Pellucida Research Articles

Diversity and distribution of arbuscular mycorrhizal fungi in maize (Zea mays) cropping fields in South Kivu, Democratic Republic of Congo

Arbuscular Mycorhizal Fungi (AMF) occur naturally in agroecosystems and interact symbiotically with crops, facilitating nutrition. This study aimed at assessing the occurrence, abundance and diversity of AMF communities in the maize cropping system and their relation to soils properties in two agroecological zones in South Kivu, eastern DR Congo. Soil samples were collected from eight sites, with 4 sites in highland at 1400 to 2000 m altitude above sea level (in Katana, Kavumu, Mulamba and Mugogo) and four in the lowland at <1000 m (in Luberizi, Bwegera, Luvungi and Kamaniola). Spores were extracted from the field soils, morphologically identified and counted. AMF spores occurrence, abundances, species richness, and diversity were determined. A total of 38 AMF morphotypes distributed in 11 genera were obtained with the majority being from Gigasporaceae, Acaulosporaceae, and Glomeraceae families. This is the first report on the occurrence of these species in the eastern of DR Congo. Acaulospopra excavata, Acaulospopra bireticulata, Densitscutata erythropa, Funneliformis mosseae and Scutellospora pellucida were ubiquitous in all the agroecologies. Spores densities were higher in the highland with the highest recorded in Mulamba. Soil pH and phosphorus content influenced AMF distribution. The many different ubiquitous species indicate adaptation to a wide range of physicochemical environments and could reduce the cost of AMF inoculants production for the region. Maize agroecosystems are rich in AMF diversity and selection of appropriate fungal species from the Gigasporaceae, Acaulosporaceae and Glomeraceae as biofertilizer could contribute in improving crops production. Key words: Arbuscular mycorrhizal fungi, diversity, occurence, agroecological zones, maize, South Kivu.

137 Cs contaminations in wild-growing medicinal plants of Zhytomyr polissya forests: A 34 years after Chernobyl accident

The contamination of 137Cs in wild-growing medicinal plants was studied with regards to the species, type of forest vegetation conditions and soil specific activity of 137Cs in Zhytomyr polissya forest phytocenoses (Ukraine). The research was carried out in two plots planted in the most region typical edatopes - fresh and wet subor (Querceto-Pinetum). Species composition of phytocenoses was analyzed and the plants that can be harvested for pharmaceutical industry were identified on these test plots,. The herbal substance samples were tested to determine the specific activity of 137Cs. The studies have shown significant variability in the content of radionuclides due to differences in the level of radioactive contamination in the soil and accumulative capacity of plant species. For European blueberry, the peculiarities of 137Cs accumulation in vegetative and generative organs were studied. Comparison of the content of 137Cs in pants of different edatopes showed more higher intensity of radionuclide accumulation in wet suborbs compared to fresh suborbs. The average values of concentration factors were calculated and their rankings were built according to the intensity of 137Cs accumulation for the plants in each edatope. All studied medicinal plants were divided into four groups according to the contration factor: with weak accumulation, moderate accumulation, strong accumulation, and very strong accumulation. Based on the permissible content of 137Cs in wild-growing medicinal plants (600 Bq/kg), the maximum permissible level of soil contamination with radionuclides was determined for each medicinal plant species.

Полное удаление zona pellucida бластоцисты перед переносом в крио-цикле не влияет на результативность программ ЭКО

Полное удаление zona pellucida бластоцисты перед переносом в крио-цикле не влияет на результативность программ ЭКО

Outcome of modified intracytoplasmic sperm injection using zona pellucida-bound spermatozoa in male infertility

