Protein synthesis by isolated pachytene spermatocytes in the absence of Sertoli cells (original) (raw)
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Regulation of survival of rat pachytene spermatocytes by lactate supply from Sertoli cells
Journal of reproduction and fertility, 1982
During incubation of fragments of seminiferous tubules in the absence of glucose, pachytene spermatocytes and round spermatids died within 24 h, while Sertoli cells were still viable. The germ cells survived for at least 72 h in seminiferous tubule fragments which were incubated in the presence of glucose. Lactate rather than glucose is essential for [3H]uridine incorporation and survival of isolated pachytene spermatocytes. However, if the spermatocytes were incubated in the presence of Sertoli cells, glucose maintained the incorporation of [3H]uridine into the germ cells. Sertoli cells secreted lactate in the presence of glucose and the lactate secretion was stimulated 2--4-fold by FSH. It is concluded that the activity and survival of pachytene spermatocytes in vitro can be regulated by the supply of lactate from Sertoli cells.
Reproduction, 1983
Sertoli cells were obtained from 3\p=n-\6-week-oldrats, which were sterile after prenatal irradiation. The production of lactate by these Sertoli cells, measured 24\p=n-\48h after isolation during incubation in the absence of hormones, increased with age of the rats from 3 to 6 weeks. At all ages investigated, the production of lactate was enhanced in the presence of FSH plus testosterone, but the stimulation was most pronounced at 4 weeks of age. Lactate production was increased by FSH alone but testosterone had no effect in the presence or absence of FSH. Sertoli cells from 4-week-old rats produced both pyruvate and lactate, which accumulated in the incubation medium in a ratio of 1:4. The stimulation of the production of pyruvate and lactate by FSH was dose\x=req-\ dependent (ED50 at~10 ng NIH-FSH-S13/ml). The production of pyruvate and lactate was stimulated 2-fold by insulin, 4-fold by FSH and > 6-fold by dibutyryl cyclic AMP (in the presence of 3-isobutyl-1-methylxanthine). The effects of FSH (0\m=.\5\ g=m\ g NIH-FSH-S13/ml) and insulin (5 \g=m\g/ml) were not additive. Leucine incorporation into isolated pachytene spermatocytes and round spermatids was stimulated by exogenous pyruvate and lactate in a dose-dependent way : maximal incorporation was obtained with 0\m=.\2 mM-pyruvate or 2 mM L-lactate. Spent medium from incubated Sertoli cells (from 4-week-old rats) stimulated the leucine incorporation into isolated pachytene spermatocytes and round spermatids 4\p=n-\8-fold.This effect could be explained by the amounts of pyruvate and lactate present in the spent medium. It is suggested that pyruvate and lactate are major products from Sertoli cells which can support synthetic activities in germ cells, and the present results indicate that pyruvate and lactate may play a role in the hormonal regulation of spermatogenesis.
Development, Growth and Differentiation, 1989
The effect of lactate on synthesis of new proteins in isolated sperrnatids and spermatocytes of rats was examined. Lactate ~timulated[~~S]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 (1 D and 2D-PAGE) and autoradiography. The syntheses of several stagespecific and non-specific proteins were observed. When spermatids and sperrnatocytes 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 sperrnatids and spermatocytes, but did not stimulate the synthesis of the Mr 24 kD protein in the membrane-rich fraction. In addition, after 1 D 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.
International Journal of Andrology, 1989
Maintenance and development of spermatocytes and round spermatids was studied in an in-vitro incubation system. This system consisted of open tubule fragments from 26-day-old rat testes, obtained after collagenase treatment. The tubule fragments contained Sertoli cells and spermatogenic cells up to and including a small number of early round spermatids. The number of primary spermatocytes and round spermatids in the tubule fragments was estimated using flow-cytometric analysis, immediately after isolation and after 72 h of incubation. In addition, the activity of LDH-C4 in the tubule fragments was measured. After 72 h of incubation, the percentage of spermatocytes was reduced by 70-80%, but the percentage of spermatids was doubled. The total LDH-C4 activity per well was increased 2-3-fold during 72 h of incubation of the fragments. A modest improvement of the culture results was observed when a combination of FSH, insulin, retinol and testosterone was added to the medium. LDH-C4 activity was investigated to see whether it could be used as a quantitative marker of isolated and cultured spermatocytes and spermatids. It was observed that LDH-C4 activity per cell was decreased when spermatocytes and spermatids were isolated and/or incubated at 4°C. However, the cellular enzyme activity returned to control values during subsequent incubation of the cells at 32"C, either in the absence or presence of a protein synthesis inhibitor. Cellular LDH-C4 activity may be influenced not only by temperature, but possibly also by other cell isolation conditions. It is concluded that LDH-C4 activity may not be a reliable quantitative marker for the presence of spermatocytes and spermatids in culture, but should be used in combination with other analytical methods such as DNA estimation and DNA flow cytometry.
