M.J. Illera

Blockade of the αvβ3 Integrin Adversely Affects Implantation in the Mouse

Abstract The role of endometrial and embryonic integrins during implantation remains unresolved although work in animal models and in humans supports their involvement in this process. Temporal and spatial distribution of the αvβ3 integrin on both embryo and endometrium in women and mice coincides with the time of initial attachment during implantation. In mice, the endometrial and embryonic αvβ3 integrin is present at the time of implantation, as shown by reverse transcription-polymerase chain reaction and immunohistochemistry. In situ hybridization demonstrates the presence of the αvβ3 integrin on the subluminal stromal cells of the uterus. Functional blockade of this integrin on the day of implantation by intrauterine injection of neutralizing monoclonal antibodies against αv or β3 integrin subunits, arg-gly-asp (RGD)-containing peptides, or of the disintegrin echistatin, reduced the number of implantation sites compared to controls receiving BSA. These studies demonstrate that, like the human, the murine αvβ3 integrin is expressed at the time of implantation in the endometrium and on the blastocyst, and may play a critical role in the cascade of events leading to successful implantation.

Effect of peritoneal fluid from women with endometriosis on implantation in the mouse model

OBJECTIVE To investigate the potential role of peritoneal fluid (PF) from women with or without endometriosis in implantation in mice with use of the delayed implantation model. DESIGN A murine experimental model with markers of uterine receptivity and prospective comparison of the effects of human PF on implantation. SETTING Academic university and hospital program. INTERVENTION(S) PF collected from women with and without endometriosis was injected intraperitoneally into recently mated mice. MAIN OUTCOME MEASURE(S) Implantation sites were counted in treated and untreated animals, and the alphavbeta3 integrin was measured in the pregnant mouse uterus by immunohistochemistry with in situ hybridization. Leukemia inhibitory factor and the beta3 subunit of alphavbeta3 were measured by Northern blot during early pregnancy and after injections of PF. RESULT(S) Animals receiving PF from infertile women with endometriosis had a reduction in the number of implantation sites compared with animals that received PF from fertile women or from patients with recently treated endometriosis. In the mouse, expression of alphavbeta3 and leukemia inhibitory factor peaked at the time of implantation and was reduced by injections of human PF from infertile patients with endometriosis. CONCLUSION(S) Leukemia inhibitory factor and alphavbeta3 are coexpressed at the time of implantation in the mouse. PF from women with endometriosis has a detrimental effect on embryo implantation, perhaps by adversely affecting uterine receptivity.

A Role for αvβ3 Integrin During Implantation in the Rabbit Model

Abstract The study of implantation has been facilitated by the identification of specific biomarkers that are associated with uterine receptivity. The αvβ3 integrin is a cell surface adhesion receptor, whose expression has been shown to be elevated in the endometrium at the time of implantation in both humans and other mammalian species; however, the distribution of αvβ3 in the rabbit model is unknown. The rabbit has been shown to be an excellent model for the study of implantation. As an obligate ovulator, the timing of pregnancy can be precisely established, and embryonic attachment occurs through specialized trophoblast-endometrial structures known as trophoblastic knobs. In the present study, the expression of αvβ3 integrin subunit in the rabbit uterus was examined by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and in situ hybridization. Expression of the αvβ3 integrin was examined in Day 6.5 embryos, flushed from pregnant does. Immunofluorescence demonstrated strong immunostaining on the rabbit blastocyst (Day 6.5). RT-PCR analyses showed higher levels of mRNA for β3 subunit at the implantation site, with reduced expression in nonimplantation sites and in nonpregnant adult and immature endometrium. Immunohistochemistry demonstrated little, if any, β3 immunoreactivity on the endometrial epithelium. In contrast, in situ hybridization showed expression of the β3 integrin subunit mRNA in the uterine myometrium and on the trophoblast. To further determine the functional significance of αvβ3 integrin expression during implantation, pregnant female rabbits that underwent ventral laparotomy on the morning of Day 6 received intrauterine injection of the following into the right uterine horn: 1) the monoclonal αvβ3 neutralizing antibody (LM609), 2) arg-gly-asp (RGD) hexapeptides (GRGDSP), 3) non-RGD hexapeptides (GRGESP), and 4) IgG isotype matched control antibody. The left horn served as a control and received only saline injections. A significant reduction in the number of implantation sites was observed in the horns receiving anti-αvβ3 antibody (P < 0.001) and the RGD peptides (P = 0.03). In the rabbit, the αvβ3 integrin is present on the embryo and trophoblast and appears to be involved in early embryo-maternal interaction.

