Dysregulation of follicle development in a mouse model of premature ovarian insufficiency.

Premature ovarian insufficiency (POI) occurs in 1% of reproductive-age women. The ovarian manifestation ranges from the presence of a variable population of follicles (follicular) to the absence of follicles (afollicular), and in the majority of cases the cause is unknown. A transgenic mouse model of follicular POI, the Double Mutant (DM), arises from oocyte-specific deletion of Mgat1 and C1galt1 required for the generation of O- and N-glycans. DM females are subfertile at 6 weeks, infertile by 9 weeks and exhibit POI by 12 weeks of age. In this study we investigate the cause of the reduced fertility at 6 weeks and infertility at 9 weeks of DM females. Ovary sections were used to analyse follicle and corpora lutea (CL) numbers, apoptosis, and levels of laminin and 3ß-hydroxysteroid dehydrogenase using immunohistochemistry. After POI, DM females unexpectedly remained sexually receptive. At both 6 and 9 weeks, DM ovaries contained more primary follicles, however, at 9 weeks DM follicles were proportionally healthier, revealed by TUNEL analysis compared with Controls. In 9 week DM ovaries (collected post-mating), secondary follicles had theca and basal lamina structure abnormalities, whilst preovulatory follicles failed to ovulate resulting in the presence of numerous luteinised unruptured follicles, indicative of ovulation failure. Finally, DM ovaries contained more regressing CL with decreased luteal cell apoptosis indicative of a defect in CL regression. Identifying these follicular modifications have provided insight into the aetiology of a model of POI and highlight targets to investigate with the hope of developing new fertility treatments.

Reduced amounts and abnormal forms of phospholipase C zeta (PLCzeta) in spermatozoa from infertile men.

BACKGROUND: In mammals, oocyte activation at fertilization is thought to be induced by the sperm-specific phospholipase C zeta (PLCzeta). However, it still remains to be conclusively shown that PLCzeta is the endogenous agent of oocyte activation. Some types of human infertility appear to be caused by failure of the sperm to activate and this may be due to specific defects in PLCzeta. METHODS AND RESULTS: Immunofluorescence studies showed PLCzeta to be localized in the equatorial region of sperm from fertile men, but sperm deficient in oocyte activation exhibited no specific signal in this same region. Immunoblot analysis revealed reduced amounts of PLCzeta in sperm from infertile men, and in some cases, the presence of an abnormally low molecular weight form of PLCzeta. In one non-globozoospermic case, DNA analysis identified a point mutation in the PLCzeta gene that leads to a significant amino acid change in the catalytic region of the protein. Structural modelling suggested that this defect may have important effects upon the structure and function of the PLCzeta protein. cRNA corresponding to mutant PLCzeta failed to induce calcium oscillations when microinjected into mouse oocytes. Injection of infertile human sperm into mouse oocytes failed to activate the oocyte or trigger calcium oscillations. Injection of such infertile sperm followed by two calcium pulses, induced by assisted oocyte activation, activated the oocytes without inducing the typical pattern of calcium oscillations. CONCLUSIONS: Our findings illustrate the importance of PLCzeta during fertilization and suggest that mutant forms of PLCzeta may underlie certain types of human male infertility.

Changes in calmodulin immunocytochemical localization associated with capacitation and acrosomal exocytosis of ram spermatozoa.

