Regulation and localization of insulin-like growth factor binding protein-5 gene expression in the uterus and placenta of the cyclic and early pregnant ewe

The insulin-like growth factor (IGF) system plays an important role in the regulation of uterine function and placental growth. However, there is little information regarding the localization and regulation of IGF binding protein-5 (IGFBP-5) in the reproductive tract. The distribution of this IGFBP was therefore investigated using in situ hybridization in sections of utero-placental tissue obtained throughout the estrous cycle, up to Day 55 of gestation, and on Days 16-17 from both horns of ewes with unilateral pregnancies that followed uterine transection. In nonpregnant ewes, IGFBP-5 mRNA was present at high concentrations in the maternal caruncles and luminal epithelium, and at moderate levels in myometrium. In these regions IGFBP-5 mRNA showed cyclic variations, with concentrations peaking around ovulation, whereas low expression in the endometrial stroma remained constant. During pregnancy, there was additional localization to the endometrial glands; and in all regions, with the exception of the caruncles, concentrations increased significantly with gestational age. In transected uteri, concentrations in the luminal epithelium of the pregnant horn were significantly higher than those in the nonpregnant horn. In the caruncles, IGFBP-5 mRNA formed an intense band just below the tips of the invading fetal villi. Below this band, IGFBP-5 mRNA localized to form a series of rings, which could create a route to allow the fetal villi access into the caruncular stroma for nutrient exchange. In conclusion, IGFBP-5 is abundantly expressed in the ovine reproductive tract, with both the concentration and localization differentially regulated during the cycle and pregnancy.     

Enzyme histochemistry of the sheep uterus during the oestrous cycle

Enzyme histochemical techniques were applied to frozen sheep uteri from different stages of the oestrous cycle. The localization and activities of succinate, lactate, glucose-6-phosphate, and isocitrate (NADP+) dehydrogenases and acid and alkaline phosphatases were studied in the luminal and glandular epithelia, caruncle and myometrium. Enzyme activity in the sections was scored on a scale of 0--5. In general the enzyme activity in the uterine caruncles and epithelia was higher than in the myometrium. The myometrium did not show any alkaline phosphatase activity and isocitrate dehydrogenase (NADP+) activity was negligible. The low activities of acid phosphatase and lactate dehydrogenase and the moderate levels of glucose-6-phosphate and succinate dehydrogenases in the myometrium were constant. The caruncular tissue showed high levels of phosphatases and glucose-6-phosphate dehydrogenase, moderate levels of lactate and succinate dehydrogenases, and low levels of isocitrate dehydrogenase (NADP+) throughout the oestrous cycle. Much lower phosphatase and isocitrate dehydrogenase (NADP+) levels were found in the epithelium of deep glands compared with superficial glands. The high activity of acid and alkaline phosphatases in the luminal epithelium and the superficial glands was constant from mid-cycle to ovulation, but a significant decrease was observed immediately after ovulation. The level of dehydrogenases in epithelia was generally high and did not change during the oestrous cycle.     

Involution and regeneration of the endometrium following parturition in the ewe

Involution and regeneration of the endometrium after parturition in the ewe, were studied by light- and electron microscopy. The luminal epithelium in intercaruncular regions of the endometrium remained intact at all stages, but degeneration and death of many glandular epithelial cells were observed on the day after parturition. Glandular regeneration had commenced by 8 d post partum, and the glands were substantially regenerated by 15 d. Caruncular epithelial cells on the maternal side of the placentomes, between the bases of the maternal septa, persisted during the period of degeneration of the foetal and maternal tissues of the placentomes. Epithelial cells from this source contributed to the regeneration of the caruncular epithelium following shedding of plaques of degenerate placental tissue from the caruncles, which commenced after 8 d and was completed before 31 d. Thus, ingrowth of epithelium from the edges of the caruncles, as previously proposed, was not the sole source of new caruncular epithelium. The additional source of regenerating epithelium identified here may account for the rapidity with which epithelium appears in the centres of some caruncles, several millimetres in diameter, during endometrial regeneration. However, in some caruncles, regeneration of the epithelium was not completed until after 31 d post partum.     

