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Mammalian spermatozoa have been dissected by a variety of chemical techniques to yield free heads, tails with attached midpieces, and tails without mitochondria. By brief exposure to trypsin, mouse and rat spermatozoa were cleaved at the junction of the head and the tail, while human, guinea pig and rabbit spermatozoa were cleaved by trypsin only after prior incubation with a sulphhydryl reducing agent. Treatment with acid or base cleaved spermatozoa of all species examined. In contrast, exposure of spermatozoa to 1% sarkosyl NL-97 resulted in the quantitative cleavage of mouse cells without noticeable effect on the spermatozoa of the other species. Mitochondria were removed from the midpiece of intact sperm and isolated tails by gentle shaking after treatment with reducing agents. Homogeneous populations of spermatozoan subcellular components were obtained by density gradient centrifugation. Ultrastructural analysis showed that cleavage of mouse spermatozoa by trypsin occurs at a specific location in the neck of the cell without trypsin occurs at a specific location in the neck of the cell without observable damage to other cell structures. The basal plate remained attached to the head structures. In contrast cleavage of spermatozoa by sarkosyl or acid left the basal plate attached to the spermatozoan midpiece. Sarkosyl also removed the plasma membrane and extracted mitochondrial components. Treatment with acid or base also resulted in vesiculation of the plasma membrane and dissolution of the acrosome. Molecular probes have also been used to facilitate mapping of the cell surface. Each mouse spermatozoon has about 10-7 receptors for the lectin concanavalin A. Binding of fluorescein-labelled concanavalin A indicated that the majority of the receptors is in the acrosomal region; this polar distribution was confirmed by measurement of the number of sites on purified heads and tails. In addition, the low molecular weight probe ANS bound to the plasma membrane of spermatozoa from all species examined, with immediate immobilization of the cells. Ethidium bromide bound to the spermatozoan head without affecting motility.
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PMID:Chemical dissection of mammalian spermatozoa. 23 23

Infertility in men and women with spermagglutinins is the result of disturbed penetration and migration of spermatozoa in the cervical mucus. In ejaculates with partial spermagglutination caused by autoimmunization, the progressive propulsion of the sperm was changed into stationary, shaking movement the moment the sperm came into contact with cervical mucus. The same alteration in spermatozoal motility pattern also occurred when spermatozoa from a normal, fertile ejaculate came into contact with cervical mucus of a woman whose serum contained sperm antibodies. This shaking phenomenon was visualized in a simple test, the sperm-cervical mucus contact test. We demonstrated that sensitized spermatozoa exhibit the shaking phenomenon after contact with the glycoprotein fraction of the cervical mucus and not after contact with the aqueous fraction. Therefore, the hypothesis is introduced that the shaking phenomenon is due to an interaction between sensitized spermatozoa and the glycoprotein micelles in cervical mucus.
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PMID:The sperm-cervical mucus contact test: a preliminary report. 125 29

Sperm (spermatozoa and the various secretions of the accessory glands) with its very complex antigenic system is only produced from puberty and thus at a time when the body has already had a for a long time the facility to respond to an antigenic stimulation with an immune reaction. Because of this, the spermatozoa, in contrast to other cells of the organism, are considered as strangers and may behave as auto-antigens. However, the existence of a haemato-testicular barrier, that prevents all contact between the sperm and the immune system must be recognized in man, its rupture has the consequence of formation of anti-spermatozoa antibodies. It is estimated that 5% of cases of sterility are of immunological origin and linked to the presence in man and/or woman of anti-sperm antibodies. The production is greater in man than in women because of the roles, in the first of macrophages and in the second of polynuclear neutrophils in the destruction of the spermatozoa. Anti spermatozoal antibodies play a part in sterility by: Spontaneous agglutination in ejaculation, thus preventing their progression in the female genital tract. Free spermatozoa, charged with antibody are retained at the level of the cervical secretion = SHAKING phenomenon. The anti-spermatozoal antibodies mask some antigens, so preventing penetration of the spermatozoa into the ovule. Contact of spermatozoa with antibody fixed to the uterine tissue is followed by secretion of histamine and this encourages the expulsion of an implanted egg. This is before the existence of: Spontaneous agglutination in ejaculation, Oligospermatosis, Indeterminate cause sterility, Immobilisation of the spermatozoa in the cervical secretion, Failure of repetition.
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PMID:[Immunological causes of male infertility]. 206 84

