However, other lipid classes and subclasses also may differ between PGC sperm and washed sperm. Unique to mammalian sperm and testicular germ cells is the selective existence of the phospholipid molecular species with Chighly unsaturated fatty acyl chains [ 15 — 24 ]. The contents of Chighly unsaturated fatty acids HUFAs -containing phospholipids appear to correlate with mammalian sperm maturity and motility [ 15 , 19 , 21 , 25 , 26 ].
These phospholipids have low phase-transition temperatures [ 15 ] and contribute to high membrane fluidity [ 27 ], which is required for hyperactivated motility and fusion-related events during gamete interaction including the zona pellucida [ZP]-induced acrosome reaction and gamete plasma membrane fusion.
Direct correlation between the contents of CHUFA-containing phospholipids and fertilizing ability of PGC sperm, however, has yet to be demonstrated. Sulfogalactosylglycerolipid SGG; also known as seminolipid is another lipid that exists selectively in mammalian sperm and male germ cells [ 28 — 30 ]. It possesses ZP-binding ability [ 32 , 33 ], and the majority of SGG on the sperm surface is present in the sperm lipid rafts, which have ZP-binding ability unpublished results.
It therefore is possible that PGC capacitated sperm also have a higher content of SGG than washed capacitated sperm have. In the present study, we have shown, to our knowledge for the first time, that the contents of both SGG and docosahexaenoic acid DHA; Cn-3 -containing phospholipids are higher in mouse PGC capacitated sperm than in washed capacitated sperm.
These findings would explain, in part, the higher fertilizing ability of PGC capacitated sperm, which also is reported herein. In all experiments described herein, these mice were fasted overnight and killed the following day by cervical dislocation. The KRB-Hepes consisted of One of these media was employed in all sperm preparations except when sperm were subjected to in vitro fertilization experiments, in which case KSOM medium was used.
The KSOM medium consisted of Sperm from the same mice were divided into two halves, each for the preparation of washed capacitated sperm and PGC capacitated sperm samples. A; Sigma, St. The remainder was placed in a glass vial, flushed with a stream of N 2 , and capped tightly. Alternatively, antioxidants 0. To assess sperm viability, the sperm samples were treated with 0.
A drop of the sperm samples was placed onto a glass slide, topped with a coverslip, and viewed under a Zeiss IM35 Microscope Zeiss Canada, Toronto, ON using a rhodamine filter.
Sperm that excluded the fluorescent dye, propidium iodide, were scored as viable. Washed capacitated and PGC capacitated sperm were evaluated for their ability to bind to cumulus-free, ZP-intact mature eggs prepared as described by Hogan et al. Student t -test was used to analyze significant differences in the numbers of sperm bound per egg between the washed capacitated and PGC capacitated sperm samples in each experiment as well as the average data from the mean values of these numbers obtained from three experimental days.
Fertilized eggs were those containing two pronuclei. Data were expressed as percentages of total eggs fertilized in the coincubation drop. To assess the level of parthenogenesis, eggs were incubated under the same conditions but without sperm addition. The Student t -test was used to analyze significant difference in the average data for the percentage of eggs fertilized by washed capacitated sperm versus PGC capacitated sperm on three experimental days.
Chlortetracycline CTC staining of mouse sperm was done according to the method described by Lee and Storey [ 35 ] with minor modifications. To this mixture, The slides were examined within 2 h under a Zeiss i.
At least sperm on each slide were assessed for the noncapacitated, capacitated, intermediate between capacitated and acrosome-reacted , and acrosome-reacted patterns as described previously [ 35 ]. Experiments were repeated three times on different days. Antigen-antibody recognition was then probed using secondary horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G antibody Bio-Rad, Hercules, CA at dilution and an enhanced chemiluminescence Western blot detection kit Amersham Pharmacia Biotech, Piscataway, NJ.
Sperm DNA was quantified by a modification of the method outlined by Labarca and Paigen [ 38 ] as previously described [ 12 ], which was based on the binding of Hoechst to DNA of live cells treated with a high salt concentration 2 M NaCl to allow separation of DNA strands and, thus, intercalation of the Hoechst dye.
Specifically for sperm cells, mM dithiothreitol DTT was added to the sperm suspension to decondense the highly compact sperm chromatin by disrupting protamine disulfide bonds as well as to minimize the adherence of sperm to the surface of the assay tubes. Calf thymus DNA Sigma was used as a standard. All sperm DNA samples were assayed in duplicate. Lipids were extracted from all sperm samples following a modification of the method outlined by Bligh and Dyer [ 39 ] as described by Kates [ 40 ].
