Retinoic acid treatment alters germ cell heterogeneity and testicular echotexture in prepubescent ram lambs.

Testicular echotextural attributes are closely associated with spermatogenic development; however, precise characterisation of specific germ cell types is difficult due to tremendous germ cell heterogeneity. Recently, retinoic acid (RA) administration in neonatal mice was found to induce highly synchronised spermatogenesis as adults. A RA-treatment protocol was tested in 17 ram lambs treated with or without RA at 8 weeks of age, with scrotal ultrasonography and blood samples collected until castration 24h or 2.5 weeks later. At 8.2 weeks of age, the nuclear:seminiferous tubule (ST) area was higher in the treated compared with the control group. Serum testosterone concentrations and numerical pixel values (NPVs) of the testicular parenchyma reached a peak at 9 weeks of age in both groups of ram lambs studied. At 10.5 weeks of age, the percentage of ST cross-sections with different germ cells as the most mature germ cell type was lower and the inter-tubular heterogeneity and NPVs were also lower in the treated compared with the control animals. RA manipulation of spermatogenesis in prepubertal ram lambs may provide a suitable model for further investigation of the echotextural characteristics of specific germ cell types and critical developmental events.

Correlations among ultrasonographic and microscopic characteristics of prepubescent ram lamb testes.

The onset of spermatogenesis during prepubertal development is accompanied by dynamic changes in testicular microstructure. Computer-assisted analysis of scrotal ultrasonograms may allow us to track these changes in a noninvasive manner; however, the echotextural characteristics of different histomorphological variables remain unclear. Hence the objective of this study was to compare echotextural and microscopic attributes of the testis over the first wave of spermatogenesis in prepubescent ram lambs. Bi-weekly ultrasound examinations and weekly testicular biopsies were carried out in 22 ram lambs from 9.5-10 weeks of age or the attainment of 15 cm(3) in testicular volume, respectively, to the first detection of elongated spermatids (ESt). Testicular echogenicity was highly variable with age; however, after the alignment of data to the first detection of ESt, there was an initial increase followed by a decline, corresponding to the mitotic and postmitotic phases of spermatogenesis in prepubescent ram lambs. Testicular echotextural attributes (mean numerical pixel values and pixel heterogeneity) correlated with seminiferous tubule (ST) diameter, the number of degenerating cells/ST cross-section (XS), and the number of ubiquitin C-terminal hydrolase L-1 (a marker for prespermatogonia and undifferentiated spermatogonia) staining cells/ST XS during the mitotic and postmitotic phases. Additionally, in the postmitotic phase, significant correlations were recorded between the quantitative echotextural characteristics and ST cell density, nuclear:ST area and percentages of STs with different spermatogenic cells as the most mature germ cell type present. These results indicate that ram testes exhibit distinctive echotextural characteristics during the mitotic and postmitotic phases of germ cell differentiation. It is concluded that scrotal ultrasonography in conjunction with computerized image analysis holds potential as a noninvasive alternative to testicular biopsy in monitoring the reproductive status throughout different stages of testicular development.

Subcellular localization of protein kinase C delta and epsilon affects transcriptional and post-transcriptional processes in four-cell mouse embryos.

During mouse preimplantation development, two isozymes of protein kinase C (PKC), delta and epsilon, transiently localize to nuclei at the early four-cell stage. In order to study their functions at this stage, we altered the subcellular localization of these isozymes (ratio of nuclear to cytoplasmic concentrations) with peptides that specifically activate or inhibit translocation of each isozyme. The effects of altering nuclear concentration of each isozyme on transcription (5-bromouridine 5'-triphosphate (BrUTP) incorporation), amount and distribution of small nuclear ribonucleoproteins (snRNPs), nucleolar dynamics (immunocytochemistry for Smith antigen (Sm) protein) and the activity of embryonic alkaline phosphatase (EAP; histochemistry) were examined. We found that nuclear concentration of PKC epsilon correlated with total mRNA transcription. Higher nuclear concentrations of both PKC delta and epsilon decreased storage of snRNPs in Cajal bodies and decreased the number of nucleoli, but did not affect the nucleoplasmic concentration of snRNPs. Inhibiting translocation of PKC delta out of the nucleus at the early four-cell stage decreased cytoplasmic EAP activity, whereas inhibiting translocation of PKC epsilon increased EAP activity slightly. These results indicate that translocation of PKC delta and epsilon in and out of nuclei at the early four-cell stage in mice can affect transcription or message processing, and that sequestration of these PKC in nuclei can also affect the activity of a cytoplasmic protein (EAP).