Expression and localization of transient receptor potential channels in the bovine uterus epithelium throughout the estrous cycle

Transient receptor potential (TRP) channels are expressed in the endometrium but it is unknown if they are modulated through the estrous cycle (EC). This study was undertaken to identify the modulation of the TRPC gene and protein isoforms in bovine uterine epithelium, as a model for human, throughout the EC. Changes in the expression of TRPC genes in bovine uterine epithelium throughout the EC were measured using Real-Time PCR, while immunohistochemistry and immunocytochemistry were used to determine the localization of these channels. Out of the 7 members of the TRPC family, TRPC1, 2, 3, 4 and 6 genes were expressed in bovine uterine epithelial tissue and TRPC 5 and 7 were not. Gene expression levels of all TRPC isoforms underwent cyclical changes throughout the EC. Moreover, cyclical changes were detected in the protein levels of TRPC1 and TRPC6 throughout the EC. These findings show that TRPC channels are modulated through the EC and therefore may have a role in reproductive events.

     

Validation of a mathematical model of the bovine estrous cycle for cows with different estrous cycle characteristics

A recently developed mechanistic mathematical model of the bovine estrous cycle was parameterized to fit empirical data sets collected during one estrous cycle of 31 individual cows, with the main objective to further validate the model. The a priori criteria for validation were (1) the resulting model can simulate the measured data correctly (i.e. goodness of fit), and (2) this is achieved without needing extreme, probably non-physiological parameter values. We used a least squares optimization procedure to identify parameter configurations for the mathematical model to fit the empirical in vivo measurements of follicle and corpus luteum sizes, and the plasma concentrations of progesterone, estradiol, FSH and LH for each cow. The model was capable of accommodating normal variation in estrous cycle characteristics of individual cows. With the parameter sets estimated for the individual cows, the model behavior changed for 21 cows, with improved fit of the simulated output curves for 18 of these 21 cows. Moreover, the number of follicular waves was predicted correctly for 18 of the 25 two-wave and three-wave cows, without extreme parameter value changes. Estimation of specific parameters confirmed results of previous model simulations indicating that parameters involved in luteolytic signaling are very important for regulation of general estrous cycle characteristics, and are likely responsible for differences in estrous cycle characteristics between cows.     

Characteristics of ovarian follicular dynamics throughout the estrous cycle of Egyptian buffaloes

Data of 56 normal and 9 abnormal estrous cycles were collected from 9 Egyptian buffaloes (Bublus bublis) to describe the follicular growth wave pattern. Heat was checked twice daily while, ovaries were scanned daily to monitor the patterns of follicular waves. Day of ovulation was determined when the largest follicle was replaced by corpus haemorrhgicum (CH). Number of waves/cycle, day of emergence of the follicular wave, characteristics of the dominant follicle and corpus luteum (CL) growth features were monitored. Buffaloes displayed mainly two types of follicular waves; two (46.4%) and three (53.6%). In cycles of three wave pattern, time of emergence of the 1st wave post-heat was longer (P < 0.05) and number of recruited follicles/wave were larger (P < 0.05) compared to the corresponding values of the two wave pattern. Number of recruited follicles in early follicular waves (1st or the 2nd) had larger number (P < 0.05) compared to the subsequent ones. Follicles that reached ovulation in both types of estrous cycle had shorter life-span (P < 0.05) than the previous ones. Life-span of CH, growing and regressed CL were 3.6 ± 0.6, 11.2 ± 0.8 and 4.4 ± 0.5 days, respectively with no difference in both types of follicular wave. Three types of ovarian disorders were observed. Follicular waves and CL growth features showed unique pattern for each individual. These results demonstrate that buffaloes display two main types of follicular waves with dominance of three wave type.     

