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Summary. Isolated Leydig cells were prepared from adult rat testes by (1) mechanical dissection, (2) collagenase dispersion or (3) mechanical dissection followed by collagenase dispersion, and their functional characteristics were assessed. Compared with Methods 2 and 3, mechanical isolation alone resulted in the purest preparation of Leydig cells but the lowest yield. Leydig cells isolated by any of the 3 methods had similar numbers of LH- and LH-RH receptors, but cells isolated by Methods 1 and 3 showed poor testosterone responsiveness compared to cells isolated by Method 2. This reduced response was evident following stimulation with hCG, dibutyryl cyclic AMP or an LH-RH agonist, and could not be accounted for simply on the grounds of diminished cell viability. It is concluded that Leydig cells in the rat testis are particularly sensitive to mechanical intrusion, and this is an important factor to bear in mind when preparing Leydig cells or when comparing results between laboratories.

Summary. The characteristics of the fetal and adult populations of Leydig cells from postnatal rat testes were compared by Percoll gradient centrifugation. A single peak of hCG binding, due to the presence of fetal Leydig cells, was obtained after purification of intertubular cells from 8-day-old animals. Two peaks of specific hCG binding were obtained after purification of intertubular cells from 15-day-old rats: it was confirmed by autoradiographic techniques that the hCG was bound by adult-like Leydig cells in one peak and fetal Leydig cells in the other. Similarly, intertubular cell preparations from 21- and 25-day-old rats resolved into two peaks of hCG binding; adult-like Leydig cells were observed in the first peak, but fetal Leydig cells were rarely observed in the second of these peaks. These results demonstrate the separation of two Leydig cell populations from intertubular cells obtained from animals aged up to 15 days. Thereafter the pattern of the hCG binding profile is similar but is not due to the presence of the same cell types. Therefore these results emphasize the necessity for morphological identification of cell types to permit the correct interpretation of the corresponding biochemical data.

Summary. Treatment of immature, hypophysectomized male rats with 50 μg ovine FSH (NIH-FSH-S12) twice a day for 5 days stimulated the maximum quantity of 17β-hydroxyandrogen produced by isolated Leydig cells in response to hCG. Pretreatment of the FSH preparation with an LH antiserum in one study markedly reduced and in another study completely abolished this stimulatory effect of FSH, but only slightly impaired the capacity of the hormone to stimulate the Sertoli cell in vivo (epididymal androgen-binding protein). Administration of another highly potent FSH preparation (LER-1881) had no discernible effects on the dose–response characteristics of the Leydig cells but was superior to the NIH-FSH-S12 in its capacity for stimulating the Sertoli cell. When all hormone preparations were tested for their ability to stimulate steroid secretion from normal Leydig cells in vitro, a close correlation was obtained between their Leydig cell-stimulating activity (a measure of LH contamination) and their capacity to alter Leydig cell responsiveness after in-vivo treatment. FSH treatment had no effects on specific LH binding per 10⁶ Leydig cells. It is concluded that the stimulatory influence of FSH on rat Leydig cells may to some extent be a result of the LH contaminating the hormone preparation.

Summary. Recent findings on Leydig cell function and its regulation are discussed. Regulatory mechanisms at different organizational levels, i.e. at the level of the pituitary, the testis and the intracellular elements, are briefly reviewed as well as the transmission and the modulation of the hormonal signal at the Leydig cell membrane. Special emphasis is given to local paracrine and autocrine regulatory interactions operating at the level of the testis.

Summary.

The use of the Leydig cell/Sertoli cell (LC/SC) ratio as a representative index for the number of Leydig cells present in the testis provides an objective method for their quantification. It reflects changes in the number of Leydig cells that might occur as their activity is depressed or stimulated by various agents. The Sertoli cell number in the adult male does not appear to be altered by administration of drugs, hormones or irradiation in dosages typically used in clinical investigations of germinal and Leydig cells. Thus, the Sertoli cell may be utilized as a constant.

