in incisors, and reduced mandibular bone in HPP vs. WT sheep, accurately replicating odonto-HPP. Skeletal muscle biopsies revealed aberrant fiber size and disorganized mitochondrial cristae structure in HPP vs. WT sheep. These genetically engineered sheep accurately phenocopy human HPP and provide a novel large animal platform for the longitudinal study of HPP progression, as well as other rare human bone diseases.

Oxygen-induced alterations in the expression of chromatin modifying enzymes and the transcriptional regulation of imprinted genes.

Embryo culture and assisted reproductive technologies have been associated with a disproportionately high number of epigenetic abnormalities in the resulting offspring. However, the mechanisms by which these techniques influence the epigenome remain poorly defined. In this study, we evaluated the capacity of oxygen concentration to influence the transcriptional control of a selection of key enzymes regulating chromatin structure. In mouse embryonic stem cells, oxygen concentrations modulated the transcriptional regulation of the TET family of enzymes, as well as the de novo methyltransferase Dnmt3a. These transcriptional changes were associated with alterations in the control of multiple imprinted genes, including H19, Igf2, Igf2r, and Peg3. Similarly, exposure of in vitro produced bovine embryos to atmospheric oxygen concentrations was associated with disruptions in the transcriptional regulation of TET1, TET3, and DNMT3a, along with the DNA methyltransferase co-factor HELLS. In addition, exposure to high oxygen was associated with alterations in the abundance of transcripts encoding members of the Polycomb repressor complex (EED and EZH2), the histone methyltransferase SETDB1 and multiple histone demethylases (KDM1A, KDM4B, and KDM4C). These disruptions were accompanied by a reduction in embryo viability and suppression of the pluripotency genes NANOG and SOX2. These experiments demonstrate that oxygen has the capacity to modulate the transcriptional control of chromatin modifying genes involved in the establishment and maintenance of both pluripotency and genomic imprinting.

Histone-lysine N-methyltransferase SETDB1 is required for development of the bovine blastocyst.

Transcripts derived from select clades of transposable elements are among the first to appear in early mouse and human embryos, indicating transposable elements and the mechanisms that regulate their activity are fundamental to the establishment of the founding mammalian lineages. However, the mechanisms by which these parasitic sequences are involved in directing the developmental program are still poorly characterized. Transposable elements are regulated through epigenetic means, where combinatorial patterns of DNA methylation and histone 3 lysine 9 trimethylation (H3K9me3) suppress their transcription. From studies in rodents, SET domain bifurcated 1 (SETDB1) has emerged as the core methyltransferase responsible for marking transposable elements with H3K9me3 and temporally regulating their transcriptional activity. SETDB1 loss of function studies in mice reveal that although extraembryonic tissues do not require this methyltransferase, establishment of the embryo proper fails without it. As the bovine embryo initiates the processes of epigenetic programming earlier in the preimplantation phase, we sought to determine whether suppressing SETDB1 would block the formation of the inner cell mass. We report here that bovine SETDB1 transcripts are present throughout preimplantation development, and RNA interference-based depletion blocks embryo growth at the morula stage of development. Although we did not observe alterations in global histone methylation or transposable element transcription, we did observe increased global levels of H3K27 acetylation, an epigenetic mark associated with active enhancers. Our observations suggest that SETDB1 might interact with the epigenetic machinery controlling enhancer function and that suppression of this methyltransferase may disrupt the bovine developmental program.

Reshaping the transcriptional frontier: epigenetics and somatic cell nuclear transfer.

Somatic-cell nuclear transfer (SCNT) experiments have paved the way to the field of cellular reprogramming. The demonstrated ability to clone over 20 different species to date has proven that the technology is robust but very inefficient, and is prone to developmental anomalies. Yet, the offspring from cloned animals exhibit none of the abnormalities of their parents, suggesting the low efficiency and high developmental mortality are epigenetic in origin. The epigenetic barriers to reprogramming somatic cells into a totipotent embryo capable of developing into a viable offspring are significant and varied. Despite their intimate relationship, chromatin structure and transcription are often not uniformly reprogramed after nuclear transfer, and many cloned embryos develop gene expression profiles that are hybrids between the donor cell and an embryonic blastomere. Recent advances in cellular reprogramming suggest that alteration of donor-cell chromatin structure towards that found in an normal embryo is actually the rate-limiting step in successful development of SCNT embryos. Here we review the literature relevant to the transformation of a somatic-cell nucleus into an embryo capable of full-term development. Interestingly, while resetting somatic transcription and associated epigenetic marks are absolutely required for development of SCNT embryos, life does not demand perfection.

