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C F Millette

Publications and source records attributed to C F Millette.

53 records · Page 3Linked to original sources
PubMed1981

Elevated cholesterol and dolichol synthesis in mouse pachytene spermatocytes.

Results are presented here which demonstrate that the rates of [14C]acetate incorporation into cholesterol and dolichol increased 4- to 5-fold as mouse spermatocytes matured from the preleptotene to prepuberal pachytene stages. The rate of acetate incorporation into cholesterol then decreased in late pachynema, remained low at all subsequent stages of meiosis, and was very low in mature sperm. In contrast, the rate of acetate incorporation into dolichol remained elevated in late pachytene spermatocytes and round spermatids, then decreased and remained low in mature sperm. The ratio of the rate of [14C]acetate incorporation into dolichol to the rate of incorporation into cholesterol increased during late meiotic prophase and remained high in round spermatids; this altered ratio is further evidence of independent regulation of dolichol and cholesterol synthesis in testes. It was shown previously that normal adult mouse testes incorporated acetate into dolichol at a much higher rate (1.8 to 2.4% of the rate of incorporation into cholesterol) than did testes from sterile W/Wv mice (0.02%) or X-irradiated mice (0.24%). This high rate of acetate incorporation into dolichol in adult testes is now attributed to differentiating spermatocytes, with particularly high rates being observed during pachynema.

Acetates↗
PubMed1981

Cell surface marker proteins during mouse spermatogenesis: two-dimensional electrophoretic analysis.

Purified plasma membranes isolated from separated highly homogenous populations of mouse pachytene spermatocytes, round spermatids (step I-8), and residual bodies have been compared using 2-dimensional polyacrylamide gel electrophoresis. Two polypeptides apparently specific to pachytene spermatocytes have been identified. Component Pa has a molecular weight of 90 k daltons (K) and pI of 5.6. Component Pb has a molecular weight of 56.5 K and a pI of 6.0. Four polypeptides detected only in plasma membranes of round spermatids have been identified as follows: RSa, 90-95 K and pI 5.9; RSb, also 90-95 K and pI 5.9; RSc, approximately 88 K and pI 5.5; RSd, 58 K and pI 6.0-6.3. No polypeptides unique to residual body membranes were identified. Short-term culture experiments have established that separated adult mouse spermatogenic cells survive short-term culture in vitro. These cells actively synthesize numerous cellular proteins as determined by the incorporation of [3H]leucine. Investigations concerning the effect of the cell separation procedure on mouse spermatogenic cell membranes indicate that only 7 of 110-120 total plasma membrane constituents are degraded enzymically during cell purification. Only one of these constituents may correspond to the presumptive cell differentiation markers described for pachytene spermatocytes and round spermatids. These results indicate, therefore, that plasma membranes obtained immediately after cell separation are suitable for the detailed biochemical analysis of the most integral surface proteins during spermatogenesis in the mouse.

Animals↗
PubMed1980

Isolation of plasma membranes from purified mouse spermatogenic cells.

Plasma membranes have been prepared from purified pachytene spermatocytes, round spermatids and residual bodies of the adult mouse testis using procedures modified from other authors'. Isolated membranes have been examined using electron microscopy, lectin binding and enzymic assays. Ultrastructural observation reveals smooth unit-membrane vesicles from 0.4-1.7 micrometer diameter. No contamination by nuclei, mitochondria or lysosomes is detected microscopically. Radiolabelled lectin-binding experiments [125I-RCAI, 125I-green pea lectin] indicate that cell surface label cofractionates with material identified morphologically as plasma membrane. Estimates of total recovery of membrane, based upn the lectin data, average 33%. Biochemical analysis of subcellular markers reveal that no detectable DNA and only 1.2% of the total cellular RNA cofractionate with membranes. A variety of enzyme assays suggests little contamination by cytosol enzymes, Golgi material or mitochondria. Assays of 5'-nucleotidase (E.C. 3.1.3.5) indicate that this enzyme is not a major component of developing mouse spermatogenic cell membranes. Instead, Sertoli cells represent the most important source of this enzyme in the adult seminiferous tubule. Polyacrylamide gel analysis of membranes isolated from purified germ cells reveals significant differences in the protein compositions of pachytene spermatocyte and round spermatid membranes. The preparation of highly purified plasma membranes from homogeneous populations of spermatogenic cells should facilitate the biochemical characterization of cell surface antigens specific to developing male germ cells.

