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.

Toxic effects of octylphenol on cultured rat spermatogenic cells and Sertoli cells.

Alkylphenols, including the estrogenic 4-tert-octylphenol (OP), are environmental pollutants. Because administration of OP to adult male rats impairs spermatogenesis and OP has been shown to be toxic to aquatic animals and to mammalian splenocytes in vitro, we studied whether OP exerts direct toxic effects on cultured spermatogenic cells and Sertoli cells isolated from male rats. Cell viability was assessed with a Live/Dead Eukolight viability/cytotoxicity kit. Culture of mixed spermatogenic cells from adult rats with 10(-8) M OP or Sertoli cells from 19- to 21-day-old rats with 10(-12) M OP, but not with 0.08% EtOH (vehicle) or 10(-6) M 17beta-estradiol (E2) or dexamethasone (DEX), significantly decreased the percentage of viable cells after 24 h of treatment. None of the treatments significantly altered total cell number. Flow cytometric analyses of spermatogenic cells revealed that exposure to 10(-4) or 10(-6) M OP yielded abnormal relative ploidy classes. Four hours of treatment with 10(-6) M OP, but not with 10(-6) M E2 or DEX, caused significant chromatin condensation in Sertoli cells as observed with acridine orange staining. The decreased percentage of viable cells after 24 h of exposure to 10(-6) M OP remained when Sertoli cells were cultured in Ca2+-free medium. Sertoli cells contained nuclei of reduced size and labeled 3'-OH DNA ends as detected by microscopic analyses when the cells had been incubated for 24 h with 10(-6) M OP but not with vehicle or 10(-6) M E2 or DEX. The results demonstrate that OP, but not E2 or DEX, is directly toxic to cultured rat spermatogenic cells and Sertoli cells and suggest that this toxic effect in Sertoli cells is exerted through Ca2+-independent apoptosis.

Gli family members are differentially expressed during the mitotic phase of spermatogenesis.

The Gli family of DNA binding proteins has been implicated in multiple neoplasias and developmental abnormalities, suggesting a primary involvement in cell development and differentiation. However, to date their specific roles and mechanisms of action remain obscure, and a drawback has been the lack of a model system in which to study their normal function. Here we demonstrate that Gli family members are differentially expressed during spermatogenesis in mice. Specifically, Gli and Gli3 mRNAs were detected in mouse germ cells, while Gli2 was not. Further, both Gli and Gli3 exhibited stage-dependent patterns of expression selectively in type A and B spermatogonia. Gli expression was somewhat higher in type B spermatogonia while the abundance of Gli3 transcripts was similar in type A and B cells. Gel-shift analyses also demonstrated the enrichment of DNA binding activity specific for the Gli target sequence in spermatogonial cells. These results indicate a selective role for Gli and Gli3 during mitotic stages of male germ cell development. Spermatogenesis may thus provide a unique opportunity to identify downstream targets and explore the normal function of Gli family proteins.

Multiple fucosyltransferases and their carbohydrate ligands are involved in spermatogenic cell-Sertoli cell adhesion in vitro in rats.

We have identified multiple fucosyltransferases (FTs) (alpha[1-2]-, alpha[1-3]-, alpha[1-4]-FTs) on cells of the rat seminiferous epithelium as demonstrated by fucose incorporation into phenyl-beta-D-galactoside (Ph-beta-D-Gal), 2'-fucosyllactose (2'-FL), and lacto-N-fucopentaose-l (LNF I), respectively. Now, using fluorescence laser scanning cytometry, we report that multiple FTs are implicated in germ cell-Sertoli cell adhesion in vitro. Sertoli cells were isolated from 19- to 21-day-old CD rats and cultured for 6-10 days. Mixed germ cells were obtained by enzymatic dispersion of adult rat testis and cultured overnight before labeling with 10 microM acetoxymethyl ester derivative of the fluorescent indicator, calcein. The adherent cell analysis and sorting 570 interactive laser cytometer was used to determine the number of labeled adherent germ cells on Sertoli cell monolayers in the presence or absence of a variety of low molecular weight acceptors for fucose. Coincubation of labeled germ cells with Sertoli cell monolayers in the presence of GDP-fucose, UDP-galactose, Ph-beta-D-Gal, 2'-FL, LNF I, and Lewis-X and 3'-sialyl-Lewis-X oligosaccharides resulted in significant reduction of germ cell binding when compared to that of the untreated controls or of control samples incubated with cellobiose, melibiose, and alpha-D-mannopyranose, which do not serve as fucose acceptors. Our results suggest that multiple FTs and their lectin/selectin ligands are involved in mediating germ cell-Sertoli cell adhesion to form a cohesive epithelium and thus aid germ cell adluminal translocation within the seminiferous epithelium.