Effects of early pregnancy diagnosis by palpation per rectum on pregnancy loss in dairy cattle.

OBJECTIVE: To determine the effect of palpation per rectum (PPR) by use of 1 or 2 fetal membrane slips (FMSs) for pregnancy diagnosis during early gestation on pregnancy loss in dairy cattle. DESIGN: Controlled, randomized block design. ANIMALS: 928 healthy pregnant cattle. PROCEDURES: All cattle were determined to be pregnant by use of transrectal ultrasonography at approximately day 31 after estrus and randomly allocated into 2 groups (control group [n = 476 cows] and palpation group [452]). The control group was not subjected to pregnancy diagnosis via PPR. The palpation group was subdivided into 2 groups (PPR FMS 1 [n = 230 cows] and PPR FMS 2 [222]), which involved PPR and pregnancy diagnosis via 1 or 2 FMSs, respectively, during the same examination, which was performed by 1 veterinarian between days 34 and 43 after estrus. All cattle were reevaluated by use of transrectal ultrasonography on days 45 and 60 to determine viability of the embryo and fetus, respectively. RESULTS: Overall pregnancy loss between days 31 and 60 was 14.1%. Pregnancy loss for the control, PPR FMS 1, and PPR FMS 2 groups from days 31 to 60 was 14.5%, 12.6%, and 14.9%, respectively. Embryonic pregnancy loss for the control, PPR FMS 1, and PPR FMS 2 groups was 12.4%, 9.1%, and 9.5%, respectively. Fetal pregnancy loss for the same groups was 2.4%, 3.8%, and 5.9%, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Pregnancy diagnosis via 1 or 2 FMSs performed during PPR in early gestation did not increase pregnancy loss in dairy cattle.

Examination of DNA methyltransferase expression in cloned embryos reveals an essential role for Dnmt1 in bovine development.

In studies of somatic cell nuclear transfer (SCNT), the ability of factors within the oocyte to epigenetically reprogram transferred nuclei is essential for embryonic development of the clone to proceed. However, irregular patterns of X-chromosome inactivation, abnormal expression of imprinted genes, and genomic DNA hypermethylation are frequently observed in reconstructed embryos, suggesting abnormalities in this process. To better understand the epigenetic events underlying SCNT reprogramming, we sought to determine if the abnormal DNA methylation levels observed in cloned embryos result from a failure of the oocyte to properly reprogram transcription versus differential biochemical regulation of the DNA methyltransferase family of enzymes (DNMTs) between embryonic and somatic nuclei. To address this question, we conducted real-time quantitation of Dnmt transcripts in bovine preimplantation embryos generated though in vitro fertilization (IVF), parthenogenic activation, and SCNT. By the 8-cell stage, transcripts encoding Dnmt1 become significantly down-regulated in cloned embryos, likely in response to the state of genomic hypermethylation, while the de novo methyltransferases maintain an expression pattern indistinguishable from their IVF and parthenote counterparts. Depletion of embryonic/maternal Dnmt1 transcripts within IVF embryos using short-interfering RNAs, while able to lower genomic DNA methylation levels, resulted in developmental arrest at the 8/16-cell stage. In contrast, SCNT embryos derived from a stable, Dnmt1-depleted donor cell line develop to blastocyst stage, but failed to carry to term. Our results indicate an essential role for Dnmt1 during bovine preimplantation development, and suggest proper transcriptional reprogramming of this gene family in SCNT embryos.

Cloning of GJA1 (connexin43) and its expression in canine ovarian follicles throughout the estrous cycle.

