Estimation of genetic parameters for bull conception rate and its genetic correlations with semen production traits in Japanese Black bulls.

The P of achieving pregnancy is an important trait of bull fertility in beef cattle and is defined as the bull conception rate (BCR). This study aimed to clarify and better understand the genetic architecture of the BCR calculated using artificial insemination and pregnancy diagnosis records from a progeny testing program in Japanese Black bulls. In this study, we estimated the genetic parameters of the BCR and their correlation with semen production traits. In addition, we assessed the correlated responses in BCR by considering the selection of semen production traits. Nine hundred and sixteen Japanese Black bulls were selected based on fertility, with 28 869 pregnancy diagnostic records from the progeny testing program. Our results showed that the heritability estimate was 0.04 in the BCR at the first service and 0.14 in BCR for the three services, and an increase in the inbreeding coefficient led to a significant decrease in BCR. The phenotypic trend of BCR remained almost constant over the years, whereas the genetic trend increased. In addition, the changes in the progeny testing year effect showed a similar tendency to the phenotypic trends, suggesting that the phenotypic trends could be mainly due to non-genetic effects, including progeny testing year effects. The estimated genetic correlation of BCR with sperm motility traits was favorably moderate to high (ranging from 0.49 to 0.97), and those with sperm quantity traits such as semen volume were favorably low to moderate (ranging from 0.23 to 0.51). In addition, the correlated responses in BCR at the first service by selection for sperm motility traits resulted in a higher genetic gain than direct selection. This study provides new insights into the genetic factors affecting BCR and the possibility of implementing genetic selection to improve BCR by selecting sperm motility traits in Japanese Black bulls.

A potent allele marker related to low bull conception rate in Japanese Black bulls.

Over the years, there has been considerable variation in the bull conception rate (BCR) of Japanese Black cattle; moreover, several Japanese Black bulls with a low BCR of ≤10% have been identified. However, the alleles responsible for the low BCR are not determined yet. Therefore, in this study, we aimed to identify single-nucleotide polymorphisms (SNPs) for predicting low BCR. To this end, the genome of Japanese Black bulls was comprehensively examined by a genome-wide association study with whole-exome sequencing (WES), and the effect of the identified marker regions on BCR was determined. The WES analysis of six sub-fertile bulls with a BCR of ≤10% and 73 normal bulls with a BCR of ≥40% identified a homozygous genotype for low BCR in Bos taurus autosome 5 in the region between 116.2 and 117.9 Mb. The g.116408653G > A SNP in this region had the most significant effect on the BCR (P-value = 1.0 × 10-23), and the GG (55.4 ± 11.2%) and AG (54.4 ± 9.4%) genotypes in the SNP had a higher phenotype than the AA (9.5 ± 6.1%) genotype for the BCR. The mixed model analysis revealed that g.116408653G > A was related to approximately 43% of the total genetic variance. In conclusion, the AA genotype of g.116408653G > A is a useful index for identifying sub-fertile Japanese Black bulls. Some positive and negative effects of SNP on the BCR were presumed to identify the causative mutations, which can help evaluate bull fertility.

Genome-wide detection of non-additive quantitative trait loci for semen production traits in beef and dairy bulls.

Semen production traits are important aspects of bull fertility, because semen quantity leads to direct profits for artificial insemination centres, and semen quality is associated with the probability of achieving a pregnancy. Most genome-wide association studies (GWASs) for semen production traits have assumed that each quantitative trait locus (QTL) has an additive effect. However, GWASs that account for non-additive effects are also important in fitness traits, such as bull fertility. Here, we performed a GWAS using models that accounted for additive and non-additive effects to evaluate the importance of non-additive effects on five semen production traits in beef and dairy bulls. A total of 65 463 records for 615 Japanese Black bulls (JB) and 50 734 records for 873 Holstein bulls (HOL), which were previously genotyped using the Illumina BovineSNP50 BeadChip, were used to estimate genetic parameters and perform GWAS. The heritability estimates were low (ranged from 0.11 to 0.23), and the repeatability estimates were low to moderate (ranged from 0.28 to 0.45) in both breeds. The estimated repeatability was approximately twice as high as the estimated heritability for all traits. In this study, only one significant region with an additive effect was detected in each breed, but multiple significant regions with non-additive effects were detected for each breed. In particular, the region at approximately 64 Mbp on Bos taurus autosome 17 had the highest significant non-additive effect on four semen production traits in HOL. The rs41843851 single nucleotide polymorphism (SNP) in the region had a much lower P-value for the non-additive effect (P-value = 1.1 × 10-31) than for the additive effect (P-value = 1.1 × 10-8) in sperm motility. The AA and AB genotypes on the SNP had a higher phenotype than the BB genotype in HOL, and there was no bull with the BB genotype in JB. Our results showed that non-additive QTLs affect semen production traits, and a novel QTL accounting for non-additive effects could be detected by GWAS. This study provides new insights into non-additive QTLs that affect fitness traits, such as semen production traits in beef and dairy bulls.

