Clinical aspects of sperm DNA fragmentation detection and male infertility

Over the past 25 years, various methods have been developed to measure sperm DNA strand breaks in situ. Currently, there are four major tests of sperm DNA fragmentation, including the Comet, Tunel, sperm chromatin structure assay (SCSA) and the acridine orange test (AOT). The Comet assay is a light microscope technique where the sperm cells are mixed with melted agarose and then placed on a glass slide. The cells are lysed and then subjected to horizontal electrophoresis. The Tunel assay, another light microscope technique, transfers labeled nucleotide to the 3'OH group of a broken DNA strand with the use of terminal deoxynucleotidyl transferase. The fluorescence intensity of each scored sperm is determined as a "yes" or "no" for sperm on a light microscope slide or by channels of fluorescent intensity in a flow cytometer. The light microscope-based AOT, uses the metachromatic properties of acridine orange to stain sperm cells. The SCSA treats sperm with low pH to denature DNA at the sites of DNA strand breaks, followed by acridine orange (AO) staining of green for native DNA and red for denatured DNA as measured by flow cytometry (FCM) as well as % sperm with high DNA stainability (HDS: immature sperm with intact DNA related to decreased fertilization rates). The SCSA method has defined a 27-30% DNA fragmentation index (DFI) as the point in which a man is placed into a statistical category of taking a longer time to in vivo pregnancy, intra uterine insemination (IUI) and more routine in vitro fertilization (IVF) cycles or no pregnancy. Current data suggest that intracytoplasmic sperm injection (ICSI) may help overcome the diminished pregnancy prognosis with high DFI over the other ART or natural methods.     

Antibodies to flowering-plant sperm

Summary The mechanisms by which sperm cells recognize and fuse with the egg and central cell during double fertilization in flowering plants are unknown. To identify membrane surface molecules that might function in fertilization, we immunized mice with isolated sperm ofBrassica campestris and screened the polyclonal sera and monoclonal hybridoma supernatants by immunocytochemistry for binding to isolated sperm cells. We identified three cell lines producing hybridoma supernatants which bind to sperm cell surfaces inB. campestris and further analyzed the properties of one of these, BRSP1. The molecular mass of the epitope to BRSP1 was 54 kDa, and was not glycosylated. Although the antibodies were immunoglobulin M, neither removal of carbohydrates nor competition with antibodies which recognize arabinogalactan decreased binding. BRSP1 recognized sperm ofPlumbago zeylanica, Nicotiana tabacum. Arabidopsis thaliana, andEruca vesicaria and generative cells ofLilium longiflorum and ofNarcissus tazetta but did not recognize sperm ofHelianthus annuus, Gerbera jamesonii, orZea mays. Antibodies to plant sperm allow us to probe sperm membranes for functional components.     

Ultrastructural aspects of infertility.

Organ culture studies have demonstrated that 17beta oestradiol can cause the rapid deterioration and dissolution of pre-existing nuclear channel systems in the human endometrial glandular cell. The importance of the nuclear channel system in implantation is emphasized and the implications of secretory activity in the endometrium of an infertile woman are discussed.     

Antibodies to semen diluent and infertility in rabbits

Parenteral immunization with semen, with Nagase egg-yolk diluent (NEYD) or with semen in NEYD depressed fertility in does artificially inseminated with semen in NEYD. Although fertilization rates were significantly reduced in the animals which had been immunized with semen alone (P < 0.05) or with diluent alone (P < 0.05), the greatest reduction occurred in the animals which had been immunized with semen plus diluent (P < 0.01). Both the anti-semen antibodies and the anti-diluent antibodies contributed to the reduction in fertility of the animals which had been immunized with semen plus diluent.     

