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Oviduct epithelial cell co-culture of early porcine embryos.

One- to 16-cell porcine embryos were cultured in either Whittens medium supplemented with bovine serum albumin and fetal calf serum (WM) or in the same medium with porcine oviduct epithelial cell co-culture (WM-Poec). All stages of embryos cultured in WM-POEC had higher cell counts after 144-168 h of development than did embryos in WM. There was however, no significant difference in blastocyst formation rate of embryos cultured in WM-POEC over those cultured in WM. A high proportion of the embryos entering culture at the 1-2-cell were able to pass the 4-cell block stage in both WM and WM-POEC, 81% and 77%, respectively. In both media, most of the 1-2-cell embryos arrested their development at the compacted morula stage and failed to blastulate while embryos initiating culture at the 4- and 8-16-cell embryos formed blastocysts in culture at a rate of 80-90%.

Animals

Characterization of deoxyribonucleic acid synthesis and the transition from maternal to embryonic control in the 4-cell porcine embryo.

These studies were conducted to identify the point during the 4-cell stage at which the porcine embryo begins to control development. Reproductive tracts of gilts were flushed 48 h after the onset of estrus to obtain 1- and 2-cell embryos. To determine the duration of the 4-cell stage in vitro, development of 29 embryos was timed from cleavage to the 4-cell stage and from cleavage to the 8-cell stage. The average duration of the 4-cell stage was 50.5 h. The duration of the 4-cell stage was positively correlated (p < 0.01) with culture time in vitro before cleavage to the 4-cell stage. DNA content was determined by using the Feulgen's reaction and quantified with micro-densitometry. Staining units (SU; density x area) were calculated at 0, 2, 4, 6, 8, 10, 12, 16, 20, 24, 30, and 36 h post-cleavage to the 4-cell stage (P4C). Results revealed a possible G1 phase (< 2 h) with DNA synthesis starting within 2 h P4C. DNA synthesis was completed by 16 h P4C, and was followed by an extended G2 phase. Embryos were evaluated for uptake and incorporation of [35S]methionine and for qualitative changes in protein profiles specific to time points during the 4-cell stage (2, 10, 14, 16, 18, 24, 30, and 40 h P4C). Methionine uptake and incorporation into protein followed similar patterns, both decreasing until 16-18 h P4C, followed by a steady increase through the 4-cell stage. Protein profiles revealed qualitative changes beginning at 14 and 16 h P4C.(ABSTRACT TRUNCATED AT 250 WORDS)

Amanitins

Influence of sexual maturity of donor on in vivo survival of transferred porcine embryos.

A study was conducted to determine whether embryos recovered from first-estrous (pubertal) and second-estrous gilts differed in survival when transferred to first- or third-estrous recipients. Embryos were recovered surgically from first- and second-estrous donors 48-72 h postmating and 6-10 normal embryos/zygotes (1-4 cells) were transferred to oviducts (3-5 embryos/ampulla) of nonmated synchronous first- (n = 40) or third- (n = 15) estrous recipients. Blood samples were collected from the jugular vein of recipient gilts on Days 3, 12, and 30 of gestation and the sera were analyzed for progesterone and free (unconjugated) estrogens by use of radioimmunoassays. Recipient gilts were subsequently slaughtered between Days 30 and 40 to assess embryonic losses. Mean number of ovulations was lower among first-estrous vs. third-estrous recipients (8.9 +/- 0.7 vs. 11.4 +/- 0.7; p < 0.05). Percentage of recipients that maintained pregnancy was similar between first- and third-estrous gilts (67.5 vs. 60.0%) and recovery of total conceptuses (normal and degenerating) resulting from transfer of one-cell- and cleavage-stage embryos did not differ among first- vs. third-estrous gilts (76.1 vs. 78.2%). Similarly, percentage of viable fetuses in first-estrous gilts that were pregnant from transfer of one-cell- and cleavage-stage embryos was not different from that of third-estrous gilts (69.3 vs. 75.6%). Percentages of total conceptuses and viable fetuses in first- and third-estrous gilts that were recipients of cleavage-stage embryos only also did not differ (p > 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors

SET domain bifurcated histone lysine methyltransferase 1 regulates histone modification and DNA damage response during zygotic genome activation in pigs.