Genetic analysis of semen production traits of Japanese Black and Holstein bulls: genome-wide marker-based estimation of genetic parameters and environmental effect trends.

The semen production traits of bulls from 2 major cattle breeds in Japan, Holstein and Japanese Black, were analyzed comprehensively using genome-wide markers. Weaker genetic correlations were observed between the 2 age groups (1 to 3 yr old and 4 to 6 yr old) regarding semen volume and sperm motility compared with those observed for sperm number and motility after freeze-thawing. The preselection of collected semen for freezing had a limited effect. Given the increasing importance of bull proofs at a young age because of genomic selection and the results from preliminary studies, we used a multiple-trait model that included motility after freeze-thawing with records collected at young ages. Based on variations in contemporary group effects, accounting for both seasonal and management factors, Holstein bulls may be more sensitive than Japanese Black bulls to seasonal environmental variations; however, the seasonal variations of contemporary group effects were smaller than those of overall contemporary group effects. The improvement of motilities, recorded immediately after collection and freeze-thawing, was observed in recent years; thus, good management and better freeze-thawing protocol may alleviate seasonal phenotypic differences. The detrimental effects of inbreeding were observed in all traits of both breeds; accordingly, the selection of candidate bulls with high inbreeding coefficients should be avoided per general recommendations. Semen production traits have never been considered for bull selection. However, negative genetic trends were observed. The magnitudes of the estimated h were comparable to those of other economically important traits. A single-step genomic BLUP will provide more accurate predictions of breeding values compared with BLUP; thus, marker genotype information is useful for estimating the genetic merits of bulls for semen production traits. The selection of these traits would improve sperm viability, a component related to breeding success, and alleviate negative genetic trends.

A genetic analysis of meat compositions in Japanese Black cattle: Genetic parameters and sex influence.

Meat composition in beef is related to eating quality and food functionality. Genetic parameters for several meat compositions including free amino acid, peptide and sugar, however, remain poorly described. In this study, we estimated genetic parameters for 51 meat components, including free amino acids, peptides, sugars and fatty acid compositions, and two carcase traits in 1,354 heifers and 1,797 steers of Japanese Black cattle. Heritability estimates were generally equivalent to or moderately greater than those in previous studies of this breed. Genetic correlations between free amino acids, peptides and sugars and carcase traits were often negative, suggesting a trade-off between traits. Using two-trait animal models that treat records from the two sexes as different traits, we estimated sex-specific heritabilities and cross-sex genetic correlations which indicate the sex differences in genetic architecture. In these analyses, 12 traits showed significant heritability differences between sexes and cross-sex genetic correlations occasionally deviated from unity. These results could be used to inform future breeding schemes and investigations of the genetic architecture of meat compositions in beef.

Whole-genome prediction of fatty acid composition in meat of Japanese Black cattle.

Because fatty acid composition influences the flavor and texture of meat, controlling it is particularly important for cattle breeds such as the Japanese Black, characterized by high meat quality. We evaluated the predictive ability of single-step genomic best linear unbiased prediction (ssGBLUP) in fatty acid composition of Japanese Black cattle by assessing the composition of seven fatty acids in 3088 cattle, of which 952 had genome-wide marker genotypes. All sires of the genotyped animals were genotyped, but their dams were not. Cross-validation was conducted for the 952 animals. The prediction accuracy was higher with ssGBLUP than with best linear unbiased prediction (BLUP) for all traits, and in an empirical investigation, the gain in accuracy of using ssGBLUP over BLUP increased as the deviations in phenotypic values of the animals increased. In addition, the superior accuracy of ssGBLUP tended to be more evident in animals whose maternal grandsire was genotyped than in other animals, although the effect was small.

