Stimulation of Tubulin Synthesis by Lactate in Isolated Spermatogenic Cells

The effect of lactate on synthesis of new proteins in isolated spermatids and spermatocytes of rats was examined. Lactate stimulated[³⁵S]methionine ([³⁵S]met) incorporation into both spermatids and spermatocytes. The rate of protein synthesis was positively correlated with the intracellular level of ATP. The [³⁵S]met-labeled proteins in the two types of cells were compared by one and two dimensional polyacrylamide gel electrophoresis (1D and 2D-PAGE) and autoradiography. The syntheses of several stagespecific and non-specific proteins were observed. When spermatids and spermatocytes were cultured in medium without lactate, two major proteins of molecular weight (Mr) 43 kD and 55 kD were detected in the water-soluble fraction (105,000 g supernatant), and one major protein of Mr 24 kD was observed in the membrane-rich fraction. Addition of lactate to the incubation medium dramatically increased the synthesis of six proteins (Mr 14 kD, 16 kD, 43 kD, 55 kD, 84 kD and 135 kD) in the water-soluble fractions of spermatids and spermatocytes, but did not stimulate the synthesis of the Mr 24 kD protein in the membrane-rich fraction. In addition, after 1D and 2D-PAGE and electrophoretic transfer to nitrocellulose, two proteins of Mr 43 kD and 55 kD were identified as actin and tubulin, respectively, on the basis of their reactivities with specific antisera. Tubulin was also produced by in vitro translation using a spermatid lysate. These results suggest that lactate may play an important role in changing the cell structure and shape during spermatogenesis by regulating the syntheses of actin and tubulin.     

Acrosomal constituents identified with a monoclonal antibody are modified during late spermiogenesis in the mouse

Monoclonal antibody 1D4, a mouse immunoglobulin M raised against CD-1 mouse spermatogenic cell membranes, recognizes acrosomal constituents in the mouse, rabbit, and guinea pig. In the mouse, acrosomes of round and condensing spermatids were labeled with 1D4 by indirect immunofluorescence on isolated cells and by immunohistochemistry on paraffin sections. During the terminal steps of spermiogenesis, however, acrosomal labeling in mouse germ cells was lost. Little or no 1D4 immunoreactivity was detected by enzyme-linked immunosorbent assays in prepubertal testes, Sertoli cells, or several somatic tissues. To identify antigens recognized by 1D4, mouse spermatogenic cell proteins were separated by one- (1D) and two-dimensional (2D) polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunostained. Multiple antigens larger than 200,000 relative molecular weight (Mr) were resolved on 1D immunoblots from round and condensing spermatids isolated by sedimentation velocity at unit gravity. A smaller antigen (Mr 85,000 isoelectric point approximately 5.7) was also detected on 1D and 2D immunoblots of round spermatid proteins. These antigens can be labeled biosynthetically with [3H] glucosamine and immunoprecipitated, suggesting that they are a set of glycoconjugates that share a common epitope recognized by 1D4. This determinant is no longer detectable in late spermatids, indicating that biochemical modifications of acrosomal constituents occur during the terminal steps of germ cell differentiation.     

Radio-iodination of plasma membrane polypeptides from isolated mouse spermatogenic cells

Cell surface polypeptides of mouse pachytene spermatocytes and round spermatids (steps 1–8) have been iodinated using 1,2,3,6,tetracholoro-3α, 6α-diphenylglycouril (IODOGEN). Labeled proteins have been assayed using two-dimensional polyacrylamide electrophoresis and radioautography. Purified plasma membranes, prepared from both spermatocytes and spermatids after the iodination of intact cells, exhibit 25–30 polypeptides which label reproducibly. No significant qualitative differences are noted in the labeled polypeptide map obtained from each of the purified cell types. Iodinated proteins range in molecular weight from greater than 100k daltons to approximately 40k daltons. The isoelectric points of labeled constituents range from pI 5.7 to 7.2. Three polypeptides represent the major iodinated species: p 94/5.8, p 75/5.9, and p 53/7.1. Comparison with total plasma membrane constituents assayed using Coomassie brilliant blue indicates that many of the radioactively labeled proteins are not present in quantities sufficient to allow ready detection without isotopic techniques. As a result, many of the proteins identified autoradiographically represent newly described surface components of mouse pachytene spermatocytes and round spermatids. The preparation of purified plasma membrane fractions prior to electrophoresis ensures that all iodinated species are in fact cell surface components. Furthermore, experiments designed to assess the vectorial nature of the IODOGEN-catalyzed labeling procedure suggest that most, if not all, of the iodinated species are exposed on the external side of the cell plasma membrane. Therefore, these studies have (1) identified hitherto unrecognized plasma membrane components of mouse pachytene spermatocytes and round spermatids and (2) provided the first available biochemical data concerning the molecular orientation of particular proteins in the surface membranes of developing mouse spermatogenic cells.     