Functional differentiation of thymocytes induced by macrophages: cellular, humoral and genetic aspects

The cellular, humoral and genetical mechanisms of induction of T-effectors of the graft vs. host reaction (GHR) were studied in a double-cell culture of phagocytizing mononuclears with thymocytes. Experiments were carried out on mice of inbred and recombinant strains. The GHR intensity was estimated by the increase in the number of cells in the popliteal lymph node, regional with reference to the introduction of parental thymocytes into the F1 hybrid. For the induction of the GHR T-effectors from the immature population of thymocytes to be realized, a direct physical contact and identity by the H-2K locus of the major histocompatibility complex between the cooperating cells in culture are indispensable. An antiserum containing antibodies against the H-2K locus products prevents the induction. At the same time antibodies against antigens controlled by loci of I-region or the H-2D locus do not affect the accumulation of T-effectors. Contact interaction of phagocytizing mononuclears with thymocytes results in accumulation of a 65,000 D humoral factor in the culture medium. Incubation of the intact thymocytes with this factor ensures functional transformation of immature thymocytes to corresponding effector cells. For the humoral induction of T-effectors to be successfully realized, identity by the T-2K locus between the factor producents and intact thymocytes is indispensable, as well as in the conditions of direct intercellular interaction. It is suggested that H-2K specificity is incorporated into the factor structure.     

Antibodies to guinea pig lymphokines. VII. Reactivity with products of lymphoid and nonlymphoid cells.

It was shown previously, that an antiserum directed against highly purified fractions of migration inhibitory factor inhibits delayed hypersensitivity reactions in vivo and in vitro. Using radiolabeling techniques we determined that the anti-lymphokine serum reacted primarily with three lymphocyte activation products (m.w. 60,000, 45,000, and 30,000) all of which had a similar isoelectric point of 5.2. The cellular origin of this material was investigated. It was found that activated B cells, B leukemia cells (L2C), and growing fibroblasts produced material of a similar m.w. as analyzed on SDS-PAGE. No cross-reaction was found with radiolabeled products of activated murine and human lymph node cells and of SV 40-infected African green monkey kidney cells. The isoelectric point of the reactive material from B cells, leukemia cells, and fibroblasts was determined at 5.2. In addition to material with pI 5.2, lymph node cells also produced material with pI 3.5 to 4.5, which focused at pH 5.0 to 5.4. After neuraminidase treatment macrophage migration inhibitory activity in fibroblast culture supernatants could be absorbed specifically to insolubilized anti-lymphokine antibody. These findings suggests that lymphoid and nonlymphoid cells are capable of producing molecules whose physicochemical and functional properties appear to be identical.     

Anti-tetanus immunization. Complexity and polymorphism of humoral and cellular manifestations

In general, anti-tetanus vaccination is well tolerated by the recipients, but the repeated inoculations of the vaccine might result in immediate or delayed type of hypersensitivity. The more recent knowledge of the epitopic construction of the toxin defined by tools offered by molecular biology, has helped understand the complexity of humoral and cellular responses as a consequence to vaccination. We have analyzed in this work major antitetanus post-vaccinal manifestations in normal subjects, as well as in patients suffering from different pathological disorders, with regard to immunoglobulin class expression, idiotypic profile, HLA-susceptibility and in vitro cellular responses.     

Playing with cellular and humoral immunity

An interesting and easy game for the teaching of immunology was developed by the authors. Previously distributed figure cards are used by students that have to ask questions of each other. Most students considered the game to be interesting (97.7%) and declared that it helped them to understand the subject. The authors emphasise the importance of improving the quality of teaching using visual memory, fun and competition.     

Cellular aspects of tolerance. VII. Inheritance of the resistance to tolerance induction

The half-lives of elimination ($\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) of ¹³¹I-RGG from the body of normal A or Balb/c animals was much longer than the $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of SJL mice. At all ages, the $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of normal hybrids (A × SJL, SJL × A, Balb/c × SJL) was similar to or longer than that of the A or Balb/c parents. Thus, in terms of the $\text{T}\text{1}\text{2}$ of normal animals, the SJL responsiveness to ¹³¹I-RGG appeared to be a recessive trait. Tolerance could be induced in newborn animals and, in terms of $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, the degree of unresponsiveness at the age of 6 weeks, was the same in A, Balb/c, A × SJL, and Balb/c × SJL animals but was much shorter in SJL mice. Thus, in neonatally induced tolerance, the duration of tolerance was recessive for the SJL type. The average $\text{T}{}_{\mathrm{<mtext>built1</mtext><mtext>2</mtext>}}$ after tolerance induction in 3-week-old hybrids (A × SJL, SJL × A, Balb/c × SJL) was similar to that of the A or Balb/c parent, but by the 8th and 12th week it approached the average $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of the SJL parent. Comparing 8-week-old hybrids, the average $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ was longest in A × SJL hybrids and identical in SJL × A and Balb/c × SJL mice. An examination of $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ distribution in various 8- and 12-week-old crosses and backcrosses revealed a fairly large proportion of individuals with a $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ which was intermediate between SJL and the other parent. There was a tendency for this number to decrease in 12 weeks as compared to 8-week-old mice. In 8-week-old mice, the number of animals with intermediate $\text{T}{}_{\mathrm{<mtext>built1</mtext><mtext>2</mtext>}}$ was smallest when SJL was the maternal animal [(SJL × A); SJL × (A × SJL); SJL × (SJL × A)]. There was no link between $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of tolerant animals and either the immunoglobulin allotype (MuAl/MuA2) or the C5 eniotype (MuB1 positive/MuB1 negative).     

