P. Peña

Alpha-glucosidase in the human epididymis: topographic distribution and clinical application

Summary.  α‐Glucosidase activity (EC.3.2.1.20) is present in human seminal plasma, and the neutral form of the enzyme originates almost exclusively from the epididymis. In this study, the specific immunocytochemical location of α‐glucosidase in the human epididymis was evaluated using a polyclonal antibody. Furthermore, a spectrophotometric assay was employed to assess epididymal obstruction in infertile patients. The enzymatic activity of α‐glucosidase free of prostate isoform (AGFPI) was determined spectrophotometrically at 405 nm. According to AGFPI activity, patients with leucocytospermia, oligozoospermia and azoospermia were recorded as having normal values or low values indicating epididymal obstruction. Specific immunochemistry staining was demonstrated in the cytoplasmic cells at the epithelial level, in the transition area and in the efferent ducts. The values of the three groups and the control were as follows (mean ± SEM): normozoospermia (control): 20.2 ± 1.4 mU ml−1; azoospermia: normal value: 17.6 ± 2.2 mU ml−1, low value: 7.4 ± 1.8 mU ml−1; oligozoospermia: normal value: 22.3 ± 2.5 mU ml−1, low value: 7.3 ± 0.7 mU ml−1; leucocytospermia: increase value: 38.9 ± 3.7 mU ml−1, low value: 11.1 ±1.3 mU ml−1. This study suggests that determination of α‐glucosidase might be helpful to evaluate functions of the epididymis and particularly to exclude epididymal obstruction.

Migration/sedimentation sperm selection method used in bovine in vitro fertilization: Comparison with washing/centrifugation

Sperm selection methods are usually considered for in vitro fertilization (IVF) programs. To achieve a population of viable, motile and morphologically normal spermatozoa, seminal plasma and contaminants must be removed. In this study 2 sperm selection methods were compared with regard to conventional parameters: 1) repeated washing/centrifugation, and 2) migration/sedimentation. Their effect on the fertilization of oocytes aspired from ovaries of slaughtered cows was determined. Frozen-thawed semen was used for all assays (n = 8). The sperm concentration was adjusted to 1.0 × 106 cells/ml for in vitro insemination. Selected oocytecumulus complexes were matured in vitro for 24 h and were co-incubated with spermatozoa prepared by the 2 different methods. Presumptive zygotes were co-cultered in TCM-199. The percentage of motile, viable and morphologically normal spermatozoa was markedly higher (p < 0.05) with the migration-sedimentation method. Triple stain showed that the integrity of the acrosome was significantly better maintained after migration/sedimentation (68.3%) than after washing/centrifugation (53.2%; p < 0.05). In accordance with these results, a high fertilisation rate was achieved after application of the migration/sedimentation method (92.5 vs 69.1%;p < 0.05). It is concluded, that this method is very promising for obtaining spermatozoa with optimal fertilization capacity.

Evidence for the synthesis and secretion of a CBG-like serpin by human cumulus oophorus and fallopian tubes.

Summary The acrosome reaction (AR)‐inducing effect of follicular cells, like that of the cumulus oophorus and granulosa cells, has been described previously. In addition to the well known steroid secreting activity of cumulus cells, the results obtained here demonstrate the secretion of a corticosteroid‐binding globulin (CBG)‐like protein. An AR‐inducing effect was shown with the culture medium of human cumulus oophorus. This effect could be eliminated by treating the sample with monoclonal antibodies against CBG. Moreover, Western blotting after SDS‐PAGE of the culture medium strongly indicates that human cumulus cells actively express and secrete a CBG‐like protein. This might give an indication as to the origin of the acrosome reaction‐inducing substance found in follicular fluid. Furthermore, AR‐inducing activity and the elimination of this activity by antibodies against CBG was shown for oviductal fluid. With immunohistochemical techniques the CBG‐like protein was localized in the epithelial lining of the fallopian tubes, giving possible evidence for the involvement of this molecule in fallopian tube function.

Evaluation of the acrosomal membrane in bovine spermatozoa: effects of proteinase inhibitors.

Thawed bovine spermatozoa are characterized by a lack of homogeneity in the acrosomal membrane. Therefore, it is difficult to visualize the acrosome to assess morphology. Synthetic proteinase inhibitors were tested on thawed bovine semen for their effect on the integrity of acrosomal membranes. The proteinase inhibitors 4-nitrophenyl-4-guanidinobenzoate (NPGB) and N-L-p-tosyl-L-lysine-chloromethylketone (TLCK) were added to a medium containing spermatozoa separated on a percoll gradient. After incubation for 30 min at 38 degrees C in 5% CO(2), 95% air (final concentration 1 mM), the action of these inhibitors was controlled by measuring the activity of acrosome proteinases. The acrosomal membrane was evaluated by means of a dual stain procedure (trypan blue, Giemsa). In contrast to spermatozoa that had been incubated with proteinase inhibitor-free solution, samples that had been incubated with TLCK showed homogeneity in 90% of the acrosomal membranes and excellent visualization of the acrosome itself; in the NPGB-treated samples, homogeneous staining was observed in 83% of spermatozoa (P < 0.0005). It is concluded that alteration of the acrosomal membrane in thawed semen is not directly caused by freezing-thawing, but may be due to activation of acrosomal proteinases, which is increased during staining procedures. The addition of proteinase inhibitors before staining offers a new possibility for improved assessment of the acrosome in bovine spermatozoa.

