Interaction between FGF2 and ER in breast cancer

La adquisición de independencia hormonal en carcinomas mamarios con expresión de receptores para hormonas esteroides es un fenómeno frecuente con gran relevancia clínica. En este trabajo, abordamos el estudio de un posible mecanismo de activación ligando independiente del receptor de estrógenos alfa (REα). Nuestra hipótesis se desprende de observaciones anteriores del laboratorio en las cuales observamos que el factor de crecimiento fibroblástico 2 (FGF2), podía activar al receptor de progesterona (RP) a través del receptor para dicho factor (FGFR-2) en el modelo experimental murino C4 y en células de cáncer de mama humano T47D. Por otro lado, habíamos comprobado que el REα era necesario para que el RP fuera transcripcionalmente activo. Con estas evidencias decidimos investigar si existía un interacción entre la vía de FGF2 y el REα. Se eligieron para este trabajo las líneas celulares de cáncer de mama humano MCF-7, por ser la línea más frecuentemente utilizada para evaluar la funcionalidad del REα, y la T47D, más utilizada para evaluar la función del RP, aunque también se estimulan con estrógenos. Al tratar ambas líneas celulares con FGF2, encontramos un aumento en su proliferación, que fue revertido tanto con inhibidores de FGFRs, como con el antiestrógeno puro ICI 182780 (ICI). Dicho incremento estaba acompañado por un aumento de la fosforilación de las serinas 118 y 167 del REα en correlación con un aumento de la activación de las quinasas responsables de fosforilar a estas serinas, MAPK y Akt respectivamente. Más aún, experimentos de silenciamiento del REα confirmaron que el mismo estaba involucrado en el efecto estimulatorio de la proliferación celular del FGF2. Con estos resultados, exploramos posteriormente la funcionalidad del REα en respuesta a FGF2, demostrando que el REα era capaz de inducir la expresión de un gen reportero luego del tratamiento con FGF2 y que no sólo era capaz de interactuar con su secuencia consenso en una construcción de ADN recombinante, sino que también se unía a esta secuencia en el ADN endógeno, induciendo la expresión de c-MYC y pS2. En particular la expresión de este último gen es considerado un indicador confiable de la activación del REα. Luego de evaluar la interacción de las vías de FGF2 y REα en células MCF-7 y T47D, desarrollamos un modelo con activación constitutiva del FGFR-2 al que denominamos MCF-7 R2CA (R2CA). Estas células fueron seleccionadas hasta obtener una transfección estable, y la línea resultante presentó una morfología similar a las células control. La línea R2CA expresó mayores niveles de MAPKs fosforiladas y al evaluar la fosfoserina 118 del REα, si bien se vio un aumento de su proporción con respecto al control de carga, al relativizarlo con los niveles de REα totales no se evidenció un aumento específico ya que las células R2CA expresaron mayores niveles de REα total. Sin embargo, el efecto final resultó en una mayor activación de esta vía corroborada además, no sólo por la inducción del RP en condiciones basales, sino por un aumento en la expresión del gen pS2, marcador de actividad transcripcional del REα. Curiosamente, no se observaron modificaciones en pAkt lo que sugiere que otros FGFR estarían más asociados a la activación de Akt por FGF2, mientras que FGFR-2 involucraría a Erk. Evaluamos también el crecimiento in vivo del modelo R2CA en comparación con el control que no crece sin el aporte hormonal exógeno. Demostramos que la activación constitutiva del FGFR-2 le confiere una ventaja proliferativa sobre las células control ya que se pudieron establecer como xenoinjertos aunque su crecimiento fue mayor con el tratamiento con estrógenos, resultados que se condicen con los estudios in vitro en los cuales se observó que se activa la vía de Erk pero no la de Akt. Posteriormente, se evaluó la relación entre las vías de REα y FGF2 en un modelo murino de cáncer de mama. En nuestro laboratorio se desarrolló un modelo de tumores por administración prolongada de acetato de medroxiprogesterona (MPA) en hembras vírgenes de ratones BALB/c. Se establecieron líneas que necesitan la administración de MPA para crecer, denominadas hormono-dependientes, entre ellas la C4-HD, variantes que no necesitan la hormona para crecer (hormono-independientes) como la variante C4-HI y variantes independientes de hormonas y resistentes a antiprogestágenos (hormono-independientes resistentes) como la C4-HIR. Lo interesante de este modelo es que mientras en