Since glucocorticoids decrease and protein kinase C (PKC) activators increase amniotic PGE₂ production, the possibility that they regulate the activity of prostaglandin endoperoxide H synthase (PGHS), the rate-limiting enzyme of prostaglandin synthesis from arachidonate, was investigated. Glucocorticoids inhibited the production of PGE₂ from exogenous arachidonate specifically and in a concentration dependent fashion. Furthermore, cortisol decreased PGHS activity and the amount of PGHS protein in amnion microsomes, and reduced the rate of recovery of PGHS after acetylsalicylic acid (ASA) pretreatment. Actinomycin D blocked the inhibition of PGHS recovery by cortisol, but did not suppress the spontaneous recovery of the enzyme, indicating that the glucocorticoid induced a post-transcriptional inhibitor of PGHS synthesis. PKC-activating phorbol esters, such as 12-tetradecanoyl phorbol 13-acetate (TPA) increased the synthesis of PGE₂ from exogenous arachidonate, also in a specific and concentration dependent manner. PGHS recovery after ASA treatment was enhanced by TPA. PGHS activity and protein concentrations were increased by phorbol ester treatment; however, this was apparent only in tissues in which the concentrations of PGHS were initially low. These results show that the synthesis of PGHS is positively and negatively regulated in the human amnion by PKC and glucocorticoids, respectively, and suggest that effectors using these pathways may regulate the enzyme in vivo.

The objective of this study was to purify cells in the Leydig cell lineage following regeneration after ethane dimethanesulfonate (EDS) treatment and compare their steroidogenic capacity. Regenerated progenitor (RPLCs), immature (RILCs), and adult Leydig cells (RALCs) were isolated from testes 21, 28 and 56 days after EDS treatment respectively. Production rates for androgens including androsterone and 5α-androstane-17β, 3α-diol (DIOL), testosterone and androstenedione were measured in RPLCs, RILCs and RALCs in media after 3-h in vitro culture with 100 ng/ml LH. Steady-state mRNA levels of steroidogenic enzymes and their activities were measured in freshly isolated cells. Compared to adult Leydig cells (ALCs) isolated from normal 90-day-old rat testes, which primarily produce testosterone (69.73%), RPLCs and RILCs primarily produced androsterone (70.21%) and DIOL (69.79%) respectively. Leydig cells isolated from testes 56 days post-EDS showed equivalent capacity of steroidogenesis to ALCs and primarily produced testosterone (72.90%). RPLCs had cholesterol side-chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase 1 and 17α-hydroxylase but had almost no detectable 17β-hydroxysteroid dehydrogenase 3 and 11β-hydroxysteroid dehydrogenase 1 activities, while RILCs had increased 17β-hydroxysteroid dehydrogenase 3 and 11β-hydroxysteroid dehydrogenase 1 activities. Because RPLCs and RILCs had higher 5α-reductase 1 and 3α-hydroxysteroid dehydrogenase activities they produced mainly 5α-reduced androgens. Real-time PCR confirmed the similar trends for the expressions of these steroidogenic enzymes. In conclusion, the purified RPLCs, RILCs and RALCs are similar to those of their counterparts during rat pubertal development.

The majority of ovarian cancers (>90%) are believed to derive from the ovarian surface epithelium (OSE); a single layer covering the entire surface of the ovary. At ovulation, the OSE cell layer undergoes an inflammatory response, involving cell death and growth, in order to overcome ovarian surface rupture. Abnormalities during these processes are believed to contribute to the development of tumours. Using primary cultures of OSE cells, we have compared anti-inflammatory and proliferative responses directly between human and ovine OSE cells to further establish the use of ovine OSE cells as a suitable model system for the study of human OSE cells. In order to compare effects of inflammatory stimulation, expression and activity of 11βhydroxysteroid dehydrogenase (11βHSD) type 1 was measured in OSE cells in response to interleukin (IL)-1α. As previously identified in human OSE cells, treatment of ovine OSE cells with IL-1α stimulated a concomitant increase of 11βHSD type 1 mRNA (31-fold; P < 0.05) and oxoreductase activity, indicating an increased production of anti-inflammatory cortisol. To compare the growth of human and ovine OSE cells, OSE cell number was measured in response to treatment with gonadotropins or growth factors. In the presence of FSH, LH or human chorionic gonadotropin (hCG), ovine and human OSE cell growth was similarly stimulated >1.2-fold (P < 0.05). In the presence of connective tissue growth factor (CTGF) and more significantly insulin growth factor I (IGF-I), human and ovine OSE cell growth was also similarly stimulated >1.2-fold (P < 0.05) and >1.5-fold (P < 0.01), respectively. The induction of both human and ovine OSE cell growth by IGF-I or hCG was further shown to be dependent on activation of the MAP kinase/extracellular-signal-regulated kinase (ERK) pathway. Stimulation of ovine OSE cell growth by hepatocyte growth factor (HGF) was similarly shown to be ERK-dependent; however, for human OSE cells, HGF only mildly stimulated ERK phosphorylation and failed to stimulate OSE cell growth. The demonstration that human and ovine OSE cells share similarities at the level of cell signalling, gene expression and cellular growth supports the use of ovine OSE cells as a suitable model for the study of human OSE cells.

