Progesterone sperm chemoattraction may be modulated by its corticosteroid-binding globulin carrier protein.

Progesterone, the main steroidal component secreted by the cumulus cells that surround the egg, chemotactically guides human spermatozoa. The aim of this work was to evaluate whether the carrier protein corticosteroid-binding globulin also participates in the sperm P chemotactic response. By means of videomicroscopy and image analysis, we observed that corticosteroid-binding globulin modulates the chemotactic activity of P, when a solution of corticosteroid-binding globulin + P is at the nanomolar range.

Molecular mechanism for human sperm chemotaxis mediated by progesterone.

Sperm chemotaxis is a chemical guiding mechanism that may orient spermatozoa to the egg surface. A picomolar concentration gradient of Progesterone (P), the main steroidal component secreted by the cumulus cells that surround the egg, attracts human spermatozoa. In order to elucidate the molecular mechanism of sperm chemotaxis mediated by P, we combine the application of different strategies: pharmacological inhibition of signaling molecules, measurements of the concentrations of second messengers and activation of the chemotactic signaling. Our data implicate a number of classic signal transduction pathways in the response and provide a model for the sequence of events, where the tmAC-cAMP-PKA pathway is activated first, followed by protein tyrosine phosphorylation (equatorial band and flagellum) and calcium mobilization (through IP(3)R and SOC channels), whereas the sGC-cGMP-PKG cascade, is activated later. These events lead to sperm orientation towards the source of the chemoattractant. The finding proposes a molecular mechanism which contributes to the understanding of the signal transduction pathway that takes place in a physiological process as chemotaxis.

Detección de receptores de estrógeno, progesterona y de proteína ligadora de corticosteroides en el tracto genital de hembras caninas (Cannis familiaris): estudio inmunohistoquímico / Detection of estrogen, progestorone receptors and corticosteroid binding globulin in the canine (Cannis familiaris) reproductive female tract: immunohistochemistry study

Los receptores esteroidales sexuales del tracto genital de la hembra tienen importancia dado que, a través de ellos, actúan las hormonas responsables de su desarrollo y de sus cambios morfofuncionales. En su mecanismo, uno de los factores a considerar son las posibles diferencias entre las distintas especies. El objetivo del presente estudio fue evaluar, en la especie canina (N=8), la presencia de receptores de estrógenos (RE), progesterona (RP) y la presencia de proteína ligadora de corticosteroides (CBG) en ovario, oviducto y útero de 3 hembras prepúberes (N=3) y 5 hembras adultas (N=5). La evaluación morfológica se realizó con tinción de hematoxilina-eosina (H-E) e inmunocitoquímica, según la técnica de Stemberger (1979). Los resultados revelaron en animales ciclando, inmunorreactividad (IR) positiva, RE en útero, oviducto y ovario siendo marcada en oviducto. La IR para RP fue leve y varió según el estadio del ciclo. En hembras prepúberes, los RE y RP no fueron evidenciados. La CBG mostró positividad en el tracto durante el ciclo y fue negativa en prepúberes. Se concluye que la presencia de RE y RP es detectable en útero, oviducto y ovario de la hembra canina adulta habiendo variaciones de concentración según el estadio del ciclo estral, siendo su presencia en las prepúberes no evidenciables, a diferencia de otras especies (oveja), donde son detectables en este estadio del desarrollo. La presencia de CBG constante durante el ciclo estral y variable en los otros estadios indica su posible participación en los procesos reproductivos.

The sexual steroid receptors of the genital tract of the female have significant importance since though them the hormones responsible acting. In their mechanism one of the factors to be kept in mind is the possible differences among the diverse species, the objective of this study was to evaluate in canine (N= 8) the presence of receptors of estrogens (ER), progesterone (PR) and the presence of corticosteroid binding globuline (CBG) in ovaries, oviduct and uterus of prepuberal female (N=.3) and matures: (N=5) It was used H-E and immunohistochemical study according to the technique of Stenberger (1979). The results during the oestrus showed: Immune reactivity (IR) positive, ER in uterus and ovary being much stronger in oviduct. The PR varied according the stage of oestrus cycle. In puberal female dog ER and PR were undetectable. The CBG revealed positive in reproductive tract of cycling females but negative in prepubertal. It was concluded that the presence of ER and PR are detectable in uterus, oviduct and ovary of mature female dogs varying their concentration according to the cycle. In prepuberal, its presences come undetectable differing from other species (ovine), in which they are detected at this stage of development. The constant of CBG presence during oeustrus cycle and such variation in other stages of the cycle, it might suggest its participation in the several reproductive activities.

