Seminal Plasma Biochemistry I: Preliminary Report: A Possible Mechanism for the Liquefaction of Human Seminal Plasma and its Relationship to Spermatozoal Motility (original) (raw)
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Human sperm hyperactivation and capacitation as parts of an oxidative process☆
Free Radical Biology and Medicine, 1993
Capacitation of spermatozoa is essential for fertilization and is visually characterized by hyperactivated motility. Previous reports have shown that foetal cord serum (FCS) and superoxide anion, 02, can trigger human sperm hyperactivation (HA) and capacitation and that superoxide dismutase (SOD) could prevent these processes. We investigated further the role of 02-and FCS components in human sperm HA and capacitation. Percoll-washed spermatozoa were incubated, at 37°C, in Ham's F-l0 medium with 7.5% of FCS, dialyzed FCS (> 12 kD), ultrafiltrate from FCS (FCSu; < 3 kD), or xanthine + xanthine oxidase + catalase (X + XO + cat). Spermatozoa incubated with FCSu were also supplemented with catalase to prevent the loss of motility often observed after 2-3 h of incubation. FCS and dialyzed FCS induced significant levels of HA (10 +_ 1% and 7.7 _+ 0.7%, respectively) that were, however, lower than those observed with FCSu (19 +_ 1%) or X + XO + cat (16 _+ 2%). Similar results were obtained when the lysophosphatidylcholine-induced acrosome reaction (LPC-AR, a measure of sperm capacitation) was evaluated. The presence of SOD in the incubation medium blocked the induction of HA and capacitation by FCS, FCSu, X + XO + cat, as well as the spontaneous HA and capacitation. The enzymatic activity of SOD was needed for the prevention of these processes. Desferrioxamine, up to 100 t~M, had no effect on HA and LPC-AR induced by FCSu and X + XO + cat. Addition of SOD to already hyperactivated spermatozoa reversed the HA. These data suggest that spermatozoa need a sustained O~-generation to maintain HA and proceed to capacitation. We hypothesize that FCSu or the O~-generated by X + XO + cat activate enzymes, possibly a reduced nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase at the level of sperm membrane.
Testicles were isolated from thirty five apparently healthy dromedary camels (Camelus dromedarius), aged between 5 to 18 years, in a local slaughterhouse during the rutting season. Epididymal fluid was collected from one epididymis for determination of twelve biochemical and antioxidant parameters using ELISA commercial kits. Spermatozoa were harvested from each region of the other epididymis (head, body and tail) and stored in SHOTOR ® , Green buffer ® ϩ 20% egg yolk and INRA-96 ® extenders at 5 and 30°C. Results revealed that, in the epididymal fluid, concentrations of testosterone, glucose, albumin, total protein, cholesterol, fatty acids, iron, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were 5.19 Ϯ 1.69 ng/mL, 3.10 Ϯ 0.41 mmol/L, 6.26 Ϯ 1.26 g/dL, 0.50 Ϯ 0.07 mg/dL, 1.74 Ϯ 0.09 mmol/L, 6.62 Ϯ 0.81 nmol/ul, 926.20 Ϯ 100.18 ug/dL, 51.17 Ϯ 7.74 mIU/ml, and 143.16 Ϯ 18.67 mIU/ml, respectively. The antioxidants activities of catalase, glutathione peroxidase (GPx) and superoxide dismutase (SOD) in the epididymal fluid were 121.55 Ϯ 6.57 nmol/min/ml, 59.35 Ϯ 10.98 nmol/min/ml and 0.18 Ϯ 0.03 U/ml, respectively. Epididymal sperm motility and concentration were higher (P Ͻ 0.05) in the body and tail than the head. The viability indices of total and forward sperm motility, at 5 and 30°C, obtained from the tail region were superior (P Ͻ 0.05) in both SHOTOR ® and INRA-96 ® extenders than Green buffer extender. It may be concluded that INRA-96 ® extender is the best for storing dromedary epididymal spermatozoa at 5 and 30°C. Crown
Enzyme Comparative Study of Spermatozoa and Seminal Plasma in Normal and Subfertile Man
Systems Biology in Reproductive Medicine, 1979
A semiquantitative colorimetric micromethod (APIZYM) was used to study the enzyme profiles of seminal plasma and of spermatozoa. Reactions with 65 different substrates are simultaneously tested in a single specimen. These substrates (principally naphtolic) allow the detection of hydrolytic enzymes (esterases, phosphatases, and peptidases) and of dehydrogenases potentially involved in sperm metabolism and in the process of fertilization. The usual sperm enzymes were regularly observed: C,-C, esterases, amino acid arylamidases, acrosine, phosphatases, glutamyl transpeptidase, and various osidases. Among the dehydrogenases we observed a striking predominance of the enzymes of the hexose monophosphate shunt and of LDH. Seminal plasma has an enzyme pattern very similar to that of spermatozoa except for the absence of acrosine and of some dehydrogenases. This unexpected similarity is discussed. The gametes from subfertile donors do not at first sight differ in their enzyme pattern from those from fertile donors. Moreover, we found no marked differences between zymograms of seminal plasma from normal, subfertile, or even azoospermic patients. Deep freezing does not modify the hydrolytic enzymes of human sperm either quantitatively of qualitatively, but the dehydrogenases of the hexose monophosphate shunt are adversely affected (60% loss of activity for G 6 PDH, and 30% for 6 PGDH); LDH is not affected. The consequences on fertilizing capacity of frozen semen are discussed.