Objective To investigate the treatment outcome of modified intracytoplasmic sperm injection(ICSI) using zona pellucida(ZP)-bound sperm. Methods 82 patients with less, weak, abnormal sperm disease who were conformed to ICSI, were divided into traditional ICSI group and the group of modified ICSI using ZP-bound sperm according to ICSI case number.The results of normal fertilization rate, cleavage rate, high-quality embryo rate, planting rate, clinical pregnancy rate and early abortion rate were compared. Results The women's age, the sterility year, mature egg rate, normal fertilization rate, cleavage rate in the two groups had no statistically significant differences (all P>0.05). The planting rate and clinical pregnancy rate of the observation group (46.6%, 63.3%) were higher than those of the control group(38.5%, 53.6%), but there were no statistically significant differences(all P>0.05). The using embryo rate and high-quality embryo rate of the observation group (73.9%, 51.0%) were significantly higher than those of control group(65.8%, 38.6%), the differences were statistically significant(χ2=5.84, χ2=11.6, all P<0.05). Conclusion Modified ICSI using ZP-bound sperm can effectively improve the embryos quality in ICSI. Key words: Spermatozoa; Zona pellucida; Intracytoplasmic sperm injection; Embryo culture techniques

Chemical composition and acaricidal activity of essential oils from Peperomia pellucida Kunth. against Tetranychus urticae

Normal 0 21 false false false MicrosoftInternetExplorer4 Os oleos essenciais obtidos por hidrodestilacao das folhas e caules de Peperomia pellucida coletadas de um fragmento de Mata Atlântica no estado da Bahia, Brasil, foram analisados utilizando cromatografia gasosa e cromatografia gasosa-espectrometria de massa. Independentemente da parte da planta, sesquiterpenos foram a classe de compostos mais abundantes nos oleos, seguido de fenilpropanoides. Os principais constituintes do oleo das folhas foram ? -gurjuneno (11,34%), 1,10-di- epi -cubenol (11,27%), ( E )-cariofileno (8,71%) e dillapiole (8,50%), enquanto que carotol (9,77%), dillapiole (9,18%), trans - ? -guaieno (9,05%) e ( E )-cariofileno (8,53%) foram os principais constituintes do oleo de caule. A atividade dos oleos contra Tetranychus urticae foi avaliada por 24 h. O oleo do caule (LC 50 = 1 x 10 -2 µL/L of air) foi quatro vezes mais toxico do que o oleo de folha (LC 50 = 4 x 10 -2 µL/L of air), mas 2,5 vezes menos ativo do que o eugenol, o qual foi utilizado como o controle positivo .

Morphological and morphometric study of early-cleavage mice embryos resulting from in vitro fertilization at different cleavage stages after vitrification.

The aim of this study was to examine the possible morphological and morphometric changes resulting from vitrification of embryos at the cleavage stage. In this study, 30 mice early-cleavage embryos at different stages of cleavage, resulting from in vitro fertilization (IVF) techniques, were examined before and after vitrification. Digital images were taken from embryos before and after vitrification. Zona pellucida thickness, differences in zona pellucida thickness, and diameter and volume of blastomeres and embryos as morphometric parameters and current rating of appearance of embryos as morphological parameters, have been studied. According to our findings, there were significant mean differences in all morphometric parameters of the two groups except in the zona pellucid thickness (P≤0.05). With regard to the morphological parameter, the decrease in embryo quality was observed but it was not significant. According to the results, although little quantitative change observed is not necessarily synonymous with harmful intracellular damage, it seems that it is better to examine vitrification method more accurately. Because by making subtle changes in concentration and type of consumed solutions or techniques used, the changes may be minimized.

Effects of abnormal zona pellucida on fertilization and pregnancy in IVF/ICSI-ET

Objective To analyze the fertilization rate, embryo development and clinical outcome of oocytes with abnormal zona pellucida after in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Methods A retrospective analysis included a total of 43 cycles (27 IVF cycles and 16 ICSI cycles) in which oocytes displaying abnormal zona pellucida were retrieved between January 2006 and December 2011. The fertilization rate, embryo quality, and the cumulative clinical pregnancy rate were analyzed. Results Rescue ICSI was applied in 27 IVF cycles in which failed extrusion of the second polar body after conventional IVF was observed, and of them, complete failure to fertilize occurred in 23 IVF cycles. The fertilization rate and the normal fertilization rate for IVF (64.83% and 59.32%, respectively) were significantly lower than those for ICSI (85.19% and 79.01%, respectively), whereas the cleavage rate (94.12%) with IVF did not differ significantly from that with ICSI (95.65%, P>0.05). The percentages of good-quality embryos in IVF group (52.67%) and ICSI group (43.75%) also did not differ significantly (P>0.05). Although the rates of implantation and pregnancy appeared to be greater in IVF group (33.33% and 40.00%, respectively) compared with those in ICSI group (25.00% and 35.71%, respectively), the differences were not significant (P>0.05). Conclusions ICSI should be carried out for oocytes with abnormal zona pellucida, for which the risk of IVF failure is high. Rescue ICSI improves the likelihood of fertilization of oocytes with abnormal zona pellucida, but cannot improve the clinical outcome.

