Genetic diagnosis of band 3 deficiency using a quenching probe (QProbe)-PCR assay in bovine embryos.

The present study was conducted to develop a simple and rapid procedure to determine the genotype of band 3 deficiency in bovine embryos by a novel real-time PCR method using a fluorescent quenching-based probe (QProbe-PCR). QProbe-PCR successfully distinguished wild type and R664X mutant alleles by melting curve analysis. Minimal amounts of DNA template were required for the detection of wild type/wild type alleles, mutant/mutant alleles, and wild type/mutant alleles; their amounts were 10 pg, 25 pg, and 50 pg, respectively. When 10 blastomeres were used as a DNA sample, accuracies of genotyping by QProbe-PCR were 100% and 89% in embryos homozygous for the wild type allele and heterozygous for the wild type and mutant alleles, respectively. QProbe-PCR takes approximately 2 h for genotyping and requires lesser time than the conventional method using PCR-RFLP, which requires digestion with a restriction enzyme and electrophoresis. Our data showed that QProbe-PCR is a useful method for rapid analysis of the genetic deficiency in preimplantation embryos. Reduction in the time required for genotyping enabled the transfer of genetically selected embryos to recipient cows on the day of embryo collection. These results suggest that determination of the genotype for the genetic deficiency in embryos is useful to select animals free from the genetic disease, and it also makes it possible to produce an animal model homozygous for the mutation.

Enhancement of maternal recognition of pregnancy with parthenogenetic embryos in bovine embryo transfer.

This study was performed to elucidate the changes in IFNT messenger RNA (mRNA) levels in in vivo-fertilized and parthenogenetic bovine embryos and their interferon-τ (IFNT) secretion amounts during the elongation phase. We assessed the induction capability of maternal recognition of pregnancy by parthenogenetic embryos and attempted cotransfer of in vivo-fertilized and parthenogenetic embryos. The expression level of IFNT mRNA in in vivo-fertilized embryos peaked on Day 18 after estrus, and the highest amount of uterine IFNT was observed on Day 20. Transfer of 10 parthenogenetic embryos produced a detectable amount of uterine IFNT. Transfer of one or three parthenogenetic embryos inhibited luteolysis. An increase in ISG15 mRNA levels in peripheral granulocytes was induced by the transfer of three parthenogenetic embryos. Cotransfer of three parthenogenetic embryos significantly improved the pregnancy rate on Day 40 in code 3 in vivo-fertilized embryos compared with single transfer without parthenogenetic embryos (65% vs. 35%). However, the pregnancy rate on Day 90 (35%) in cotransfer of code 3 in vivo-fertilized embryos did not differ from that upon single transfer (29%), because the cotransfer group had a higher incidence of pregnancy loss than with single transfer (47% vs. 17%) after Day 40. Cotransfer did not affect the pregnancy rate of code 2 in vivo-fertilized embryos. The incidence of pregnancy loss was higher in cotransfer of code 2 in vivo-fertilized embryos than in single transfer (30% vs. 7%). In conclusion, parthenogenetic embryos in the elongation phase secreted IFNT, enabling induction of maternal recognition of pregnancy. The present study revealed that enhancement of the maternal recognition of pregnancy using parthenogenetic embryos promoted the viability of poor-quality embryos until Day 40 of gestation. However, the incidence of pregnancy loss increased after Day 40 in the cotransfer of parthenogenetic embryos. A technique for promoting the full-term survival of poor-quality embryos is needed.

Embryo sexing and sex chromosomal chimerism analysis by loop-mediated isothermal amplification in cattle and water buffaloes.