Catalase-like and superoxide dismutase-like activities in human seminal plasma
Urological Research, 2002
Human spermatozoa are highly susceptible to oxidative injury but are naturally protected from such injury by the antioxidant properties of seminal plasma. We measured catalase-like and superoxide dismutase (SOD)-like activities in the seminal plasma of fertile and vasectomized men in order to gain insight into the potential source(s) and function(s) of these antioxidants in semen. Semen samples were obtained from fertile men (n=11) and men post-vasectomy (n=16). Catalase-like activity was measured by the decrease in hydrogen peroxide concentration after incubation with seminal plasma. SOD-like activity was measured as the inhibition of nitroblue tetrazolium reduction due to superoxide anion generation by xanthine plus xanthine oxidase. Mean seminal catalase-like activity (±1SD) in the fertile group was not significantly different from that of the post-vasectomy group (389±163 and 325±119 U/ml, respectively). Similarly, mean seminal SOD-like activity in the fertile group was not significantly different from that of the post-vasectomy group (37±10 and 36±10 U/ml, respectively). Our data suggest that the testis and epididymis are not an important source of catalase-like and SOD-like activities in semen. These findings indicate that antioxidants in semen are primarily of post-testicular origin and probably serve to protect ejaculated spermatozoa from oxidative stress such as that which occurs in the female reproductive tract.
The Journal of urology, 2002
Semenogelin (Sg), the major protein of the human semen coagulum, is present at high concentrations in seminal vesicle secretions. It is degraded by the prostate-specific antigen (PSA) to generate peptides of various biological activities that were found on and inside spermatozoa. Our aim was to determine the effect of Sg on capacitation, which is the series of transformations that spermatozoa must undergo to become fertile. At concentrations of 0.1 to 1.0 mg/mL (600-to 20-fold lower than those of semen), Sg did not affect sperm motility (%) but completely prevented capacitation induced by fetal cord serum ultrafiltrate; a partial inhibition of capacitation was noted with 0.03 mg Sg/mL. There was also a dose-dependent decrease in the tyrosine phosphorylation of fibrous sheath proteins and in the O 2 Ϫ •-related chemiluminescence. Ribonuclease (RNase), which has as high an isoelectric point (pI ϭ 9.7) as Sg (pI ϭ 9.5), also prevented sperm capacitation and O 2 Ϫ •-related chemiluminescence but to a lower extent, suggesting that one mechanism of Sg action on spermatozoa could be related to its positive charge at physiological pH. Sg at 1, but not 0.3 or 0.1 mg/mL, scavenged the O 2 Ϫ • generated by the mix of xanthine ϩ xanthine oxidase and modified the kinetics of the reaction; RNase did not have such effects. Therefore, Sg is a potential scavenger for O 2 Ϫ • but probably also affects the sperm oxidase. Spermatozoa rapidly processed Sg; a high proportion of Sg was degraded after 15 minutes of incubation. The resulting polypeptide patterns were reminiscent of those obtained with PSA as a proteolytic enzyme. These data suggest that Sg, its degradation products, or both may be natural regulators of sperm capacitation and could prevent this process from occurring prematurely. One mechanism by which Sg acts could involve an interference with the O 2 Ϫ • that is normally generated during this process.