Molecular glass wool filtration as a new tool for sperm preparation.

BACKGROUND: Magnetic-activated cell sorting (MACS) using annexin V-conjugated microbeads in a liquid phase eliminates apoptotic spermatozoa based on the externalization of phosphatidylserine (EPS) residues. The procedure allows the enrichment of a sperm population free of apoptosis markers, giving higher fertilization potential. Our aim was to determine if the annexin V binding principle can be transferred onto a glass wool filter system in order to produce a solid phase filter. METHODS: Semen samples (n = 42) were subjected to a molecular glass wool filter system using glass surfaces coated with annexin V and compared with aliquots separated by conventional glass wool, as well as with annexin V-MACS. The extent of apoptosis was assessed by measuring levels of activated caspase 3 using fluorescein-labelled inhibitors of caspase, alterations in mitochondrial membrane potential (MMP) using a lipophilic cationic dye, and EPS using a fluorescein isothiocyanate-coupled monoclonal antibody. RESULTS: Annexin V-negative sperm filtered out by the newly developed molecular glass wool filtration (GWF) system displayed superior quality in terms of high MMP integrity, as well as, to a small extent, caspase 3 activation and EPS. CONCLUSIONS: The effect of traditional GWF can be further improved by combination with annexin V binding. This newly developed solid phase molecular filter system has been proven to enrich spermatozoa free of apoptosis markers to the same extent as the annexin V magnetic separation technique. The selection of spermatozoa free of apoptosis markers by molecular glass wool filters may enhance the results of IVF.

Presencia de cbg (corticosteroid binding globulin) y receptores de estrógeno (fracción alfa) y progesterona en el sistema reproductor de ovejas en distintos estadios del ciclo reproductivo. Estudio inmunohistoquímico / Presence of CBG (Corticosteroid binding globulin) Estrogen Receptors Alpha and Progesterone Receptors Along the Reproductive Tract in Sheep During Different Stages of the Reproductive Cycle. Inmunohistochemestry Study

Las hormonas esteroidales sexuales actúan a través de sus receptores permitiendo el desarrollo del tracto genital y determinando su estado morfofuncional. El objetivo del presente estudio fue estudiar la presencia de CBG (corticosteroid binding globulin) y de los receptores de estrógenos alfa y progesterona en el útero, oviducto y ovario de ovejas cíclicas (n = 3), gestantes (n = 3), y en anestro (n = 3). La evaluación morfológica se realizó con tinción de hematoxilina - eosina (H-E) e inmunohistoquímica, según técnica de Sternberger. Los resultados revelaron para receptores de estrógenos en oveja cíclicas, inmunorreactividad positiva moderada en el epitelio glandular y estroma endometrial y en epitelio superficial y corión de la mucosa oviductal; siendo la inmunorreactividad leve en ovejas gestantes y anéstricas. Los receptores de progesterona mostraron en los 3 órganos inmunorreactividad positiva moderada en ovejas cíclicas e inmunorreactividad leve o negativa en animales acíclicos. La inmunorreactividad para CBG en endometrio fue marcada en ovejas cíclicas, y leves o negativa en gestantes y anéstricas. En ovario se observó en ovejas durante el ciclo, reacción inmunopositiva a la CBG en células foliculares, en el fluido folicular y en células estromales llenas de material granular inmunopositivo dispuestas en forma grupal o aislada. Se concluye que la presencia de los receptores de estrógenos alfa y de progesterona es siempre detectable en útero, oviducto y ovario variando su concentración según el estadio. La presencia de CBG fue constante en los tres órganos durante el ciclo estral y variable en los otros estadios, siendo su participación dentro del proceso reproductivo tema de próximos estudios.

The steroids hormones operate through their receptors in the development of the genital tract and determine it morphofuncional state. The objective of the present study was to investigate the presence of CBG (corticosteroid binding globulin), estrogens alpha and progesterone receptors in the uterus, oviduct and ovary of sheep during oestrous cycle (n = 3), pregnancy (n = 3), and in anoestrous (n = 3). Cross sections were stained with Hematoxylin-eosin and Inmunohistochemical (ICQ) study was made according to the technique of Sternberger. The results showed for estrogens receptors in sheep on cycle estral, moderate positive immunoreaction in the estroma and on the epithelial cells of endometrial glands and in oviductal superficial epithelium and estroma; the immunoreaction in pregnant and anestrics sheep was low. The progesterone receptors had in the 3 organs moderate immunorreactivity in cycling sheep and immunorreactivity low or negative in acycling animals. The CBG endometrial immunorreaction was high in cycling sheep and low or negative in pregnant and anestrics. The CBG in sheep during the cycle demonstrated a positive reaction at ovarian level in follicular cells, inside follicular liquid, and in stromal cells filled with granulate material disseminated in groups or isolated. In conclusion, the presence of estrogens alpha and progesterone receptors are always detectable in uterus, oviduct and ovary varying their concentration according to the stage The presence of CBG was constant in the three organs during the oestral cycle and variable in the other stages, its participation in the reproductive process is topic for next studies.