Localization and correlation between NADPH-diaphorase and nitric oxide synthase isoforms in the porcine uterus during the estrous cycle

Nitric oxide (NO), a highly reactive free radical is involved in vasodilation, neurotransmission, hormone secretion, and reproduction. Since all known nitric oxide synthase (NOS) isoforms possess NADPH-diaphorase (NADPH-d) activity, NADPH-d histochemistry was used as a commonly accepted procedure for NOS identification. The aim of our study was to determine the cellular localization of NADPH-d, eNOS, and iNOS in the porcine uterus and the correlation between NADPH-d and NOS activity in the early, middle, late luteal, and follicular phase of the estrous cycle. Light-microscopic observations of the sections revealed the differential expression of the NADPH-d in the analyzed stages of the estrous cycle. The most intense staining was observed in the luminal epithelium in the late luteal phase and in some groups of the endometrial glands in all studied stages. Positive reaction was also found in the endothelial cells of blood vessels and in the myometrium itself. Immunostaining for eNOS was observed in the luminal and glandular epithelium in all studied stages, but no clear fluctuations were observed. The endothelium of both endometrial and myometrial blood vessels displayed pronounced eNOS immunostaining. Strong iNOS staining was observed in the luminal epithelium in the late luteal and follicular phase and in selected groups of endometrial glands. Thus, only NADPH-d and iNOS undergo cyclic changes in the studied stages of the estrous cycle. The differential expression of NADPH-d/NOS in the porcine uterine horn during the estrous cycle suggests a role for NO in modulating uterine function.     

Sialomucin complex (Muc4) expression in the rat female reproductive tract

Previous studies in our laboratory demonstrated the presence of sialomucin complex (SMC)/Muc4 covering the rat uterine luminal epithelium. SMC/Muc4 expression in the uterus is regulated by estrogen and progesterone and lost at the time of receptivity. In contrast to this hormonal regulation at the uterine luminal surface, SMC/Muc4 in the uterine glandular epithelium, oviduct, cervix, and vagina was constitutively expressed at all stages of the estrous cycle. Furthermore, SMC was expressed in the cervix and vagina of the ovariectomized rat, even though it is not found in the uterine luminal epithelium. Both soluble and membrane-bound forms of SMC were present in these tissues. Immunohistochemical analyses showed distinctive localization patterns of SMC in the various tissues during the estrous cycle. Moreover, the previously unreported expression of SMC/Muc4 in the isthmus, ampulla, and infundibulum of the oviduct suggests potential functions in gamete development. These results indicate that SMC/Muc4 is expressed in most tissues of the female reproductive tract, in which it may have multiple functions. However, hormonal regulation appears to be restricted to the uterine luminal epithelium.     

Expression of prostaglandin transporter in the bovine uterus and fetal membranes during pregnancy

Uteroplacental prostaglandins (PGs) play pivotal roles in the maintenance and termination of pregnancy in mammals. In the present study, we have characterized the expression of prostaglandin transporter (PGT) in placentome caruncles, intercaruncular tissues, fetal membranes, and utero-ovarian plexus during pregnancy in cattle. Pregnant bovine uteri were collected and classified into six groups covering the entire gestational length. In caruncles and intercaruncular tissues, PGT mRNA (also known as SLC02A1) and PGT protein were highly expressed at the late stage of pregnancy compared to the early and mid stages, whereas the level of expression is constant and low in fetal membranes throughout pregnancy. PGT mRNA and PGT protein were expressed at a constant level in the utero-ovarian plexus both ipsilateral and contralateral to corpus luteum throughout the course of pregnancy. Overall, the relative expression of PGT mRNA and PGT protein were higher in caruncles than in intercaruncular tissue and fetal membranes, whereas no differences were detected between intercaruncular tissues and fetal membranes at any stage of gestation. Immunohistochemistry indicated that PGT was preferentially expressed in caruncular epithelial cells of placentomes and endometrial luminal epithelial and myometrial smooth muscle cells of the intercaruncular regions. The level of PGT expression was comparatively higher in maternal components than in fetal components. In conclusion, differential spatiotemporal tissue-specific expression of PGT in uterine and intrauterine tissues suggests a role for this transporter in the exchange of PGs between the maternal and the fetal compartments, as well as for intrauterine metabolism of PGs during pregnancy.     

