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C F Millette

Publications and source records attributed to C F Millette.

At least 37 records · Page 2Linked to original sources
PubMed1989

Mitochondrial gene expression in male germ cells of the mouse.

Inheritance of the mitochondrial genome is known to be exclusively maternal. To determine whether the loss of paternal mitochondria could be due to a deficiency of RNA in the spermatozoal mitochondria, the expression of mitochondrial genes was studied in testicular cells at various stages of spermatogenesis and in epididymal spermatozoa. The presence of mitochondrial transcripts was examined by Northern blot analysis using probes for the following mitochondrially encoded genes: 12 S and 16 S ribosomal RNAs and a group of mRNAs including cytochrome oxidase subunits I and II (COI-COII), cytochrome b (cyt b), adenosine triphosphatase (ATPase) subunits 6 and 8, and subunit 1 of the respiratory chain NADH dehydrogenase (ND1). Comparison of total testicular RNA preparations from prepuberal (6, 8, 12, 16, 18, 20, 22, and 30 days old) and sexually mature (45 days old) mice revealed no major qualitative or quantitative differences in the levels of the mitochondrial transcripts described above. Similar results were observed from enriched preparations of type A and B spermatogonia and interstitial cells obtained from the testes of 8-day-old mice. Transcripts for COI-COII, ATPase 6, or ND1 were reduced in amount in the enriched preparations of pachytene spermatocytes, round spermatids, and residual bodies when compared to the amount in total testis or liver RNA. Transcripts of all the mitochondrial genes analyzed were present in RNA preparations isolated from sperm midpiece tails obtained after sonication of epididymal spermatozoa. These studies demonstrate that (a) during testicular development the levels of mitochondrial RNA in total testicular extracts show no major qualitative and quantitative differences; (b) the mitochondrial transcripts in enriched populations of type A and type B spermatogonia are not different from those obtained from total testes extracts; (c) mitochondrial transcript levels gradually decrease in enriched preparations of pachytene spermatocytes, round spermatids, and residual bodies; and (d) the mitochondrial rRNAs and mRNAs encoded by several mitochondrial genes can be isolated from sperm midpiece tails.

Animals↗
PubMed1988

Egg-penetration ability and structural properties of human sperm prepared by Percoll-gradient centrifugation.

Human sperm with a high zona-free hamster egg-penetration ability were obtained by centrifuging freshly ejaculated sperm through a discontinuous two-step (47% and 90%) Percoll gradient at 600g at room temperature for 30 min. Highly motile sperm with good penetration ability were recovered in the pellet fraction (Per-sperm), whereas those with low penetration ability were in the gradient interface. The increased penetration ability of Per-sperm, as compared to sperm capacitated by other methods such as a single-tube swim-up or multiple-tube swim-up preparation, was not due to an increased proportion of acrosome reacted sperm. Rather, transmission electron microscopy indicated that Per-sperm were devoid of coating envelopes, which were present around both the head and tail regions of noncapacitated and single-tube swim-up sperm. Changes to the surface of Per-sperm were demonstrated by their decreased interaction with UEA I lectin, which binds specifically to fucose residues. Removal of the coating envelopes as well as other changes on the sperm surface may lead to an enhanced binding of Per-sperm to the oocyte. In addition, 99% of Per-sperm contained chromatin that was fully condensed. By contrast, about 15% of swim-up sperm still possessed incompletely condensed chromatin. With a higher penetration ability, "clean" appearance, and homogeneity of condensed chromatin, Per-sperm are recommended for insemination and studies of human sperm capacitation.

Centrifugation, Density Gradient↗
PubMed1988

Pachytene spermatocyte and round spermatid binding to Sertoli cells in vitro.