Transformation of sperm histone during formation and maturation of rat spermatozoa.

Changes of chromosomal basic proteins of rats have been followed during transformation of spermatids into spermatozoa in the testis and during maturation of spermatozoa in the epididymis. Rat testis chromatin has been fractionated on the basis of differing sensitivity to shearing, yielding a soluble fraction and a condensed fraction. The sperm histone is found in the condense fraction. Somatic-type histones are found in both fractions. The somatic-type histones in the condensed fraction contains much more lysine-rich histone I, than does the somatic-type histones in the soluble fraction. This may suggest that the lysine-rich histone I is the last histone to be displaced during the replacement of somatic-type histones by sperm histone. After extensive shearing followed by sucrose centrifugation, the condensed portion of testis chromatin can be further fractionated into two morphologically distinctive fractions. One is a heavy fraction possessing an elongated shape typical of the head of late spermatids. The other is a light fraction which is presumably derived from spermatids at earlier stages of chromatin condensation and which is seen as a beaded structure in the light microscope. Sperm histone of testis chromatin can be extractable completely by guanidinium chloride without a thiol, wheras 2-mercaptoethanol is required for extraction of sperm histone from caput and cauda epididymal spermatozoa. The light fraction of the condensed testis chromatin contains unmodified and monophospho-sperm histone. The sperm histones of the heavy fraction is mainly of monophospho and diphospho species, whereas unmodified and monophosphosperm histones are found in caput and cauda epididymal spermatozoa. Labeling of cysteine sulfhydryl groups of sperm histone releases by 2-mercaptoethanol treatment shows that essentially all of the cysteine residues of sperm histone in testis chromatin are present as sulfhydryl groups, while those of sperm histone isolated from mature (cauda epididymal) spermatozoa are present as disulfide forms and approximately 50% of the cysteine residues of sperm histone obtained from caput epididymal spermatozoa are in disulfide forms. These results suggest that phosphorylation of sperm histone is involved in the process of chromatin condensation during transformation of spermatozoa in the epididymis.     

Immunolocalization of sperm protein 17 in human testis and ejaculated spermatozoa.

Sperm protein 17 (Sp17) is a highly conserved mammalian protein whose primary function is still poorly understood. Immunohistochemistry (IHC) in the human testis reveals the presence of Sp17 in some spermatocytes and abundantly in spermatids. All spermatogonia, Sertoli cells, and Leydig cells appear to be immunonegative for Sp17, whereas some interstitial cells are immunopositive. IHC recognized two distinct populations (immunopositive or not for Sp17) in the ejaculated spermatozoa. Although it will be necessary to clarify why some ejaculated spermatozoa do not contain Sp17, its distribution suggests that this protein may be associated with some phases of germinal cell differentiation.     

Molecular and cellular aspects of immunologic tolerance.

