Individual differences in the distribution of sperm acrosome-associated 1 proteins among male patients of infertile couples; their possible impact on outcomes of conventional in vitro fertilization.

The aims of this study were to show the existence of individual differences in the distribution of sperm acrosome-associated 1 (SPACA1) among male patients of infertile couples and to examine their possible impact on the outcomes of conventional in vitro fertilization (IVF). The spermatozoa were collected from male patients of infertile couples, washed by centrifugation, collected by the swim-up method, and then used for clinical treatments of conventional IVF. The surplus sperm samples were fixed and stained with an anti-SPACA1 polyclonal antibody for the immunocytochemistry. In the clinical IVF treatments, fertilization rates and blastocyst development rates were evaluated. The immunocytochemical observations revealed that SPACA1 were localized definitely in the acrosomal equatorial segment and variedly in the acrosomal principal segment. Specifically, the detection patterns of SPACA1 in the acrosomal principal segment could be classified into three categories: (A) strong, (B) intermediate or faint, and (C) almost no immunofluorescence. The SPACA1 indexes were largely different among male patients with the wide range from 13 to 199 points. The SPACA1 indexes were significantly correlated with developmental rates of embryos to blastocysts (r = 0.829, P = 0.00162), although they were barely associated with fertilization rates at 19 h after insemination (r = 0.289, P = 0.389). These results suggest that the distribution of SPACA1 in sperm affects the outcomes of conventional IVF. In conclusion, this study provides initial data to promote large-scale clinical investigation to demonstrate that the SPACA1 indexes are valid as molecular biomarkers that can predict the effectiveness of conventional IVF of infertile couples.

Roles of extracellular Ca(2+) in the occurrence of full-type hyperactivation in boar ejaculated spermatozoa pre-incubated to induce the cAMP-triggered events.

There are species differences in the regulatory system for sperm capacitation and subsequent hyperactivation between livestock and laboratory animals. In livestock spermatozoa, it is poorly understood when and how extracellular Ca(2+) is necessary for hyperactivation, although it has been demonstrated that the [Ca(2+) ]i increase is indispensable to occurrence of hyperactivation. In this study, we examined necessity of extracellular Ca(2+) for the initiation and maintenance of hyperactivation and then sought possible target molecule of Ca(2+) that was involved in hyperactivation of boar spermatozoa. Boar ejaculated spermatozoa were pre-incubated with a cell-permeable cyclic adenosine monophosphate (cAMP) analog 'cBiMPS' and without CaCl2 to induce the cAMP-triggered events including capacitation-associated changes. Subsequently, they were incubated with CaCl2 to induce hyperactivation and then used for motility assessment. Many of the spermatozoa after the incubation exhibited full-type hyperactivation which was characterized by high-amplitude and extremely asymmetrical beating of whole middle piece and principal piece. The initiation of full-type hyperactivation required the millimolar concentration of CaCl2 in the medium. However, CaCl2 of the medium was less necessary for maintenance than initiation of full-type hyperactivation, as hyperactivated spermatozoa were barely affected by the incubation with the Ca(2+) -chelating reagent. On the other hand, the pre-treatment with the inhibitor for Ca(2+) -dependent protease 'calpain 1 and 2' clearly suppressed the occurrence of CaCl2 -induced hyperactivation without influences on the percentages of motile spermatozoa. Western blotting and indirect immunofluorescence showed distribution of calpain 2 in the middle and principal pieces in which full-type hyperactivated spermatozoa exhibited extremely asymmetrical beating. On the basis of these results, we conclude that the millimolar concentration of extracellular Ca(2+) is necessary for the initiation, but not for the maintenance of full-type hyperactivation in boar spermatozoa that beforehand undergo the cAMP-triggered events including capacitation-associated changes. Moreover, we suggest possible involvement of calpain 2 in the intracellular Ca(2+) signal transduction leading to full-type hyperactivation.

Detection of the activator cAMP responsive element modulator (CREM) isoform ortholog proteins in porcine spermatids and sperm.