Spermatogenic cells differentiate in vivo while in continuous contact with the Sertoli cell. During differentiation, the spermatogenic cells and Sertoli cells form a number of morphologically distinct stage-specific adhesions. We describe an in vitro assay system for studying the adhesion of spermatogenic cells to Sertoli cell monolayers. Mixed populations of spermatogenic cells or enriched fractions of pachytene spermatocytes and round spermatids were labelled with the vital dye, fluorescein diacetate, prior to their addition to Sertoli cell monolayers so that the adhesion of viable spermatogenic cells could be quantified. Using this assay system, the number of pachytene spermatocyte and round spermatid binding sites on the Sertoli cell monolayer were similar, but the kinetics of binding were different. Pachytene spermatocytes were able to inhibit significantly round spermatid binding, while round spermatids did not significantly inhibit pachytene spermatocyte binding. After coculture for 24-48 h, spermatocytes form junctional structures with Sertoli cells that are similar to desmosome-like junctions. These results suggest that pachytene spermatocytes and round spermatids bind to Sertoli cells by different mechanisms.

Animals↗
PubMed1988

Identification of the protein product of the c-mos proto-oncogene in mouse testes.

The mouse c-mos proto-oncogene RNA is expressed primarily in mouse gonadal tissues and embryos. Until now, the c-mos protein has not been identified. Utilizing two different site-directed affinity purified anti-peptide antibodies, we have identified a 43 kDa c-mos protein in mouse testes and in germ cell preparations derived from testes. This 43 kDa testicular protein was found to be structurally related to a bacterially expressed c-mos protein by peptide mapping. Immunoblots of whole mouse sections were employed to establish that the c-mos protein is expressed primarily in the testes.

Animals↗
PubMed1987

Quantitation of macromolecular binding using size exclusion filters: application to a fucosyltransferase assay.

A method to quantify the covalent attachment of small radiolabeled substrates to macromolecules on the basis of molecular weight using size exclusion filters in an Amicon Centrifree micropartition system is described. GDP-[14C]-L-fucose was covalently attached to asialofetuin in a fucosyltransferase reaction catalyzed by mouse spermatogenic cell extracts. Radiolabeled product was separated from unreacted substrate by centrifuging 200-400 microliter of cell extract through a 10-kDa size exclusion filter at 1000 g for 10 to 20 min. After 10 washes with an appropriate buffer, no detectable radioactivity was found in the eluant and the membrane-bound radiolabeled product was counted in a scintillation vial. Using this method the fucosyltransferase activity of mouse spermatogenic cells was approximately 17 pmol/mg protein/min which is essentially identical to values obtained using size exclusion chromatography. This technique provides a rapid, efficient, and inexpensive alternative for the isolation and detection of acceptor-substrate complexes.

Animals↗
PubMed1987

Expression of c-mos RNA in germ cells of male and female mice.

We have investigated the cell types in mouse testis and ovary in which the c-mos protooncogene is normally transcribed. Blot hybridization analysis of electrophoretically fractionated RNAs from testes of mice with defects in germ-cell development and from prepubertal and adult mice indicated that c-mos was transcribed during male germ-cell development. Analysis of purified populations of spermatogenic cell types detected c-mos RNA in the earliest haploid postmeiotic germ cell, the round spermatid, indicating that c-mos was expressed transiently during spermatogenesis. c-mos RNA was detected by blot hybridization in the ovaries of prepubertal mice and decreased in relative concentration following gonadotropin-stimulated proliferation of granulosa cells. These results suggested that c-mos was transcribed in oocytes and were confirmed by detection of high levels of c-mos RNA in isolated grown oocytes. Thus, c-mos is expressed in both male and female germ cells, suggesting possible roles for this protooncogene in meiosis, germ-cell development, fertilization, and early embryogenesis.

Age Factors↗
PubMed1987

Fucosylation events during mammalian spermatogenesis.

