Staining For Epidermal Growth Factor Research Articles

Wound Healing Efficacy of Cucurbitaceae Seed Oils in Rats: Comprehensive Phytochemical, Pharmacological, and Histological Studies Tackling AGE/RAGE and Nrf2/Ho-1 Cue.

The Cucurbitaceae family includes several edible species that are consumed globally as fruits and vegetables. These species produce high volumes of seeds that are often discarded as waste. In this study, we investigate the chemical composition and biological activity of three seed oils from Cucurbitaceae plants, namely, cantaloupe, honeydew, and zucchini, in comparison to the widely used pumpkin seed oil for their ability to enhance and accelerate wound healing in rats. Our results showed that honeydew seed oil (HSO) was effective in accelerating wound closure and enhancing tissue repair, as indicated by macroscopic, histological, and biochemical analyses, as compared with pumpkin seed oil (PSO). This effect was mediated by down-regulation of the advanced glycation end products (AGE) and its receptor (RAGE) cue, activating the cytoprotective enzymes nuclear factor erythroid 2 (Nrf2) and heme oxygenase-1 (HO-1), suppressing the inflammatory mediators tumor necrosis factor (TNF)-α, nuclear factor kappa B (NF-κB), and nod-like receptor protein 3 (NLRP3), and reducing the levels of the skin integral signaling protein connexin (CX)-43. Furthermore, immunohistochemical staining for epidermal growth factor (EGF) showed the lowest expression in the skin after treatment with HSO, indicating a well-organized and complete healing process. Other seed oils from cantaloupe and zucchini exhibited favorable activity when compared with untreated rats; however, their efficacy was comparatively lower than that of PSO and HSO. Gas chromatographic analysis of the derivatized oils warranted the superior activity of HSO to its high nutraceutical content of linoleic acid, which represented 65.9% of the fatty acid content. This study's findings validate the use of honeydew seeds as a wound-healing fixed oil and encourage further investigation into the potential of Cucurbitaceae seeds as sources of medicinally valuable plant oils.

Blockade of epidermal growth factor and its receptor and axial elongation in experimental myopia.

To examine the influence of epidermal growth factor (EGF) and its receptor (EGFR) on axial ocular elongation, we intraocularly injected an EGF antibody and an EGFR antibody into young guinea pigs with lens-induced axial elongation (myopization). Mean axial elongation was reduced in the eyes injected with the EGF/EGFR-antibody compared with the contralateral control eyes injected with PBS (phosphate-buffered solution) (0.43±0.13mm vs 0.53±0.13mm; P<.001). The intereye difference in axial length increased (P=.005) as the doses of the EGF antibody and EGFR antibody increased. As a corollary, the thickness of the retina at the posterior pole was dose-dependently increased in the injected eyes compared to the contralateral control eyes. Immunohistochemical staining for EGF and the relative mRNA expression of EGF and EGFR were the highest in eyes not injected with the EGF antibody or EGFR antibody and decreased (P<.05) as the dose of EGF antibody or EGFR antibody increased. In an in vitro study, EGF had a stimulating effect and the EGF antibody had an inhibitory effect on the proliferation and migration of RPE cells. The findings showed that the intravitreal application of an EGF antibody and EGFR antibody is associated with a dose-dependent reduction in lens-induced axial elongation in young guinea pigs. The EGFR family may play a role in axial elongation of the eye and in the development of myopia.

Diminished salivary epidermal growth factor secretion: a link between Sjögren’s syndrome and autoimmune gastritis?

Objectives: Healthy human labial salivary glands produce epidermal growth factor (EGF). In Sjögren’s syndrome (SS), EGF staining is diminished. SS is also associated with chronic autoimmune corpus gastritis. We therefore hypothesized that EGF secretion would be diminished in SS and that this could affect gastric target cells.Methods: Salivary EGF secretion in SS was compared to that in healthy controls using an enzyme-linked immunosorbent assay (ELISA). EGF receptor (EGFR) immunoreactive cells in the gastric corpus of healthy human subjects were analysed using immunostaining.Results: Salivary secretion of EGF was diminished in SS patients (232.4, range 52.6–618.4, vs. 756.6, range 105.3–1631.6 pg/min, p = 0.002). Proton-pump positive parietal cells were mostly EGFR immunoreactive whereas very few pepsinogen I (PGI)-positive cells were EGFR positive.Conclusions: As EGF is relatively acid resistant, salivary gland-derived EGF might participate in an exo/endocrine mode of parietal cell maintenance in the gastric corpus. Deficiency of salivary gland-derived EGF in SS patients may cause impairment of gastric parietal cells resulting in exposure of immunogenic cryptic antigens and loss of immunological self-tolerance.