It is necessary to obtain basic information on transcription factors expressed in the spermatids of livestock to determine mechanisms of defective spermiogenesis. In this study, we characterized the activator cAMP responsive element modulator (CREM) ortholog isoforms in porcine testicular germ cells and ejaculated sperm. At least two kinds of porcine activator CREM τ family ortholog mRNAs were more strongly expressed in the testis than in kidney or liver. The activator CREM isoform ortholog proteins were localized in nuclei of round spermatids and around nuclei of elongated spermatids. Furthermore, approximately 34% of ejaculated sperm had the activator CREM isoform ortholog proteins at their connecting piece. This is apparently the first report demonstrating the expression and localization of the activator CREM isoform ortholog proteins in spermatids and ejaculated sperm of a livestock species.

Exogenous hyaluronic acid enhances porcine parthenogenetic embryo development in vitro possibly mediated by CD44.

Exogenous hyaluronic acid (HA) has been reported to improve early embryo development in vitro in pigs and cows. Although early embryo development in vitro is improved by exogenous HA, the mechanism mediating the action of HA is not clearly defined. In the present study, two possible HA actions on early embryo development were proposed to understand interactions between HA and the embryos using porcine parthenotes. We hypothesized that improvement of early embryo development mediated by HA would be caused by embryo-derived growth factors due to the high molecular weight of HA or cellular response through its receptor (CD44). We examined the effects of HA molecular weight on parthenogenetic embryo development, permeability of HA into the zona pellucida, expression of CD44 in porcine parthenotes at various stages, and blocking interactions between HA and CD44 by monoclonal anti-CD44 antibody (mCD44Ab). As a result, although development of porcine parthenotes to the blastocyst stage was significantly enhanced by exogenous HA with various molecular weights, there was no difference in blastocyst formation among the various molecular weights (P < 0.05). Immunofluorescence revealed that exogenous HA was accessible to CD44 through the zona pellucida, irrespective of the oocyte activation and that CD44 was also expressed in both oocytes and parthenotes at all developmental stages. In addition, development of parthenotes was partially blocked by mCD44Ab. In conclusion, we demonstrated that exogenous HA enhanced development of porcine parthenotes in vitro. This improvement mediated by exogenous HA on parthenogenetic embryo development was possibly caused by cellular response via CD44.

Stage-specific effects of the osmolarity of a culture medium on the development of parthenogenetic diploids in the pig.

The objective of this study was to investigate the effects of osmolarity of culture media on the development of porcine parthenogenetic diploids. Oocyte-cumulus-granulosa cell complexes were collected from ovaries and then in vitro-cultured for 48 h. The mature oocytes were subjected to a single electro-stimulation (El-St; 100 micros, 1500 V/cm), treated with 5.0 microg/ml Cytochalasin B for 4h and then cultured under various conditions as described below. In Experiment 1, the diploids were cultured for 168 h after El-St in modified Whitten's medium with 256 mOsmol (mWM256), mKRB with 309 mOsmol, and mWM with 309 mOsmol (mWM309), in which the osmolarity was adjusted by addition of NaCl or mannitol, or by reduction of distilled water. In Experiment 2, the diploids were cultured in the five media used in Experiment 1 for the first 48 h, and then in mWM256 until 168 h after El-St. In Experiment 3, the diploids were cultured for the first 48 h in mWM with osmolarity adjusted from 256 to 330 mOsmol by addition of NaCl for the first 48 h and then in mWM256 until 168 h after El-St. In Experiment 4, the diploids were cultured in mWM with 290 mOsmol (mWM290) for the first period of 24, 48, or 72 h, and then in mWM256 until 168 h after El-St. In Experiment 5, after diploids were cultured in mWM290 for the first 48 h, the obtained 4-cell diploids were transferred to mWM with osmolarity adjusted from 200 to 310 mOsmol by addition of NaCl, then cultured until 168 h after El-St. All media were supplemented with 0.5mg/ml hyaluronic acid and 4.0mg/ml bovine serum albumin. The results obtained in Experiments 1-5 indicate that the osmolarity of a medium, but not the Na(+)/K(+) ratio, exerts effects on the development of diploids to the blastocyst stage. The change of osmolarity of the culture media after the 4-cell stage increased the rate of expanded blastocyst formation in porcine diploids. The optimal osmolarities of culture medium for the first 48 h after El-St (before the 4-cell stage) were 290 and 280-320 mOsmol, and those for the later period (after the 4-cell stage) were 256 and 220-270 mOsmol, respectively.