The aim of this study was to determine the localization of calmodulin (CaM) in ram sperm and the possible changes during in vitro capacitation (CA) and the ionophore-induced acrosome reaction (AR). Likewise, changes in intracellular calcium levels ([Ca(2+)](i)) were also analysed by using flow cytometry. CA was induced in vitro in a medium containing BSA, CaCl(2), NaHCO(3), and AR by the addition of the calcium ionophore A23187. The acrosomal status was assessed by the chlortetracycline-fluorescence (CTC) assay. Flow cytometry (FC) analyses were performed by loading samples with Fluo-3 AM, that emits fluorescence at a high [Ca(2+)](i), combined with propidium iodide (PI) that allowed us to discriminate sperm with/without an integral plasma membrane both with high/low [Ca(2+)](i). Immunocytochemistry localized CaM to the flagellum, and some sperm also contained CaM in the head (equatorial and post-acrosomal regions). CA and AR resulted in a slight increase in the post-acrosomal labelling. The treatment of sperm with increasing concentrations of two CaM antagonists, W7 and calmidazolium (CZ), accounted for an increase in capacitated and acrosome-reacted CTC-sperm patterns. CZ induced a significant reduction in the content of three protein tyrosine-phosphorylated bands of approximately of 30, 40 and 45kDa. However, W7 showed no significant effect at any of the studied concentrations. Neither of them significantly influenced protein serine and threonine phosphorylation. FC analysis revealed that the main subpopulation in the control samples contained 70% of the total sperm with integral plasma membrane and a medium [Ca(2+)](i). After CA, 67.1% of the sperm preserved an integral membrane with a higher [Ca(2+)](i). After AR, only 7.2% of the total sperm preserved intact membranes with a very high [Ca(2+)](i). These results imply that CaM appears to be involved in ram sperm capacitation, and both treatments increased its localization in the post-acrosomal region.

The pattern of localization of the putative oocyte activation factor, phospholipase Czeta, in uncapacitated, capacitated, and ionophore-treated human spermatozoa.

BACKGROUND: Recent studies suggest that in mammals, oocyte activation at fertilization is triggered by a sperm-specific phospholipase C, PLCzeta. We investigated PLCzeta localization in human spermatozoa. METHODS: A polyclonal antibody was generated against human PLCzeta and used in immunoblotting and immunofluorescence studies of ejaculated human sperm in uncapacitated and capacitated states. An ionophore was also used to induce the acrosome reaction in vitro. RESULTS: After verifying specificity of the anti-PLCzeta antibody by immunoblotting, immunofluorescence studies showed that the predominant localization of PLCzeta in uncapacitated sperm was in the equatorial region, a pattern maintained following capacitation and ionophore treatment. The analysis of pooled samples showed approximately 88% of uncapacitated sperm expressed PLCzeta in the equatorial region, whereas approximately 35% and approximately 21% of sperm expressed additional populations of PLCzeta in the acrosomal or post-acrosomal region, respectively. One population of PLCzeta was observed in the post-acrosomal region of approximately 12% of sperm. The proportion of cells with post-acrosomal PLCzeta increased following capacitation and ionophore treatment (P < 0.05). The same tendency was found in individual samples. There was a strong correlation (r = 0.716, P < 0.0001) between presence of an intact acrosome and proportion of sperm immunoreactive to PLCzeta in the acrosomal region. CONCLUSIONS: PLCzeta was variably detectable in three localities within the sperm head: the equatorial segment and acrosomal/post-acrosomal region. Variability in PLCzeta localization in sperm from fertile males may reflect differences in oocyte activation capabilities between individuals or within an ejaculate. This approach may help in investigating the possible links between PLCzeta and certain types of male infertility.

Sperm survival and heterogeneity are correlated with fertility after intrauterine insemination in superovulated ewes.