Epithelial cells are active participants in vocal fold wound healing: an in vivo animal model of injury.

Vocal fold epithelial cells likely play an important, yet currently poorly defined, role in healing following injury, irritation and inflammation. In the present study, we sought to identify a possible role for growth factors, epidermal growth factor (EGF) and transforming growth factor-beta 1 (TGFβ1), in epithelial regeneration during wound healing as a necessary first step for uncovering potential signaling mechanisms of vocal fold wound repair and remodeling. Using a rat model, we created unilateral vocal fold injuries and examined the timeline for epithelial healing and regeneration during early and late stages of wound healing using immunohistochemistry (IHC). We observed time-dependent secretion of the proliferation marker, ki67, growth factors EGF and TGFβ1, as well as activation of the EGF receptor (EGFR), in regenerating epithelium during the acute phase of injury. Ki67, growth factor, and EGFR expression peaked at day 3 post-injury. Presence of cytoplasmic and intercellular EGF and TGFβ1 staining occurred up to 5 days post-injury, consistent with a role for epithelial cells in synthesizing and secreting these growth factors. To confirm that epithelial cells contributed to the cytokine secretion, we examined epithelial cell growth factor secretion in vitro using polymerase chain reaction (PCR). Cultured pig vocal fold epithelial cells expressed both EGF and TGFβ1. Our in vivo and in vitro findings indicate that epithelial cells are active participants in the wound healing process. The exact mechanisms underlying their roles in autocrine and paracrine signaling guiding wound healing await study in a controlled, in vitro environment.

The expression of epidermal growth factor (EGF) and its receptor (EGFR) during post-natal testes development in the yak.

Growth factors play critical role in cell proliferation, regulate tissue differentiation and modulate organogenesis. Several growth factors have been identified in the testes of various mammalian species in last few years. In present investigation, the objective was to determine the expression of epidermal growth factor (EGF) and the epidermal growth factor receptor (EGFR) in yak testicular tissue by relative quantitative real time polymerase chain reaction (RT-PCR), Western blot (WB) and immunohistochemistry (IHC) from mRNA and protein levels. The testicular tissues were collected from male yak at 6 and 24months old. Results of RT-PCR and WB showed that the expression quantity of EGF and EGFR at 24months of age was higher than at 6months, and the increase rate of EGFR on mRNA and protein levels was higher than the increase rate EGF during post-natal testes development. Positive staining for EGF and EGFR was very low and mainly localized to Leydig cells testes at 6months of age with immunohistochemistry, and seminiferous tubules were not observed. At 24month of age, both the EGF and EGFR could be detected in Leydig cells, peritubular myoid cells, sertoli cells and germ cells of the yak testes. However, EGF and EGFR were localized to preferential adluminal compartment and basal compartment in the seminiferous tubules, respectively. In conclusion, the findings in present studies suggest that EGF and EGFR as important paracrine and/or autocrine regulators in yak testes development and spermatogenesis.

The role of epidermal growth factor in the healing tympanic membrane following perforation in rats

Epidermal Growth Factor (EGF) has been identified as playing a critical role in the wound healing process. The objective of this study is to investigate the role that EGF plays in rat tympanic membrane (TM) wound healing using two techniques, microarray and immunohistochemistry. The tympanic membranes of rats were perforated using a sterile needle and sacrificed at time points during 2weeks following perforation. The normalized signal intensities at the time points for EGF and associated genes are presented. The rat EGF mRNA did not change significantly between time points. Five associated proteins, including heparin-binding EGF-like growth factor were found to be differentially expressed above a two fold threshold at 12 h following perforation. EGF staining was found at low levels in the uninjured TM. Levels of EGF staining increased at 24 h in the basal keratinocyte layer, became diffusely elevated in the specimen at 36 h, before a second peak in staining of the keratinocyte layer at Day 4. The staining of EGF corresponds to its multiple roles in TM wound healing.

