This study was carried out to identify the possible effects of epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) on cumulus expansion and nuclear maturation in bovine oocytes matured in vitro in serum-free media. Bovine follicular oocytes were matured in vitro and were then classified as cumulus cell-enclosed oocytes or denuded oocytes. They were then cultured for 24 h in TCM-199 (tissue culture medium-199) with EGF (50 ng ml-1), IGF-I (100 ng ml-1) and EGF plus IGF-I (50 and 100 ng ml-1, respectively). At the end of the culture period, the morphology of oocytes was evaluated for cumulus expansion and nuclear stage of maturation. In Expt 1, percentages of oocytes reaching maximum cumulus expansion were: 12.5% (control), 46.5% (EGF), 15.2% (IGF-I) and 52.8% (EGF plus IGF-I). In Expt 2, the respective rates of nuclear maturation were: 35.6%, 52.1%, 45.5% and 61.4% for cumulus cell-enclosed oocytes, and 35.3%, 46.6%, 45.4% and 42.5% for denuded oocytes; the same growth factor treatments were used in both cases. There was no significant effect of IGF-I on cumulus expansion. Maximum rates of cumulus expansion and nuclear maturation were obtained in the presence of both growth factors. These results lead to the following conclusions: (i) EGF, either alone or together with IGF-I, stimulates cumulus expansion; and (ii) both growth factors, acting alone or together, enhance nuclear maturation in oocytes surrounded by compact cumulus cells.

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 alpha(v)beta(3) 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 alpha(v)beta(3) 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 alpha(v)beta(3) 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 alpha(v) or beta(3) 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 alpha(v)beta(3) 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.

The study of implantation has been facilitated by the identification of specific biomarkers that are associated with uterine receptivity. The ␣ v ␤ 3 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 ␣ v ␤ 3 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 trophoblastendometrial structures known as trophoblastic knobs. In the present study, the expression of ␣ v ␤ 3 integrin subunit in the rab- embryo, implantation, uterus

Canine inflammatory mammary cancer (IMC) shares epidemiologic, histopathological and clinical characteristics with the disease in humans and has been proposed as a natural model for human inflammatory breast cancer (IBC). The aim of this study was to characterize a new cell line from IMC (IPC-366) for the comparative study of both IMC and IBC. Tumors cells from a female dog with clinical IMC were collected. The cells were grown under adherent conditions. The growth, cytological, ultrastructural and immunohistochemical (IHC) characteristics of IPC-366 were evaluated. Ten female Balb/SCID mice were inoculated with IPC-366 cells to assess their tumorigenicity and metastatic potential. Chromosome aberration test and Karyotype revealed the presence of structural aberration, numerical and neutral rearrangements, demonstrating a chromosomal instability. Microscopic examination of tumor revealed an epithelial morphology with marked anysocytosis. Cytological and histological examination of smears and ultrathin sections by electron microscopy revealed that IPC-366 is formed by highly malignant large round or polygonal cells characterized by marked atypia and prominent nucleoli and frequent multinucleated cells. Some cells had cytoplasmic empty spaces covered by cytoplasmic membrane resembling capillary endothelial cells, a phenomenon that has been related to s vasculogenic mimicry. IHC characterization of IPC-366 was basal-like: epithelial cells (AE1/AE3+, CK14+, vimentin+, actin-, p63-, ER-, PR-, HER-2, E-cadherin, overexpressed COX-2 and high Ki-67 proliferation index (87.15 %). At 2 weeks after inoculating the IPC-366 cells, a tumor mass was found in 100 % of mice. At 4 weeks metastases in lung and lymph nodes were found. Xenograph tumors maintained the original IHC characteristics of the female dog tumor. In summary, the cell line IPC-366 is a fast growing malignant triple negative cell line model of inflammatory mammary carcinoma that can be used for the comparative study of both IMC and IBC.

Undefined follicular factors that may influence nuclear maturation and/or cytoplasmic maturation are required during in vitro maturation of pig oocytes. Epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), and dialysed porcine follicular fluid (dpFF) were evaluated for their effects on porcine oocyte nuclear maturation in vitro. In Experiment I, eight different maturation media were made in a split-plot factorial design with dpFF (0% vs. 10% v/v dialyzed pFF) as the whole plot component, and EGF (0.0 vs. 50 ng/ml) and/or IGF-I (0.0 vs. 100 ng/ml) as the factorial subplot component. Experiment II was a complete factorial design with dpFF and EGF. Pig follicular granulosa-cumulus-oocyte complexes (GCOC) were obtained from slaughterhouse ovaries, washed, and cultured at 38.5 degrees C in a humidified incubator with 5% CO2 in air for 42 h. Following culture, GCOC were mechanically stripped of granulosa-cumulus cells and evaluated for nuclear maturation by light microscopy. In Experiment I, the percentage of Metaphase II oocytes for control, IGF-I, EGF, and IGF-I+EGF treatments without pFF were 50.7%, 52.6%, 80.9%, and 84.3% (control and IGF-I groups significantly less, P < .001). The same treatments in the presence of pFF were similar and high (84.2, 84.9, 82.1, and 86.8%, respectively). Experiment II gave similar results. These results demonstrate that EGF, in the absence of pFF, promotes a similar level of oocyte nuclear maturation as does pFF alone or pFF with EGF and/or IGF-I. IGF-I does not appear to influence nuclear maturation of GCOC.

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The effects of different combinations of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on cumulus expansion and meiotic maturation were examined in rabbit oocytes. Selected rabbit follicular oocytes were matured in vitro and were classified as cumulus-oocyte complexes or denuded oocytes. They were cultured in TCM 199, and were treated with growth factors at different concentrations: EGF at 0, 1, 10, 50 and 100 ng ml-1, IGF-I at 0, 50, 100 and 200 ng ml-1 and EGF plus IGF-I at 10 + 50; 10 + 100; 50 + 50 and 50 + 100 ng ml-1, respectively. After 6 h of culture, the oocytes were assessed for nuclear maturation and after 16 h of culture, for cumulus expansion and maturation stage. After culture for 6 h, the incidence of germinal vesicle breakdown was higher (P < 0.05) in all of the growth factor treatments tested compared with controls. After culture for 16 h, EGF enhanced the incidence of cumulus expansion at all of the concentrations tested. Cumulus expansion was greatest with 50 mg EGF ml-1 plus 100 ng IGF-I ml-1 (72.0% versus 2.4% in controls). Treatment with IGF-I significantly increased (P < 0.05) the incidence of metaphase II stage, and maximum stimulation occurred at 100 ng IGF-I ml-1 (84.5% versus 31.1% in controls). However, IGF-I did not affect cumulus expansion. When denuded oocytes were used, no positive effects on nuclear maturation rates were observed for any treatment. These results suggest that: (1) EGF, either alone or with IGF-I, stimulates cumulus expansion; (2) the addition of IGF-I or EGF plus IGF-I significantly enhances nuclear maturation in immature rabbit oocytes; and (3) this effect is mediated by the presence of cumulus cells.

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