It will be necessary to conduct further studies to establish more precisely the localization of FT on mouse male germ cells. Antibodies to FTs are not yet available, so an immunocytochemical approach is not currently feasible. Additional cell fractionation protocols can be designed to compare plasma membrane fractions with enriched fractions of Golgi apparatus and to compare directly the activities of multiple glycosyltransferase enzymes and Golgi-specific markers in these preparations. Schachter et al. and Nyquist and colleagues have already provided experimental techniques for the isolation of Golgi fractions of good purity from rodent pachytene spermatocytes and spermatids. Ample opportunity exists, then, for a detailed analysis of the number, specificity, and localization of FT enzymes during mammalian spermatogenesis. All available data imply that these enzymes will prove to be vital components in the differentiation of cells within the seminiferous epithelium.

Animals↗
PubMed1986

Expression of proenkephalin messenger RNA by mouse spermatogenic cells.

The presence of proenkephalin mRNA in germ cells purified from adult mouse testis were examined using RNA gel- and dot-blot analyses. Both pachytene spermatocytes and round spermatids were shown to contain concentrations of this transcript that are 2- to 3-fold greater than that found for whole mouse testis, on a per microgram of polyadenylylated RNA basis. The detection of proenkephalin mRNA in purified spermatocytes and spermatids could not be accounted for by contamination by either Leydig or Sertoli cells. No proenkephalin mRNA was detectable in extracts of mature sperm. These data suggest that developing germ cells may be a major site of proenkephalin synthesis in the adult testis and that proenkephalin-derived peptides may function as germ cell-associated hormones or autocrine/paracrine factors.

Animals↗
PubMed1986

Sertoli cell binding to isolated testicular basement membrane.

We have examined the adhesion of primary Sertoli cells to a seminiferous tubule basement membrane (STBM) preparation in vitro. The STBM isolation procedure (Watanabe, T.K., L.J. Hansen, N.K. Reddy, Y.S. Kanwar, and J.K. Reddy, 1984, Cancer Res., 44:5361-5368) yields segments of STBM that retain their histotypic form in both three-dimensional tubular geometry and ultrastructural appearance. The STBM sleeves contain two laminae: a thick, inner basal lamina that was formed in vivo between Sertoli cells and peritubular myoid cells; and a thinner, outer basal lamina that was formed between myoid cells and sinusoidal endothelial cells. Characterization by immunofluorescence and SDS PAGE revealed that the isolated STBM retained fibronectin, laminin, and putative type IV collagen among its many components. When the STBM sleeves were gently shaken with an enriched fraction of primary Sertoli cells, the Sertoli cells bound preferentially to the lumenal basal lamina at the ends of the STBM sleeves. Few Sertoli cells bound to either the outer basal lamina of the STBM sleeves or to vascular extracellular matrix material which contaminated the STBM preparation. 3T3 cells, in contrast, bound to all surfaces of the STBM sleeves. Pretreatment of the STBM sleeves with proteases, 0.1 M Na metaperiodate, 4 M guanidine HCl, or heating to 80 degrees-90 degrees C inhibited lumenal Sertoli cell binding, but binding was not inhibited by chondroitinase ABC, heparinase, hyaluronidase, or 4 M NaCl. The lumenal Sertoli cell binding occurred in the presence or absence of added soluble laminin, but not fibronectin. The addition of soluble laminin, but not fibronectin, restored random binding of Sertoli cells to trypsinized STBM sleeves. Our in vitro model system indicates that Sertoli cells recognize differences in two basal laminae produced in vivo on either side of myoid cells.

Animals↗
PubMed1986

The development of regionalized lipid diffusibility in the germ cell plasma membrane during spermatogenesis in the mouse.