Genomic prediction in Japanese Black cattle: application of a single-step approach to beef cattle.

The implementation of genomic selection for Japanese Black cattle, known for rich marbling of their meat, is now being explored. Although multiple-step methods are often adopted for dairy cattle, they present shortcomings such as bias and loss of information in addition to operational complexity. These can be avoided using single-step genomic BLUP (ssGBLUP) based on the relationship matrix H, which is constructed from the numerator relationship matrix (A) augmented by the genomic relationship matrix (G). This study assessed the use of ssGBLUP for 3 economically important traits in Japanese Black cattle. Three aspects of ssGBLUP that are important for practical use were examined specifically: the mixing proportions of blending G with A, selection of subsets of genotyped animals used for constructing H, and prediction ability for ungenotyped animals. Different mixing proportions were tested to assess the influence of these proportions on variance component estimation and prediction accuracy. For all traits, the highest or nearly highest accuracy was obtained when the adopted mixing proportion provided heritability closest to that inferred based on A. However, the accuracy did not increase greatly under adjustment of the mixing proportion, thereby suggesting that the influence of the mixing proportion on the accuracy was limited. Genotype data of influential bulls showed a greater contribution to accuracy than that of bulls that were less influential. Genotyping animals with phenotypic records increased the accuracy. It can be prioritized over genotyping bulls that are not influential on the population. These results are expected to present good guides to the future expansion of genotyped populations. Even for animals without genotype data but with genotyped sires, ssGBLUP provided more accurate prediction than BLUP did. For both phenotype and breeding value prediction, ssGBLUP provides more accurate prediction than BLUP, suggesting its usefulness in genomic selection in Japanese Black cattle.

Small heat shock protein 27 (HSP27) associates with tubulin/microtubules in HeLa cells.

One of the monoclonal antibodies raised against mitotic HeLa cells (termed as mH3) recognized a 27-kDa protein and stained microtubules in the mitotic spindles of HeLa cells. Immunoscreening of a HeLa cDNA library revealed that mH3 antigen is a small heat shock protein, HSP27. Immunoprecipitation analysis using mH3 suggested that both alpha- and beta-tubulin are associated with HSP27. Further, sucrose-cushioned ultra centrifugation revealed that HSP27 is co-sedimented with taxol-stabilized microtubules. These results indicate that HSP27 associates with tubulin/microtubules in HeLa cells.

Biological action of keratinocyte growth factor in BeWo cells, a human choriocarcinoma cell line.

Previously, we reported that the expression of keratinocyte growth factor (KGF) is enhanced in secretory phase endometrial and decidual cells in early pregnancy as compared with the expression of KGF in proliferative phase endometrial cells, in humans. In order to clarify the role of KGF in embryo-endometrial interaction, we analyzed the in vitro effect of KGF on the human chorionic gonadotropin (hCG) secretion and on DNA synthesis in chorionic villi which are in close contact with the endometrium/decidua in the early stage of pregnancy. In this study, we used the BeWo cell line, a human choriocarcinoma cell line that possesses the biological features of secreting various placental hormones including hCG. Furthermore, we investigated the expression of KGF receptor (KGF-R) in these cells. KGF significantly stimulated hCG secretion in cultured BeWo cells but did not affect [3H]-thymidine incorporation. KGF-R mRNA was detected in BeWo cells by reverse transcriptase-polymerase chain reaction. These results suggest that the expression of KGF, which is induced in endometrial/decidual cells by progesterone, plays an important role in the embryo-endometrial/ decidual interaction by stimulating hCG secretion rather than affecting cell proliferation.

Ovarian strumal carcinoid with severe constipation: immunohistochemical and mRNA analyses of peptide YY.

Functioning ovarian carcinoid tumors are well known to cause carcinoid syndrome. Recently, strumal and trabecular ovarian carcinoid tumors are reported to cause severe constipation possibly because of tumor-producing peptide YY (PYY). We studied a case of primary ovarian strumal carcinoid who had had severe constipation until the tumor was removed by surgical operation. Immunohistochemically, many tumor cells were strongly positive for PYY. By Northern blot and reverse transcription polymerase chain reaction analyses, PYY mRNA was expressed in a complete form as detected in normal human colon mucosa. From these findings, an ovarian strumal carcinoid is strongly suggested to express complete PYY mRNA and therefore complete PYY protein that results in severe constipation.