In domestic animals of the family Bovidae, sex preselection of offspring has been demanded for convenience of milk/beef production and animal breeding. Development of the nonsurgical embryo transfer technique and sexing methods of preimplantation embryos made it possible. Sexing based on detection of Y chromosome-specific DNA sequences is considered the most reliable method to date. PCR enables amplification of a target sequence from a small number of blastomeres. However, it requires technical skill and is time consuming. Furthermore, PCR has the risk of false positives because of DNA contamination during handling of the PCR products in duplicate PCR procedures and/or electrophoresis. Therefore, for embryo sexing to become widely used in the cattle embryo transfer industry, a simple, rapid and precise sexing method needs to be developed. Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification method, and the reaction is carried out under isothermal conditions (range, 60 to 65 C) using DNA polymerase with strand displacement activity. When the target DNA is amplified by LAMP, a white precipitate derived from magnesium pyrophosphate (a by-product of the LAMP reaction) is observed. It is noteworthy that LAMP does not need special reagents or electrophoresis to detect the amplified DNA. This review describes the development and application of an embryo sexing method using LAMP in cattle and water buffaloes.

Changes in the expression patterns of the genes involved in the segregation and function of inner cell mass and trophectoderm lineages during porcine preimplantation development.

In mouse embryos, segregation of the inner cell mass (ICM) and trophectoderm (TE) lineages is regulated by genes, such as OCT-4, CDX2 and TEAD4. However, the molecular mechanisms that regulate the segregation of the ICM and TE lineages in porcine embryos remain unknown. To obtain insights regarding the segregation of the ICM and TE lineages in porcine embryos, we examined the mRNA expression patterns of candidate genes, OCT-4, CDX2, TEAD4, GATA3, NANOG, FGF4, FGFR1-IIIc and FGFR2-IIIc, in blastocyst and elongated stage embryos. In blastocyst embryos, the expression levels of OCT-4, FGF4 and FGFR1-IIIc were significantly higher in the ICM than in the TE, while the CDX2, TEAD4 and GATA3 levels did not differ between the ICM and TE. The expression ratio of CDX2 to OCT-4 (CDX2/OCT-4) also did not differ between the ICM and TE at the blastocyst stage. In elongated embryos, OCT-4, NANOG, FGF4 and FGFR1-IIIc were abundantly expressed in the embryo disc (ED; ICM lineage), but their expression levels were very low in the TE. In contrast, the CDX2, TEAD4 and GATA3 levels were significantly higher in the TE than in the ED. In addition, the CDX2/OCT-4 ratio was markedly higher in the TE than in the ED. We demonstrated that differences in the expression levels of OCT-4, CDX2, TEAD4, GATA3, NANOG, FGF4, FGFR1-IIIc and FGFR2-IIIc genes between ICM and TE lineages cells become more clear during development from porcine blastocyst to elongated embryos, which indicates the possibility that in porcine embryos, functions of ICM and TE lineage cells depend on these gene expressions proceed as transition from blastocyst to elongated stage.

Differences in apoptotic status in the bovine placentome between spontaneous and induced parturition.

We conducted this study to analyze apoptotic changes in the bovine placentome at spontaneous and induced parturition. Cows delivered i) after the administration of dexamethasone followed by prostaglandin F(2α) and estriol, ii) after the administration of prostaglandin F(2α) and estriol or iii) spontaneously. Prepartum changes in plasma progesterone and estradiol-17ß concentrations were similar between spontaneous and induced parturition. Messenger RNA of BCL2-related protein A1 (BCL2A1), an antiapoptotic gene, was expressed by trophoblast binucleate cells and caruncular epithelial cells. Quantitative RT-PCR showed that the expression of BCL2A1 mRNA in cotyledonary and caruncular portions was significantly lower in spontaneous parturition than induced parturition. The expression of BCL2-associated X protein (BAX) mRNA, a proapoptotic gene, was significantly higher in cotyledons at spontaneous parturition than parturition induced without dexamethasone. Caspase-3 (CASP3) mRNA and pre-activated CASP3 protein were predominantly detected in caruncular epithelial cells regardless of how parturition proceeded. Activated CASP3 protein was found in trophoblast uninucleate cells and binucleate cells rather than caruncular epithelial cells. In spontaneous parturition, intense staining of activated CASP3 was detected in caruncular epithelial cells. Spontaneous and dexamethasone-induced parturition increased apoptotic cells in the placentome compared with parturition induced without dexamethasone. The number of binucleate cells was significantly decreased in spontaneous parturition. The present results suggest that although the clinical dose of dexamethasone induces apoptosis in the placentome at term, neither dexamethasone nor prostaglandin F(2α) evoke normal physiological changes in the placentome during delivery such as a change in the balance of apoptosis-related genes and disappearance of binucleate cells.

