Follicle Stimulating Hormone Regulation of Protein Kinase Activity and Protein Synthesis in Testis

Incubation of testis tubules containing predominantly Sertoli cells (SCE) with Follicle Stimulating Hormone results in a rapid activation of cyclic AMP-dependent protein kinase. Multiple forms of soluble protein kinase were shown to exist in Sertoli cell cytosol by DEAE chromatography. Evidence suggests thst only one of the two cyclic AMP-dependent forms is activated by FSH. In addition FSH stimulates testicular protein synthesis and induces the accumulation of a specific Sertoli cell protein, ABP. Kinetic studies reveal this induction to be selective. Moreover, stimulation of ABP can also be accomplished by injection of a derivative of cyclic AMP. These effects are inhibited by prior treatment of the animals with cycloheximide or actinomycin D. Thus, FSH appears to induce de novo synthesis of ABP in a manner which requires prior stimulation of a transcriptional event. Finally, FSH is shown to increase the concentration of Sertoli cell mRNA assayed in a heterologous cell-free protein synthesizing system. Comparison of the time-course of stimulation of mRNA and ABP in response to FSH leads to the possibility that the former is required for the latter. The significance of these events with respect to the biochemical actions of peptide hormones is discussed. It is now well accepted that peptide hormones initiate a temporal sequence of biochemical events in a target cell by first binding to specific membrane-associated receptors. This hormone-receptor interaction usually triggers an increase in the activity of adenylate or guanylate cyclase which results in a change in the intracellular concentration of the respective cyclic nucleotide. It is then generally assumed that the cyclic nucleotide modulates a variety of other biochemical reactions which ultimately define the molecular actions of the peptide hormones. If the precise mechanism by which a peptide hormone alters the function of its target cell is to be elucidated, two criteria must be met. First, a sensitive and accurate marker must be found which can serve as an end point by which to assess the efficacy of the hormone. Secondly, changes in the levels of cyclic nucleotides must be demonstrated to result in an alteration in the specific end point marker. Such criteria have been reported to be established for hormones such as LH and ACTU which stimulate steroidogenesis (Catt and Dufau, 1973; Gill, 1972). In these systems secretion of the steroid hormone has been used as an end point of the physiologic actions of the peptide hormones. Moreover, cyclic AMP will mimic the stimulation of steroid synthesis (Gill, 1972; Hall, 1970). In many instances, however, such convenient markers do not exist. Consequently, another intracellular event must be sought which will serve the same purpose. The activation of cytoplasmic cyclic AMP-dependent protein kinase by peptide hormones is one of the most popular intracellular responses being measured at the present time. The manner by which cyclic AMP activates protein kinase has now been thoroughly investigated in a number of systems in which hormones alter the level of cyclic AMP (Krebs, 1972; Bitensky and Gorman, 1973; Soderling et al., 1973; Means et al., 1974). The state of activation of cytoplasmic protein kinase can be given by the equation: ERC] + [cAMPI [RcAMPJ + [CI (1) Newly synthesized cyclic AMP results in increased binding to the regulatory subunit of the inactive protein kinase ERCI. This allows dissociation of the holoenzyme into the regulatory subunit-cyclic AMP complex (Rcyclic AMP) and active catalytic subunit [CI. The activation of protein kinase presumably results in phosphorylation of enzymes or structural proteins which result in an alteration of function. These changes presumably lead to some of the biological actions attributed to the hormone in question. In any given cell a fraction of the protein FSH AND SERTOLI CELLS 55 TABLE 1. Activation of Sertoli cell protein kinase in response to FSH. Time after FSI-I (mm) Protein kinase activity ratio (-cAMP/+cAMP) 0 .200 2 .235 5 .275 10 .360 15 .390 20 .420 45 .500 60 .500 Tubules (100 mg) from SCE-testis of 16 day old rats were incubated for the times indicated with 500 ng/ml of highly purified oFSH (Papkoff). Following incubation protein kinase activity was measured in the absence and presence of 1(T6 M cAMP as previously described (Means et al., 1974). kinase will exist as the active enzyme [CI. One can assay this fraction of enzyme by performing a straight forward protein kinase assay in the absence of exogenous cyclic AMP ( C). In order to determine the total cyclic AMP-dependent protein kinase activity, the assay is repeated but this time in the presence of an excess of cyclic AMP. This will shift equation 1 to the right resulting in all of the cyclic AMP-dependent protein kinase now existing in the form of active enzyme ( RC+C). The data then can be expressed as a protein kinase activity ratio, that is the activity obtained in the absence of cyclic AMP divided by the activity obtained in the presence of excess of cyclic AMP, (CIRC+C). This value (the protein kinase ratio) when multiplied times 100 yields an estimate of the percent of the total protein kinase activity that is present in the active form. Thus, an increase in intracellular cAMP, i.e., by hormone, should result in an increase in the activity ratio. Follicle Stimulating Hormone binds to membrane-associated receptors present on the surface of the testicular target cell, the Sertoli cell (Means and Vaitukaitis, 1972;Means and I-luckins, 1974). The association of hormone with receptor activates adenylate cyclase which rapidly results in an increased cyclic AMP level within the cell (Means et al., 1974; Means and Vaitukaitis, 1972; Means and Huckins, 1974; Means, 1973). Application of the protein kinase activity ratio assay to the FSH Sertoli cell model system is shown in Table 1. It can be seen that in the absence of FSH, the protein kinase activity ratio (or the percent catalytic subunit) is 20 percent. Within 2 mm after incubation of testis seminiferous tubules which contain only Sertoli cells (SCE testis) with FSH, an increase in the protein kinase activity ratio can be seen. A time-dependent stimulation occurs reaching a maximum of 50 percent by 45 mm. These data then suggest that half of the protein kinase activity present in the cytosol is now being expressed. If physiologic increases in intracellular cyclic AMP result in an activation of protein kinase, a correlation should exist between incremental increases in intracellular cyclic AMP and the protein kinase activity ratio. These data are shown in Table 2. Again, incubation of testis with FSH results in a time-dependent increase in the protein kinase activity ratio. In these experiments, tissue levels of cyclic AMP were measured in the same tissue sample. It can be seen that initial levels of cyclic AMP are approximately 1 picomole/mg of tissue. FSI-I stimulates the intracellular levels of cyclic AMP with maximal values being reached after 30 mm in vitro. Moreover, the incremental increase in cyclic AMP corresponds TABLE 2. FSI-I stimulation of protein kinase and cyclic AMP in vitro. Time after FSH (mm) Protein kinase activity ratio (-cAMP/+cAMP) Cyclic AMP (pmole/mg tissue) 0 .195 1.14 5 .260 1.63 10 .385 2.01 20 .410 3.51 30 .505 3.80 Testis tubules (100 mg) from 16 day old SCE-rats were incubated with 500 ng/ml of oFSU (Papkoff). Protein kinase was measured as previously described (Means et al.. 1974) and cyclic AMP was assayed by the binding procedure described by Gilman (1970).