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We have examined the distribution of lipid droplets and mitochondria in relation to the cytoskeletons of Leydig cells in primary culture by using light and electron microscopy on living, intact and detergent-extracted cells. After mild extraction with Triton X-100 lipid droplets and mitochondria retained their original distribution within the cell. Double immunofluorescent microscopy showed that both structures co-localise with intermediate filaments. Transmission electron microscopy of intact (unextracted) and mildly extracted Leydig cells showed that intermediate filaments are closely associated with mitochondria and lipid droplets. By examination of stereo pairs, intermediate filaments were shown to establish direct contact with mitochondria and lipid droplets. The association of droplets and mitochondria with intermediate filaments suggests possible mechanisms by which the transport of cholesterol takes place from droplets to mitochondria where this substrate enters the steroidogenic pathway.

Summary. Rat testicular interstitial fluid and hydroxycholesterol both stimulated testosterone production by isolated Leydig cells in vitro in a dose-dependent manner, but the dose—response lines were not parallel. The addition of cycloheximide blocked the stimulation by interstitial fluid but not that of hydroxycholesterol. Use of the compounds SU 10603 and cyanoketone (which inhibit 3β-hydroxysteroid dehydrogenase and 17α-hydroxylase respectively) or aminoglutethimide (which acts on the cholesterol side-chain cleavage enzyme) showed that the stimulatory factor(s) in interstitial fluid stimulated steroidogenesis at the cholesterol side-chain cleavage enzyme, before the conversion of pregnenolone. This enzyme is rate-limiting in the synthesis of testosterone by Leydig cells and a site of action of LH; therefore, these results support the view that an interstitial fluid factor may be involved in the paracrine regulation of testicular steroidogenesis.

Summary. The responsiveness of decapsulated testes and isolated Leydig cell preparations from rats (30–80 days of age) to a constant dose of 3 ng hCG/2 ml was assessed by comparison of the production of testosterone and 'total 17β-hydroxy androgen' (17β-HA). When testosterone secretion was used as the index of response, there was a marked increase in the production with age by decapsulated testes and also by equal numbers of Leydig cells. When 17β-HA was taken as the response parameter this increase was only marginal for the decapsulated testes and there was an age-dependent decrease when expressed per 10⁶ cells. These differences probably reflect changes in the metabolism of testosterone to 5α-reduced products with increasing age because 80% of androgen secreted at 30 days is 3α-androstanediol and 86% is secreted as testosterone at 80 days. We conclude that for studies on hCG responsiveness and the steroidogenic capacity of immature rat Leydig cells (a) testosterone is an inappropriate response parameter and (b) this response undergoes a decrease rather than an increase during prepubertal development.

Summary. Tumour Leydig cells and normal mature Leydig cells lost their steroidogenic response (pregnenolone and testosterone secretion) to LH after 24 h of culture. Immature cells showed a 2-fold increase in the basal pregnenolone secretion and no change in the LH-dependent pregnenolone secretion after 24 h of culture, whereas the LH-dependent steroidogenesis decreased after 48 h. None of the 3 cell preparations showed morphological signs of degeneration during a culture period of more than 7 days. Histochemical 3β-hydroxysteroid dehydrogenase activity in isolated immature Leydig cells disappeared during the first 2 days of the culture period and re-appeared after 5–7 days. Testosterone production by mature Leydig cells decreased during the first hours after exposure to LH, whereas pregnenolone secretion remained constant. From these results it was concluded that Leydig cells attached to plastic can be used for investigation of acute actions of LH on steroidogenesis. A perifusion technique for cells attached to plastic was developed and was applied to the kinetics of LH action on steroidogenesis in tumour and immature Leydig cells. The first stimulation of pregnenolone secretion occurred within 5 min, but a full stimulation was only obtained after 20–30 min. This was followed by a gradual decrease in the stimulated steroid secretion to ∼50% after 60 min.

Summary. Daily treatment of adult cynomolgus monkeys with 450 i.u. hCG for 16 days resulted in a significant 163% increase in the number of Leydig cells, and a 9-fold rise in plasma testosterone concentrations. The number of proliferating Leydig cells was very low, even after 16 days of treatment with hCG. Daily FSH administration (2 injections of 15 i.u. per day) did not have any effect on the number of Leydig cells or plasma testosterone values. It can be concluded, therefore, that in adult cynomolgus monkeys daily hCG treatment results in an increase in the number of Leydig cells, which is mainly caused by the differentiation of precursor cells. Since plasma testosterone concentrations were increased to an even higher extent, the steroid production per Leydig cell was also stimulated.