SET domain bifurcated histone lysine methyltransferase 1 (SETDB1) is a key epigenetic regulator that catalyzes histone H3 lysine 9 trimethylation (H3K9me3), a mark essential for transcriptional repression and heterochromatin formation. Here, we investigated the role of SETDB1 during zygotic genome activation (ZGA) in porcine embryos. SETDB1 knockdown (KD) was induced by microinjecting double-stranded RNA (dsRNA), and its impact on early embryonic development was evaluated. SETDB1 KD decreased H3K9me3 levels, markedly increased H3K9ac, and downregulated ZGA-associated genes. These epigenetic alterations were accompanied by impaired cleavage, reduced blastocyst formation, and a lower total cell number. Upon etoposide-induced DNA double-strand breaks, SETDB1 KD embryos showed reduced expression of key DNA repair proteins, failed to efficiently restore DNA integrity, and exhibited increased apoptosis, indicating a compromised DNA damage response and repair process. SETDB1 KD also reduced HDAC3 expression, suggesting that SETDB1 may regulate HDAC3 to maintain histone acetylation balance. Consistently, HDAC3 inhibition increased H3K9ac, decreased H3K9me3, and reduced SETDB1 protein levels, supporting a reciprocal regulatory relationship. Together, these findings indicate that SETDB1 is important for porcine embryonic development by coordinating histone modifications and safeguarding genomic integrity during ZGA, and they suggest that the interplay between SETDB1 and HDAC3 constitutes a potentially important epigenetic axis for proper histone modification dynamics and developmental competence.

Animals

Porcine conceptus proteins: antiviral activity and effect on 2',5'-oligoadenylate synthetase.

Interferon-like proteins synthesized by conceptuses of domestic ruminants inhibit luteolysis during early pregnancy. Although pig conceptuses secrete trophoblast interferons during the period of CL maintenance, estrogen is involved with maintenance of the CL. The principal purposes of this work were to confirm production of trophoblast interferons by porcine conceptuses and to compare the effect of trophoblast interferons on endometrium of pigs and cattle. When measured using Madin-Darby bovine kidney (MDBK) cells challenged with vesicular stomatitis virus, antiviral activity in uterine flushings from cyclic gilts was not detectable throughout the estrous cycle; however, in pregnant gilts, antiviral activity increased from undetectable amounts to 4-11 x 10(3) U on Days 14, 16, and 18. Porcine embryos in culture produced 1,100 U/embryo/ml/24 h. Porcine conceptus secretory proteins induced 2',5'-oligo(A) synthetase in MDBK cells and in endometrial explants of cows but had no measurable effect on 2',5'-oligo(A) synthetase activity of endometrial explants of pigs. Similarly, endometrial 2',5'-oligo(A) synthetase of pregnant pigs was unaffected in vivo during the period of maximal synthesis of conceptus secretory proteins. Porcine conceptus secretory proteins produced no detectable increase in serum antiviral activity or 2',5'-oligo(A) synthetase activity of blood mononuclear leukocytes in utero-ovarian venous blood. These results suggest that conceptus interferons of pigs play different roles in the establishment of pregnancy compared to their roles in ruminants.

2',5'-Oligoadenylate Synthetase

[Effect of cycloheximide on the ultrastructure and several metabolic processes of the cells of an SPEV culture. II].

The action of cyclohexamide (in doses of 1 and 10 mkg/ml in the course of 24 hours) on porcine embryo kidney cells in culture is accompanied by a powerful suppression of protein and DNA synthesis, mitotic activity, a weaker suppression of RNA synthesis, a lowering of the activity of succinate-, lactate- and alpha-glycerophosphate-dehydrogenase, which leads to disorders in the ultrastructure of the cells. After incubation of cells in a fresh medium (in the course of 18, 24 hours) there occurs a total restoration of the ultrastructure of the nuclei, granular endoplasmatic reticulum, mitochondria, due to the repairing and strong intensification of synthetic processes, respiration and glycolysis, mitotic activity of the cells.

Animals

[Effect of mitomycin C on SPEV cell cultures].

Mitomycin C (in doses of 0.5-1.5 mkg/ml during 24-72 hours) significantly changes the ultrastructure and morphology of porcine embryo kidney cells (condensation of chromatin and the mitochondrial matrix, expansion of small channels in the endoplasmatic reticulum, total hypertrophy of the nuclei and cells). In the given case, mytomycin C sharply inhibits DNA synthesis and mitotic activity, considerably more weakly reduces RNA and protein synthesis, raises the activity of lactate and alpha-glycerophosphate-dehydrogenases. The disbalance of syntheses leads to protein accumulation in the cells, and general enlargement of nuclei and cells. As the action of the antibiotics increases, the ultrastructural changes progress and lead to the destruction of a considerable part of cells in the culture.

