Unbound Sperm Research Articles

Heparin and Ca 2+-free medium can enhance release of bull sperm attached to oviductal epithelial cell monolayers

The success of assisted reproductive techniques, such as IVF, could be enhanced by being able to select the most competent spermatozoa in a sample. Attachment and subsequent release of spermatozoa from oviductal epithelial cells (OEC) could provide populations of functionally superior spermatozoa for use in these protocols. The objective of the present study was to investigate the ability of heparin and Ca 2+-free medium to induce spermatozoa release from bovine OEC. Epithelial cells were grown to confluence in 24-well plates and pooled frozen bull semen was added to a final concentration of 1 × 10 6 spermatozoa/well. Spermatozoa were allowed to bind to OEC for 2 h. Medium with unbound spermatozoa was removed and replaced by Sperm-TALP, only (control), with heparin (5, 10, or 15 IU/mL), or Ca 2+-free with 2 mM EGTA. Treatments were left on sperm-OEC co-cultures for 0.5, 1, 2, 3, or 5 h. At each time, the media were recovered and spermatozoa from each treatment were counted and evaluated for acrosome integrity and motility. The total number of spermatozoa attached to OEC after 2 h of co-culture was considered 100%. Spermatozoa release is expressed as percentage of the total number of sperm cells bound to OEC after 2 h of co-culture. Data were analyzed by ANOVA and results are expressed as mean ± SEM from three independent replicates. Beginning at 0.5 h, more sperm cells (P < 0.05) were released from OEC in the heparin groups (10 and 15 IU/mL, 77.3 ± 6.2% and 84.0 ± 6.2%, respectively) as compared to the control (46.4 ± 6.2%). The Ca 2+-free medium also induced spermatozoa release when compared with the control, but the effect was not significant until 3 h (38.2 ± 1.9% vs 59.5 ± 6.9%; P < 0.05). The percentage of acrosome reacted spermatozoa was not affected by heparin treatment. Heparin at 10 IU/mL increased (P < 0.05) the percentage of motile spermatozoa, whereas Ca 2+-free medium caused the opposite effect at 0.5 h after addition of treatments. We conclude that both heparin and Ca 2+-free medium are able to promote spermatozoa displacement from OEC attachment. Based on motility and acrosome status data, we predict that released sperm cells may be used for IVF and other assisted reproductive techniques.

Exposure of human, boar, or bull sperm to a synthetic peptide increases binding to an egg-membrane substrate.

We evaluated the effects of in vitro exposure of sperm to synthetic FertPlus peptide, which represents a 60-amino acid sequence within rat prosaposin, using a microwell sperm-binding assay (SBA), in which an extract of hen's egg served as the binding substrate. Sperm suspensions were incubated with FertPlus peptide (six to eight concentrations; 0 and 20-1,280 pM) at 37 degrees C for 10 minutes, diluted > or = 20 times, and placed onto SBA plates. After 60 minutes at 37 degrees C, unbound sperm were washed away and the DNA of bound sperm was quantified. Percentage of sperm bound was independent of the percentage of motile sperm, but immotile sperm did not bind. For fresh human sperm (25 ejaculates), the percentage of sperm bound was increased by exposure to 640 pM peptide (P < 0.01). For 11 of 25 samples, the percentage of sperm bound for the aliquot exposed to 640 pM peptide was > or = 1.4 times the value for a 0 pM control aliquot. With frozen-thawed human sperm, for six of seven samples, binding was > or = 1.4 times greater after exposure to 640 pM peptide. For boar sperm held for approximately 24 hours at approximately 18 degrees C before use (28 ejaculates), there was a higher percentage of sperm bound for aliquots previously exposed to 1,280 pM peptide than there was for control aliquots (P < 0.01). For 16 of 28 samples, exposure to peptide increased the percentage of sperm bound by > or = 1.4 times. For frozen-thawed bull sperm, percentage of sperm bound was > or = 1.4 times greater for 4 of 10 samples that were briefly exposed to 160 pM peptide. Clearly, human, boar, and bull sperm were beneficially modified by brief in vitro exposure to FertPlus peptide, so that for many samples a greater percentage of sperm was bound in vitro. As presented in an accompanying paper, fertility of bull sperm was increased by brief exposure to FertPlus peptide.