Histological changes in bovine endometrium during the estrous cycle

Endometrial biopsy specimens were obtained from 46 normally cyclic heifers at known stages of their estrous period to show precise characteristic changes. These tissues were embedded in paraffin, sectioned, and stained with hematoxylin and eosin. The following histological changes were observed during the estrous cycle. Metrorrhagia was observed on Days 0 to 1 (estrus = Day 0). Mitoses in glandular epithelium occurred on Day 5. Basal vacuolation in the surface epithelium occurred on Days 5 to 6. Leukocyte invaded the functional layer on Day 7. Stromal mitoses were observed on Days 9 to 12. The results indicate that clincians need to be aware that histological evaluation is important for the diagnosis of endometrial function and that biopsy is useful for this purpose.     

Changes in uterine oxytocin receptor concentrations throughout the estrous cycle of the cow

The amount of oxytocin (OT) bound by plasma membrane fractions from the bovine myometrium and endometrium on different days of the estrous cycle was determined. In the myometrium, OT was bound to a single class of independent sites with an apparent KD (+/- SE) of 1.2 +/- 0.16 nM (n = 34). There were no differences in the apparent KD for different days of the cycle. The amount of OT bound per mg of membrane protein, however, was greatest near the time of estrus and the least in the luteal phase. Nearly ten times more OT was bound to myometrial membranes on Day 21 than on Day 7 (p less than 0.05). OT was bound to endometrial plasma membranes with KD values ranging from 1.3 to 25.7 nM. The mean apparent KD (+/- SE) with 11 samples was 14.9 +/- 3.2 nM. No consistent differences in KD values were detected among days of the cycle. Because of the large variation in the apparent KD values, we were unable to determine if there were differences in the amount of OT bound to endometrial membranes at different stages of the cycle. The correspondence between myometrial OT receptor concentrations and sensitivity to OT during the estrous cycle suggests that the myometrial response to OT in vivo is regulated at the receptor level as well as or rather than by circulating OT concentrations.     

Peripheral plasma levels of lh in the cow throughout the estrous cycle.

Peripheral plasma levels of LH during the bovine estrous cycle were measured in 5 cows and one heifer by using double antibody radioimmunoassay. The sources of variability of basal LH-levels were analyzed in detail. The range of basal levels of LH WAS 0.3--3.5 Ng/ml. Between days 11 and 14 of the cycle the mean daily levels were lower in all cows by about 28% as compared with the remaining parts of the cycle. This did not seem to be directly influenced by changes in progesterone levels. A single peak of LH was observed around estrus. The magnitude of that preovulatory peak varied from 6.7 to 16.0 mg/ml, and lasted only a few hours.     

Effect of repeated laparoscopic surgery on the bovine estrous cycle

The effects of repeated laparoscopic surgery on the length of the bovine estrous cycle, estrus, ovulation and corpus luteum function were determined after one estrous cycle of normal duration (18 to 24 days). Five, Angus x Hereford cows were subjected to laparoscopy on days 5, 13, 18 and 20 (estrus = day 0) of the subsequent cycle. Blood was collected daily during the cycle in which laparoscopy was performed (surgical cycle) and during the next cycle (postsurgical cycle). Lengths of the surgical and postsurgical cycles (22.3 +/- .5 days and 21.5 +/- .6 days, respectively) did not differ (P>.05) from that of the presurgical cycle (21.8 +/- .2 days). Average concentrations (ng/ml) of LH and progesterone in serum were similar during the surgical and postsurgical cycles (1.2 +/- .1, 2.2 +/- .2 vs 1.3 +/- .2 and 2.3 +/- .1). Progesterone concentrations remained above 1 ng/ml for 17 and 16 days during the surgical and postsurgical cycles, respectively. A pre-ovulatory rise in LH, along with estrus and ovulation was confirmed in all animals. Follicular development, characterized by follicular volume, increased progressively from days 5 to 20, with the largest increase occurring between days 13 and 18. These results indicate that laparoscopy, used at the times and frequency specified, does not alter reproductive function of cyclic cows and can provide information on ovarian activity.     