Panel of monoclonal antibodies to sperm surface proteins as a tool for monitoring sperm-zona pellucida receptor localization and identification

Panel of monoclonal antibodies to sperm surface proteins as a tool for monitoring sperm-zona pellucida receptor localization and identification

Absence of the Egg's Zona Pellucida: A Newly Identified Cause of Infertility

In 15% to 30% of infertile couples, no apparent cause is determined. Many experts believe that unexplained infertility results from defects manifested at or after fertilization or implantation. Now, investigators report an autosomal recessive form of infertility associated with abnormal eggs in a Chinese family. In eggs of the index patient, absence of …

Ultrastructural and morphometric evaluation of aged cumulus-oocyte-complexes

Maternal age is one of the most significant factors influencing oocyte quality (1). 35 years of age seems to be a watershed in reproductive potential. The aim of this study was to reveal the amount and distribution of specific ultrastructural organelles in human mature cumulus-oocyte-complexes belonging to women of different ages (<35 years old; ≥35 years old/ reproductive aging) and to evaluate their different response during 24 hours prolonged culture (defined as in vitro aging) (1). The samples were studied by light and transmission electron microscopy; a morphometric analysis of TEM data was performed (2). In all aged samples, the amount of mitochondria- smooth endoplasmic reticulum aggregates, cortical granules and microvilli decreased (p<0,05), while the amount of mitochondria-vesicle complexes increased up (p<0,05). Occasional vacuoles were found in oocytes from older women after in vitro aging. A significant (p<0,05) increase of zona pellucida thickness was linked to the donor age but not to in vitro aging. A re-compaction of cumulus cells was seen in in vitro aged samples. Morphometric data strongly confirmed our preliminary results (3) revealing that: i) reproductive aging and in vitro aging share specific ultrastructural features ii) In vitro aging can be consider a model for reproductive aging iii) young oocytes seem to be less sensitive to in vitro aging than older ones. The above results may represent a reliable background for further multidisciplinary studies regarding aged oocytes and may be also useful in clinical settings.

Morphofunctional aspects of in vitro matured oocytes from prepubertal and adult sheep

The aim of this study was to analyze the morphofunctional aspects of prepubertal and adult sheep oocytes subjected to in vitro maturation (IVM). The structural and ultrastructural morphology was examined by light and transmission electron microscopy (LM and TEM) while the metabolic competence, as determined by the distribution of active mitochondria, was assessed by confocal microscopy (CM). Cumulus-oocyte complexes underwent IVM in standard condition for 24 h. Half of the mature metaphase II (MII) stage oocytes were processed for LM and TEM observations. The other half was subjected to immunostaining with MitoTracker Red (to stain mitochondria with functionally active membrane potential), SNARF-1 (a pH sensitive fluoroprobe), Hoechst 33342 (to label DNA) and analyzed by CM. Immature germinal vesicle (GV) stage oocytes, retrieved at 0 h IVM, were used as controls. By LM and TEM all the oocytes were regularly rounded, covered by microvilli and surrounded by an intact zona pellucida. Numerous rounded, oval or hooded mitochondria appeared either isolated or grouped in the ooplasm. The GV was usually rounded in prepubertal oocytes. In the adult, the GV often appeared flattened against the oolemma, with a crescent-shaped outline (an early sign of meiotic resumption). Scattered cortical granules (CGs) were rarely found in the ooplasm of both prepubertal and adult GV oocytes. After 24 h of IVM, CGs became abundant and distributed in a single row under the oolemma, particularly in adult oocytes. CM showed a homogeneous fine-to-granular mitochondrial distribution in prepubertal GV and MII oocytes. In adult GV oocytes, the mitochondrial distribution pattern was granular while in MII oocytes mitochondria were arranged in heterogeneous clusters. Thus, both prepubertal and adult oocytes completed maturation after 24 h in culture and showed an overall good preservation after IVM. However, the diverse distribution patterns of mitochondria in prepubertal oocytes reflect their low developmental competence.

