Extracellular calcium is involved in egg yolk-induced head-to-head agglutination of bull sperm.

Head-to-head agglutination of bull sperm occurs when semen is highly diluted in an egg yolk-citrate diluent without streptomycin. The objectives were to investigate causes of sperm agglutination and the underlying mechanism. Aliquots of bull semen were diluted in a base diluent (BD) supplemented with various test components and the percentage of agglutinated sperm (% AggSp) was quantified at 1, 5, 24, 48, and 72 h of incubation. When sperm were incubated at 22 °C, no agglutination was observed in BD for up to 72 h, whereas the % AggSp was 5.0, 41.7, 72.2, 91.1, and 92.8% in BD + 5% egg yolk (BD + EY) at 1, 5, 24, 48 and 72 h, respectively. However, no sperm agglutination was observed in BD + EY if incubation temperature was 37 °C. Addition of 5 or 10 mM ethylenebis (oxyethyleneni-trilo) tetra-acetic acid to BD + EY reduced the % AggSp from 95% to <5% at 72 h (P < 0.001), but addition of 5 mM CaCl(2) to BD failed to induce sperm agglutination in the absence of egg yolk, implicating calcium and other factors in egg yolk. Addition of the citrate-soluble fraction (CSF) of egg yolk to BD induced sperm agglutination similar to whole egg yolk, whereas water- and saline-soluble fractions of egg yolk were ineffective. The sperm-agglutinating efficacy of CSF (the % AggSp = 95% at 72 h) was reduced by dialysis (20%; P < 0.05), partially restored by addition of 5 mM CaCl2 (70%; P < 0.05), but the calcium effect was neutralized by addition of 5 mM ethylenebis (oxyethyleneni-trilo) tetra-acetic acid (1.7%; P < 0.05), again implicating calcium. Addition of 30 µM of a protein kinase A inhibitor (H-89) to an agglutinating diluent failed to inhibit sperm agglutination, whereas addition of 2 mM of a cAMP analogue, dbcAMP, to a nonagglutinating diluent failed to induce sperm agglutination. Agglutination status had no effect on sperm plasma membrane/acrosome status and mitochondrial membrane potential. In conclusion, calcium and other component(s) in the CSF of egg yolk induced head-to-head agglutination of bull sperm in a time- and temperature-dependent manner. Although the mechanism of agglutination was not determined, the cAMP- protein kinase A signaling pathway was not involved.

TCM treatment of male immune infertility--a report of 100 cases.

OBJECTIVE: To observe the therapeutic effect of Yikang Tang (Yikang Decoction) for male immune infertility. METHODS: 100 cases of male immune infertility in the treatment group were treated with Yikang Decoction, while 100 cases treated with prednisone as the controls. Physical exam, routine semen and prostate exams, and exams for presence of anti-sperm antibody (AsAb) and mycoplasma in the serum or seminal plasma were carried out. RESULTS: 1) The serum and seminal plasma AsAb levels decreased significantly (P < 0.01) in both the groups after treatment, with a more remarkable effect in the treatment group. 2) The sperm density and percentage of motile spermatozoa increased significantly in the two groups, but more significantly in the treatment group after treatment. The pregnancy rate of their wives was higher in the treatment group than that in the control group (P < 0.01). 3) The sperm agglutination rate in the two groups decreased, but more significantly in the treatment group after treatment. 4) The improvement rate of the symptoms and the stability of the therapeutic effect were more dramatic in the treatment group than that in the control group (P < 0.01) after termination of drug administration. CONCLUSION: The Yikang Decoction has a more stable effect for male immune infertility than prednisone.

Calcium (hydrogen-1-malate) hexahydrate on Echeveria gibbiflora leaves and its effect on sperm cells.

Echeveria gibbiflora is a plant widely used for its contraceptive activity in traditional Mexican medicine. Data on calcium crystals in plants are not outstanding. In the case of the Echeveria gibbiflora leaves, however, its quality, quantity, and salt type are quite surprising; one striking result of its X-ray crystallographic data shows the presence of calcium bis (hydrogen-1-malate) hexahydrate [2(C4H5O(5)1), Ca(1)2+, 6(H2O1)]. This highly soluble compound might explain the rapid shape changes of calcium crystals. Because SEM-EDS analysis shows that calcium malate crystals were obtained in a highly pure state and the immobilization and agglutination pattern that OBACE show on human and bull spermatozoa are not found even when high concentrations of calcium bis (hydrogen-1-malate) hexahydrate salt are present it is not feasible to involucrate molecules as calcium malate as part of the OBACE contraceptive activity.

Relationship between bicarbonate and cyclic nucleotide in the promoting effects on head-to-head agglutination in boar spermatozoa.

