STK3/4 Expression Is Regulated in Uterine Endometrial Cells during the Estrous Cycle

The uterus is dynamically regulated in response to various signaling triggered by hormones during the estrous cycle. The Hippo signaling pathway is known as an important signaling for regulating cellular processes during development by balancing between cell growth and apoptosis. Serine/threonine protein kinase 3/4 (STK3/4) is a key component of the Hippo signaling network. However, the regulation of STK3/4-Hippo signaling in the uterus is little known. In this study, we investigated the regulation and expression of STK3/4 in the uterine endometrium during the estrous cycle. STK3/4 expression was dynamically regulated in the uterus during the estrous cycle. STK3/4 protein expression was gradually increased from the diestrus stage and reached the highest in the estrus stage. STK3/4 was exclusively localized in the luminal and glandular epithelial cells of the uterus, and phosphorylated STK3/4 was also increased at the estrus stage. Moreover, the increase of STK3/4 expression in uteri was induced by administration of estradiol, but not by progesterone injection in ovariectomized mice. Pretreatment with an estrogen receptor antagonist ICI 182,780 reduced estrogen-induced STK3/4 expression and its phosphorylation. The estrogen-induced STK3/4 expression was related to the increase in phosphorylation of downstream targets including LATS1/2 and YAP. These findings suggest that STK3/4-Hippo signaling acts a novel signaling pathway in the uterine epithelium and STK3/4-Hippo is one of key molecules for connecting between the estrogen downstream signaling pathway and the Hippo signaling pathway leading to regulate dynamic uterine epithelium during the estrous cycle.

Ultrasonographic and vaginal cytological diagnostics of the Queen

Ultrasonography is a diagnostic method to image the conditions of reproductive organs and it could be supported by vaginal cytology to identify the activities of the ovaries by the types of vaginal exfoliate cells. The aims of this study was to observe reproduction organ through ultrasonography with supportive diagnostic with vaginal cytological assessment. A total of 10 individual queens were used in this study and then grouped into intact group (n=5) and spayed (ovariohysterectomy) group (n=5) based on the anamneses or their medical history. The vagina, cervix, uterus body and horns, and ovaries were imaged and measured by ultrasound. Vagina, uterine body and horn seem as pipe-like structures with hyperechoic outer lines. The lumen in uterine body and horn seem as a hyperechoic structure. The ovaries seem as round- or oval-shaped structures with anechoic follicles. The corpus luteal has thick wall and seen as anechoic in its centre part. The corpus albicans seems as a hyperechoic structure. The vagina of spayed queens seemed more corrugated than those intact queens. The cervix is seen as a hyperechoic structure linking the vagina and uterine body. Exfoliate vaginal epithelial cell types were then also be identified and counted on each queens. The results of vaginal cytology showed that proestrus occured in 3 intact queens, late metestrus in 1 intact and 3 spayed queens, anestrus in 1 spayed queen, and unidentifiable estrus stage in 1 intact and 1 spayed queens. Moreover, the morphology of cervix and uterine was affected by the activity of ovary.

Dynamics of arborization of secretions of mucous membranes for the stimulated sexual cycle of cows

The changes of arborization of secretions of mucous membranes during stimulated sexual cycle in cows have been investigated. It is established that the dynamics of arborization from the oral mucous membranes, the vestibule of the vagina for hormonal stimulation of the sexual cycle has a direct effect on the level of estrogens in cows. Starting from the third day of research, the number of animals in which we observed a clear arborization increased to four (40%), which indicates an increase in estrogen levels. Instead, six animals (60%) lacked arborization. On the fourth day in the estrus stage (insemination day), the number of cows in the smears we noticed a clear arborization increased to 7 (70%). This was the highest rate during the studies and suggests that only 7 cows had the highest estrogen levels at their peak. From the fifth to the 10th day of the study, a gradual decrease in the number of animals in smears was marked by clear arborization. This, in its turn, is associated with the formation on the site of an ovulatory follicle of the yellow body that begins to produce a progesterone, indicating the stage of the meteor shower. At the 12th of observation in smears of one animal was marked a clear crystallization while in the other nine it was absent. Beginning from the 13th to the 21st day, arboretization in the smears from the mucous membrane of prickly vagina was absent. Only on 14, 15, 17, and 20th day it was noticed singular or not clear arborization. At the 22–24th day of observation in one cow, signs of the next sexual cycle were recorded. Namely, in smears, we noticed a clear arborization, which confirms the renewal of sexual cycle and lack of fertilization. Mucus mucous membranes of spit vagina and saliva of cows for spontaneous want have a longer-term character and a clear figure in the form of a leaf of fern than in the induced sexual cycle, where the drawing was less intense and short-lived in the stage of the estrus. According to our data, one can sometimes observe single crystallization in the stage of metetrusus and diestrus. This is what we associate with the selection of smears from the oral cavity after feeding the animals with concentrated foods, acidic silage, and in smears from mucus, vinegar vinegar – functional disorders in the sexual system.