The details of this procedure have been described previously for sperm [ 12 , 13 ]. All phospholipid standards and cholesterol were purchased from Doosan Serdary Research Laboratories Englewood Cliffs, NJ , and the mole amounts of phospholipid standards were quantified using the phosphorus assay described below. All lipids were stained blue.
Because these lipid classes and phospholipid subclasses of the same mole amounts were not stained equally with Coomassie brilliant blue G, a standard curve was constructed for each lipid by plotting the analyzed optical density values of the lipid bands versus the lipid amounts in moles. The mole amounts of all phospholipid standards were determined from the phosphate assay see below. Cholesterol in extracted sperm lipids was quantified by the modified fluorometric method described by Gamble et al.
Cholesterol was oxidized first by cholesterol oxidase to 4-cholesteneone and H 2 O 2. The H 2 O 2 and p -hydroxyphenylacetic acid then served as substrates of peroxidase to generate a stable fluorescence product. In the present study, the assay volume was reduced so that the assay could be performed in a microtiter plate. Briefly, sperm lipids in chloroform were dried under N 2 and then resuspended in methanol. The reaction mixture was made by combining 4 ml of 0. All sperm lipid samples were assayed in triplicate.
The sensitivity of the assay was 0. Phospholipids were quantified according to the method described by Duck-Chong et al. The assay was based on phospholipid digestion by magnesium nitrate at high temperature to inorganic phosphate followed by its color reaction with molybdate and malachite green. An aliquot of sperm lipids extracted from approximately 0.
The amount of inorganic phosphorus was quantified from a standard curve of KH 2 PO 4 0—0. The only sulfolipid in mouse sperm [ 9 ], SGG was quantified according to the method described by Kean [ 46 ] as modified by Weerachatyanukul et al. An aliquot of sperm lipid in chloroform, extracted from approximately 20 million sperm, was used for each assay point.
A standard curve was constructed using SGG purified from ram testis [ 41 ]. All sperm lipid samples were assayed in duplicate.
However, the extracted lipid was applied to the TLC plate as a thin, cm band. Individual phospholipid classes were located by staining the plate in Rhodamine 6G Sigma solution 0.
The PC and PE bands were scraped gently from the plate. This treatment hydrolyzed the ester bonds of glycerophospholipids, generating fatty acid methyl esters FAMEs. Mammalian sperm also contain an appreciable amount of plasmalogens [ 48 ].
The sn-1 chain of plasmalogens is an alkylenyl ether linkage, whereas the sn-2 chain possesses a fatty acyl chain. The acid methanolysis procedure releases a FAME from the sn-2 chain and a fatty aldehyde dimethylacetal DMA from the sn-1 chain from a plasmalogen [ 40 ]. However, this 0. The GC column was a DB-5 narrow-bore capillary column length, 30 m; inner diameter, 0. The linear velocity of the helium carrier gas was The mass range scanned was 40— atomic mass units with the automatic gain control setting to obtain the total ion spectrum.
Blanks were analyzed along with samples to detect any cross-contamination between analyses. The cis -4,7,10,13,16,DHA Cn-3 methyl ester and cis -7,10,13,16,docosapentaenoic acid Cn-6 methyl ester were purchased from Sigma. Methylarachidonate was from Doosan Serdary Research Laboratories. Fatty acid methyl ester peaks were identified by both their GC retention times as compared to those of standards and by their mass spectra.
They were then quantified by comparison of their GC peak areas with that of an internal standard methylheptadecanoate.
However, the ability of PGC capacitated and washed capacitated mouse sperm to bind to the egg ZP and to fertilize eggs has not, to our knowledge, been compared previously. Therefore, the higher fertilizing ability of PGC capacitated sperm, as observed by the increase in the number of sperm bound per egg and in the in vitro fertilization rates, would be attributed to possibilities other than the percentage sperm motility.
First, the hyperactivated motility patterns showing an elevated beat frequency of the sperm tail and a larger amplitude of the sperm movement were more obvious in the PGC capacitated sperm sample than in the capacitated washed sperm preparation when assessed under a light microscope. Second, the PGC sample may have a higher population of capacitated gametes.
CTC has been used widely to assess microscopically the percentages of noncapacitated F pattern , capacitated B pattern , intermediate between capacitated and acrosome-reacted, S pattern , and acrosome-reacted AR pattern gametes in a sperm sample. However, the percentage of the noncapacitated sperm population F pattern was the same in both samples. Nonetheless, the slight increase in the capacitated sperm population in the PGC sample could not account entirely for their significant increases in ZP-binding ability and in vitro fertilization compared with the washed sample Fig.