el tumor C4-HD el tratamiento con progestágeno activa al REα y es completamente sensible al tratamiento con antiestrógenos, la variante C4-HI muestra una activación basal del REα medida por fosforilación en las serinas 118 y 167, y es moderadamente sensible al tratamiento con el antiestrógeno Fulvestrant. En concordancia, cuando tratamos a los tumores con los inhibidores específicos de los FGFRs los tumores son parcialmente sensibles y los tumores tratados muestran una disminución en la fosforilación de las dos serinas mencionadas del REα, indicando una interacción cruzada de estas vías en el modelo. Otra área de interés en el laboratorio es la regulación epigenética en relación con la adquisición de hormono independencia en cáncer de mama. En trabajos previos de nuestro grupo se estudió el rol de la metilación en los mecanismos de resistencia a antiprogestágenos. En este trabajo de tesis decidimos estudiar el papel de los miRNAs (miRNAs) en la progresión hacia la resistencia endócrina. Se analizó la expresión de más de 300 miRNAs en un microarray, para las tres variantes mencionadas de la familia C4. Como contábamos con la información del transcriptoma de C4-HD y C4-HI, decidimos cruzar los datos con los obtenidos del array de miRNAs. Analizamos todos los genes potencialmente regulados por algún miRNA sobre o sub expresado en cada comparación, y evaluamos cómo era su expresión en el transcriptoma conocido. De los genes obtenidos, se destacaban tanto FGFR-2 como STAT5. En particular STAT5 es un gen que codifica para una proteína que ya habíamos estudiado en el modelo experimental y podría formar parte de un complejo con FGFR-2 y mediar efectos transcripcionales a través de RP. Encontramos también como candidato interesante a PTEN, una fosfatasa involucrada en la vía de señalización de Akt, que tiene relevancia en la activación de REα ligando independiente. Nos centramos en 5 miRNAs, de los cuales el miR-1 se destaca por ser el único sobre expresado en C4-HIR al compararlo con C4-HI, aunque no se pudo validar; los miRNAs 335 y 222, para los cuales no observamos diferencias significativas entre los tres tipos de tumores; y los miRNAs 31 y 155, los cuales aumentaban su expresión en las distintas variantes tumorales con estadios más avanzados de progresión tumoral: C4-HI mostró mayores niveles que C4-HD y C4-HIR que C4-HI (p<0.001). Ambos miRNAs se encuentran involucrados en la vía de señalización de FGF2 sugiriendo su participación en la progresión hacia la resistencia hormonal en este modelo, a través de la regulación de esta vía. Estos resultados también sugieren que la expresión de estos miRNAs podría ser utilizada como marcador de progresión tumoral. En conclusión FGF2 es capaz de activar tanto a REα como a RP en forma ligando independiente y la activación de esta vía de señalización participaría en la progresión del cáncer de mama.The acquisition of hormone independence in breast cancer has a wide implication in patient therapeutics. In this thesis, we studied a possible mechanism of ligand-independent activation of the estrogen receptor alpha (ERα). Our hypothesis is based on previous observations, mainly from our laboratory, in which it was determined that Fibroblast Growth Factor 2 (FGF2) could activate the progesterone receptor (PR), through FGFR-2, in the C4 murine breast cancer model and in human breast cancer T47D cells. On the other hand, we found that ERα was necessary to mediate PR transcriptional activity. With this evidence we decided to investigate whether there was an interaction between FGF2 and ERα signaling pathways. In this PhD thesis we used the emblematic cell line used to study ERα responses in breast cancer, the MCF-7 cells, as well as the T47D cells, which although estrogen responsive, their proliferation is triggered mainly by the PR. An increase in the proliferation rate was observed in both cell lines when treated with FGF2 that was reversed by incubation with the pure antiestrogen ICI182.780 (ICI) or by two FGFRs inhibitors, PD173074 and BGJ398. Next, we measured the phosphorylation status of the ERα, and found increased phosphorylation of serines 118 and 167, which correlated with increased MAPK and Akt activation, the kinases responsible for the phosphorylation of the mentioned sites respectively. Moreover, ERα silencing experiments confirmed that this receptor was involved in FGF2-induced cell proliferation, at more extent in MCF-7 than in T47D cells. We then explored the functionality of the ERα in response to