Electrophoresis of NADH-dependent oxidoreductase (diaphorase) in boar spermatozoa: Elektrophorese der NADH-abhängigen Oxydoreduktase (Diaphorase) in Eberspermatozoen

Two forms of NADH-dependent oxidoreductase (diaphorase [EC.1.6.99.-]) are established in boar spermatozoa. The first form is typical for soluble proteins with a varying electrophoretic profile, while the other form for sedimental proteins with a specific, slowly-moving fraction, which is not common for the soluble form. The two enzyme forms have a close isoelectric point (pI5.5-6.0) and they can not be inhibited by dicumarol 10(-5) mol l-1 and FAD 10(-4) mol l-1. The molecular mass of the soluble form of the enzyme is 28, 37, 46 and 67 kD, while of the sedimental form it is 220, 250 and 260 kD, respectively.     

Gonadal and Extragonadal Sperm Reserves of the Brazilian Nelore Zebu (Bos indicus)

Summary: Gonadal and extragonadal sperm reserves were estimated through hemocytometric method in six Nelore zebu bulls, aging 4–6 years, with normal spermatogenesis, and kept at sexual rest. Gonadal sperm reserve was estimated to be 47.8 ± 5.8 times 10⁶ sperm cells/g testis parenchyma and 9.8 ± 1.7 times 10⁹ sperm cells/testis. Using a time divisor of 4.94 days the daily sperm production was estimated to be 10.0 ± 0.9 times 10⁶ sperm cells/g testis parenchyma/day and 2.0 ± 0.3 times 10⁹ sperm cells/testis/day. Epididymal sperm reserve amounted 11.9 ± 1.6 times 10⁹ spermatozoa/organ, distributed as follows: 35.3 ± 3.6% in the head, 16.9 ± 1.7% in the body and 47.7 ± 3.7% in the tail.
Zusammenfassung: Gonadale und extragonadale Spermareserven des brasilianischen Nelore-Zebu (Bos indicus)
Bei sechs Nelore-Zebubullen im Alter von vier bis sechs Jahren mit normaler Spermatogenese und unter sexueller Karenz wurden mit einer haemocytometrischen Methode die gonadalen und extragonadalen Spermareserven bestimmt. Für die gonadale Spermareserve wurden Werte von 47.8 ± 5.8 times 10⁶ Spermatozoen/g Hodenparenchym und 9.8 ± 1.7 times 10⁹ Spermatozoen/Hoden gefunden. Unter Benutzung eines Zeitdivisors von 4.94 Tagen berechnet sich die tägliche Spermaproduktion zu 10.0 ± 0.9 times 10⁶ Spermatozoen/g Hodenparenchym/Tag und 2.0 ± 0.3 times 10⁹ Spermatozoen/Hoden/Tag. Die Spermareserve im Nebenhoden betrug 11.9 ± 1.6 times 10⁹ Spermatozoen/Nebenhoden in folgender Verteilung: 35.3 ± 3.6% im Nebenhodenkopf, 16.9 ± 1.7% im Nebenhodenkörper und 47.7 ± 3.7% im Nebenhodenschwanz.     

Relation Between Motility and Adenosinetriphosphate (ATP) in Human Spermatozoa

Zusammenhang zwischen Motilität und Adenosintriphosphat (ATP)-Gehalt menschlicher Spermatozoen
Die ATP-Bestimmung beweglicher und unbeweglicher Spermatozoen aus dem Sperma fertiler Männer ergab signifikant höhere Werte für die beweglichen Zellen. Dies deutet darauf hin, daß zur Bereitstellung der von den Gameten benötigten Energie die Aufrechterhaltung eines bestimmten ATP-Spiegels wichtig ist.     

Aspartate transaminase/alanine transaminase (De Ritis ratio) predicts survival in major burn patients