Epidermal growth factor, transforming growth factor-α, and epidermal growth factor receptor expression and localization in the canine endometrium during the estrous cycle and in bitches with pyometra

Gene expression and immunohistochemical localization of epidermal growth factor (EGF), transforming growth factor-α (TGF-α), and epidermal growth factor receptor (EGF-R) were compared between the endometrium of bitches ( Canis familiaris) with pyometra accompanied by cystic endometrial hyperplasia (CEH) and that of healthy bitches at similar stages of the estrous cycle. In normal bitches, endometrial TGF-α mRNA levels were highest at proestrus and gradually decreased as the cycle progressed to anestrus. Epidermal growth factor receptor mRNA levels were not significantly affected by the stage of the estrous cycle. Epidermal growth factor mRNA levels were higher at Day 35 of diestrus than at other stages of the estrous cycle (P < 0.05). In bitches with pyometra, endometrial TGF-α and EGF-R mRNA levels did not differ significantly from those at diestrus in normal bitches, but EGF mRNA levels were lower than those at Day 35 of diestrus in normal bitches (P < 0.05). In normal bitches, positive immunohistochemical staining for TGF-α, EGF, and EGF-R was mainly present in the glandular and luminal epithelial cells of the endometrium. In contrast, in bitches with pyometra, immunoreactivity for EGF was clearly present in endometrial stromal cells. Inflammatory cells that had infiltrated the endometrial stroma stained strongly for TGF-α and EGF-R. Luminal and glandular epithelial cells also stained positive for EGF-R. In conclusion, expression of TGF-α by inflammatory cells and a low level of expression and differential localization of EGF may be involved in aberrant growth of endometrial glands and development of CEH.

Antigen expression of human eccrine sweat glands

The proliferating abilities of sweat glands are very limited, so researches on the repair and regeneration of sweat glands are important. First of all, we must find out reliable and specific antigen markers of sweat glands. To investigate the antigen expression of human eccrine sweat glands. The development of eccrine sweat glands was investigated by hematoxylin and eosin staining, and the antigen expression was detected by immunohistochemical techniques. Human eccrine sweat glands expressed cytokeratin (CK) 7, CK8, CK14, CK18, CK19 and epithelial membrane antigen (EMA). Carcinoembryonic antigen (CEA) was only expressed in sweat glands in the adult skin. Developing and developed sweat glands all had some cells expressing Ki67 and p63 antigens. Epidermal growth factor (EGF) was mainly localized in the secretory cells and ductal cells. Some myoepithelial cells were also labeled with anti-EGF antibody. In the older fetus, positive staining for EGF was seen in the lumen of the secretory portion. EGF receptor (EGFR) was expressed in the ducts. Human eccrine sweat glands express CK7, CK8, CK14, CK18, CK19, CEA, EMA, Ki67, p63, EGF and EGFR. In skin, CEA can be used as a specific immunological marker of sweat glands.

Localization of epidermal growth factor (EGF) and its receptor (EGFR) during postnatal testis development in the alpaca ( Lama pacos)

The objective of the present studies was to determine the localization of epidermal growth factor (EGF) and the epidermal growth factor receptor (EGFR) in testicular tissue collected from male alpacas at 12 and 24 months of age. In the testes of 12-month-old alpacas, positive staining for EGF was not detected. EGFR was localized to Leydig cells within the 12-month-old alpaca testis, but staining was absent within seminiferous tubules. At 24 months of age, EGF was localized to Leydig cells, peritubular myoid cells, Sertoli cells and germ cells of the alpaca testis, with a preferential adluminal compartment staining within the seminiferous tubules. EGFR was also localized to the Leydig cells, peritubular myoid cells, Sertoli cells and germ cells within the 24-month-old alpaca testis, but staining within the tubules was primarily within the basal compartment. Results indicate distinct temporal and spatial regulation of EGF and EGFR in the alpaca testis and support a potential role for EGF and its related ligands in alpaca testis development and spermatogenesis.

Epithelial Phenotypes in the Developing Human Prostate

An intermediate population has been identified among prostate glands called transiently amplifying (TA) cells, which are characterized by coexpression of basal and luminal cytokeratins (CKs), high proliferation, and lack of p27 expression. These cells are rare in the normal adult prostate and increase in pretumoral conditions, but their importance in the developing gland remains unknown. We analyzed fetal prostates for the expression of CKs (5/6, 18, 19) and factors involved in proliferation and apoptosis: p63, Ki67, p27, epidermal growth factor (EGFR), Bcl2, androgen receptor (AR). Immunostaining was performed on a tissue microarray, including 40 prostates from fetuses aged 13-42 weeks and normal prostate tissue from 10 adults. In both solid buds and the basal compartment of canalized glands, cells expressed p63, CK5/6, CK19, CK18, BCL2, EGFR and were p27 negative. Luminal cells of fetal canalized glands continue to express CK19, EGFR, and BCL2, without p27 expression. In contrast, adult epithelial luminal cells showed diffuse AR and p27 expression, without CK19, BCL2, and EGFR staining. Proliferation was high and diffuse in fetal glands and rare and restricted to basal cells in adult glands. These results indicate that most fetal epithelial prostatic cells exhibit the phenotype of TA cells, suggesting their regulatory function in prostate development.

