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Summary.

Pilocarpine (50 mg) administered to the boar 20 min before semen collection had no detectable effect on either ejaculation or semen composition. However, the volume of fluid semen, the total volume of semen, and the time required for ejaculation differed significantly (P<0·01) between breeds of boars.

Leydig cells in the rat testis can be specifically ablated with ethane dimethane sulfonate (EDS) and will subsequently re-generate. In this study, we have characterized Leydig cell re-generation and expression of selected cell-signaling molecules in a germ cell-free model of EDS action. This model offers the advantage that re-generation occurs on a stable background without confounding changes from the regressing and repopulating germ cell population. Adult rats were treated with busulfan to remove the germ cell population and Leydig cells were then ablated with EDS. Testicular testosterone levels declined markedly within 24 h of EDS treatment and started to recover after 8 days. After EDS treatment there were marked declines in levels of Leydig cell-specific mRNA transcripts coding for steroidogenic enzymes cytochrome P450 11a1 (Cyp11a1), cytochrome P450 17a1 (Cyp17a1), 3β-hydroxysteroid dehydrogenase type 1 (Hsd3b1), 17β-hydroxysteroid dehydrogenase type 3 (Hsd17b3) and the LH receptor. Levels of all transcripts recovered within 20 days of EDS treatment apart from Hsd17b3, which remained undetectable up to 20 days. Immunohistochemical localization of CYP11A1 during the phase of early Leydig cell re-generation showed that the Leydig cell precursors are spindle-shaped peritubular cells. Studies on factors which may be involved in Leydig cell re-generation showed there were significant but transient increases in platelet-derived growth factor A (Pdgfa), leukemia inhibitory factor (Lif), and neurofilament heavy polypeptide (Nefh) after EDS, while desert hedgehog (Dhh) levels declined sharply but recovered by 3 days. This study shows that the Leydig cell precursors are peritubular cells and that expression of Pdgfa and Lif is increased at the start of the re-generation process when precursor proliferation is likely to be taking place.

It has been shown that testicular germ cell development is critically dependent upon somatic cell activity but, conversely, the extent to which germ cells normally regulate somatic cell function is less clear. This study was designed, therefore, to examine the effect of germ cell depletion on Sertoli cell and Leydig cell transcript levels. Mice were treated with busulphan to deplete the germ cell population and levels of mRNA transcripts encoding 26 Sertoli cell-specific proteins and 6 Leydig cell proteins were measured by real-time PCR up to 50 days after treatment. Spermatogonia were lost from the testis between 5 and 10 days after treatment, while spermatocytes were depleted after 10 days and spermatids after 20 days. By 30 days after treatment, most tubules were devoid of germ cells. Circulating FSH and intratesticular testosterone were not significantly affected by treatment. Of the 26 Sertoli cell markers tested, 13 showed no change in transcript levels after busulphan treatment, 2 showed decreased levels, 9 showed increased levels and 2 showed a biphasic response. In 60% of cases, changes in transcript levels occurred after the loss of the spermatids. Levels of mRNA transcripts encoding Leydig cell-specific products related to steroidogenesis were unaffected by treatment. Results indicate (1) that germ cells play a major and widespread role in the regulation of Sertoli cell activity, (2) most changes in transcript levels are associated with the loss of spermatids and (3) Leydig cell steroidogenesis is largely unaffected by germ cell ablation.

This study was designed to identify genes that regulate the transition from FSH- to LH-dependent development in the bovine dominant follicle (DF). Serial analysis of gene expression (SAGE) was used to compare the transcriptome of granulosa cells isolated from the most oestrogenic growing cohort follicle (COH), the newly selected DF and its largest subordinate follicle (SF) which is destined for atresia. Follicle diameter, follicular fluid oestradiol (E) and E:progesterone ratio confirmed follicle identity. Results show that there are 93 transcript species differentially expressed in DF granulosa cells, but only 8 of these encode proteins known to be involved in DF development. Most characterised transcripts upregulated in the DF are from tissue development genes that regulate cell differentiation, proliferation, apoptosis, signalling and tissue remodelling. Semiquantitative real-time PCR analysis confirmed seven genes with upregulated (P≤0.05) mRNA expression in DF compared with both COH and SF granulosa cells. Thus, the new genes identified by SAGE and real-time PCR, which show enhanced mRNA expression in the DF, may regulate proliferation (cyclin D2; CCND2), prevention of apoptosis or DNA damage (growth arrest and DNA damage-inducible, β; GADD45B), RNA synthesis (splicing factor, arginine/serine rich 9; SFRS9) and unknown processes associated with enhanced steroidogenesis (ovary-specific acidic protein; DQ004742) in granulosa cells of DF at the onset of LH-dependent development. Further studies are required to show whether the expression of identified genes is dysregulated when abnormalities occur during DF selection or subsequent development.

Two-pore domain K⁺ channels are an emerging family of K⁺ channels that may contribute to setting membrane potential in both electrically excitable and non-excitable cells and, as such, influence cellular function. The human uteroplacental unit contains both excitable (e.g. myometrial) and non-excitable cells, whose function depends upon the activity of K⁺ channels. We have therefore investigated the expression of two members of this family, TWIK (two-pore domain weak inward rectifying K⁺ channel)-related acid-sensitive K⁺ channel (TASK) and TWIK-related K⁺ channel (TREK) in human myometrium. Using RT-PCR the mRNA expression of TASK and TREK isoforms was examined in myometrial tissue from pregnant women. mRNAs encoding TASK1, 4 and 5 and TREK1 were detected whereas weak or no signals were observed for TASK2, TASK3 and TREK2. Western blotting for TASK1 gave two bands of approximately 44 and 65 kDa, whereas TREK1 gave bands of approximately 59 and 90 kDa in myometrium from pregnant women. TASK1 and TREK1 immunofluorescence was prominent in intracellular and plasmalemmal locations within myometrial cells. Therefore, we conclude that the human myometrium is a site of expression for the two-pore domain K⁺ channel proteins TASK1 and TREK1.