Effect of body temperature on threshold for retinal light damage.

Body temperature is known to influence the threshold for retinal light damage, but the magnitude of the effect has varied substantially between previous studies. The purpose of the present study was to establish a quantitative relation between body temperature in the range of 30-42 degrees C and dose of radiation for a just visible change in fundo. Anesthetized pigmented rats were exposed to 380 nm radiation for 10 min. Four intensities were simultaneously presented. Funduscopic changes were noted 2-3 d after exposure. At 30 degrees C, threshold dose was 6 J/cm2; at 42 degrees C, it was about 1 J/cm2. A fair fit to the data could be obtained with a linear regression between log threshold dose and temperature. The slope was -0.067. In an additional experiment, threshold dose at 500 nm and 41 degrees C body temperature was established at 400 J/cm2. These results agree with data in monkey and rabbit, but they vary from earlier data in rats that show a slope of -0.8. Exposure time, damage criterion, and the chromophores involved in retinal light damage are possible factors in the discrepancy.

Regulation of inhibin alpha- and beta B-subunit mRNA levels in rat Sertoli cells.

The hormonal regulation of the expression of the inhibin alpha-subunit and beta B-subunit genes was studied in cultured rat Sertoli cells. The alpha-subunit mRNA level increased during incubation of the cells in the presence of follicle-stimulating hormone (FSH), reaching maximal levels within 1.5 h. This stimulation was mimicked by addition of dibutyryl-cyclic AMP, indicating that FSH action on the alpha-subunit gene is exerted via cyclic AMP. Inhibition of translation by cycloheximide (CX) caused upregulation of the alpha-subunit mRNA, and did not block the effect of FSH on the level of this mRNA. In FSH-stimulated cells, the half-life of the alpha-subunit mRNA was 6 h, and this half-life was prolonged by inhibition of transcription using actinomycin D (AD). It is concluded that the effect of FSH on alpha-subunit mRNA expression represents a direct effect on the alpha-subunit gene, and that alpha-subunit mRNA levels are influenced by a short-lived mRNA destabilizing protein. The levels of two beta B-subunit mRNAs (4.2 kb and 3.5 kb) were not affected by FSH or dbcAMP. However, these mRNAs were also upregulated by CX treatment. Experiments using AD showed that the 4.2 kb mRNA is less stable than the 3.5 kb mRNA. The differential regulation of the inhibin alpha- and beta B-subunit mRNAs is discussed.

Quantitative evaluation of the maintenance and development of spermatocytes and round spermatids in cultured tubule fragments from immature rat testis.

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 degrees C. However, the cellular enzyme activity returned to control values during subsequent incubation of the cells at 32 degrees 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.

Effects of FSH and testosterone on highly purified rat Sertoli cells: inhibin alpha-subunit mRNA expression and inhibin secretion are enhanced by FSH but not by testosterone.

The effects of FSH and testosterone on inhibin mRNA expression and inhibin production by highly purified Sertoli cell preparations were examined. Sertoli cells were isolated from testes of 22-day-old rats by sequential trypsin, collagenase and hyaluronidase treatments, with subsequent osmotic shock treatment on day 3 of culture. Contamination by peritubular and germ cells was less than 0.5 and 1-3% respectively. Intracellular and secreted inhibin levels were measured by radioimmunoassay, using Sertoli cells which were incubated for 24 h in the absence or presence of FSH and testosterone from days 4 to 5 of culture. FSH stimulated the cellular inhibin content and the secreted inhibin level by four- and sevenfold respectively, with a half-maximal effective dose of 5-50 ng/ml. Under the present incubation conditions, testosterone (1 mumol/l) had no effect on immunoreactive inhibin levels in either the presence or absence of FSH. Similarly, the expression of inhibin alpha-subunit mRNA was increased following FSH stimulation, whereas testosterone had no effect. The expression of inhibin beta B-subunit mRNAs was not influenced by FSH or testosterone. It is concluded that highly purified Sertoli cell preparations, with a very low number of peritubular or germ cells, are fully responsive to FSH with respect to inhibin mRNA expression and inhibin production.

Effects of FSH and IGF-I on immature rat Sertoli cells: inhibin alpha- and beta-subunit mRNA levels and inhibin secretion.

Effects of follicle-stimulating hormone (FSH) and insulin-like growth factor-I (IGF-I) on inhibin production by cultured Sertoli cells from 21- to 23-day-old rats were studied. The expression of inhibin alpha- and beta-subunit mRNAs, and inhibin immunoreactivity and in vitro bioactivity were estimated. Using a cDNA probe corresponding to the alpha-subunit of bovine inhibin, specific hybridization with a 1.5-1.7 kilobase (kb)mRNA species was observed. Addition of FSH to the cultured Sertoli cells for 24 h markedly increased the level of this mRNA in a dose-dependent way. IGF-I had no effect on the intensity of the hybridization. Using a cDNA probe corresponding to the beta B-subunit of human inhibin, 3.5 and 4.2 kb mRNA species were detected. FSH and IGF-I had no effect on the hybridization signal. No hybridization was observed with a cDNA probe corresponding to the beta A bovine inhibin subunit. Inhibin activity was detected in cells and medium by immunoassay, and in the medium by in vitro bioassay. FSH stimulated both immunoreactivity and in vitro bioactivity, whereas IGF-I had no effect at all. The present effect of FSH on inhibin alpha-subunit mRNA expression in cultured Sertoli cells indicates that regulation of inhibin production by FSH includes an effect at the transcriptional level. However, this does not exclude additional translational and posttranslational effects.

Transport of transferrin-bound iron into rat Sertoli cells and spermatids.

Transferrin (Tf), a major secretory protein of Sertoli cells, may transport iron to spermatogenic cells. This was assessed by measuring the uptake of Fe from 59Fe-125I-labelled rat Tf by Sertoli cells and round spermatids in vitro. Uptake of Fe from labelled Tf by Sertoli cells after a 72-h pre-incubation period was linear for 20 h (approximately 18 pmol/10(6) cells/20 h), whereas the uptake of Fe from labelled Tf by round spermatids after a 16-h pre-incubation period reached a plateau by 2 h (approximately 5 pmol/10(6) cells/2 h). The corresponding net uptake of Tf by both cell types was less than 0.1 pmol. High speed supernatants prepared from Sertoli cells and spermatids labelled with 59Fe-125I-Tf were fractionated by gel permeation chromatography. Separate peaks of protein-bound 59Fe and 125I-Tf were observed. Protein bound 59Fe could be precipitated with an antiserum to rat ferritin. It is concluded that iron from exogenous Tf is transported into Sertoli cells and round spermatids in vitro, and is complexed to intracellular ferritin. However, the present results do not exclude the possibility that Sertoli cell Tf may serve purposes other than iron transport.