Interstitial Vascular Rarefaction and Reduced VEGF-A Expression in Human Diabetic Nephropathy

Diabetic nephropathy (DN) is a frequent complication in patients with diabetes. Although the majority of DN models and human studies have focused on glomeruli, tubulointerstitial damage is a major feature of DN and an important predictor of renal dysfunction. This study sought to investigate molecular markers of pathogenic pathways in the renal interstitium of patients with DN. Microdissected tubulointerstitial compartments from biopsies with established DN and control kidneys were subjected to expression profiling. Analysis of candidate genes, potentially involved in DN on the basis of common hypotheses, identified 49 genes with significantly altered expression levels in established DN in comparison with controls. In contrast to some rodent models, the growth factors vascular endothelial growth factor A (VEGF-A) and epidermal growth factor (EGF) showed a decrease in mRNA expression in DN. This was validated on an independent cohort of patients with DN by real-time reverse transcriptase-PCR. Immunohistochemical staining for VEGF-A and EGF also showed a reduced expression in DN. The decrease of renal VEGF-A expression was associated with a reduction in peritubular capillary densities shown by platelet-endothelial cell adhesion molecule-1/CD31 staining. Furthermore, a significant inverse correlation between VEGF-A and proteinuria, as well as EGF and proteinuria, and a positive correlation between VEGF-A and hypoxia-inducible factor-1alpha mRNA was found. Thus, in human DN, a decrease of VEGF-A, rather than the reported increase as described in some rodent models, may contribute to the progressive disease. These findings and the questions about rodent models in DN raise a note of caution regarding the proposal to inhibit VEGF-A to prevent progression of DN.

EPITHELIAL PHENOTYPES IN THE DEVELOPING HUMAN PROSTATE

SUMMARY An intermediate population has been identified among prostate glands called transiently amplifying (TA) cells, which are characterized by coexpression of basal and luminal cytokeratins (CKs), high proliferation, and lack of p27 expression. These cells are rare in the normal adult prostate and increase in pretumoral conditions, but their importance in the developing gland remains unknown. We analyzed fetal prostates for the expression of CKs (5/6, 18, 19) and factors involved in proliferation and apoptosis: p63, Ki67, p27, epidermal growth factor (EGFR), Bcl2, androgen receptor (AR). Immunostaining was performed on a tissue microarray, including 40 prostates from fetuses aged 13–42 weeks and normal prostate tissue from 10 adults. In both solid buds and the basal compartment of canalized glands, cells expressed p63, CK5/6, CK19, CK18, BCL2, EGFR and were p27 negative. Luminal cells of fetal canalized glands continue to express CK19, EGFR, and BCL2, without p27 expression. In contrast, adult epithelial luminal cells showed diffuse AR and p27 expression, without CK19, BCL2, and EGFR staining. Proliferation was high and diffuse in fetal glands and rare and restricted to basal cells in adult glands. These results indicate that most fetal epithelial prostatic cells exhibit the phenotype of TA cells, suggesting their regulatory function in prostate

A role of LIM kinase 1/cofilin pathway in regulating endocytic trafficking of EGF receptor in human breast cancer cells