Gene expression and specific binding of platelet-derived growth factor and its effect on DNA synthesis in human decidual cells.

To clarify the biological significance of platelet-derived growth factor (PDGF) in human decidual cell function, which is important for the maintenance of pregnancy, we investigated gene expression of the PDGF subunits, PDGF-A and PDGF-B, specific binding of the PDGF isoform, and the effect of PDGF dimers on DNA synthesis in human decidual cells. We detected in decidua from early pregnancy the expected DNA bands of PDGF-A and PDGF-B by reverse transcriptase-polymerase chain reaction (RT-PCR) as well as mRNAs of each PDGF subunit by Northern blot hybridization, demonstrating that both PDGF subunits exist in this tissue. Scatchard plot analysis showed that decidual cells had both PDGF-alpha and PDGF-beta receptors. PDGF-AA, -AB and -BB stimulated [3H]-thymidine incorporation in cultured decidual cells in a dose-dependent manner. These results indicate the importance of PDGF in human decidua.

Gene expressions of keratinocyte growth factor and its receptor in the human endometrium/decidua and chorionic villi.

Keratinocyte growth factor (KGF) is secreted from mesenchymal fibroblasts and has a mitogenic specificity for epithelial cells in a paracrine fashion. In order to clarify the biological significance of KGF in the human endometrium which undergoes dynamic changes under the influence of sex steroid hormone, we investigated the gene expressions of KGF and its receptor (KGF-R) in the human endometrium in various sex steroid hormone milieus and chorionic villi, by RT-PCR and Northern blot hybridization. The secretory phase endometrium had a KGF mRNA level 10-fold greater than that of the proliferative phase endometrium. Similarly abundant KGF mRNA was found in decidua and pseudopregnant endometrium compared with proliferative phase endometrium. The KGF-R mRNA was detected by RT-PCR in chorionic villi from early pregnancy. These results indicate that the gene for KGF expressed in the human endometrium is mainly regulated by progesterone and that KGF might have a role in the interaction between decidua and chorion in early pregnancy in man.

Gene expression of gonadotropin-releasing hormone, but not its receptor, in human endometrium and decidua.

Gonadotropin-releasing hormone (GnRH) has been reported to exist in extrahypothalamic tissues such as the placenta, gonads and mammary glands. While we have reported the presence of GnRH-mRNA in the rodent uterus, there have been no reports concerning gene expression of GnRH and its receptor (GnRH-R) in human endometrial tissue. In order to investigate the role of GnRH as a local regulator in the human endometrium, we examined the gene for GnRH and GnRH-R in non-pregnant endometrium and decidua of early pregnancy. Using reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot analysis we found GnRH-mRNA but not GnRH-R-mRNA transcripts in the human endometrium and decidua at 7-9 weeks gestation. This is the first report that suggests GnRH gene expression in the human endometrium/decidua.

Continuous stimulation of gonadotropin-releasing hormone (GnRH) receptors by GnRH agonist decreases pituitary GnRH receptor messenger ribonucleic acid concentration in immature female rats.

Although it is well recognized that continuous administration of gonadotropin-releasing hormone agonist (GnRHa) induces pituitary desensitization, the precise molecular mechanism of this phenomenon is still unclear. To test the hypothesis that pituitary gonadotroph desensitization is mediated by a change in GnRH receptor (GnRH-R) gene expression, the GnRH-R mRNA concentration was analyzed in immature female rats during GnRHa treatment. Northern blot hybridization was used to determine the GnRH-R mRNA concentration several times after an injection of TAP-144-SR, a slow releasing GnRHa. The GnRH-R mRNA readings were 92.7 +/- 9.5%, 49.9 +/- 5.0%, 35.7 +/- 2.3% and 73.8 +/- 5.7% (Mean +/- SD) compared to each control value at 1, 2, 4 and 8 weeks, respectively, after a single injection of 0.94 mg TAP-144 SR. These changes in GnRH-R mRNA coincided with the changes in gonadotropin secretion and LH-beta mRNA in response to GnRH in the results of our previous report. The present results indicate that the reduction of the number of pituitary GnRH-R sites induced by continuous stimulation with GnRHa is regulated at a transcriptional level.