FGF2. We found that ERα was not only able to induce a reporter gene expression upon FGF2 treatment, but it was also able to interact with the native sequence in the DNA, inducing the expression of both c-MYC and pS2, being the latter gene considered as a trade mark of gene expression in response to estrogen receptor activation. Then, we stably transfected MCF-7 cells with a constitutive activated FGFR-2. These cells, named R2CA MCF-7 (R2CA), express higher levels of phosphorylated MAPKs in basal conditions than the control counterpart, showing no changes in Akt activation. Accordingly, an increase in total serine 118 pERα was observed which correlated with an increase in PR and pS2 expression. The fact that FGF2 activates both Akt and Erk pathways suggests that other FGF receptors in addition to FGFR-2 participate in FGF2-mediated actions in this model. We evaluated the ability of R2CA cells to bypass estrogen dependency in vivo. The constitutive activation of FGFR-2 gave these cells a proliferative advantage over control cells which do not grow without estrogen supply. Tumors were established although they reached a small size that did not increase with time unless estrogen was administered. These results are in agreement with the previous data suggesting that activation of FGFR-2 partially mimics FGF2 effects on cell signaling. In our laboratory we have developed a murine mammary tumor model by prolonged administration of medroxyprogesterone acetate (MPA) in female virgin BALB/c mice. These tumors originally needed MPA administration to grow and were considered as hormone-dependent being C4-HD the prototype tumor of this class. Sporadically, hormone-independent variants arise, as C4-HI, that do not need exogenous administration of hormones to grow but they still respond to antiprogestin treatment. Upon selective pressure induced by antiprogestin treatment, we were able to develop resistant variants such as C4-HIR. Interestingly, serine 118/167 pERα is highly expressed in hormone-independent tumors which partially respond to fulvestrant (antiestrogen) or to FGFR inhibitors, being the latter able to inhibit pERα expression, indicating an interaction between these pathways in the murine model. Another area of interest in our Laboratory is the epigenetic regulation in relation to the acquisition of hormone resistance. In this thesis we encouraged the study of the role of miRNAs that might be associated with the FGF2 pathway. The expression of more than 300 miRNAs was analyzed in a microarray for the three mentioned variants of the C4 tumors. From this analysis all differentially expressed miRNAs were obtained in all possible comparisons between tumor variants. As we already had information about the transcriptome of C4-HD and C4-HI, we decided to cross the data with that obtained from the miRNA array. We examined all genes potentially regulated by a miRNA over- or under-expressed in each comparison, and their expression was evaluated in the known transcriptome. Two genes that stood out were FGFR-2 and STAT5. In particular, STAT5 was already studied in C4 tumors as being part of a complex with FGFR-2 mediating the transcriptional effects of PR. PTEN phosphatase, was also found as an interesting candidate involved in the Akt signaling pathway, which is relevant in the ligand independent ERα activation. We focused in five miRNAs to further assess their relevance to mechanisms linked to tumor progression. miR-1 was the only overexpressed miRNA in C4-HIR when compared to C4-HI in the array, however we were unable to validate this data. miRNAs 335 and 222 were similarly expressed in all tumors and miRNAs 31 and 155, which are possible FGF2-regulated miRNAs, both showed increased expression in C4-HI as compared with C4-HD and in C4-HIR as compared with C4-HI, a model that represents different stages of tumor progression. These results are in agreement with those which suggest a role for FGF2 pathway in the acquisition of hormone-independence and endocrine resistance. These results also indicate that these miRNAs might also be used as markers of tumor progression in breast cancer. In conclusion, FGF2 activates ERα, as well as PR, suggesting that alterations in key members of the FGF signaling pathway may be participating in the onset of hormone independence and/or resistance.Fil: Guillardoy, Tomás. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Crescimento do câncer de mama receptor de hormônio dependente: contribuições de um modelo experimental

