Mixture Of Sperm Research Articles

A double method sperm wash for artificial insemination.

Recently, there have been concerns regarding the presence of reactive oxygen species (ROS) produced during sperm processing for insemination. However, the sperm wash methods that yielded low ROS levels also had low sperm recovery after processing. The objective of this study was to compare sperm recovery after swim-up from pellet, overlay, and 2-layer Percoll wash methods with the recovery after the double method wash. The latter method consisted of a combination of 2 sperm wash methods, namely, the overlay and the Percoll method. Motile sperm were first collected through the overlay method. The leftover semen was then processed through the 2-layer Percoll method to scavenge motile sperm and the resultant pellet combined with the pellet from the overlay method. In this manner, the level of ROS was kept to a minimal, sperm recovery was improved, and a mixture of sperm with different surface properties was produced as a result of using different processing methods. The results indicated an improvement in sperm recovery and in total sperm motility in noncryopreserved sperm after using the double method wash when compared with the other wash methods. The study suggests that the double method wash is a feasible method for processing sperm for insemination.

Antigens on rat spermatozoa with a potential role in fertilization

Eight monoclonal antibodies (McAbs), directed against antigens on rat cauda epididymal spermatozoa, were tested for their capacity to interfere with fertilization in vitro as a means of identifying molecules with a potential role in sperm-egg recognition and fusion. Antigens recognized by the McAbs were visualized on live spermatozoa by indirect immunofluorescence (IIF) and characterized by immunoblotting. Five McAbs (designated 1B5, 2C4, 4B5, 5B1, and 8C4) recognized antigens specifically on the sperm acrosome and three (designated 2B1, 2D6, and 6B2) bound to the flagellum. Of the eight McAbs investigated, three (2B1, 2C4, and 6B2) were effective in blocking fertilization in vitro when added as culture supernatants to mixtures of sperm and eggs. McAb 6B2 was inhibitory due to its ability to agglutinate spermatozoa. McAbs 2B1 and 2C4 did not agglutinate capacitated spermatozoa, had no observable effect on motility, and yet blocked fertilization in a dose-dependent manner. McAb 2C4 did not give a reaction on immunoblots, but the 2B1 antigen was identified as an Mr 40 kD glycoprotein. McAb 2B1 appeared to block fertilization at the level of zona binding, whereas the effects of 2C4 were directed more against zona penetration and/or fusion with the vitellus. When sperm-egg complexes were stained with 2C4 or 2B1 McAbs and viewed by IIF, all spermatozoa that were attached to the zona showed fluorescence on the head. These results suggest that different antigens on the rat sperm head participate in different aspects of the fertilization process and that during capacitation there is either exposure of these antigens or else they migrate to their site of action from the flagellum.

Use of a monoclonal antibody to evaluate integrity of the plasma membrane of stallion sperm.

Transmission electron microscopy was used to confirm that a monoclonal antibody (F79.3E2; class IgG1 kappa) was specifically localized to an antigen in the acrosomal ground substance of stallion sperm. This antibody was used to develop and validate an indirect immunofluorescent procedure to evaluate integrity of the plasma-acrosomal membranes of stallion sperm. The concept was that primary monoclonal antibody would be "shielded" from its acrosomal antigen by an intact plasma membrane. Conversely, sperm with damaged plasma-acrosomal membranes would exhibit green acrosomal fluorescence when viewed with an epifluorescence microscope. A lipophilic counterstain (red fluorescence) was used to insure that all sperm were visualized. Sperm in fresh-extended or frozen-thawed semen were incubated with hybridoma supernatant containing monoclonal antibody for 30 min at 37 degrees C, then a second antibody (rabbit anti-mouse IgG-FITC) was added for 30 min at 37 degrees C. Unbound antibody was removed by dilution and centrifugation. Sperm were resuspended in phosphate-buffered saline containing Evan's blue as a counterstain. All sperm fluoresced bright red, regardless of the status of cell membranes, except that in cells with damaged plasma-acrosomal membranes, the green fluorescence associated with antibody was overriding for the rostral portion. By counting fluorescent and nonfluorescent "acrosomes", the percentage of sperm with intact plasma-acrosomal membranes was easily determined. Evaluation of five mixtures of undamaged and damaged sperm by this procedure gave a correlation of 0.91 between the percentage of damaged sperm in a mixture and the percentage of sperm with a fluorescent acrosome. Intra- and interassay coefficients of variability were less than 6%.

Different outcomes of sperm competition in right and left sides of the female reproductive tract revealed by thymidine-3H-labeled spermatozoa in the rat.

We attempted to record directly the results of sperm competition between two male rats in samples from female reproductive tracts. Gametes of 7 male rats were labeled in vivo by repeated injections of thymidine-6-3H. 33 females were mated successively with one of the injected males and with a noninjected male, or vice versa. Samples of the resulting sperm mixtures were recovered from both the uterine horns and the oviducts of the females. Pure samples of the injected males' sperm were obtained from uterine horns of females mated solely to them. Smears of samples were evaluated autoradiographically. Except for one male, the injections resulted in a broad peak of sperm labeling with maximum intensity on days 56-58 postinjection. This labeling enabled the assessing of contribution of either male to uterine and tubal sperm in the competitive matings. The injections did not influence competitive ability of male gametes in terms of sperm transport to horns or oviducts. The outcomes of sperm competition were significantly different in the right and left sides in most female subjects. Nevertheless, the outcomes were usually similar in a horn and a tube on the same side of the body. In vivo sperm labeling appears to be a useful method in the study of rodent sperm competition.

