Expression and regulation of the proenkephalin gene in rat Sertoli cells.

Previous studies have shown that multiple forms of proenkephalin RNA are present in the rat and mouse testis that are cell type specific: a 1700-nucleotide transcript, unique to the testis, found in spermatogenic cells, and a 1450-nucleotide form of somatic cell origin. In the present studies, rat Sertoli cells are shown to contain the 1450-nucleotide form of proenkephalin RNA. The concentration of this transcript is quite low immediately after Sertoli cell isolation, but increases after 1 day in culture to a constant level. Proenkephalin RNA levels are stimulated severalfold by FSH and cAMP, but are not significantly affected by LH or testosterone. These studies demonstrate that Sertoli cells are at least one source of the somatic, 1450-nucleotide form of proenkephalin RNA in the rat testis. The expression of opioid peptide genes by Sertoli cells as well as by germ and Leydig cells is further evidence for the postulated autocrine or paracrine functions of these peptides in the testis.

Widespread organ expression of the rat proenkephalin gene during early postnatal development.

The opioid peptides have been implicated as potential regulators of cell development in nervous and reproductive tissues. A survey of proenkephalin gene expression during rat development showed that the mRNA for this opioid precursor is present at substantial concentrations in several developing tissues (kidney, liver, skin, skeletal muscle, and lung) that have essentially undetectable levels in adults. In neonatal rats, skeletal muscle has greater concentrations of this transcript than brain. Polysomal analysis further demonstrated that proenkephalin mRNA is actively translated in skeletal muscle from newborn rats. These results raise the possibility that proenkephalin and its products perform a general regulatory role in cell proliferation or differentiation.

Translational status of proenkephalin mRNA in the rat reproductive system.

The mRNA for the opioid peptide precursor proenkephalin is widely distributed throughout the male and female reproductive systems of rodents. In the present studies, the concentrations of proenkephalin-derived peptides in selected reproductive tissues of the rat have been determined. When compared with previously characterized tissues such as brain, the peptide contents in reproductive tissues were unexpectedly low relative to the abundance of proenkephalin mRNA. This suggested that either translation of proenkephalin mRNA is relatively inefficient in reproductive tissues or that the turnover of proenkephalin products occurs at a higher rate, or both. To distinguish between these possible mechanisms, the polysomal distributions of proenkephalin mRNA in different rat reproductive tissues and in rat brain were determined. In adult rat testis, in which the predominant proenkephalin RNA is the 1700-nucleotide form present in spermatogenic cells, the transcript was found to be mainly associated with translationally inactive ribonucleoprotein fractions. In contrast, the 1450-nucleotide form of proenkephalin mRNA appeared to be translated to a similar extent in rat brain, epididymis, ovary, and somatic cells of the immature rat testis. It therefore appears that inefficient translation of proenkephalin mRNA in spermatogenic cells is a major determinant of the low ratio of proenkephalin peptides to RNA in the adult rat testis, while posttranslational mechanisms (most likely peptide turnover) are involved in the rat epididymis, ovary, and presumably other reproductive tissues. These findings also indicate that mRNA and/or translation product concentrations within a given tissue are not always accurate indicators of the level of peptide or protein production.

The influence of temperature on the functions of cultured Sertoli cells.

Three functions of Sertoli cells were examined with cells from rats aged 13 and 25 days at 34 C, 38 C, and 40 C, namely protein synthesis (incorporation of amino acids into material precipitated by trichloroacetic acid), transport of alpha-aminoisobutyric acid, and production of lactate. A fourth function, namely production of cAMP was examined at two temperatures (34 C and 38 C) in cells from rats aged 25 days. In all cases, activity was greater at 38 C than at 34 C and greater at 40 C than at 38 C. Lysate of Sertoli cells showed similar differences in protein synthesis at the three temperatures, again at both ages. Protein synthesis was higher at 38 C than at 34 C in Sertoli cell-enriched tubules from rats aged 25 days and in Sertoli cells from normal rats of the same age cultured on rat tail collagen. The difference in protein synthesis at the two temperatures was seen whether the cells were cultured, before the experiment, for 7 days at 38 C or at 34 C. Production of cAMP by Sertoli cells was greater at 38 C than at 34 C. It is concluded that Sertoli cells do not show in vitro any inhibitory effect of body temperature on any of the functions tested, but on the contrary, are more active at body than at scrotal temperature. The possible significance of the differences seen is discussed.

Occurrence of spectrin-like protein in Y-1 adrenal tumor cells.