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.

Biochemical characterization of sialoprotein "anti-agglutinin" purified from boar epididymal and seminal plasma.

Sialoprotein "anti-agglutinin," previously shown to inhibit sperm head-to-head agglutination, is found in both boar epididymal and seminal plasma. The present report characterizes anti-agglutinin by mass spectrometry, by N-terminal amino acid sequence analysis, and by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and Western blotting techniques to assess phosphate content of the molecule. Anti-agglutinin had the SDS-PAGE mobility of approximately 25 kDa. By electrospray ionization-mass spectrometry, however, mass spectra of anti-agglutinin were characterized by two major peaks (19,379-19,382 Da and 19,395-19,397 Da) and several minor peaks. Mass spectrometry of tryptic peptide fragments of deglycosylated anti-agglutinin and amino acid sequence analysis revealed that the protein has a unique peptide-mass fingerprinting of fragments (12,668 Da, 5,209 Da, 1,226 Da, and 1,168 Da) and a novel N-terminal amino acid sequence (KTDDY AISGA KEEEF YDYME ELYAV), respectively. Additionally Western blot techniques, using commercially available monoclonal antibodies, were used to detect presence of phosphothreonine and phosphoserine substituents, but two different monoclonal antibodies did not detect phosphotyrosine. Moreover, treatment with two different alkaline phosphotases converted the molecule, as assessed by SDS-PAGE and detection by silver stain, from the parent form of about 25 kDa to forms of approximately 19 kDa (similar to that assigned by mass spectrometry) and/or 15 kDa. Original antiserum generated toward, and reacting with native anti-agglutinin, reacted only with 19 kDa form. These results are consistent with the conclusion that the native anti-agglutinin may be a novel protein that is phosphorylated at serine and/or threonine residues.

Immunolocalization of anti-agglutinin for spermatozoa in boars.

A boar "anti-agglutinin," which inhibits head-to-head agglutination of spermatozoa, has been identified as a 25-kDa sialoprotein contained in epididymal and seminal plasma. This study was conducted to determine the location of the anti-agglutinin on spermatozoa and in various organs, including epididymides, by indirect immunofluorescence and Western blotting techniques. Ejaculated boar spermatozoa were washed and subjected to immunocytochemical observation. Epididymal plasma was recovered from three different regions of epididymides and subjected to sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and Western blotting. Twelve kinds of organs (testis, epididymis, seminal vesicle, prostate, heart, liver, kidney, spleen, stomach, small intestine, lung, and muscle) were recovered from boars. The unilateral epididymides were fixed, cut into 10-microm frozen sections, and subjected to immunohistochemical observation. The other organs were homogenized and used for SDS-PAGE and Western blotting. Immunocytochemical observations revealed that the antiserum strongly recognized the acrosomal region and equatorial segment on unfixed and methanol-fixed spermatozoa. Immunohistochemical observations revealed that the epithelia of the epididymal ducts were recognized by the antiserum mainly in the corpus epididymides. Moreover, the antiserum reacted with the luminal contents of the corpus and cauda epididymides. However, no specific reaction was detected in the caput epididymides. Western blotting showed that the antiserum selectively recognized a band of the anti-agglutinin in the corpus and cauda epididymal plasma, although no band was detected in the caput epididymal plasma. In the extracts from various organs, the single band was detected in the corpus and cauda epididymides at the same mobility as the anti-agglutinin, but not in the other organs. Based on these results, the following matters concerning the anti-agglutinin are discussed: (1) the importance of its association with the acrosome of spermatozoa in inhibiting sperm head-to-head agglutination; (2) its origin in the epididymis; and (3) its tissue specificity.

Changes in epididymal protein anti-agglutinin on ejaculated boar spermatozoa during capacitation in vitro.

