Immunohistochemical study of immune cells in the bovine endometrium at different stages of the oestrous cycle

Normal uteri were collected from 34 maiden heifers at slaughter and their stage of the oestrous cycle was determined from the ovarian structures. Cells expressing major histocompatibility complex ( ) class II were widely distributed in the epithelium and lamina propria of the endometrium and were the most abundant population studied. The expression of class II increased in the endometrium during the follicular phase of the cycle. Macrophages occurred throughout the stratum compactum and stratum spongiosum and their density did not appear to vary with the stage of the cycle. CD4+ cells were found mainly in the stratum spongiosum, whereas CD8+ cells were present mainly within the luminal and glandular epithelia and in the stratum compactum. The density of the populations of CD4+ and CD8+ cells increased markedly during the mid-to-late luteal and follicular phases, respectively, of the cycle. In the endometrium lymphoid aggregates were observed only rarely and no B cells were identified in any of the heifers.     

Cell-specific localization of oestrogen receptor beta (ESR2) mRNA within various bovine ovarian cell types using in situ hybridization

The localization of oestrogen receptor beta (ESR2) mRNA, in this article denominated as (ERbeta) mRNA, was examined using in situ hybridization in the ovaries of randomly selected cows, irrespective of the cycle stage of the animals. A 602-bp fragment of ERbeta mRNA was cloned, sequenced and digoxigenin (DIG)-labelled. Semi-quantitative evaluation showed that the scores for ERbeta mRNA were moderate to high in the follicle cells of both primordial and primary follicles, but lower in granulosa cells of secondary follicles. In vital tertiary follicles, the total ERbeta mRNA expression was low but varied between the different animals. In both obliterative and cystic atretic follicles, high to moderate ERbeta mRNA scores were noticed in the granulosa cells. The stroma cells surrounding primordial and primary follicles and the theca cells of secondary follicles showed moderate ERbeta mRNA levels, whereas the ERbeta mRNA score in theca interna and theca externa cells of vital tertiary follicles was distinctly higher. In the theca cells of atretic follicles the score was even higher. Cells of corpora hemorrhagica and corpora lutea had moderate ERbeta mRNA scores, while higher scores were seen in cells of corpora albicantia. Cells of the surface epithelium had a moderate score for ERbeta mRNA, whereas cells of the tunica albuginea and deep stroma showed high ERbeta mRNA scores. The present findings have clearly established a cell-specific localization of ERbeta mRNA in several cell types in the bovine ovary.     

Tissue concentrations of eosinophilia in the bovine oviduct and uterus of different stages of the oestrous cycle

Numbers of eosinophils in the bovine oviduct and uterus were determined during the oestrous cycle. The eosinophil numbers in the oviduct (ampulla and isthmus) and horn of the uterus during oestrus were significantly higher than during dioestrus. The number of eosinophils in the uterine cervix was lower than in the uterine horn for all stages of the oestrous cycle. In the oviduct, eosinophils accumulated in the lamina propria of the tunica mucosa, in the tunica muscularis and in the connective tissue of the tunica serosa. In the uterus, they were concentrated mainly in the upper parts of the stroma in the endometrium. Degranulation of eosinophils was observed during oestrus when they increased in number in the oviduct and uterus.     

Chemoattractant properties of conditioned medium from equine corpora lutea collected at various stages of the oestrous cycle

This study investigated the chemotactic activity of equine CL at different stages of the oestrous cycle. The purpose of this was to ascertain whether luteal tissue itself contributes to the massive influx of leucocytes around the time of natural and induced luteal regression. Corpora lutea were collected at different stages of dioestrus and after treatment with PGF2alpha. Culture medium harvested after incubation of luteal tissue for 20 h was chemotactic for both polymorphonuclear and mononuclear cells in late dioestrus (before functional regression) as well as after natural and induced luteal regression. By contrast, midluteal tissue showed no chemotactic activity. This is the first report of the ability of equine luteal tissue actively to recruit inflammatory cells in vitro and supports our earlier findings that this infiltration starts prior to functional luteolysis. We hypothesise that this early influx of inflammatory cells may play an active role in luteal regression. Further research is needed to identify the specific chemotactic factor(s).     

Hormonal control of the bovine oestrous cycle

The role of hormones in controlling the estrous cycle of cows is reviewed in the context of current research, specifically on endocrinal functions. This is central as the follicles grow on the ovaries in response to the pituitary's follicular stimulating hormone. When the ovaries produce sufficient estrogens, an ovulatory surge of luteinizing hormone is released by the pituitary. This causes the ovulation of 1 of the follicles and the growth of a corpus luteum in the follicular cavity. This corpus luteum secretes progesterone for about 2 weeks until its activity is terminated by uterine luteolysin. Control of the estrous cycle thus depends on alternate signals from the pituitary and ovaries with the end of the cycle caused by the nonpregnant uterus.     