Detection of a CBG-like protein in human Fallopian tube tissue*

The acrosome reaction (AR), a modified exocytotic process, is prerequisite for successful mammalian fertilization. The protein component that is responsible for the AR‐inducing activity of human follicular fluid, has been found to be immunologically identical with corticosteroid‐binding globulin (CBG), which is well characterized and serves as a transport protein for progesterone and cortisol in the plasma. Our findings have shown that the CBG‐like protein is expressed by endothelial cells of the Fallopian tube depending on the hormonal cycle. In the culture medium of human epithelial tubal cells, the CBG‐like protein was detected by Western blot analysis. The protein was also found in biologically active form in human tubular fluid. Our investigations strongly indicate that human Fallopian tube cells actively express and secrete a CBG‐like progesterone‐binding protein, which might play a role in the in vivo modulation of human sperm AR.

Evaluation of the acrosome reaction in human spermatozoa: comparison of cytochemical and fluorescence techniques.

Summary.  The acrosome reaction, which is essential for fertilization, includes fusion and vesiculation of the plasma membrane with the outer acrosomal membrane of spermatozoa, thereby releasing the acrosomal content. Determination of the ability of spermatozoa to undergo the acrosome reaction has proved to be a useful parameter in evaluation of infertile patients. The objective of this study was to compare cytochemical techniques, such as double stain (Giemsa/trypan blue) and triple stain (Bismarck brown/rose bengal/trypan blue), with a fluorescence method using Pisum sativum agglutinin fluorescein conjugate and Hoechst dye N°33258 (double fluorescence). Whereas the cytochemical methods are easy to perform in general laboratories, the fluorescence technique requires special and costly instrumentation. In semen obtained from fertile donors, spermatozoa were selected by the swim‐up technique and the acrosome reaction was induced by incubation at low temperature. The percentages of vital and acrosome‐reacted spermatozoa were determined after incubation at 4 °C and at room temperature. No statistically significant difference was found between double fluorescence (viability 86.3%, acrosome reaction 14.7%) and triple stain (viability 85.3%, acrosome reaction 17%) (P > 0.05). On the other hand, the double stain technique showed different values for viability (70.3%) and acrosome reaction (42.5%) (P < 0.05). In conclusion, triple stain yielded results similar to those obtained by the fluorescence technique in evaluating the acrosome reaction and can therefore easily be used in general or research laboratories.

Determinación de células peroxidasa positivas en líquido seminal: ¿es un parámetro confiable para el diagnóstico de infección genital asintomática?

Background: The presence of leukocytes, detected by peroxidase test in semen, can be a good indicator of infections in the male genital tract. Peroxidase positive cells have been positively correlated with elevated values of elastase, one of the major proteases liberated by granulocytes at the inflammation place. However, seminal granulocytes may not be adequately detected by the peroxidase test in comparison with immunological methods. Aim: To correlate the determination of peroxidase positive cells with the elastase level in the seminal plasma. Material and methods: Seminal plasma from 64 patients with a high number of round cells (>106/ml) in semen, was studied. Correlation analysis was done using the Pearson correlation coefficient. Results: No correlation between the level of granulocyte elastase and the number of peroxidase positive cells (r=0.2237, p >0.05), or even the number of round cells (r=0.03934, p >0.05) was observed. Conclusions: Our results suggest that the determination of peroxidase positive cells is not a reliable indicator of leukocytes in the seminal plasma and their absence do not discard a silent genital tract infection (Rev Med Chile 2003; 131: 613-616)

Endometrio con reacción decidual idiopática en una mujer postmenopáusica: evaluación inmunocitoquímica de receptores de estrógeno, de progesterona y de su proteína transportadora (CBG)

Endometrial decidual reaction in postmenopausal women without an evident exogenous or endogenous progesterone stimulus, is uncommon. The morphological findings are decidual transformation of the endometrial stroma, necrosis, cellular pleomorphism and in some cases hyperplastic glands that exhibit focal atypia raising the possibility of malignancy. We report the case of a 55 years old woman with an idiopathic endometrial decidual reaction. In this woman, immunocytochemistry of endometrial tissue demonstrated the absence of cytoplasmatic and nuclear estrogen receptors. Staining of nuclear progesterone receptors and cytoplasmatic corticoid binding globulin, exceeded the immunoreactivity observed in any period of the menstrual cycle (Rev Med Chile 2002; 130: 425-429)