This study is a detailed investigation of changes in epididymal protein anti-agglutinin on ejaculated boar spermatozoa during an incubation designed to promote capacitation in vitro. Ejaculated spermatozoa were collected from six mature boars, washed, and incubated to promote capacitation. Sperm samples were subjected to Western blotting-densitometric analyses, flow cytometry after immunostaining and immunocytochemical observation by indirect immunofluorescence. An antiserum to anti-agglutinin was raised in a rabbit by subcutaneous injection of a purified antigen, as described previously (Harayama et al. 1999). Western blotting-densitometric analyses revealed an approximate halving of the amount of sperm-bound anti-agglutinin during the first 45-min incubation, followed by a gradual decrease thereafter. Comparison between immunostained sperm samples by flow cytometry before and after incubation confirmed this decrease in sperm-bound anti-agglutinin during the incubation. Microscopic characterization established that this decrease occurred mainly on the acrosome. Supplementation with seminal plasma (5% or 10%, v/v) attenuated the decrease. These findings are consistent with the conclusion that a large portion of the anti-agglutinin bound to sperm acrosomes is released at an early stage of the capacitation process in vitro.

Influence of calcium on proliferation and phenotype alteration of cardiomyocyte in vitro.

An accelerated weight gain is noted in the heart of Ca-deficient, hypertensive chick embryos maintained in a shell-less culture in vitro. We previously observed that the Ca handling property of cardiomyocytes isolated from the shell-less embryo is altered, i.e., faster Ca uptake, suggesting a requirement for adequate Ca supply and/or proper Ca handling in embryonic cardiac development. In this study, we have examined the function of Ca on cardiomyocytes by analyzing the effects of 1) various Ca concentration in the culture medium (NCa, 1.8 mmol/ L; HCa, 2.8 mmol/L; LCa, 0.9 mmol/L), and 2) various modulators of Ca handling on cell proliferation and phenotype regulation in chick embryonic cardiomyocytes. The analytical parameters included cell number, DNA content, expression of cell cycle-specific and cardiomyocyte-specific proteins, and creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) enzyme activities. Cell number and total DNA were significantly larger (P < 0.01) in LCa cultures compared with those in NCa. The level of LDH was elevated (P < 0.01), but that of CPK was lowered in LCa. Expression of the G1-S-specific protein PCNA was raised, but that of the contractile proteins myosin and tropomyosin was substantially suppressed in LCa; in HCa, the cells did not proliferate as well, whereas the level of contractile proteins was higher. Thapsigargin, a sarcoplasmic reticulum (SR)-specific, Ca-ATPase inhibitor, simulated the effects of LCa by enhancing cell proliferation and lowering the expression of tropomyosin. These results suggest that culturing in low Ca concentration and inhibition of SR Ca pumping enhance myocardial cell proliferation and suppress sarcomeric protein expression, perhaps by inducing cellular de-differentiation. The in vitro effects of medium Ca concentration and Ca handling modulators on cardiomyocytes also suggest that the in vivo cardiomegaly of the SL embryos is a direct result of Ca-deficiency, and that Ca is important in the phenotype regulation of cardiomyocytes.

Effects of calcium and bicarbonate on head-to-head agglutination in ejaculated boar spermatozoa.

The present study was conducted to reveal the effects of calcium and bicarbonate on the occurrence of head-to-head agglutination in ejaculated boar spermatozoa in vitro. Boar spermatozoa were washed and incubated in a modified Krebs-Ringer bicarbonate (mKRB) in a 37 degrees C CO2 incubator (5% CO2 in air) for 1-5 h. Before and after the incubation, aliquots of each sperm sample were fixed, smeared on glass slides, and stained with a phosphate-buffered solution of Giemsa to assess the percentages of head-to-head agglutinated spermatozoa. Before the incubation, only 5-12% of the spermatozoa were agglutinated. After the 1-h incubation, however, the percentage of head-to-head agglutinated spermatozoa rose to approximately 50%, followed by only minor increases thereafter. This rise was dependent on the concentrations of calcium chloride contained in the mKRB and was attenuated by the addition of 2 mM [ethylenebis(oxyethylenenitrilo)]tetra-acetic acid (EGTA) to the medium. Moreover, the replacement of sodium bicarbonate with 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (Hepes) in the medium and treatment with ruthenium red, which have both been shown previously to inhibit calcium uptake by boar spermatozoa, significantly reduced the rise. Based on these findings, it was concluded that extracellular calcium and bicarbonate are key factors regulating head-to-head agglutination in boar spermatozoa. The possible relationship between agglutinability and the fertilizing ability of boar spermatozoa is also discussed.