BackgroundAlthough treatment of burn patients has significantly improved in recent decades, major burns remain fatal. Therefore, the evaluation of the death risk of the patients with extensive burns is very important. The ratio between the serum levels of aspartate transaminase and alanine transaminase (De Ritis ratio) was an independent predictor of poor outcomes in patients with acute ischemic stroke, cardiac surgery, non-metastatic renal cell carcinoma, and upper urinary tract urothelial carcinoma. Our aim was to determine whether the ratio between the serum levels of AST and ALT (De Ritis ratio) was useful to assess prognosis in extensively burned patients.MethodsWe conducted a single-center cohort study at the Burns Department of Changhai Hospital. This retrospective observational analysis was performed based on the clinical data of major burn patients admitted between May 1, 2005 and April 30, 2018. Univariate and multivariate logistic regression analyses were performed on variables such as age, sex, total body surface area (TBSA), De Ritis ratio, and serum albumin level, which may affect mortality in major burn patients. We assessed their diagnostic value and found the cut-off value by receiver operative characteristic (ROC) curve analysis. We used the Kaplan–Meier curve to display the impact of the De Ritis ratio and serum albumin level on survival in burn patients.ResultsA total of 351 patients with extensive burns were included in the study. The cohort predominantly consisted of males (74.64%), and most of the patients (78.35%) had been burned by a flame. Age, TBSA, inhalation, and the De Ritis ratio were found to be independent risk factors for the 30-days mortality of major burn patients, while age, TBSA, inhalation, and the De Ritis ratio were independent risk factors for 90-day mortality. Further, the De Ritis ratio was a better mortality predictor than serum albumin in severely burned patients, whose area under ROC for 30-day and 90-day mortality was 0.771 (95% confidence intervals [CI], 0.708–0.835) and 0.750 (95% CI, 0.683, 0.818).ConclusionsThe De Ritis ratio was useful as a prognostic indicator for major burn patients, which can be conveniently obtained through blood examination. Regardless of whether the prediction was for 30-day or 90-day mortality, the accuracy remained high. Moreover, compared to serum albumin level, the De Ritis ratio was superior in assessing the prognosis of extensively burned patients.     

Adenosine 5''-Triphosphate (ATP) Concentration and Acrosin Activity in Human Spermatozoa. Their Relation with the Sperm Penetration Assay (SPA)

Fertility of men depends on the quality of semen. The aim of the present paper is to determine both the acrosin activity by radioimmunoassay and ATP concentration by bioluminescence in human spermatozoa, and evaluate these results in those samples with normal or low sperm penetration according to SPA test. Ejaculates obtained from 42 untreated men, were studied one hour after the obtention. These materials were divided into two groups:20 human semen samples with "in vitro" potentiality to penetrate zone-free hamster ova, between 15% to 98% and 22 human semen with SPA test between 0% to 14%. When we compare the group with normal penetration response vs that group with low or absent penetration ones, a significant decrease of ATP and acrosin concentrations was observed (P less than 0.001). Nevertheless no significant difference was observed in relation with percentage of motility, volume (ml), sperm concentration (10(6)/ml), percent of quick progressive spermatozoa and number of gametes capable of migrating into the medium layer (10(6)), between the group with low or absent penetration test against that one with normal zona-free hamster egg test.     

Adenosinetriphosphate (ATP) in Human Spermatozoa

The Fisher's dispersion analysis test was employed to establish the accuracy limits of spermatozoa concentrations for the evaluation of ATP content of spermatozoa. It was established that the Bücher's method working between 4 times 10⁸ and 1 times 10⁸ spermatozoa, showed no significant variation of the results. With less than 1 times 10⁸ spermatozoa results are significantly different and therefore they are not reliable.     

Inhibition of the acrosome reaction (AR) and fertilization capacity of mouse spermatozoa by norethisterone A-ring reduced metabolite (5alpha-NET)

The contra gestational effects of norethisterone and its main metabolites, 5alpha-NET and 3beta5alpha-NET, has been demonstrated in several species. However, the focus has been mainly on their effects in the uterus. We previously reported that 5alpha-NET inhibits the progesterone-induced AR in pig spermatozoa and induces severe morphological damage to fertilized mouse oocytes. In the present study, we analysed the effects of these compounds on the fertilization process in vitro. Oocytes and spermatozoa were obtained from Balb/c female and C57BL/6J male mice, respectively. Both, the AR assays and the fertilization experiments were performed under different steroid treatment schemes using progesterone as a control. Results showed that norethisterone induced the AR, while 5alpha-NET reduced the percentage of spermatozoa that had undergone progesterone-induced AR. Both 17beta-estradiol and 3beta5alpha-NET induced the AR in a considerably lower percentage of spermatozoa than progesterone. In addition, when 5alpha-NET was added to the medium simultaneously with progesterone at the moment of fertilization, the percentage of fertilized oocytes (two-cell stage) decreased by as much as 77% as compared with the control progesterone-treated group. All results suggest that these compounds can have important effects on the fertilization process.     