The lipids and proteins of sperm cells are highly regionalized in their lateral distribution. Fluorescence recovery after photobleaching studies of sperm membrane component lateral diffusibility have shown that the sperm plasma membrane is also highly regionalized in the extents and rates of diffusion of its surface components. These studies have also shown that regionalized changes in lateral diffusibility occur during the differentiative processes of epididymal maturation and capacitation. Unlike mammalian somatic cells, sperm cells exhibit large nondiffusing lipid fractions. In this paper, we will show that both regionalized lipid diffusibility and nondiffusing lipid fractions develop with the morphogenesis of cell shape during spermatogenesis in the mouse. Pachytene spermatocytes and round spermatids show diffusion rates and the nearly complete recoveries (80-90%) typical of mammalian somatic cells. In contrast, stage 10-11 condensing spermatids, testicular spermatozoa, cauda epididymal spermatozoa, as well as the anucleate structures associated with these later stages of spermatogenesis (residual bodies and the cytoplasmic droplets of condensing spermatids and testicular spermatozoa), exhibit large nondiffusing fractions. Both the diffusion rates and diffusing fractions observed on the anterior and posterior regions of the head of stage 10-11 condensing spermatids are the same as the values obtained for these regions on testicular spermatozoa. Possible mechanisms of lipid immobilization and possible physiological implications of this nondiffusing lipid are discussed.

Animals↗
PubMed1986

Stage-specific synthesis and fucosylation of plasma membrane proteins by mouse pachytene spermatocytes and round spermatids in culture.

Little is known about the ability of mammalian spermatogenic cells to synthesize plasma membrane components in the presence or absence of Sertoli cells. In this study, purified populations (greater than 90%) of pachytene spermatocytes or round spermatids were isolated by unit gravity sedimentation and cultured for 20-24 h in the presence of [35S]methionine or [3H]fucose. Cell viabilities remained over 90% during the course of these experiments. Plasma membranes were purified from these cells and analyzed by two-dimensional gel electrophoresis. Qualitatively, the same plasma membrane proteins were synthesized by both cell types with the exception of the major Concanavalin A-binding glycoprotein, p151; the synthesis of p151 is greatly diminished or inhibited after meiosis. [3H]Fucose was incorporated into at least 6 common glycoproteins of both cells. Eight components fucosylated with molecular weights from 35,000 to 120,000 were specific to pachytene spermatocyte membranes. One fast-migrating fucosylated component may represent an uncharacterized lipid whose synthesis is terminated after meiosis. Round spermatids specifically fucosylated two components with molecular weights of 45,000 and 80,000. These results demonstrate the viability of germ cells of the male mouse in short-term culture and show that they are capable of synthesizing and fucosylating plasma membrane components in the absence of Sertoli cells.

Animals↗
PubMed1984

Transformation of the Hprt gene with DNA from spermatogenic cells. Implications for the evolution of X chromosome inactivation.

DNA-mediated transformation of hypoxanthine guanine phosphoribosyl transferase (HPRT)-deficient cells was used to assess the state of the chromosome Hprt gene in spermatogenic cells. It had been shown previously that DNA from the inactive X chromosome of somatic cells functions poorly or not at all in HPRT transformation, indicating that DNA modification is involved in somatic cell X chromosome inactivation (XCI). In contrast, DNA from mature sperm does function in HPRT transformation suggesting that DNA modification may not be the basis of XCI in mature sperm. In this paper, transformation of HPRT- mouse and hamster cells has been performed to test the nature of XCI during earlier stages of spermatogenesis. DNA from these developing murine germ cells was shown to be capable of HPRT transformation, extending the observation that XCI in sperm does not appear to involve a DNA modification. We also show here that DNA from mature sperm of marsupials functions in HPRT transformation, a result consistent with a role for sperm XCI in the evolution of somatic X inactivation.

Animals↗
PubMed1984

Stage-specific expression of three cell surface carbohydrate antigens during murine spermatogenesis detected with monoclonal antibodies.