Increased activity associated with the MAST205 protein kinase complex during mammalian spermiogenesis.

The morphological and biochemical changes that occur in the haploid male germ cell during spermiogenesis facilitate the natural delivery of the paternally imprinted chromosomes into oocytes. Despite the obvious morphological changes, little is known about the molecular events underlying spermiogenesis. We recently cloned a novel 205-kDa manchette microtubule-associated serine/threonine protein kinase (MAST205) from mouse testis. The objective of this study was to further delineate the role of MAST205 in mammalian spermiogenesis. While MAST205 RNA levels were similar in pachytene spermatocytes, round spermatids, and residual bodies, MAST205 protein could be detected only in round spermatids and residual bodies. Kinase activity associated with MAST205 immunoprecipitates was low in pachytene spermatocytes, high in round spermatids, and maximal in residual bodies, indicating that MAST205-associated kinase activity is modified during spermatid maturation. Furthermore, MAST205 protein and the associated kinase activity were not detected in epididymal spermatozoa, indicating that MAST205 protein is either excluded from, or degraded in, the latter cell type. Multiple heterologous protein species were seen in immunoprecipitates from 35S-labeled mouse seminiferous tubules using an affinity-purified MAST205 antiserum. Consistent with this observation, MAST205 eluted as part of a 1-2 x 10(6) dalton protein complex when extracts of mouse testis were fractionated by Superose 6 column chromatography. MAST205 mRNA was detected in human testis indicative of conservation in other mammalian species. Taken together, these results indicate that the MAST205 complex functions in spermatid maturation in mammals.

Transcription factor Sp1 is expressed by three different developmentally regulated messenger ribonucleic acids in mouse spermatogenic cells.

Gene expression during spermatogenesis is highly cell- and stage-specific and involves the complex interplay of multiple developmentally regulated transcription factors. Recent evidence suggests that the DNA-binding protein Sp1 functions as an important trans-activator during cell development and differentiation. In the present study, the developmental expression of Sp1 was characterized during mouse spermatogenesis. Three distinct Sp1 transcripts were detected in mouse spermatogenic cells, each with a distinct developmental pattern; an 8.2-kilobase (kb) messenger RNA (mRNA) identical in size to the somatic mRNA expressed in spermatogonial cells, a larger mRNA approximately 8.8 kb in size present in meiotic cells, and a 2.4 kb mRNA in meiotic and postmeiotic germ cells. The 8.8- and 2.4-kb Sp1 transcripts were not observed in somatic cells and, thus, are male germ cell specific. Northern, ribonuclease protection, and RT-PCR assays revealed that the 2.4-kb Sp1 transcript is truncated in both the 5'- and 3'-untranslated regions relative to the somatic mRNA and lacks a short segment of the N-terminal coding region. Polysome analysis further indicated that these germ cell-specific Sp1 mRNAs are translated, albeit with a lower efficiency than the somatic transcript. Consistent with these results, spermatogenic cells were shown to contain approximately 9-fold lower concentrations of Sp1 proteins that are approximately the same size as the somatic form. Of particular interest, the apparent affinity of Sp1 DNA-binding activity in nuclear extracts from mouse germ cells was 5-fold greater than that in extracts from mouse somatic tissues. This may reflect the existence of mechanisms within mouse spermatogenic cells that compensate for the lower nuclear concentrations of Sp1 protein. These results suggest that cell- and stage-specific regulation of Sp1 gene expression and activity may be an important component of the mouse spermatogenic cell developmental program.

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.

Presence of multiple fucosyltransferases in rat Sertoli cells and spermatogenic cells.