Morphometric analysis of llama (Lama glama) sperm head

Llama production in Argentina has increased, as the international interest in breeding this type of animals has grown in the last years. Considering the great polymorphism that llama spermatozoa present at evaluation using light microscopy, the aim of this study was to objectively evaluate llama sperm head morphometry using digital morphometric analysis. Five ejaculates from each of eight males were obtained to evaluate morphometric parameters of 8000 sperm heads stained with Tinción 15^®. The following average results were obtained for each parameter: size parameters: area 20.09 μm², length 6.60 μm, width 4.14 μm, equivalent circle diameter 5.06 μm, curve length 5.79 μm and curve width 3.48 μm; boundary parameters: perimeter 18.54 μm and convex perimeter 17.34 μm; and shape parameters: roundness 1.28 and elongation 1.59. Morphometric parameters of sperm head were compared between ejaculates of the same male and between males. Significant differences between ejaculates of the same male were found for all parameters evaluated (P < 0.01). Significant differences between males were found for all morphometric parameters (P < 0.01) except for curve length, curve width and perimeter. The differences detected would indicate that there is not a single morphometric pattern for Lama glama sperm head, because parameter values cannot be standardised.     

Activity and Localization of NADH-dependant Oxidoreductase (Diaphorase) in Boar Spermatozoa

Investigations were carried out into the activity and localization of NADH-dependant diaphorase in boar spermatozoa. Semen samples were collected from healthy boars, used in A.I. centers. The enzyme was extracted with distilled water and Triton X-100. Two forms of diaphorase were found-water-soluble and Triton X-100 soluble, showing low activity-0.36 U/ml and 0.26 U/ml. The enzyme was localized in the mitochondria, manifesting different intensities of reaction between sperm cells in the same ejaculate. It was found, that a part of the mitochondria and outer doublets showed positive reaction. It is suggested that the enzyme regulates the ratio between reduced and oxidized forms of NADH, takes part in the energy balance and possibly in the mechanism of sperm motility.     

Fluorescence Supravital Stain of Human Sperm: Correlation with Sperm Motility Measured by a Transmembrane Migration Method/Supravital-Fluoreszenz-Färbung von menschlichen Spermatozoen: Korrelation zur Spermatozoenmotilität (Messung mittels Transmembran-Migration-Methode)

We developed a supravital stain of human sperm with fluorescent dyes. Either Hoechst 33,258 or fluorescein isothiocyanate could be used, the former stained sperm head while the later stained the whole sperm. Sperm vitality assessed with any of these two fluorescent dyes correlated well with that determined by eosin-nigrosin counterstain. When sperm vitality was compared with sperm motility measured with a transmembrane migration method, we found that many vital sperm were immotile because sperm vitality was higher than sperm motility in tested samples.     

Deoxyribonucleic Acid (DNA) Content of Morphologically Different Sperm Types from Normal and Subfertile Human Males*

Desoxyribonukleinsäure-Gehalt morphologisch unterschiedlicher Spermatozoentypen bei normalen und subfertilen Männern 20 männliche Patienten (G2), bei denen man eine idiopathische Oligozoospermie festgestellt hatte, und 10 normale, fertile Männer (G***1) wurden untersucht. Der DNA-Gehalt von 50 nach Feulgen gefärbten Spermienköpfen pro Person wurde mit Hilfe eines Einzelzellphotometers gemessen. In einem zweiten Arbeitsgang wurde die Fläche, der Umfang, die Länge und die Breite der gleichen Spermienköpfe mit Hilfe eines halbautomatischen Bildanalyse-gerätes gemessen. Bei einem Vergleich zwischen G1 und G2 unter Berücksichtigung von nur morphologisch normalen Köpfen ist der mittlere DNA-Gehalt nur leicht reduziert in G2. Ganz im Gegen-teil dazu ist die DNA-Variation stark erhöht in G2. Unglücklicherweise, zeigen auch morphologisch defekte Köpfe eine erhöhte DNA-Variation. Bei einer Bestimmung der DNA-Variation in einem Samen mit vielen morphologisch defekten Köpfen ist deshalb eine gemeinsame Bestimmung von DNA-Gehalt und Kopfmorphologie notwendig. Dies kann nur mit Hilfe der Einzelzellzytophotometrie erreicht werden. Auf dieses Problem bezogen ist deshalb die Einzelzellzytophotometrie der Durchflußzytophotometrie überlegen. Viele Sper-mienköpfe mit stark abnormalem DNA-Gehalt zeigen keine abnormen Morphologiemerk-male. Diese subzellulären Veränderungen können nur mit einer DNA-Bestimmung aufge-zeigt werden.     