Animals↗
PubMed1977

Spermatogenic cells of the prepuberal mouse. Isolation and morphological characterization.

A procedure is described which permits the isolation from the prepuberal mouse testis of highly purified populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, leptotene and zygotene primary spermatocytes, pachytene primary spermatocytes and Sertoli cells. The successful isolation of these prepuberal cell types was accomplished by: (a) defining distinctive morphological characteristics of the cells, (b) determining the temporal appearance of spermatogenic cells during prepuberal development, (c) isolating purified seminiferous cords, after dissociation of the testis with collagenase, (d) separating the trypsin-dispersed seminiferous cells by sedimentation velocity at unit gravity, and (e) assessing the identity and purity of the isolated cell types by microscopy. The seminiferous epithelium from day 6 animals contains only primitive type A spermatogonia and Sertoli cells. Type A and type B spermatogonia are present by day 8. At day 10, meiotic prophase is initiated, with the germ cells reaching the early and late pachytene stages by 14 and 18, respectively. Secondary spermatocytes and haploid spermatids appear throughout this developmental period. The purity and optimum day for the recovery of specific cell types are as follows: day 6, Sertoli cells (purity>99 percent) and primitive type A spermatogonia (90 percent); day 8, type A spermatogonia (91 percent) and type B spermatogonia (76 percent); day 18, preleptotene spermatocytes (93 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent).

Age Factors↗
PubMed1977

Temporal expression of membrane antigens during mouse spermatogenesis.

The temporal expression of cell surface antigens during mammalian spermatogenesis has been investigated using isolated populations of mouse germ cells. Spermatogenic cells at advanced stages of differentiation, including pachytene primary spermatocytes, round spermatids, and residual bodies of Regaud and mature spermatozoa, contain common antigenic membrane components which are not detected before the pachytene stage of the first meiotic prophase. These surface constituents are not detected on isolated populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, or leptotene and zygotene primary spermatocytes. These results have been demonstrated by immunofluorescence microscopy, by complement-mediated cytotoxicity, and by quantitative measurements of immunoglobulin (Ig) receptors on the plasma membrane of all cell populations examined. The cell surface antigens detected on germ cells are not found on mouse thymocytes, erythrocytes, or peripheral blood lymphocytes as determined by immunofluorescence and by cytotoxicity assays. Furthermore, absorption of antisera with kidney and liver tissue does not reduce the reactivity of the antibody preparations with spermatogenic cells, indicating that these antigenic determinants are specific to germ cells. This represents the first direct evidence for the ordered temporal appearance of plasma membrane antigens specific to particular classes of mouse spermatogenic cells. It appears that at late meiotic prophase, coincident with the production of pachytene primary spermatocytes, a variety of new components are inserted into the surface membranes of developing germ cells. The further identification and biochemical characterization of these constituents should facilitate an understanding of mammalian spermatogenesis at the molecular level.

Animals↗
PubMed1975

Chemical dissection of mammalian spermatozoa.

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.

Animals↗
PubMed1973

Chemical dissection of mammalian spermatozoa.

Spermatozoa from several mammalian species have been dissected by chemical methods to yield free heads, tails with attached midpieces, and tails from which the mitochondrial components of the midpiece were removed. Mouse and rat spermatozoa were cleaved by brief treatment with trypsin to yield free heads and tails, while human, guinea pig, and rabbit spermatozoa were cleaved by trypsin only after incubation with 2-mercaptoethanol or dithiothreitol. Spermatozoa were also cleaved at the junction of the head and the tail by treatment with acid and base. Mitochondria were removed from intact spermatozoa or isolated tails by mechanical shear after treatment with 2-mercaptoethanol or dithiothreitol. The dissected components of spermatozoa were fractionated with good yield and high purity by density gradient centrifugation. Ultrastructural analysis indicates that proteolytic cleavage to yield separated heads and tails occurs at a specific location in the neck of the spermatozoon, leaving the basal plate attached to the head of the cell. In contrast, after acid cleavage the basal plate remains with the midpiece. Proteolytic treatment has no apparent effect on any other spermatozoan structures, whereas acid or base treatment results in damage to the plasma membrane, the acrosome, and other structures. The specificity of the proteolytic cleavage suggests that a particular protein or group of proteins may be responsible for the linkage between the sperm head and tail.