This suggested that the capacitated sperm population, as detected by CTC staining B pattern , of the two sperm preparations differed in their biochemical and functional properties. Sperm fertilizing ability of PGC capacitated sperm and washed capacitated sperm. A Sperm-ZP binding ability. B In vitro fertilizing ability.
A Distribution of chlortetracycline staining patterns in PGC capacitated sperm and washed capacitated sperm. Four staining patterns F, Noncapacitated; B, capacitated; S, intermediate; AR, acrosome reacted [ 35 ] were observed in both sperm types. B Sperm tyrosine phosphorylation patterns of PGC capacitated sperm and washed capacitated sperm.
Lane 1: washed capacitated sperm; Lane 2: PGC capacitated sperm. Molecular masses of protein standards kDa are shown on the right. Proteins extracted from an equal number of PGC capacitated or washed capacitated sperm 0.
Sperm protein tyrosine phosphorylation has been shown to be highly correlated with capacitation [ 49 , 50 ]. Figure 2B shows that both the PGC capacitated sperm sample and the washed capacitated sperm sample possessed a similar pattern of protein tyrosine phosphorylation, as revealed by immunoblotting using 4G antiphosphotyrosine antibody.
The immunoreactive bands included sperm proteins of molecular weights ranging from approximately 45 to kDa. The immunoreactive intensity of only two phosphorylated proteins i. These results suggested that the capacitation stage of PGC sperm may be slightly more advanced than that of washed sperm, although it may not account completely for the observed increases in ZP binding and in vitro fertilization competence of PGC capacitated sperm.
Because mouse sperm tend to adhere to the tube wall surface within 10 min in suspension, we used the amount of DNA instead of the sperm number as a denominator of the sperm lipid content. The DNA assay involved treating sperm with DTT, which prevented sperm adherence to the tube wall, thereby allowing the assay to be carried out at a later time [ 12 ].
Levels and molar ratios of various lipid classes In washed capacitated sperm and PGC capacitated sperm. All lipid quantification was performed with five replicate samples. Denotes the lipid content as a percentage of total lipids in washed capacitated sperm or PGC capacitated sperm.
It is possible that the marked decrease of phospholipids in PGC capacitated sperm compared to washed capacitated sperm was confined to specific phospholipid subclasses. However, a minor lipid band, migrating slightly faster than SM, was present in washed capacitated but not in PGC capacitated sperm. This lipid did not stain purple with the orcinol dye data not shown , indicating that it was not a glycolipid.
Another Coomassie blue-stained lipid with a higher mobility than SGG also was observed in both PGC capacitated and washed capacitated sperm. The identity of both lipids each denoted by a question mark symbol in Fig. Staining of lipids was performed using Coomassie brilliant blue G Two bands, labeled with a question mark, with one present only in the washed capacitated sperm samples, are unidentified lipids.
The PGC sperm immediately acquired hyperactivated motility patterns on suspension in medium. This suggested that their plasma membrane may have a higher fluidity than that of washed sperm. In addition, a greater ability of PGC capacitated sperm to fertilize eggs as compared with that of washed capacitated sperm Fig. Because lipids play an important role in regulating membrane fluidity, we characterized further the lipid profile of PGC capacitated sperm in comparison with that of washed capacitated sperm.
The phospholipid molecular species in PGC sperm may be different from those of washed sperm. In particular, a higher proportion of phospholipid species containing HUFA likely would contribute to high membrane fluidity [ 27 ], which is required for hyperactivated motility and membrane fusion events.
This indicated that the hydrocarbon chains of PC and PE of both sperm samples were linked mainly to the glycerol backbone through ester linkages, whereas minor amounts of PC and PE existed as plasmalogens.
This indicated that this hydrocarbon chain was present at the sn -1 position of PE plasmalogen. The results corresponded with the greater degree of hyperactivated movement of PGC capacitated sperm observed visually under a light microscope.
Sperm tyrosine phosphorylation has been shown to correlate with capacitation [ 49 , 50 , 53 — 55 ]. The mechanism of this sperm protein modification is through the cAMP and protein kinase A pathway, which is initiated by cholesterol efflux and the increase in intracellular bicarbonate levels [ 49 , 50 , 54 , 55 ]. The PGC capacitated sperm showed a slight increase in sperm protein tyrosine phosphorylation Fig. The number of parasites captured was calculated by haemocytometer determination, as with the Percoll treatment.