GJA1 (also known as connexin43 or Cx43) is the most abundant gap junction protein in mammalian tissues including the ovary. Here, it facilitates intercellular communication among granulosa cells and growing oocytes, thereby connecting the developing gamete to the hormonal axis as well as to the essential network of supporting granulosa cells. To date, the pattern of follicular GJA1 expression has not yet been defined for canines, a species with unique reproductive physiology including delays in follicle development, ovulation, oocyte maturation and fertilization. Here, we report the complete mRNA sequence for canine GJA1 and identify not only increases (P<0.05) in GJA1 mRNA expression in follicles at the secondary stage and larger, but also differences in expression levels between estrous cycle stages in both secondary and antral stage follicles. Expression of GJA1 mRNA in secondary follicles during proestrus was higher than in anestrus or estrus (P<0.01), and at diestrus (P<0.10). Antral follicles obtained during estrus expressed lower levels of GJA1 mRNA than any other cycle stage (P<0.01). GJA1 mRNA expression in primary and large antral follicles was similar across the estrous cycle. Despite the extensive length of the canine estrous cycle as compared with that of other mammals, the GJA1 mRNA and protein expression profiles are not significantly different from those reported for other species and suggests that mechanisms regulating GJA1 transcription are not likely to contribute to the extended delays in follicle and oocyte development in the dog.

Early pregnancy diagnosis by palpation per rectum: influence on embryo/fetal viability in dairy cattle.

The objective was to estimate the effect of palpation per rectum (for early pregnancy diagnosis) on embryo/fetal viability in dairy cattle. A controlled, randomized block-design experiment with two blocks, one by category, and the other by number of embryos, was conducted. Five-hundred-and-twenty pregnant dairy cows and heifers with a viable embryo detected by transrectal ultrasonography (TRUS) between days 29 and 32 after AI were included. The pregnant females were randomly allocated into two nearly equal groups: palpation per rectum (PAL group; n=258) and no palpation per rectum (NPAL group; n=262). The PAL group was submitted to palpation per rectum (PPR) using the fetal membrane slip (FMS) technique once between days 34 and 41 of pregnancy. The fetal membrane slip consisted of compressing the pregnant uterine horn and allowing the chorioallantoic membrane to slip between the fingers. Both groups were submitted to two additional TRUS at days 45 and 60 of pregnancy, to monitor the potential immediate and delayed deleterious effects of PPR on embryo and fetal viability, respectively. A diagnosis of embryo/fetal death was made when there was no embryo/fetal heart beat or the absence of positive signs of pregnancy in an animal previously diagnosed pregnant, or the presence of signs of embryo/fetal degeneration. The overall rate of embryo/fetal death was 14.0% (73/520). Embryonic death (10%; 52/520) was higher than fetal death (4.5%; 21/468; P<0.001). Embryo/fetal mortality was higher in cows (16.4%; 59/360) than in heifers (8.8%; 14/160; P<0.025) and in cattle with twin (25.5%; 12/47) versus singleton pregnancies (12.9%; 61/473; P<0.025), but was not different (P>0.05) between PAL (14.7%; 38/258) and NPAL (13.4%; 35/262). In conclusion, PPR between days 34 and 41 of pregnancy using the fetal membrane slip technique did not affect embryo/fetal viability.

Cloning of exotic/endangered species: desert bighorn sheep.

Cloning using somatic cell nuclear transfer (SCNT) may be a useful tool for conserving genetic diversity and for propagating exotic and/or endangered animal species. Somatic cells can be obtained easily, expanded in culture, cryopreserved, and thawed at a later date for use in NT. Significant challenges relevant to using SCNT for cloning wild and endangered animal species include the need for using interspecies NT and interspecies embryo transfer. Animal care and welfare issues raised that are unique to exotic and endangered species also are raised. In this chapter, the methods used in attempts to clone the wild animal species of Desert Bighorn Sheep are described.

Early pregnancy diagnosis by transrectal ultrasonography in dairy cattle.

The objective of the present study was to determine differences in time of detection of pregnancy between heifers and cows and the interval after insemination at which the maximum sensitivity and negative predictive value of transrectal ultrasonography were obtained. One-thousand-four-hundred transrectal ultrasonographies (TRUS-1; 1,079 in cows and 321 in heifers) were performed using a 5-MHz linear-array transducer. The cattle were randomly assigned to have TRUS performed once between days 24 and 30 (estrus=day 0) in cows or between days 21 and 27 in heifers. Every TRUS diagnosis was subsequently compared with a second TRUS diagnosis (TRUS-2), performed 3-8 days later, after day 30 (range 31-38) for cows and after day 27 (range 28-35) for heifers. The sensitivity and specificity between cows and heifers for the common days of TRUS (from 24 to 27) were compared. In cows, sensitivity increased gradually from 74.5% at day 24 to 100% at day 29 (P<0.01). Specificity increased from days 24-25 and reached a plateau of 96.6% on day 26 (P<0.01). In heifers, sensitivity increased from 50% at day 21 to 100% at day 26 (P<0.01). Specificity increased from 87.5% at day 21 and remained steady at 94% starting on day 23 (P>0.05). The sensitivity for cows and heifers was 89.2 and 96.8%, respectively (P<0.05) and the specificity was 93.0 and 93.4% (P>0.05). In this study, heifers were diagnosed pregnant earlier than cows, and the maximum sensitivity and negative predictive value were obtained 3 days earlier in heifers than cows (days 26 and 29, respectively).

