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.

Prospective ultrasonographic and endocrine predictors of spermatogenic onset in ram lambs.

The objective of this study was to examine testicular ultrasonographic characteristics and endocrine profiles in prepubescent ram lambs for correlations with the age at first detection of elongated spermatids (ESt age). Bi-weekly ultrasound examinations and weekly testicular biopsies began at 10 weeks of age or at the time that testicular volume reached 15cm3, and continued until 1-2 weeks after Est's were first detected by histological examination of testicular biopsies in twenty-two spring-born Rideau Arcott×Polled Dorset lambs. Computer-assisted analysis of testicular ultrasonograms was performed using commercially available image analytical software. Blood samples were drawn before each ultrasonographic examination and were used for measurements of free triiodothyronine (fT3) and thyroxine (fT4), and follicle-stimulating hormone (FSH) concentrations. The mean (±SEM) age at first detection of ESts was 15.9±0.5 weeks. Testicular volumes recorded between 10 and 12 weeks of age correlated inversely with the ESt age (r=-0.44 to -0.50, P≤0.05). Statistically significant correlations were recorded between the ESt age and numerical pixel values of testicular parenchyma at 10 (r=-0.48, P=0.05) and 15 (r=0.52, P=0.05) weeks of age, and between the ESt age and testicular pixel heterogeneity in ram lambs aged 14.5 weeks (r=0.60, P=0.007). Lastly, circulating FSH concentrations at 10 weeks (r=-0.43, P=0.05), serum fT3 concentrations at 13 weeks (r=0.44, P=0.04) and fT4 concentrations at 11.5 weeks of age (r=0.48, P=0.03) were all correlated with the ESt age. The present results show that testicular volume has the most stable relationship with pubertal onset; however, testicular echotexture as well as circulating concentrations of FSH and free fractions of thyroid hormones at specific ages may be indicative of more intricate developmental events heralding puberty.

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.

Relationships of serum thyroid hormones and follicle-stimulating hormone concentrations to Sertoli cell differentiation during the first wave of spermatogenesis in euthyroid ram lambs.

The main purpose of this study was to determine if temporal relationships exist between serum concentrations of free fractions of thyroxin (fT4) and triiodothyronine (fT3), follicle-stimulating hormone (FSH) levels, and Sertoli cell differentiation in euthyroid ram lamb testes. Additionally, testicular thyroid hormone (TH) receptors (TRs) were identified using immunohistochemistry and Western blot analysis. Weekly testicular biopsies and jugular blood samples were collected from 12 ram lambs over the 9 weeks of study. Hormone concentrations and the numbers of dividing Sertoli cells per seminiferous tubule (ST) area were analyzed relative to chronological age of animals and the two distinctive stages of Sertoli cell differentiation: (a) tight junction/ST lumen formation and (b) the onset of support mechanisms for the development of multiple germ cell types (presence of primary spermatocytes in >95% STs). Circulating FSH concentrations increased (p<0.05) immediately after first detection of ST lumen and reached a nadir (p<0.05) just prior to the end of the first wave of spermatogenesis. A decline in both fT4 and fT3 levels (p<0.05) occurred after Sertoli cells had formed the ST lumen and began supporting germ cell differentiation. There was a positive correlation between the numbers of proliferating Sertoli cells and serum fT4 (r=0.51, p<0.001) and fT3 (r=0.52, p<0.001) concentrations. TRs were expressed throughout the study period; however, prior to the formation of ST lumen, two isoforms were detected while only one TR isoform was present by the end of the first wave of spermatogenesis. Overall, the exit of Sertoli cells from the cell cycle that presages their final differentiation begins when THs and FSH levels are high, suggesting a permissive role of these hormones in the maturation of STs in prepubertal ram lambs.