Occurrence of vacuolization in human mature oocytes subjected to slow cooling

Ooplasm vacuolization is a sign of cryoinjury. In this study human mature oocytes, obtained from consenting IVF patients, were cryopreserved by slow cooling using PrOH and sucrose as cryoprotectants. Oocytes were fixed at sampling (controls) and at different time intervals during thawing (1st, 2nd and 3rd passage in the thawing solutions: groups A, B and C, respectively). These oocytes were then observed by light and transmission electron microscopy (LM and TEM), to determine at what stage of the procedure the vacuoles may form and/or increase in number. By LM, both fresh and cryopreserved oocytes were rounded, 90-100 μm in diameter, provided with a homogenous ooplasm and surrounded by a continuous zona pellucida. Areas in which staining and matter consistency appeared reduced, identified as vacuoles, were only occasionally detected in the ooplasm of fresh controls. On the contrary, vacuoles of different sizes and shapes were numerous in the cytoplasm of the cryopreserved oocytes belonging to all experimental groups (A, B, C). Morphometric analysis revealed that the differences between fresh control and cryopreserved samples were statistically significant (P<0.05). In particular, the mean number ± SD of vacuoles larger than 0.5 μm in diameter per 100 μm2 was 1.06±0.18 (control), 7.2±1.5 (group A), 17.05±5.5 (group B), 9.5±5.2 (group C). Thus, vacuoles appeared to increase during thawing, reaching a maximum amount in group B (difference between A and B was statistically significant, P=0.033), and then undergoing a partial recovery at the end of the thawing process (difference between A and C was not statistically significant, P=0.5). By TEM vacuoles appeared as empty spaces lined by a membrane often interrupted. These data: 1) evidence that vacuolization may be a recurrent form of cell damage during slow cooling; 2) reveal that vacuoles primarily form during freezing but may further increase in number at thawing, particularly during the PrOH step-wise dilution.

Morphometric Evaluation of Mouse Embryos In Vitro Cultivated

Variation of the zona pellucida thickness and the embryo diameter are associated with the pregnancy rates and the degree of polispermy. The morphometric parameters gained a major importance for embryo quality evaluation in all in vitro procedures, since the software accessibility made this operation easy to apply. The aim of this paper was a comparative study of several parameters of the in vivo obtained embryos recovered in the morula and blastocyst stage versus in vivo obtained embryos in 2, 4 and 8 cells stage in vitro cultivated up to the morula and blastocyst stage. The parameters taken in consideration in our experiments were: the zona pelucida thickness, inner diameter, outer diameter, inner and outer perimeter of the embryo. The measurements were made with Quick Photo Micro 2.2 software. The experimental variants were: in vivo embryos obtained after 72 hours from the vaginal plug, in vivo embryos obtained after 48 hours of in vitro cultivation in specific media, in vivo embryos in vitro cultivated for 48 hours after vital staining. From the obtained data’s was observed that the parameters taken under study varied. At the in vitro cultivated embryos has been observed a tendency of increasing the zona pelucida thickness from 9.8±1.7 μm at the morula stage embryos recovered at 72 hours from the vaginal plug to 11.1±1 μm for the embryos, stained prior in vitro cultivation. A similar variation was observed for the others parameters taken under study: inner diameter (from 98.4±3.3 μm to 111.6±3.2 μm), outer diameter (from 116.7±2.5 μm to 129.4±4.8 μm), inner perimeter (from 302±0.5 μm to 338±0.4 μm) and outer perimeter (from 325±0.8 μm to 391±0.4 μm). A similar difference was observed for the blastocyst stage embryos (from 6.2±0.5 μm for the embryos recovered at 72 hours from the vaginal plug to 8.7±1 μm for the embryos stained before in vitro cultivation). The embryos assessed by staining prior in vitro cultivation had higher values comparing to in vitro cultivated embryos without staining, the differences had no statistical significance. In vitro cultivation of mouse embryos on synthetic media induces a smooth increase of the zona pellucida thickness. Values obtained varied from 9.8±1.7 μm for the embryos in morula stage recovered at 72 hours from the vaginal plug up to 11.1±1 μm for in vitro cultivated embryos vital stained prior cultivation. Differences were not statistical significant (p>0.05).