AIM: To clarify the relationship between bicarbonate and cAMP in the promoting effects on the sperm agglutination. METHODS: Spermatozoa were collected from mature boars, washed and resuspended in a modified Krebs-Ringer HEPES lacking calcium chloride (mKRH). The sperm suspensions were incubated in a water bath (38.5 degrees C) for 60 min and then the percentage of head-to-head agglutinated spermatozoa was determined. RESULTS: Supplementation of the mKRH with sodium bicarbonate (5-10 mM) significantly raised the percentage of head-to-head agglutinated spermatozoa in the samples. The addition of selective inhibitors for calcium/calmodulin-dependent phosphodiesterases (type 1: 8-methoxymethyl-IBMX and vinpocetine, 25-50 micro M) or for cAMP-specific phosphodiesterases (type 4: Ro20-1724 and rolipram, 25-50 microM) enhanced the effect of bicarbonate on sperm agglutination as highly as did the addition of non-selective inhibitors for phosphodiesterases (IBMX and papaverine, 25-50 microM). A calmodulin antagonist (W-7, 2 microM), that potentially blocks the stimulator of the calcium/calmodulin-dependent phosphodiesterases, significantly enhanced the effect of bicarbonate on sperm agglutination. Moreover, a phosphodiesterase-resistant cAMP analogue (cBiMPS, 0.1 mM) markedly induced agglutination in more spermatozoa (76%) after the incubation without bicarbonate and phosphodiesterase inhibitors than did a less potent cAMP analogue (dibutyryl cAMP, 1 mM) (21%), while three kinds of cGMP analogues (0.1-1 mM) had no effect on sperm agglutination. In addition, a cAMP antagonist (Rp-cAMPS, 1 mM) significantly reduced the sperm agglutination resulting from the actions of bicarbonate and IBMX. On the other hand, the effect of bicarbonate was abolished by a change of incubation temperature from 38.5 degrees C to 25 degrees C. CONCLUSION: These findings demonstrate that the bicarbonate-induced agglutination of boar spermatozoa is controlled via the cAMP-mediated, temperature-dependent signaling cascade. This cascade is suppressed by the action of the phosphodiesterase (at least types 1 and 4).

Sperm aggregation by water extracts from two Bursera species.

Some plants have more than the common utility value, as is the case of some members of the Bursera species such as the Mexican copal, a plant used for worship. Water extracts of several plants have vaginal contraceptive properties. The authors evaluated the sperm agglutinating activity of two Bursera species on human and boar sperm. Extracts from stems and leaves were obtained. Capacitated sperm samples were used in all cases. There were different agglutinating capacities, which were not observed in the vehicle-only samples. The most frequent sperm agglutination response was that involving the heads. Agglutinating activity was higher from stem- than leaf-derived extracts. The results indicate that proteins present in the extracts are responsible for the aggregation of sperm heads.

Role of cyclic adenosine 3',5'-monophosphate and serum albumin in head-to-head agglutination of boar spermatozoa.

It has previously been shown that when boar spermatozoa are incubated in a modified Krebs-Ringer bicarbonate (mKRB), head-to-head agglutination occurs in many cells. The aim of the present study was to investigate the effects of cyclic adenosine 3',5'-monophosphate (cAMP) and serum albumin on sperm agglutination and to discuss a possible mechanism for sperm agglutination. Spermatozoa were collected from four mature boars, washed and incubated in mKRB. After a 1-h incubation, a sample of each sperm suspension was smeared gently on a separate glass slide, dried and stained in a phosphate-buffered solution of Giemsa to assess the percentage of head-to-head agglutinated cells in each suspension. In the samples incubated in mKRB, approximately 50% of the spermatozoa were agglutinated with one another at the acrosome. However, the percentages of head-to-head agglutinated spermatozoa were greatly reduced by a lack of calcium chloride in mKRB, but were recovered by the addition of dibutyryl cAMP (dbcAMP, a cAMP analogue) in a dose-dependent manner between 1 and 1000 microM. Addition of 3-isobutyl-1-methylxanthine (IBMX, 100 and 500 microM) instead of dbcAMP also significantly increased the percentages of head-to-head agglutinated spermatozoa. Moreover, the effects of adding dbcAMP were attenuated by treatment with Rp-adenosine 3',5'-cyclic monophosphorothioate triethylamine salt (0.25-1.0 mM, a cAMP antagonist) or H-89 (5 microM, a protein kinase-A inhibitor), but were enhanced by treatment with okadaic acid (500 nM) and calyculinA (500 nM) (inhibitors of protein serine/threonine phosphatase). In sperm samples incubated in mKRB containing 0.1% polyvinyl alcohol (mKRB-P) or mKRB-P lacking calcium chloride and supplemented with 1 mM dbcAMP, a lack of bovine serum albumin (BSA) resulted in a significant decrease in the percentages of head-to-head agglutinated spermatozoa. Addition of porcine serum albumin (PSA, 1-4 mg mL(-1)) or methyl-beta-cyclodextrin (MBC, 5-10 mg mL(-1)) instead of BSA was as effective as BSA (4 mg mL(-1)) in enhancing sperm agglutination. However, the effects of BSA (4 mg mL(-1)) or MBC (5 mg mL(-1)) were reduced by pre-mixing these reagents with cholesterol 3-sulfate (a cholesterol analogue, 5 microg mL(-1) for BSA and 375 microg mL(-1) for MBC). In addition, a protein 'anti-agglutinin' inhibiting sperm agglutination, was extracted from spermatozoa incubated with serum albumin or MBC and detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting techniques. The obtained Western blots revealed that sperm-bound anti-agglutinin was detected less in the samples incubated with either BSA (4 mg mL(-1)) or MBC (5-10 mg mL(-1)), compared with control samples. Moreover, pre-mixing MBC (5 mg mL(-1)) with cholesterol 3-sulfate (375 microg mL(-1)) reduced this reagent's effects on the loss of sperm-bound anti-agglutinin. Additionally, the assay of sperm agglutination and a chlortetracycline staining assay revealed that the percentages of head-to-head agglutinated spermatozoa were positively correlated with those of spermatozoa classified into B pattern (capacitated spermatozoa). These results are consistent with the following suggestions: (i) an adenylyl cyclase-cAMP-protein kinase system mediates a signalling pathway leading to head-to-head agglutination; and (ii) loss of anti-agglutinin from the spermatozoa may be modulated by changes in the plasma membrane induced by actions of serum albumin or MBC contained in a medium.