Fluorometric detection of platelet activating factor receptor in cultured oviductal epithelial and stromal cells and endometrial stromal cells from bovine at different stages of the oestrous cycle and early pregnancy.

During the oestrous cycle and early pregnancy, the oviduct and uterus undergo a variety of morphological and physiological modifications in which the platelet activating factor receptor (PAF-R) plays an important role. PAF-R levels were quantified in bovine oviductal epithelial and stromal cells and endometrial stromal cells at days 2 to 4, 12, and 20 of the estrous cycle and during early pregnancy. Cells were grown in vitro and their intracellular PAF-R concentration was measured by flow cytometry using a polyclonal anti-PAF-R antibody system. A significant increase (P < 0.05) in the portion of PAF-R-positive oviductal epithelial and stromal cells was detected in both non-pregnant and pregnant cattle on days 2 to 4 in comparison to day 12 and 20. In endometrial stromal cells derived from day 20 pregnant bovine, a significant increase (P < 0.05) in PAF-R staining was observed in comparison to the day 20 non-pregnant and days 2 to 4 or 12 pregnant and non-pregnant animals. The PAF-R was detected in oviductal cells by using immunoblotting and immuno-gold postembedding method. Positive binding of the anti-PAF-R antibody was found on the cell membrane and in the cytoplasm. We concluded that the increased PAF-R concentration measured in cultured oviductal epithelial and stromal cells of cyclic and pregnant heifers on days 2 to 4 was hormonally regulated. The increased PAF-R in endometrial stromal cells on day 20 of pregnant heifers was a pregnancy-specific effect and may mediate a local increase in endometrial vascular permeability known to precede the implantation.     

Insulin-like growth factor binding proteins in granulosa and thecal cells from bovine ovarian follicles at different stages of development

Because IGFBP inhibit IGF-stimulated cellular proliferation and differentiation, it is hypothesized that variations among IGFBP in individual follicles might contribute to the regulation of recruitment, selection, dominance, and turnover of ovarian follicles. Sources of IGFBP in fluid of bovine follicles are not well established; thus, objectives of this study were to determine levels of IGFBP binding activities and messenger RNA (mRNA) in granulosa and theca interna cells at different stages of follicular development (small [< 6 mm], medium [6 to < 8 mm], and large [> or = 8 mm]) and to characterize associations of these levels measured in the cells with levels of IGFBP and steroids in follicular fluid. Thecal and granulosa cells from large healthy follicles contained two- to twentyfold less (P < 0.05) IGFBP-2, -3, and -5 than cells from small, medium, and large atretic follicles. Thecal cells from small, medium, and large atretic follicles contained more (P < 0.05) IGFBP-3 and -4 than granulosa cells from these follicles, whereas granulosa cells from these follicles contained more IGFBP-2 activity than thecal cells. Differences in IGF binding activity were paralleled by differences in levels of mRNA for the respective IGFBP. Developmental differences in IGFBP activity in follicular fluid were positively associated with activity in granulosa and/or thecal cells, with the exception of IGFBP-4, which was low in fluid from large healthy follicles but markedly increased (mRNA and binding activity) in granulosa cells from these follicles. It is concluded that developmental changes in follicular fluid IGFBP-2 and -5 binding activities seem to be controlled in part by alterations in synthesis of these IGFBP by granulosa and thecal cells, whereas diminished IGFBP-4 in fluid from large healthy follicles occurs concomitantly with increased levels of IGFBP-4 mRNA and activity in granulosa cells, implicating posttranslational regulation by specific proteases.     

Apoptotic cell death in the porcine endometrium during the oestrous cycle

It has been reported that apoptosis plays an essential role in controlling the physiological cell kinetics in the human and rodent endometrium but this type of death has never been studied in the porcine endometrium. The aim of this study was to investigate the apoptotic cell death in the porcine endometrium during the middle (Days 9-11) and late (Day 13) luteal phase, during the luteolysis (Day 15) and early follicular phase (Days 17-19) of the oestrous cycle. Apoptotic cells were identified by in situ DNA 3'-end labelling method. It was revealed that the greatest number of apoptotic cells in the luminal and glandular epithelium was found on Days 17-19 and on Day 15 of the oestrous cycle, respectively. In the stroma, the greatest number of these cells was found on Days 9-11. Our data have shown that in the porcine endometrium, both epithelial and stromal cells undergo apoptosis and that the number of apoptotic cells varies depending on the phase of the oestrous cycle.     

