W. A. King

Copy number variation of testis-specific protein, Y-encoded (TSPY) in 14 different breeds of cattle (Bos taurus).

Multi-copied gene families are prevalent in mammalian genomes, especially within the Y chromosome. Testis specific protein Y-encoded (TSPY) is present in variable copy number in many mammalian species. Previous studies have estimated that TSPY ranges from 50–200 copies in cattle. To examine TSPY localization on the Y chromosome we employed fluorescence in situ hybridization (FISH) and fiber-FISH. The results show a strong signal on the short arm of the Y chromosome (Yp). To investigate TSPY copy number we used relative real-time polymerase chain reaction (PCR) to analyze the DNA of 14 different cattle breeds. Variation both within and between breeds was observed. All breeds show significant variation in TSPY copy number among individual members. Brown Swiss (161 copies, CI = 133–195) had higher average levels of TSPY and Western Fjord Cattle (63 copies, CI = 45–86) had lower levels than some breeds. Overall, however, most breeds had a similar average TSPY copy number. The pooled average was 94 copies (CI = 88–100). The significance of the TSPY array remains uncertain, but as the function of TSPY is unraveled the purpose of the array may become clearer.

Equine Disorders of Sexual Development in 17 Mares Including XX, SRY-Negative, XY, SRY-Negative and XY, SRY-Positive Genotypes

We described the clinical, cytogenetic and molecular findings of 17 clinical equine cases presented for abnormal sexual development and infertility. Six horses with an enlarged clitoris had an XX, SRY-negative genotype, which displayed male-like behavior (adult individuals). Bilateral ovotestes were noted in 2 of those cases, while another case showed increased levels of circulating testosterone. Six horses with a female phenotype, including normal external genitalia, had an XY, SRY-negative genotype. These individuals had small gonads and an underdeveloped internal reproductive tract. Four horses with normal appearing external genitalia had an XY, SRY-positive genotype, 3 of them had hypoplastic testes and male-like behavior. In addition, one young filly with enlarged clitoris and hypoplastic testes had the same genotype but did not show male-like behavior due to her age. Three of these horses were related with 2 being siblings. These findings demonstrate the diversity of disorders of sexual development seen in the horse. Furthermore, they emphasize the need for further research to identify genes involved in abnormal sex determination and differentiation in the horse.

Thyroid hormone supplementation improves bovine embryo development in vitro

BACKGROUNDnEarly embryo development (EED) forms the basis of assisted reproductive technologies (ARTs), which are used to treat human infertility and to propagate other mammalian species. Thyroid hormones (THs) play an important role in the post-implantation development of the embryo in mammals; however, the effects of THs on pre-attachment embryos are not known. Currently utilized in-vitro embryo production media are devoid of THs and hence our main objective was to examine whether THs affected EED in a bovine model.nnnMETHODSnTo determine if THs are present at the site of fertilization and EED in cattle, we evaluated the presence of the hormones in oviductal and uterine horn tissues. To assess the outcome of free TH supplementation (50 ng/ml of each hormone: triiodothyronine-T3 and thyroxin-T4), embryos were followed through standard and TH-supplemented in-vitro procedures, and evaluated for the cleavage rates, blastocyst formation rate and hatching rates. Embryo quality was assessed using TUNEL assay and post-cryopreservation survival was also evaluated.nnnRESULTSnAlthough TH levels in in-vitro culture media were found to be approximately 60% of the administered doses, the TH-treated embryos exhibited significant increases in blastocyst formation and hatching rates (P < 0.05). Embryo quality was significantly improved in the treated groups as demonstrated by greater total cell counts and reduced proportions of apoptotic cells (P < 0.05). Finally, TH supplementation was associated with improved post-cryopreservation viability, defined by blastocyst re-expansion and hatching rates after frozen embryos had been thawed and cultured (P < 0.05).nnnCONCLUSIONSnThese findings not only provide a way of optimizing ART efficiency, but also further our understanding of how THs influence embryonic development in mammals.

Ovarian Responses, Hormonal Profiles and Embryo Yields in Anoestrous Ewes Superovulated with Folltropin®‐V after Pretreatment with Medroxyprogesterone Acetate‐releasing Vaginal Sponges and a Single Dose of Oestradiol‐17β