Regulated Expression of Osteopontin in the Peri-Implantation Rabbit Uterus

Abstract Blastocyst attachment to the lining of the mammalian uterus during early implantation involves the initial apposition of the trophoblast to the uterine epithelial surface. Osteopontin (OPN) is a glycoprotein component of the extracellular matrix that is secreted by the glandular epithelium of mammalian uteri at the time of implantation. This protein is recognized by several members of the integrin family and promotes cell-cell attachment and adhesion. In the present study, rabbit uteri were examined using Northern and in situ hybridization to evaluate the temporal and spatial distribution of OPN mRNA during early pregnancy. Northern blot analysis demonstrated a dramatic increase in OPN expression on Days 4–7 of pregnancy, corresponding to the rise in circulating progesterone and the time of initial embryo attachment in this species. In situ hybridization analysis revealed OPN mRNA expression on Day 6.75 of pregnancy, which was most prominent on endometrial epithelium. Using immunofluorescence, OPN protein was present on the glandular epithelium on Day 6.75 of pregnancy, but was absent on blastocysts. Further, no expression of OPN mRNA or protein was found in the nonpregnant endometrium. Induction of endometrial OPN expression was observed in unmated rabbits treated with progesterone alone and was prevented by cotreatment with the antiprogestin ZK137.316. Estradiol-17β had no effect on OPN expression by itself, and estrogen priming was not necessary to demonstrate the stimulatory effect of progesterone. In The rabbit uterus, as in other mammalian species studied, OPN is expressed in a stage-specific manner by the endometrial glands during the peri-implantation period and is regulated by progesterone.

The effects of different anaesthetic treatments on the adreno-cortical functions and glucose levels in NZW rabbits.

The effects of five anaesthetics on the corticosterone, cortisol and glucose concentrations were investigated in the NZW rabbit. Sixty animals were assigned to 6 treatment groups (n=10 per group): control (iv saline solution injection), ketamine (10 mg/kg iv) with either xylazine (3 mg/kg iv) or diazepam (2 mg/kg iv), pentobarbitone (30 mg/kg iv), thiopentone (20 mg/kg iv) and fentanyl/droperidol (1 mg/kg sc). Plasma glucocorticoids were measured by competitive enzymeimmunoassay EIA and glucose by an autoanalyzer, previously validated for this species in both cases. Blood samples were obtained at 6 time-points: before injection, at 10, 30, 60, 120 min and 24 h after injection of the anaesthetics/saline. A significant decrease of plasma glucocorticoids at 10–60 min was observed in the pentobarbitone and fentanyl/ droperidol groups, whereas the administration of ketamine/diazepam or thiopentone stimulated plasma glucocorticoid release, principally in the recovery period. However, in the ketamine/xylazine group no changes were observed in the glucocorticoid levels, except for a significative increase of cortisol at 60–120 min. Glucose levels significantly increased after ketamine/diazepam administration and principally, after ketamine/xylazine treatment. The present data suggest that ketamine/xylazine has little effect on glucocorticoid levels and provides an adequate level of surgical anaesthesia, hence it would be the anaesthetic of choice, although the hyperglycaemic effect after injection has to be considered for any experimental procedures in rabbits.ResumenSe estudian los efectos de 5 anestésicos sobre las concentraciones de corticosterona, cortisol y glucosa en el conejo NZW. Se utilizan 60 animales divididos en 6 grupos (n=10 por grupo): control (inyección de solución salina iv), fentanil/droperidol (1 mg/kg sc), ketamina (10 mg/kg iv) junto a xilazina (3 mg/kg iv) o diacepán (2 mg/kg iv), pentobarbital (30 mg/kg iv) y tiopental (20 mg/kg iv). Las concentraciones plasmáticas de glucocorticoides se miden mediante la técnica EIA de competición y la glucosa con un autoanalizador. Las muestras de sangre se recogen a los 0, 10, 30, 60, 120 min y 24 horas tras la administración de los anestésicos/solución salina. En los grupos tratados con pentobarbital o fentanil/ droperidol se observa una disminución significativa de los glucocorticoides plasmáticos a los 10–60 min, mientras que la administración de ketamina/diacepán o del tiopental estimula la liberación de glucocorticoides, principalmente durante la recuperación. Sin embargo, tras la administración de ketamina/ xilazina no se observan cambios en los niveles de glucocorticoides, excepto un aumento significativo de cortisol a los 60–120 min. Los niveles de glucosa aumentan significativamente tras la administración de ketamina/diacepán y principalmente, de ketamina/xilazina. Los datos presentes sugieren que la ketamina/xilazina apenas afecta a los niveles de glucocorticoides y alcanza un adecuado nivel de anestesia quirúrgica, por lo que sería el anestésico de elección, aunque el efecto hiperglicémico tras el tratamiento ha de ser considerado en cualquier experimento realizado en conejos.