Histomorphometrical and proliferative aspects of placenta and uterus of the collared peccary (Tayassu tajacu)

The histomorphometric and proliferative characteristics of the collared peccary (Tayassu tajacu) placenta and uterus were analyzed. The material was examined by standard histological techniques and histochemistry (PAS, Perls and Alcian Blue pH 0.5 and 2.5%) and the cellular proliferation by AgNORs and flow cytometry. All the analyzed morphometric variables differed between pregnant and non-pregnant uteri in the luteal phase using the Dunnet test. Height and gland diameter of uterine glands increased linearly during pregnancy, with an intense positive PAS and Perls reaction in all stages. The cells with more than seven AgNORs per nuclei and the cells in the G2M cell cycle phase in the maternal tissue also increased after 70 days of pregnancy. The uteroplacental ridges had a linear increase in size with two distinct areas, base and top, with uterine epithelium and trophoblastic cells changing their morphology following the placental ridge development. Flow cytometry analysis showed the percentage of cells in each cell cycle phase with a quadratic behavior for stages G2/M in the maternal tissue, suggesting an increase in proliferative capacity of maternal tissue after 65 days of pregnancy. The same quadratic effect was observed in the G0/G1 phase in both maternal and fetal tissues. Cells in apoptosis showed cubic behavior in both tissues. The morphometric and cellular dynamic aspects observed in this study have not been previously described and they extend our knowledge of functions relating to maternal-fetal dynamics in this species.     

Structural changes in the luminal epithelium of the porcine uterus between days 10 and 19 of the estrous cycle

Light and electron microscopy were used to study the morphology of uterine luminal epithelium from 4 or 5 gilts slaughtered on each of days 10, 13, 16, and 19 of the estrous cycle. Ultrastructural evidence indicated that metabolic activity and accumulation of glycogen by the uterine epithelium increased between days 10 and 16 of the estrous cycle. This phase of high synthetic activity had been terminated by day 19, as evidenced by the reduction or absence of glycogen deposits and decreased incidence of organelles associated with synthetic activity. Diffuse degeneration of epithelial cells occurred throughout the period of study but was maximal between days 16 and 19. Mitotic activity indicated that cell replacement also occurred between day 16 and day 19.     

Malate dehydrogenase and glucose-6-phosphate dehydrogenase, key markers for studying the genetic diversity of Actinobacillus actinomycetemcomitans

Abstract Cell-free extracts of strains belonging to the 5 serotypes of A. actinomycetemcomitans were screened for several enzymes. Enzymes representative of the pentose phosphate pathway/hexose monophosphate shunt and the TCA cycle were present. Of these glucose-6-phosphate dehydrogenase (G6PDH) and malate dehydrogenase (MDH) were the most readily detected and stable. MDH and G6PDH retained more than 50% of their activities at alkaline pHs (10–11) for up to 6 h and 3 h at 25°C, respectively, while at pH 6.5, 50% of their activities were lost within 2–3 h. The K _m for malate oxidation catalysed by MDH was 5.8×10⁻⁴ M while that for glucose-6-phosphate oxidation was 2.0×10⁻⁴ M. The pH optima for MDH and G6PDH oxidation activities were 10 and 9.5, respectively. Among the 5 designated serotypes of A. actinomycetemcomitans three groups were delineated by multilocus enzyme electrophoresis using MDH and G6PDH.     