In ruminants, superovulatory treatments started at the time of follicular wave emergence result in greater and less variable ovulatory responses and embryo yields compared with the treatments begun in the presence of a large growing antral follicle(s) from the previous waves. The progesterone-oestradiol treatment is routinely used for follicular wave synchronization in cattle. The main objective of this study was to characterize the ovarian responses, hormonal profiles and in vivo embryo production in anoestrous Rideau Arcott ewes (May-June), which were superovulated after pretreatment with medroxyprogesterone acetate (MAP)-releasing intravaginal sponges and a single dose of oestradiol-17beta (E(2)-17beta). Six days after insertion of MAP sponges, eight ewes were given an i.m. injection of 350 microg of E(2)-17beta (E(2)-17beta-treated ewes); 10 ewes were given an i.m. injection of vehicle (control ewes). Multiple-dose Folltropin-V treatment, followed by the bolus injection of GnRH (50 microg i.m.), began 6 days after E(2)-17beta/vehicle injection. Transrectal ovarian ultrasonography revealed that: (i) the interval between E(2)-17beta/vehicle injection and regression of all follicles > or =5 to 3 mm in diameter was shorter (p < 0.01; 2.6 +/- 0.4 vs 4.8 +/- 0.6 days respectively); and (ii) the interval between injection and emergence of the next follicular wave was longer (p < 0.05; 5.4 +/- 0.3 vs 1.2 +/- 0.4 days, respectively) in E(2)-17beta-treated than in control ewes. During the 6 days after injection, the mean FSH peak concentration and basal FSH concentration were lower (p < 0.01) in E(2)-17beta-treated ewes. The mean ovulation rate and the number of recovered embryos did not differ (p > 0.05) between the two groups of ewes. However, the number of luteinized unovulated follicles per ewe, and the variability in the number of luteal structures and overall embryo yield were less (p < 0.05) in E(2)-17beta-treated compared with control ewes. In conclusion, the MAP-E(2)-17beta pretreatment significantly reduced the variability in ovarian responses and embryo yields, without affecting the embryo production in superovulated anoestrous ewes.

Spatial distribution of histone isoforms on the bovine active and inactive X chromosomes.

The inactive X chromosome (Xi) in female mammals serves as an important model for studying the role of histone isoforms in directing specific nuclear processes leading to inherited differences in transcription. In the present study, we investigated the distribution of some histone isoforms known to be involved in the process of human X inactivation on their bovine counterparts. To ascertain the identity of active and inactive X chromosome, their distribution was investigated on the X chromosomes in a cell line derived from a bovine female carrying an X;autosome translocation rcp(Xp+;23q–) which allowed the recognition of the maternal (translocated) and paternal (normal) X chromosome. The distribution patterns of histone H3 trimethylated at lysine 9 (H3K9me3) and trimethylated at lysine 27 (H3K27me3), and histone macroH2A1 and macroH2A2 (isoforms specific to heterochromatin) were determined by immunocytochemistry and compared to the temporal pattern of replication using BrdU pulse labeling prior to staining. Immunostaining revealed that H3K9me3, H3K27me3, and macroH2A1 are preferentially concentrated on the Xi, whereas the histone variant macroH2A2 is not a marker for this chromosome. H3K9me3, H3K27me3, and macroH2A1 were consistently located in bands along the Xi, while H3K9me3, macroH2A1 and macroH2A2 localized in the pericentromeric regions of the autosomes. H3K27me3 identified two intense bands on the Xi at Xp22 and Xq31, representing the early replication regions of the chromosome. H3K27me3 and macroH2A1 overlapped in the Xq31 region. It was concluded that different heterochromatin regions on the bovine inactive X chromosome can be identified by their histone isoform composition.

GTG Mutation in the Start Codon of the Androgen Receptor Gene in a Family of Horses with 64,XY Disorder of Sex Development

Genetic sex in mammals is determined by the sex chromosomal composition of the zygote. The X and Y chromosomes are responsible for numerous factors that must work in close concert for the proper development of a healthy sexual phenotype. The role of androgens in case of XY chromosomal constitution is crucial for normal male sex differentiation. The intracellular androgenic action is mediated by the androgen receptor (AR), and its impaired function leads to a myriad of syndromes with severe clinical consequences, most notably androgen insensitivity syndrome and prostate cancer. In this paper, we investigated the possibility that an alteration of the equine AR gene explains a recently described familial XY, SRYu200a+ disorder of sex development. We uncovered a transition in the first nucleotide of the AR start codon (c.1A>G). To our knowledge, this represents the first causative AR mutation described in domestic animals. It is also a rarely observed mutation in eukaryotes and is unique among the >750 entries of the human androgen receptor mutation database. In addition, we found another quiet missense mutation in exon 1 (c.322C>T). Transcription of AR was confirmed by RT-PCR amplification of several exons. Translation of the full-length AR protein from the initiating GTG start codon was confirmed by Western blot using N- and C-terminal-specific antibodies. Two smaller peptides (25 and 14 amino acids long) were identified from the middle of exon 1 and across exons 5 and 6 by mass spectrometry. Based upon our experimental data and the supporting literature, it appears that the AR is expressed as a full-length protein and in a functional form, and the observed phenotype is the result of reduced AR protein expression levels.

