W.A. King

Genetic regulation of embryo death and senescence

The survival of the preimplantation mammalian embryo depends not only on providing the proper conditions for normal development but also on acquiring the mechanisms by which embryos cope with adversity. The ability of the early conceptus to resist stress as development proceeds may be regulated by diverse factors such as the attainment of a cell death program and protective mechanisms involving stress-induced genes and/or cell cycle modulators. This paper reviews the recent research on the genetic regulation of early embryo cell death and senescence focussing on the bovine species where possible. The different modes of cell death will be explained, clarifying the confusing cell death terminology, by advocating the recommendations set forth by the Cell Death Nomenclature Committee to extend to the embryology research field. Specific pro-death and anti-death genes will be discussed with reference to their expression patterns during early mammalian embryogenesis.

The sex ratios of bovine embryos produced in vivo and in vitro

The male:female ratio of developing bovine embryos produced and allowed to develop in vitro and in vivo was determined retrospectively from the cytogenetic analysis of 804 embyos. The overall male:female ratio of the 307 (38%) embryos that could be sexed was 162 (52.8%):145 (47.2%) and did not differ (P>0.05) from the expected 1:1 ratio. Among premorula stage embryos produced in vivo (n = 66) and in vitro (n = 30), the ratios were 1.2:1 and 0.76:1, respectively. Among morulae and blastocysts produced in vivo (n = 74), produced and cultured in vitro (n = 106, and produced in vitro and cultured in vivo (n = 31), the ratios were 1.11:1, 1.3:1 and 0.94:1, respectively, none of which differed significantly from 1:1. There was no difference (P>0.05) in the number of cells or mitotic index between male and female morulae and blastocyst, respectively.

Endocrine and behavioral events of estrous cyclicity and synchronization in wood bison ( Bison bison athabascae )

Abstract This study was designed to noninvasively characterize estrous cycles and evaluate the effectiveness of estrous cycle synchronization protocols in wood bison. Daily urine and fecal samples and behavioral observations were collected over 2 breeding seasons from a captive herd of 18 adult, female wood bison. All samples were analyzed by enzymeimmunoassay for pregnanediol-glucuronide (PdG: urine) or progesterone (P4: feces). In Year 1, animals were divided into 3 treatment groups: 1) natural estrous controls, n = 6; 2) PG, 2 injections of cloprostenol, 11 d apart, n = 6; 3) SMB, Syncro-Mate-B implants left in place for 9 d with an estradiol valerate injection at the time of implantation, n = 6. In Year 2, control animals remained the same and synchronization regimens were repeated, but with each animal receiving alternate treatments. In both years, 11 d after treatment cessation, females were subjected to ultrasound examination to evaluate ovarian activity. Combined endocrine and behavioral data from natural estrous cycles demonstrated seasonal effects, with mean cycle lengths of 20.8 ± 0.3, 21.5 ± 0.5 and 21.1 ± 0.4 d, based on behaviors, PdG and P4 respectively. Females which demonstrated behavioral estrus did so within 4 (SMB, 11 12 ) or 5 (PG, 10 12 ) days after treatment. The SMB-treated animals exhibited estrous behavior within a 3-d span, with 55% exhibiting estrus 3 d after treatment ended, while the onset of estrus in PG-treated animals occurred over 4 d, with only 40% occurring on a single day. At the second estrus, 77% of SMB-treated animals and 74% of PG-animals demonstrated estrous behavior within 4 d, while at the third estrus, 88 and 90% of treated animals showed estrous behavior within 3 or 4 d, respectively. Ultrasound assessment revealed that only 50% ( 12 24 ) of treated animals possessed corpora lutea (CL), 67% in PG-treated females and 33% in SMB-animals. The total number of CL tended to be greater in PG- (10) than in SMB-(5) treated animals. Females with no CL demonstrated attenuated progestin profiles compared to those with ovulations. Additionally, a total of 28 follicles was detected, with a greater proportion (P

Serum-Starved Apoptotic Fibroblasts Reduce Blastocyst Production but Enable Development to Term after SCNT in Cattle

Cell cycle synchronization by serum starvation (SS) induces apoptosis in somatic cells. This side effect of SS is hypothesized to negatively affect the outcome of somatic cell nuclear transfer (SCNT). We determined whether apoptotic fibroblasts affect SCNT yields. Serum-starved, adult, bovine fibroblasts were stained with annexin V-FITC/propidium iodide to allow apoptosis detection by flow cytometry. Positive and negative cells sorted by fluorescence activated cell sorting (FACS) and an unsorted control group were used as nuclear donors for SCNT. Reconstructed embryos were cultured in vitro and transferred to synchronized recipients. Apoptosis had no effect on fusion and cleavage rates; however, it resulted in reductions in blastocyst production and quality measured by apoptotic index. However, reconstructed embryos with apoptotic cells resulted in pregnancy rates similar to that of the control on day 30, and generated one live female calf. In conclusion, we showed that apoptotic cells present in serum-starved cultures negatively affect embryo production after SCNT without compromising full-term development. Further studies will evaluate the ability of the oocyte to reprogram cells in specific phases of apoptosis.