This review seeks to explain the most exciting recent data concerning the nature of self/non-self discrimination by the immune system in a manner accessible to a biochemical readership. The nature of recognition in the two great lymphocyte families, B cells and T cells, is described with special emphasis on the nature of the ligands recognized by each. The history of the field of immunologic tolerance is surveyed, as are the key experiments on conventional mice which provided a conceptual framework. This suggested that tolerance was essentially due to 'holes' in the recognition repertoires of both the T and B cell populations so that lymphocytes competent to react to self antigens were not part of the immunologic dictionary. There were essentially two ways to achieve this situation. On the one hand, self antigens might 'catch' developing lymphocytes early in their ontogeny and delete the cell, a process of clonal abortion. On the other hand, self antigens might signal lymphocytes (particularly immature cells) in a negative manner, reducing or abolishing their capacity for later responses, without causing death. This process is referred to as clonal anergy. Evidence for both processes exists. Special emphasis is placed on a wave of experimentation beginning in 1988 which imaginatively uses transgenic mouse technology to study tolerance. Transgenic manipulations can produce mice which synthesize foreign antigens in a constitutive and/or inducible manner, sometimes only in specific locations; mice which possess T or B lymphocytes almost all expressing a given receptor of known specificity; and mice which are an immunologic time bomb in that the antigen is present and so too are lymphocytes all endowed with receptors for that antigen. These experiments have vindicated the possibility of both clonal abortion and clonal anergy in both T and B cell populations, the choice of which phenomenon occurs depending on a number of operational circumstances. For T cell tolerance, clonal abortion occurs if the self antigenic determinant concerned is present within the thymus; if not, clonal anergy is more likely. For B cell tolerance, the strength of the negative signal and therefore the choice between abortion and anergy depends on the molar concentration of the self antigen, the capacity for multivalent presentation to a B cell, and the affinity of the B cell's receptor for the antigen in question. Some B cells with low affinity for self antigens certainly escape censorship and remain capable of secreting low affinity anti-self antibodies, which however do no harm.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytotoxic activity of lymphocytes. VII. cellular origin of alpha-lymphotoxin.

To detect the cellular origins of alpha-lymphotoxin (alpha-LT), we cultured various subpopulations of human blood lymphocytes separated by erythrocyte-rosetting techniques with various mitogens. T cell-enriched subpopulations responded to PHA by increased 3H-thymidine uptake into DNA and large amounts of alpha-LT production. SPL and Con A-Sepharose stimulated DNA synthesis in T cell-enriched cultures if the macrophage content was greater than 1.5%; however, alpha-LT production was not induced by these two mitogens even when reconstituted with 10% macrophages. B and/or null cell-enriched populations severely depleted of T cells (less than 0.7% did not respond to PHA, SPL, or Con A-Sepharose. However, reconstitution to 5 or more percent in E-RFC allowed all three mitogens to stimulate DNA synthesis and alpha-LT production. The LT made by all cell populations 5 and 7 days after stimulation were equally neutralized by a heterologous antiserum to alpha-LT. These results show that human T and B and/or null cells, when appropriately stimulated, can produce alpha-LT.     

Neonatal exposure to idiotype induces Schistosoma mansoni egg antigen-specific cellular and humoral immune responses.

Exposure of neonatal mice to appropriate, cross-reactive Id (CRI) preparations alters immune responsiveness, ameliorates pathology, and prolongs survival of animals upon subsequent Schistosoma mansoni infection. However, because schistosome infections profoundly affect host immunobiology, which responses are effected by neonatal Id exposure alone and which responses are influenced by infection is unclear. To directly examine the schistosome soluble egg Ag (SEA)-specific immune responses altered by CRI exposure, neonatal mice were injected with CRI-expressing (CRI+) SEA-specific Ab preparations, SEA-specific Abs that did not express CRI (CRI-), or normal mouse Ig. At 9 wk of age, only mice that were neonatally exposed to CRI+ anti-SEA Abs displayed significant SEA-specific IgG serum levels and spleen cell proliferative responses. SEA-stimulated spleen cells from these CRI+-exposed mice also produced IFN-gamma, although not at significantly higher levels than mice receiving CRI- Id or normal mouse Ig. If CRI+-exposed mice were also injected with SEA at 8 wk of age, the 9-wk IFN-gamma responses were significantly higher than those of the other neonatal injection groups. The presence of both CRI and anti-CRI in the sera of animals neonatally injected with CRI, but receiving no exposure to S. mansoni Ags or infection, suggested a functional idiotypic network led to these responses. These data demonstrate that appropriate idiotypic exposure induces B and T cell responsiveness to the Ag recognized by the Id and support the hypothesis that neonatal idiotypic exposure can be an important immunoregulatory factor in schistosomiasis.