Enrichment of non–apoptotic human spermatozoa after cryopreservation by immunomagnetic cell sorting

Cryopreservation increases the rate of apoptotic spermatozoa withdecreased capability to fertilise oocytes. In order to optimise thefertilisation rates, especially in assisted reproduction the use of apoptoticsperms should be avoided. Early events of apoptosis in cryopreservedspermatozoaare not detectable by conventional methods. However, the surface of apoptoticspermatozoa is characterised by externalisation of phosphatidylserine (PS),which has a high affinity to Annexin V. Therefore, colloid paramagneticAnnexin-V-conjugated microbeads (AN-MB) were tested fortheir ability to eliminate apoptotic spermatozoa from a total of 40 fresh andinTEST yolk buffer cryopreserved semen samples which were provided by 15 healthyvolunteers. By passing through a magnetic field (MiniMACS, Miltenyi Biotec) thesperm suspensions were divided into 2 sperm fractions depending on boundmagnetic Annexin V-microbeads (AN-MB) to spermatozoa. Asadditional markers of apoptosis CD95 (Fas, APO-1) on the sperm surfaceand activated caspases in the cytosol were detected in both fractions.Supplementary investigations comprised eosin-supravital staining andcomputer assisted sperm motion analysis. The separation was supervised by flowcytometric analysis of spermatozoa labelled with FITC-conjugated antiAnnexin V-antibodies.Analyses of the magnetic inactive sperm fraction (AN-MB-negative)showed CD95 on 0.6 ± 0.3% (X ± SEM) of spermatozoa andonly3.2 ± 0.5% were stainable with eosin, whereas, 40.6 ±6.7% of the remaining cells in the column appeared to be CD95 positiveand 99.8 ± 0.1% stainable with eosin after cryopreservation.Indeed the overall amount of CD95 positive spermatozoa did not significantlyincrease after cryopreservation (2.5 ± 0.5% vs. 4.3 ±1.2%; p > 0.05). Activated caspases were found in 21.8 ±2.6% of the spermatozoa in fresh and in 47.7 ± 5.8% ofcryopreserved semen samples (p < 0.01). The separation procedure of thecryopreserved spermatozoa reduced significantly the quantity of thosecontainingactivated caspases to 9.3 ± 2.2% within theAN-MB-negative fraction. In contrast 89.1 ± 2.3% ofAN-MB-positive sperms showed activation of these proteolyticenzymes. Flow cytometric analyses using FITC-conjugated anti AnnexinV-antibodies for monitoring of AN-MB-binding to spermatozoashowed 5.2 ± 1.0% labelled spermatozoa in the AN-MBnegative fraction and 72.6 ± 2.7% labelled spermatozoa in theAN-MB positive one. There was no significant influence of the separationcolumn and the magnetic field on the sperm functions. The passage through thecolumn led to a sperm loss of 0.8 ± 1.2%.Conclusion: The binding of paramagnetic AnnexinV-conjugated microbeads is an excellent method to eliminate spermatozoaat early apoptotic stages from cryopreserved semen samples. A deleteriousinfluence of the separation column and the magnetic field on the spermatozoawasnot observed.     

Acrosome reaction in spermatozoa from hagfish (Agnatha) Eptatretus burgeri and Eptatretus stouti: acrosomal exocytosis and identification of filamentous actin

Spermatozoa of the hagfishes Eptatretus burgeri and Eptatretus stouti, caught in the sea near Japan and North America, respectively, were found to undergo the acrosome reaction, which resulted in the formation of an acrosomal process with a filamentous core. The acrosomal region of spermatozoa of E. stouti exhibited immunofluorescent labeling using an actin antibody. The midpiece also labeled with the antibody. The acrosomal region showed a similar labeling pattern when sperm were probed with tetramethylrhodamine isothyocyanate (TRITC)-phalloidin; the midpiece did not label. Following induction of the acrosome reaction with the calcium (Ca2+) ionophore ionomycin, TRITC-phalloidin labeling was more intense in the acrosomal region, suggesting that the polymerization of actin occurs during formation of the acrosomal process, as seen in many invertebrates. The potential for sperm to undergo acrosomal exocytosis was already acquired by late spermatids. During acrosomal exocytosis, the outer acrosomal membrane and the overlying plasma membrane disappeared and were replaced by an array of vesicles; these resembled an early stage of the acrosome reaction in spermatozoa of higher vertebrates in which no formation of an acrosomal process occurs. It is phylogenetically interesting that such phenomena occur in spermatozoa of hagfish, a primitive vertebrate positioning between invertebrates and high vertebrates.     

