Pgk Isozymes During Spermatogenesis in the Mouse.
Kramer, James Milton
1981
Abstract
The regulation of the phosphoglycerate kinase isozymes, PGK-1 (X-linked) and PGK-2 (autosomal), was studied in spermatogenic cells of the mouse. The aim of these studies was to investigate two major facets of spermatogenesis: X-chromosome inactivation and post-meiotic gene expression. Total protein synthesis was found to decline in middle-late spermatids. By two-dimensional gel analysis of soluble and membrane proteins, however, it appeared that the complexity of proteins synthesized did not appreciably decline. Indeed, some 50 proteins were found only in post-meiotic cells. One of these proteins was identified as the testis-specific isozyme PGK-2. Enzyme activity studies show that a small amount of PGK-2 first appears in the developing testis coincident with the appearance of early spermatids. A significant amount of PGK-2 is not seen until late-middle stage spermatids appear however. Starting at this time a five-fold increase in the total PGK activity of the testis is initiated, which is entirely due to increasing PGK-2 specific activity. This indicates that late-middle stage and more mature spermatids have very high levels of PGK-2 relative to other testicular cells. PGK-2 was localized to the post-meiotic germ cells of the testis by the technique of immunofluorescence. The earliest cell in which PGK-2 was detectable was the Stage 12 spermatid (late-middle stage). The intensity of fluorescence increased in later stage spermatids implying that the amount of PGK-2 increases. Specific immunofluorescence for PGK-1 was only detectable in interstitial and Sertoli cells. In order to study the rates of synthesis of the PGK isozymes, a rapid, minature PGK purification technique was devised. Cells were labeled in vitro for 1 hour with ('3)H-leucine. The resulting cell lysate was passed through a 25 microliter ATP-agarose affinity column. The affinity matrix with bound PGK was then directly transferred to the first dimension isoelectric focusing gel of a two-dimensional gel system. After completion of 2-D electrophoresis, the spots corresponding to PGK-1 and PGK-2 were exercised and counted. In total testicular cell suspensions, PGK-2 synthesis was not detectable prior to 24 days of age. From 24 to 30 days PGK-2 accounted for about 0.02% of total protein synthesis, from 30 to 60 days it increased to about 0.1%. After 70 days of age, the rate of synthesis of PGK-2 in some animals reached to 0.5%. PGK-1 synthesis was relatively constant at all ages, but was 50-100 fold lower (0.005%) than PGK-2. Testicular cells were fractionated into specific stages of spermatogenesis by the use of centrifugal elutriation. PGK-2 synthesis was low to non-detectable in pachytene spermatocytes, 0.07% of total protein synthesis in early spermatids, and about 0.7% in late spermatids. PGK-1 synthesis was not significantly different among the cell fractions but, again, was at very low levels (0.002-0.010%). The levels of PGK-1 synthesis were too low to allow detection of a decline in PGK-1 synthesis after meiosis, as would be predicted by the hypothesis of X-chromosome inactivation at meiosis. Thus, no conclusions about X-chromosome inactivation can be drawn. The analysis of expression of PGK-2 reveals that it is restricted to post-meiotic cells and that its synthesis increases after meiosis. This is of particular interest in regard to the control of post-meiotic gene expression, because the increase in PGK-2 synthesis appears to occur in cells in which no RNA synthesis has been previously detected. It is now of interest to know if this increase in PGK-2 synthesis arises from ongoing RNA synthesis or could be the result of activation of "stored mRNA".Types
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