The sperm flagella 2 (SPEF2) gene is essential for development of normal sperm tail and male fertility. In this study, we characterized first the splice variants, promoter and its methylation, and functional single-nucleotide polymorphisms (SNPs) of the SPEF2 gene in newborn and adult Holstein bulls. Four splice variants were identified in the testes, epididymis, sperm, heart, spleen, lungs, kidneys, and liver tissues through RT-PCR, clone sequencing, and western blot analysis. Immunohistochemistry revealed that the SPEF2 was specifically expressed in the primary spermatocytes, elongated spermatids, and round spermatids in the testes and epididymis. SPEF2-SV1 was differentially expressed in the sperms of high-performance and low-performance adult bulls; SPEF2-SV2 presents the highest expression in testis and epididymis; SPEF2-SV3 was only detected in testis and epididymis. An SNP (c.2851G>T) in exon 20 of SPEF2, located within a putative exonic splice enhancer, potentially produced SPEF2-SV3 and was involved in semen deformity rate and post-thaw cryopreserved sperm motility. The luciferase reporter and bisulfite sequencing analysis suggested that the methylation pattern of the core promoter did not significantly differ between the full-sib bulls that presented hypomethylation in the ejaculated semen and testis. This finding indicates that sperm quality is unrelated to SPEF2 methylation pattern. Our data suggest that alternative splicing, rather than methylation, is involved in the regulation of SPEF2 expression in the testes and sperm and is one of the determinants of sperm motility during bull spermatogenesis. The exonic SNP (c.2851G>T) produces aberrant splice variants, which can be used as a candidate marker for semen traits selection breeding of Holstein bulls.

This study aimed to determine if short-term nutrient alteration affects (1) ovarian morphology, (2) plasma and ovarian antioxidant capability and (3) cell apoptosis and AKT signaling within the ovary. After estrus synchronization, 24 Hu sheep were assigned to three groups based on the nutrient requirement recommended for maintenance (M): 1 × M (Control), 1.5 × M (S) and 0.5 × M (R) during days 7–14 of their estrous cycle. The results indicated that undernourishment significantly increased the counts and volume of follicles <2.5 mm and decreased the counts and volume of follicles ≥2.5 mm (P < 0.05). Feed restriction altered the plasma and follicular redox balance within follicles ≥2.5 mm by inhibiting total antioxidant capacity, increasing malondialdehyde concentration (P < 0.05) and reducing the mRNA expression levels of superoxide dismutase 2 (SOD2) and glutathione peroxidase (GSH-PX), as well as the activities of total SOD and GSH-PX. Feed restriction also attenuated B-cell lymphoma-2 (BCL2) but enhanced Bcl-2-associated X protein (BAX) and BAX/BCL2 transcription and translation levels in granulosa cells (P < 0.05). Uniform staining intensities of AKT and P-AKT-Ser473 were observed in each follicle stage, whereas weaker P-AKT-Thr308 staining in the antral follicle than in the pre-antral follicle suggested possible involvement of P-AKT-Thr308 during the beginning of follicle development. P-AKT-Ser473 levels in follicles ≥2.5 mm was significantly reduced in the R group (P < 0.05). The results presented in this study demonstrate that suppressed folliculogenesis caused by feed restriction might be associated with attenuated AKT signaling, reduced follicular antioxidant capacity and enhanced granulosa cells apoptosis.