Characterization of Antiestrogenic Activity of the Chinese Herb, Prunella vulgaris, Using In Vitro and In Vivo (Mouse Xenograft) Models

Abstract Prunella vulgaris (PV), a commonly used Chinese herb, also known as Self-heal, has a wide range of reported medicinal activities. By screening multiple herbs using the endometrial cancer cell line, ECC-1, and an alkaline phosphatase detection assay, we found that PV displayed significant antiestrogenic activity. We investigated the possible usefulness of antiestrogenic activity using both in vitro and in vivo models of endometrial function. Using the well-differentiated, hormone-responsive endometrial cell line, ECC-1, PV extract, at concentrations that were not toxic to the cells, significantly reduced alkaline phosphatase activity and cell proliferation in response to estrogen in a dose-dependent manner. The expression of CYR61, an estrogen-induced protein, was blocked in ECC-1 cells by both the antiestrogen ICI 182 780 and PV extract. Interestingly, PV extract did not appear to directly inhibit estrogen signaling. Rather, we found that its activities were probably related to an ability to function as an aryl hydrocarbon receptor (AHR) agonist in ECC-1 cells. In support of this hypothesis, we noted that PV induced CYP1A1, CYP1B1, and AHR repressor expression in a dose-dependent manner—responses that were blocked by small interfering RNA treatment to reduce AHR and specific AHR antagonists. Ovariectomized immunodeficient RAG-2/gamma(c) knockout mice implanted with human endometrial xenografts developed implants only when treated with estrogen. Mice treated with estrogen and PV tea in their drinking water had fewer and smaller xenograft implants compared with their estrogen-treated counterparts that drank only water (P < 0.05). Analysis of the resulting implants by immunohistochemistry demonstrated persistent estrogen receptor (ER), but reduced proliferation and CYR61 expression. Mouse uterine tissue weight in PV-treated mice was not different from controls, and cycle fecundity of intact C57 female mice was unaffected by PV tea treatment. PV, or Self-heal, exhibits significant antiestrogenic properties, both in vitro and in vivo. This activity is likely due to the ability of PV-activated AHR to interfere with estrogen. This herb may be useful as an adjunct for the treatment of estrogen-dependent processes like endometriosis and breast and uterine cancers. Full characterization of this herb will likely provide new insights into the crosstalk between AHR and ESR1, with potential for therapeutic applications in women.

Influence of epidermal growth factor on mammalian oocyte maturation via tyrosine-kinase pathway

Epidermal growth factor (EGF) has been reported to promote different functions in mammalian ovaries, including oocyte maturation. The aim of the present study was to establish: that EGF influences oocyte maturation in ovine and equine, that a tyrosine kinase-dependent intracellular mechanism mediates EGF effect and, that EGF-R receptor is detectable in ovarian follicles by immunohistochemistry methods. Selected ovine and equine oocytes were aspirated from 2–5 mm (ovine) or 25 mm (equine) follicles and cultured in TCM 199 for 22 (ovine) or 36 hours (equine). They are then subjected to culture with EGF and two specific tyrosine-kinase inhibitors (TKIs, tyrphostins A-23 y A-47). Maturation was determined as the percentage of oocytes at metaphase II stage after culture. Treatments with EGF significantly increased incidences of metaphase II stage compared to controls (86.2% vs. 55% and 70.4% vs, 22.5% in ovine and equine oocytes, respectively). Tyrphostins A-23 and A-47 were effective in suppressing EGF-effect on oocytes. EGF-receptor was localized in follicles, being more prominent in cumulus and granulosa cells. These results confirm that EGF has a physiological role in the regulation of oocyte maturation via tyrosine-kinase pathway.ResumenEl factor de crecimiento epidérmico (EGF) promueve divers as funciones en el ovario de mamífero, incluyendo la maduración de los oocitos. El objetivo del presente trabajo consiste en establecer que el EGF es un regulador de la maduración de los oocitos mediante un mecanismo en el que están implicadas enzimas tirosina-quinasas y la presencia del receptor de EGF en los folículos ováricos mediante técnicas imunocitoquímicas. Para el estudio, se seleccionan oocitos ovinos y equinos tras la aspiración de los contenidos foliculares y se cultivan para su maduraciónin vitro, en TCM 199 durante 22 (ovinos) o 36 horas (equinos), en presencia de EGF y/o de tirfostinas A-23 y A-47, dos inhibidores selectivos de la actividad tirosina-quinasa (TKIs). La maduración de los oocitos se expresa como porcentaje de los que alcanzan el estadío nuclear de metafase II. Los oocitos cultivados en presencia de EGF presentan mayor porcentaje de maduración que los controles. La presencia de TKIs en los medios con EGF da lugar a tasa de maduración similar a las de los controles. El receptor para el EGF se ha localizado en folículos ováricos, siendo su presencia más acusada en el cúmulo celular y en las células de la granulosa. En conjunto, los resultados obtenidos confirman que el EGF tiene un papel importante en la regulación fisiológica de la maduració n del oocito mediante enzimas tirosina-quinasas.