Differential expression of fucosyltransferases (FTs) on Sertoli cell and germ cell surfaces and their function as ectoenzymes may be important in the process of spermatogenesis. To determine the glycosidic linkage specificity of FTs present in cultured Sertoli cells and in germ cells, we quantified FT activities by thin-layer chromatography using both high and low molecular weight acceptors in the presence of GDP-[14C]-L-fucose. Analysis of the acceptor substrate specificity of the FTs indicated that alpha(1-2), alpha(1-3), alpha(1-4)-FTs are expressed as demonstrated by fucose incorporation into phenyl-beta-D-galactoside, 2'-fucosyllactose, and lacto-N-fucopentaose-I, respectively. In Sertoli cells, the ratios of the three FTs examined were the same for whole-cell extracts and samples of purified plasma membranes. Higher relative FT activity was observed in plasma membranes from mixed germ cells than in Sertoli cell membranes. Furthermore, alpha(1-3)-FT and alpha(1-4)-FT activities were higher in mixed germ cell membranes. Spermatogenic stage specificity of FT expression was assessed in purified populations of germ cells. With calculation on a per-cell basis, all three alpha-FTs exhibited a quantitative decrease during the transition between pachytene spermatocytes and round spermatids. The decrease in alpha(1-3)-FT activity was particularly significant. In rat germ cells, all three alpha-FT activities associated with the cell surface in pachytene spermatocytes and round spermatids were 34-53% and 52-53%, respectively, of the total cell FT activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Surface-associated glycosyltransferase activities in rat Sertoli cells in vitro.

We have previously demonstrated fucosyltransferase (FT) activity on mouse germ cell surfaces at different stages of spermatogenesis. To complement these findings, here we report FT activity on the Sertoli cell (SC) surface. SC isolated and cultured from 20-day-old rat testes displayed FT activity with a Vmax of 12.5 pmoles/mg protein/min and a Km of 22 microM, while purified Sertoli cell plasma membranes (SCPM) showed FT activity with a Vmax of 10 pmoles/mg protein/min and a Km of 18.2 microM for GDP-[14C]-L-fucose. Fucosyltransferase activities were 16.7 and 2.6 pmoles/mg protein/min in SC and SCPM, respectively; approximately 16% of FT activity is, therefore, on the cell surface. To test whether the expression of FT activity in SC was regulated by hormones and growth factors, SC were cultured in serum-free medium supplemented with insulin, transferrin, sodium selenite, and epidermal growth factor (medium 4F) or in 4F plus follicle-stimulating hormone, testosterone, hydrocortisone, and vitamin E (medium 8F). We found that FT activity in SC is not modulated by these hormones or growth factors (4F or 8F). For comparison with FT, galactosyltransferase (GalTase) activities in SC and SCPM were also determined. SC displayed GalTase activity with a Vmax of 50 pmoles/mg protein/min and a Km of 38.5 microM, while SCPM showed GalTase activity with a Vmax of 25 pmoles/mg protein/min and a Km of 20.8 microM for UDP-[3H]-galactose. Galactosyl-transferase activities were 29.2 and 9.6 pmoles/mg protein/min in SC and SCPM, respectively. Therefore, approximately 33% of the total cell GalTase activity was detected on the surface membranes of rat Sertoli cells.(ABSTRACT TRUNCATED AT 250 WORDS)

N-cadherin mediates Sertoli cell-spermatogenic cell adhesion.

The complex topological association of Sertoli cells and spermatogenic cells in the testis suggests the existence of cell surface adhesion molecules that regulate cellular interactions within the seminiferous epithelium. The recent report of N-cadherin mRNA expression in the mouse testis implies the involvement of this known adhesion molecule in testicular cell binding. Accordingly, here we report that (1) N-cadherin is found on the surface membranes of rat spermatogenic cells and on Sertoli cells, and (2) that N-cadherin is a partial mediator of Sertoli cell-germ cell adhesion as tested in an vitro cell-cell binding assay. Antiserum directed against the N-cadherin cell adhesion recognition sequence was used for Western blot anlaysis of purified plasma membranes from Sertoli cells and from spermatogenic cells. Both membrane preparations exhibited reactivity at an appropriate M(r) of about 130 kDa. In addition, immunofluorescence assays demonstrated that both germ cells and Sertoli cells were labeled by anti-N-cadherin. Finally, the antiserum was included in a cytometer-assisted cell-cell binding test to determine its inhibitory ability. The antiserum consistently reduced specific testicular cell-cell adhesion by 30%-50%. This is the first demonstration that antibodies directed against the cadherin cell adhesion recognition sequence are capable of inhibiting cell-cell interactions. Pre-incubation of either rat Sertoli cells or spermatogenic cells alone was sufficient to achieve statistically significant inhibition of intercellular adhesion. We conclude, therefore, that N-cadherin is expressed by both Sertoli cells and spermatogenic cells and that N-cadherin is one of a number of regulatory molecules mediating local cellular associations in the mammalian seminiferous tubule.