Erythrocyte osmotic response test on malignant hyperthermia-susceptible pigs

Erythrocytes harvested from a group of 17 young Belgian Landrace male pigs, of which io were classified as malignant hyperthermia susceptible by exposure to halothane anesthetic and were classified as non-susceptible, were exposed to osmotic shock in saline solutions at o. 9 percent, 0.8 percent and 0.7 percent. The results show an increased rate of hemolysis in susceptible pigs as compared to non-susceptible ones. This is in favour of the hypothesis of a membrane defect being at the origin of malignant hyperthermia, an abnormality which is generally associated with an increased muscular development. Similar results obtained in cattle by KiNG et al (197 6) indicate that the same defect occurs in so-called double-muscled animals.

Osmotic response tests on erythrocytes for the detection of double muscle carriers in cattle

Erythrocyte harvested from heparinized whole blood of a group of cattle of various breeds including overtlydouble muscled animals and more or less normal carriers of the trait revealed an increased rate of hemolysis on exposure to osmotic shock as compared to normal Charolais cattle. There is also a clearcut distinction between the rate of hemolysis of double muscled animals and normal carriers detected because they have some affinity with the double muscled and have transmitted the trait. They all seem in our samples to be heterozygotes in the generally admitted hypothesis that double muscling is due to a simple mendelian factor. It is hence suggested that the erythrocyte fragility test described in this report may be used for the identification of carriers which are often difficult to distinguish from normal animals on the basis of morphologic criteria.

Testis-specific protein Y-encoded copy number is correlated to its expression and the field fertility of Canadian Holstein bulls.

Testis-specific protein Y-encoded (TSPY) is present in varying copy number in both human (20–76 copies) and cattle (37–200 copies), and some studies have linked this variation to semen quality in men. The purpose of this study was to determine if TSPY copy number is associated with fertility in bulls by using adjusted non-return rates, a commonly used measure of field fertility in Canada. In addition, we investigated the associations between TSPY copy number and its expression as well as specific semen parameters, such as average sperm concentration, sperm count, ejaculate volume, and motility. In 2 independent trials, TSPY copy number was shown to be positively correlated to adjusted non-return rates (trial #1: Spearman r = 0.34, p < 0.05; trial #2: Spearman r = 0.77, p < 0.01). Furthermore, TSPY copy number was inversely correlated to TSPY mRNA expression in the testis (Pearson r = –0.71, p < 0.0001). There were no correlations of TSPY copy number or expression with the semen parameters measured. Therefore, TSPY copy number might represent a potential marker of bull fertility, but its mechanism does not appear to be directly related to the semen characteristics analyzed as part of this study.

Systemic Concentrations of Endogenous and Exogenous FSH in Anoestrous Ewes Superstimulated with Folltropin®-V

The synchronization of follicular waves with medroxyprogesterone acetate (MAP) and oestradiol-17beta (E(2)-17beta) prior to ovarian superstimulation in anoestrous ewes reduces the variability in superovulatory responses by an unknown mechanism. Follicle stimulating hormone (FSH) is a primary promoter of antral follicular development, but the relevance of circulating FSH concentrations to the superovulation performance in ewes has not been examined. Eighteen anoestrous Rideau Arcott ewes (May-June) were superovulated with Folltropin-V (porcine FSH), with (n = 8; treated ewes) or without (n = 10; control ewes) a single i.m. dose of 350 microg of E(2)-17beta, given on the sixth day of a 14-day treatment with MAP-releasing intravaginal sponges (60 mg). The superovulatory treatment, begun 6 days after E(2)-17beta injection, consisted of six i.m. applications of Folltropin-V given twice daily (at 08:00 and 16:00 h), followed by an i.m. injection of GnRH (50 microg). Blood samples collected every 8 h throughout the 3-day treatment, were analysed by radioimmunoassays for concentrations of ovine and porcine FSH, using species-specific standards and primary antibodies. Serum concentrations of oFSH were greater (p < 0.05) in the controls compared to treated ewes at 40, 64 and 72 h and the variability in mean oFSH concentrations was greater (p < 0.05) in control ewes at 40, 48, 64 and 72 h after the 1(st) Folltropin-V injection. There were no differences (p > 0.05) between the two groups in serum concentrations of pFSH. Significant correlations were recorded between the number of corpora lutea (CL) and oFSH concentrations at 8 h (r = 0.72, p < 0.05), 16 h (r=0.63, p < 0.05) and 64 h (r = 0.84, p < 0.01) after the 1(st) Folltropin-V injection. The total number of recovered embryos was positively correlated to oFSH concentrations at 56 h (r = 0.69, p < 0.05). We concluded that changes in endogenous FSH concentrations during ovarian superstimulation with pFSH might contribute to the variability in superovulatory responses in ewes.