LOCALIZACION INMUNOCITOQUIMICA DE LA SERPINA CBG-símil EN EL SISTEMA REPRODUCTOR DE MAMÍFEROS HEMBRA

La participacion de la globulina que une corticoides (CBG) en el proceso de la induccion de la reaccion acrosomal, ha sido claramente demostrada in vitro en espermatozoides humanos. Esta molecula fue aislada primariamente desde fluido folicular y su presencia, detectada inmunologicamente, ha sido demostrada por nosotros en foliculos ovaricos, epitelio tubular uterino y en endometrio de humanos y bovinos. Estos resultados nos llevaron a ampliar el estudio de la probable presencia de esta molecula en otras especies mamiferas, con la hipotesis de que si esta involucrada en mecanismos previos a la fecundacion en humanos y bovinos, su presencia podria estar tambien jugando un rol modulatorio en especies con procesos de interaccion gametica basicamente comparables. El objetivo del presente trabajo fue determinar inmunocitoquimicamente la presencia y distribucion de la CBG-simil en el sistema reproductor de cerdos, perros, gatos, conejos y ratas. Las muestras fueron obtenidas, en los distintos estadios del ciclo reproductivo, desde piezas quirurgicas, a excepcion de las de cerdo que se obtuvieron desde un matadero local. Muestras de ovario, tuba uterina y utero fueron procesadas para la inmunocitoquimica (ICQ) con anticuerpos (Ac) poli y monoclonales anti hCBG que reconocieron antigenos comunes dentro de las distintas especies animales. En todas ellas la ICQ revelo una intensa reaccion positiva a nivel de los foliculos ovaricos, en las celulas secretoras del epitelio de la mucosa tubarica y en las celulas epiteliales de las glandulas y de la mucosa endometrial. En general, la inmuno tincion se manifesto muy intensa hacia el periodo ovulatorio y muy escasa en los periodos de niveles esteroidales bajos. Sin embargo, existieron diferencias particulares entre los distintos animales debido, probablemente, a la variacion antigenica por la lejania de la especie con la molecula generadora del Ac. Muy interesante resulto el estudio del animal en el cual se produjo el Ac policlonal, este confirmo la presencia de la CBG endogena, determinada por una intensa inmunorreactividad en los preparados controles (sin Ac especifico), y que por tanto, la unica fuente de ellos fue la proveniente de la generacion de sus propios Ac. Nuestros resultados nos permiten sugerir que en las especies estudiadas existe CBG-simil, la cual se distribuye en areas morfologicas del sistema reproductor, similares a las observadas en humanos y en bovinos, lo cual puede estar indicando que esta molecula tambien podria estar involucrada en sus correspondientes procesos reproductivos. Sin embargo, su directa participacion en mecanismos fisiologicos, como por ejemplo la Reaccion Acrosomal, hasta el presente, no ha sido demostrada

TERATOMA DERMOIDE QUÍSTICO CÓRNEO-CONJUNTIVO EN UN PERRO OVEJERO ALEMÁN (Canis familiaris): CASO CLÍNICO

El teratoma dermoide quistico (TDQ) es una neoplasia proveniente de celulas totipotenciales, que han quedado secuestradas en regiones remotas, y cuya expresion dara origen a tejidos ectopicos. Su localizacion es variada y, a nivel del bulbo ocular, es muy poco frecuente. Su importancia clinica radica en que cuando afecta al bulbo ocular, produce irritacion cronica y dificultades visuales severas que obligan su pronta extirpacion. Los dermoides localizados a nivel del ojo y estructuras adyacentes, pueden afectar tanto a animales como al Hombre, y por lo general se alojan en el limbo, tercer parpado, cornea, en parte sobre la conjuntiva, y, en casos raros, con extension hacia el tegumento palpebral (JENSEN, 1971; WALDE et al., 1990; SLATTER, 1992; MOORE, 1999; STADES et al., 1999). El objetivo del presente trabajo fue presentar los analisis clinico e histopatologico de un caso de TDQ, de localizacion corneo conjuntival, en un canino Ovejero Aleman. La extraccion del tumor se realizo por reseccion quirurgica, segun tecnica descrita por KASSEN & HASSIEB (1986) y modificada por SLATTER, 1992 y, posteriormente, por STADES et al.; cirugia con la cual el animal recupero totalmente la vision. El estudio histopatologico del tejido extraido arrojo el diagnostico de teratoma dermoide quistico corneo conjuntival. Esta neoplasia benigna presenta generalmente tejidos derivados del ectodermo (piel y anexos), como fue el presente caso, pero, ademas de la expresion de piel con todos sus componentes, se observo la presencia de cartilago hialino y fibras musculares lisas, que son elementos de origen mesodermico. El particular origen embrionario del TDQ del presente caso, su desarrollo y localizacion corneo conjuntival, la especie animal y su escasa frecuencia de aparicion, unido al exito de la tecnica quirurgica y la recuperacion total del paciente, entre otras, hacen al presente reporte de particular interes