The effects of temperature and glucose on protein biosynthesis by immature (round) spermatids from rat testes

A method is described for the preparation of highly purified fractions (greater than 80% pure) of immature spermatids (round, steps 1--8) from rat testes by centrifugal elutriation in sufficient yields for biochemical studies when four rat testes are used. Electron microscopy established the identity of the cells and demonstrated that the cell membrane is intact. Some cells develop nuclear and cytoplasmic vacuoles during the 2 h required for preparation. Immature spermatids prepared by this method use glucose with an increase in oxygen consumption, lactate production, and protein synthesis over control levels (no glucose). The testicular cell suspension from which spermatids are separated, like whole testis and spermatids themselves, show higher incorporation of amino acids into TCA-precipitable material at 34 degrees C than at 38 degrees C and in the presence of glucose. A subcellular system prepared from immature spermatids with excess ATP shows greater incorporation of amino acids into TCA-precipitable material at 34 degrees C than at 38 degrees C. This difference does not result from increased breakdown of protein. It is concluded that body temperature (38 degrees C) inhibits some aspect(s) of protein synthesis in addition to previously reported effects on amino acid transport and production of ATP (Means and Hall. 1969. Endocrinology. 84:285--297.).     

OBSERVATIONS ON HUMAN MONOCYTE KINETICS AFTER PULSE LABELING

Monocyte kinetics were studied in seven hematologically normal individuals using in vivo pulse labeling with tritiated thymidine. Although occasional labeled cells appear in the peripheral blood within 4 or 5 hr of the administration of label, a significant outflow from the marrow begins 13–26 hr later. This interval is occupied by the G₂ and M phases of the mitotic cycle since mitotic cells are not observed in the peripheral blood. The duration of the DNA synthesis phase of monocytes is measured at 34 hr ≈ 1.8 hr. Cells do not enter this phase while circulating since exposure of circulating cells to tritiated thymidine does not result in any uptake. If monocytes are not 'end'cells which have completed their mitotic activity before leaving the marrow they must at least be inhibited from further proliferative activity until they are permanently sequestered in other tissues.
The generation time is probably not less than 40 hr and data derived from the mean grain counts of labeled cells suggest that it is often more than 70 hr. The total daily output of monocytes in man is 9.4 × 10⁸ cells per 24 hr ≈ 3.3 × 10⁸.
Cells leave the bloodstream with a half-time of about 71 hr thereby proving themselves to be considerably more durable than neutrophils which have a half-life in the neighborhood of 6 hr.     

Labeling of hepatic glycogen after short- and long-term stimulation of glycogen synthesis in rats injected with 3H-galactose

The effects of short- and long-term stimulation of glycogen synthesis elicited by dexamethasone were studied by light (LM) and electron (EM) microscopic radioautography (RAG) and biochemical analysis. Adrenalectomized rats were fasted overnight and pretreated for short- (3 hr) or long-term (14 hr) periods with dexamethasone prior to intravenous injection of tracer doses of 3H-galactose. Analysis of LM-RAGs from short-term rats revealed that about equal percentages (44%) of hepatocytes became heavily or lightly labeled 1 hr after labeling. The percentage of heavily labeled cells increased slightly 6 hr after labeling, and unlabeled glycogen became apparent in some hepatocytes. The percentage of heavily labeled cells had decreased somewhat 12 hr after labeling, and more unlabeled glycogen was evident. In the long-term rats 1 hr after labeling, a higher percentage of heavily labeled cells (76%) was observed compared to short-term rats, and most glycogen was labeled. In spite of the high amount of labeling seen initially, the percentage of heavily labeled hepatocytes had decreased considerably to 55% by 12 hr after injection; and sparsely labeled and unlabeled glycogen was prevalent. The EM-RAGs of both short- and long-term rats were similar. Silver grains were associated with glycogen patches 1 hr after labeling; 12 hr after labeling, the glycogen patches had enlarged; and label, where present, was dispersed over the enlarged glycogen clumps. Analysis of DPM/mg tissue corroborated the observed decrease in label 12 hr after administration in the long-term animals. The loss of label observed 12 hr after injection in the long-term pretreated rats suggests that turnover of glycogen occurred during this interval despite the net accumulation of glycogen that was visible morphologically and evident from biochemical measurement.     