Efficient animal production involves accurate estimations of fertilizing ability. One key factor is the plasma membrane of the sperm cell, which is actively involved in the cascade of events before oocyte fusion. Many methods are used to analyze the characteristics of this membrane, including partition in aqueous two-phase systems which is an efficient method to analyze sperm surface changes accounting for loss of viability and different functional states. Centrifugal countercurrent distribution (CCCD) analysis can also be used in an aqueous two-phase system to determine the relationship between sperm parameters and in vivo fertility in ewes. In a previous work, we found a significant correlation between two post-CCCD parameters (heterogeneity and recovered viability) and field fertility when the same sample was used after cervical AI. The present study was intended to find out whether the control of several external factors that affect reproductive efficiency is able to increase the correlation coefficient between post-CCCD parameters and fertility. Thus, 90 Rasa aragonesa ewes were controlled on the same farm and received intrauterine inseminations using the same technical equipment. The fertilizing ability of the raw semen and sperm samples selected by a dextran/swim-up process was compared using a low number of spermatozoa per insemination (7 x 10(7)) to enhance possible fertility differences. A new post-CCCD parameter was considered; the loss of viability (LV) occurred during the CCCD process. This variable denotes the sperm surviving ability and corresponds to the difference between the total number of viable cells loaded and recovered after the CCCD run. The mean fertility of eight sperm control samples was 60% (range: 25-76%), and there was no significant correlation between standard parameters and in vivo fertility. LV ranged from 2 to 69% (average 27%) and was negatively correlated with fertility (r = -0.914, P < 0.01). Ejaculate heterogeneity (H) ranged from 20 to 47% and was positively, but not significantly, correlated with fertility (r = 0.391). A predictive equation for fertility was deduced by multiple analysis with a very high correlation coefficient (r = 0.967), and level of significance (P < 0.005): predictive fertility PF = 52.546 - 0.594 LV + 0.665 H. The mean fertility of 13 swim-up selected samples was 63% (range: 25-86%). Again, only parameters derived from the CCCD analysis were highly correlated with fertility, especially LV and H (P < 0.05).

Ram sperm selection by a dextran/swim-up procedure increases fertilization rates following intrauterine insemination in superovulated ewes.

The objective of this study was to compare the efficacy of 2 dextran/swim-up media to increase the sperm quality parameters and the maintenance of these parameters at 15 degrees C and 30 degrees C over 6 hours. Additionally, this study examined whether differences in sperm quality reflect different reproductive efficiencies following intrauterine insemination in superovulated ewes. The study involved 2 selected samples (SS) obtained by dextran/swim-up, performed either with (SS+) or without (SS-) capacitating compounds, and a control sample consisting of raw semen diluted in the same medium. The efficacies of the swim-up sperm selection procedures were similar in both media, and no significant differences were found among the evaluated parameters. Conversely, we found important differences between selected and control samples. Sperm motility, viability (as assessed by carboxifluorescein diacetate/propidium iodide [PI] staining), and mitochondrial activity (as assessed by rhodamine 123/PI) were significantly higher in the selected samples than in the control. Additionally, following incubation at 15 degrees C, the preservation of sperm quality was significantly better in the selected samples than in the control samples. After 6 hours of incubation at 15 degrees C, selected samples had a motility value of 46%, which was significantly (P < .001) higher than the value observed in control samples (27%). The percentage of viable cells observed after 6 hours of incubation at 15 degrees C was significantly (P < .0001) higher in selected samples than in the control samples. Furthermore, after 2 hours of incubation at 30 degrees C, swim-up samples had viability values that were significantly (P < .0001) higher than those of the control samples. SS+ and SS- samples did not differ significantly in spermatozoa yield, sperm quality, or survival. Differences between selected samples and controls were reflected in the fertilization rate obtained following intrauterine insemination in superovulated ewes that experienced a 52-hour interval between progestagen removal and artificial insemination. A restricted criterion for fertilization rate evaluation was established, and only the percentage of embryos recovered from the uterine horns 6 days after insemination was considered with respect to the total number of corpora lutea counted in the ovaries. The fertilization rate of SS- samples (50%) was significantly higher (P > .001) than those of the SS+ (2%) and control samples (5%).

Different functional states of ram spermatozoa analysed by partition in an aqueous two-phase system.