Effect of coculturing spermatozoa with oviductal cells on the incidence of polyspermy in pig in vitro fertilization

It is known that oviductal cells play an important role in fertilization in vivo. We were interested in the effect of those cells on spermatozoa and their influence on the incidence of polyspermy in pig in vitro fertilization (IVF) when cocultured with spermatozoa. Oviductal cells are believed to select a highly fecund population of spermatozoa. By coculturing spermatozoa with oviductal cells it is possible to reduce the number of spermatozoa confronting the eggs at fertilization, reducing the incidence of polyspermy. In our study, spermatozoa were cocultured with oviductal cells for 30 min so that they could bind to the oviductal cells. Both bound and unbound spermatozoa were used for fertilization. The results show that the spermatozoa that were bound to the oviductal cells were capable of fertilizing the eggs, but the nonbound spermatozoa had a reduced penetration incidence. With the bound sperm, polyspermy incidence decreased and the two-pronuclei proportion increased. We also found that with time the spermatozoa released from the cells had better motility than those that did not bind. Therefore, it is our belief that oviductal cells can be used for porcine IVF resulting in a lower polyspermy incidence and higher pronuclei incidence.

An investigation using lectins of glycocomponents of mouse spermatozoa during capacitation and sperm-zona binding.

Ten different lectins conjugated to fluorescein isothiocyanate (FITC) were used to study the distribution of surface carbohydrates on mouse spermatozoa, and to monitor the possible changes of their distribution during capacitation in vitro and sperm-egg interaction. Most of the lectins gave a restricted pattern of binding to fixed or unfixed epididymal spermatozoa. Binding was highly specific because no staining occurred in the presence of appropriate monosaccharides. Binding of UEA I, DBA and Con A was unaffected by the type of fixative used, but it was influenced by mild centrifugation. While unwashed spermatozoa showed binding mainly over the acrosomal cap and equatorial or postacrosomal regions, spermatozoa washed by mild centrifugation showed a change in the staining of the equatorial segment. Binding of 5 different lectins to spermatozoa did not change during capacitation in vitro. In contrast, capacitated spermatozoa bound to the zona pellucida exhibited a UEA I binding pattern which was strikingly different from that of the capacitated but unbound spermatozoa. We conclude that glycocomponents of specific regions of mouse spermatozoa do not change dramatically during capacitation, but do alter significantly during binding to the zona pellucida.

An ultrastructural study of epididymal mouse spermatozoa binding to zonae pellucidae in vitro: sequential relationship to the acrosome reaction.

Mouse sperm bind to the zona pellucida of the egg prior to penetration of the zona and entry into the perivitelline space. The question then arises: when does the acrosome reaction occur relative to these processes? An ultrastructural study of mouse epididymal sperm bound to the surface of the zona and in the privitelline space was undertaken to clarify this point. Cumulus-free mouse eggs were inseminated in either a complete defined culture medium capable of supporting in vitro fertilization or in Tris/NaCl buffer containing Ca+2. Both media support sperm binding to the zona to the same extent; binding is complete in 15 minutes. Unbound sperm were removed by a step gradient density centrifugation to yield a preparation of eggs with sperm firmly bound. All sperm in the perivitelline space had undergone the acrosome reaction. Sperm bound at the surface of the zonae pellucidae of eggs recovered at ten minutes after insemination all had intact acrosomes. At 40 minutes after insemination, half of the sperm were intact; the other half were in the initial stages of the acrosome reaction. At 90 minutes after insemination, 12% of the sperm had undergone the full acrosome reaction and were starting to penetrate the zona; of the balance, half were in various stages of the acrosome reaction, while half were still intact. These findings support the hypothesis that the sequence of the early reactions leading to fertilization in the mouse is: intact sperm binding to zona; acrosome reaction at the zona surface; penetration of the zona.

Methods for quantitating sea urchin sperm-egg binding

Two simple photometric methods are described for determining the average number of bound sea urchin spermatozoa per egg between 0 and 60 sec after insemination. One method is based on stopping gamete interaction with formaldehyde and then, after the eggs settle with sperm bound to their surfaces, measuring the turbidity of sperm remaining in suspension. An alternative method involves removal of the dead, unbound sperm from the formaldehyde fixed eggs by repeated washing in sea water. The bound sperm are then released from the egg surface by pronase digestion and the turbidity of the sperm suspension measured and related to sperm concentration by direct cell counts. Two phases of gamete interaction exist (1) the binding phase, from 0 to 20 or 25 sec, during which time sperm continuously bind to the egg surfaces; (2) the unbinding phase, from 20 or 25 to 50 sec, during which time the sperm are unbound from the eggs and return to the suspension. The results of experiments in which the method is used to assess sperm binding at different pH values and at different calcium concentrations are presented. Data are presented suggesting that a definite number of sperm binding sites may exist on the vitelline layer.