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.     

Expression of Oestrogen Receptor α and Oestrogen Receptor β in the Uterus of the Pregnant Swine

The uterus is a well‐known target of endocrine, paracrine and autocrine acting molecules among which steroid hormones are of special importance. The objective of our work was to localize oestrogen receptors (ERα and ERβ) mRNA and protein in the pig uterus throughout pregnancy (10, 18, 32, 50, 71, 90 days post coitum) using RT‐PCR, Western‐blot and immunohistochemistry. The present study is the first one to demonstrate the presence of ERs protein in the porcine uterus not only at the beginning but also at mid‐ and late pregnancy. In the pregnant swine, ERα was immunolocalized in the luminal epithelium (LE) and glandular epithelium (GE) and the myometrium of the uterus with differences in the intensity of staining at different stages of pregnancy studied. The LE and GE of pregnant swine stained for ERβ regardless of the day of pregnancy examined, whereas only a few cells within the myometrium showed a weak immunoreactivity. Western blot analysis confirmed the presence of ERα and ERβ proteins on all investigated days of gestation. The expression of ERα and ERβ mRNA was detected by RT‐PCR in all examined samples corresponding to each of the consecutive stages of pregnancy. The obtained results show that ERα is more abundant in comparison to ERβ within the porcine pregnant uterus. The presence of ERα and ERβ in all compartments of the pig uterus during pregnancy may indicate direct action of oestrogens on proliferation and differentiation of these cells.     

The Activity and Localization of 3β-hydroxysteroid Dehydrogenase/Δ5-Δ4 Isomerase and Release of Androstenedione and Progesterone by Uterine Tissues During Early Pregnancy and the Estrous Cycle in Pigs

Steroid hormones are produced by the porcine uterus. We hypothesized that the uterus in pigs possesses active 3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴ isomerase (3β-HSD) responsible for progesterone and androstenedione production, that uterine steroids may supplement the amount of steroid hormones produced by embryos and corpus luteum and that these steroids are necessary for maintenance of pregnancy. In this study, we examined 1) endometrial and myometrial expression of 3β-HSD mRNA, 2) uterine 3β-HSD protein activity and 3) in vitro production of A₄ and P₄ by uterine slices harvested from pigs on days 10 to 11, 12 to 13 and 15 to 16 of pregnancy and the estrous cycle. The expression of 3β-HSD and the presence and activity of 3β-HSD protein were different in the endometrium and the myometrium during the examined periods of pregnancy and the estrous cycle. Production of A₄ by the endometrium and myometrium was highest on days 12 to 13 of pregnancy and the estrous cycle. Endometrial secretion of P₄ did not differ in the course of early pregnancy and on the respective days of the estrous cycle. The gravid myometrium was the highest source of P₄ in pregnant pigs on days 12 to 13. The release of P₄ by the cyclic myometrium rose during the examined days of the estrous cycle. The steroidogenic activity of the uterus, as described in this study, may support early pregnancy or the luteal phase of the estrous cycle in pigs.     

Nongonadal LH receptors, their involvement in female reproductive function and a new applicable approach

Luteinising hormone (LH) and human chorionic gonadotropin (hCG) share a common receptor in gonadal cells. The receptors have also been detected in several nongonadal but reproduction-associated tissues of pigs, cattle, and other species including the uterus (myometrium, endometrium), oviduct, cervix, blood vessels, mammary gland and other tissues. The main role of LH/hCG receptors in the myometrium is stimulation of growth and hyperplasia, and relaxation of uterine motility; hCG also boosts blood flow in the uterine artery. LH and hCG can increase production of prostaglandins in the endometrium, oviduct, and blood vessels. We suggest that the preovulatory surge of LH plays an important role in controlling oviductal contractions. Awareness of LH binding to many tissues of the female reproductive tract and integration with embryonic factors may lead to the elaboration of new strategies for improved reproductive efficiency in domestic species. Mammary glands also possess LH/hCG receptors through which gonadotropins can affect the metabolism of steroid hormones and could play an inhibitory role in mammary carcinogenesis and in the growth of breast tumours. A novel approach to target and ablate carcinoma cells through LH receptors is described.     