For light microscopy readings, Giemsa-stained thin smears were prepared from each well and the number of ring-infected erythrocytes in 1, erythrocytes was counted. The microscopist was always blinded to the experimental group designation of each slide.
Each experiment was set up in duplicate or triplicate wells, and invasion rates were averaged between all the wells. The percentage of efficiency of invasion compared to the MCB method is the result of the following formula:. A paired t-test analysis was performed, where each sample from Percoll and from MBC done on the same day was compared to each other for their invasion percent into new red blood cells. Table 1 shows the results of the experiments. In 10 paired assays, the mean percent invasion of intact red cells after overnight culture was 5.
It is evident that schizonts purified by magnetic bead column separation were more viable than those purified by Percoll gradient centrifugation. The reasons for this are not clear. However, there are several possible explanations.
It hs been reported that permeabilization of the host erythrocyte membrane appears s early as 6 h after parasite invasion and increases gradually with parasite maturation [ 15 ]. Since the collection of parasites for next day invasion need collection of schizont forms, the erythrocyte is at its peak of permeability.
This suggests that molecules could diffuse in and out of the schizont-infected erythrocyte with relative ease, perhaps leading to increased susceptibility to toxic substances that could be present in Percoll. Another possible explanation for the decreased viability after Percoll centrifugation is the leakage of ions across the membrane due to the hyper osmotic effect of Percoll.
Bates et al [ 16 ] reported that a group of laboratory established strains of P. However, they reported that when they performed the same analysis with field isolates their results were' similar to those we show here, with the MCB treated ones invading at higher rates. In view of the present experiments, these results are better explained by the difference in susceptibility to external chemicals and solvents inherent to different strains.
Reed et al [ 11 ] reported that the laboratory-adapted strain W2 mef could not be isolated using the Percoll method, which would also point out to different strains behaving differently to the Percoll treatment, for reasons that are still unknown, and that could include different membrane porosity and osmotic pressure sensitivity. In the case of 7G8, the isolation is possible but the invasion efficiency is reduced, something that could be avoided using the magnetic beads based technique.
In addition to increased viability, magnetic bead column separation offers the advantage that the parasitized red cells remain in an iso-osmotic environment. This means that the red cells can be introduced back into the culture media without concern for abrupt changes in the osmolarity, which could affect some strains more than others. Magnetic column enrichment of schizont and late stage trophozoite-infected red cells is a very viable alternative to Percoll gradient centrifugation especially for the purpose of preparing red cell invasion assays with the 7G8 strain.
This technique can be recommended for several reasons: the procedure itself has the advantage of not being invasive on the parasites since it involves no contact with any foreign chemical reagent to the culture media, it requires no expensive centrifuges, and 7G8, as well as some other strains seem to be affected by the use of Percoll gradient centrifugation.
Jensen JB: Concentration from continuous culture of erythrocytes infected with trophozoites and schizonts of Plasmodium falciparum.
Am J Trop Med Hyg. Exp Parasitol. Article PubMed Google Scholar. Ann Trop Med Parasitol. J Parasitol. Nat Protoc. Parasitol Res. Google Scholar. Nillni EA, Londner MV, Spira DT: A simple method for separation of uninfected erythrocytes from those infected with Plasmodium berghei and for isolation of artificially released parasites.
Z Parasitenkd. Malar J. PLoS Pathog. Correspondence and Footnotes. Many hypotheses for the various processes leading to conception in vivo are extrapolated from studies on other eutherian mammals. The methods used in animal models have to be under constant revision and development due to limitations that should not be overlooked. The highest purity of germ cells described in the literature was obtained by Meistrich et al.
TP2 is only present in elongating spermatids steps 2. SGP1 is a specific marker for rat Sertoli cells 3. COX II is present in mitochondria of all germ cells and Sertoli cells, but its highest expression occurs in pachytene spermatocytes 4.
Labeling of extracted mRNA with a 32 P cDNA probe was detected using a PhosphorImager and the data were analyzed using computer software, thus reducing subjective bias. The molecular approach for assessing contamination of germ cell preparations is novel and should be more rigorous than morphological identification which has been used routinely in the literature.
Eight adult male Wistar rats weighing g and aged days were used for each experiment. The rats were killed by CO 2 asphyxiation and cervical dislocation. Following the first enzymatic digestion, 15 ml of dissection medium was added. The supernatant containing interstitial cells was removed and discarded. Enzyme solution B was added to the pelleted tubules 7. Following the second enzymatic digestion, a further 10 ml of dissection medium was added to the suspensions. The supernatant was discarded, and the pellet was resuspended in 1 ml of dissection medium and aspirated with a ml syringe.
