Heat shock protein HSP90 immunoexpression in equine endometrium during oestrus, dioestrus and anoestrus.

Heat shock proteins play a crucial role in cellular development, proliferation, differentiation and apoptosis. Heat shock protein 90 (HSP90) has been localised in the human endometrium, where its immunoexpression changes during the menstrual cycle. Similar studies have not been done for the equid species, so the present study aimed to describe endometrial HSP90 immunoexpression in mare endometrium. Endometrial biopsies were formalin-fixed and paraffin-embedded, and sections were stained with haematoxylin-eosin in preparation for HSP90 immunohistochemistry. Immunostaining and morphometric analyses were performed on the epithelial lining, endometrial glands and connective stroma during oestrus, dioestrus phase and anoestrus period (n = 7 per phase or period). Immunoexpression was localised in the basal region of the epithelial cells lining the lumen. Immunoexpression was greater during oestrus than during either dioestrus or anoestrus. During anoestrus, there was little immunostaining in the endometrium, suggesting that HSP90 is involved in the functional modulation of sex steroid receptors in cyclic mares. Indeed, the function of HSP90 as a chaperone in the folding of proteins, such as steroid receptors, might explain the greater intensity of immunostaining during the oestrus and dioestrus phases, compared the anoestrus period. We conclude that, in the mare, HSP90 plays a role in endometrial function and that further studies are needed to test whether it is important in pathological conditions as endometritis.

Sperm transport and endometrial inflammatory response in mares after artificial insemination with cryopreserved spermatozoa.

This study aimed to determine whether the insemination site and dose with cryopreserved sperm of reproductively normal mares affect the sperm population in uterine tubes and the intensity of endometrial inflammatory response. Experimental subjects were estrous mares inseminated, in the mid-uterine body (Body) or the tip of the uterine horn (Tip), ipsilateral to the dominant follicle, with one 0.5 mL straw with 50 × 106 sperm (50) or with eight straws with 50 × 106 sperm/straw (400). Mares were slaughtered 2 h, 4 h and 12 h after artificial insemination (AI) and randomly assigned to following groups: Body 50 (n = 19) (2 h, 4 h or 12 h); Tip 50 (n = 29) (2 h, 4 h, or 12 h); Body 400 (n = 24) (2 h, 4 h, or 12 h); Tip 400 (n = 21) (2 h, 4 h, or 12 h). A Control group (n = 16) was not inseminated. After slaughter, uterine tubes were separated from uterus, and uteri and tubes flushed with phosphate-buffered saline (PBS). After flushing, an endometrial sample was collected from ipsilateral and contralateral horns and mid-uterus body for further histopathological examination. A sample of each uterine tube flushing was examined for sperm count, and a sample of each uterine flushing was used for polymorphonuclear neutrophils (PMNs) count. Data were analyzed using PROC GLM from SASv9.4. Insemination time, site, sperm dose, and their interactions were considered independent variables and sperm and PMNs numbers dependent variables. Deep horn insemination increased ipsilateral uterine tube sperm number without an increase in the inflammatory reaction compared with the uterine body insemination. The higher the insemination dose, the higher the uterine tubes' sperm number and inflammatory reaction, with a quicker resolution. In conclusion, the insemination site and dose affected sperm in the uterine tubes, while post-insemination time and dose influenced the inflammatory reaction.

Histomorphometric and vascular changes in equine endometrium after the infusion of conceptus fragments.

This experiment aimed to verify if the proteins present in a 13th day conceptus induce changes in the equine endometrial ultra-structure, histology, and vascularization, two days after its infusion. Ten healthy cyclic mares were used. Once estrus was confirmed, mares were examined daily to detect ovulation (day 0). After ovulation, mares were examined daily until day seven by transrectal palpation and B-mode and Doppler ultrasonography. In this first cycle, intrauterine biopsies were collected at day seven after ovulation, constituting the Cyclic group (n = 10). In the second cycle, the same mares daily were examined until ovulation was detected. After ovulation, mares were examined daily by transrectal palpation and B-mode and Doppler ultrasonography until day 7. On day 5, after ovulation, fragments from previously collected 13-day-old concepti were infused into the uterus of each mare. Intrauterine biopsies were collected at day 7 in all mares (n = 10), constituting the Fragment group. The percentage of ciliated and flattened cells decreased in the Fragment group. Protruded cells, superficial and intraglandular secretion, glandular lumen and diameter, blood vessel diameter, endometrial vascularization, and immune cells were higher in the Fragment group than in the Cyclic group. In summary, proteins of 13th day equine conceptus fragments infused at day five after ovulation signaled histological and vascular changes in the endometrium at the 7th day after ovulation.