[The sperm-agglutinating activity of the fractions of saline root extracts from Arum maculatum]. / Spermoaglutinirashta aktivnost na fraktsii ot korenishten vodno-solevi ekstrakt na Arum maculatum.

Our previous investigations have demonstrated that saline extract of Arum maculatum contains lectin (sperm agglutinin) which agglutinates human ejaculated spermatozoa of the type tail to tail and head to tail. By means of speed liquid chromatography (Mono S XP 5/5) eight fractions (I-VIII) were obtained. The sperm-agglutinating activity of the fractions was investigated against frog testicular spermatozoa and testicular and epididymal spermatozoa of man, rabbit, rat and mouse. The fractions did not agglutinate frog spermatozoa. Fraction I agglutinated rabbit, rat and murine spermatozoa; fraction II-rabbit and rat spermatozoa; fraction VII-only human spermatozoa, and fraction VIII-human, as well as animal spermatozoa. The fractions III, IV, V, VI had no sperm-agglutinating activity. These data could be explained by the assumption that the differences in the sperm-agglutinating activity of the fractions are due to isoforms (called also isolectins) of the phytospermagglutinin, contained in the saline extract of Arum maculatum.

[Agglutinating and immobilizing activity of an ethanol extract of Bursera fagaroides on human and other mammalian spermatozoa]. / Actividad aglutinante e inmovilizante del extracto etanólico de Bursera fagaroides sobre espermatozoides de humano y de otros mamíferos.

Human spermatozoa and those obtained from mouse epididymis incubated in ethanolic from mouse epididymis incubated in ethanolic extracts of the cortex of Bursera fagaroides became agglutinated and immobilized in 100% with 30% of viability decrease. This double effect occurred in 50% of pig'ssperm and 50% of rabbit sperm when the concentration was increased tenfold. The pattern of progressive motility was affected 80% of rat's spermatocytes without agglutination. The values remained the same when the extracts were partially purified with salts of Zn++ and Ba++ or by filtration in a sephadex g-10 column. Analysis with thin layer chromatography of both purified extracts produced 3 compounds in comparison with the ethanolic unpurified extract. The extract of the plant Bursera fagaroides contains 3 compounds, which apparently are glycosides with a potent activity upon agglutination -immobilization and a low effect upon spermatocytes viability, which might be used as contraceptives .

Apparent effect of ascorbic acid medication on semen metal levels.

The apparent effect of ascorbic acid therapy for nonspecific spermagglutination on semen levels of ascorbic acid as well as macro- and micrometals was determined in 20 men (ages 25 to 38). Pretreatment diagnosis was based on infertility and relatively low ratings in sperm density, motility, motility index, and semen volume, and were associated with large numbers of abnormal sperm, sperm precursors, and leukocytes. The pretreatment levels of ascorbic acid, sodium, iron, potassium, zinc, manganese, lead, magnesium, and copper were measured in each patient's semen and compared with levels following 60 days of dietary vitamin C supplementation (1.0 gm/day). Analysis of the vitamin C preparation prescribed revealed that each subject was given an impure ascorbic acid medication to supplement a normal diet. Therefore, the significant increases in levels of ascorbic acid and metals in semen following therapy could not be attributed to ascorbic acid alone, nor, similarly, the improved physical parameters of each subject's semen following therapy; no apparent spermagglutination and restored fertility may be due to the interaction of ascorbic acid with cations found in semen.