We have identified three germ cell surface carbohydrate antigens that exhibit a common, stage-specific pattern of expression during spermatogenesis in the mouse. IgM-class monoclonal antibodies designated "J1," "C6," and "A5" were absorbed by adult testis, but not by any adult somatic tissue tested. In indirect immunofluorescence assays using collagenase-dissociated prepuberal and adult testicular cells, these antibodies labeled the surfaces of early and late pachytene spermatocytes and round spermatids. Gonocytes from fetal and neonatal testes were not labeled. In paraffin sections of prepuberal and adult testes, sialidase treatment exposed antigens recognized by antibodies C6 and A5 on preleptotene, leptotene, and zygotene spermatocytes located near the perimeter of seminiferous tubules. The determinants recognized by antibodies J1, C6, and A5 were characterized partially using a sugar hapten inhibition assay. The binding of J1 to adult testicular cells was inhibited specifically by N-acetylglucosamine and the binding of both C6 and A5 was inhibited by N-acetyllactosamine. The glycoconjugates recognized by J1, C6, and A5 eluted from gel filtration columns with an apparent molecular weight greater than 1 X 10(6) and were sensitive to endo-beta-galactosidase (keratanase) treatment. The apparent high molecular weight of these glycoconjugates was confirmed by immunolabeling Western blots of testis extracts separated by SDS-polyacrylamide gel electrophoresis. The results suggest that polylactosamine (keratan) glycoconjugates of high molecular weight are associated with the plasma membranes of meiotic and haploid male germ cells. The effects of sialidase on antibody labeling patterns suggest that changes in cell surface sialylation accompany the transition of early meiotic germ cells to pachytene spermatocytes during spermatogenesis.

Animals↗
PubMed1984

Identification and immunochemical characterization of spermatogenic cell surface antigens that appear during early meiotic prophase.

Three spermatogenic cell populations isolated from prepuberal mice--type B spermatogonia, preleptotene spermatocytes, and leptotene/zygotene spermatocytes--were used to elicit distinct polyclonal antisera. Surface binding specificities were determined for purified IgGs by indirect immunofluorescence and rosette assays on live cells. Binding activities were assayed both before and after absorptions with a variety of somatic and spermatogenic cells. Each of these antisera binds to surface antigens that are present on germ cells throughout spermatogenesis and are not shared by splenocytes, thymocytes, and erythrocytes. Only the antiserum raised against leptotene and zygotene spermatocytes (ALZ) recognizes a stage-specific subset of surface determinants. After appropriate absorptions, ALZ binds to the surface of early pachytene spermatocytes and germ cells at subsequent stages of differentiation, including vas deferens spermatozoa. Antigens which react with this absorbed IgG are not detected on the surface of spermatogonia or meiotic cells prior to pachynema, including leptotene and zygotene spermatocytes. The observed binding specificities may result from the synthesis of one or more surface molecules during the early meiotic stages, followed by delayed insertion into the plasma membrane during the pachytene stage of meiotic prophase. Stage-specific antigens recognized by ALZ, including both protein and probably lipid, have been localized immunochemically on nitrocellulose blots from one-dimensional SDS gels. A dithiothreitol-sensitive constituent (Mr approximately 39,000) recognized by ALZ has been identified as the major protein determinant present in early meiotic cells but absent in 8-day-old seminiferous cell suspensions containing spermatogonia and Sertoli cells. This determinant is present in populations of preleptotene, leptotene/zygotene, and early pachytene spermatocytes isolated from 17-day-old animals, an observation consistent with the hypothesis of delayed insertion into the plasma membrane.

Animals↗
PubMed1984

Generation of flagella by cultured mouse spermatids.

During the short-term culturing of mouse spermatogenic cells, flagella were generated by round spermatids previously lacking tails. Unseparated germ cells were obtained by enzymatic treatments and round spermatids (greater than 90% pure) were purified by unit gravity sedimentation. As determined by Nomarski or phase-contrast microscopy, no cells had flagella immediately after isolation; flagella were first clearly detected after 6 1/2 h of culture in Eagle's minimal essential medium containing 10% fetal bovine serum and 6 mM lactate. After 24 h, approximately 20% of round spermatids had formed flagella. Multinucleated round spermatids often formed multiple flagella, the number never exceeding the number of nuclei per symplast. Round spermatids were the only spermatogenic cells capable of tail formation. Flagella elongation was blocked by 1 microM demecolcine, an inhibitor of tubulin polymerization. Indirect immunofluorescence localized tubulin in the flagella. As seen by scanning electron microscopy, flagella developed as early as 2 h after culture and continued to elongate over the next 20 h, reaching lengths of at least 19 micron. Transmission electron microscopy demonstrated that flagella formed in culture resembled flagella from Golgi-phase round spermatids in situ; the flagella consisted of "9+2" axonemes lacking other accessory structures such as outer dense fibers and the fibrous sheath. As determined by acridine orange staining of the developing acrosomes, all spermatids that formed flagella in culture were Golgi-phase spermatids. By these criteria, the structures are indeed true flagella, corresponding in appearance to what others have described for early mammalian spermatid flagella in situ. We believe this is the first substantiated report of limited in vitro differentiation by isolated mammalian spermatids.