Prediction of superovulatory response in Japanese Black cattle using ultrasound, plasma anti-Müllerian hormone concentrations and polymorphism in the ionotropic glutamate receptor AMPA1/GRIA1.

The aim of this study was to improve the reliability of predicting the superovulatory response in Japanese Black cattle. Follicle counts and plasma anti-Müllerian hormone concentrations were analyzed within four days prior to the initiation of superovulation. The single nucleotide polymorphism (guanine or adenine) of the ionotropic glutamate receptor AMPA1 was determined. The plasma anti-Müllerian hormone concentration was positively correlated (P<0.001) with the numbers of all follicles and small (<5 mm) follicles and with the numbers of ova/embryos (P<0.001), fertilized embryos (P<0.001) and transferable embryos (P=0.005). There was no significant difference in follicle counts and superovulatory responses between donor cows bearing guanine/adenine or guanine/guanine alleles of AMPA1. Donor cows with a high plasma anti-Müllerian hormone concentration and homozygous for the guanine-containing allele of AMPA1 were most responsive to superovulation. The results suggest that physiological and genetic markers of superovulation have a synergistic effect on the accuracy of predictions of responsiveness.

Epigenetic status and full-term development of bovine cloned embryos treated with trichostatin A.

We examined the comprehensive epigenetic status, including histone H3 and H4 acetylation, DNA methylation and level of mRNA transcripts of bovine somatic cell nuclear transfer (SCNT) embryos treated with trichostatin A (TSA), along with their full-term developmental efficacy. Treatment with 50 nM TSA enhanced early developmental competence; increased acetylation of two histones, H3K9K14 and H4K8, at the blastocyst stage; and maintained the DNA methylation status of the satelliteI sequence in bovine SCNT embryos. The difference in IGFBP-3 transcript levels between in vivo and SCNT embryos disappeared in SCNT embryos after treatment with 50 nM TSA. Pregnancy, full-term developmental competence and body weight at birth of offspring did not differ between SCNT embryos treated with 50 nM TSA and untreated embryos. These results suggest that treatment with TSA improves preimplantation development and changes the epigenetic status but does not promote the full-term development competence in bovine SCNT embryos.

Cloned cows with short telomeres deliver healthy offspring with normal-length telomeres.

Dolly, the first mammal cloned from a somatic cell, had shorter telomeres than age-matched controls and died at an early age because of disease. To investigate longevity and lifetime performance in cloned animals, we produced cloned cows with short telomeres using oviductal epithelial cells as donor cells. At 5 years of age, despite the presence of short telomeres, all cloned cows delivered multiple healthy offspring following artificial insemination with conventionally processed spermatozoa from noncloned bulls, and their milk production was comparable to that of donor cows. Moreover, this study revealed that the offspring had normal-length telomeres in their leukocytes and major organs. Thus, cloned animals have normal functional germ lines, and therefore germ line function can completely restore telomere lengths in clone gametes by telomerase activity, resulting in healthy offspring with normal-length telomeres.

DNA methylation status of bovine blastocyst embryos obtained from various procedures.

DNA methylation is an important factor for the regulation of gene expression in early embryos. It is well known that the satellite I sequence is more heavily methylated in bovine somatic cell nuclear transfer (NT-SC) embryos than in embryos derived from in vitro fertilization (IVF). However, the methylation status of bovine embryos obtained by other procedures is not well known. To clarify DNA methylation levels of bovine embryos obtained from various procedures, we examined satellite I sequences in bovine blastocyst (BC) embryos derived from NT-SC, NT using embryonic blastomeres (NT-EM), in vivo (Vivo), IVF and parthenogenetic treatment (PA). Furthermore, in order to evaluate the efficacy of DNA demethylation by the NT procedure, we determined the DNA methylation levels in bovine embryos in which NT was recapitulated (Re-NT). Although the DNA methylation levels in the NT-SC embryos were higher than those in the other embryos, the NT-EM embryos exhibited lower DNA methylation levels. The satellite I sequence in the NT-SC embryos was more demethylated than that in the donor cells. Although the DNA methylation level in the individual NT-SC embryos showed variation, the full-term developmental efficacy of these embryos were not different. These findings suggest that the methylation level of the satellite I sequence at the BC stage is not related to the abnormalities of bovine embryos produced by NT-SC. There was no difference in methylation levels between Re-NT and NT-SC embryos. Our results indicated that the DNA methylation status differed among embryos produced by various methods and that at least some of the demethylation of the donor cell genome occurred in the recipient cytoplast after NT-SC, but the demethylation ability of the NT procedure was noted in the first NT but not in the second NT.