Animals↗
PubMed1972

Specific fractionation of immune cell populations.

Antigen-binding cells from spleens of immune and nonimmune mice were isolated by the method of fiber fractionation. Binding of the lymphoid cells to derivatives of nylon fibers made with various antigens was prevented by the presence of the respective free antigen, as well as by antibodies to mouse immunoglobulins. Antigen-binding cells specific for dinitrophenyl groups were separated from direct and indirect plaque-forming cells of the same specificity. Spleen cells from immune and nonimmune animals were fractionated according to their relative affinities for antigen, and the percentage of antigen-binding cells in the spleens of nonimmune animals was estimated. A comparison of the numbers and relative affinities of immunoglobulin receptors of immune and nonimmune populations indicated that after immunization only those antigen-binding cells of higher affinities were increased in number. This finding suggests that the specificity of clonal selection depends not only upon the binding of antigen to a lymphoid cell but also upon the capacity of that cell to be triggered to mature and replicate.

Animals↗
PubMed1971

Molecular probes of spermatozoan structures.

Several methods have been devised for the isolation and labeling of structural components of spermatozoa. Rodent spermatozoa were cleaved rapidly and specifically at the junction of the heads and tails by treatment with various proteases, and the separate components were isolated by density-gradient centrifugation. Treatment with reducing agents released the mitochondrial membranes from the midpiece, exposing the underlying tail structures. Mouse spermatozoa were found to contain about 10(7) sites per cell that bind concanavalin A; most of the sites appear to be on the head, for fluorescein-labeled conjugates of concanavalin A were bound mainly to the acrosomal region. Binding of concanavalin A resulted in rapid agglutination of spermatozoa; mixed agglutinates could be formed with somatic cells, as well as with spermatozoa of other species. Fluorescent probes (naphthalenesulfonic acids) bound to the sperm plasma-membrane and caused an immediate loss of motility. In contrast, ethidium bromide bound to the nuclear structures, but did not cause immediate immobilization. These isolation and probing procedures should facilitate detailed chemical analysis of the major components of mammalian spermatozoa.

Agglutination↗
PubMed1971

Cell fractionation and arrangement on fibers, beads, and surfaces.

A new method, fiber fractionation, has been used to isolate and separate cells. The cells are adsorbed to fibers covalently coupled to molecules such as antigens, antibodies, and lectins which can bind specifically to cell-surface components. The cells are then removed mechanically by plucking the taut fibers. Alternatively, competitive inhibitors of binding may be used to remove the cells at a lesser rate. Successful fractionations have been achieved by varying the degree of derivatization of the fibers by the lectin concanavalin A. Lymphoid cells have been separated by the use of different antigens coupled to the fibers. The method may also be used for specific fixation and manipulation of viable cell populations in culture. In addition to fibers, beads and surfaces have been specifically derivatized and used to achieve different geometrical arrangements of the cells.

Animals↗
PubMedDate not supplied

Toxic effects of polychlorinated biphenyls on cultured rat Sertoli cells.