蛋白激酶H11基因在小鼠子宫中的表达及其性激素调节

为研究蛋白激酶H11基因在生殖系统中的作用,我们采用半定量RT-PCR和原位杂交方法,研究了蛋白激酶H11基因在小鼠中的组织特异性表达,在妊娠初始期胚胎植入位点、妊娠期子宫和胎盘以及正常动情周期子宫中的表达及其受性激素的调节。结果发现:蛋白激酶H11基因在小鼠多种组织中都有表达,在卵巢及子宫等一些生殖相关的组织中表达水平较高;妊娠初始期,蛋白激酶H11基因在小鼠子宫内膜植入位点处有明显的高表达,其mRNA定位于腔上皮细胞和基质细胞中。在动情周期中,蛋白激酶H11基因在动情前期子宫中表达水平较低;卵巢切除模型显示雌激素和孕激素均可显著上调蛋白激酶H11基因的表达。以上结果提示蛋白激酶H11可能参与了胚胎植入过程中腔上皮细胞凋亡和基质细胞增殖与蜕膜化以及动情周期小鼠子宫内膜细胞的功能调节[动物学报51(3):462-468,2005]。     

Fluctuations of lactoferrin protein and messenger ribonucleic acid in the reproductive tract of the mouse during the estrous cycle.

The physiological role of lactoferrin (LF), the major estrogen-inducible protein in the murine uterus, is unclear; however, LF may be a useful marker for the study of estrogen action in the uterus. Thus, the expression of LF mRNA and the localization of the protein in genital tract tissues and secretions of female mice (6-8 wk old) at different stages of the estrous cycle were investigated. Uterine luminal fluid (ULF) was analyzed for LF by means of gel electrophoresis and Western blot techniques; LF mRNA and protein were identified in reproductive tract tissues through in situ hybridization and immunocytochemistry. At diestrus, the level of LF mRNA was low, and staining for the protein was very light in uterine epithelial cells; LF was undetectable in ULF. At proestrus, LF mRNA and protein increased in the uterine epithelium and LF was readily detectable in ULF. LF mRNA and protein reached the highest levels at estrus. At early metestrus as compared to estrus, LF mRNA and protein were detected in decreasing amounts in uterine epithelial cells; the protein was undetected in ULF. By late metestrus and diestrus, LF mRNA and protein returned to a low level, and the protein was undetectable in ULF. LF protein was also demonstrated by immunocytochemistry in the epithelium of the oviduct, cervix, and vagina. LF protein fluctuation similar to that observed in the uterus was seen in these tissues; however, the uterus demonstrated the most dramatic changes in the number of epithelial cells involved in LF production during the estrous cycle. In summary, LF mRNA and its expression in uterine epithelial cells of the mouse varied with the stage of the estrous cycle. These results, combined with previously reported findings that LF is a major constituent of mouse ULF under the influence of estrogen, suggest that LF may play an important role in normal reproductive processes.     

Steroidal control mechanism of cell proliferation in mouse uterine epithelium

The percentage of labeled cells in the uterine luminal epithelium of cycling mice showed the different zonal distributions at each stage of estrous cycle after cumulative labeling with 3H-thymidine for 36 hr. It was estimated that the proliferating fraction in the epithelium at proestrus, estrus, metestrus, and diestrus was 100%, 100%, 40% and 5%, respectively. The percentage of labeled cells in the uterine luminal epithelium of cycling mice treated with progesterone remained below 10% level for at least 20 hr after injections of progesterone. Total labeling was attained in the uterine epithelium of castrated mice by the administration of estradiol-17beta. On the other hand, the cell proliferation in the uterine epithelium of castrated mice treated with estradiol and progesterone was markedly suppressed and the percentage of labeled cells remained approximately at 35%. The remaining cell population, however, still showed the mitotic potency when mice received estradiol. It is suggested from this study that the effect of progesterone is to suppress the epithelial cell proliferation and transfer cells into resting cell fraction which is still evoked to proliferate as the effect of estradiol and that a key factor controlling epithelial proliferation in mouse uterus during the estrous cycle is proliferating fraction rather than cell cycle time.     