Prepartum maternal plasma glucose concentrations and placental glucose transporter mRNA expression in cows carrying somatic cell clone fetuses.

In this study, the plasma glucose concentrations of cows carrying a somatic cell clone fetus during late pregnancy and placental glucose transporter (GLUT) mRNA levels at parturition were examined. Parturition was induced using dexamethasone, prostaglandin F(2α) and estriol in cows bearing a clone (Clone) or a fetus fertilized in vivo as a control (DEX). Plasma glucose concentrations were measured in the cows (days 257 and 271 of pregnancy and at parturition) and newborn calves. Cotyledon and caruncle tissues removed just after parturition were used for mRNA extraction. Expression of mRNA was also analyzed in control cows that were induced to undergo parturition without dexamethasone (PG) or that spontaneously delivered (SP). The glucose concentrations of the Clone group were significantly low at all points examined, but those of the calves were normal. The increase in the maternal glucose concentration from day 257 to parturition was significantly lower in the Clone group. Glucose concentrations were negatively correlated with birth weight for clones (day 257; r=-0.584, day 271; r=-0.286, parturition; r=-0.549). There was no difference in mRNA levels in the cotyledons among the animals examined. In the caruncles, the Clone and PG groups showed significantly higher GLUT1 and GLUT3 mRNA levels than the SP group, and the GLUT3 mRNA level was significantly higher in the Clone group than in the DEX group. The glucocorticoid receptor α mRNA level was significantly lower in the SP group than in the DEX group. Although spontaneous parturition and administration of dexamethasone suppressed the placental GLUT mRNA levels, the action was not observed in clone pregnancy. These results raise the possibility of facilitation of glucose transportation through the placenta to meet increased nutritional requirements of overgrown clone fetuses.

The possibility of a false positive arising from sperm DNA in genetic diagnosis of bovine embryos.

The present study was conducted to evaluate the effect of accessory sperm cells that adhered to the zona pellucida or blastomeres on the accuracy of genetic diagnosis of preimplantation embryos. The properties of sperm cells as a template for DNA amplification were examined using bovine sperm cells frozen-thawed or incubated in PBS after thawing for 7 days at 39 C. Sexing by loop-mediated isothermal amplification (LAMP) and claudin-16 genotyping by polymerase chain reaction (PCR) were performed using 10, 50 and 100 sperm cells. When sexing based on LAMP was performed, no amplified DNA was detected in 10 sperm-derived samples, whereas male-specific (10-60%) and gender-natural DNA (30-100%) sequences were detected in 50 and 100 sperm-derived samples. The detection rates for gender-natural DNA sequences were higher in incubated sperm samples than in sperm samples immediately after freeze-thawing. The detection rates for claudin-16 were low (7-13%) regardless of the concentration of sperm cells and the period of incubation after thawing. The present results showed that male-specific DNA, gender-natural DNA and claudin-16 sequences were not usually amplified from a small number of sperm cells (< or =10 cells). However, when a large number of sperm cells (> or =50 cells) were present, male-specific and gender-natural DNA sequences were amplified at a high rate, and claudin-16 DNA sequences were also occasionally detected. These results raise the possibility that accessory sperm cells may reduce the accuracy of the genetic diagnosis of bovine embryos. Therefore, steps to prevent the contamination of sperm cells, such as removal of the zona pellucida and washing of sample blastomeres, are necessary to obtain an accurate result.