[A case of multiple sclerosis with galactorrhea-amenorrhea syndrome].

Patients with multiple sclerosis sometimes show subthalamic lesions presenting syndrome of inappropriate secretion of ADH (SIADH), hypothermia, hyperprolactinemia, weight loss, and cachexia. Hyperprolactinemia also has been found in the patients with active systemic lupus erythematosus, because prolactin can be produced from human activated lymphocytes. We described a case of multiple sclerosis showing galactorrhea-amenorrhea syndrome with hyperprolactinemia. A 31-year-old woman showed a high level of prolactin in the serum (79.6 ng/ml) during remission stage 5 months after the onset of multiple sclerosis. She showed galactorrhea-amenorrhea syndrome 3 years later. She showed dysesthesia in her limbs, relapsing monoparesis, visual disturbance and Gd-enhanced plaques in Brain MRI for 6 years. She was admitted to our hospital on November 24, 1995. A neurological examination showed hyporeflexia of the upper extremities, hyperreflexia of the lower extremities, bilateral ankle clonus, truncal ataxia, and neurogenic bladder. Laboratory tests revealed increased level of serum prolactin, exaggerated secretion of serum prolactin after intravenous injection of 500 micrograms TRH, and marked suppression after oral administration of 2.5 mg bromocriptine. Brain MRI showed demyelinating lesions near the lateral ventricle, and cervical MRI (T2 image) showed high signal intensity lesions in the spinal cord from C2 to C5. In the previous case, galactorrhea-amenorrhea syndrome was found during the exacerbation stage of multiple sclerosis. Hyperprolactinemia may be caused from subthalamic lesions or by activated lymphocytes in multiple sclerosis. We considered that hyperprolactinemia and galactorrhea-amenorrhea syndrome in our patient might be caused from subthalamic lesions because lymphocytes were not activated during the remission stage of multiple sclerosis.

Function of FK506 binding protein (FKBP) in chick embryonic cardiac development.

FK506 binding protein (BP) 12, an immunophilin of FK506-binding proteins, is involved in intra-cellular signal transduction through the calcineurin-nuclear factor pathway. FKBP12 is reported to be associated with the ryanodine-receptor and IP3 Ca2+ channels, and to regulate cell proliferation via binding transforming growth factor (TGF)-beta receptor and cyclin dependent kinase (CDK). To elucidate the function of FKBP12 in cardiac development, we analyzed the temporal profile and regulation of FKBP12 expression in chick heart and in cultured cardiomyocytes. FKBP12 is expressed in embryos as early as day 4 and is predominantly associated with cardiomyocytes and osteo-chondrocytes. Tissue FKBP level in the heart increases with development. Immunohistochemically, the distribution and levels of FKBP12 appear to be related to sarco-endoplasmic reticulum Ca-ATPase 2 (SERCA2) but not to sarcomeric proteins. In proliferating cells, FKBP12 expression correlates with cellular mitosis, but not with DNA synthesis. In earlier embryos (< day 8), suppressing the activity of FKBP by FK506 administration is lethal, and induces cardiomegaly at later stages. In cultured cardiomyocytes, FK506 reduces the level of contractile proteins and inhibits cell proliferation. These results show that FKBP12 is enriched in cell types involved in dynamic Ca handling, and is likely an important molecule for cardiac development. FKBP12 most likely functions by affecting cellular Ca handling, since its effects are modified by modulators of Ca handling by sarcoplasmic reticulum.

[P300 findings in patients with corticobasal degeneration].

An auditory discrimination paradigm was employed to elicit the P300 component of the event-related potentials from three patients with clinically diagnosed corticobasal degeneration (CBD). Presenile and senile dementia of Alzheimer type (DAT), vascular dementia (VD) and age-matched control subjects were also examined. All patients with CBD manifested limb kinetic apraxia, hemiparkinsonism and mild dementia. Brain MRI revealed marked parietal atrophy, which was predominant in contralateral to the extremities with severe motor dysfunction. 123I-IMP SPECT analysis showed asymmetric reduction of cerebral blood flow, in accordance with the MRI findings. The P300 latencies in the two CBD patients were markedly prolonged compared to those of DAT and VD patients, although they were not as severely demented as DAT and VD patient. It should be noted that the P300 was not detected in one patient with CBD. The latencies of the N100 were not significantly different among the all groups. These abnormal findings of the P300 may be characteristic in CBD, and, furthermore, may be associated with the parietal lobe atrophy and low blood flow in cortex and thalamus revealed by brain MRI and SPECT.