Estrous Cycle Alters Micturition Pattern in Conscious Rat

Background The present study aims to investigate the influence of estrous cycle on micturition pattern in conscious spontaneously hypertensive rat. Methods Micturition pattern was evaluated by measuring void volume, void interval and void pressure in each stage of the estrous cycle by cystometry. Results A higher bladder capacity and a decrease in the frequency of micturition occurred in the pro-estrus and estrus stage, while a lower bladder capacity and an increase in the frequency of micturition occurred in the diestrus stage. Conclusions It is suggested that these remarkable variations in the physiology of the micturition pattern during different stages of the estrous cycle might be due to the effects of estrogen and progesterone because the level of these hormones fluctuate during each stage of the cycle. Therefore, monitoring the estrous cycle prior to any cystometry experiment in conscious rat is recommended for a better understanding of the effects of these hormones on bladder function.

229CHARACTERIZATION OF CONNEXIN 43 IN THE CANINE OVARIAN FOLLICLE

Connexin 43 (Cx43) is the most abundant gap-junction protein in ovarian follicle cell interactions and has been well characterized for several mammalian species. However, the involvement of Cx43 in canine follicular development has not been extensively investigated. The objective of this study was to 1) characterize the spatial patterns of Cx43 localization within the canine ovarian follicle at various physiological states and during the estrous cycle (prepubertal, anestrus, proestrus, estrus and diestrus), and 2) to characterize the canine Cx43 mRNA transcript. The spatial expression pattern of Cx43 protein was evaluated by immunofluorescence microscopy in canine ovaries. Cx43 was not detected in primordial follicles, but was detected in primary and secondary follicles at each physiological state at all granulosa cell borders. In secondary follicles, definitive, punctate staining patterns were localized along mural granulosa cells and also in the surrounding granulosa-cumulus cell borders. Notably, more intense staining was observed in the corona radiata cells immediately surrounding the oocyte, as well as in trans-zonal projections and at the perivitelline membrane. Patterns of localization were most similar between proestrus and diestrus, and between prepubertal and anestrus in secondary follicles. Estrus-stage follicles showed a decrease in localization at the corona-oocyte cell borders as compared to proestrus and diestrus. In large, healthy antral follicles from proestrus and estrus stages, Cx43 was present in the stroma, theca, and granulosa layers. However, antral follicles from estrus-stage ovaries showed more intense staining in the mural granulosa and theca layers, and less intense in the stroma as compared to those of proestrus stage. The most intense pattern of punctate staining was observed in the corpora lutea of diestrus-stage ovaries. Additionally, gene-specific primers were designed from highly conserved regions of Cx43 mRNA among bovine, human and mouse. RNA was isolated from canine uterus and used as a template for RT-PCR. The PCR products were then sequenced and verified in GenBank to assess homology. The sequenced coding region for canine Cx43 mRNA shares 93% sequence homology with bovine vascular smooth muscle. Canine specific primers for this sequence have been designed, and expression analysis studies in canine ovarian follicles are currently underway. These results indicate that the pattern of localization of Cx43 is similar to that reported for the cow and the pig, except that in canine ovarian preantral follicles, Cx43 is also localized to the peri vitelline membrane. Additionally, these results suggest that the localization of Cx43 is dependent on the physiological state of the ovary, and is likely necessary for folliculogenesis and subsequent oocyte development in canines.

Effect of ovarian hormones on lysosomal acid hydrolase activities in rat myometrium.

The activities of the lysosomal acid hydrolases-cathespin D, acid phosphatase, beta-N-acetylglucosaminidase, and beta-glucuronidase-were measured in rat myometrium under the following hormonal conditions: during the estrus stage of the estrous cycle (NE); at 1,2, and 3 wk after ovariectomy; and in 3-wk postovariectomized females after hormone replacement therapy with 17 beta-estradiol (E2), progesterone (P), or E2 + P. Activities per milligram protein and per milligram DNA of the enzymes were significantly decreased after ovariectomy and were restored to the NE level or above after injecting E2 or E2 + P. Lysosomal enzyme activities did not change with hormonal state in hypophysectomized rats, suggesting that other hormones are required for mediation of enzyme activity. Acid hydrolase activities in other tissues and nonlysosomal enzyme activites in the myometrium did not fluctuate with hormonal state. Studies of lysosomal membrane integrity suggested that one population of lysosomes richer in cathepsin D and acid phosphatase and another rich in beta-N-acetylglucosaminidase and beta-glucuronidase may be present in rat myometrium. Estrogen seemed to labilize the lysosomal membrane of at least the latter of the two proposed populations of myometrial lysosomes.