Si bien los estrógenos han demostrado tener un rol protagónico en el cáncer de mama, hoy se sabe que la progesterona, así como sus derivados sintéticos, ejercen roles proliferativos tanto en la glándula mamaria normal como neoplásica. Utilizando un modelo experimental de cáncer mamario murino, iniciado en la Academia Nacional de Medicina y trasladado al IBYME en el año 1995, demostramos la importancia de los fibroblastos asociados a tumor en la provisión de factores de crecimiento que activan en forma ligando-independiente a los receptores de progesterona (RPs) en las células tumorales. En este artículo mencionamos la importancia por un lado de la proporción de isoformas del RP en la determinación de la respuesta al tratamiento hormonal, y por otro, los mecanismos por los cuales el factor de crecimiento fibroblástico 2 (FGF2) estromal participaría en el crecimiento tumoral imitando la acción de la hormonaIt is well known that estrogens are key players regulating breast cancer growth. In addition, there is compelling evidence pointing out that progestins induce proliferative effects in normal and in neoplastic mammary glands. Using a murine breast cancer model, first developed in the National Academy of Medicine in Buenos Aires, and then moved to the IBYME in 1995, we demonstrated that carcinoma associated fibroblasts provide growth factors such as fibroblast growth factor 2 (FGF2), which are involved in the ligand independent activation of progesterone receptors (PR) of tumor cells. This article briefly describes, on one hand, the relevance of the evaluation of the PR isoform ratio to predict hormone responsiveness, and on the other hand, the mechanisms by which stromal FGF2 mimics the effects of progesterone to stimulate tumor growth.Embora os estrógenos tenham demonstrado ter um papel protagônico no câncer de mama, hoje se sabe que a progesterona, bem como seus derivados sintéticos, exerce papéis proliferativos tanto na glândula mamária normal quanto neoplá- sica. Utilizando um modelo experimental de câncer mamário murino, iniciado na Academia Nacional de Medicina e trasladado ao IBYME no ano 1995, demonstramos a importância dos fibroblastos associados a tumor na provisão de fatores de crescimento que ativam em forma ligando-independente os receptores de progesterona (RPs) nas células tumorais. Neste artigo mencionamos a importância, de um lado, da proporção de isoformas do RP na determinação da resposta ao tratamento hormonal, e do outro, dos mecanismos pelos quais o fator de crescimento fibroblástico 2 (FGF2) estromal participaria no crescimento tumoral imitando a ação do hormônio.Fil: Lanari, Claudia Lee Malvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Novaro, Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Lamb, Caroline Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Fabris, Victoria Teresa. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Rojas, Paola Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Giulianelli, Sebastian Jesus. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Gorostiaga, Maria Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Wargon, Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Guillardoy, Tomás. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Polo, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Riggio, Marina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Sequeira, Gonzalo Ricardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Sahores, Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Pampena, María Betina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentin

Interaction between FGFR-2, STAT5, and progesterone receptors in breast cancer

Fibroblast growth factor (FGF) receptor 2 (FGFR-2) polymorphisms have been associated with an increase in estrogen receptor and progesterone receptor (PR)-positive breast cancer risk; however, a clear mechanistic association between FGFR-2 and steroid hormone receptors remains elusive. In previous works, we have shown a cross talk between FGF2 and progestins in mouse mammary carcinomas. To investigate the mechanisms underlying these interactions and to validate our findings in a human setting, we have used T47D human breast cancer cells and human cancer tissue samples. We showed that medroxyprogesterone acetate (MPA) and FGF2 induced cell proliferation and activation of ERK, AKT, and STAT5 in T47D and in murine C4-HI cells. Nuclear interaction between PR, FGFR-2, and STAT5 after MPA and FGF2 treatment was also showed by confocal microscopy and immunoprecipitation. This effect was associated with increased transcription of PRE and/or GAS reporter genes, and of PR/STAT5-regulated genes and proteins. Two antiprogestins and the FGFR inhibitor PD173074, specifically blocked the effects induced by FGF2 or MPA respectively. The presence of PR/FGFR-2/ STAT5 complexes bound to the PRE probe was corroborated by using NoShift transcription and chromatin immunoprecipitation of the MYC promoter. Additionally, we showed that T47D cells stably transfected with constitutively active FGFR-2 gave rise to invasive carcinomas when transplanted into NOD/SCID mice. Nuclear colocalization between PR and FGFR-2/STAT5 was also observed in human breast cancer tissues. This study represents the first demonstration of a nuclear interaction between FGFR-2 and STAT5, as PR coactivators at the DNA progesterone responsive elements, suggesting that FGFRs are valid therapeutic targets for human breast cancer treatment. ©2011 AACR.Fil: Cerliani, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Guillardoy, Tomás. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Giulianelli, Sebastian Jesus. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Vaque, José P.. National Institutes of Health; Estados UnidosFil: Gutkind JS. National Institutes of Health; Estados UnidosFil: Vanzulli, Silvia. Academia Nacional de Medicina de Buenos Aires; ArgentinaFil: Martins, Rubén. Clínica Bancaria; ArgentinaFil: Zeitlin, Eduardo. Clínica Bancaria; ArgentinaFil: Lamb, Caroline Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Lanari, Claudia Lee Malvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

Fibroblast Growth Factor Receptors (FGFRs) in Human Sperm: Expression, Functionality and Involvement in Motility Regulation.

Fibroblast growth factors receptors (FGFRs) have been widely characterized in somatic cells, but there is scarce evidence of their expression and function in mammalian gametes. The objective of the present study was to evaluate the expression of FGFRs in human male germ cells, to determine sperm FGFR activation by the FGF2 ligand and their participation in the regulation of sperm motility. The expression of FGFR1, 2, 3 and 4 mRNAs and proteins in human testis and localization of these receptors in germ cells of the seminiferous epithelium was demonstrated. In ejaculated sperm, FGFRs were localized to the acrosomal region and flagellum. Sperm exposure to FGF2 caused an increase in flagellar FGFR phosphorylation and activation of extracellular signal-regulated kinase (ERK) and protein kinase B (PKB or Akt) signaling pathways. Incubation with FGF2 led to a significant increase in the percentage of total and progressive sperm motility, as well as in sperm kinematics. All responses were prevented by sperm preincubation with BGJ398, a specific inhibitor of FGFR tyrosine kinase activity. In addition to confirming the expression of FGFRs in germ cells of the human testis, our study describes for the first time the presence, localization and functionality of human sperm FGFRs, and provides evidence of the beneficial effect of FGF2 upon sperm motility

Localization of FGFRs in human seminiferous epithelium.

<p>Immunohistochemical analysis of FGFRs in human testis using anti FGFR antibodies; rabbit IgG was included as control. The specimens were counterstained with hematoxylin. S: Sertoli cell, Sg: spermatogonia, Sc: spermatocyte, St: spermatid. Arrows indicate immunoreactivity for FGFRs in the flagellum of elongating/elongated spermatids and the arrow head indicates FGFR4 immunoreactivity in spermatid acrosome. Bar: 20 μm.</p

Activation of FGFR-related intracellular pathways in sperm exposed to FGF2.

