The present studies aimed to understand the interrelationships between stress, hormones and heat shock proteins (HSPs) in the ovary. We examined (1) whether HSP70.2, HSP72 and HSP105/110 can be produced and accumulated in porcine ovarian tissue, (2) whether these HSPs could be indicators of stress, i.e. whether two kinds of stress (high temperatures and malnutrition/serum deprivation) can affect them, and (3) whether some hormonal regulators of ovarian functions (insulin-like growth factor (IGF)-I, leptin and follicle-stimulating hormone (FSH)) can affect these HSPs and response of ovaries to HSP-related stress. We analysed the expression of HSP70.2, HSP72 and HSP105/110 mRNA (by using real-time reverse transcriptase polymerase chain reaction) in porcine ovarian granulosa cells, as well as the accumulation of HSP70 protein (by using sodium dodecyl sulphate polyacrylamide gel electrophoresis–Western) in either whole ovarian follicles and granulose cells cultured at normal (37.5°C) or high (41.5°C) temperature, with and without serum and with and without IGF-I, leptin and FSH. Expression of mRNA for HSP70.2, HSP72 and HSP105/110 in ovarian granulosa cells and accumulation of HSP70 protein in whole ovarian follicles and granulosa cells were demonstrated. In all the groups, addition of either IGF-I, leptin and FSH reduced the expression of HSP70.2, HSP72 and HSP105/110 mRNA. Both high temperature, serum deprivation and their combination resulted in increase in mRNAs for all three analysed HSPs. Additions of either IGF-I, leptin and FSH prevented the stimulatory effect of both high temperature and serum deprivation on the transcription of HSP70.2, HSP72 and HSP105/110. In contrast, high temperature reduced accumulation of peptide HSP70 in both ovarian follicles and granulosa cell. Serum deprivation promoted accumulation of HSP70 in granulosa cells, but not in ovarian follicles. Addition of IGF-I, leptin and FSH was able to alter accumulation of HSP70 in both follicles and granulosa cells. The present observations suggest (1) that HSPs can be synthesised in ovarian follicular granulosa cells; (2) that hormones (IGF-I, leptin and FSH) can inhibit, whilst stressors (both high temperature and malnutrition/serum deprivation) can stimulate transcription of HSP70.2, HSP72 and HSP105/110 genes, whilst heat stress, but not malnutrition, can promote depletion of HSP70 in ovarian cells, and (3) that hormones (IGF-I, leptin and FSH) can prevent stress-related changes in HSPs. The application of HSPs as indicators and mediators of stress and hormones on ovarian functions, as well as use of hormones and HSPs as anti-stressor molecules, are discussed.

Flow cytometry becomes a common method for analysis of spermatozoa quality. Standard sperm characteristics such as viability, acrosome and chromatin integrity, oxidative damage (ROS) etc. can be easily assess in any animal semen samples. Moreover, several fertility-related markers were observed in humans and some other mammals. However, these fertility biomarkers have not been previously studied in ram. The aim of this study was to optimize the flow-cytometric analysis of these standard and novel markers in ram semen. Ram semen samples from Slovak native sheep breeds were analyzed using CASA system for motility and concentration and were subsequently stained with several fluorescent dyes or specific antibodies to evaluate sperm viability (SYBR-14), apoptosis (Annexin V, YO-PRO-1, FLICA, Caspases 3/7), acrosome status (PNA, LCA, GAPDHS), capacitation (merocyanine 540, FLUO-4 AM), mitochondrial activity (MitoTracker Green, rhodamine 123, JC-1), ROS (CM-H₂DCFDA, DHE, MitoSOX Red, BODIPY), chromatin (acridine orange), leukocyte content, ubiquitination and aggresome formation, and overexpression of negative biomarkers (MKRN1, SPTRX-3, PAWP, H3K4me2). Analyzed semen samples were divided into two groups according to viability as indicators of semen quality: Group 1 (viability over 60%) and Group 2 (viability under 60%). Significant (p < 0.05) differences were found between these groups in sperm motility and concentration, apoptosis, acrosome integrity (only PNA), mitochondrial activity, ROS production (except for DHE), leukocyte and aggresome content, and high PAWP expression. In conclusion, several standard and novel fluorescent probes have been confirmed to be suitable for multiplex ram semen analysis by flow cytometry as well as several antibodies have been validated for the specific detection of ubiquitin, PAWP and H3K4me2 in ram spermatozoa.