Free Radical Biology and Medicine, 2020
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Clinical Chemistry and Laboratory Medicine, 2000
Activities of total lactate dehydrogenase, which plays an important role in providing energy for cell metabolism, lactate dehydrogenase-X, an isoenzyme of lactate dehydrogenase supposed to be specific for germinal epithelium activity, diluted semen absorbance and some other properties have been measured in 51 seminal plasma samples. Aiming to study its clinical use as a marker of seminiferous epithelium activity, determination of isoenzyme-X and investigation of correlation between it and the spermiogram properties were carried out. Besides lactate dehydrogenase and lactate dehydrogenase-X activity, their ratio and diluted semen absorbance were correlated with the different properties of the spermiogram, the best correlation of the enzymes being obtained with the total sperm count and motile sperm count. Correlation of diluted semen absorbance with the total sperm count and motile sperm count was also noted, whereas it did not correlate with sperm concentration and motility rate. The data suggests the clinical utility of these properties as reliable markers for both germinal activity and spermatozoid quality when "total sperm count" and "motile sperm count" are used to define sperm properties instead of just sperm concentration and motility rate.
Effects of antioxidants on human sperm preparationtechniques
Int J Androl, 1994
The effect of two different sperm preparation techniques, Percoll gradient centrifugation and swim-up from a washed pellet were tested on the functional competence of the selected spermatozoa. Percoll gradient centrifugation brought about an improvement in sperm motility parameters such as curvilinear velocity and straight-line velocity, an increase in the rates of hyperactivation and the acrosome reaction and an increase in the percentage of motile spermatozoa after 24 h of incubation compared to the centrifugation, swim-up procedure. The effects of antioxidants such as dithiothreitol (DTT) o r reduced glutathione (GSH), and reactive oxygen species-scavenging enzymes such as catalase or superoxide dismutase (SOD) during the stage of centrifugation before the swim-up procedure were also studied. Though all of these agents prevented the fall in sperm motility after 24 h incubation, only DTT and SOD improved the rates of both hyperactivation and the acrosome reaction. GSH also improved the acrosome reaction, whereas catalase was without significant effect on the rates of hyperactivation or the acrosome reaction. These results indicate that Percoll gradient centrifugation selects spermatozoa with better functional competence than does centrifugation swim-up. The damage caused by the centrifugation can be prevented by the addition of antioxidants, suggesting that the differences noted with the Percoll gradient method was due to better protection against peroxidative damage due to the centrifugation of unselected spermatozoa.' However, the use of DTT is limited by virtue of the fact that this sulphydryl reducing agent leads to destabilization of the sperm chromatin. In contrast, GSH and SOD could have therapeutic potential.
Int J Androl, 1993
Capacitation of spermatozoa is essential for fertilization, and can be induced by various agents or biological fluids. Previous reports have shown that foetal cord serum (FCS) and the superoxide anion trigger human sperm hyperactivation and capacitation, and that superoxide dismutase (SOD) prevents these processes. We investigated: (1) the capacity of seminal plasma (SP) and follicular fluid (FF) (whole, or fractionated into high and low molecular weight components), in the presence or absence of SOD, to induce the spontaneous acrosome reaction (no stimulant needed, AR) and capacitation (as measured by the lysophosphatidylcholine-induced AR, LPC-AR); (2) a possible relationship between the levels of AR and capacitation obtained with these biological fluids and the superoxide scavenging capacity of the same fluids. The highest levels of LPC-AR were obtained with FF ultrafiltrate (48 f 6%), followed by SP ultrafiltrate (31.9 f 0.8%), FF (30 k 5%), dialysed FF (27 k 4%), and finally, by FCS ultrafiltrate (23 f lo/.), SP (21 f 1%) and dialysed SP (18.9 k 0.8%). A similar order ofpotency for the fluids existed when sperm AR was studied, the levels of AR observed ranging from 26 f 2% to 5.3 f 0.8% after incubation with FF ultrafiltrate and SP respectively. None of these treatments had detrimental effects on sperm motility. In the presence of SOD, there was always an important reduction (52-86%) of the AR and LPC-AR observed. A highly significant inverse linear relationship was observed between the SOD-like activity of the fluids tested and the AR (Y = 0.86, p < 0.001) and LPC-AR (Y= 0.91, p < 0.001) observed in the presence of these fluids. The results suggest that biological fluids contain inducers of the AR and capacitation, and that these probably act by a common mechanism, possibly by direct or indirect induction of an NADPH oxidase in the sperm membrane. The data suggest strongly that the SOD-like activity of a specific fluid is probably one of the most important factors that will determine its capacity to induce the AR and capacitation. Superoxide and human sperm capacitation 259