Progesterone at the picomolar range is a chemoattractant for mammalian spermatozoa.

By means of a videomicroscopy system and a computer image analysis, we performed chemotaxis assays to detect true chemotaxis in human spermatozoa, in parallel to immunohistochemistry detection of progesterone inside the cumulus cells. Progesterone indeed chemotactically guides mammalian spermatozoa at very low hormone concentrations, and the cumulus oophorus could be a potential place for sperm chemotaxis mediated by progesterone in vivo.

Influence of reactive oxygen species produced by activated leukocytes at the level of apoptosis in mature human spermatozoa.

Human spermatozoa incubated with polymorphonuclear leukocytes and Escherichia coli showed a significant increase in the annexin V binding (P<.05). This increase was similar to that obtained when spermatozoa were incubated with polymorphonuclear leukocytes activated by phorbol-12-myristate-13-acetate.

[Determination of peroxidase positive cells in semen: is it a secure parameter for the diagnosis of silent genital infections?]. / 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.

Expression and location of the bradykinin B2 receptor in rat testis.

To investigate the possible role of the local tissue kallikrein-kinin system in spermatogenesis, we analyzed gene expression and cellular distribution of the bradykinin subtype-2 receptor (B(2) receptor) in the rat testis. Reverse transcription-polymerase chain reaction revealed B(2) receptor expression in testis and primary cultures of Sertoli cells and peritubular cells isolated from immature and mature rats. In situ hybridization of the B(2)-receptor mRNA showed intense labeling of cells on the base of the seminiferous tubule, whereas the autoradiographic signals gradually decreased toward the lumen. Immune histochemistry using testicular sections of pubertal and adult rats showed specific staining for the B(2)-receptor protein in cells of the adluminal compartment of the seminiferous tubules, especially on pachytene spermatocytes and spermatids. This immunostaining varied with the stages of the seminiferous cycle. The receptor protein was also observed on peritubular cells of pubertal rats. In conclusion, we demonstrated a stage-specific expression of the bradykinin B(2) receptor in different cells of the seminiferous tubules of the rat testis. The results point to a possible function of the tissue kallikrein-kinin system in the local regulation of spermatogenesis.

[Idiopathic endometrial decidual reaction in a woman in postmenopause: immunocytochemical evaluation of estrogen and progesterone receptors and its corticoid binding globulin]. / 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.

Endometrio con reacción decidual idiopática en una mujer postmenopáusica: evaluación inmunocitoquímica de receptores de estrógeno, de progesterona y de su proteína transportadora, CBG / Immunocytochemical localization of estrogen and progesterone receptors and corticoid binding globulin in a woman with idiopathic endometrial decidual reaction

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

Efecto de enzimas proteolíticas y de la abstinencia sexual en la cuantificación bioquímica de alfa glucosidasa en plasma seminal humano / Effects of proteolytic enzymes and sexual abstinence on alfa glucosidase levels in human seminal plasma

Background: a-glucosidase is found in human seminal plasma as an acid form, located in accessory glands, and as a neutral form secreted almost exclusively by the epididymis. Quantification of a-glucosidase activity is a marker of the secretory function of the epididymis and indemnity of the sperm transport pathway Aim: To obtain reference values for a-glucosidase in normal samples of seminal plasma, to evaluate its behavior in serial samples and to determine the effect of proteolytic enzymes. Material and methods: Fifty donors, with normal semen analysis according to the criteria of the World Health Organization, were evaluated. For the study with alpha-quimotrypsin, 0.1 to 10 mg/ml of the enzyme was added to the seminal plasma from a group of donors. a-glucosidase was also measured in semen obtained from nine patients at different time intervals. Results: Normal a-glucosidase values ranged from 14.52 to 25.69 µU/ml. Concentrations up to 10 mg/ml of alpha-quimotrypsin (10 times of that usually used in the liquefaction of the semen) did not alter the quantification of a-glucosidase. Serial determinations revealed oscillations in their magnitude, which stayed in each patient's characteristic range. However a subgroup presented a marked reduction of the activity of a-glucosidase as the abstinence diminished (40 percent). Conclusions: Evaluation of a-glucosidase in seminal plasma gives reliable information of the secretor state of the epididymis and especially replaces invasive methods used to evaluate the indemnity of the spermatic transport from the epididymis to the anterior urethra