We have previously shown that overexpression of LIM kinase1 (LIMK1) resulted in a marked retardation of the internalization of the receptor-mediated endocytic tracer, Texas red-labeled epidermal growth factor (EGF) in low-invasive human breast cancer cell MCF-7. We thereby postulate that LIMK1 signaling plays an important role in the regulation of ligand-induced endocytosis of EGF receptor (EGFR) in tumor cells by reorganizing and influencing actin-filament dynamics. In the present study, we further assessed the effect of wild-type LIMK1, a kinase-deficient dominant negative mutant of LIMK1 (DN-LIMK1) and an active, unphosphorylatable cofilin mutant (S3A cofilin) on internalization of EGF-EGFR in MDA-MB-231, a highly invasive human breast cancer cell line. We demonstrate here that a marked delay in the receptor-mediated internalization of Texas red-labeled EGF was observed in the wild-type LIMK1 transfectants, and that most of the internalized EGF staining were accumulated within transferrin receptor-positive early endosomes even after 30 min internalization. In contrast, the expression of dominant-negative LIMK1 mutant rescued the efficient endocytosis of Texas red-EGF, and large amounts of Texas red-EGF staining already reached LIMPII-positive late endosomes/lysosomal vacuoles after 15 min internalization. We further analyzed the effect of S3A cofilin mutant on EGFR trafficking, and found an efficient delivery of Texas red-EGF into late endosomes/lysosomes at 15-30 min after internalization. Taken together, our novel findings presented in this paper implicate that LIMK1 signaling indeed plays a pivotal role in the regulation of EGFR trafficking through the endocytic pathway in invasive tumor cells.

Epithelial proliferation markers in dysplasias and carcinomas associated with human papillomavirus

Proliferative verrucous leukoplakia (PVL) is a disorder characterized by the progressive development of epithelial dysplasia eventuating in carcinoma. The majority of these lesions have been associated with human papillomavirus (HPV). The purpose of this study was to evaluate 2 epithelial proliferation markers in HPV-associated oral dysplasia and carcinoma to determine the level of epithelial activation and to assess the possibility of the epithelial marker-guided prediction of prognosis for patients with PVL. Forty archival cases of paraffin-embedded HPV-positive lesions diagnosed as either epithelial dysplasia, verrucous hyperplasia, verrucous carcinoma, or squamous cell carcinoma obtained from patients with the clinical presentation of PVL were retrieved from the files of the University of North Carolina Oral and Maxillofacial Pathology Laboratory. Controls consisted of epithelium surfacing irritation fibromas. Immunostaining with primary antibodies to epidermal growth factor (EGFr) and proliferating cell nuclear antigen was performed by using the avidin-biotin-peroxidase technique with diaminobenzidine as a chromogen. EGFr positivity was graded subjectively on a scale from 0 to 3+, whereas proliferating cell nuclear antigen positivity was evaluated by examining sections under 100× oil immersion and determining the ratio of labeled-to-unlabeled cells from a count of 1000 cells per section. The results revealed a mean of 0.53 PCNA labeling in controls and a mean of 0.72 in all experimental groups combined (t test: P < .0001). EGFr staining of lesional epithelium revealed similar elevation over controls. We conclude that these proliferation markers have potential as prognostic indicators of the progression of PVL.

Effect of diethylstilbestrol on cell proliferation and expression of epidermal growth factor in the developing female rat reproductive tract.

To evaluate mechanisms of cell proliferation in the fetal female rat reproductive tract, diethylstilbestrol (DES) effects on cell division and estrogen receptor (ER), epidermal growth factor (EGF) and EGF receptor (EGF-R) expressions were determined from gestational day (GD) 15.5 to 21.5. Reproductive tracts were evaluated within three regions along the Müllerian duct axis; these were proximal, middle and caudal, which differentiate into oviduct, uterus and upper vagina respectively. In fetuses from non-treated dams, epithelial and mesenchymal proliferation, as evaluated by 5-bromo-2'-deoxyuridine incorporation, was decreased with development in all regions of the Müllerian duct. EGF levels were determined by immunohistochemistry. Müllerian epithelial EGF immunoreactivity was intense in the proximal and middle regions on GDs 15.5 and 17.5. EGF staining remained intense only in the proximal epithelia by GD 19.5 and was weak in the caudal epithelium, but substantially reduced throughout epithelia in all regions by GD 21.5. Thus, decreased cell proliferation correlated with decreased EGF expression in the developing Müllerian duct. DES (100 microg/kg body weight) was injected from GD 15 to 19 and female fetuses were collected on GD 19.5. DES increased Müllerian duct cell proliferation in the proximal epithelium and mesenchyme but decreased it in the caudal epithelium compared with oil-treated controls. No proliferative DES effect was observed in any cell type in the middle region. Müllerian duct EGF immunoreactivity was suppressed by DES compared with oil. Competitive RT-PCR indicated DES also decreased mRNAs for EGF, ERbeta1 and ERbeta2, but not ERalpha and EGF-R. These results indicate EGF may be an important regulatory factor of Müllerian duct cell proliferation, and that DES may alter cell proliferation by disrupting normal EGF, ERbeta1 and ERbeta2 expression in the developing female rat reproductive tract.