Electrophoretic patterns of spermatozoal nucleoproteins (NP) in fertile men and infertility patients and comparison with NP of somatic cells

During spermatogenesis in mammals the evolutionary early histones are replaced step by step by more species specific nucleoproteins (protamines). The protamines protect the genetic information of the male against mutagenic or other damaging influences and possibly play a role in male fertility. A method was presented to study the nucleoproteins (NP) of single human ejaculates. This procedure was checked concerning its usability and validity by bull spermatozoa. The NP of cryopreserved human spermatozoa of fertile men and of infertility patients were investigated and compared with the electrophoretic patterns of NP of somatic hepatocytes of rat and mouse. A total of 48 semen samples were studied. Ten of these samples were obtained from semen donors of proven fertility and 38 samples were collected from male partners of infertile couples. Photodensitometrically it could be distinguished between percentages of non-protamines (PNP) and percentages of protamines (PP). The PP varied intra-individually with a mean standard deviation of 34%, analogously to the alterations of classical semen parameters. The PP of fertile men did not significantly differ from the infertility patients with normal spermiogram parameters but showed a significantly higher value compared with the semen samples with pathological spermiograms. On average the PP of all infertile patients were significantly lower than the PP of fertile semen donors.     

谷氨酸脱羧酶发酵工艺的优化

用大肠杆菌AS1.505进行液态发酵生产谷氨酸脱羧酶并优化培养基,考察了碳源、氮源、复合营养物质、起始pH及发酵时间对酶活的影响,确定最佳产酶培养基组成为:葡萄糖1.0 g/dL,蛋白胨3.0 g/dL,氯化钠0.3 g/dL,磷酸氢二钾0.1 g/dL,硫酸镁0.02 g/dL,L-谷氨酸0.01g/dL,玉米浆1.5 g/dL,生物素30 t,g/L,麸皮4 g/dL;pH 6.5.在此基础上,设计发酵条件的优化实验.实验结果表明为:250 mL的三角瓶装液量25 mL,37℃,起始pH 6.5,培养18 h达到产酶高峰,产酶活力可达1 290 U/mL.     

Quantitative （stereological） study on the spermatozoal storage capacity of epididymis in rats and monkeys

Aim: To investigate the spermatozoal production rate of the testis and the spermatozoal storage capacity of the epi-didymis in monkeys and rats. Methods: The number of the late spermatids (steps 13 - 14 in the monkey or steps15 - 19 in the rat) per testis and the number of spermatozoa per epididymis were estimated in 6 normal adult monkeys(Macaca fascicularis) and 6 normal adult SD rats on 25 um-thick methacrylate-embedded sections using a contempo-rary unbiased and efficient stereological method-the optical disector. The diameter and length of the efferent ductulesand ductus epididymidis and the volume of the epididymal fluid in the tubules were also estimated. Results: The totalnumber of the late spermatids per testis was 2902 ±749 (million, x ±s) in the monkey, or 179 ±31 in the rat; thenumber of spermatozoa per epididymis was 3235 ±1835 in the monkey, or 241±76 in the rat. Conclusion: A largenumber of spermatozoa was densely packed and stored in the ductus epididymidis; the epididymal transit t     

Adenosinetriphosphate (ATP) in Human Spermatozoa II. Concentrations in Fertile Men

Adenosintriphosphat in menschlichen Spermatozoen (II) Conzentration bei fertilen Männern
Mit der Hilfe der Bucher-Methode wurden die Norm-Werte für ATP in den Spermatozoen von 24 Männern ermittelt, die ihre Fertilität nachgewiesen hatten und deren Ejakulat den Norm-Werten entsprach (3–5 ml Ejakulat-Menge; über 40 Mill. Spermatozoen/ml; über 60% Motilität; 80% Normalmorphologie; normale biochemische Daten für Fruktose, Zitronensäure und Glyzerylphosphorylcholin). Es kann gezeigt werden, daß der Norm-Wert für ATP 6,5 ± 3,4 μg S.E. pro 10⁸ Spermatozoen beträgt. Bei verschiedenen Spermatozoenpools ergab sich, daß die Inkubation im Spermaplasma bei Raumtemperatur einen progressiven Abfall von ATP in den Keimzellen bewirkt.     