Bovine Uterine,Cervical and Ovarian Cytosol Estrogen and Progesterone Receptor Concentrations in Cystic Ovarian Disease

Bovine cytosol estrogen (ERC) and progesterone receptor (PRC) concentrations were measured simultaneously in various regions of the uterus and in ovarian stromal tissue in cows with cystic ovarian disease (follicular cysts), arid the concentrations compared with those in animals with normal cycles. In cystic ovarian disease, ERC concentrations in endometrium (550 fmol/mg cytosol protein (c.p.)) and in myometrium (405) were significantly higher than in control animals. Very high PRC contents were measured in the endometrium (3115) and myometrium (2761) of cows with cystic ovarian disease. In control animals, PRC concentrations in the endometrium and myometrium were significantly lower than in diseased animals. No statistical differences were observed in ERC or PRC contents between the endometrium and the myometrium in cows with cystic ovarian disease. ERC and PRC concentrations in the uterine cervix and ovaries were low compared to those detected in the uterus. Bovine serum estradiol-17ß concentrations were higher (p<0.001) in cows with cystic ovarian disease than in control animals in postpartum anestrus or during the normal estrous cycle. Serum sex hormone-binding globulin (SHBG) concentrations were of the same magnitude as in control cows during their estrous cycles. These findings show that long standing low endogenous progesterone and elevated estradiol concentrations in serum are associated with elevated ERC and PRC concentrations in bovine uterus.     

Treatment with eCG Decreases the Vascular Density and Increases the Glandular Density of the Bovine Uterus

The uterus plays an essential role in mammalian reproduction and is a target of several hormonal protocols used to improve fertility in cattle. Many studies highlighted the importance of eCG treatment following fixed‐time artificial insemination in improving follicular growth, ovulation and pregnancy rates in cattle. Moreover, eCG has been implicated in angiogenesis, leading to important changes in uterine blood flow and vascularisation. However, there is still a lack of information regarding the specific alterations induced by eCG upon glandular and vascular characteristics of bovine uterus. To investigate the influence of eCG on: uterine thickness and area; uterine artery diameter and area; uterine vascular and gland density; and the expression of the VEGFA‐system, the uteri of crossbred beef cows were collected. All cows were submitted to follicular wave emergence synchronization. On day four of protocol, cows submitted to superovulation (n = 6) received 2000 IU eCG, on day eight, after expected follicular deviation, cows submitted to stimulatory treatment (n = 5) received 400 IU eCG. Control cows (n = 5) did not receive eCG. On day five po cows were subjected to ultrassonographic evaluation and slaughtered for uterine tissue sampling on day six po. Uterine vessels and glands were quantified by the counting point stereological method. The VEGFA‐system was localized in different cellular types, showing no qualitative or quantitative differences in the site of expression or the intensity of the positive signal among the groups. Vascular density was decreased in the endometrium of stimulated and myometrium of superovulated cows compared with the control ones, which showed higher vascular density in the myometrium and endometrium of the ipsilateral uterine horn. The uterine gland density was higher in superovulated compared with stimulated and control cows. Thus, we can infer that stimulatory or superovulatory treatments with eCG influence the vascular density in the endometrium and myometrium in cattle.     

Distribution patterns of mast cells in the uterus of pregnant Meishan pigs

The present study was performed to investigate the numerical distribution of mast cells (MCs) in the uteri of pregnant Meishan pigs to explore the functions of MCs in pig pregnancy. The uterine samples from pregnant (on days 15, 26 and 50 of gestation) pigs were obtained respectively and stained with toluidine blue. The results were as follows: MCs were constitutively located in the uterus of the Meishan pig, with the distribution varying with gestational stages. On days 15 and 26 of gestation, MCs were mainly distributed around the blood vessels and uterine glands within the endometrium. On day 50 of gestation, MCs were mostly distributed in the myometrium. These results indicated that uterine MCs possibly have versatile functions in pig pregnancy.     

Apoptosis in the porcine uterine endometrium during the estrous cycle, early pregnancy and post partum

The mammalian uterus changes dramatically during the estrous cycle, pregnancy, and involution post partum. Dynamic changes in the uterine endometrium are a type of homeostasis and proceed with proliferation and exclusion of cells. Homeostasis of the uterus is closely related to apoptosis involving various hormones and cytokines. The objective of the present study was to determine the morphological features and occurrence of apoptosis in the porcine endometrium during the estrous cycle, early pregnancy, and post partum. Cyclic changes in the morphology of the surface epithelium were observed during the estrous cycle. The heights of surface epithelia were significantly high on day 4 of the estrous cycle and the early pregnancy. The heights of the surface epithelium remained low from days 1 to 31 post partum. We then used terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end-labeling (TUNEL) of the 3'-terminal of fragmented DNA, which is effective for detection of apoptosis in various tissues. We found that apoptosis in the porcine endometrium contributed to homeostasis of the endometrium during the estrous cycle through control of cell proliferation and exclusion. Conversely, apoptosis on days 4 and 8 of gestation before the implantation window depended on the plasma estrogen and progesterone levels; however, suppressive homeostasis of apoptosis occurred at the time of implantation on days 15, 18 and 21 of gestation. Our study is the first to demonstrate apoptotic cell death in the porcine endometrium directly by TUNEL method. The results strongly suggest that uterine homeostasis is mainly controlled by apoptosis during the estrous cycle and early pregnancy.     

