Pretreatment Of Spermatozoa Research Articles

Research Note: Avian Sperm-Egg Interaction: Perivitelline Layer Possesses Receptor Activity for Spermatozoa

Sperm-egg interaction in mammals is initiated by the binding of spermatozoa to the zona pellucida (ZP), an acellular coat completely surrounding the vitelline membrane of unfertilized eggs. The perivitelline layer (PL) of the ovum of the hen is analogous to the mammalian ZP. In vitro incubation of the PL with spermatozoa results in fragmentation of the PL. The PL promotes the acrosome reaction whereby sperm acrosomal enzymes are released; the enzymes in turn dissolve the PL. The present study was conducted to determine if the PL removed from chicken ova, recovered immediately after ovulation, possess sperm-receptor activity. The results suggest that the solubilized PL does contain one or more components which behave in a manner analogous to sperm-receptors. Pretreatment of spermatozoa with solubilized PL prevented their subsequent attachment to and fragmentation of the intact PL which was observed with untreated spermatozoa.

Concanavalin A Modifies Allo-specific Sperm-Egg Interactions in the Ascidian, Ciona intestinalis: (self incompatibility/vitelline coat/concanavalin A/Ciona gametes).

In the self sterile ascidian, Ciona intestinalis, the spermatozoa rarely bind to the vitelline coat of autologous eggs and never penetrate it. We report here that concanavalin A (ConA), a lectin recognizing mannose or glucose residues of carbohydrates, can modify these self- and nonself-specific sperm-egg interactions. When eggs were pretreated with 0.1-0.5 mg/ml of ConA, about two thousand spermatozoa became attached to the autologous vitelline coat within five minutes of insemination. The effect of ConA was not modified by the addition of D-mannose or pretreatment of spermatozoa with ConA, showing that ConA does not function merely as a ligand bridging the sperm and vitelline coat. In contrast to the marked enhancement of sperm-egg binding, ConA did not facilitate the penetration of spermatozoa through the autologous vitelline coat. Even in non-autologous insemination, it blocked the sperm penetration and, consequently, fertilization did not occur, as shown by Rosati et al. (1978). D-Mannose, when mixed with ConA in advance, completely abolished this inhibitory effect of ConA. Lotus agglutinin, a fucose-binding lectin, was less effective and wheat germ agglutinin and soy bean agglutinin had no effect on sperm entry in the perivitelline space. The results of this study are discussed in relation to the possible involvement of mannosyl and/or glucosyl glycoconjugates in allo-specific sperm-egg interactions.

Significance of D-mannose as a sperm receptor site on the zona pellucida in human fertilization

The role of monosaccharides in human fertilization was studied by testing their effects on penetration of spermatozoa into mature human oocytes (zona penetration test). When oocytes were pretreated with concanavalin A, wheat germ agglutinin, or Ricinus communis agglutinin-I at a concentration of 100 micrograms/ml, no spermatozoa were found to bind to or penetrate through the zona pellucida. Penetration of spermatozoa was restored when the zona pellucida pretreated with wheat germ agglutinin and Ricinus communis agglutinin-I were rinsed with N-acetyl-D-glucosamine (wheat germ agglutinin inhibitor) and D-galactose (Ricinus communis agglutinin inhibitor), respectively. Conversely, the blocking effect of concanavalin A on sperm penetration was not reversed by treatment with D-mannose (concanavalin A inhibitor). Furthermore, pretreatment of spermatozoa with D-mannose (50 mmol/L) completely inhibited sperm penetration through the zona pellucida. However, sperm penetration was clearly demonstrated when the zona pellucida was pretreated with D-mannose. These data suggest that D-mannose residues are essential in, or sterically closely related to, the sperm receptor site on the human zona pellucida.

