Relationship between rank and plasma testosterone and cortisol in red deer males (Cervus elaphus).

The objective of this study was to assess the effect of a change in the social composition in a group of red deer males on the relationship between their rank and testosterone. A group of twelve adult red deer males (Cervus elaphus) was tested in two social settings. From April 15 to June 9 (Period 1) this group was kept separately in an enclosure. On June 10, nine 3-year-old males were added to that group of adult males. They were kept together until August 31. We performed 10 observations of the group when the agonistic interactions of the males were recorded and we took 9 blood samples per male in Period 1; 11 observations were made and 10 samples were taken in Period 2. Concentrations of testosterone and cortisol were later determined in plasma. Adding much younger and smaller sparring partners into the experimental group of adult males in Period 2 altered the agonistic behaviour of the adults even though this did not trigger any change in rank position of the experimental males except one. Adult males targeted preferentially their attacks on individuals much lower in the hierarchy. Experimental male deer with higher social rank had lower levels of testosterone in Period 1; in Period 2 it was just the opposite. In Period 1 the animals had higher cortisol levels than in Period 2. As controls we used four adult (5years old) males sharing the enclosure with four 3-year-old males. No changes in hormone concentrations were observed in the control group. Thus, changing the social environment of adult red deer males resulted in change of the relationship between rank and testosterone and cortisol concentrations.

Telomerase activity in pig granulosa cells proliferating and differentiating in vitro.

The aim of the work was to analyze the telomerase activity (TA) in two different populations of pig granulosa cells (GC) proliferating and differentiating in vitro: (a) in relatively undifferentiated granulosa cells isolated from small (1-2 mm) antral follicles and (b) in functionally advanced, differentiated cells obtained from large (5-7 mm) antral follicles. The proliferative potential in vitro of small follicle granulosa cells (SF-GC) was higher than that of large follicle granulosa cells (LF-GC). EGF stimulated significantly (p<0.01) proliferation in SF-GC as well as LF-GC. FSH did not have a stimulating effect on proliferation in both of the GC populations. Steroidogenesis was induced in both SF- and LF-GC in vitro. Significantly higher (p<0.01) levels of estradiol were measured in LF-GC cultures. In SF-GC, no significantly different effects of EGF and FSH on estradiol production were found. The production of progesterone in vitro was higher in LF-GC than in SF-GC and its production was specifically promoted by FSH in contrast to estradiol the synthesis of which in vitro was less dependent on culture conditions. Using the TRAP assay telomerase activity was detected in freshly isolated and in vitro cultured pig SF- and LF-GC. In EGF, but not FSH stimulated SF-GC, significantly enhanced (p<0.05) TA in comparison with the control was observed at an interval of 24 h of culture. After the 48 h in vitro, levels of TA in both EGF and FSH treated cells were comparable with control. In LF-GC, both EGF and FSH stimulated significantly (p<0.05) TA after the 24h of in vitro culture. At an interval of 48 h, no significant differences in the level of TA were observed between control, EGF and FSH stimulated LF-GC. Comparing the levels of TA in SF- and LF-GC, significantly higher levels of TA were found in control (p<0.05) and EGF (p<0.01) treated SF-GC after 24 h in vitro. On the other hand, absolutely, but not significantly, higher levels of TA were found in LF-GC versus SF-GC in all culture conditions after 48 h in vitro.

Molecular mechanisms of insulin-like growth factor 1 promoted synthesis and retention of hyaluronic acid in porcine oocyte-cumulus complexes.

The purpose of the present study was to elucidate signaling pathways by which insulin like-growth factor 1 (IGF1) promotes FSH-stimulated synthesis and retention of hyaluronic acid (HA) in pig oocyte-cumulus complexes (OCCs) cultured in serum-free medium. We found that IGF1 had no effects on FSH-stimulated production of cAMP and activation of protein kinase A in the OCCs. Immunoblotting with phospho-specific antibodies showed that FSH moderately phosphorylated v-akt murine thymoma viral oncogene homolog (AKT) and mitogen-activated kinase 3 and 1 (MAPK3/1) in cumulus cells. The exposure of OCCs to both FSH and IGF1 resulted in a significant (P < 0.05) increase in AKT and MAPK3/1 phosphorylation. An inhibitor of phosphoinositide-3-kinase (PIK3), LY 294002, significantly (P < 0.05) reduced the IGF1-enhanced phosphorylation of AKT, and inhibitors of AKT (SH6) and MAPK3/1 (U0126) significantly (P < 0.05) decreased the synthesis and retention of HA stimulated by concomitant exposure of OCCs to both FSH and IGF1. The IGF1-promoted synthesis of HA was not accompanied by an increase in the relative abundance of hyaluronan synthase 2 (HAS2) mRNA in the cumulus cells. We conclude that IGF1 promotes the FSH-stimulated synthesis and retention of HA in pig OCCs by PIK3/AKT- and MAPK3/1-dependent mechanisms.