CABYR, a novel calcium-binding tyrosine phosphorylation-regulated fibrous sheath protein involved in capacitation

To reach fertilization competence, sperm undergo an incompletely understood series of morphological and molecular maturational processes, termed capacitation, involving, among other processes, protein tyrosine phosphorylation and increased intracellular calcium. Hyperactivated motility and an ability to undergo the acrosome reaction serve as physiological end points to assess successful capacitation. We report here that acidic (pI 4.0) 86-kDa isoforms of a novel, polymorphic, testis-specific protein, designated calcium-binding tyrosine phosphorylation-regulated protein (CABYR), were tyrosine phosphorylated during in vitro capacitation and bound (45)Ca on 2D gels. Acidic 86-kDa calcium-binding forms of CABYR increased during in vitro capacitation, and calcium binding to these acidic forms was abolished by dephosphorylation with alkaline phosphatase. Six variants of CABYR containing two coding regions (CR-A and CR-B) were cloned from human testis cDNA libraries, including five variants with alternative splice deletions. A motif homologous to the RII dimerization domain of PK-A was present in the N-terminus of CR-A in four CABYR variants. A single putative EF handlike motif was noted in CR-A at aas 197-209, while seven potential tyrosine phosphorylation-like sites were noted in CR-A and four in CR-B. Pro-X-X-Pro (PXXP) modules were identified in the N- and C-termini of CR-A and CR-B. CABYR localizes to the principal piece of the human sperm flagellum in association with the fibrous sheath and is the first demonstration of a sperm protein that gains calcium-binding capacity when phosphorylated during capacitation.     

Creatine phosphokinase in domestic cat epididymal spermatozoa

Mammalian spermatozoa that have not completed final testicular sperm maturation have residual cytoplasm and increased creatine phosphokinase (CK) content. This study determined: (1) if CK could be detected by immunostaining cat spermatozoa from the caput, corpus, and cauda epididymis, (2) fluctuations in the proportions of spermatozoa with mature or immature CK-staining patterns during epididymal sperm transit, and (3) how well sperm maturity (as determined by a CK marker) correlated with testicular or epididymal dysfunctions associated with morphological sperm abnormalities. One epididymis was collected from each of 37 cats after orchiectomy and processed immediately to allow sperm morphology evaluations on a 'regional' basis. Sperm released from the contralateral epididymis were evaluated for motility, sperm membrane integrity, and immunostaining with CK-B antibodies. Proportions of spermatozoa with malformed or detached heads, proximal droplets and acrosomal or midpiece abnormalities decreased (P < 0.05) from the caput to the cauda epididymis. In contrast, proportions of spermatozoa that were motile, membrane-intact or with flagellar abnormalities or distal droplets increased (P < 0.05) from the caput to cauda region. Percentages of spermatozoa with an immature CK-staining pattern also decreased (P < 0.05) with epididymal transit (which differs from that reported for the human and stallion). There was no correlation (P > 0.05) between sperm morphology and the CK-staining patterns. In summary, the results reveal that some specific sperm malformations in the domestic cat are of testicular origin, whereas others develop during epididymal transit.     

Motor apparatus in human spermatozoa that lack central pair microtubules

Electron microscopic examination of the spermatozoa from a man suffering from asthenozoospermia (poor or low sperm motility) showed that approximately 92% of the sperm flagella lacked central pair microtubules but possessed dynein arms and radial spokes while a small percentage of the spermatozoa had complete flagella. The characteristics of the motor apparatus of the spermatozoa and the effects of caffeine on the sperm motility were examined, as were the reactivation of demembranated spermatozoa and the sliding of doublet microtubules. Almost all spermatozoa were immotile in a Tyrode solution while only a small percentage of spermatozoa showed slow forward movement or feeble flagellar vibration, whereas addition of caffeine to the sperm suspension induced forward swimming of approximately half of the spermatozoa. The reactivation of demembranated spermatozoa with MgATP(2-) could not succeed because of disintegration of the demembranated flagella. However, when the demembranated spermatozoa were exposed to MgATP(2-) and then treated with elastase, the microtubular doublets of approximately half the number of the flagella slid from the end or middle of the flagella. These results suggest that the motor apparatus in the sperm flagella that lack the central pair microtubules is functionally assembled and intrinsically capable of undergoing flagellar movement but not strong enough to beat normally.     