猪子宫腔液外泌体来源的TIMP2蛋白对胚胎附植的影响

旨在研究猪子宫腔液外泌体来源的TIMP2蛋白对妊娠早期胚胎附植的影响.本研究采用超速离心法分离妊娠第9天(n=3)和第12天(n=3)大白猪子宫腔液外泌体,利用Western blot(WB)检测外泌体中TIMP2蛋白的变化,并通过免疫组化试验研究TIMP2蛋白在子宫中的表达定位.进一步用外泌体与猪胚胎滋养层细胞(po...     

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.     

Immunohistochemical Studies on Oestrogen Receptor Alpha (ERα) and the Proliferative Marker Ki-67 in the Sow Uterus at Oestrus and Early Pregnancy

Oestrogen receptor alpha (ERalpha), the main subtype in the uterus, is involved in the regulation of uterine growth/proliferation. A relationship between ERalpha and proliferative activity has been shown in the cyclic sow uterus, but to our knowledge, no study has been carried out on early pregnant sows. Therefore, by means of immunohistochemistry and use of mouse monoclonal antibodies to ERalpha and a proliferative marker, Ki-67, the localization of these proteins was investigated in the sow uterus during early pregnancy. Eighteen crossbred multiparous sows were artificially inseminated once at 20-15 h before expected ovulation. After artificial insemination (AI), they were slaughtered at five different times: at oestrus, 5-6 h after AI (n = 4), 20-25 h after ovulation (n =4), 70 h after ovulation (n = 4), on day 11 (the first day of standing oestrus = day 1, n = 3) and on day 19 (n = 3). Immediately after slaughter, uterine samples were collected at the mesometrial side of the uteri, fixed in 10% formaldehyde and embedded in paraffin. Immunohistochemistry was performed by using mouse monoclonal antibodies to ERalpha (C-311) and Ki-67 (MM1). All sows slaughtered after ovulation were pregnant. In general, positive immunostaining for ERalpha and Ki-67 was found in the nuclei. Variations in staining intensity and proportion of positive nuclei were observed in different uterine compartments and stages of early pregnancy. The highest level of ERalpha presence in the surface epithelium and myometrium was found at oestrus (5-6 h after AI), and low levels of ERalpha in these compartments were observed as early as 20-25 h after ovulation. In the glandular epithelia, presence of ERalpha was highest at 70 h after ovulation. The largest number of ERalpha-positive cells in the stroma was observed at oestrus and early after ovulation. Low proliferation was observed, and with no significant difference in tissue compartments except in the glandular epithelium. High proliferative activity in the glandular epithelium at 70 h after ovulation indicated involvement in preparation for secretory activity and growth during pregnancy establishment. Significant positive correlation was found between the number of ERalpha-positive cells in the stroma and Ki-67-positive cells in the surface epithelium. In conclusion, the present study showed differences in immunolocalization of ERalpha and the proliferative marker Ki-67 in different tissue compartments of the sow uterus at oestrus and early pregnancy. In some uterine compartments, the patterns of ERalpha and Ki-67 immunostaining seemed to be influenced by insemination and the presence of embryos, in addition to the effects of steroid hormones.     

Immunohistochemical Localization of the Progesterone and Oestrogen α Receptors in the Uterine Horns of the African Giant Rat (Cricetomys gambianus)

The present study investigated the immunolocalization of the progesterone and oestrogen α receptors in the uterine horns of the African giant rat during the oestrous cycle. The progesterone and oestrogen α receptors were demonstrated in various cellular constituents of the endometrium, myometrium and perimetrium. The intensity of progesterone and oestrogen α receptor immunostaining in the endometrial and myometrial layers of the uterine horns varied during the oestrous cycle. The intensity of oestrogen α receptor immunoreactivity in the luminal epithelium was high during pro‐oestrus, oestrus and dioestrus. Progesterone and oestrogen α receptor immunoreactivity in the endometrial epithelia was absent during metoestrus. Moderate to strong immunostaining for the progesterone and oestrogen α receptors was demonstrated in the myometrial smooth muscle cells during pro‐oestrus, oestrus and dioestrus. The intensity of progesterone and oestrogen α receptor immunostaining in the myometrial smooth muscle cells was low during metoestrus. Stromal cells in the perimetrium consistently expressed progesterone and oestrogen α receptor immunoreactivity throughout the oestrous cycle. The findings of the study indicate that in the giant rat the immunolocalization of the progesterone and oestrogen α receptors, in endometrial and myometrial regions of the uterine horns, varies during the oestrous cycle.     