Animals

[Repair of damage to cells in an SPEV culture after exposure to mitomycin C].

The action of mitomycin C on a porcine embryo kidney culture (in dose of 0.5-1.5 mkg/ml in the course of 24-72 hours) is accompanied by significant changes in the ultrastructure and morphology of cells. Moreover, mitomycin C sharply inhibits DNA synthesis, mitotic activity and much more weakly suppresses protein and RNA syntheses. After a prolonged (48 hours) washing of the antibiotics only the mitochondrial ultrastructure is restored in the fresh cultural medium. DNA synthesis and mitotic activity remain suppressed, while protein and RNA syntheses increase sharply, which leads to protein accumulation in cells, and to the enlargement of nuclei, nucleoli and cells. Such changed cells are unable to keep on living and perish.

Cell Line

L-glutamine supplementation improves porcine sperm quality and early embryo development during in vitro fertilization.

L-glutamine (Gln), as a key additive in porcine sperm capacitation medium and in vitro fertilization (IVF) systems, has been shown to significantly improve sperm motility and survival rates. However, its precise roles during porcine IVF and subsequent early embryonic development remain elusive. This study utilized an IVF model in pigs to investigate the effects of glutamine on sperm quality and embryonic development. We found that Gln supplementation during sperm treatment significantly improved sperm quality, as evidenced by reduced reactive oxygen species (ROS) production and early apoptosis, while enhancing calcium ion levels and endoplasmic reticulum activity. Supplementing glutamine during embryo culture reduced polyspermy rates, promoted zygotic genome activation (ZGA) and accumulation of 5-ethynyluridine (EU) and histone modifications (H3K4me3 and H3K27ac) at the two-cell and four-cell stages, increased blastocyst formation rates and total cell numbers, while simultaneously reducing DNA damage and early apoptosis during the blastocyst stage. In summary, these findings demonstrate that Gln enhances porcine IVF outcomes by improving sperm quality, reducing polyspermy, and facilitating early embryonic development, thereby providing a basis for optimizing culture systems.

Animals

Growth, protein content and distribution of early pig embryos.

Development of porcine conceptuses included transitions of five stages: blastocysts of spherical, ovoid, and tubular forms containing an embryonic disc and trophoblast, extremely elongated filamentous blastocysts, and stages of embryogenesis between days 9 and 18 after mating. Embryonic survival was reduced by 17% during this period. In this litter-bearing species, intense alteration in distribution patterns occurred at days 11 and 12, when blastocysts rapidly elongated to filamentous forms. Increased embryo mortality did not result from rapid changes in distribution patterns of conceptuses within the same uterine horn at this time. Filamentous blastocysts quickly reached lengths often exceeding 60 cm, and these conceptuses became regularly spaced with no overlap of tubular membranes from other embryos in that horn. The number of conceptuses within a uterine horn ranged from 1 to 13. Protein in individual conceptuses was used as an indicator of growth and denoted exponential increase, but at a lower rate for blastocysts of spherical, ovoid, and tubular forms as compared with that found in filamentous blastocysts and embryogenesis stage conceptuses. Growth on a conceptus, based upon its protein content, was independent of the developmental stage or potential loss of those neighbors nearest that conceptus.

Animals

[Immunoelectrophoretic study of serum proteins of swine embryos].

During embryonic development, antigenic spectrum of serum proteins from porcine foetuses gradually become more complex, the number of antigens increasing from 4 in 1-month embryos to 12--13 in mature foetuses. Immunoelectrophoretic analysis of the serum reveals prealbumin, alpha- and beta-globulins. It was shown that with respect to its immunological properties, procine fetuin is not presented by a true stage-specific protein, since together with ESA-glogulin it contains antigens which are identical to those in the blood serum of adult animals. Immunoelectrophoresis did not reveal gamma-globulins in embryonic serum. It is suggested that porcine foetuses may synthesize the main proteins of the blood serum; however, final formation of antigenic spectrum is accomplished in postembryonic period.