Polychlorinated biphenyls (PCBs) are ubiquitous and persistent environmental contaminants. In mammals, PCBs affect spermatogenesis and may be associated with Sertoli cell changes. Therefore, our aim was to evaluate in vitro toxic effects of hydroxylated PCB (PCB-22; 2',3',4',5'-tetrachloro-4-biphenylol) and PCB congener (PCB-77; 3,3',4,4'-tetrachlorobiphenyl) on Sertoli cells isolated from 19- to 21-day-old male rats. Sertoli cells incubated for 24 hours in 10(-7) M PCB-22 and 10(-8) M PCB-77, but not in 0.05% ETOH or 10(-7) M 17beta-estradiol (E2) showed morphological changes. Sertoli cells demonstrated progressive damage with higher concentrations of PCB-77 (10(-7) M). After 24 hours, 10(-7) M PCB-22 killed 20% of the Sertoli cells and equimolar PCB-77 killed 45% of the Sertoli cells in culture. At 10(-8) M, PCB-22 did not kill any significant number of Sertoli cells, whereas PCB-77 killed 40% of cells in culture. This result showed differential effects of PCB compounds, with PCB-77 being more cytotoxic than PCB-22 as tested on Sertoli cells. Because PCB-77 produced greater toxic effects, we further tested this congener on Sertoli cell lactate production. After 24 hours, lactate production by Sertoli cells treated with 10(-7) or 10(-8) M PCB-77 was significantly increased. Finally, Sertoli cells exposed to 10(-7) M PCB-77 showed disorganized and less intense F-actin staining. The results demonstrate that PCBs, but not E2, is directly toxic to Sertoli cells in vitro, and suggest this toxic effect is independent of estrogenic action.

Actins↗
PubMedDate not supplied

Sertoli cell plasma membrane polypeptides involved in spermatogenic cell-Sertoli cell adhesion.

This study concerns Sertoli cell-spermatogenic cell adhesive interactions in the seminiferous tubule. Sertoli cell surface polypeptides involved in germ cell-Sertoli cell adhesion were identified by serological inhibition of an in vitro Sertoli-germ cell adhesion assay. This assay was modified from a previously reported adhesion assay, and employs a scanning laser cytometer for quantification of adherent cells. Reactivity of the polyclonal antiserum raised against rat Sertoli cells was also assessed via immunofluorescent microscopy. The addition of antiserum to the adhesion assay resulted in a 42% to 66% inhibition of cell-cell adhesion. Moreover, preincubation of antiserum with Sertoli cell monolayers resulted in a significant reduction of spermatogenic cell binding. Conversely, preincubation of antiserum with germ cells resulted in no reduction. Western blot analysis of the antiserum against purified Sertoli cell membranes indicated reactivity with four polypeptides. The data suggest that one or more of these polypeptides are directly involved in the adhesion of germ cells to Sertoli cell monolayers in vitro.

Animals↗
PubMedDate not supplied

Glycosidic specificity of fucosyltransferases present in rat epididymal spermatozoa.

We have recently demonstrated multiple fucosyltransferase (FT) activity in rat spermatogenic cells. To complement these findings, here we identify and partially characterize the glycosidic linkage specificity of FTs present in spermatozoa from caput and cauda epididymides. Analysis of the acceptor substrate specificity of the FTs by thin-layer chromatography indicated that both caput and cauda sperm expressed alpha(1-2)-, alpha(1-3)-, alpha(1-4)-FTs as demonstrated by fucose incorporation into phenyl-beta-D-galactoside, 2'-fucosyllactose, and lacto-N-fucopentaose-I, respectively. Spermatozoa from the cauda epididymidis exhibited significant decreases in the levels of alpha(1-2)-, alpha(1-3)-, alpha(1-4)-FTs, and of total soluble FTs in comparison to spermatozoa from the caput epididymidis. The relative ratio of alpha(1-3)-FT to total FT activity appeared to be significantly higher than those of alpha(1-2)- or alpha(1-4)-FTs, in spermatozoa both from caput and cauda epididymides. Using different types of low molecular weight acceptors and the selective inhibition of the FT by N- ethylmaleimide, we have demonstrated that at least alpha(1-2)-FT is different from alpha(1-3)- or alpha(1-4)-FTs. Kinetic studies also showed that alpha(1-2)-FT is different from alpha(1-3)- or alpha(1-4)-FTs as demonstrated by apparent Km and Vmax values. Moreover, alpha(1-3)- and alpha(1-4)-FT activities in cauda sperm were found to be highly sensitive to Mn2+ but showed differential responses to divalent cations. In contrast, both alpha(1-3)- and alpha(1-4)-FTs seemed to be relatively less sensitive to Mg2+. Thus, these results not only demonstrate the presence of multiple FTs in rat epididymal sperm but also differentiate individual FTs with regard to their kinetic properties and sensitivity to both inhibitor and divalent cations.

Animals↗