EFFECTS OF ENZYMATIC AND NONENZYMATIC PROTEINS ONARBACIASPERMATOZOA: REACTIVATION OF AGED SPERM AND THE INDUCTION OF POLYSPERMY

Arbacia sperm become inactive after dilution in sea water. We have shown that any of six proteins reactivated the aged sperm as judged by their fertilizing capacity or their motility. In suspensions of inactive sperm in which the mean fertilizing capacity was less than 3%, brief incubation with any of the proteins at 0.5 mg/ml stimulated fertilizing capacity to 70—90%. Reactivation by the proteins was detected at concentrations lower than 2 @ig/ml. All six ofthe proteins also stimulated motility ofaged sperm by 30—70%. The normal block to polyspermy may involve inactivation of sperm by sub stances released from the eggsduring the fertilization reaction. All six proteins tested on inactive sperm were also shown to induce polyspermy in mixtures of eggs and fresh sperm. Whereas in control mixtures with polyspermic cleavage of 1% of eggs, proteins at 2 mg/ml induced 5—50% polyspermic cleavage, and induction of polyspermy was detected at 5 @tg/ml. The six proteins showing activity included enzymes and also the relatively inert gelatin. The concentration dependence was upon weight/volume and not molarity. Though the mode of action is unclear, it must be rather nonspecific, and is certainly not dependent on enzymatic activity. The same mode of action is likely for activation of aged sperm and induction of polyspermy.

Modification of Ram Sperm Membranes During Epididymal Transit1

Our previous research established that membrane physical properties of testicular and cauda epididymal sperm differ in many respects. The data presented herein establish the region of the epididymis where these and other changes occur. Isoelectric focusing experiments were used to detect a change in the net charge of the glycocalyx. Evaluations of mixtures of sperm from proximal caput versus distal cauda revealed bimodal distributions with a pH 5.0 band and a band at pH 5.2-5.4. In contrast, evaluation of mixtures of central caput and distal cauda sperm gave a single population focusing at pH 5.0. Thus, the charge of the glycocalyx is altered before sperm enter the central caput. Changes in the lipid bilayer occur in the lower epididymis. Sperm from the caput and corpus resisted cold shock, sperm from proximal cauda were altered, or inconsistently damaged, and membranes of sperm from distal cauda were invariably damaged by coldshock.Changes in the plasma membrane were evaluated by lectin-induced agglutination of sperm. Ricinus Communis Agglutinin-120 (RCA) and Ulex Europeas Agglutinin Type I (UEA) induced a high degree of agglutination with caput sperm, but this capacity was lost as sperm progressed through the epididymis. Concanavalin A (ConA) induced high agglutination with caput and cauda sperm, but a lower extent of agglutination with corpus sperm. In sharp contrast, Wheat Germ Agglutinin (WGA) did not induce agglutination of sperm from the caput or corpus but did induce agglutination of sperm from the proximal and distal cauda epidiymidis. Diluted sheep serum also induced head-to-head agglutination and the extent of thisagglutination also changedassperm passed through theepididymis. Thus, continual modification of the sperm surface occurs throughout epididymal passage with specific changes at discrete locations of the duct between the proximal caput and the central cauda epididymidis.

A comparative study of the effects of freezing and frozen storage on intact and demembranated bull spermatozoa

The damage caused to bull sperm by freezing and thawing them without cryoprotectants was assessed in both intact and membrane-extracted cells. Preparations of membrane-extracted cells were produced by treating the sperm with 0.1% Triton X-100 and motility was restored with exogenously applied ATP and Mg 2+. Motile demembranated sperm showed no detectable reduction in motility after freezing and thawing. In contrast, when intact cells where subjected to freezing and thawing they lost all motility. These damaged cells were also restored to motility when exogenous ATP and Mg 2+ were added to the sperm mixture. Apparently freezing and thawing sperm cells causes damage to the plasma membrane which permits ATP and Mg 2+ to freely enter or leave the cells, but does not damage the components of the sperm cell which generate motility. The effects of storage temperature on frozen demembranated sperm were also explored. Sperm held at −20 °C showed marked structural changes and progressively decreased motility after prolonged storage. When sperm were frozen at −20 °C the mitochondrial structures were completely lost after 48 to 72 hr and ATP caused the disintegration of the flagellum rather than initiating motility. Sperm which were frozen at −76 °C retained motility after short periods of storage, but showed a significant decline in motility when thawed after 8 days. Demembranated sperm which were kept frozen at −196 °C showed no significant loss of motility when thawed after 1 year of storage.

Structural chemistry of outer dense fibers of rat sperm.