<p>Sperm were incubated for a total 4-h period and exposed to FGF2 (0, 1, 10 and 100 ng/ml) for the last 15 min. In some aliquots, sperm were incubated for 15 min with BGJ398 (0.1 μM) before the addition of FGF2. (<b>A</b>) Immunolocalization of pERK and pAkt in sperm incubated in the absence of FGF2 (Control), with 100 ng/ml FGF2, and with BGJ398 + 100 ng/ml FGF2. Sperm were processed for immunocytochemistry, stained with anti pERK or pAkt and FITC-conjugated secondary antibodies; nuclei were stained with propidium iodide. Bar: 10 μm. (<b>B</b>) Percentage of sperm cells stained with anti pERK and anti pAkt after exposure to FGF2 in the absence (<b>left</b>) or presence of BGJ398 (<b>right</b>). Results are expressed as mean ± SEM, n = 4. * <i>P</i> < 0.05; ** <i>P</i> < 0.01 compared with Control. (<b>C</b>) Phosphorylation of ERK and Akt assessed by Western immunoblotting. Protein extracts from human sperm were subjected to SDS-PAGE and Western immunoblotting using anti pERK, ERK, pAkt and Akt antibodies. The estimated molecular weights of the protein bands are indicated on the right. (<b>D</b>) Densitometric analysis of Western immunoblotting results for pERK normalized to ERK and pAkt normalized to Akt. Results are expressed as mean ± SEM, n = 5 for ERK and n = 5 for Akt. * <i>P</i> < 0.05 and ** <i>P</i> < 0.01 compared with Control.</p

Localization of FGFRs in human sperm.

<p>Sperm cells were stained with anti FGFR1, FGFR2, FGFR3 and FGFR4 or rabbit IgG and a secondary antibody labeled with Cy3. The corresponding fields stained with FITC-PSA to assess acrosomal status are shown. Bar: 10 μm. On the right, a representative image of individual sperm is depicted; (<b>A</b>) sperm stained with anti FGFR antibody and Cy3-conjugated secondary antibody, (<b>B</b>) FITC-PSA, (<b>C</b>) merge.</p

Effect of sperm incubation with FGF2 on sperm motility.

<p>Sperm were incubated with 0, 10 and 100 ng/ml FGF2 in the absence or in the presence of 0.1 μM BGJ398 and subjected to computer-assisted sperm analysis. (<b>A</b>) Percentages of progressive (Grade a + b) and total motility (Grade a + b + c) for aliquots incubated in the absence (<b>left</b>) or in the presence of BGJ398 (<b>right</b>). Results are expressed as mean ± SEM, n = 5. ** <i>P</i> < 0.01 compared with Control. (<b>B</b>) Individual recordings of the effect of sperm incubation with FGF2 (0, 10 and 100 ng/ml) on the percentage of total sperm motility in samples with low and high sperm motility. Each sample is identified with a different symbol (n = 12). (<b>C</b>) The percentages of sperm with Grade a, b, c and d motility in each condition (as defined in Materials and Methods) is depicted. * <i>P</i> < 0.05; ** <i>P</i> < 0.01 compared with the same Grade in Control (n = 12).</p

Expression of FGFRs in human testis and sperm.

<p>(<b>A</b>) Messenger RNA expression of testicular and sperm FGFRs assessed by RT-PCR. Messenger RNA extracted from MCF7 cells served as positive controls; negative controls without reverse transcriptase (RT Control) and without template (PCR Control) are shown. The amplicon sizes are indicated on the right. (<b>B</b>) Detection of testis and sperm FGFR protein forms using Western immunoblotting. Protein extracts from human testis and sperm were subjected to SDS-PAGE and Western immunoblotting using anti FGFR antibodies or rabbit IgG as control. The estimated molecular weights of the protein bands are indicated on the right. The experiments were performed at least 3 times obtaining similar results. Typical results are shown.</p