Immunocytochemical localization of relaxin in corpora lutea of sows throughout the estrous cycle

Porcine relaxin has been sought by localization in the corpus luteum of sows on Days 3, 7, 9, 11, 12, 15, 18, 19, and 21 of the estrous cycle, using the avidin-biotin immunoperoxidase method and an antiserum to purified porcine relaxin. Simultaneous localization of relaxin in corpora lutea from sows on Days 108 and 113 of pregnancy was used to compare the intensity of immunostaining with that of corpora lutea of cyclic animals. However, the antiserum dilution necessary for optimal localization differed considerably in these two states (1:10,000 in pregnancy and 1:750 in the cycle), suggesting that lower levels of antigen are present in the luteal cells of the cycle. Relaxin immunostaining was undetectable on Day 3 of the cycle but became evident by Days 7 and 9. At Day 11 staining intensity increased and persisted through Day 15. On Day 18 some stain was still evident, but by Days 19, 20, and 21 there was complete absence of immunostain. Relaxin immunostaining appeared to be located throughout the cytoplasm of the luteal cell, as clear areas in the nuclear region were often observed. The results suggest that relaxin is produced in low amounts by the luteal cells of the cyclic sow and that the levels fluctuate with stage of the cycle. Lack of evidence from radioimmunoassay for a surge of relaxin secretion into the systemic circulation prior to luteolysis in the pig estrous cycle suggests that the relaxin localized in the luteal cells of the cycle may have an intraovarian function.     

Expression of monocyte chemoattractant protein-1 and distribution of immune cell populations in the bovine corpus luteum throughout the estrous cycle.

This study characterizes the expression of monocyte chemoattractant protein-1 (MCP-1) and the relative distribution of immune cell populations in the bovine corpus luteum throughout the estrous cycle. Immunodetectable MCP-1 was evident in corpora lutea of cows at Days 6, 12, and 18 postovulation (Day 0 = ovulation, n = 4 cows/stage). Day 6 corpora lutea contained minimal MCP-1 that was confined primarily to blood vessels. In contrast, relatively intense staining for MCP-1 was observed in corpora lutea from Days 12 and 18 postovulation. MCP-1 was again most evident in the cells of the vasculature, but it was also observed surrounding individual luteal cells, particularly by Day 18. An increase in immunohistochemical expression of MCP-1 on Days 12 and 18 postovulation corresponded with increases in MCP-1 mRNA and protein in corpora lutea as determined by Northern blot analysis and ELISA. Monocytes and macrophages were the most abundant immune cells detected in the bovine corpus luteum, followed by CD8+ and CD4+ T lymphocytes. In all instances, Day 6 corpora lutea contained fewer immune cells than corpora lutea from Days 12 and 18. In conclusion, increased expression of MCP-1 was accompanied by the accumulation of immune cells in the corpora lutea of cows during the latter half of the estrous cycle (Days 12-18 postovulation). These results support the hypothesis that MCP-1 promotes immune cell recruitment into the corpus luteum to facilitate luteal regression. These results also raise a provocative issue, however, concerning the recruitment of immune cells several days in advance of the onset of luteal regression.     

Morphometric analysis of cell types in the ovine corpus luteum throughout the estrous cycle