Hormonal control of estrouscyclicity and attempted superovulation in wood bison (Bison bison athabascae)

The wood bison (Bison bison athabascae) is a threatened Canadian species that has faced extinction twice in the last 100 yr. Development of assisted reproductive technologies could help ensure the long-term propagation and genetic management of this species. The objectives of this study were to refine estrus synchronization techniques and evaluate superovulatory responses after FSH or eCG administration. In Experiment 1, females were fitted with Syncro-mate B (SMB) implants for 9 d and received an injection of either estradiol valerate (E2V; n = 9) or cloprostenol (PGF; n = 9) at implant insertion (Day-9). In Experiment 2, estrus was synchronized with SMB implants and a PGF injection of Day-9, and superovulation was attempted on Day-2 with either 2500 IU eCG (n = 5) or 400 mg Folltropin-V (n = 5). In each experiment, biosin were examined daily for estrual behavior. Ultrasonography was used during the luteal phase to detect ovulation and assess ovarian status; feces were analyzed by ELISA for immunoreactive progestogens (P) to study ovarian endocrine responses. In Experiment 1, a closer synchrony of estrus was observed between Days 2 to 4 among the PGF-treated (77.8%) than the E2V-treated (66.7%) females. Corpora lutea (CL) were detected in 55% of E2V- and PGF-treated females. In Experiment 2, neither treatment successfully induced superovulation, with only a single female per treatment producing > or = 1 CL. In both experiments, progestogen profiles were similar for each treatment (P < 0.05).

Somatic Mosaicism in Bulls Estimated from Genome-Wide CNV Array and TSPY Gene Copy Numbers.

Somatic mosaicism has become a focus in human research due to the implications of individual genetic variability in disease. Here, we assessed somatic copy number variations (CNVs) in Holstein bulls in 2 respects. We estimated genome-wide CNVs and assayed CNVs of the TSPY gene, the most variable bovine gene from the Y chromosome. Somatic tissues (blood, lung, heart, muscle, testis, and brain) of 4 bulls were arrayed on the Illumina Bovine SNP50k chip and qPCR tested for TSPY copy numbers. Our results showed extensive copy number divergence in tissues within the same animal as well as significant copy number alterations of TSPY. We detected a mean of 31 CNVs per animal among which 14 were of germline origin, as they were constantly present in all investigated tissues of the animal, while 18 were specific to 1 tissue. Thus, 57% of the total number of detected CNVs was the result of de novo somatic events. Further, TSPY copy number was found to vary significantly among tissues as well as among the same tissue type from different animals in a wide range from 7 to 224% of the calibrator. Our study shows significant autosomal and Y-chromosomal de novo somatic CNV in bulls.

The Porcine TSPY Gene Is Tricopy but Not a Copy Number Variant.

The testis-specific protein Y-encoded (TSPY) gene is situated on the mammalian Y-chromosome and exhibits some remarkable biological characteristics. It has the highest known copy number (CN) of all protein coding genes in the human and bovine genomes (up to 74 and 200, respectively) and also shows high individual variability. Although the biological function of TSPY has not yet been elucidated, its specific expression in the testis and several identified binding domains within the protein suggests roles in male reproduction. Here we describe the porcine TSPY, as a multicopy gene with three copies located on the short arm of the Y-chromosome with no variation at three exon loci among 20 animals of normal reproductive health from four breeds of domestic pigs (Piétrain, Landrace, Duroc and Yorkshire). To further investigate the speculation that porcine TSPY is not a copy number variant, we have included five Low-fertility boars and five boars with exceptional High-fertility records. Interestingly, there was no difference between the High- and Low-fertile groups, but we detected slightly lower TSPY CN at all three exons (2.56-2.85) in both groups, as compared to normal animals, which could be attributed to technical variability or somatic mosaicism. The results are based on both relative quantitative real-time PCR (qPCR) and droplet digital PCR (ddPCR). Chromosomal localization of the porcine TSPY was done using fluorescence in situ hybridization (FISH) with gene specific PCR probes.

Highly dynamic temporal changes of TSPY gene copy number in aging bulls

The Y-chromosomal TSPY gene is one of the highest copy number mammalian protein coding gene and represents a unique biological model to study various aspects of genomic copy number variations. This study investigated the age-related copy number variability of the bovine TSPY gene, a new and unstudied aspect of the biology of TSPY that has been shown to vary among cattle breeds, individual bulls and somatic tissues. The subjects of this prospective 30-month long study were 25 Holstein bulls, sampled every six months. Real-time quantitative PCR was used to determine the relative TSPY copy number (rTSPY CN) and telomere length in the DNA samples extracted from blood. Twenty bulls showed an altered rTSPY CN after 30 months, although only 9 bulls showed a significant change (4 significant increase while 5 significant decrease, P<0.01). The sequential sampling provided the flow of rTSPY CN over six observations in 30 months and wide-spread variation of rTSPY CN was detected. Although a clear trend of the direction of change was not identifiable, the highly dynamic changes of individual rTSPY CN in aging bulls were observed here for the first time. In summary we have observed a highly variable rTSPY CN in bulls over a short period of time. Our results suggest the importance of further long term studies of the dynamics of rTSPY CN variablility.