Steroid-level response to insulin-like growth factor-1 in oocytes matured in vitro.

The objective was to establish the influence of insulin-like growth factor-1 (IGF-1) on steroid production and nuclear maturation during oocyte in vitro maturation (IVM). Immature-selected rabbit follicular oocytes, divided as cumulus-oocyte complexes (COC) and denuded oocytes (DO), were cultured in Bracketts medium with different concentrations of IGF-1 at 0, 50, 100 and 200 ng/ml. After 8 and 16 h of culture, the oocytes were assessed for nuclear maturation by acetic-orcein stain, and media were analyzed by enzyme-immunoassay (EIA) for 17 beta-estradiol (E), progesterone (P), androstenedione (A) and testosterone (T) content. After culture treatments with IGF-1 significantly increased (P < 0.01) the incidence of nuclear activation (germinal vesicle breakdown stage, GVBD) and nuclear maturation (metaphase II stage); maximum stimulation occurred at 100 ng IGF-1/ml (86.9 vs. 49.3% in control). Compared to controls, the presence of IGF-1 in cultures was associated with a significant increase of E and A production by COCs (P < 0.01). However, P and T levels were not significantly influenced by the IGF-1. In addition, positive correlations between E/T and E/A ratios and nuclear maturation rates were only found in the IGF-1 treatments. Regarding the DOs, neither positive effects in nuclear maturation rates nor increase of steroid levels in culture were observed for any treatment. These results suggest that: (1) IGF-1 had a significant effect on E and A production during oocyte maturation; (2) the addition of IGF-1 enhanced nuclear maturation significantly in rabbit oocytes; and (3) all these effects are only possible in oocytes surrounded by cumulus cells.

Role of EGF, IGF-I, sera and cumulus cells on maturation in vitro of bovine oocytes

Abstract We examined the effects of epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), the presence/absence of cumulus cells, and sera (fetal calf serum, FCS or estrous cow serum, ECS) on cumulus expansion and meiotic maturation during in vitro bovine oocyte maturation. The oocytes obtained (n = 4491) were divided into cumulusoocyte complexes (COC) or denuded oocytes and were then cultured in 3 groups comprising TCM-199 and sera: Group I (without serum), Group II (10% FCS), and Group III (10% ECS). Each group was subjected to 4 predefined treatments with growth-factors: control (no growth factor), EGF, IGF-I, and EGF + IGF-I. At the end of the 24-h culture period, the oocytes were assessed for degree of cumulus expansion and metaphase II stage. Treatments with EGF enhanced the incidence of full cumulus expansion in all 3 groups. Maximal stimulation for both cumulus expansion and nuclear maturation occurred with EGF + IGF-I in all groups, mainly in the groups with serum. Regarding the denuded oocytes, no positive effects on nuclear maturation rates were observed for any treatment. These results suggest that: 1) EGF, with or without IGF-I, stimulates cumulus expansion and meiotic maturation significantly; and 2) the presence of FCS or ECS enhances the effect of these growth factors in bovine cumulus-oocyte complexes.

Amplified androstenedione enzymeimmunoassay for the diagnosis of cryptorchidism in the male horse: comparison with testosterone and estrone sulphate methods

An amplified enzymeimmunoassay (EIA) was validated for androstenedione in the serum of male horses. We will use the assay as a tool for the diagnosis of equine cryptorchidism. We will compare androstenedione EIA to the currently used methods (testosterone and estrone sulphate determinations). The study was conducted on 115 horses of pure Spanish and Arabian breeds, that included 30 geldings, 60 bilateral cryptorchids and 25 stallions. Androstenedione standard curve covered a range between 0 and 1 ng per well. Low detection limit was 1.54 pg/ml. Intra- and inter-assay coefficients of variation (CV%) were <8.2 and <9.3, respectively (n=10). Recovery rate of known androstenedione concentrations averaged from 96.62+/-2.69 to 97.63+/-1.87%. Androstenedione mean+/-S.E. serum concentrations were 10.52+/-1.36 ng/ml in stallions (n=25), 0.51+/-0.04 ng/ml in cryptorchids (n=60), and 0.03+/-0.01 ng/ml in geldings (n=30). Diagnostic validation parameters in basal samples showed for estrone sulphate the lower positive predictive value (0.85) with the higher number of false positives, and lower specificity (0.84). Testosterone showed the higher number of false negatives with a negative predictive value of 0.85, and lower sensitivity (0.85). Among the three hormones evaluated, androstenedione presented the best results with the smaller number of horses diagnosed as false positives (0.93) or negatives (0.91). This technique also resulted in higher sensitivity, specificity and efficiency over the other two methods assayed. We concluded that our amplified EIA is a highly sensitive and specific assay that provides a rapid, simple, and inexpensive alternative to other methods.