Animals↗
PubMed1984

Serological and biochemical identification of a plasma membrane antigen specific to Leydig cells.

Purified mouse Leydig cells have been prepared from interstitial cell suspensions using a Percoll gradient procedure. The isolated cells are 88-95% pure as determined by light microscopy. Staining for 3-beta-hydroxysteroid dehydrogenase indicates that over 85% of the Leydig cells recognized by differential interference microscopy are also positive for this enzyme. After separation, the Leydig cells are viable by dye exclusion assays and exhibit normal in situ morphology when examined at the ultrastructural level. Leydig cell suspensions have been used to raise a polyclonal antiserum in rabbits. This antiserum, prior to absorption, reacts in indirect immunofluorescent studies with the surfaces of isolated Leydig cells, with testicular germ cells, with spermatozoa and with somatic cells such as splenocytes. Following absorption with lymphocytes and spermatogenic cells, the antiserum binds only to Leydig cell plasma membranes. Quantitative measurements with 125I-protein A confirm the specific labeling of Leydig cells by the absorbed antiserum. Biochemical identification of the Leydig cell plasma membrane antigen has been accomplished by immunoblotting polyacrylamide gel nitrocellulose transfers. A single major band of Mr approximately 40,000 is detected on one-dimensional transfers; two weakly reactive spots of Mr 43,000 and 45,000 are detected using two-dimensional immunoblots. Blotting experiments conducted using concanavalin A have identified the major Leydig cell constituents reactive with this lectin. The Leydig cell plasma membrane antigen(s) does not bind concanavalin A.

3-Hydroxysteroid Dehydrogenases↗
PubMed1984

Identification of spermatogenic cell plasma membrane glycoproteins by two-dimensional electrophoresis and lectin blotting.

Plasma membrane glycoproteins present in purified mouse spermatogenic cells have been identified by two-dimensional polyacrylamide gel electrophoresis and lectin blotting techniques. Four membrane glycoproteins labelled with Bandeiraea simplicifolia lectin (I) have been detected, ranging in Mr from 55 000 to 76 000 and in pI from 6.0 to 6.3. Only one of these proteins, p76/6.3, is synthesized by short-term in vitro cultures of spermatogenic cells, as determined by the incorporation of [35S]methionine. Approximately 20 surface glycoproteins labelled with concanavalin A have been identified, ranging in Mr from 50 000 to 151 000 and in pI from 5.7 to 7.0. None of the components detected with B. simplicifolia lectin (I) are labelled significantly with concanavalin A. A major concanavalin A-binding protein in the membranes of both pachytene spermatocytes and round spermatids is p151/6.0. This glycoprotein has been previously shown to be exposed on the outer surface of spermatogenic cell membranes and may represent a mediator of germ cell-Sertoli cell interactions. Furthermore, two constituents identified in the present study represent stage-specific markers. Component p73/5.7 is detected with concanavalin A only in the membranes of pachytene spermatocytes. Conversely, p84/6.3 is found only in round spermatid membranes. These results, then have: (a) provided a map of membrane glycoproteins in developing mouse male germ cells; (b) identified p151/6.0 as a membrane constituent of possible functional significance; and (c) identified the first reported glycoprotein surface differentiation markers for mouse spermatogenesis.

Animals↗