Stage-specific protein synthesis by isolated spermatogenic cells throughout meiosis and early spermiogenesis in the mouse

Spermatogenic cells isolated from prepubertal and adult mice by unit gravity sedimentation have been used to examine proteins synthesized in a stage-specific manner throughout meiosis and early spermiogenesis. Preleptotene, leptotene/zygotene, and pachytene spermatocytes were isolated from 17-day-old mice. Adult pachytene spermatocytes and round spermatids were isolated from mature animals. These germ cells were then cultured in defined medium with [35S]methionine [( 35S]met) for 4-5 h. For each cell type, relative [35S]met incorporation was determined and labeled proteins were compared by two-dimensional (2D) polyacrylamide gel electrophoresis and autoradiography. Levels of [35S]met incorporation by isolated germ cells correlate closely with previous autoradiographic estimates of protein synthesis during spermatogenesis (Monesi, 1967). Pachytene spermatocytes from prepubertal mice incorporate the highest levels of [35S]met, when expressed either as cpm/-10(6) cells or cpm/mg protein. Comparisons of 2D autoradiograms indicated that many proteins, including actin and tubulins, are synthesized at approximately equal levels in all stages examined. Other proteins, including heat-shock proteins and multiple plasma membrane constituents, are synthesized in a stage-specific manner in leptotene/zygotene spermatocytes, pachytene spermatocytes, and round spermatids. These studies define conditions for monitoring protein synthesis in isolated spermatogenic cells prior to the pachytene stage of meiosis, provide a 2D map of proteins synthesized at these earlier meiotic stages, and examine the synthesis of several proteins previously identified on 2D gels with biochemical and immunological methods.     

Localization of a single sperm membrane autoantigen (RSA-1) on spermatogenic cells and spermatozoa

The rabbit sperm membrane autoantigen RSA-1 is a sialoglycoprotein of 13,000 daltons which first appears on the surface of pachytene spermatocytes. Using specific antiserum to RSA-1 the antigen has been localized by immunofluorescence and immunoperoxidase staining. On testicular cells labeled at 37°C, RSA-1 is seen in patches on the surfaces of pachytene spermatocytes, round spermatids, and over the acrosomal area of later spermatids and spermatozoa. Over the postacrosomal and middle-piece regions of late spermatids and spermatozoa the labeling appears uniform. The uniformity can be seen to stop abruptly at the equatorial segment-postacrosomal border. Labeling cells after fixation gives a uniform distribution of label over the surface where patches were seen at 37°C. The polypeptides recognized by the antiserum used for labeling were identified by immunoadsorbent chromatography and subsequent SDS-PAGE. In testicular cells anti-RSA-1 recognizes the 13,000-dalton form and another component which migrates with the dye front. In ejaculated spermatozoa anti-RSA-1 recognizes a distinct ejaculate complex of higher-molecular-weight proteins containing an 84,000-dalton major band and five minor components.     

Purine nucleoside phosphorylase synthesis and turnover in human lymphoid cells

We have studied the turnover and synthesis of purine nucleoside phosphorylase by using a polyclonal rabbit antiserum to this protein. The turnover of purine nucleoside phosphorylase was studied in the B lymphoblast cell, WI-L2, by specific immunoprecipitation of [3H]leucine-labeled proteins. The half-lives for total protein and purine nucleoside phosphorylase were 14.5 and 14.1 hr, respectively. For cells cultured in the presence of inosine the half-life of purine nucleoside phosphorylase was reduced to 11.2 hr. The synthesis of purine nucleoside phosphorylase was analyzed during phytohemagglutinin-stimulated T cell transformation by pulse labeling cells with [35S]methionine. Purine nucleoside phosphorylase synthesis increased greater than 10-fold during the first 12 hr of transformation and continued to a maximum of 30-fold. The relative rate of purine nucleoside phosphorylase labeled to total proteins was 0.04% in unstimulated T cells and increased to 0.18% 12 hr after stimulation. These studies identify some preferential synthesis of purine nucleoside phosphorylase during the early stages of T cell transformation.     