Metabolic background for establishment of the subpopulation structure in early ontogenesis in the Atlantic salmon (the case of energy of carbohydrate metabolism)

The activity of some enzymes involved in energy and carbohydrate metabolism was studied in Atlantic salmon embryos at the eyed egg stage and in salmon fingerlings (0+) from two trophic–ecological groups: the Varzuga River bed and two tributaries, the Pyatka and Sobachii rivers (Kola Peninsula). It has been demonstrated that heterogeneity of embryos was most evident in the case of cytochrome c oxidase (CO), malate dehydrogenase (MDH), glycerol-1-phosphate dehydrogenase (G1PDH), and glucose-6-phosphate dehydrogenase (G6PDH), while the lowest level of heterogeneity was observed for lactate dehydrogenase (LDH) and aldolase. A positive correlation was revealed between the activities of CO, LDH, MDH, and G1PDH. It was noted that G6PDH showed a negative correlation with almost all enzymes under study. It was found that salmon juveniles inhabiting the tributaries were characterized by high LDH, aldolase, and G1PDH activity and lower activity of G6PDH compared to the juveniles inhabiting the main river bed. Notably, the differences in the activity of the enzymes involved in aerobic metabolism between the two groups of fingerlings under analysis were observed only in the autumn.     

Microphotometric measurement of some enzymatic activities in rabbit spermatozoa during epididymal maturation

Cytochemical quantitative measurements of isocitrate dehydrogenase (ICDH), malate dehydrogenase (MDH), cytochrome oxidase, lactate dehydrogenase (LDH), glucose 6-phosphate dehydrogenase (G6PDH) and glutamate dehydrogenase (GLDH) activities were made on rabbit spermatozoa collected from the testis, the different epididymal sites and the ductus deferens. These measurements were made on individual spermatozoa (at least 100 spermatozoa for each site under consideration) using a Vickers M 85 scanning microdensitometer.The activity patterns of the enzymes involved in the tricarboxylic acid cycle (ICDH, MDH) and in the respiratory chain (cytochrome oxidase) both showed a progressive decrease in the intramitochondrial oxidative metabolism from the testis to the ductus deferens. This was in contrast to LDH activity which represents the anaerobic glycolysis pathway rather than the activity of intramitochondrial LDH. The G6PDH activity could be related to those membrane modifications which the male gamete undergoes during its epididymal maturation. Potential GLDH activity was relatively intense in the spermatozoa from the testis and from the initial and distal segments of the genital tract, suggesting an intramitochondrial synthesis of enzymes such as cytochrome oxidase or ATPase.The quantitative variations of the enzymatic activities occurring during the transit of spermatozoa along the male genital tract suggested the existence of different specific interactions between the spermatozoon and the epididymal microenvironment.     

Peptidylarginine deiminase of the mouse. Distribution, properties, and immunocytochemical localization

Peptidylarginine deiminase (protein-L-arginine iminohydrolase, EC 3.5.3.15) is widely distributed in various organs of the mouse. Activity in salivary glands, pancreas, and uterus is higher than that in the other organs. In submandibular gland and uterus, sex- and estrous cycle-related differences were observed, respectively. The activity in the submandibular gland from females was approximately four times higher than that in the male. In the uterus, the activity increased in proportion to the hyperplasia of the tissues. Peptidylarginine deiminase from the murine skeletal muscle resembles the enzyme obtained from other animal species with respect to enzymatic and chemical properties. Double immunodiffusion tests and immunoblotting analyses showed that the enzymes present in each murine tissue have the same molecular weight (81,000) and are immunologically indistinguishable. Immunohistochemical analyses of salivary glands and pancreas revealed an intense staining only of the exocrine cells. In uterus, the staining was restricted to the luminal and glandular epithelia of endometrium; the intensity of the staining changed during the course of the estrous cycle. Furthermore, immunoelectron microscopy showed that the enzyme is distributed diffusely in the cytoplasm of the exocrine cell. These observations indicate the general importance of peptidylarginine deiminase, presumably in a cytoplasmic secretory process of the exocrine cells.     

Histochemical studies of human breast tumors: Activity of alkaline phosphatase, acid phosphatase and glucose-6-phosphate dehydrogenase.

