The importance of gonadotropins and androgens for spermatogenesis is generally accepted in vertebrates, but the role played by specific hormones has not been clarified. Under cultivation conditions, male Japanese eels (Anguila japonica) have immature testes containing only premitotic spermatogonia, type A and early-type B spermatogonia. In the present study, a recently developed organ-culture system for eel testes was used to determine in vitro effects ofvarious steroid hormones on spermatogenesis. After 9 days of culture in serum-free, chemically defined medium. containing 11-ketotestosterone (10 ng/ml), a major androgen in male eels, type A and early-type B spermatogonia began mitosis, producing late-type B spermatogonia. After 18 days, zygotene spermatocytes with synaptonemal complexes appeared, indicating that melosis had already started by this time. In testis fragments cultured for 21 days, round spermatids and spermatozoa were observed with spermatogenic cells at all stages of development. Addition of 11-ketotestosterone to the culture medium also caused a marked cytological activation of Sertoli cells. No other steroid hormones tested had such stimulatory effects. These results, together with our earlier observations, suggest the following sequence for the hormonal induction of spermatogenesis Wi eel testes; gonadotropin stimulates the Leydig cells to produce 11-ketotestosterone, which, in turn, activates the Sertoli cells leading to the completion of spermatogenesis. This is, thus, an example of an animal system in which all stages of spermatogenesis have been induced by hormonal manipulation in vitro.The formation of sperm, spermatogenesis, is an extended process that begins with the proliferation of spermatogonia and proceeds through the extensive morphological changes that convert the haploid spermatid into a mature, functional spermatozoon. Although it is generally accepted that the principal stimuli for vertebrate spermatogenesis are pituitary gonadotropins and androgens, the specific role played by individual hormones has not been clarified (1-4). A number of factors complicating in vivo investigations of the mechanisms involved in the spermatogenesis can be eliminated in in vitro organ (5, 6) and cell (7-10) culture systems in which the direct effects of various factors, including hormonal influences, upon the spermatogenic cells and testes can be investigated.Under conditions of cultivation, male Japanese and European eel have immature testes containing only premitotic spermatogonia, type A and early-type B spermatogonia (11)(12)(13)(14)(15). It has been reported that in both species a single injection of exogenous human chorionic gonadotropin induces all stages of spermatogenesis in vivo (12,14,15). This injection also caused an increase in plasma levels of 11-ketotestosterone (12, 15). Thus, the eel testis provides an excellent system for studying the mechanism by which spermatogenesis is regulated. In the present study, we have used a recently developed organ culture system for eel te...

Insulin stimulates the activity of mitogen-activated protein kinase (MAPK) via its upstream activator, MAPK kinase (MEK), a dual specificity kinase that phosphorylates MAPK on threonine and tyrosine. The potential role of MAPK activation in insulin action was investigated with the specific MEK inhibitor PD98059. Insulin stimulation of MAPK activity in 3T3-L1 adipocytes (2.7-fold) and L6 myotubes (1.4-fold) was completely abolished by pretreatment of cells with the MEK inhibitor, as was the phosphorylation of MAPK and pp90Rsk, and the transcriptional activation of c-fos. Insulin receptor autophosphorylation on tyrosine residues and activation of phosphatidylinositol 3'-kinase were unaffected. Pretreatment of cells with PD98059 had no effect on basal and insulin-stimulated glucose uptake, lipogenesis, and glycogen synthesis. Glycogen synthase activity in extracts from 3T3-L1 adipocytes and L6 myotubes was increased 3-fold and 1.7-fold, respectively, by insulin. Pretreatment with 10 microM PD98059 was without effect. Similarly, the 2-fold activation of protein phosphatase 1 by insulin was insensitive to PD98059. These results indicate that stimulation of the MAPK pathway by insulin is not required for many of the metabolic activities of the hormone in cultured fat and muscle cells.

SHPTP2 is a ubiquitously expressed tyrosine-specific protein phosphatase that contains two aminoterminal Src homology 2 (SH2) domains responsible for its association with tyrosine-phosphorylated proteins. In this study, expression ofdominant interfering mutants of SHPTP2 was found to inhibit insulin stimulation of c-fos reporter gene expression and activation of the 42-kDa (Erk2) and 44-kDa (Erkl) mitogen-activated protein kinases. Cotransfection of dominant interfering SHPTP2 mutants with v-Ras or Grb2 indicated that SHPTP2 regulated insulin signaling either upstream of or in parallel to Ras function. Furthermore, phosphotyrosine blotting and immunoprecipitation identified the 125-kDa focal adhesion kinase (pp125FAK) as a substrate for insulin-dependent tyrosine dephosphorylation. These data demonstrate that SHPTP2 functions as a positive regulator of insulin action and that insulin signaling results in the dephosphorylation of tyrosine-phosphorylated ppl25FAK.The insulin receptor is a ligand-stimulated transmembrane protein-tyrosine kinase that phosphorylates itself as well as intracellular substrates on specific tyrosine residues (1, 2). One proximal intracellular target for the kinase-activated insulin receptor has been identified as a 185-kDa protein, termed insulin receptor substrate 1 (IRS1) (3,4). This molecule contains several insulin receptor-specific tyrosine phosphorylation sites that provide recognition signals for the binding of specific Src homology 2 (SH2) domain-containing proteins (5, 6). The interaction of IRS1 with signaling proteins containing multiple SH2 domains provides a mechanism by which insulin can modulate the function of several distinct pathways. For example, activation of phosphatidylinositol (PI) 3-kinase activity occurs upon the association of the p85 subunit of the PI 3-kinase with tyrosine-phosphorylated IRS1 (7-9). It has been suggested that the insulin activation of Ras function results from the interaction and/or appropriate targeting of the guanine nucleotide exchange factor Sos with IRS1 (6, 10). This is thought to occur via the constitutive association of the Src homology 3 (SH3) domains of the adapter protein Grb2 with the C-terminal domain of Sos (Grb2/Sos complex) and subsequent binding of the Grb2 SH2 domain to tyrosinephosphorylated IRS1 (10, 11).Recently, several groups have identified a ubiquitously expressed 68-kDa tyrosine-specific protein phosphatase, SHPTP2 (also termed Syp, PTP1D, SHPTP3, PTP2C, or PTPL1), that contains two N-terminal SH2 domains and a C-terminal catalytic domain (12-17). The SH2 domains of this phosphatase mediate the binding of SHPTP2 to tyrosinephosphorylated epidermal growth factor receptor, plateletderived growth factor receptor, and IRS1, resulting in the activation of protein-tyrosine-phosphatase activity (18)(19)(20)(21). Although protein tyrosine phosphatase activity is generally thought to function as the inactivating arm of receptor kinase signaling pathways, in the case of T-cell receptor signaling, the CD45 protein-tyrosi...

Our findings suggest that administration of teriparatide increased the quality of the lumbar spine bone marrow and pedicle cortex.

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