Germ cells of the mouse testis express P450 aromatase.

Estrogen production within the testis has been a subject of considerable controversy for many years. Several studies have shown that both Sertoli and Leydig cells produce estrogen during different stages of development. Therefore, we have conducted experiments to localize aromatase, a cytochrome P450 enzyme that converts androgen to estrogen, within the testis. First, P450 aromatase (P450arom) was localized in germ cells of the adult mouse testis by immunocytochemistry, using an antiserum generated against purified human placental cytochrome P450arom. In the germinal epithelium, P450arom was located primarily in the Golgi region of round spermatids, throughout the cytoplasm of elongating spermatids, and along the flagella of late spermatids. Second, localization of P450arom within the germinal epithelium was supported by Western blot analysis of isolated germ cells. Third, Northern blot analysis using a mouse P450arom cDNA probe indicated that the mRNA for the mouse P450arom was present in testicular germ cells. Fourth, P450arom activity was measured in germ cells by the 3H2O water assay. Based upon these observations, we conclude that germ cells are a site of estrogen synthesis in the adult mouse testis.

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.

Quantification of an in vitro cell-cell adhesion assay using interactive laser scanning cytometry.

We are interested in identifying cell-cell adhesion molecules on the surface of Sertoli cells that mediate Sertoli cell-spermatogenic cell adhesion. Numerous cell-cell adhesion assays employ microscopic observation, photomicroscopy or radioactive isotopes for quantification. Previously, we developed an in vitro assay for testicular cell interactions. This assay was, however, time consuming using photography for analysis. We have now modified this system using laser cytometry to quantify adherent cells. Rat testicular epithelial cells are cultured for approximately 6 days before labelling with fluorescein diactetate (FDA) to assess confluency by image scanning so that spermatogenic cell binding can be normalized to available epithelial cell surface area. Rat spermatogenic cells are labeled with FDA before addition to epithelial cell monolayers. In some studies, purified spermatogenic cell populations were isolated to determine average cell size. We found that spermatocyte area varied between 225-500 microns2, spermatids were 100-225 microns2 and residual bodies were less than 100 microns2. Using these parameters, scanning cytometry allows the differential analysis of adhesion by individual germ cell sub-classes from mixed cell suspensions, saving time, animals, and major expense. The scanning laser assisted assay is faster, more reproducible and less subjective than earlier cell-cell adhesion assays using light microscopy or isotopes. This experimental approach should facilitate any cell-cell adhesion assay in which one cell type is adherent to a substrate.

DNA methylation and demethylation events during meiotic prophase in the mouse testis.

The genes encoding three different mammalian testis-specific nuclear chromatin proteins, mouse transition protein 1, mouse protamine 1, and mouse protamine 2, all of which are expressed postmeiotically, are marked by methylation early during spermatogenesis in the mouse. Analysis of DNA from the testes of prepubertal mice and isolated testicular cells revealed that transition protein 1 became progressively less methylated during spermatogenesis, while the two protamines became progressively more methylated; in contrast, the methylation of beta-actin, a gene expressed throughout spermatogenesis, did not change. These findings provide evidence that both de novo methylation and demethylation events are occurring after the completion of DNA replication, during meiotic prophase in the mouse testis.

The testicular effects of tumor necrosis factor.