Expression of uterine sensitization-associated gene-1 (USAG-1) in the mouse uterus during the peri-implantation period

Rat uterine sensitization-associated gene-1 (USAG-1) mRNA is expressed in the uterus during the peri-implantation period, and its mRNA expression in uterine epithelial cells is highest on day 5 of pregnancy. On the other hand, since changes in USAG-1 mRNA expression in the mouse uterus are not seen during the estrous cycle, USAG-1 expression might be specifically regulated by embryonic factors rather than by the maternal environment. However, the expression pattern and function of USAG-1 in the mouse uterus have not been determined. Thus, we examined the tissue-specific USAG-1 mRNA expression in the uteri of ICR mice during peri-implantation using real-time quantitative PCR. Uterine tissues, such as the myometrium, luminal epithelium, and stroma, were collected by laser capture microdissection at 3.5-6.5 dpc. USAG-1 mRNA was expressed in the uteri of pregnant mice from 3.5 dpc to 6.5 dpc, and the highest level of expression was seen at 4.5 dpc (P<0.01). Significantly high USAG-1 mRNA expression was detected in the luminal epithelium at 4.5 dpc (P<0.05). The stroma and myometrium exhibited unchanged expression levels of USAG-1 mRNA at 3.5-5.5 dpc. USAG-1 mRNA was undetectable in blastocysts and implanting embryos. Expression of USAG-1 mRNA appears to be associated with blastocyst implantation to the luminal epithelium, suggesting that physiological or biochemical contact of the blastocyst to the uterus is required for USAG-1 expression.     

Uterine secretions of the cow contain proteins that are immunochemically related to the major progesterone-induced proteins of the sheep uterus

Proteins that cross-react with antiserum to the major progesterone-induced proteins found in the pregnant sheep uterus, the uterine milk proteins (UTM-proteins), were detected as radiolabelled secretory products of endometrium from pregnant cows. Cross-reactive proteins included a form at 57,000 molecular weight as well as other lower-molecular-weight variants found in lower amounts. Similar proteins were also detected in uterine fluid from a cow at day 270 of gestation. Using immunohistochemical procedures, proteins that cross-reacted with antiserum to sheep UTM-proteins could be localized to the epithelial cells of endometrial glands in the cow. Results indicate that UTM-protein-like molecules are secreted by the endometrium of the cow during pregnancy.     

Tumour necrosis factor in the canine endometrium: an immunohistochemical study

Tumour necrosis factor (TNF), a pleiotropic cytokine that regulates cell growth and differentiation as well as the synthesis of other cytokines, has been identified in the uterus of several species describing a cyclic pattern, eventually under ovarian steroid regulation. Information is yet limited on the presence of TNF protein in the canine endometrium during the oestrous cycle and early pregnancy. This study depicts the temporal immunolocalization of TNF in the bitch endometrium along the oestrous cycle and changes associated with the early steps of embryo invasion. TNF immunolabelling was found in both the stromal fibroblasts and epithelial components of the canine endometrium in all stages studied. Stromal immunostaining was more intense than that of the epithelia, in all the stages of the oestrous cycle. In addition, a tendency for a decrease in the surface epithelium intensity score was found in early dioestrus. A positive glandular content was only observed in anoestrus and proestrus stages. In early pregnancy (days 13-16), TNF immunolabelling was detected at the embryo-maternal surface, in the syncytium cords and the trophoblast, as well in the endometrial stroma and the basal endometrial glands, but not in the lacunar epithelium. The overall TNF immunoreactivity was higher in early pregnancy samples in comparison with those of the early dioestrus and dioestrus stages, suggesting it plays a role during implantation.     