Suppression of prion protein in livestock by RNA interference.

Given the difficulty of applying gene knockout technology to species other than mice, we decided to explore the utility of RNA interference (RNAi) in silencing the expression of genes in livestock. Short hairpin RNAs (shRNAs) were designed and screened for their ability to suppress the expression of caprine and bovine prion protein (PrP). Lentiviral vectors were used to deliver a transgene expressing GFP and an shRNA targeting PrP into goat fibroblasts. These cells were then used for nuclear transplantation to produce a cloned goat fetus, which was surgically recovered at 81 days of gestation and compared with an age-matched control derived by natural mating. All tissues examined in the cloned fetus expressed GFP, and PCR analysis confirmed the presence of the transgene encoding the PrP shRNA. Most relevant, Western blot analysis performed on brain tissues comparing the transgenic fetus with control demonstrated a significant (>90%) decrease in PrP expression levels. To confirm that similar methodologies could be applied to the bovine, recombinant virus was injected into the perivitelline space of bovine ova. After in vitro fertilization and culture, 76% of the blastocysts exhibited GFP expression, indicative that they expressed shRNAs targeting PrP. Our results provide strong evidence that the approach described here will be useful in producing transgenic livestock conferring potential disease resistance and provide an effective strategy for suppressing gene expression in a variety of large-animal models.

Activation of equine nuclear transfer oocytes: methods and timing of treatment in relation to nuclear remodeling.

Early development of embryos produced by transfer of equine nuclei to bovine cytoplasts is superior to that of intraspecies equine nuclear transfer embryos. This may be related to differences in chromatin remodeling or efficiency of activation between the two oocyte types. The pattern of donor nucleus remodeling was examined in equine-equine and equine-bovine reconstructed oocytes. Chromosome condensation occurred in equine cytoplasts by 2 h but was not seen in bovine cytoplasts until 4 h. We investigated the effect of activation of equine-equine reconstructed oocytes at <30 min or at 2 h after reconstruction. Four activation treatments were evaluated at each time point: injection of sperm extract alone, or in combination with 6-dimethylaminopurine (6-DMAP), cytochalasin B, or 1% dimethylsulphoxide. There was no significant difference in normal cleavage rate or average nucleus number of embryos between equine oocytes activated <30 min or at 2 h after reconstruction. The combination of 6-DMAP with sperm extract significantly (P < 0.01) improved cleavage rate compared with the other three treatments. Activation with sperm extract and 6-DMAP 2 h after donor nucleus injection gave the highest cleavage (79%) and the highest cleavage with normal nuclei (40%). Sperm extract and 6-DMAP also effectively activated oocytes parthenogenetically, yielding 83% cleavage and 73% cleavage with normal nuclei. These results indicate that although nuclear remodeling occurs rapidly in equine cytoplasts, early activation does not improve embryonic development after reconstruction.

Influence of maturation culture period on the development of canine oocytes after in vitro maturation and fertilization.

The objective of this study was to determine an optimum maturation period of canine oocytes for the development in vitro after in vitro fertilization (IVF). Canine oocytes larger than 110 micrometers in diameter, which were collected from ovaries at the follicular phase of the reproductive cycle, were cultured for each time (48, 72 and 96 h) in TCM 199 medium supplemented with 10% canine serum, fertilized, and then cultured in vitro for 8 days. Significantly more oocytes reached metaphase II (MII) in the 72-h culture group than in the 48-h culture group (25.6% vs. 41.0%). The percentages of oocytes that reached MII or beyond after maturation culture did not differ significantly between the 72- and 96-h culture groups, but the percentage of parthenogenetically activated oocytes in the 96-h culture group was significantly higher than that in the 72-h culture group. The percentages of cleaved embryos after IVF were significantly higher in the 48- and 72-h culture groups than in the 96-h culture group. In the 48-h culture group, 3.9% of fertilized oocytes developed to the 16-cell stage or beyond, but none of the cleaved embryos in the 72- and 96-h culture groups developed to the same stage. These results indicate that full nuclear maturation of oocytes collected from ovaries at the follicular phase occurs after 72 h of in vitro culture. However, an optimum maturation period (48 h) for the in vitro development of canine oocytes after IVF may be different from the period necessary to reach the maximal oocyte maturation rate, when based on the developmental stage of the cleaved embryos.