Evaluation of the cryoprotective effect of seminal plasma on llama (Lama glama) spermatozoa

In South American camelids, sperm survival is low after thawing and poor results are obtained when artificial insemination is performed with cryopreserved semen. The aim of this study was to evaluate the effect of different percentages (10% and 50%) of seminal plasma added prior to the process of cryopreservation and also to evaluate the absence of seminal plasma on llama sperm survival after freezing and thawing. A total of 15 ejaculates from five adult llama males (n = 5; r = 3) were evaluated. A significant decrease in sperm motility, viability, membrane function and intact acrosomes was observed in thawed samples (0%, 10% and 50%) when compared to raw semen. Neither morphology nor chromatin condensation was altered in all thawed samples (p > 0.05), but a significant increase (p < 0.05) in the percentage of spermatozoa with fragmented DNA was observed after thawing all samples compared to raw semen. Higher percentages of total and progressive sperm motility were observed when 0% and 10% of seminal plasma were used compared to 50%. However, no statistical differences were established for sperm viability, membrane function, morphology, acrosome status and DNA quality between thawed treatments. To conclude, neither of the percentages of seminal plasma used showed superiority nor cryoprotective effect on llama sperm survival.     

Effects of Methyl Xanthines upon the Spermatozoa Adenosinetriphosphate (ATP) Concentration in Normal Semen

The aim of the present study was to ascertain the effect of methyl xanthines upon the spermatozoa ATP concentration in normal semen in vitro. 26 normal semen were studied. The specimens were diluted after liquefaction with equal volume of Lopata's buffer or Lopata's buffer plus 1 or 6 mM either of caffeine or pentoxifylline. The samples of semen were incubated at room temperature during 90, 180 and 240 minutes before motility and ATP determination by the firefly luciferin-luciferase method. Significant variation was observed in sperm motility nevertheless variations in the ATP concentration was not induced by any of the methyl xanthines we used.     

Purification and characterization of hepatocyte growth factor (HGF) from human seminal plasma

Naturally occurring forms of hepatocyte growth factor/scatter factor (HGF/SF) have been purified by heparin-Sepharose chromatography, followed by cation exchange chromatography from a pool of human seminal plasma. Using an enzyme-linked immunosorbant assay, MDCK scatter assay, and Western blot analysis, it was found that, after heparin-Sepharose chromatography, human HGF/SF present in seminal plasma eluted in two different fractions, between 0.72 and 0.85 M NaCl (fraction I) and between 0.95 and 1.10 M NaCl (fraction II). Further purification of fraction I by cation exchange chromatography resulted again in two fractions which eluted at 0.2-0.4 and at 0.6-0.8 M NaCl. The fraction which eluted at 0.2-0.4 M NaCl consisted of two biologically less active heavy chains of the heterodimeric form of HGF/SF (107.1 U/ng immunoreactive HGF), with approximate molecular weights of 65 and 62 kDa under reducing conditions. The second fraction, which eluted at 0.6-0.8 M NaCl, revealed three bands with molecular weights of 87, 65 and 62 kDa, respectively. The 87 kDa form is thought to be a single chain precursor of HGF/SF devoid of biological activity. After subjecting fraction II to cation exchange chromatography, only one major peak eluted between 0.9 and 1.0 M NaCl, and consisted of two biologically active heavy chains of the heterodimeric form of HGF/SF (708.3 U/ng immunoreactive HGF), with approximate molecular weights of 65 and 62 kDa under reducing conditions. Nonreducing conditions for both fraction I and fraction II revealed only one band with a molecular weight of 68 kDa. The ratio ofpro-HGF/SF and less biologically active HGF/ SF (fraction I) over mature heterodimeric HGF/SF (fraction II) was approximately 1:3, in seminal plasma from sperm donors. In seminal plasma, pro-HGF/SF represents an 87 kDa glycoprotein which, apparently, is converted by limited proteolysis into several bands with molecular weights of 65 and 62 kDa. This is the first report showing the presence of pro-HGF/SF and heterodimeric mature HGF/SF, as well as less biologically active forms of HGF/SF in human seminal plasma.     

Forward motility protein (FMP): localization in hamster epididymal spermatozoa

The presence of FMP was investigated by immunocytochemistry in spermatozoa from the hamster caput and cauda epididymis. Spermatozoa from the caput showed no staining whereas spermatozoa pre-incubated with FMP were stained on the acrosome. Pre-treatment of the same sperm with epididymal plasma induced staining on the principal piece of the flagellum. Spermatozoa from the cauda were stained without previous incubation both on the acrosome and on the principal piece of the flagellum. These results suggest that the action of FMP, which prevents head-to-head agglutination of motile spermatozoa and allows acquisition of forward motility, may be due to at least two proteins. The first localizes to the acrosome during epididymal transit (anti-sticking factor), whilst the second localizes to the principal piece of the flagellum (forward motility initiation factor).