Structural chemistry of outer dense fibers of rat sperm.

Infection and Fertilization of Mice after Artificial Insemination with a Mixture of Sperm and Murine Cytomegalovirus

Female mice were artificially inseminated with a solution of sperm and cytomegalovirus (experimental) or sperm alone (control). Maternal infection was usually induced when virus accompanied sperm at insemination. Fertilization occurred in the presence of virus, and embryogenesis appeared normal on gross examination, although a slight reduction in the number of embryos was observed in the experimental group. Tissue cultures were prepared after gestation for 14 days from individual and pooled embryos. In one of 28 instances, virus was recovered from blind-passaged, experimental, embryonic fibroblasts. All control cultures were negative. This model may prove useful in further study of the pathogenesis of intrauterine cytomegalovirus infection.

Diversity in patterns of coital contact of mosquitoes: A scanning electron microscopic study.

Specimens, representative of each of the major taxa of mosquitoes, were fixed in copula and the external genitalia examined by scanning electron microscopy. The periphery of the basin-like everted aedeagus of Aedus aegypti precisely matches that of the everted atrial membrane of the female. These structures are appressed during coitus and sealed by pressure of the paraprocts, aedeagal pouch and proctiger. When everted, the aedeagus of male Culex pipiens reveals a ridged dome that surrounds the genital opening. This dome seals itself laterally into a gutter formed by pad-like extensions of the female's genital lips and is sealed dorsally by pressure of the aedeagal apodeme. The aedeagus of another culicine species, Wyeomyia smithii, bears the gonopore at the apex of a spined tube. This tube is inserted between the female's genital lips and is sealed within the genital atrium. The aedeagus of the toxorhynchitine species Toxorhynchitis brevipalpus is immobile and is inserted deep within the genital atrium of the female where it is sealed by pressure of the atrial walls. Males of each of these mosquitoes deliver a mixture of semen and sperm to the copulatory bursa of the female. After withdrawal of the aedeagus, sperm is transferred to the spermathecae. In contrast, sperm of Anopheles quadrimaculatus are delivered directly to the spermathecal duct. The tube-like aedeagus is positioned by its leaflets during sperm transfer and is driven deep into the atrium, where a mixture of semen and sperm is ejaculated. The significance of mechanical barriers to mating between species is discussed.

Inherited sterility in the F 1 progeny of irradiated male pink bollworms.

When individual P, male Pectinophora gossypiella (Saunders) were treated with 20 had of gamma irradiation and paired with normal females, the sex ratio of the resulting F, adults was 4 males to I female, and the F1 males were not competitive. When males were treated with 15 had, the sex ratio of the F1 adults was about 2 males to I female. Most of these F1 males mated and transferred sperm, but the sperm mixture was abnormal; in half the matings, eupyrene sperm was lacking. As with many other species of moths, the F1 males were more sterile than the irradiated male parent.

Selective fertilization following the use of sperm mixtures in the mouse.

ARTIFICIAL insemination has been carried out in the mouse using mixtures of sperm from a number of inbred lines each containing a suitable genetic marker. The lines are REB (containing the dominant hair structure gene rex, ReRe), G (coat colour and pattern tan, atat), and C3H (coat colour agouti, AA); all possible combinations of these genes are phenotypically distinct in the offspring. The first two lines are derived from inbred lines A and CBA respectively. Sperm is obtained from the vas deferens of killed males and the density of each sample estimated by haemocytometer counts. Sperm mixtures were made with equal numbers of spermatozoa from each donor; the donor males for each mixture were of approximately the same age. Four types of sperm mixture were made, namely, a triple mixture of all three types and three double mixtures containing the three possible pairs of the types. These mixtures were used for inseminating œstrous females of the same three lines. Since fertility was rather low in the inbred lines1, the same sperm mixtures have also been used on other females taken from highly fertile non-inbred strains. These are referred to as X in the tables, which give the results obtained up to the present time.

Iodine Compounds and Fertilisation

ABSTRACT If fresh and ripe spermatozoa of E. esculentus or E. miliaris are put into a solution of thyroxine in sea-water of a concentration of 1/50,000 by weight and at the hydrogen-ion concentration of sea-water (pH 8-2), they very soon come together by their heads and form clumps. The most favourable concentration of the sperm for observing this phenomenon is a mixture of sea-water and undiluted sperm in a proportion of 500 : 1 or 1000 : 1. This “agglutination” is visible under the microscope in 1-3 min. and becomes very obvious in 10 min. In a short time the larger clumps fall to the bottom and there form a flocculent precipitate, which can be collected into the centre of the dish by shaking, and is easily visible to the naked eye. The clumps are dense and at first spherical, but their shape becomes irregular as they grow larger, probably in part by the coalescence of several small clumps to form one larger one, and in part by uneven addition of spermatozoa on the outside of the clump. The sperm are active, at least for many minutes, after they are agglutinated. The agglutination is permanent. It only occurs in fresh and ripe sperm. Perfectly ripe sperm may show a slight spontaneous agglutination in seawater, but this is always much less than the agglutination in the presence of thyroxine.