Atrial natriuretic peptide accelerates proliferation of chick embryonic cardiomyocytes in vitro.

The developing embryonic heart has been reported to contain significant levels of atrial natriuretic peptide (ANP). In this study, the role of ANP in cardiac development was evaluated using cultured cardiomyocytes isolated from chick embryos. We analyzed the effect of ANP on cell number, DNA synthesis, total RNA level, the expression of cell-cycle-specific and sarcomeric proteins, and levels of lactate dehydrogenase and creatine phosphokinase. ANP increased overall DNA synthesis (measured by BrdU incorporation, P < 0.01) and enhanced cell proliferation. Morphologically, the development of the cardiomyocyte network was distinctly enhanced in the ANP-treated cells. Cellular RNA content was elevated; likewise, myosin and tropomyosin biosynthesis was significantly greater in ANP-treated cells. In addition, expression of G1/S-specific protein increased, whereas G2/M-specific protein remained unchanged by ANP treatment. An antibody against ANP and a specific ANP receptor antagonist, HS-142-1, antagonized and/or attenuated the action of ANP on both cell proliferation and protein biosynthesis. These results indicate that ANP accelerates myocardial cell proliferation by enhancing entry into S phase and by increasing DNA synthesis during S phase specifically through receptor mediated pathway. The in vitro effects of ANP on myocardial cell proliferation, together with the elevated levels of ANP seen in vivo during normal heart formation, suggest a possible autocrine function of ANP in embryonic cardiac development.

Electrophoretic characterization of boar epididymal antiagglutinin.

Boar epididymal antiagglutinin, previously shown to inhibit sperm head-to-head agglutination, was purified from cauda epididymal plasma by precipitation with ammonium sulfate, anion-exchange chromatography, and reverse-phase HPLC, and was characterized by electrophoretic and membrane blotting techniques. Blotting techniques, using the ECL Glycoprotein Detection System (Amersham Life Science, Buckinghamshire, UK) and wheat germ agglutinin (WGA)-peroxidase, established the presence of sialic acid residues on purified antiagglutinin. Removal of sialic acid residues from antiagglutinin greatly reduced its immunoreactivity with the specific antiserum. Further purification by two-dimensional PAGE established the presence of one major and two minor forms that cross-reacted with the antiserum, with only the major form reacting with WGA-peroxidase. Extracts of washed epididymal spermatozoa contained a polypeptide with the same electrophoretic mobility as the major form. Additionally, the antiserum detected cross-reacting material in seminal plasma and in extracts from ejaculated spermatozoa. When spermatozoa were incubated under conditions shown to promote capacitation, the cross-reacting material could not be detected in sperm extracts. These results are consistent with the following conclusions: 1) antiagglutinin contains sialic acid residues that may be related to its immunoreactivity and molecular heterogeneity, and 2) either sperm-bound antiagglutinin is released or its epitope recognized by the antiserum is altered after ejaculation and in vitro capacitation.

Major risk factors for atherosclerosis are manifested in experimental Ca-deficiency.

The eggshell is the major source of Ca required during growth of chick embryos. Therefore, chick embryos placed ex ovo for long-term (SL) are rendered severe systemic calcium deficiency. We report here that SL chick embryos express Ca-deficiency related atherogenic disorders, and that in vitro Ca-deficiency induces dedifferentiation, i.e. loss of cell-type specific features and accelerated proliferative activities, in the various types of cultured cells. Systemic blood pressure is significantly higher and an accelerated weight gain of the heart is noted in SL compared to normal embryos (NL) at the incubation Day-14. Plasma cholesterol was lower, while triglyceride and glucose were higher in SL. Varying Ca in the culture medium (FCa, 1.8 mM; HCa, 2.8 mM; Ca/2, 0.9 mM) clearly affected the phenotype of the cultured cardiomyocytes and vascular cells isolated from the chick embryos. The cell number and total DNA were significantly larger and the level of LDH and proliferating cell nuclear antigen (PCNA) was elevated in Ca/2 compared to FCa. On the contrary, the level of CPK and contractile proteins were lowered in Ca/2. Thus, it is indicated that Ca-deficiency induces atherogenic disorders in vivo, and accelerates cell proliferation and decelerates sarcomeric protein expression in vitro. Taken together, it is suggested that the atherogenic, developmental disorders in SL may be the integrated result of the phenotype alteration in the various cell types directly induced by Ca-deficiency.