Endometrial expression of the insulin-like growth factor system during uterine involution in the postpartum dairy cow

Rapid uterine involution in the postpartum period of dairy cows is important to achieve a short interval to conception. Expression patterns for members of the insulin-like growth factor (IGF) family were determined by in situ hybridisation at day 14 ± 0.4 postpartum (n = 12 cows) to investigate a potential role for IGFs in modulating uterine involution. Expression in each uterine tissue region was measured as optical density units and data were analysed according to region and horn. IGF-I mRNA was localized to the sub-epithelial stroma (SES) of inter-caruncular and caruncular endometrium. Both IGF-II and IGF-1R expression was detected in the deep endometrial stroma (DES), the caruncular stroma and myometrium. IGFBP-2, IGFBP-4 and IGFBP-6 mRNAs were all localised to the SES of inter-caruncular and caruncular uterine tissue, and in the DES and caruncular stroma, with IGFBP-4 mRNA additionally expressed in myometrium. IGFBP-3 mRNA was only detectable in luminal epithelium. IGFBP-5 mRNA was found in myometrium, inter-caruncular and caruncular SES and caruncular stroma. These data support a role for IGF-I and IGF-II in the extensive tissue remodelling and repair which the postpartum uterus undergoes to return to its non-pregnant state. The differential expression of binding proteins between tissues (IGFBP-3 in epithelium, IGFBP-2, -4, -5 and -6 in stroma and IGFBP-4 and -5 in myometrium) suggest tight control of IGF activity within each compartment. Differential expression of many members of the IGF family between the significantly larger previously gravid horn and the previously non-gravid horn may relate to differences in their rate of tissue remodelling.     

Bovine uterine, cervical and ovarian androgen receptor concentrations. Correlation with estrogen and progesterone receptor concentrations.

Bovine cytosol androgen receptor (ARC) concentrations were examined simultaneously in various regions of the uterus and in ovarian tissues of cows, and were related to cytosol estrogen (ERC) and progesterone receptor (PRC) concentrations and circulating steroid levels. ERC concentrations were 3-7-fold and PRC concentrations 13-29-fold those of ARC in bovine endometrial and myometrial tissues. When serum progesterone levels were low, both endometrial and myometrial ARC, endometrial ERC, and endometrial and myometrial PRC concentrations were higher (p < 0.05) than those observed during higher progesterone concentrations. Because serum 5 alpha-dihydrotestosterone (5 alpha-DHT) concentrations were higher during the luteal phase, it is possible that ARC was down-regulated by this natural ligand at this phase of the cycle. There were no differences between uterine horns in endometrial or myometrial ARC concentrations. Bovine cervical and ovarian stromal tissue also contained ARC, and the concentrations were about the same as in the endometrium and the myometrium. The relative binding affinities (RBAs) of some steroid hormones towards ARC in vitro were: the synthetic compound R1881 (146%), 5 alpha-dihydrotestosterone (100%), testosterone (75%) while estradiol-17 beta, progesterone and dexamethasone had lower RBAs (2, < 1, < 1% respectively). Cytosol androgen receptor concentrations correlated significantly with cytosol progesterone (PRC) and estrogen receptor (ERC) concentrations, both in the endometrium and myometrium. These data show that androgens, such as 5 alpha-DHT, may participate the endocrine regulation of bovine reproductive tissues.     

Old,new and the newest concepts of inhibition of luteolysis during early pregnancy in pig

In 1977 Bazer and Thatcher proposed that maternal recognition of pregnancy in the pig involves the secretion of PGF(2alpha) towards the uterine lumen (exocrine) rather than towards the uterine venous drainage (endocrine) as occurs in the non-pregnant pig during the mid to late stages of the estrous cycle. The retrograde transfer of PGF(2alpha) from the venous blood and uterine lymph into the uterus and the ability of the uterine vein and artery wall to accumulate PGF(2alpha) could constitute a part of putative mechanism of corpus luteum protection during early pregnancy. A luteotropic/anti-luteolytic effect of PGE(2) in the pig also has been frequently demonstrated and it seems that the most effective agent in changing PGE(2):PGF(2alpha) secretion is estradiol. The role for oxytocin during luteolysis and early pregnancy is controversial. It appears, however, that the main function of this hormone is autocrine and/or paracrine stimulation of PGF(2alpha) secretion. Pig trophoblastic interferons, unlike those of ruminants, do not themselves exert an anti-luteolytic effect in pigs. It is likely, that cytokines and angiogenic growth factors are involved in the initiation of luteolysis and/or maintenance of corpora lutea (CL).A discovery of functional LH receptors in porcine endometrium opened a new possibility for this hormone in luteolysis and perhaps in recognition of pregnancy in pigs. The endogenous LH pulses can provoke prostaglandin secretion from endometrium in pigs. On the other hand prolongation of up-regulation of LH receptors in endometrium of early pregnant gilts can additionally increase angiogenic factor production before the process of implantation is completed. Finally new integrated concepts of luteolysis and inhibition of luteolysis in pigs based on selectively reviewed information are presented.