Histochemical study of estradiol valerate-induced polycystic ovary syndrome in the rat

*Summary For the present study, and follow-up of histochemical changes in estradiol valerate-induced polycystic ovary syndrome (PCOS), 48 virgin 90-day-old female rats were used. The rats were divided into the treatment and control groups. For each rat of the treatment group, 4 mg estradiol valerate was injected through intramuscular route for the induction of PCOS. The control group was taken the same amount of sterile sesame oil. After 63 days of hormone administration, all rats were sacrificed and their ovaries were collected immediately and processed for histochemical studies. These studies were consisted of localization of carbohydrate using PAS method, saturated and unsaturated lipids using Oil-Red-O and Sudan Black B, lipase and alkaline phosphatase. The PAS reaction was seen in some structures of the atretic and cystic follicles such as on zona pellucida (ZP), basement membrane of granulosa cells, connective tissue fibers of ovarian stroma, follicular fluid and granulose cells. This reaction was enhanced in basement membrane of granulose cells of cystic follicles. The lipid droplets were seen in follicular structures of both the atretic and cystic follicles. The presence of macrophages was confirmed in cystic ovaries by this study. The lipase reactions were seen in granulose and theca cells of atretic and cystic follicles. The lipase reaction in the theca layer of cystic follicles was stronger than in the granulose cell layer of such follicles. The alkaline phosphatase reaction was seen in the theca and granulose cells of atretic and cystic follicles and by the progression of these processes and cystic follicle formation, the reaction was increased accordingly. We concluded that, during follicular atresia and cystic follicle formation, histochemical alterations are occurred in follicular structures.

THE MOSS FLORA OF THE COMMUNITIES OF Buxus sempervirens L. IN FIRTINA VALLEY (ÇAMLIHEMŞİN, RİZE)

This paper deals with a moss floristic study of the communities of Buxus sempervirens L. in Firtina valley, on northern slopes of the Kackar Mountains. After the identification of 85 moss specimens collected from the research area between June and August in 2005, total 47 taxa were defined. These taxa belong to 18 families and 34 genera of the bryophytes. Among them 6 taxa (Tetraphis pellucida Hedw., Dicranoweisia crispula (Hedw.) Milde, Dicranodontium denudatum (Brid.) E.Britton var. denudatum, Oncophorus virens (Hedw.) Brid., Pseudoleskeella nervosa (Brid.) Nyholm, Eurhynchium schleicheri (R.Hedw.) Jur.) are new records for A4 grid-square which was adopted by Henderson (1961). The families which have the richest species with high taxon number are as follows: Dicranaceae (7), Hypnaceae (6), Brachytheciaceae (5), Mniaceae (5), Polytrichaceae (4). The richest genera on account of the number of species, subspecies and varieties are: Plagiomnium (4), Polytrichum (3), Hypnum (3). The ecological spectrums of these taxa are considered, comprising their preferred substrate, humidity and life forms have been assessed using DierAŸen (2001) and MA¤gdefrau (1982). Key Words: Firtina Valley, Buxus sempervirens, Moss Flora, Rize

Oocyte proteomics: localisation of mouse zona pellucida protein 3 to the plasma membrane of ovulated mouse eggs.