Fructose stimulates shedding of cytoplasmic droplets from epididymal boar spermatozoa.

The aim of the present study was to establish the presence of an inducer(s) for the shedding of cytoplasmic droplets from boar spermatozoa after ejaculation. Cauda epididymal spermatozoa were incubated with seminal plasma, seminal vesicular fluid (SVF) or chemical agents at 39 degrees C for 30 min. After fixation and staining, percentages of spermatozoa without a droplet were determined. In the samples incubated with seminal plasma, SVF and a filtrate of SVF obtained after passage through an ultrafilter (molecular weight cut-off, 10,000), 43%, 60-69% and 43% of the spermatozoa were without a droplet respectively. The percentage of spermatozoa without a droplet after incubation with D-fructose (1.0 mM), which was one of the energy substrates included in SVF, was 76%. Furthermore, percentages increased to 93% and 90% with the addition of caffeine (2.0 mM) and N6, 2'-O-dibutyryl cyclic adenosine 3',5'-monophosphate (1.0 mM), respectively, but decreased to 48% with the addition of imidazole (2.0 mM). Based on these results, it is suggested that the shedding of cytoplasmic droplets from boar spermatozoa is induced by fructose originating from SVF. It also appears that this event is mediated by increasing the concentration of intracellular cyclic adenosine 3',5'-monophosphate.

Identification of anti-agglutinin for spermatozoa in epididymal boar plasma.

The present report identifies epididymal boar anti-agglutinin and examines its effect on sperm motility. Boar spermatozoa from the cauda epididymidis were washed and incubated in modified Krebs-Ringer bicarbonate at 37 degrees C (5% CO2 in air). In the samples washed three or five times and then incubated for 3-5 h, higher rates (72-79%) of spermatozoa were associated with one another at the acrosomal region, mainly in groups of 2-5 cells (head-to-head agglutination), and many cells exhibited intensively flagellant and/or circular types of movement but rarely progressive motility. The addition of epididymal plasma or 25 kDa protein purified from it markedly inhibited the occurrence of head-to-head agglutination in washed spermatozoa, whereas heat treatment and subsequent removal of insoluble materials reduced the anti-agglutination activity of epididymal plasma. The percentages of progressively motile cells in the samples incubated with epididymal plasma or 25 kDa epididymal protein rose coincident with the reduction of sperm agglutination. These findings demonstrate that the 25 kDa epididymal protein is an anti-agglutinin for the cauda spermatozoa and that it effectively functions to maintain progressive motility of the cells in vitro.

Capacity of rete testicular and cauda epididymal boar spermatozoa to undergo the acrosome reaction and subsequent fusion with egg plasma membrane.

The capacity to undergo the acrosome reaction and subsequent fusion with egg plasma membrane was examined in rete testicular and cauda epididymal spermatozoa from boars. Sperm penetration assay using zona-free hamster eggs demonstrated that the penetration rates for rete testicular spermatozoa preincubated for induction of the acrosome reaction for 2 and 3 h were 55% and 97%, respectively. However, most of the eggs (93%) were penetrated with polyspermy by cauda epididymal cells preincubated for 2 h. Results obtained by the triple-stain technique revealed the percentages of acrosome-reacted spermatozoa in the rete testicular and cauda epididymal samples preincubated for 3 h to be 61% and 74%, respectively. These results indicate that many rete testicular spermatozoa possess the capacity to undergo the acrosome reaction and subsequent fusion with egg plasma membrane in vitro, which appears to be completely established only after sperm transit through at least the proximal part of the epididymis.