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Endometritis-pyometra is the most frequent and complex pathology in domestic bitches. This process involves several immunological changes as well as molecular mechanisms responsible for inflammation in the female uterus. The various clinical stages of pyometra are associated with various symptoms. In this review, several aspects are described, including physiological and pathological mechanisms as well as molecular changes which take place during induction of endometritis-pyometra. The authors also highlight the important role of growth factors and their receptors in this process. It is well known that pyometra is a compound process which mainly involves immunological changes during inflammation. However, this review presents a new overview of this process, which includes changes at the molecular level, e.g., the altered expression of genes crucial for the development of this disease. Although pyometra is the most frequent disease of the reproductive tract in bitches, the molecular basis of this process is still not entirely understood.

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The TGFB superfamily genes are involved in several important cell functions, including proliferation and differentiation, and the role of the expression of these genes in growth and development of theca and granulosa cells is well recognised. However, the dependence between the stage of oocyte maturation or follicular size and the expression of these genes in pigs is still not entirely known. This study was aimed at investigating the expression pattern of GDF9, TGFB1, TGFB2 and TGFB3 in porcine oocytes before and after in vitro maturation (IVM) as well as in oocytes collected from follicles of different sizes. RQ-PCR was performed to analyse the expression of GDF9, TGFB1, TGFB2 and TGFB3 in oocytes before and after IVM (oocytes cultured for 44 h in TCM-199), isolated from large (> 5 mm), medium (3-5 mm) and small (< 3 mm) follicles collected from ovaries of 28 puberal crossbred Landrace gilts after slaughter. We found an increased expression of both TGFB1 and TGFB2 in oocytes before IVM collected from large as compared to medium and small follicles (P < 0.05, P < 0.001, P < 0.01, P < 0.05, respectively). In these groups of oocytes we did not observe differences in GDF9 and TGFB3 mRNA levels. However, after IVM, GDF9 protein distribution in oocytes was significantly higher in large and medium follicles as compared to small ones (P < 0.01, P < 0.001, respectively). Moreover, an increased TGFB1, TGFB2 and TGFB3 proteins pattern was observed in oocytes of large compared to small follicles. The highest GDF9 and TGFB1 mRNA levels were found in oocytes after IVM compared to those before IVM. Based on our study we can suppose that the distribution pattern of TGFB superfamily genes is associated with the stage of maturation of porcine oocytes and the follicle size. Furthermore, GDF9 and TGFB1 may serve as molecular markers of the develop-mental potential of porcine oocytes. The confocal microscopic observation revealed that TGFB1 and TGFB3 were translocated between the zona and the cytoplasm of oocytes, depending on the stage of maturation and follicle size.

The CC (cumulus cell) proliferation index in relation to the expression and distribution of Cdk4 and Cx43 proteins, which are crucial factors for oocyte maturation, was investigated. Cumulus-oocyte complexes (COCs) were recovered from pubertal crossbred Landrace gilts and treated with collagenase, and separated CCs were cultured in standard TCM199 medium for 44 h. At each step of in vitro cultivation (IVC) of CCs (0, 12, 24 and 44 h), a normalized proliferation index was assessed. Cdk4 and Cx43 protein expression and the CC-specific cellular distribution were analyzed by confocal microscopic observation. The normalized proliferation index (number of cells attached, measured by impedance) was increased in the first 12 h of IVC (P<0.01) and differed between 12 h and 24 h of cultivation (P<0.001). Later, between 24 h–44 h of IVC, the CC proliferation rate was stable, and no significant differences were observed. Based on the confocal microscopic observation, increased expression of both Cdk4 and Cx43 was found after 44 h of IVC compared with the expression of these proteins before IVC. Moreover, after IVC, a substantial translocation of Cdk4 and Cx43 was noted from the nucleus to the cytoplasm of CCs. In conclusion, it was demonstrated for the first time that CCs can be cultured in vitro separately without oocytes and that the proliferation index was significantly increased in the first 12 h of IVC, which may reflect the process of ordinary cumulus cell expansion. Furthermore, the expression of both Cdk4 and Cx43 in CCs suggested that these proteins may be regarded as markers not only of proper oocyte maturation but also of CC differentiation. Translocation of these proteins into the cytoplasm of CCs after 44 h of IVC may be related to the expansion process.

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Integrins are the major receptors within the extracellular matrix (ECM) that mediate several functions connected with cell life and metabolism, such as cell adhesion, migration, cytoskeletal organization, proliferation, survival, and differentiation. A vascular endothelial growth factor (VEGF) is one of the most important angiogenic factors. It has been suggested that the expression of this gene may play crucial physiological roles in reproductive organs.All investigated endometrial tissues were isolated on day 10-12 after mating. Control bitches, used in this study, were in metestrus, which was determined according to the vaginal cytology and progesterone level in blood. Early pregnancy was verified by flushing the uterine horns with PBS. Total RNA was isolated from the bitches endometrium by means of the Chomczyński and Sacchi method, treated by DNase I, and reverse-transcribed into cDNA. A quantitative analysis of integrins α2b, β2 and β3, VEGF 164, 182 and 188 cDNA was performed by RT-PCR.In results we have shown an increased expression of all investigated genes (integrins α2b, β2 and β3, VEGF 164, 182, and 188) in pregnant bitches uterus as compared to non-pregnant females (P<0.001).Our results indicated that the expression of genes encoding integrins and vascular endothelial growth factors is different in relation to the time of the embryo implantation and it is increased in the first period of this process. This may be associated with the induction of specific mechanisms responsible for receptivity of uterus following the embryo attachment. In addition, all of investigated genes are up-regulated in a pregnancy-specific manner and the increased expression of these genes may regulate the uterus function during the implantation of canine embryos.

Oocytes and somatic cumulus cells are connected by an extensive network of gap junctions. These connections contribute in a major way to oocyte maturation and developmental competence. Cumulus-oocyte complexes (COCs) were cultured in standard porcine IVM culture medium (TCM 199) for 44 h. The morphological classification of COCs is based on the number of cumulus cell layers and the degree of their compaction, as well as on cytoplasm composition (homogenous, heterogeneous). The obtained COCs were divided into four grades according to this classification system. By assessing the activity of glucose-6-phosphate dehydrogenase (G6PDH) using the brilliant cresyl blue (BCB) test, real-time quantitative PCR (RQ-PCR) reaction methods and confocal microscopic observations, we determined the transcript levels of connexins 43 and 45, cyclin dependent kinases (cdk5 and cdk5r), and cdk inhibitors 1 and 3 (p27kip1 and cdkn3) as well as cdk4 protein in morphologically different groups of porcine oocytes isolated from puberal gilts. To assess their nuclear status the completely denuded oocytes were subjected to DAPI staining. We found statistically increased cdkn3, cdk5 and connexin 45 mRNA levels in oocytes graded as I as compared to II, III, and IV. The cdkn1, cdk5r and connexin 43 transcript contents were higher only when comparing between oocytes graded as I, III and IV. The cdk4 protein in oocytes graded I and II is localized mainly in the zona pellucida, although in grade III COCs the expression of this protein is decreased and observed only in the cytoplasm. Grade IV COCs do not demonstrate a significant presence of cdk4 protein. With regards to nuclear maturation, the percentage of MII stage oocytes was significantly (P < 0.05) higher in grade I and II oocytes as compared to grade III and IV oocytes. Our results demonstrate for the first time that cdk4 protein localization and all of the investigated transcript levels are associated with COC morphology and may be related to further maturation ability as well as developmental competence of oocytes.