In order to gain a deeper understanding of the molecular underpinnings of sperm-egg interaction and early development, we have used two-dimensional (2D) electrophoresis, avidin blotting and tandem mass spectrometry to identify, clone and characterise abundant molecules from the mouse egg proteome. Two-dimensional avidin blots of biotinylated zona-free eggs revealed an abundant approximately 75-kDa surface-labelled heterogeneous protein possessing a staining pattern similar to that of the zona pellucida glycoprotein, mouse ZP3 (mZP3). In light of this observation, we investigated whether mZP3 specifically localises to the plasma membrane of mature eggs. Zona pellucidae of immature mouse oocytes and mature eggs were removed using acid Tyrode's solution, chymotrypsin or mechanical shearing. Indirect immunofluorescence using the mZP3 monoclonal antibody (mAb) IE-10 demonstrated strong continuous staining over the entire surface of immature oocytes and weak microvillar staining on ovulated eggs, regardless of the method of zona removal. Interestingly, in mature eggs, increased fluorescence intensity was observed following artificial activation and fertilisation, whereas little to no fluorescence was observed in degenerated eggs. The surface localisation of ZP3 on mature eggs was supported by the finding that the IE-10 mAb immunoprecipitated an approximate 75-kDa protein from lysates of biotinylated zona-free eggs. To further investigate the specificity of the localisation of mZP3 to the oolemma, indirect immunofluorescence was performed using the IE-10 mAb on both CV-1 and CHO cells transfected with full-length recombinant mZP3 (re-mZP3). Plasma membrane targeting of the expressed re-mZP3 protein was observed in both cell lines. The membrane association of re-mZP3 was confirmed by the finding that biotinylated re-mZP3 (approximately 75 kDa) is immunoprecipitated from the hydrophobic phase of Triton X-114 extracts of transfected cells following phase partitioning. Immunoprecipitation assays also demonstrated that surface re-mZP3 was released from transfected CV-1 in a time-dependent manner. These results demonstrate that ZP3 is specifically associated with the surface of mature eggs and its subsequent release from the cell surface may represent one mechanism by which ZP3 is secreted. Furthermore, the increase in ZP3 surface expression following fertilisation suggests that ZP3 may have a functional role during sperm-oolemma binding and fusion. These results also validate the usefulness of using the 2D proteomic approach to identify and characterise egg-surface proteins.

Examination of the binding ability of bovine spermatozoa to the zona pellucida as an indicator of fertility.

Despite the development of many new techniques, laboratory assays still do not predict male fertility accurately. To identify targets for laboratory assessment, we first need to determine which steps in fertilization are most often defective in subfertile males. We developed a competitive in vitro fertilization assay in which spermatozoa from 2 different males, stained with different lipophilic dyes, are incubated together with oocytes in a droplet. By exposing mixed spermatozoa to the same oocytes, this assay controls for many of the variables of in vitro fertilization and should allow identification of the most common faulty steps in fertilization. The relationship of zona-binding ability to fertility is controversial. Therefore, as a first step, we determined if zona pellucida-binding ability, measured by this competitive assay, was related to bovine spermatozoal fertility. Fertility data were collected from 2 groups of bulls by 2 means of evaluation, nonreturn to estrus rates postinsemination and competitive insemination. In the nonreturn to estrus study, semen samples from 15 bulls were effectively ranked by zona-binding ability, using pairwise competitive in vitro zona-binding assays (R(2) = 0.84). However, this ranking was not significantly correlated with nonreturn rates (r = -0.04). In the competitive insemination study, semen samples from 8 bulls were effectively ranked by pairwise comparison using the competitive zona-binding assay (R(2) = 0.67). Again, this ranking was not significantly correlated to the competitive insemination index calculated for these bulls (r = 0.29). In the third study, we tested 3 bulls to determine if in vivo zona binding, assessed by the number of accessory spermatozoa, was correlated with in vitro zona binding. The number of accessory spermatozoa on oocytes recovered from cows after mating was not correlated with in vitro competitive binding of the spermatozoa. In conclusion, in vitro competitive zona binding was not correlated with bovine fertility or binding of accessory spermatozoa to oocytes in vivo.

In vitro fertilization with intracytoplasmic sperm injection is an effective therapy for male factor infertility related to subnormal hypo-osmotic swelling test scores.

The aim of the study was to determine if intracytoplasmic sperm injection (ICSI) would be an effective therapy for mates with subnormal hypo-osmotic swelling test (HOST) scores, a condition known to prevent implantation of embryos despite allowing normal fertilization and embryo formation. Couples in whom the male partner had a HOST score of <50% and failed to achieve a pregnancy despite at least 3 cycles of intrauterine insemination with chymotrypsin-galactose-treated sperm were treated with in vitro fertilization (IVF) with ICSI. The clinical and viable pregnancy rates were 49.0% and 45.3% (n = 53). The implantation rate was 27.1%. These data thus demonstrate that ICSI is an effective therapy for infertile couples in whom the male partner has a subnormal hypo-osmotic swelling (HOS) score. Previous studies using conventional IVF without ICSI when HOS was subnormal found normal fertilization rates but a marked reduction in pregnancy and implantation rates. The very acceptable pregnancy and implantation rates demonstrated in this study with ICSI is consistent with the hypothesis that the defect associated with sperm that have subnormal HOST scores is not related to the single spermatozoon that is responsible for fertilizing the oocyte but may be related to a toxic factor associated with the supernumerary sperm attached to the zona pellucida.