Animals

Establishment of the origin of a porcine cell line by chromosome banding techniques and scanning.

Use of cultured cells in diagnostic virology implies the necessity to establish the genetic origin of the cells. Chromosome analysis, performed on a cell line of pig embryo kidney cells (PEK), made the porcine origin of the cells plausible. Definite proof, however, was furnished by chromosome banding and scanning. A comparison was made between PEK-cells and normal pig chromosomes. One of the chromosomes No. 1 had an interstitial deletion, which provided an useful marker for identification purposes.

Animals

Rapid increase in relaxin gene expression in early pregnancy in the pig.

Relaxin mRNA concentrations in porcine corpora lutea were examined during the peri-implantation period and throughout pregnancy using Northern and slot blot analysis. Total RNA was extracted from corpora lutea obtained from pigs of known breeding dates and pregnancy was confirmed by embryo recovery. A 32P-labelled porcine relaxin cDNA probe identified the 1.0 kilobase relaxin transcript. Slot blots were subsequently used to quantify relaxin mRNA concentrations. Relaxin mRNA was detectable in the corpus luteum of the regular cycle and was also present at similar low levels in corpora lutea of days 10, 11 and 12 of pregnancy. In corpora lutea from day 16 of pregnancy onwards 100-fold greater quantities of relaxin mRNA were observed. The intensity remained similar in samples between days 16 and 102 of pregnancy. These studies indicate that elevated relaxin gene expression commences very early in pregnancy and is first detectable in the peri-implantation period.

Animals

Possible association of oncornavirus type D with some forms of human cancer.

The method of molecular hybridization of nucleic acids of oncornavirus type D produced by HEp2 cells with cellular nucleic acids was applied. No sequences homologous to the viral nucleic acids were found in normal human embryo cells as well as in bovine, porcine, murine, and chicken cells. No such sequences were found in tissues of malignant lymphomas. Sequences homologous to the viral nucleic acids were found in some cancer cells, predominantly in hormone-associated tumors of females.

Animals

Stage-specific remodeling of wingless-related integration sites (WNT) signaling during oocyte-to-embryo transition in pigs.

The WNT signaling pathway is a central regulator of cell polarity, adhesion, cytoskeletal dynamics, and lineage specification during early embryonic development. Although its roles have been extensively studied in murine and human models, the temporal regulation and pathway architecture of WNT signaling during early porcine development remain poorly defined. Here, we performed a comprehensive transcriptomic analysis to characterize WNT pathway dynamics across key stages of pig in vitro development, including immature oocytes (IMO), mature oocytes (MO), zygotes (ZY), cleaved embryos (2-4 cells; CL), and blastocysts (BL). Global analyses revealed major transcriptomic transitions (FDR <0.05; |Fold Change| &#x2265;2) during oocyte maturation and blastocyst formation, whereas zygotes and cleaved embryos exhibited highly similar expression profiles. Module-based and gene-level analyses showed that oocyte maturation is associated with increased expression of extracellular WNT antagonists and components of the &#x3b2;-catenin destruction complex, together with selective regulation of Frizzled receptors, consistent with tight control of canonical WNT signaling at the MII stage. Following fertilization, this inhibitory configuration was partially relieved, alongside transient upregulation of specific WNT ligands, transcriptional mediators, and adhesion-related components during zygotic genome activation and early cleavage. At the blastocyst stage, WNT signaling became increasingly associated with planar cell polarity and epithelial organization modules. Together, the data reveal a highly dynamic and stage-specific restructuring of WNT signaling during early porcine development. Our findings indicate that precise temporal modulation-rather than uniform activation-of WNT pathway components accompanies the porcine oocyte-to-embryo transition, providing a molecular framework to better understand early developmental regulation and offering insights relevant to reproductive biotechnology and developmental biology.

Wnt Signaling Pathway

Prenatal infection following maternal exposure to porcine parvovirus on either the seventh or fourteenth day of gestation.

Intranasal and oral exposure of two gilts to porcine parvovirus on either the seventh or 14th day of gestation resulted in prenatal infection. Normal appearing fetuses and necrotic remnants of what were believed embryos and extraembryonic membranes were found when the gilts were necropsied seven weeks after exposure. The presence of masses of porcine parvovirus antigen throughout necrotic tissues of six of seven embryos, but not in any of the nine normal appearing fetuses suggested that embryonic death was due to porcine parvovirus.

Animals