Seventeen couples (13%) were selected from a group of 129 infertile patients according to the following criteria: (i) unexplained infertility for 3 years and (ii) greater than 50% shaking spermatozoa during SCMC testing. The couples were tested for sperm antibodies after a complete diagnostic work-up schedule. Post-coital tests were performed during the first menstrual cycle of the wife, followed by SCMC and sperm antibody titre testing. Ten males and seven females were thus treated with 96 mg methylprednisolone. Nine (52%) of the 17 with sperm antibodies achieved a pregnancy. The results of the SCMC test were in all the cases indicative of the actual sperm antibody titre. Reduction of the antibody titre and a decrease in the percentage of shaking spermatozoa as detected by the SCMC test correlated well with the pregnancy rate amongst the patients.
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PMID:The SCMC test: a reliable monitor for antispermatozoal antibodies. 317 Jul

Trout testes at various stages of maturation were dissociated by perfusion at 12 degrees C with collagenase plus pronase and then with collagenase alone, followed by slight shaking overnight in 1% bovine albumin. This step provided a suspension of isolated somatic and germ cells, clusters of interstitial cells, and either intact spermatogenetic cysts (meiotic testes) or clusters of Sertoli cells (other testes). Most of the spermatozoa were removed from the testis cell suspension by centrifugation in Percoll (density 1.065 g/ml). Sertoli and Leydig cells were prepared by a two-step separation method: 1) the testis cell suspension was separated by sedimentation at unit gravity into "isolated cell" and "cell cluster" populations; 2) these populations were fractionated by isopyknic centrifugation in Percoll gradients. In terms of somatic cell composition, a nearly pure Sertoli cell (clusters) population was obtained between 1.017 and 1.033 g/ml and a Leydig cell (clusters) enriched population of between 1.033 and 1.048 g/ml (testes resuming spermatogenesis) or 1.048 and 1.062 g/ml (other testes). These various cell populations were cultured in modified Leibovitz L15 medium for 10-15 days. When seeded, the Sertoli cells had a normal ultrastructure that remained unchanged for at least 10 days, and the steroidogenic activity of Leydig cells could be stimulated by salmon gonadotropin. Leydig cells remained 3 beta-HSD positive and produced progesterone and 17 alpha, 20 beta-OH progesterone for at least 11 days. This study points out that viable and differentiated trout somatic testicular cells can be prepared and cultured for several days.
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PMID:Trout Sertoli and Leydig cells: isolation, separation, and culture. 323 52

The acrosomal matrix of hamster spermatozoa was enriched and characterized. Acrosomal matrices were released from spermatozoa with shaking in a pH 5.2 buffer containing Triton X-100 and protease inhibitors, and enriched on a glass-bead column. Phase-contrast microscopy indicated that 70-80% of the acrosomal matrices were released from the spermatozoa and only minor contamination from sperm heads was detected. Transmission electron microscopy confirmed the low level of contamination in the preparation and revealed a bilaminar structure similar but not identical to that of guinea-pig acrosomal matrix. One- and two-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed the acrosomal matrix to be a complex structure enriched for several polypeptides. Proteinase activity was demonstrated by gelatin-SDS-PAGE. The major activity corresponded to bands of relative molecular masses (Mr) of 56,000, 51,000 and 48,000 with two minor bands of Mr 30,000 and 28,000. The lectin Pisum sativum agglutinin (PSA) bound to the anterior head of spermatozoa and isolated acrosomal matrix as judged by fluorescence microscopy using FITC-PSA. Western blots of spermatozoa and acrosomal matrices followed by overlay with biotinylated PSA indicated that there are at least two PSA-binding glycoproteins of Mr 60,000 and 72,000.
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PMID:Characterization of isolated acrosomal matrices from hamster spermatozoa. 329 98