Immunohistochemical localization of proliferating cell nuclear antigen (PCNA) in the pig ovary.

The aim of the study was to determine the expression of proliferating cell nuclear antigen protein (PCNA) in the pig ovary. The localization of PCNA was demonstrated in paraffin sections of pig ovarian tissue using primary mouse monoclonal anti-PCNA antibody. In primordial follicles, no remarkable staining for PCNA either in granulosa cells or in the oocytes was observed. In primary to secondary follicles, positive staining in oocytes and in some granulosa cells was detected. The advanced preantral and particularly actively growing small to large antral follicles showed extensive PCNA labeling in the layers of granulosa and theca cells and in the cumulus cells encircling the oocyte. PCNA labeling was expressed in nuclei of oocytes in preantral and small antral follicles. In atretic follicles, the level of PCNA protein expression was dependent on the stage of atresia. Follicles demonstrating advanced atresia showed only limited or no PCNA labeled granulosa and theca cells. The results of the study demonstrate that follicular growth and development in pig ovary may be effectively monitored by determining the granulosa cell expression of PCNA.

Aromatic hydrocarbon receptor (AhR) in the porcine theca and granulosa cells: effect of TCDD, PCB 126 and PCB 153 on the expression of AhR.

OBJECTIVE: In our previous papers, we demonstrated that TCDD and PCBs (both coplanar PCB 126 and non-coplanar PCB 153) caused the decreased estradiol secretion in cultured pig ovarian follicles. However, the mechanism of action is not clearly understood. Moreover, to our knowledge, the expression of AhR in pig follicular cells was not described yet. METHODS: In order to elucidate if TCDD, PCB 126, and PCB 153 can disrupt ovarian steroidogenesis via AhR-dependent mechanism, granulosa and theca cells were isolated from pig ovarian small follicles and subsequently treated with 32 pg/ml of TCDD, 100 ng/ml of PCB 153, 100 pg/ml of PCB 126 in an in vitro culture. After 3-hour exposure to the chemicals, cells were prepared for immunohistochemical analysis or collected for immunobloting, and the effects of TCDD and PCBs on the levels of AhR protein were studied. RESULTS: Under basal conditions, AhR staining showed a diffuse cytoplasmic pattern of distribution as well in granulosa and theca cells. However, immunocytochemical labeling of AhR in theca cells was less evident and only few cells displayed cytoplasmic staining. Immunoblotts prepared from granulosa and theca cell lysates detected a strong band of 120 kDa indicating AhR protein expression. The level of AhR expression detected by Western blot analysis was higher in granulosa cells than in theca cells. Intensive cytoplasmic and nuclear labeling corresponding to AhR protein in TCDD and PCB 126 treated granulosa cells was observed. In PCB 153 treated cells, the level of AhR staining was comparable with control. Western blot analysis showed a strong band in TCDD-treated grnulosa cells and only slightly differences compared with control in those exposed to PCB 126. The effect of both PCBs on immunocytochemical AhR staining in theca cells was less evident. Immunoblott analysis did not reveal any substantial differences between the control and TCDD- and PCBs-treated theca cells. CONCLUSIONS: This study has shown different AhR expression in porcine theca and granulosa cells, in respect of both its intracellular localization and cellular distribution. Both, dioxin and dioxin-like PCB activated AhRs in granulosa but not in theca cells. Differences in AhR-responsiveness of granulosa and theca cells to TCDD and dioxin-like PCB 126 are probably connected with the differences of these cells in estradiol secretion and different proliferative potential.

Comprehensive comparison of the cytotoxic activities of onconase and bovine seminal ribonuclease.

Onconase (ONC) and bovine seminal ribonuclease (BS-RNase) are homologs of bovine pancreatic ribonuclease (RNase A). Unlike RNase A, ONC and BS-RNase can evade the cytosolic ribonuclease inhibitor protein and are potent cytotoxins. Here, the endogenous cytotoxic activities of ONC and BS-RNase are compared in a wide variety of assays. Injections of ONC into one or both testes of mice and rats evokes a stronger aspermatogenic activity than does the injection of BS-RNase. Epididymides exposed to ONC lose mass and all sperm. Testicular tissue is gradually colonized by immunite and fibrocytic cells. Yet, Leydig cells are always present and functional in the ligamented parts of testicles injected with ONC or BS-RNase. ONC is likewise more toxic to mouse embryos than is BS-RNase, both in vitro and in vivo. The antiproliferative effect of ONC on human tumor cell line ML-2 and lymphocytes in a mixed lymphocyte culture is also more pronounced than is that of BS-RNase. The number of granulocyte-macrophage colony-forming units is repressed almost completely by ONC, whereas a five-fold higher dose of BS-RNase does not cause substantial inhibition. In mice, ONC is less immunogenic than BS-RNase but more immunogenic than RNase A. Together, these data indicate that ONC is a pluripotent cytotoxin, and serves as the benchmark with which to gauge the cytotoxicity of other ribonucleases.