The cellular composition of ovine corpora lutea obtained during the early (Day 4), mid (Days 8 and 12), and late (Day 16) stages of the estrous cycle was determined by morphometric analysis. Individual corpora lutea were collected via midventral laparotomy from a total of 19 ewes. A center slice from each corpus luteum was processed for electron microscopy and subsequent morphometric analysis of the numbers and sizes of steroidogenic and nonsteroidogenic cells. Luteal weight progressively increased throughout the estrous cycle (p less than 0.05). Corpora lutea collected on Day 16 were assigned to one of two subgroups on the basis of gross appearance and weight: nonregressed (NR, 542 +/- 25 mg) or regressed (R, 260 +/- 2 mg). There were no significant changes in the proportion of the corpus luteum occupied by small luteal cells (19 +/- 2%) or large luteal cells (36 +/- 1%) throughout the estrous cycle. The total number of steroidogenic cells per corpus luteum increased from 21.8 +/- 3.7 (X 10(6)) on Day 4 to 61.7 +/- 5.4 (X 10(6)) on Day 8 (p less than 0.05) and remained elevated thereafter. The number of small luteal cells was 10.0 +/- 2.7 (X 10(6)), 39.7 +/- 1.4 (X 10(6)), 46.1 +/- 5.8 (X 10(6)), 49.0 +/- 13.7 (X 10(6)), and 29.9 +/- 8.6 (X 10(6)) on Days 4, 8, 12, 16 (NR), and 16 (R), respectively (p less than 0.05, Day 4 vs. Days 8, 12, 16 NR). In contrast, the number of large luteal cells was 11.8 +/- 1.5 (X 10(6)) on Day 4 and did not vary significantly during the remainder of the estrous cycle. The numbers of nonsteroidogenic cell types increased (p less than 0.05) from Day 4 to Day 16 (NR) but were decreased in regressed corpora lutea (Day 16 R). Regression was characterized by a 50% decrease (p less than 0.05) in the total number of cells per corpus luteum from 243 +/- 57 ( X 10(6)) on Day 16 (NR) to 125 +/- 14 ( X 10(6)) on Day 16 (R) (p less than 0.05). Small luteal cells remained constant in volume throughout the entire estrous cycle (2520 +/- 270 microns 3), whereas large luteal cells increased in size from 5300 +/- 800 microns 3 on Day 4 to 16,900 +/- 3300 microns 3 on Day 16 (NR) (p less than 0.05). In summary, small luteal cells increased in number but not size throughout the estrous cycle, whereas large luteal cells increased in size but not number.     

Immunohistochemical localization of estrogen receptors in the ovine corpus luteum throughout the estrous cycle

Using immunohistochemistry and Western blot analysis we attempted to identify the estrogen receptors in ovine luteal cells at different stages of the estrous cycle. Monoclonal antibody against estrogen receptors was used for immunolocalization of estrogen receptor-alpha in corpora lutea sections. Generally, the most intense cytoplasm staining was present in large luteal cells. On the 6th day of the estrous cycle, weak immunostaining of estrogen receptors was observed in large luteal cells as well as in the connective tissue. Luteal cells from regressing corpora lutea expressed the weakest immunostaining. The most intense immunoreactivity for estrogen receptors was found in sections of corpora lutea collected on the 9th day of the cycle. Both, cytoplasmic and nuclear localization was observed depending on cell types in the ovine corpus luteum. Our studies demonstrated the presence of the estrogen receptor-alpha in the luteal cells and suggested an autocrine/paracrine role of estrogen in the regulation of estrous cycle in sheep.     

Monosaccharides are not detected in whole or isthmic bovine oviductal fluid collected throughout the estrous cycle, as analyzed by HPLC

In the bovine oviduct, monosaccharides may play a role in the preparation of gametes for fertilization. Sperm are sequestered in the isthmic region of the oviduct where capacitation, requisite biochemical changes in sperm membranes, may take place. Retention of spermatozoa in the oviductal isthmus is dependent on a carbohydrate recognition system between oviductal epithelium and sperm membrane lectins. The monosaccharide, fucose, has been found to be important to this recognition system. However, both gametes and epithelium are also bathed in oviductal fluid (ODF), and fucose or other monosaccharides may be constituents of ODF and so may be important to sperm binding to oviductal epithelium and subsequent preparation for fertilization. In this study, ODF from dairy cows was analyzed by HPLC for the presence of 5 monosaccharides (fucose, galactose, glucosamine, mannose and xylose). Both whole ODF, collected by cannulation of the entire oviduct of 1 cow over a complete estrous cycle, and regional staged ODF, collected and pooled from 13 cows from the isthmic region only at estrus, were analyzed. We report negligible concentrations of all 5 monosaccharides in both types of ODF analyzed. Because the detection limit of our assay was 10(8) times lower than fucose concentrations found to be physiologically important in earlier in vitro studies, we conclude that bovine ODF does not contain physiologically active levels of free fucose or other, similar monosaccharides at any time of the estrous cycle.     