Analysis of DNA (cytosine 5) methyltransferase mRNA sequence and expression in bovine preimplantation embryos, fetal and adult tissues.

Mammalian preimplantation development is a critical stage for establishment of the genomic methylation pattern and proper function of the enzymes responsible for this appear essential for normal development. To date, the vast majority of work concerning the developmental expression of the DNA cytosine 5-methyltansferases (Dnmts) has been conducted in mice. Here we report the sequence and expression of the Dnmt family during bovine preimplantation and fetal development. Bovine Dnmt mRNAs display strong sequence homology to those of human and mouse and similar to other species, exist as multiple isoforms. Two of these splice variants, which have been termed Dnmt2gamma and Dnmt3a4, represent previously unreported sequence combinations. Work presented here demonstrates early bovine embryos express mRNA coding for the somatic form of Dnmt1 and that this transcript fractionates with the ribosome. Unlike the murine model, mRNA encoding the de novo methyltransferases, Dnmt3a and 3b are present during preimplantation development and can also be found in the ribosomal subcellular fraction. Further, results of Real Time PCR analysis indicate significant differences in Dnmt mRNA expression levels exist among different tissue types as well as between fetal and adult stages. Recently, it has been postulated that the cause of abnormal methylation observed in cloned embryos may be due in part to misexpression of the Dnmt1o isoform during preimplantation development. Work presented here raises new and significant hypotheses that must be considered both regarding the cadre of DNA methyltranferases that direct epigenetic programming during normal development and regarding the implication of abnormal DNMT expression in cloned embryos.

Effect of co-culture with theca interna on nuclear maturation of horse oocytes with low meiotic competence, and subsequent fusion and activation rates after nuclear transfer.

We conducted this study to examine whether or not co-culture with theca cells improves the maturation rate of horse oocytes with compact cumuli and to evaluate the cytoplasmic competence of oocytes after maturation by assessing fusion, activation and cleavage rates after nuclear transfer. We collected oocytes by scraping follicles from slaughterhouse-derived ovaries and classified them as having an expanded or a compact cumulus. Expanded oocytes were matured in M199 supplemented with 10% FBS and 5 microU/ml FSH for 24 h: compact oocytes were cultured in the same medium, or they were co-cultured in the same medium with theca interna explants, for 24 or 42 h. Oocytes were held with or without 10 microg/ml cytochalasin B, before washing and micromanipulation. and they were fused with donor fibroblasts by electrical pulse. Fused oocytes were activated with Ca ionophore/cycloheximide, cultured for 5 days, and stained with Hoechst to assess nuclear development. We considered oocytes with an enlarged nucleus, or having cleavage with multiple nuclei, to be activated. There was no significant difference in overall maturation rate between compact oocytes cultured with theca and compact controls. When these two groups were combined, there was a significant increase in the proportion of oocytes in MII between 24 and 42 h (P < 0.05). Expanded oocytes had a significantly higher rate of maturation than did compact oocytes (64% versus 25-30%; P < 0.001). There were no significant differences in rates of successful enucleation, fusion, activation or cleavage between compact control and compact + theca oocytes, nor between compact and expanded oocytes; however, expanded oocytes treated with cytochalasin B had a significantly higher survival rate after enucleation than did untreated expanded oocytes (P < 0.05). Three embryos developed from recombined oocytes, with maximum cleavage to 10 cells. The results of this study indicate that co-culture with theca cells does not increase either nuclear or cytoplasmic maturation of compact oocytes. Cytochalasin B is helpful in increasing survival of horse oocytes during enucleation. In vitro matured equine oocytes have the potential to develop into embryos after nuclear transfer; this is the first full report of production of cloned embryos in this species.