Histochemical studies of human breast tumors were performed with particular emphasis on the activity of alkaline phosphatase (AIP), acid phosphatase (AcP) and glucose-6-phosphate dehydrogenase (G6PDH). Enzyme activities in benign and malignant lesions were compared. AIP was prominent in normal mammary epithelium, limited to the myoepithelial layer in benign tumors and was absent in cords of malignant cells. AcP activity was faintly detected in normal mammary epithelium, increased in canalicular epithelium of fibroadenomas and was marked in malignant cells. G6PDH exhibited marked activity in neoplastic epithelium and the stroma of nearly all carcinomas studied, whereas in benign tumors, G6PDH activity was strictly limited to the connective tissue. The study suggests a strong correlation between G6PDH activity and malignancy. The different results obtained by various workers in this field are critically reviewed, and discussed in the light of the results of the present study.     

Expression of insulin-like growth factor binding protein-4, insulin-like growth factor-I receptor, and insulin-like growth factor-I in the mouse uterus throughout the estrous cycle

Recent evidence suggests that a regulated insulin-like growth factor (IGF) system mediates the effects of estrogen, promoting the proliferation and differentiation of specific uterine cell types throughout the estrous cycle and during gestation in the rodent. Previous studies have shown that IGFs are differentially expressed in the mouse uterus during the periimplantation period. In the current study, we examined the expression of IGF binding protein-4 (IGFBP-4), IGF-I receptor (IGF-IR), and IGF-I in the mouse uterus throughout the estrous cycle. Ligand blot analysis was conducted on uterine homogenates using [125I]IGF-I. IGFBP-4 was detected in all uterine homogenates, varying in intensity throughout the estrous cycle. In situ hybridization studies at metestrus and diestrus demonstrated an intense IGFBP-4 mRNA signal in antimesometrial stromal cells between the luminal epithelium and the myometrium, but at proestrus and estrus, no IGFBP-4 signal was detected. No IGF-I mRNA was detected at any stage of the estrous cycle by in situ hybridization. However, by RT-PCR analysis, IGF-I mRNA was detected at all stages of the estrous cycle. RT-PCR analysis also showed IGF-IR mRNA throughout the estrous cycle. Using immunohistochemistry, IGF-IR immunostaining was detected throughout the estrous cycle and on days 2-7 of gestation, but was restricted to the glandular epithelium. These results suggest that uterine IGFBP-4 expression may not be dependent on uterine IGF-I expression. They also suggest that IGFBP-4 may play a role in uterine physiology independent of the inhibition of IGF-I action, and that IGF-IR is constitutively expressed in the mouse uterus.     

Characterization and immunolocalization of bovine uterine retinol-binding protein.

Endometrial explants obtained from cows between Days 13 and 29 of pregnancy were cultured for 24 h in modified minimum essential medium in the presence of [35S]methionine or [3H]leucine. Proteins synthesized and released into medium were analyzed by two-dimensional polyacrylamide gel electrophoresis and fluorography. Uterine luminal flushings were obtained from cyclic cows (Days 2-20 of estrous cycle) and early pregnant cows (Days 17-22). Endometrial tissues from cows on Days 17 and 29 of pregnancy were prepared for immunocytochemistry. A uterine secretory protein, which consisted of five isoelectric variants (pI 5.3-6.1) of identical molecular mass (23,000 Da), was shown to react immunologically with antiserum raised against bovine placental retinol-binding protein (bpRBP). Limited N-terminal sequence analysis of two major isoforms showed that the protein had nearly complete homology with bovine placental and plasma retinol-binding protein (RBP) over the first 25 amino acids. Through use of bpRBP antiserum, immunoreactive RBP was detected in uterine flushings collected from cows in the late luteal phase of the estrous cycle and early pregnancy by Western blotting, and in medium conditioned by uterine explants prepared at Days 13-29 of pregnancy by immunoprecipitation. Immunoreactive RBP was localized in endometrial surface and glandular epithelium on Days 17 and 29 of pregnancy by immunocytochemistry. These results demonstrate that RBP is a product of bovine uterine tissues. The uterine RBP may play an important role in vitamin A transport between maternal tissues and developing embryos.