Changes in the DNA methylation status of bovine embryos from the blastocyst to elongated stage derived from somatic cell nuclear transfer.

The epigenetic reprogramming of the donor cell nucleus is an important factor in the development of embryos and production of normal offspring derived by somatic cell nuclear transfer (NT-SC). During early development, a dramatic reduction in methylation levels occurs in mouse. In early embryos, this process makes it possible to erase gamete-specific methylation patterns and induce de novo methylation at defined developmental time-points. To clarify changes in DNA methylation in bovine NT-SC embryos, we examined satellite I sequences in bovine embryos derived in vivo (Vivo) and by NT-SC at the blastocyst (BC) and elongated (EL) stages. Because the EL stage embryo consists of the embryo disc (ED) and trophectoderm (TE), the methylation status of each part was analyzed with respect to the progress of differentiation. DNA methylation levels in Vivo embryos were increased during the elongation stage. In contrast, DNA methylation levels in NT-SC embryos remained unchanged in the ED and significantly decreased in the TE. Real-time PCR analysis showed that Dnmt-1 expression in BC embryos derived by NT-SC was significantly lower than that in Vivo embryos; thus, differences in the DNA methylation status may reflect transcript levels of Dnmt-1. Our results suggest that the aberrant methylation level of bovine NT-SC embryos in the satellite I region is corrected as a result of demethylation and retention of methylation as the embryo develops and differentiates.

Excess estrogen sulfoconjugation as the possible cause for a poor sign of parturition in pregnant cows carrying somatic cell clone fetuses.

We conducted this study to elucidate a factor causing a poor sign of parturition and prolonged gestation, which is frequently observed in cows carrying somatic clone fetuses. Pre-partum rises in concentrations of plasma estrone and estradiol-17beta in the recipient cows pregnant with clones were subtle. By contrast, the plasma concentration of estrone sulfate in clone pregnancies increased gradually from pre-initiation of parturition induction whereas control cows that received in vivo-derived embryos showed a significant increase at parturition. Therefore, in clone pregnancies, the ratio of estrone/estrone sulfate was low during the pre-partum period compared with control. Messenger RNA expression of estrogen sulfotransferase (SULT1E1) in the placenta at parturition was significantly higher in clone pregnancies than control pregnancies and was localized in binucleate cells (BNC). SULT1E1 mRNA abundance was negatively and positively correlated with concentrations of maternal estrone and estrone sulfate at parturition respectively. Messenger RNA expressions of estrogen sulfatase (STS) and aromatase (CYP19) were similar between clone and control pregnancies and were localized in BNC and caruncular epithelial cells. STS and CYP19 mRNA abundances showed positive correlations with maternal estradiol-17beta concentration. The population of BNC in the placenta did not differ between clone and control pregnancies. Plasma cortisol concentration of vaginally delivered newborn clone calves was comparable with those of control, although cesarean section delivered clone calves showed a low concentration. These results suggest that excess estrogen sulfoconjugation is the reason for the perturbed low ratio of active to inactive estrogens and the resulting hormonal imbalance contributes to the lack of overt signs of readiness for parturition in cows pregnant with clones.

Changes in the mRNA transcripts of insulin-like growth factor ligand, receptors and binding proteins in bovine blastocysts and elongated embryos derived from somatic cell nuclear transfer.

The objective of this study was to determine changes in the transcription of insulin-like growth factor (IGF)-related genes in blastocyst (BC)- and elongated (EL)-stage embryos produced by nuclear transfer using somatic cells (NT-SC). Bovine BC (day 7)- and EL (day 15)-stage embryos were obtained from NT-SC or in vivo production (Vivo). The relative abundance of mRNA was examined by RT- real-time PCR. The transcript of IGF-II was only detected at the EL stage in both the NT-SC and Vivo embryos. The level of transcription of the IGF-I receptor (r) in the NT-SC embryos was decreased at the EL stage and was significantly (P<0.05) lower than at the BC stage. In contrast, the IGF-IIr levels did not differ significantly between the NT-SC and Vivo embryos, regardless of the developmental stage. IGF-binding protein (IGFBP)-2 levels were markedly decreased in the NT-SC and Vivo embryos at the EL stage (P<0.05). The IGFBP-3 level in Vivo was significantly (P<0.05) increased at the EL stage compared with at the BC stage. However, the IGFBP-3 levels in NT-SC embryos were unchanged and lower (P<0.05) than in the Vivo embryos at the EL stage. These results suggest that there are differences in the levels and changes in the transcription of IGF-related genes in bovine embryos produced by NT-SC compared with those produced by Vivo.