The cyclic GMP-specific phosphodiesterase inhibitor, sildenafil, stimulates human sperm motility and capacitation but not acrosome reaction.

Capacitation is the series of transformations that spermatozoa undergo in the female genital tract in order to bind to the zona pellucida, initiate the acrosome reaction, and fertilize an egg. Cyclic adenosine monophosphate (cAMP) plays an important role in this process and its levels are regulated by 2 key enzymes, adenylyl cyclase and cyclic nucleotide phosphodiesterase (PDE), the latter being involved in cAMP degradation. Evidence was provided for the involvement of PDE in sperm motility and capacitation. Of the 10 gene families of PDE that exist in mammalian tissues, the calcium-calmodulin-dependent (type 1) and the cAMP-specific (type 4) have been found in human spermatozoa. Using sildenafil, we investigated a highly potent cyclic guanosine monophosphate (cGMP)-specific PDE (type 5) inhibitor and whether this PDE is present in human spermatozoa and is involved in sperm functions. Sildenafil inhibited PDE activity of Percoll-washed spermatozoa with an IC50 of 97+/-3 and 33+/-3 microM when cAMP and cGMP, respectively, were used as substrates. Because the IC50 of sildenafil obtained for PDE type 5 is much lower (2 to 6 nM) than that obtained with sperm PDE, the data suggest that PDE type 5 represents only a small fraction of the whole PDE activity of spermatozoa. Sildenafil causes dose-dependent increases in sperm cAMP levels and capacitation, which are associated with an increase in the levels of tyrosine phosphorylation of 2 fibrous sheath proteins (p105/81). Sperm velocity, amplitude of lateral head displacement, and hyperactivation were increased at 30-180 minutes. Sildenafil did not trigger the acrosome reaction in capacitated spermatozoa. These results suggest that under our experimental conditions, sildenafil triggers human sperm motility and capacitation, probably via its inhibitory action on PDE activity other than type 5 with a resultant rise in cAMP levels.

Calpain and calpastatin are located between the plasma membrane and outer acrosomal membrane of cynomolgus macaque spermatozoa.

Mammalian sperm must undergo an acrosome reaction prior to penetration of the zona pellucida and subsequent fusion with an oocyte. Sperm gain the capability to acrosome react after a period of capacitation, which primarily involves biochemical changes in the sperm membranes. The morphological events of the acrosome reaction have been well-documented, but the underlying cellular mechanisms that regulate capacitation and the acrosome reaction remain unclear. Antibodies to the 2 ubiquitous calpains, mu and m, as well as the small subunit, which associates with both calpains, were localized at the ultrastructural level to the region between the plasma membrane and the outer acrosomal membrane of cynomolgus macaque sperm. After the acrosome reaction, all of the anti-calpain antibodies labeled the acrosomal shroud, suggesting that calpains are located throughout the cytoplasmic area between the 2 outer sperm membranes. Calpastatin is an endogenous modulator of calpain activity and is also localized within the same cytoplasmic region as calpains. The antibodies used for ultrastructural localization were also used to probe Western blots of sperm extracts. Antibodies to either the mu- or m-calpain recognized an 80-kd protein, which is similar to the molecular weights of other ubiquitous calpains described. The small subunit (30 kd) was also recognized with a specific monoclonal antibody. An antibody to calpastatin recognized a major band at 78 kd and a lighter band at 45 kd, while the antibody to the testis-specific isoform of calpastatin (TCAST) recognized a 110-kd protein. We hypothesize that this cysteine protease system may be functional in cynomolgus macaque sperm during capacitation, the acrosome reaction, or both.