Steven S. Witkin

Information Transfer and Sperm Uptake by Mammalian Somatic Cells

Publisher Summary This chapter describes that sperm are endowed by their specific differentiation to interact with and to deliver their genetic components into ova for subsequent activation and function. Whether analogous genetic information can be transferred by sperm to mammalian somatic cells is unknown. The chapter describes that mammalian sperm interact with mammalian somatic cells as evidenced by actual penetration, the appearance of changes in morphology and growth characteristics, and the production of new gene products. These changes may be consequences of the direct acquisition and genetic expression of either the sperm chromatin itself or the reverse transcriptase containing complex within the sperm heads. The interaction of sperm with somatic cells that leads to fetal antigen expression and to changes resembling oncogenic transformation may involve a recapitulation, at least in part, of the program of postfertilization gene expression. The normal function of sperm is fertilization; the presence of its various components in somatic cells might very well induce changes analogous to those that occur in embryological and fetal development. It has been estimated that the amount of unique-sequence DNA that is transcribed in the rabbit blastocyst but no longer in the midgestation embryo is enough to specify some 23,000 different proteins; the corresponding difference between normal and virus-transformed mouse cells is regarded as sufficient to specify some 200,000 different proteins. To sort out the many steps involved in embryogenesis, fetal development, and oncogenesis in the face of such complexity is indeed a prodigious task.

An enzyme-linked immunoassay for the detection of antibodies to the mouse mammary tumor virus: Application to human breast cancer

An enzyme-linked immunoassay (ELISA) was developed, using the mouse mammary tumor virus (MMTV) fixed to wells of a microtiter plate, for the determination of antibodies to MMTV. The intensity of the final color change was dependent upon virus or viral antibody concentration. MMTV antibody was readily detectable in sera diluted as much as 1 : 2800. Fixed MMTV bound antibodies to an internal viral protein (p 28) as well as to viral envelope components (gp 52, gp 34), demonstrating that the virus was rendered permeable by our procedure. Applying this assay to human sera, significant differences (P less than 0.005) in IgG binding to MMTV were detected between sera of breast cancer patients, benign breast disease patients and healthy individuals. 26% of breast cancer-derived sera contained MMTV binding antibody; 10% of benign sera or 8% of normal sera were also positive. The reactivity of human IgG with MMTV was blocked by prior incubation of the virus with antisera to gp 34 or, to a lesser extent, with gp 52. The results demonstrate that MMTV antibodies can be quantitated by this simple, rapid and inexpensive procedure.

Isolation of a nuclear DNA synthesizing complex from human sperm.

Summary A simplified procedure is described for isolating a human sperm nuclear DNA-synthesizing complex which does not involve the use of detergent or necessitate the prior purification of membrane-free nuclei. Selective disruption of sperm heads is accomplished by the use of dithiothreitol, trypsin, and deoxyribonuclease. The particulate complex is freed of soluble components by sedimentation through a solution of 19% (w/v) Metrizamide and further purified by buoyant density centrifugation. The characteristics of this endogenously templated nuclear DNA polymerase differ from other sperm-associated polymerases that have been reported.

Sperm immobilization by sera from unimmunized guinea pigs: requirements for immunoglobulin and complement

Abstract The components in serum from non-immunized guinea pigs responsible for the immunological immobilization of human spermatozoa have been elucidated. Heating the serum at 56°C for 30 min, pretreatng the serum with purified cobra venom factor or the inclusion aggregated human IgG in the immobilization test all eliminated its immobilizing capacity, suggesting the involvement of complement in this process. Using purified guinea pigs components, it was established that complement components C1-9 as well as guinea pig IgG were required for immobilization. Rabbit IgG, but not human IgG, could be substituted for guinea pig IgG without affecting the degree of immobilization. Absorption of guinea pig IgG with human spermatoza to its addition to C1-9 and motile spermatoza led to the loss of all immobilizing activity. Thus, it appears that ‘natural’ antibody to spermatoza plus all nine complements are required for sperm immobilization.

Effects of Vasectomy and Antisperm Antibodies on Human Seminal Fluid Deoxyribonucleic Acid Polymerase Activity

DNA-synthesizing complexes possessing a sucrose buoyant density of 1.24 to 1.25 gm/ml were identified in cell-free human seminal fluids prior to and 1 to 3 months following division of the vasa deferentia, performed for fertility control in otherwise normal males. After vasectomy, there was a 3.5-fold decrease in endogenous DNA polymerase activity per ejaculate. Partial purification of the seminal fluid DNA polymerase by gel filtration revealed a similar 3.5-fold decrease in dT12--18-poly (rA)-templated DNA polymerase activity postvasectomy. Immunoglobulin G, isolated from a rabbit immunized with nuclei derived from detergent-treated ejaculated human spermatozoa, inhibited both sperm- and seminal fluid-derived DNA polymerase activity. The decrease in seminal fluid enzyme activity following vasectomy might be due to its inhibition by sperm autoantibodies induced after vas division.

A particulate DNA polymerase activity in adult rat brain

Abstract A particulate fraction of adult rat brain (sucrose buoyant density 1.24 gm/ml) catalyzed the incorporation of [ 3 H]dTTP into an acid-insoluble product in an endogenously templated reaction sensitive to ribonuclease pretreatment. Upon fractionation, this activity was identified in the cerebellum, pons, frontal lobes and base. The DNA polymerase present in these brain fractions exhibited a strong preference for the synthetic template dT 12–18 ·poly rA rather than dT 12–18 ·poly dA; dT 10 was completely inactive. Purification and equilibrium Cs 2 SO 4 gradient centrifugation of the [ 3 H]DNA product-endogenous template complex suggested that RNA was serving as primer for endogenous DNA synthesis.