Levels and 75Se-labeling of specific proteins as a consequence of dietary selenium concentration in mice and rats

Selenium-labeled proteins (SLP) distinct from glutathione peroxidase (GSH-PX) recently have been purified and partially characterized. Antisera to two SLP, a 56-kDa and a 14-kDa protein, were generated in rabbits and used to examine expression of these proteins as a consequence of dietary selenium concentration (0.02, 0.2, 2.0 ppm) in mice and rats. Additionally, the kinetics of 75Se labeling in plasma, liver, kidney, and mammary gland were examined over a 40-hr time period as a function of dietary selenium concentration. A plasma 57-kDa protein was labeled by 30 min after 75Se injection and reached maximum labeling by 4 hr. The cellular 56-kDa and 14-kDa proteins, as well as GSH-Px, labeled progressively over 40 hr starting between 1 and 4 hr after injection. In general, the 56-kDa and GSH-Px followed similar labeling patterns, whereas the 14-kDa protein was labeled less and was not labeled in discernible quantities until 40 hr. The extent of labeling of all proteins was inversely proportional to the dietary selenium concentration and was probably a reflection of different endogenous selenium body pools. The most important observation was generated by the immunoblot data. The amount of 56-kDa and 14-kDa proteins as detected and measured on immunoblots was not a function of dietary selenium concentration. This result suggests that the synthesis and maintenance of the 56-kDa and 14-kDa proteins are not selenium dependent, a characteristic which distinguishes the two proteins from GSH-Px. The single exception to the above results was the 40% decrease of liver 14-kDa protein concentration in carcinogen-treated rats fed 2.0 ppm of selenium. An organic selenium compound, selenobetaine, did not lead to a decrease under similar conditions. In 15 rat mammary tumors induced by 7,12-dimethylbenzanthracene and analyzed on immunoblots, the SLP-56 was undetected in 5 cases and appeared as two bands (56,000 Da, 50,000 Da) in 10 cases. This latter result raises the possibility that the expression of SLP-56 may be altered in mammary tumors as compared with normal mammary gland.     

Lobular and cellular patterns of early hepatic glycogen deposition in the rat as observed by light and electron microscopic radioautography after injection of 3H-galactose

Very low hepatic glycogen levels are achieved by overnight fasting of adrenalectomized (ADX) rats. Subsequent injection of dexamethasone (DEX), a synthetic glucocorticoid, stimulates marked increases in glycogen synthesis. Using this system and injecting 3H-galactose as a glycogen precursor 1 hr prior to sacrifice, the intralobular and intracellular patterns of labeled glycogen deposition were studied by light (LM) and electron (EM) microscopic radioautography. LM radioautography revealed that 1 hr after DEX treatment, labeling patterns for both periportal and centrilobular hepatocytes resembled those in rats with no DEX treatment: 18% of the hepatocytes were unlabeled, and 82% showed light labeling. Two hours after treatment with DEX, 14% of the hepatocytes remained unlabeled, and 78% were lightly labeled; however, 8% of the cells, located randomly throughout the lobule, were intensely labeled. An increased number of heavily labeled cells (26%) appeared 3 hr after DEX treatment; and by 5 hr 91% of the hepatocytes were intensely labeled. Label over the periportal cells at this time was aggregated, whereas centrilobular cells displayed dispersed label. EM radioautographs showed that 2 to 3 hr after DEX injection initial labeling of hepatocytes, regardless of their intralobular location, occurred over foci of smooth endoplasmic reticulum (SER) and small electron-dense particles of presumptive glycogen, and in areas of SER and distinct glycogen particles. After 5 hrs of treatment with DEX, the intracellular distribution of label reflected the glycogen patterns characteristic of periportal or centrilobular regions.     

Germ cell-specific DNA and RNA binding proteins p48/52 are expressed at specific stages of male germ cell development and are present in the chromatoid body

Proteins homologous to the Xenopus oocyte mRNA binding proteins mRNP₃₊₄ and designated p48/52 have been identified in male mouse germ cells (1993: Dev Biol 158:90–100). Western and Northwestern blots of extracts from testes and isolated germ cells indicate that p48/52 are present during meiosis but reach their highest levels postmeiotically at a time when many mRNAs are stored. Here we analyze the cellular and subcellular distribution of p48/52 in rat and mouse testes by LM and EM immunocytochemistry using an anti-mRNP₃₊₄ antibody. Immunolabeling was found to be predominantly cytoplasmic and specific to germ cells at certain periods during their development. p48/52 were first detected in early pachytene spermatocytes at stage V of the seminiferous cycle and progressively increased during the remainder of meiotic prophase to a post-meiotic peak in steps 1–8 round spermatids; thereafter, labeling gradually declined as elongated spermatids underwent nuclear condensation and elongation. A proportionally higher concentration of cytoplasmic immunolabeling was found within the lacunae of the anastomotic granulofilamentous network of the chromatoid body. The pattern of synthesis of these mRNA binding proteins together with their association with the chromatoid body suggests a role as germ cell-specific mRNA stabilizing and/or storage proteins. © 1996 Wiley-Liss, Inc.     