Human sperm chromosomes in zona‐free hamster eggs: Pretreatment of spermatozoa with calcium ionophore facilitates later examination of chromosomes

AbstractFreshly ejaculated human spermatozoa exposed to 10 μM calcium ionophore A23187 for 30 minutes quickly bound to and penetrated into zona‐free hamster eggs. When these eggs were cultured for 13 hours in the presence of low concentrations of vinblastin and podophyllotoxin, then fixed and spread with the use of a cold fixation technique, haploid sets of sperm chromosomes were demonstrated. The number of spermatozoa that were attached to the egg surface at insemination influenced the success rate of chromosome demonstration. The best results (an average of about 0.7 chromosome set per egg) were obtained when insemination was controlled so that an average of seven spermatozoa attached to each egg at the end of a 15–30‐minute insemination period. This improved method for demonstrating human sperm chromosomes would be useful in the analysis of paternal genetic contributions in reproduction as well as in assessing the effects of environmental genotoxic agents or conditions on spermatozoa.

The effect of semen extension, cAMP and caffeine on [formula omitted][formula omitted] fertilization of bovine oocytes

A previously reported in vitro system that used epididymal spermatozoa for fertilizing bovine follicular oocytes (1) has been expanded to include ejaculated semen as the sperm source. Frequency of fertilization was higher when semen was extended 1:1 prior to transport to the laboratory rather than transport as neat semen. Pretreatment of spermatozoa with cAMP, caffeine or both prior to insemination of oocytes did not increase frequency of either acrosome reactions or fertilization after sperm/oocyte incubation.

Induction of the shaking phenomenon by pretreatment of spermatozoa with sera containing antispermatozoal antibodies

Sera containing sperm-agglutinating and/or complement-dependent sperm-immobilizing activity were tested for their ability to induce a shaking phenomenon in the sperm-cervical mucus contact (SCMC) test. Donor spermatozoa were treated with the sera in a one-step incubation and washing procedure. The percentage of motile pretreated spermatozoa showing the shaking phenomenon (S%) was determined in the SCMC test. In addition, the immunoglobulin class of antibodies present on the pretreated spermatozoa was determined in mixed antiglobulin reaction tests for IgG, IgM, and IgA. An S% of at least 80 was always observed with spermatozoa pretreated in sera with a sperm agglutination titer of at least 32, provided that IgG was detected on more than 90% of the motile spermatozoa. The high S% was also obtained with spermatozoa pretreated in purified IgG from sera containing antispermatozoal antibody activity. It was concluded that the reduced cervical mucus penetration capacity of spermatozoa pretreated with sera containing antispermatozoal IgG could be ascribed, at least partially, to the occurrence of a shaking phenomenon.

The effect of human spermatozoa on antigen and mitogen induced blastogenesis.

In an attempt to identify factors capable of specifically or nonspecifically modulating results in an in vitro blastogenic assay system, studies were performed evaluating cell mediated immune response to human spermatozoa. Spermatozoa were capable, on a dose dependent basis, of both inhibiting and stimulating normal lymphocyte DNA synthesis as well as suppressing mitogen-induced response. Pretreatment of spermatozoa with neuraminidase and alpha-methyl-D-mannoside abrogated suppressive properties on spontaneous and/or mitogen-induced stimulation. Such suppressive activity on mitogen-induced response was observed using B and T-cell enriched populations. Inhibitory properties were not evident using intact cell populations stimulated by specific antigen. It is suggested that: (1) receptors on spermatozoa bind lectin; and (2) such receptors can be inactivated by enzymes having glycoprotein specificity.

Ouabain effect on bovine spermatozoan motility and testosterone binding.

SummarySpermatozoan motility of whole bull semen incubated aerobically at 37° in 0.9% NaCl saline was inhibited by 10-3 to 10-7M ouabain, an effect opposite to that observed for testosterone. Preincubation for 30 to 45 min with ouabain blocked the effect of testosterone, while preincubation with testosterone only partly blocked the effect of ouabain. When equimolar ouabain and testosterone were incubated together motility of spermatozoa initially tended to be intermediate between the individual effects of ouabain and testosterone. However, by the second hour of incubation ouabain inhibition was similar to that observed in ouabain treatment alone. Ouabain showed a synergistic effect when testosterone was added at only one-third the molar concentration of ouabain.The ability of ouabain and testosterone to block the action of each other suggests that their mechanisms are interrelated. However, they do not appear to have common binding sites because pretreatment of spermatozoa with ouabain (which is irreversi...