Immunolocalisation of oestrogen receptors alpha (ERalpha) and beta (ERbeta) in porcine embryos and fetuses at different stages of gestation

The sites of oestrogen action can be shown by the localisation of their receptors in the target tissues. The aim of the present study was to show the localisation of oestrogen receptors in porcine embryos and fetuses obtained on days 18, 22, 32, 40, 50, 60, 71 and 90 post coitum (p.c.). The visualisation of proteins was conducted in embryos and various fetal organs such as gonads, uterus, lung, kidney, intestine and adrenal gland. Both ERs were observed in the blastocysts on day 18 p.c. In the male, ERbeta was detected in the testis and epididymis, whereas ERalpha was present in the efferent ductules. In the female, ERbeta was detected in the ovarian stromal cells investing the oocyte nests, while ERalpha protein was detected in the surface epithelium. In the uterus, ERs were present in the stromal cells, while ERbeta was present in the luminal epithelium. In the non-reproductive fetal porcine tissues ERbeta was localised in the lungs, kidneys, adrenal glands and in the umbilical cords. Both ERs were observed in the intestine. It is possible that ERbeta may play important roles in the development of the adrenal gland, testis, kidney and lungs, while both ERs are involved in the development of the ovary, uterus, epididymis and intestine of the porcine fetus.     

Expression of oestrogen receptor,androgen receptor and progesterone nuclear receptor in sheep uterus during the oestrous cycle

Oestrogen, androgen and progesterone are involved in the regulation of uterine physiological functions, with the participation of the following proteins: oestrogen receptor (ER), androgen receptor (AR) and progesterone nuclear receptor (PGR). In this study, we used immunohistochemistry to detect the localization of ERα, ERβ, AR and PGR in sheep uterus. Additionally, we used real‐time polymerase chain reaction (RT‐qPCR) and Western blot technique to analyse their expression profiles at different stages of sheep oestrous cycle in the endometrium and myometrium. Immunohistochemical analysis showed that ERα, ERβ, AR and PGR were present in sheep uterus in oestrus, mainly in the uterine luminal epithelium, stroma, gland and myometrium. Real‐time polymerase chain reaction results showed that in the endometrium, ERα expression level was highest in oestrus. ERβ and PGR, instead, were highly expressed in pro‐oestrus. In the myometrium, ERα was highly expressed in both oestrus and pro‐oestrus, and ERβ was highly expressed in oestrus and dioestrus. Progesterone nuclear receptor expression was highest in oestrus, followed by metoestrus. In the endometrium, both receptors ERα and ERβ were abundant in pro‐oestrus, while the maximum AR protein content was found in oestrus. At this stage of the oestrous cycle, PGR protein concentration in the myometrium was significantly lower than those observed in other stages. These results suggest that these receptors are important for sheep reproductive function, as their expression at mRNA and protein levels exhibits particular time‐ and tissue‐specific profiles along the oestrous cycle.     

Matching of embryo stages and grades with recipient oestrous synchrony in bovine embryo transfer

The efficacy of matching embryos with recipients on the basis of embryo stage and grade and donor-recipient oestrous synchrony was investigated using the records of 13,663 embryos that were collected and transferred at a commercial embryo transfer centre. The selection of early blastocysts for exact oestrous synchrony cows was effective and resulted in the highest pregnancy rates. Selection of early morulae was effective for recipients in oestrus after the donor but not when transferred into exact and negative recipients. The matching of late morulae with recipients in oestrus after the donor was not effective and had no influence on pregnancy rates. The selection of late, hatched and collapsed blastocysts for transfer into recipients in oestrus before the donor was ineffective and pregnancy rates were higher in exact and +12 hour recipients. Pregnancy rates declined 23.6 per cent in quality grades 1 to 4 whereas the range between stages was 13.3 per cent. Higher quality embryos of all stages gave the highest pregnancy rates. Examination of pregnancy rates of grades within stages suggested that the more developed the embryo the more difficult it is to grade. The difference in pregnancy rates between exact and -24 (6.9 per cent) and +24 (4.8 per cent) hour recipients was small and declined a further 4.7 per cent and 9.8 per cent in -36 and +36 hour recipients. Grade 3 and 4 embryos tolerated asynchrony better than grade 1 and 2, and early morulae tolerated asynchrony better than the other stages. It was concluded that the matching of certain embryo stages with the donor-recipient oestrous synchrony is advantageous but not always possible.