Keywords: Leydig cells; hCG; FSH; adult cynomolgus monkey

Summary. Male Cape horseshoe bats were studied in the Cape Province of South Africa (33°17′S, 26°25′E) between January 1983 and June 1985. The reproductive cycle is characterized by reactivation of the seminiferous tubules in early summer (October) after a 4-month (June to September) period of winter inactivity. Spermiogenesis occurred between January and April, and spermatozoa were released to the epididymides in April and May. Spermiogenesis was associated with Leydig cell activity and increasing plasma testosterone concentrations. At this time components of the reproductive accessory glands became secretorily active or showed increasing secretory activity. During winter Leydig cells were secretorily inactive and plasma testosterone concentrations dropped, but components of the accessory complex remained active. There was a second period of Leydig cell secretory activity and increasing and peak plasma testosterone values in late winter/early summer which may be associated with copulation or the initiation of a new cycle of spermatogenesis.

Summary. A mouse Leydig cell bioassay was established and validated to determine whether the bovine conceptus (age: 16–20 days after oestrus) synthesized and secreted a substance having luteinizing hormone (LH) activity. The response measured was testosterone production after incubating Leydig cells with various doses of LH. The assay was specific for LH as compared to other protein hormones, precise (an interassay CV of 21%) and sensitive (minimal detectable level 15-5 pg NIH-LHB4/ml). The application of this assay to bovine conceptus homogenates or media in which similar conceptuses had been cultured for 0-5 to 20 h resulted in no LH activity being detected. Furthermore, concentrating the conceptus homogenate or culture media by freeze-drying and reconstitution in a minimum amount of buffer resulted in a similar finding. Because this bioassay is more specific and sensitive to the presence of LH-like activity than are other assays, and yet no activity was detected when conceptuses were manipulated in several ways, it is concluded that the bovine conceptus probably does not produce an LH-like substance at this stage of development.

Summary. Crossbred boars were (a) immunized against GnRH conjugated to human serum globulin (200 μg GnRH-hSG) in Freund's adjuvant at 12 weeks of age and boosted at weeks 18 and 20 (N = 10), (b) served as controls and received hSG only in adjuvant (N = 10), or castrated at weaning (N = 10). At 24 weeks of age (immediately before slaughter), the boars were challenged with saline or pig LH (1 μg/10 kg body weight). After slaughter, fresh testicular fragments were incubated with pig LH (0·05 and 0·2 ng/2 ml medium) to assess the effects of immunization on Leydig cell function. Pituitary contents of LH and FSH, and testicular LH receptor content were also measured. The results indicated that plasma LH and testosterone concentrations, pituitary LH content, testicular LH receptor content, testis and sex accessory organ weights were significantly reduced in GnRH-immunized boars compared to hSG-adjuvant controls. However, plasma and pituitary FSH content were not affected by high antibody titres generated against GnRH. The testicular testosterone response to exogenous LH in vivo and in vitro was significantly reduced (P < 0·05) in GnRH-immunized boars. These results indicate that active immunization against GnRH impairs pituitary and Leydig cell functions in boars.

Keywords: GnRH immunization; testosterone; LH; FSH; LH receptor; boars

Departamento de Investigación Científica, del Centro Médico Nacional del Instituto Mexicano del Seguro Social, México City, Mexico

(Received 18th March 1975)

Vasectomy is widely used as a method for sterilization, and recent surgical techniques have shown that the procedure can be reversed (Mehta & Ramani, 1970; Pardanani et al., 1973, 1974). It is therefore mandatory to evaluate the function of the gonads following vasectomy. Flickinger (1972) described the effects on the testis of the rat up to 9 months after vasectomy and found no morphological changes in the seminiferous tubules; only brief mention was made of the morphology of the Leydig cells. The present work was therefore designed to examine possible modifications in the number and/or morphology of these cells in the rat after vasectomy.

Forty-five adult Wistar male rats weighing 200 to 250 g were used. All animals were subjected to 12 hr light/12 hr dark and given Purina