Tumor necrosis factor (TNF) is a cytokine that mediates many of the metabolic responses after endotoxemia, septicemia, and tissue injury. The effect of TNF on testicular function was determined in a series of studies in which rhTNF (0, 2, and 4 X 10(5) units/kg/24 hours) was administered by continuous infusion to male Wistar rats maintained on total parenteral nutrition adequate for growing rats. Testicular weight and histology, and plasma luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone levels were determined at 1, 3, and 6 days. Testicular weight decreased within 24 hours and this was associated with a fall in plasma testosterone and increased LH and FSH levels. These changes persisted for 6 days, indicating a loss of testosterone-mediated negative feedback on gonadotropin release. Histologic examination demonstrated significant damage to the germ cells in the adluminal compartment of the seminiferous epithelium; extensive exfoliation of spermatocytes and spermatids occurred at day six. However the primary spermatogonia in the basal compartment were relatively spared. Damage to the seminiferous epithelium at earlier times was noted in some tubules. The decrease in testosterone concentration and increase in gonadotropin levels suggest that TNF interferes with Leydig cell function. Germ cell damage may be a direct effect of TNF on these cells or may occur through secondary mechanisms involving Leydig or Sertoli cell dysfunction.

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.

Specific expression of nuclear proto-oncogenes before entry into meiotic prophase of spermatogenesis.

The expression of proto-oncogenes representative of several functional categories has been investigated during development of mouse male germ cells. The c-raf proto-oncogene and three members of the c-ras gene family were expressed in mitotically active stem cells, throughout the prophase of meiosis and to varying extents in post-meiotic cell types. In contrast, the nuclear proto-oncogenes c-fos, c-jun, and c-myc were specifically expressed at high levels in type B spermatogonia. High levels of c-myc and c-jun RNAs were also detected in spermatocytes early in the prophase of meiosis. The type B spermatogonia represent the last mitotic cell division before entry into meiotic prophase; therefore, these nuclear proto-oncogenes may be involved in altering programs of gene expression at this developmental transition.

Thin-layer chromatographic method for the determination of glycosyltransferase activity.

To survey glycosyltransferase activities and specificities we have developed a TLC method to separate various nucleotide sugars from both high- and low-molecular-weight sugar acceptors. Here, we report details of the procedure and its application for galactosyltransferase and fucosyltransferase detected in mouse spermatogenic cells. The assay method involves sample separation using polyethyleneimine cellulose plastic-backed thin-layer plates, developed in sodium phosphate buffer for 30 min. Nucleotide sugars, including UDP-Gal, GDP-Fuc, CMP-NeuNAc, and GDP-Man, remain at the origin, while both high- and low-molecular-weight sugar acceptors migrate within 2 cm of the solvent front. Assays for galactosyltransferase and fucosyltransferase are linear with time and yield results comparable to other methods such as gel permeation chromatography and micropartitioning filtration. The TLC protocol should be useful for determinations of many different glycosyltransferases.

Protein carboxyl methyltransferase activity specific for age-modified aspartyl residues in mouse testes and ovaries: evidence for translation during spermiogenesis.

An antiserum prepared against the purified protein carboxyl methyltransferase (PCMT) from bovine brain has been used to compare testicular and ovarian levels of the enzyme and to study the regulation of PCMT concentrations during spermatogenesis. The PCMT, which specifically modifies age-damaged aspartyl residues, is present at a significantly higher concentration in mature mouse testis than in ovary. However, the PCMT is present at nearly equal concentrations in extracts of germ cell-deficient ovaries and testes obtained from mutant atrichosis/atrichosis mice. In normal testis, the concentration of the PCMT increases severalfold during the first 4-5 weeks after birth, paralleling the appearance and maturation of testicular germ cells. Both immunochemical and enzymatic measurements of PCMT specific activities in purified spermatogenic cell preparations indicate that PCMT levels are twofold and 3.5-fold higher in round spermatids and residual bodies, respectively, than in pachytene spermatocytes. The results are consistent with the enhanced synthesis and/or stability of the PCMT in spermatogenic cells and with the continued translation of the PCMT during the haploid portion of spermatogenesis. The relatively high levels of PCMT in spermatogenic cells may be important for the extensive metabolism of proteins accompanying spermatid condensation or for the repair of damaged proteins in translationally inactive spermatozoa.