Murine serum cytokines throughout the estrous cycle, pregnancy and post partum period

Cytokines are pleiotropic glycoproteins participating in many aspects of mammalian reproductive physiology. Although murine models have been established to study normal and pathological pregnancy, the small volume of retrievable sample has hampered investigations into the role of cytokines in these processes. These problems were overcome by using fluid-phase multiplex immunoassays to monitor the serum profiles of 18 cytokines in single animals throughout normal murine reproduction: estrus, diestrus, post coitum, preimplantation, implantation, mid-pregnancy, late pregnancy and post partum. Most cytokines were detectable throughout all stages studied. Modest changes in profile were associated with estrous cyclicity and early pregnancy while virtually all cytokine levels increased markedly in mid- to late pregnancy and either fell slightly or levelled off post partum. The functional interrelationships between the various cytokines and the hormonal milieu are discussed with respect to gestational stage. Although certain profiles supported the 'conventional' Th1:Th2 cytokine paradigm of pregnancy, many of the changes recorded were orchestrated around IL-12 (p40) and (p70). The present findings suggest that the traditional cytokine dichotomy poorly describes complex immunological processes like pregnancy.     

Effects of tumor necrosis factor-alpha on secretion of prostaglandins E2 and F2alpha in bovine endometrium throughout the estrous cycle

To determine the physiological significance of tumor necrosis factor-alpha (TNFalpha) in the regulation of endometrial prostaglandin (PG) release in cattle, we investigated the effects of TNFalpha on the secretion of PGE2 and PGF2alpha by bovine endometrium during the estrous cycle. Bovine uteri were classified into six stages (estrus: Day 0, early luteal 1: Days 2 to 3, early luteal 11: Days 5 to 6, mid-luteal: Days 8 to 12, late luteal: Days 15 to 17 and follicular: Days 19 to 21). After 1 h of pre-incubation, endometrial tissues (20 to 30 mg) were exposed to 0 or 0.6 nM TNFalpha for 4 h. The PGE2 concentrations in the medium were higher in the luteal stages than in the follicular stage and in estrus. In contrast, PGF2alpha concentrations were higher in the follicular stage and in estrus than in the luteal stages. The ratio of the basal concentrations of PGE2 and PGF2alpha (PGE2/PGF2alpha ratio) was higher in the luteal stages than in the follicular stage and in estrus. Although TNFalpha stimulated both PGE2 and PGF2alpha secretion during the entire period of the estrous cycle, the level of stimulation of TNFalpha on PGE2 output by the bovine endometrium does not show the same cyclical changes as that shown on PGF2alpha output. The stimulation of TNFalpha resulted in a decrease in the PGE2/PGF2alpha ratio only in the late luteal stage. Furthermore, TNFalpha stimulated PGE2 secretion in stromal, but not epithelial cells. The overall results suggest that TNFalpha is a potent regulator of endometrial PGE2 secretion as well as PGF2alpha secretion during the entire period of estrous cycle, and that TNFalpha plays different roles in the regulation of secretory function of bovine endometrium at different phases of the estrous cycle.     

Expression of superoxide dismutases in the bovine oviduct during the estrous cycle

The superoxide dismutases (SODs) are first-line enzymatic antioxidants that dismute superoxide anion (O(2)(-)) to produce hydrogen peroxide (H(2)O(2)). The primary objective was to characterize, by western blot analysis, the expression of two SODs, the cytosolic (Cu,ZnSOD or SOD1) and the mitochondrial (MnSOD or SOD2) forms in three sections of the oviduct, i.e. isthmus (I), ishtmic-ampullary junction (IA), and ampulla (A), during the estrous cycle. The Cu,ZnSOD and MnSOD proteins were mostly expressed in the ampulla (I相似文献