The conditions used to optimize both purity and yield of cells according to the diameter of the tubes used in the present study are as outlined below:. Fraction 2 was discarded and the others were pelleted in elutriation medium at g for 10 min in an Omnispin R.
The first chamber was filled only with dissection medium at a density of 1. A density of 1. A ml continuous linear gradient was poured into a ml Falcon tube. The gradients formed had four steps for the load fraction 1. These gradients were designed to separate the spermatids and pachytene spermatocytes based on the buoyant density of these cells as described in the literature 1.
The pelleted cell fractions from the centrifugal elutriator were gently layered above the gradients using a plastic Pasteur pipette. The cells were centrifuged at g for 30 min at room temperature in an Omnispin R. The bands formed in the continuous gradients were collected as follows. The second and third bands from the top of the gradient were collected from the load gradients and in the other fractions the band formed in the middle of the gradient was aspirated. In the discontinuous gradients, the band at a density of 1.
These fractions were removed using a Pasteur pipette. The top part of the gradient was removed until the desired band was reached. An unused Pasteur pipette was used to aspirate the band. Special care was taken to avoid contamination from different fractions in the continuous gradients. Only 1 ml, containing the pelleted cells, was left in the bottom of the tube. The cells were identified according to their morphology based on cell size and nuclear appearance and by the presence of the acrosome.
The classical description of Meistrich et al. Figure 1 shows photomicrographs of some of the germ cells. The criteria used for cell identification in smears was as follows:. In the basal portion, four spermatogonia are attached. Magnification, X. Spermatogonia Figure 1 H. The nucleus of these cells was variable in size and contained deeply stained chromatin. The nucleus was surrounded by a thin rim of cytoplasm. Spermatocytes Figure 1 E,F.
The spermatocytes are formed by a continuous series of cells that differ in size, but have a similar nuclear appearance and consist of cells from leptotene to secondary spermatocyte stages. The nucleus of leptotene and zygotene cells was deeply stained with woolly masses of chromatin. The nucleus of pachytene spermatocytes Figure 1 E was large and contained separate clumps or thick strands of chromatin corresponding to the chromosomes.
The cytoplasm was more abundant than in previous stages and increased proportionally in subsequent stages. The secondary spermatocytes were identified by the presence of dusty and fine chromatin inside the nucleus.
The cytoplasm was also abundant. Divisions of primary to secondary spermatocytes were often seen. Spermatids Figure 1 A-D. The classical division of the steps from 1 to 19 given by Meistrich et al. Instead, the classification adopted was early and late spermatids. Early spermatids Figure 1 D were characterized by abundant cytoplasm with a small and deeply stained nucleus.
In preparations of good quality, the Golgi zone and the acrosomic system could be seen. The early spermatids were equivalent to steps 1 to 8 of spermiogenesis. Late spermatids Figure 1 A-C were determined by the discrete amounts of cytoplasm and by the dark stained and elongated nucleus. The late spermatids were equivalent to steps 9 to 19 of spermiogenesis. Sertoli cells Figure 1 I. These cells were characterized by the presence of an irregular and elongated cytoplasm and the large nucleus contained a fine pale-staining chromatin and were often seen attached to other cells or in clumps.
Cytoplasmic fragments and residual bodies Figure 1 G. Pale-stained cytoplasmic fragments without any nucleus were seen and identified as residual bodies. Large fragments with late spermatid nuclei were identified as Sertoli cell cytoplasm. The slides were coded randomly and examined by an experienced cytologist for cell identification. Four fields at X magnification were counted. Following this procedure, cell counts were performed by an operator who did not know the results obtained by the previous cytologist.
The results were then compared to check the ability for cell recognition. The methodology followed was a slight modification of the manufacturer instructions.
The cells were lysed by vortexing. RNA was separated by electrophoresis according to standard methods 11 on gels containing 2. Double-stranded DNA for labeling was usually prepared by amplification of the cloned cDNA insert from a plasmid vector using polymerase chain reaction PCR 12 according to methods described elsewhere Membranes were prehybridized at 65 o C for 3 h in buffer solution containing 0.
Hybridization was allowed to continue for 18 h. Oligonucleotides mers were synthesized according to methods described elsewhere Following hybridization and detection of the signals from the labeled double-stranded DNA, the membranes were washed with solution containing 40 mM sodium phosphate, pH 7. The membranes were checked in the PhosphorImager to confirm the removal of the radiolabeled probe and then prehybridized with SSC plus Denhardt's solution for oligonucleotide probes at 65 o C for 4 h.
0コメント