Uterine infusion of conceptus fragments changes the protein profile from cyclic mares.

This experiment aimed to compare at day seven after ovulation, the protein profile of uterine fluid in cyclic mares with mares infused two days before with Day 13 conceptus fragments. Experimental animals were ten healthy cyclic mares, examined daily to detect ovulation (Day 0) as soon as estrus was confirmed. On day seven, after ovulation, uterine fluid was collected, constituting the Cyclic group (n = 10). The same mares were examined in the second cycle until ovulation was detected. On day five, after ovulation, fragments from a previously collected concepti were infused into each mare's uterus. Two days after infusion, uterine fluid was collected, constituting the Fragment group (n = 10). Two-dimensional electrophoresis technique processed uterine fluid samples. A total of 373 spots were detected. MALDI-TOF/TOF and NanoUHPLC-QTOF mass spectrometry identified twenty spots with differences in abundance between the Cyclic and Fragment group. Thirteen proteins were identified, with different abundance between groups. Identified proteins may be related to embryo-maternal communication, which involves adhesion, nutrition, endothelial cell proliferation, transport, and immunological tolerance. In conclusion, conceptus fragments signalized changes in the protein profile of uterine fluid seven days after ovulation in comparison to the observed at Day 7 in the same cyclic mares.

Ceruloplasmin, serotransferrin and albumin presented different abundance in mares' uterine fluid five days after insemination.

Characterization of the uterine proteome before the entry of the conceptus to the uterus is essential to know the factors involved in the physiological events of gestation. The objective of the study was to compare proteomic profile of uterine fluid collected on day 5 post ovulation of cyclic and inseminated mares. Samples of endometrial secretion were recovered over 2 cycles during the fifth day post ovulation. The first cycle constituted the Cyclic group and in the following cycle, the same mares were inseminated and considered as the Inseminated group. All the samples were subjected to two-dimensional electrophoresis (2D-PAGE). A total of 107 spots were visualized by 2D-PAGE. Three spots with differences in abundance between the inseminated and cyclic mares and with presence in at least 80% in one of the groups were selected and identified. The selected spots were extracted, digested by trypsin and analyzed by matrix assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry (MS) for protein identification. Three proteins were identified: ceruloplasmin (CP) serotransferrin (TF) and albumin (ALB). The identified proteins in this study were related to iron metabolism and immunological tolerance suggesting that changes in their abundance during the 5 days after ovulation are probably a signaling mechanism of the potential equine conceptus to the maternal immune system for its immunological recognition. Probably changes in abundance of CP, ALB and TF represent a mechanism of endometrial preparation for the maternal recognition, attachment and development of a potential equine embryo. There is also evidence to support an alternative hypothesis suggesting that protein changes are inflammatory events, resulting from a previous inflammation due to residual seminal effects.

Tissue preservation and cell culture of Przewalskii´s horse (Equus przewalskii): an endangered species / Preservação de tecidos e cultura de células do cavalo de Przewalski (Equus przewalskii): uma espécie em extinção

The Przewalskii´s horse or Mongolian wild horse (Equus przewalskii, Poljakov, 1881) is presently the only species of wild horse in existence. Originally from Asia, it is, classified as in extremely high risk of extinction which puts the species in the seriously threatened category. The aim of this work was the preservation of tissues and the development of cell cultures from tissue samples obtained from a Przewalskii´s horse after its death. Biopsies of skin, skeletal and cardiac muscle, and ear cartilage were removed from a recently dead horse, added to Phosphate Buffer Saline (PBS) and refrigerated until processing. Some of the samples were frozen in liquid nitrogen and the other was grown as explants to generate fibroblast cell monolayers. The cell cultures obtained, were subsequently propagated with low passages, and frozen in liquid nitrogen, thus avoiding genetic and phenotypic alterations. The tissues and cell cultures were thawed to ascertain their viability by checking its progressive grow in a flask. It was not possible to obtain cultures from cardiac muscle. A bank of tissues and cells from the single Przewalskii´s horse that existed in Uruguay was generated, and can be used for scientific purposes and for the conservation of the species in the future