Nature of the proteins which form disulfide bonds during the maturation of rat spermatozoa
International Journal of Andrology, 1982
The proteins which form disulfide bonds during the maturation of rat spermatozoa were studied by comparing the [14C]carboxamidomethylated products between the caput and caudal spermatozoa. The total incorporation of [14C]iodoacetamide into 106 spermatozoa from the caput epididymis was 13.4 nmole, and those from the caudal epididymis was 3.6 nmole. The proteins extractable from the caput and caudal spermatozoa showed no detectable difference in SDS polyacrylamide gel electrophoretic patterns. Similarly, no detectable change was observed when the proteins were extracted and analysed on polyacrylamide gel containing the cationic detergent cetyltrimethylammoniumbromide (CTAB). However, when the [14C]carboxamidomethylated products were compared, large differences were evident. There were 4 major carboxamidomethylated products on proteins with molecular weights of 32.000; 29.000; 22.000; and 13.000. The 32.000 and 29.000 dalton proteins were carboxamidomethylated about 15 times in the caput over the caudal spermatozoa. The results suggest that these 4 proteins constitute the majority of those which form disulfide bonds during the maturation of rat spermatozoa. The probable origins of those proteins were briefly discussed.
Synthesis and amino acid composition of basic proteins in mammalian sperm nuclei
Developmental Biology, 1975
The basic chromosomal proteins (SCP) of human, mouse, rabbit and guinea pig sperm nuclei were characterized by polyacrylamide gel electrophoresis and amino acid analysis. Spermatozoa were decapitated with 1% SDS and the nuclei recovered by density gradient centrifugation. Examination by Nomarski and electron microscopy revealed the nuclei to be intact and 99% pure. The basic proteins were extracted from nuclei, aminoethylated and purified by ion exchange chromatography and gel filtration chromatography. The SCP of human, rabbit and guinea pig gave single protein bands with similar mobilities when subjected to polyacrylamide gel electrophoresis. In contrast, aminoethylated mouse SCP consisted of two proteins, SCP.AE, and SCP.AE,, which had different electrophoretic mobilities. The SCP of these mammalian species were characteristically rich in arginine (47~54.4%) and cysteine (7.7-X2.2%). Major differences existed in the amino acid compositions of these proteins. Mouse and human SCP were rich in histidine (12.2 and 7.7%, respectively) and guinea pig was high in tyrosine (11.7%) and phenylalanine (3.5%). Valine was detected only in rabbit SCP and proline in human and guinea pig. Aspartic acid, methionine and tryptophan were not detected in all four species. Studies on the incorporation of 13H]arginine into mouse SCP demonstrated that these basic proteins are synthesized during the terminal stages of spermatogenesis and are subsequently conserved.
Hydrolytic Enzymes During Spermatogenesis in Rat an Electron Microscopic and Histochemical Study
Journal of Histochemistry & Cytochemistry, 1968
The localization of lipids and the activities of nonspecific esterase, aryl sulfatase and acid phosphatase were studied in different stages of spermatogenesis in rats. In addition, the distribution of acid phosphatase activity was demonstrated electron histochemically. The spermatogenetic cycle was divided into two phases—corresponding to the first and the last four stages of Roosen-Runge-Giesel (RG) classification. Spermatids in the first phase contained abundant endoplasmic reticulum with rosette formation and well developed Golgi apparatus with numerous vesicles. They displayed high activity of hydrolytic enzymes but contained no appreciable amount of lipids. The Sertoli cells contained large lipid granules but showed minimal enzyme activity. During the second phase reduction of the cytoplasm of spermatids with fragmentation of the endoplasmic reticulum and Golgi lamellae, accumulation of lipids, aggregation of ribonucleo-protein particles, formation of residual bodies and marked...
Journal of Endocrinology, 2002
This study investigates the effects of spermatogenic germ cells on inhibin alpha-subunit and beta B-subunit expression, and inhibin alpha-subunit and inhibin B production by rat Sertoli cells in vitro. Sertoli cells isolated from 19-day-old rats were cultured for 48 h at 32 degrees C, in the presence or absence of FSH (2.3-2350 mIU/ml), and in the presence of pachytene spermatocytes, round spermatids or cytoplasts of elongated spermatids purified from adult rat testis by elutriation and density gradient separation. Sertoli cell secretion of inhibin alpha-subunit and inhibin B, as measured by immunoassay, was dose-dependently stimulated by FSH (maximal stimulation 13- and 2-fold, respectively). Round spermatids or cytoplasts co-cultured with Sertoli cells had no effect on basal or FSH-induced secretion of inhibin alpha-subunit or inhibin B. When Sertoli cells were co-cultured with pachytene spermatocytes, inhibin alpha-subunit secretion was unaltered, while inhibin B secretion was su...
The Golgi apparatus of rat pachytene spermatocytes during spermatogenesis
The Anatomical Record, 1991
A morphological and immunocytochemical study of the Golgi apparatus in pachytene spermatocytes was performed in an effort to correlate the structure and function of this organelle during meiotic prophase. In stages 1-111 of the cycle, the Golgi complex of pachytene spermatocytes is a flattened discoid, 0.5-1 pm in diameter, composed of vesicles interspersed with classically described Golgi cisternae. During subsequent maturation of pachytene spermatocytes (stages IV-XIII), the size of the Golgi complex increases significantly, attaining a size of 2-3 pm. However, unlike pachytene spermatocytes of stages 1-111, the majority of the Golgi complex of more mature spermatocytes is characterized by an abundance of distinct stacks of cisternae interspersed with numerous vesicles and tubules. The composition of the Golgi complex was also studied by using two