Thirty-eight testicular biopsies were taken from nineteen normal men. Quantification of the biopsies, using the LC/SC ratio method revealed that: (1) no statistically significant variation could be found in biopsies taken from the same testis of the same subject at different times; (2) biopsy ratios between subjects ranged from 0·19 to 0·72; and (3) a statistical difference in ratios between right and left testis of the same subject occurred in four out of eleven subjects.

It is concluded that the LC/SC ratio is an accurate and reproducible quantitative measure for comparing a given subject against his own control when a further biopsy is taken from the same testis.

Summary.

The activity pattern and functional significance of leucine aminopeptidase (LAP) have been studied in the testes of oestrogen-treated, cryptorchid and HCG-treated guinea-pigs. The enzyme was present only in the Leydig cells and showed marked fluctuations in its activity pattern, which correlated closely with alterations in the functional activities of the Leydig cells. Hyperfunctioning Leydig cells exhibited enhanced LAP activity, whereas hypofunctioning of these intertubular cells resulted in a diminution in the enzyme activity. The LAP activity in the intertubular Leydig cells may be regarded as a cytochemical marker enzyme in the assessment of Leydig cell function in the guinea-pig testes under varying experimental conditions.

Summary. The response of rat Leydig cells to hCG in vitro was examined using decapsulated testes and enriched Leydig cell suspensions of a known purity. The secretion of testosterone and 5α-androstan-3α,17β-diol was used as the response and medium extracts were assayed before and after celite chromatography. At least 4 important phases in the development of steroidogenic function of the Leydig cells were detected. (1) A period after birth up to 10 days of age when the Leydig cell preferentially secretes testosterone. The cells may constitute the remnants of a fetal Leydig cell population. (2) A period which begins between 10 and 15 days of age and continues until 35–40 days of age, when the major products of the Leydig cell are 5α-reduced metabolites of testosterone, especially 5α-androstan-3α,17β-diol. (3) A phase at 20–30 days of age when responsiveness of the Leydig cell to hCG in vitro undergoes a distinct increase (the quantity of androgen produced with maximum hCG stimulation) and attains adult levels, although the sensitivity of the Leydig cells to hCG (the dose of hCG eliciting a half-maximal response, ED₅₀) is gradually decreased after Day 20. (4) A period from 35–40 days of age into adulthood when testosterone increasingly becomes the major secretory product of the Leydig cell.

Hypophysectomy for 8 days decreased the quantity of androgens produced per 10⁶ Leydig cells in response to hCG to an extent dependent on age. At 28 days of age steroidogenic function was impaired to a much greater extent (reduced by some 80–90%) than at 38 or 58 days (reduced by 40–50%). At all ages the sensitivity of the Leydig cells to hCG was increased after hypophysectomy.

Summary. Testes from 47 adult (4–20 years) stallions obtained in November–January (non-breeding season) and 41 adult stallions obtained in May–July (breeding season) were perfused with glutaraldehyde, placed in osmium and embedded in Epon 812. Percentage Leydig cell cytoplasm or nuclei in the testis was determined by point counting of 0·5 μm sections under bright-field microscopy. Testes from 6 randomly selected horses per season were processed for electron microscopy. The volume (ml) of SER/testis was calculated from the % SER in the cytoplasm % Leydig cell cytoplasm, and parenchymal volume. Number of Leydig cells was calculated from the % nuclei, parenchymal volume, histological correction factor, and volume of single nucleus. Intratesticular testosterone content was determined from the contralateral testis by radioimmunoassay. The volume of SER/g and testosterone/g tended to be higher in the breeding than non-breeding season. Leydig cell number/g, volume of SER/testis, testosterone/testis, and Leydig cell number/testis were significantly greater in the breeding than in the non-breeding season. Volume of SER/testis and testosterone/testis were related significantly to the cell number/testis, and SER/testis was related (P < 0·05) to testosterone/testis. These results emphasize the importance of seasonal changes in the number of Leydig cells on the amount of SER available to produce testosterone and on testosterone content/testis in the stallion.