Growth Kinetics, Cell Shape, and the Cytoskeleton of Primary Astrocyte Cultures

Abstract: We examined correlations among growth kinetics, cell shape, and cytoskeletal protein content in rat astrocytes grown in primary culture. Cell suspensions from brains of newborn rats were seeded at densities from 0.2 to 3 × 10⁵/cm². At initial densities above 1 × 10⁵ the population increased to reach confluency by 10–12 days, after which cell number remained stable for many weeks. At low initial densities, 0.2–0.4 × 10⁵/cm², cells did not increase in number. Final density increased with increasing plating densities. High-density cells had small perikarya and several long cytoplasmic processes; low-density cells appeared flat and polygonal. All cultures were almost entirely astrocytic, as judged by immunofluorescent staining with antiserum against glial fibrillary acidic protein (GFAP). Cytoskeletal proteins were analyzed by gel electrophoresis after extraction from cells with nonionic detergent. Relative amounts of the proteins differed, in that low-density cells contained large amounts of cytoskeletal actin relative to the intermediate filament (IF) proteins vimentin and GFAP, whereas high-density cells contained relatively less actin and more IF proteins. Such differences in cytoskeletal proteins between the high- and low-density cultures were mirrored in the relative rates of synthesis of the cytoskeletal proteins. In the low-density cells amino acid incorporation into cytoskeletal-associated actin was more active than that into the IFs, whereas in the high-density cells higher rates of IF protein synthesis were observed.     

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.     

The synthesis of acidic chromosomal proteins during the cell cycle of HeLa S-3 cells. II. The kinetics of residual protein synthesis and transport

The kinetics of acidic residual chromosomal protein synthesis and transport were studied throughout the cell cycle in HeLa S-3 cells synchronized by 2 mM thymidine block and selective detachment of mitotic cells. Pulse labeling the cells with leucine-³H for 2 min and then "chasing" the radioactive proteins for up to 3 hr showed that the amount of protein synthesized, transported, and retained in the acidic residual chromosomal protein fraction is greater immediately after mitosis and later in G₁ than in the S or G₂ phases of the cell cycle. During S, only 20–25% of the proteins synthesized and transported to the acidic residual chromosomal protein fraction are chased during the first 2 hr after pulse labeling, whereas up to 40% of the material entering the residual nuclear fraction in mitosis, G₁, and G₂ leaves during a 2 hr chase. Polyacrylamide gel electrophoretic profiles of these proteins, at various times after pulse labeling, reveal that the turnover of individual polypeptides within this fraction has kinetics of synthesis and turnover which are markedly different from one another and undergo stage-specific changes.     

Purification and characterization of the rat spermatid basic nuclear protein TP4.

Following elongation of spermatids in mammals, the histones are replaced by a set of basic nuclear transition proteins; in the rat there are four, named TP1-TP4. Of these, TP1 and TP2 are well characterized. Here we report the purification to homogeneity of TP4 from rat spermatids. It is a low molecular mass (about 13-20 kDa) basic protein with arginine and lysine constituting 24 mol % and histidine 2.2 mol %. Its 25 N-terminal amino acids were sequenced, and no sequence homologies with any known protein were found. Polyclonal antibodies raised against it in rabbit did not cross-react with other transition proteins, protamines, or histones. The presence of TP4 during sperm development was monitored by cell separation studies. No TP4 was detected in round spermatids, and along with TP1 and TP2, it is present in step 13-15 spermatids and its amount decreased in steps 16-19. Trace amounts of TP4 were also detected in epididymal sperm. A possible role for TP4 in spermatid and sperm chromatin structure is discussed.     