The surface of spermatozoa plays a critical role in many stages involved in fertilisation. The plasma membrane undergoes important alterations in the male and female reproductive tract, which result in the ability of spermatozoa to fertilise eggs. One of these membrane modifications is sperm capacitation, a process by which sperm interacts with the zona pellucida receptors leading to the acrosome reaction. It has been proposed that the freezing process induces capacitation-like changes to spermatozoa, and that this premature capacitation could explain the reduction in longevity and fertilising capacity of cryopreserved mammalian spermatozoa. Our research focused on the relationship between membrane alterations occurring throughout freezing-thawing and the processes of capacitation and acrosome reaction. We used centrifugal countercurrent distribution (CCCD) analysis to compare the partition behaviour of ram spermatozoa that was either subjected to cold-shock or frozen-thawed with capacitated and acrosome reacted samples. In addition, the effect of the induced acrosome reaction on membrane integrity of ram spermatozoa was studied using biochemical markers and electron microscopy scanning. The CCCD analysis revealed important similarities between the surface characteristics of capacitated and cold-shocked sperm as well as between acrosome-reacted and frozen-thawed sperm. Cold-shocked and capacitated sperm showed an increased cell affinity for the lower dextran-rich phase as well as a decreased heterogeneity. Likewise, the induction of the acrosome reaction resulted in a loss of viability and an important decrease in cell surface heterogeneity compared to the untreated-control sample. Similar surface changes were found when semen samples were frozen with either Fiser or milk-yolk extender. These results confirm those obtained for membrane integrity by fluorescence markers. Thus, the high cell viability value found in the control sample (74.5%) was greatly decreased after cold-shock (22.2%), cryopreservation (26.38% Fiser medium, 24.8% milk-yolk medium) and acrosome reaction (6.6%), although it was preserved after inducing capacitation (46.7%). The study using electron microscopy scanning revealed dramatic structural alterations provoked by the induction of the acrosome reaction.

[Wilms' tumor in the adult. A new case and review of the literature]. / Tumor de Wilms en el adulto. Un nuevo caso y revisión de la literatura.

The nephroblastoma is a rare tumour in the adult and there are 240 cases reported in the world literature. We offer a new case and then review the literature, analysing the major features of diagnosis, treatment and evolution of this tumour.

[Primary meningioma of the nose and the paranasal sinus]. / Méningiome primitif nasosinusien.

Primary extracranial and extraspinal meningiomas are not very common, less than 1% out of meningiomas and with a pathogeny not well defined. Less than 60 cases have been described so far. The histopathological diagnosis can be really difficult. All the cases published were diagnosed after an anatomopathological study. We reviewed all publications about these tumors, which belong to the group of those rare neoplasms the otorhinolaryngologist must have in mind whenever he faces a paranasal sinus neoformation.

[Wegener's granulomatosis in the elderly. Report of 3 cases]. / Granulomatosis de Wegener del anciano. Descripción de tres casos.

Wegener's Granulomatosis (W.G.) is a systemic vasculitis which the usual age of presentation is the fourth and fifth decades. It seldom appears in the aged patients and it often exists a greater delay in the diagnosis time and in the beginning of therapy in them. We present three cases of W.G. in aged patients (66, 79 and 80 years). One case was diagnosed in the autopsy and the two others had a favourable evolution after therapy. We insist on the need of using all the available tools in order to confirm the W.G. diagnostic, in spite of the aging. The therapy of these patients must be as vigorous as in the young patients in order to avoid the development of renal failure, the most important cause of death in this disease.

[Kikuchi's necrotizing lymphadenitis. Clinical and pathological study of a new case]. / Linfadenitis necrotizante de Kikuchi. Estudio clínico-patológico de un nuevo caso.

Contribution to the topic with the case of a woman, 24, admitted in our ENT Department for exam and treatment of multiple right sided adenopathies of the neck. The histopathological study was consistent with diagnosis of Kikuchi's necrotizing lymphadenitis. This is a new clinicopathologic entity first described, 1972, by Kikuchi, of unknown etiology, an a picture characterized for painful cervical lymphadenitis presented in young women and healing spontaneously after 2-3 months course. We want to emphasize in this paper the scarcity of cases published and the difficulties arising when dealing with the differential diagnosis with lympho-proliferative malignancies as well.