Rapid sex chromosomal chimerism analysis in heterosexual twin female calves by Loop-mediated Isothermal Amplification.

We attempted to apply an embryo sexing kit with Loop-mediated Isothermal Amplification (LAMP) to sex chromosomal chimerism analysis in heterosexual twin female calves. Peripheral blood was used for the amplification of male-specific DNA, derived from XY leukocytes. When blood samples were diluted 1:1000 in LAMP reaction mixture, hemoglobin or blood coagulation did not influence the turbidity measurement of the reaction mixture for detection of amplified DNA. This procedure detected the existence of XY leukocytes of 0.01% in female blood. Furthermore, all heterosexual twin female calves, bearing sex chromosomal chimerism based on karyotyping and PCR, showed male-specific DNA from peripheral blood by LAMP. These results indicated that the embryo sexing kit with LAMP was available for sensitive detection of sex chromosomal chimerism. This procedure made it possible to detect easily Y-chromosome specific DNA in a short interval compared with PCR, and was convenient for field application of freemartin diagnosis.

Genetic diagnosis of band 3 deficiency and sexing in bovine preimplantation embryos.

Band 3 deficiency with hereditary spherocytosis and hemolytic anemia in Japanese black cattle, band 3(Bov.Yamagata), is caused by a total lack of band 3 protein with an autosomal dominant inheritance. Genotyping for band 3 deficiency and sexing were successfully achieved in biopsied embryo cells with efficiencies of 98.4% and 97.4%, respectively. Transfer of the embryo that was determined as homozygous for the mutant allele into a recipient cow resulted in the production of a fetus exhibiting the genotype and red cell phenotypes characteristic of band 3(Bov.Yamagata). These results demonstrate that our procedure is reliable and applicable to produce animals free from or homozygous for the mutant allele by breeding carrier animals.

Rapid sexing of water buffalo (Bubalus bubalis) embryos using loop-mediated isothermal amplification.

Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification method that amplifies a target sequence specifically under isothermal conditions. The objective of this study was to identify a Y chromosome-specific sequence in water buffalo and to establish an efficient procedure for embryo sexing by LAMP. The homologues of a Y chromosome-specific sequence, bovine repeat Y-associated.2, in swamp and river buffalo were cloned, and designated swamp buffalo repeat Y-associated.2 and river buffalo repeat Y-associated.2, respectively. Sexing by LAMP was performed using primers for swamp buffalo repeat Y-associated.2. A 12S rRNA was also amplified by LAMP as a control reaction in both male and female. The minimal amount of the template DNA required for LAMP appeared to be 0.1-10 pg. The sensitivity was further examined using swamp buffalo fibroblasts as templates. When fibroblasts were lysed with NaOH, the minimal cell number required for detection of both male-specific and male-female common DNA appeared to be two cells, whereas correct determination of sex could not be achieved using fibroblasts lysed by heat denaturation. Embryo sexing was also performed using blastomeres from interspecies nuclear transfer embryos. The sex determined by LAMP for blastomeres corresponded with the sex of nuclear donor cells in analyses using four or five blastomeres as templates. The LAMP reaction required only about 45 min, and the total time for embryo sexing, including DNA extraction, was about 1 h. In conclusion, the present procedure without thermal cycling and electrophoresis was reliable and applicable for water buffalo embryos.

Analysis of mRNA transcripts for insulin-like growth factor receptors and binding proteins in bovine embryos derived from somatic cell nuclear transfer.