With the aid of two monospecific antibodies raised in rabbits (antimouse erythrocyte spectrin and antimouse brain spectrin), the presence of a spectrin-like protein was demonstrated in mouse adrenal tumor (Y-1) cells. Y-1 cells contain two large polypeptides, with mol wt characteristic of nonerythroid spectrin alpha- and beta-subunits (240,000 and 235,000). When proteins from plasma membranes of Y-1 cells were electrophoretically transferred to a nitrocellulose membrane, two polypeptides with mol wt of 240,000 and 225,000 were specifically stained with antimouse erythrocyte (rbc) spectrin immunoglobulin G (IgG). The rbc spectrin antibody was used to immunoprecipitate Y-1 spectrin from a neutral detergent (physiological ionic strength) cell extract. The 240,000 (alpha)- and 235,000 (beta)-dalton polypeptides were immunoprecipitated in a 1:1 molar ratio, despite the fact that the antibody recognizes only the alpha-subunit. Two-dimensional chymotryptic peptide-mapping analysis indicated that the 240,000- and 235,000-dalton subunits of Y-1 adrenal tumor spectrin are structurally unique and share limited homology with mouse rbc spectrin alpha- and beta-subunits, but are nearly identical to the mouse brain spectrin 240,000-dalton alpha-subunit and 235,000-dalton beta-subunit. Indirect immunofluorescence with anti-rbc or antibrain spectrin IgG and goat antirabbit IgG conjugated with rhodamine demonstrated intense staining at the plasma membrane and throughout the cytoplasm of Y-1 cells, with little staining within the nucleus.

Plasma membranes from rat Sertoli cells: purification and properties.

A method is reported for preparing surface (plasma) membranes from rat Sertoli cells. The procedure is based upon homogenization in hypotonic buffer, extraction in a two-phase system, and sedimentation through two sucrose density gradients. The purified membranes consist of large sheets of membrane. The identity and purity of the membranes was demonstrated by electron microscopy, enzyme markers, and functional activities associated with the membranes (binding of follicle-stimulating hormone [FSH] and production of cyclic adenosine 5'-monophosphate [cAMP]. Electron microscopy showed membranes with small fragments of cytoplasm attached to the inside of the membrane sheets. Marker enzymes for plasma membrane (5'-nucleotidase and alkaline phosphatase) showed more than 16- and 6-fold enrichment, respectively, and other enzymes showed that contamination by nuclei, mitochondria, endoplasmic reticulum, or cytosol was negligible. Binding of FSH was found to be specific, with KD 1.2 nM and the equivalent of 7500 sites per cell. This binding was enriched 20-fold compared to whole homogenate. Production of cAMP by membranes was increased by addition of FSH and by forskolin to the purified membranes in vitro.

Proenkephalin products are stored in the sperm acrosome and may function in fertilization.

Previous studies have shown that spermatogenic cells are a major source of testicular RNA encoding the opioid peptide precursor proenkephalin, suggesting that proenkephalin-derived peptides may function as intratesticular paracrine factors produced by male germ cells. However, direct evidence for the production of proenkephalin by spermatogenic cells has been lacking. In this report, we have used polysome profile analysis, peptide quantitation, and immunocytochemistry to show that proenkephalin products are synthesized during spermatogenesis and are retained within spermatozoa of humans, hamsters, rats, and sheep. We further show that these peptides are stored in the sperm acrosome and are depleted from sperm following the acrosome reaction, an exocytotic event required for fertilization. Proenkephalin products thus may serve a dual function as sperm acrosomal factors released during the fertilization process as well as intratesticular regulators secreted by spermatogenic cells.

Identification of a spectrin-like protein in Sertoli cells.

Sertoli cells prepared from rats ages 15 and 25 days were shown to contain a spectrin-like protein. Indirect immunofluorescence with monospecific antimouse erythrocyte immunoglobulin G (IgG) and with monospecific antimouse brain spectrin IgG revealed specific staining in Sertoli cells. Both antibodies precipitated two spectrin-like peptides of 240,000 and 235,000 daltons from cells solubilized with octyl glucoside. Proteins from Sertoli cell membranes were separated by electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate and electrophoretically transferred to nitrocellulose membrane. Incubation of nitrocellulose membrane with either of the two antibodies, followed by horseradish peroxidase conjugated to second antibody, revealed only the larger, or alpha, spectrin subunit (Western blots). Both antibodies were used to provide immunoautoradiographic identification of the spectrin-like protein. In this procedure, spectrin and Sertoli cell membranes were shown to compete with [125I]-labeled spectrin from mouse erythrocytes for binding to antimouse erythrocyte spectrin IgG. Finally, two-dimensional proteolytic mapping of the 240,000- and 235,000-dalton peptides demonstrated limited spot homology with rat erythrocyte spectrin. However, subcellular fractions from Sertoli cells all contained a spectrin-like protein showing high homology from fraction to fraction. It is concluded that Sertoli cells contain a spectrin-like protein that is seen in cell fractions prepared by centrifugation, i.e., mitochondria, microsomes, nuclei, cytoplasm, and plasma membranes. Although homology with spectrin from erythrocytes or brain is not seen in peptide maps, the alpha subunit shares antigenic determinants with spectrin from erythrocytes. The beta subunit is believed to be precipitated by antispectrin as the result of binding to the alpha subunit, since the beta subunit shows no detectable antigenic homology with that of spectrin.