Pseudocarcinomatous change in lymphomatoid papulosis and primary cutaneous CD30+ lymphoma: A clinicopathologic and immunohistochemical study of 6 patients

We report 6 cases of pseudoepitheliomatous hyperplasia (PEH) mimicking squamous cell carcinoma in association with an atypical CD30+ dermal infiltrate. Three patients had lymphomatoid papulosis type A, and 3 patients had cutaneous CD30+ lymphoma. All 6 cases showed histologic evidence of PEH with keratinocyte atypia. In 4 cases there was significant atypia to prompt a diagnosis of squamous cell carcinoma. Three of these received treatment with wide local excision and 2 had been engrafted. Immunohistochemical staining for epidermal growth factor (EGF) and transforming growth factor α (TGF-α) showed similar expression in lesional and perilesional skin. Epidermal growth factor receptor (EGFR) expression by the proliferating epithelium was similar to that of the suprabasal adjacent normal epidermis. There was no aberrant expression of EGF, TGF-α, and EGFR by atypical lymphocytes. These cases demonstrate that PEH associated with CD30+ lymphoproliferative disease may closely resemble squamous cell carcinoma, thereby leading to inappropriate diagnosis and treatment. (J Am Acad Dermatol 2001;44:239-47.)

Nuclear localization of epidermal growth factor and epidermal growth factor receptors in human thyroid tissues.

Epidermal growth factor (EGF) has widespread growth effects, and in some tissues proliferation is associated with the nuclear localization of EGF and epidermal growth factor receptor (EGFR). In the thyroid, EGF promotes growth but differs from thyrotropin (TSH) in inhibiting rather than stimulating functional parameters. We have therefore studied the occurrence and cellular distribution of EGF and EGFR in normal thyroid, in Graves' disease, where growth is mediated through the thyrotropin receptor (TSHR), and in a variety of human thyroid tumors. In the normal gland the staining was variable, but largely cytoplasmic, for both EGF and EGFR. In Graves' disease there was strong cytoplasmic staining for both EGF and EGFR, with frequent positive nuclei. Nuclear positivity for EGF and particularly for EGFR was also a feature of both follicular adenomas and follicular carcinomas. Interestingly, nuclear staining was almost absent in papillary carcinomas. These findings document for the first time the presence of nuclear EGF and EGFR in thyroid. Their predominant occurrence in tissues with increased growth (Graves' disease, follicular adenoma, and carcinoma) may indicate that nuclear EGF and EGFR play a role in growth regulation in these conditions. The absence of nuclear EGF and EGFR in papillary carcinomas would suggest that the role played by EGF in growth control differs between papillary carcinoma and follicular adenomas/carcinomas of the thyroid.

Cholecystokinin does not affect the pancreatic contents of epidermal growth factor or its receptor.

Cholecystokinin (CCK) is a hormone with well-known secretory and trophic effects on the pancreas. This also is true for epidermal growth factor (EGF), which acts in a paracrine and autocrine way. The aim was to study the influence of CCK on cell proliferation in rat pancreas with special reference to the expression of EGF, the EGF receptor, and phosphorylated tyrosine. Twenty-four male Sprague-Dawley rats received either one single injection, or injections twice daily for 3 days of 6 microg sulfated CCK-8 (CCK-8S) subcutaneously in the neck. The same number of rats received injections of 1% bovine serum albumin (BSA) in the same way. The rats were killed 1, 3, or 6 hours after the last injection. One hour before killing, they received 50 mg/kg of bromodeoxyuridine (BrdU) intraperitoneally. Plasma was collected for analysis of CCK. The pancreas was dissected, and in situ hybridization using a probe for EGF mRNA was performed for semiquantification of gene expression. Immunocytochemistry using antibodies against the EGF receptor and phosphotyrosine was performed to examine the expression of the proteins, and against BrdU for measuring the cell proliferation. A single injection of CCK-8S led to hyperCCKemia at 1 and 3 hours afterward. After 6 hours, plasma CCK had returned to the same levels as in control rats. The cell proliferation was unaffected. The rats that received CCK-8S injections for 3 days still had hyperCCKemia 6 hours after the last injection. The cell proliferation was increased by CCK, as indicated by the BrdU labeling. However, neither body weight nor pancreatic weight was affected. In controls, EGF was expressed all over the gland, but its receptor and phosphotyrosine were expressed only in ductal cells and in the islet cells of endocrine pancreas. There was no difference in the pancreatic staining of EGF, its receptor, or phosphotyrosine at the different time points studied. There was no difference in the staining of EGF and its receptor between CCK-8S- and BSA-treated animals, but phosphotyrosine staining was detectable in acinar cells after 3 days of CCK-8S injections. Thus CCK-8S causes hyperCCKemia with ensuing enhanced cell proliferation in rat pancreas. This effect on the cell proliferation seems to be a direct effect of CCK and not mediated by changes in the tissue levels of EGF or its receptor.