The low efficiency of animal production using somatic cell nuclear transfer procedures is considered to be the result of an incomplete reprogramming of donor cell nucleus, which leads to abnormal expression of developmentally important genes. The objective of this study was to determine the abundance of gene transcripts of insulin-like growth factor (IGF)-related genes in cloned bovine embryos reconstructed with somatic cells. Single embryos derived from nuclear transfer reconstructed with somatic cells (NT-SC) or embryo blastomeres (NTEM), in vitro fertilization (IVF), in vivo production (Vivo), and parthenogenetic treatment (PA) were analyzed. The relative abundance of mRNA was examined by real-time PCR. Transcripts of the IGF-1 receptor (r) and IGF binding protein (BP)-2 were detected in all embryos, regardless of origin. IGF-IIr and IGFBP-3 transcripts signals in NT-SC embryos were detected with significantly lower frequencies of 25 and 50%, respectively. Although IGF-Ir and IGFIIr transcript levels were not significantly different in NT-SC, NT-EM, IVF, Vivo, and PA embryos, the relative abundance in individual embryos indicated large variation in NT-SC. IGFBP-2 and IGFBP-3 levels were high in the Vivo embryos compared with NT-SC, NT-EM, IVF, or PA embryos. These results suggest differences in levels of transcripts of IGF-related genes in the bovine embryos produced by NT compared with IVF, Vivo, and PA.

Establishment of a specific radioimmunoassay for bovine interferon tau.

A radioimmunoassay (RIA) was developed for quantification of bovine interferon (bIFN) tau, conceptus secretory protein, which allows for the maintenance of the corpus luteum during early pregnancy. A cDNA coding bIFN tau was derived from cultured trophoblast cells (TBs). Recombinant (r) bIFN tau was produced in a baculovirus expression system with two different viruses. The RIA was a double-antibody competitive binding assay that used anti-bIFN tau antiserum (raised in rabbits) as the primary antibody, a radioiodinated derivative of bIFNtau as the radioactive tracer, and goat anti-rabbit IgG as the secondary antibody. The antibody did not cross-react with rbIFN alpha, recombinant human IFN beta or recombinant ovine IFN tau. The correct recovery of amounts of rbIFN tau indicated good accuracy. Serially concentrated TB conditioned media, paralleled the standard curve for bIFN tau. The intra-assay and inter-assay coefficients of variation at bIFN tau levels of 7.8 and 15.6 ng/mL were 7.1 and 8.1%, and 11.0 and 8.5%, respectively. bIFN tau was directly detected in uterine flushings obtained from cows at Day 16 of pregnancy. In summary, this assay was suitable for the measurement of bIFN tau.

A female pseudohermaphrodite Holstein heifer with gonadal mosaicism.

An 11-month-old Holstein heifer diagnosed as a female pseudohermaphrodite (PH) was subjected to clinical, hormonal, histological, and cytogenetic examinations. The urogenital sinus and external genitalia of the heifer were virilized and no vulval orifice was present. A male urethra-like tubular structure was palpable in the perineal area; urination occurred through a urethral fossa at the distal end of the urethra (cranial aspect of the mammary tissue). Bilateral ovaries and uterine horns, ovarian follicular development, ovulation and CL formation (with increasing blood P4 concentration) were similar to a normal heifer. Testosterone (T) concentrations of all samples collected both before and after hCG challenge were <0.1 ng/mL (absence of a responsive endogenous androgen source). Histologically, the ovary, uterus, oviduct and vagina resembled the normal female reproductive tract. Although the corpus cavernosum was present, the penile urethra and corpus spongiosum penis were hypoplastic. A normal female karyotype (60, XX) was detected in metaphase spreads obtained from cultured peripheral lymphocytes. Moreover, Y-chromosome-specific sequences were not detected in genomic DNA from lymphocytes. By PCR analysis of genomic DNA (from the tissues of urogenital organs), Y-chromosome-specific sequences (both SRY and Amelogenin (Amel) locus) were detected. In conclusion, this was a female PH heifer due to gonadal mosaicism, with normal female genitalia and ovarian activity. This is the first report of an SRY-positive, mosaic female PH heifer.