Determinación de la variabilidad en la condensación de la cromatina espermática y alfa-glucosidasa seminal con relación al espermiograma / Chromatin condensation and seminal alpha glucosidase variability and their relationship to spermiogram

Background: Sperm functional tests as an addition to semen analysis have been used to study the fertilization ability of spermatozoa. Besides the usual variability of the seminal analysis an individual variability in the results of functional tests has been recently found. Aim: to evaluate in a three months period, the individual variability of sperm parameters and sperm maturation using the chromatin condensation test and epidydime a-glucosidase (that allows to discriminate obstructive processes). Material and method: The evaluation was carried out in two donors (12 samples) apparently in good health. One of them presented evident semen analysis alterations (donor 1) and the other was considered normal under the WHO standards (donor 2). Results: The averages for donor 1 were: Sperm count 24x106 sperm/ml (range 10-58x106 sperm/ml), morphology 31.8 percent (range 30-35 percent), total motility 33 percent (range 20-42 percent), sperm maturation 38 percent (range 28-78 percent), a-glucosidase 8.65 (U/ml (range 5-10 (U/ml). The averages for donor 2 were: Sperm count 96x106 sperm/ml (range 50-140x106 sperm/ml), morphology 32.2 percent (range 30-35 percent), total motility 69 percent (range 58-78 percent), sperm maturation 17 percent (range 7-30 percent), a-glucosidase 36.9 (U/ml (range 20-82 (U/ml). Conclusions: These results show that significant variations can be found in the sperm parameters and in seminal plasma a-glucosidase; however these variations are generally maintained at the normal or abnormal ranges for each individual, except the sperm morphology that was constant and with low variation in both donors. The determination of the chromatin condensation in the semen analysis gives an additional information about the grade of sperm maturation and would be of great value for differentiating between sperm samples that show similar morphology values

Evidencias inmunocitoquímicas sobre la presencia de la serpina CBG-símil en el sistema reproductor de la hembra en bovinos / Inmunocytochemistry evidence on the presence of the serpina CBG-like in the reproductive system of the female bovine

La reacción acrosomal es un proceso esencial para la fecundación en mamíferos. Recientemente, ha sido demostrado que la globulina que une corticoides (CBG), asociada a progesterona, induce la reacción acrosómica en niveles fisiológicos, in vitro. En la búsqueda de un componente morfológico in situ, utilizando rastreo inmunocitoquímico, nuestro grupo ha demostrado su presencia en células secretoras de ciertas regiones del sistema reproductor femenino humano. Si esta molécula, a travéz de la reacción acrosomal, esta involucrada en los mecanismos que llevan finalmente a la fecundación en humanos, parece razonable suponer que tiene una participación similar en otras especies mamíferas con procesos reproductivos básicamente comparables. El objetivo del presente trabajo fue determinar, a travéz de la inmunocitoquímica (ICQ), la presencia y distribución de la CBG-símil en el sistema reproductor de bovinos hembras, en los distintos estadios del ciclo reproductivo. Muestras de ovarios, útero, tuba uterinay células epiteliales tubáricas en cultivo fueron procesadas para la ICQ. Para ello, se utilizaron anticuerpos policionales contra CBG humana (comercial y otro producto de laboratorio) capaz de reconocer determinantes antigénicos comunes. La ICQ demostró, a nivel ovárico una reacción positiva preferentemente en células de folículos en estadios superiores de desarrollo. A nivel de la tuba uterina la inmunotinción se presentó claramente en células no ciliadas del epitelio tubárico. En el endometrio la reacción se concentró en las células epiteliales de las glándulas endometriales, sin observarse inmunorreacción a nivel estromal. La inmunotinción fue muy intensa en los períodos de mayor actividad estrogénica para, prácticamente, desaparecer en vacas preñadas. Las células de cultivo oviductales revelaron una población con reacción intensamente positiva y otra claramente negativa. Nuestros resultados nos permiten sugerir que el bovino existe la presencia de CBG-símil, la cual se distribuye en áreas morfológicas similares a las observadas en humano, y su concentración depende del ciclo ovárico. Tales elementos permitirían sugerir que esta particular molécula juega un rol similar en la especie bovina