Some immunological aspects of male infertility are discussed, including the mechanism of induction of auto-antibodies to sperm-specific antigens. Tests to determine antispermatozoal antibodies in serum are discussed. Since there is no direct relation with infertility, more attention is focused on the presence of antispermatozoal antibodies in semen. These antibodies affect male fertility by reducing the capacity of the spermatozoa to penetrate cervical mucus. This penetration inhibition is caused by autoagglutination of the spermatozoa in the ejaculate and by the shaking phenomenon. The sperm-cervical mucus contact test, based on the shaking phenomenon is described. The use of the Mixed Antiglobulin Reaction tests, to detect IgG and IgA antibodies on spermatozoa is discussed. Finally, the effect of antispermatozoal antibodies on the fertilization process is reviewed.
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PMID:Immunological aspects of male infertility. 331 Jul 56

A fertile man had sperm-agglutinating activity in his serum (titers 1:16-1:128) and in his seminal plasma (titers 1:128-1:2048). The antibodies in the seminal plasma could be absorbed with anti-IgA antiserum but not with anti-IgG antiserum. A fresh ejaculate showed strong auto-agglutination of the spermatozoa. With mixed antiglobulin reaction tests (MART) and/or immunobead tests (IBT), IgA and IgG were detected on almost all motile spermatozoa; the erythrocytes, in the MART, and the latex spheres, in the IBT, adhered mainly to the tip of the tail. After mixing the fresh semen with cervical mucus, only 40% of the spermatozoa were locally shaking. The spermatozoa showed excellent penetration of cervical mucus in vitro. This case shows that IgA coating of the tails of the spermatozoa does not necessarily lead to adherence of these spermatozoa to the micelles of the cervical mucus and that the sperm cervical mucus contact test has a better predictive value than the sperm agglutination titer in the seminal plasma.
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PMID:A fertile man with a high sperm agglutination titer in the seminal plasma: a case report. 342 77

Thirteen couples with a history of primary infertility for at least 3 years were selected according to the following criteria: a positive sperm/cervical mucus contact (SCMC) test, the presence of sperm-agglutinating antibodies in the cervical mucus, and/or seminal fluid of one of the partners. Nine of the males and 4 of the 13 females were accordingly treated with high dosages of corticosteroids for 6 months. Among 4 (44%) males and 2 (50%) females a pregnancy ensued. Four (80%) of the 6 patients in whom a pregnancy was reported showed not only a decline in their sperm antibody titer but also a decrease in the percent shaking spermatozoa during SCMC testing.
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PMID:Sperm/cervical mucus contact test for sperm antibodies. 382 86

Mouse spermatozoa recovered from the caput, corpus, or cauda epididymidis were examined for their ability to bind the vitro to zonae pellucidae. Since spermatozoa from the caput epididymidis do not display progressive motility as compared with more mature spermatozoa, direct comparison of the different sperm populations may not measure zona binding ability validly. To equalize the motile properties of the spermatozoa, a method was developed to immobilize vigorously motile corpus and cauda spermatozoa. Reversible immobilization was achieved by incubation in 25 microM La3+ which resulted in a twitching, nonprogressive type of motility. La3+ incubation did not appear to affect the spermatozoa adversely, since vigorous motility (equivalent to the controls) of corpus and cauda sperm was displayed upon subsequent incubation in standard La3+-free culture medium. Moreover, cauda spermatozoa preincubated for 90 min in La3+ displayed levels of fertilization in vitro equivalent to their control counterparts. Using this La3+-immobilization technique, the zona binding ability of the different sperm population could be assayed. Gamete collision was insured under these conditions by shaking the gamete-containing dishes at 100 cycles/min. Regardless of the extent of sperm motility, a similar zona-binding pattern emerged: cauda sperm bound in high numbers, corpus sperm bound at some intermediate level (an average of 24% of cauda binding level), and caput sperm bound rarely (2% of cauda binding level). Thus it appears that, for mouse spermatozoa, the onset of fertility during epididymal transit parallels the ability to bind to zonae pellucidae. Unlike the interaction between spermatozoa and zonae, La3+ was unable to support sperm binding in to egg plasma membrane, supporting the view that mouse sperm may have different sites for interaction with the zonae pellucida and the egg plasma membrane.
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PMID:Development of the ability to bind to zonae pellucidae during epididymal maturation: reversible immobilization of mouse-spermatozoa by lanthanum. 720 Aug 7


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