Developmental program of PGK-1 and PGK-2 isozymes in spermatogenic cells of the mouse: Specific activities and rates of synthesis

The specific activities and synthesis of the ubiquitous isozyme, PGK-1, and the testis-specific isozyme, PGK-2, have been quantitated and localized in spermatogenic cells of the mouse. There is a fivefold increase in total PGK specific activity between immature and adult testes which begins at approximately 30 days of age, coincident with the appearance of late-middle stage spermatids. The increase in total PGK is entirely due to the appearance and increase of the PGK-2 isozyme. Rates of PGK synthesis were measured by labeling testicular cells in vitro with [³H]leucine and purifying the PGK isozymes. When total testicular cells were examined, PGK-2 synthesis was detectable after 22 days of age at very low levels and increased in older testes to a level of 0.5% of total protein synthesis. PGK-1 synthesis remained relatively constant at all ages at a level 100-fold lower (0.005%). Testicular cells were separated into highly enriched fractions of particular spermatogenic stages by centrifugal elutriation. The PGK-1 synthesis rates were, again, very low and not significantly different between the various spermatogenic stages. PGK-2 synthesis was low to nondetectable in pachytene spermatocytes, increased to 0.07% in early spermatids and represented 0.7% of total protein synthesis in late spermatids. This increased rate of PGK-2 synthesis appears to require an increase in the amount of PGK-2 mRNA in late spermatids, cells in which no active RNA synthesis is detectable.     

Heat-shock cognate protein (hsc71) and related proteins in mouse spermatogenic cells

A monoclonal antibody (13D3) has been developed that recognizes a 71 kilodalton (71 kDa) protein on two-dimensional immunoblots of proteins extracted from a mixture of mouse spermatogenic cells (mainly pachytene spermatocytes and spermatids). This protein was shown by immunoblotting and adenosine triphosphate (ATP)-binding characteristics to be identical to a 71 kDa mouse heat-shock cognate (hsc) protein, hsc71, present in 3T3 cells. Along with a 70 kDa heat-shock inducible protein (hsp70), and a 74 kDa heat-shock cognate protein (hsc74), hsc71 is a product of the mouse HSP70 multigene family. Although antibody 13D3 reacted strongly with hsc71, it reacted only faintly with hsp70 in 3T3 cells, and not at all with hsc74 or a germ cell-specific hsp70-like protein (P70) on immunoblots of mixed germ cells. Antibody 13D3 is unique among known antibodies in its pattern of reaction with these heat-shock proteins. In immunofluorescence studies on isolated germ cells, 13D3 reacted uniformly with the cytoplasm of pachytene spermatocytes, round spermatids, and residual bodies, but only with the midpiece of spermatozoa. Antibody 13D3 recognizes other proteins in addition to hsc71 on two-dimensional immunoblots of condensing spermatids and spermatozoa. Two of the proteins (70 kDa/pI 6.4 and 70 kDa/pI 6.5) were present in condensing spermatids and spermatozoa, and another protein (69 kDa/pI 7.0) was detected only in spermatozoa. The new proteins also were recognized by monoclonal antibody 7.10, which reacts specifically with hsp70, hsc71, hsc74, and P70. Although [35S]methionine was incorporated into the new proteins in condensing spermatids, hsc71, hsc74, and P70 were not labeled. These results suggest that unique heat-shock proteins are synthesized late in spermatogenesis.     

Synthesis of sperm-specific basic nuclear proteins (SPs) in cultured spermatids from Xenopus laevis

The accumulation and synthesis of sperm-specific basic nuclear proteins (SPs) in Xenopus spermatids in vitro were studied by acid-urea-Triton polyacrylamide gel electrophoresis and fluorography. In synchronous cultures of round spermatids, the amount of SP2 and SP3-5 accumulated almost linearly with time, while that of SP1 remained almost constant. Fluorography showed that round spermatids incorporated [14C]arginine mostly into SP1 and SP3-5, very little into SP2, and none into histones. When [14C]arginine was incorporated into cells for 24 h on Days 0, 3, and 6, followed by immediate extraction of basic nuclear proteins, the SP1 band was detected faintly on Day 0 and the intensity increased to the maximum level by Day 3 and remained constant on Day 6; the SP3-5 bands were first detected on Day 3 and their intensity increased by Day 6. Thus, SP1 and SP3-5 were synthesized differentially during the culture period. When [14C]arginine or [14C]lysine was incorporated into round spermatids on Days 0, 3, and 6 for 15 h and chased for 3-12 days, the intensity of the SP2 band increased significantly, while the intensity of the SP1 band decreased concomitantly. This result indicates that SP2 was processed from a precursor protein which is probably SP1.