Mouse lipocalin as an enhancer of spermatozoa motility

The 24p3 protein is a 25 kDa glycoprotein that is secreted into the uterine fluid during the proestrous phase of mice. We assessed the effects on spermatozoa motility and on the functions of mouse spermatozoa using the computer-assisted sperm analysis method, cytochemical staining and detection of the protein tyrosine phosphorylation pattern. Compared with the control cells, sperm motility was stimulated by the addition of 24p3 protein into the medium. Introducing 24p3 protein enhanced progressive motility but did not promote the appearance of hyperactivated movement. The presence of 24p3 protein in the medium did not allow the cells to undergo the capacitated protein tyrosine phosphorylation pattern and acrosome reaction. The tyrosine phosphorylation pattern shows phosphoproteins in the range of Mr 50000–106000 correlated with the sperm progressive motility after the addition of 24p3 protein into the medium. Using flow cytometry, we assessed the changes in the intracellular pH and measured the intracellular cAMP concentration with an immunodetection kit. The results indicated that the elevation in intracellular pH from 6.67 to 6.89, increase of intracellular cAMP accumulation, and protein tyrosine phosphorylation might be the factors in enhancement of sperm motility as the 24p3 protein bound to the spermatozoa. The 24p3 protein may have a role in regulating flagellar motility.     

Reduction of intramembranous particles in the periacrosomal plasma membrane of boar spermatozoa during in vitro capacitation: a statistical study

Membrane remodeling in the periacrosomal plasma membrane (PAPM) of boar spermatozoa during incubation in capacitation medium was examined by the freeze-fracture technique. In the preservation medium (PM) group, the major small (about 8 nm) intramembranous particles (IMP) and the minor large (> 10 nm) IMP were distributed evenly in the PAPM. The IMP-free area increased during capacitation. To correct the IMP-free area, arithmetically redistributed (ARD)-IMP density was used for statistical analysis. In the PM group, the mean density +/- SD of large IMP was 379 +/- 64 and 266 +/- 58/microm2, and that of small IMP was 1450 +/- 155 and 672 +/- 252/microm2 in protoplasmic (P) and external (E) faces, respectively. During capacitation, the significant (P < 0.01) reduction of large IMP density was encountered only in the E face of a few incubation groups, while that of the small IMP density occurred in the P face by 2 h. Consequently, reduction of the total IMP density of both faces was not significant in the large IMP, but it was significant (P < 0.01) in the small IMP. One-fifth of the total small IMP density reduced by 2 h. Filipin-sterol complexes (FSC) were numerous in the PAPM, and FSC-free areas also increased during capacitation. The mechanism of IMP-free area formation and the behavior of the small IMP in the PAPM during capacitation were discussed in relation to membrane stability.     

The ageing phenomenon of turbot spermatozoa: effects on morphology, motility and concentration, intracellular ATP content, fertilization, and storage capacities

The deleterious effect of the ageing phenomenon of turbot spermatozoa was investigated in relation to the sampling date. Spermatozoa with a low or highly condensed chromatin and a middle piece containing numerous or a few vesicles were observed simultaneously 80 and 47 days before the beginning of the spawning period of the females. The middle piece of spermatozoa contained few vesicles, 39 days after the end of the reproductive period. At the same date, some spermatozoa appeared in which the plasma membrane was broken. Sperm motility, assessed just after collection in terms of arbitrary motility scores from 0 to 5, was significantly increased both at 10 and 60 s post-activation, for samples collected 18 days after, 25 days before and 9 days after the beginning of the spawning period of the females, respectively compared to samples collected 6 days before, 55 days after and 88 days after the end of this period. A lower short-term storage capacity was recorded at 10 and 60 s post-activation for sperm samples collected 6 days before and 88 days after the end of the reproductive period, respectively compared to 18 days and 9 days after the beginning of the spawning period. At 60 s post-activation, a higher motility of thawed spermatozoa was observed for samples collected 5 days before the beginning of the spawning period (motility recovery index: 86.4 ± 19.4%) compared to 71 days after the end of this period (55.0 ± 12.0%). The fertilizing capacity of sperm samples collected 61 days after the end of the spawning season (66.1 ± 14.6%) was significantly lower than that recorded for samples collected 34 days after the beginning of the spawning period (75.2±9.6%). On the contrary, there was no significant decrease in endogenous ATP content (31 days after the beginning of the spawning period, 14.53 ± 0.84; 48 days after the end of this period, 10.75 ± 5.26 nmol 10^{? 8} spermatozoa). Furthermore, sperm concentration significantly increased between the same dates (respectively 3.3 ± 0.8–9.4±4.8×10⁹ spermatozoa ml^?1).