Expression of transforming growth factor-alpha, epidermal growth factor, and epidermal growth factor receptor in follicles of human ovarian tissue before and after cryopreservation

Objective: To study the expression of transforming growth factor-alpha (TGF-α), epidermal growth factor (EGF), and EGF receptor in follicles of human ovarian tissue. Design: A retrospective, controlled comparative study. Setting: In vitro fertilization laboratory of a university hospital. Patient(s): Fifteen women with regular menstrual cycles who underwent laparoscopy and the biopsy of ovarian tissue. Intervention(s): Paraffin sections were prepared from ovarian tissues, followed by immunohistochemical staining of TGF-α, EGF, and EGF receptor. Main Outcome Measure(s): Immunostaining for TGF-α, EGF, and EGF receptor in follicles of fresh and frozen ovarian tissues. Result(s): Immunoreactivities for TGF-α and EGF receptor were observed simultaneously in the oocytes of primordial, primary, preantral, and antral follicles. Strong staining for TGF-α and EGF receptor was present in thecal cells. The TGF-α and EGF receptor was also expressed in some granulosa cells of primary to antral follicles. The EGF only stained weakly in the oocytes of primordial and primary follicles and in thecal cells. There was no difference in staining patterns for TGF-α, EGF, and EGF receptor between fresh and frozen ovarian tissues. Conclusion(s): The TGF-α and EGF receptor was expressed in primordial to antral follicles, indicating a role of TGF-α in regulating follicular development through binding to the EGF receptor. Freeze-thawing did not substantially alter immunoreactivites for TGF-α, EGF, and EGF receptor in frozen ovarian tissue.

Transforming growth factor-alpha, epidermal growth factor receptor, and PCNA immunoexpression in uterine leiomyosarcomas and leiomyomas in B6C3F1 mice

The role of growth factors in the development of murine uterine mesenchymal tumors is unknown. In this study, immunohistochemical expression of transforming growth factor alpha (TGF-alpha) and its receptor epidermal growth factor (EGF-R) was assessed in spontaneous uterine leiomyomas and leiomyosarcomas in B6C3F1 mice. Cell proliferation, which has been induced by some growth factors, was evaluated by immunohistochemical detection of an endogenous marker of cell proliferation, proliferating cell nuclear antigen (PCNA). PCNA labeling indices were determined and compared to the intensity and distribution of TGF-alpha staining in sequential sections of control myometrium or tumor tissue. Results showed uterine leiomyosarcomas had positive cytoplasmic staining for TGF-alpha; however, all uterine leiomyomas evaluated were negative. Positive EGF-R staining was also observed in the uterine leiomyosarcomas, but not in the leiomyomas. EGF-R immunoexpression was detected primarily within the cytoplasm of the leiomyosarcoma cells, with occasional nuclear immunoreactivity. Immunohistochemical staining for PCNA was more intense and there were increased numbers of positively staining nuclei in the leiomyosarcomas compared to samples of control myometrium or leiomyomas. The mean labeling index for the uterine leiomyosarcomas (7.40%) was significantly (p < 0.01) higher than that of leiomyomas (0.29%) and control uterine myometrium (0.13%). We conclude, that TGF-alpha and its receptor, EGF-R, are expressed more intensely in uterine leiomyosarcomas, compared to leiomyomas in B6C3F1 mice. Immunoexpression of TGF-alpha may be an important biomarker of malignancy in uterine smooth muscle tumors in mice. Futhermore, TGF-alpha may play a critical role in increased proliferation of uterine smooth muscle tumor cells as suggested by increased immunolocalization of PCNA in rodent leiomyosarcomas expressing TGF-alpha, although other factors regulating cell replication can not be ruled out.