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Summary. Seasonal changes of the testicular interstitial tissue were studied by electron microscopy. During the breeding season in spring, clusters of Leydig cells are surrounded by wide lymphatic sinusoids. In sexually quiescent moles, these sinusoids collapse, and the abundant Leydig cells become closely packed and occupy most of the testis. During sexual activity, the Leydig cells contain abundant smooth endoplasmic reticulum (SER), mitochondria with tubular cristae, and lipid droplets. Some areas of the cytoplasm are occupied exclusively by tubular SER, arranged in parallel. During regression the SER appears tortuous, and large lipid droplets are found in the cytoplasm, although these gradually become smaller. During the long period of sexual quiescence, the size and abundance of Leydig cells and the appearance of SER, lipid droplets and mitochondria were similar to those observed during sexual activity.

Summary. During spermatogenesis in sexually mature ground squirrels Leydig and Sertoli cells were morphologically well differentiated. For Leydig cells the most prominent organelles were lipid droplets, mitochondria with tubulo-vesicular cristae and abundant agranular reticulum organized as a mass of anastomosing tubules. These morphological criteria suggest that the Leydig cells were steroidogenically active. Sertoli cells exhibited a topographical distribution of certain organelles with basal regions containing stacks of granular reticulum, and large areas of agranular reticulum. The cytoplasm surrounding maturing germ cells contained numerous microtubules, and an adluminal layer of spermatids at a certain stage of spermiogenesis became enveloped by Sertoli cytoplasm containing an enormous proliferation of agranular reticulum. The presence of these organelles in Sertoli cells suggests that during spermatogenesis they are active in the synthesis of proteins and steroids. In particular the mass of agranular reticulum surrounding late stage spermatids indicates that steroids may be required for spermatid maturation and/or spermiation. By contrast Leydig and Sertoli cells observed during testicular regression, when only spermatogonia remain in the seminiferous tubules, had undergone structural changes. Leydig cells were still numerous and large with abundant agranular reticulum that was now organized as a loose assemblage of single unbranched tubules. Sertoli cells were drastically reduced in both cytoplasmic volume and content of organelles.

Summary. Daily treatment of immature (30-day-old) male rats for 40 days with 50 ng of an agonist of LH-RH impaired normal development of Leydig cell function. This treatment partly or completely inhibited maturational increases in (1) the serum levels of testosterone, (2) seminal vesicle weight, (3) the in-vitro steroidogenic responsiveness of the testis, and (4) the in-vitro testicular binding of¹²⁵I-labelled hCG. In contrast, twice-weekly treatment of immature or adult rats with 50 ng LH-RH agonist had only minor effects on Leydig cell function, although hCG-binding was always significantly reduced. Testicular growth in immature rats was unaffected by daily injection of LH-RH agonist whereas twice-weekly treatment caused a small reduction in weight. None of the treatments had any major consistent effect on the pituitary or serum levels of gonadotrophins and prolactin, although daily treatment with the LH-RH agonist clearly reduced the responsiveness of the pituitary to the agonist, in terms of the amounts of LH and FSH released.

Summary. Spermatogenesis in Schreibers' long-fingered bat from ∼ 33° S in South Africa was seasonal, and occurred in the 3 months (February–April) preceding ovulation. The ultrastructure of the Leydig cells indicated a period of increased steroidogenesis at this time, and plasma testosterone concentrations were elevated from March to May (10·3 ng/ml). The reproductive accessory glands were secretorily active between March and May, and copulation occurred at the end of this period of activity. Changes in LH-β immunoreactivity suggest that the LH gonadotrophs were secretorily active 1 month before the onset of spermatogenesis and that peak activity coincided with peak plasma testosterone concentrations, spermiogenesis and spermiation. During winter (May–August) there was no reproductive activity and the bats remained active, only entering prolonged periods of torpor during particularly cold spells. A secondary elevation in plasma testosterone concentration, during reproductive inactivity (October; 9·3 ng/ml), was not accompanied by any change in Leydig cell ultrastructure, and the biological significance of this peak is unknown. Such synchronous activity of the pituitary, Leydig cells, seminiferous epithelium and accessory glands is associated with the typical reproductive cycle of long-fingered bats in which copulation and fertilization are restricted to a brief period at the end of summer, and in which neither sperm storage nor a prolonged period of copulation occur.

Keywords: plasma testosterone; Leydig cell ultrastructure; gonadotroph activity; bat

Summary. Gonadotrophin binding to rat Leydig cells after a single administration of ethylene dimethanesulphonate (EDS) (75mg/kg i.p.) was followed by using intratesticular microdoses of ¹²⁵I-labelled hCG, whilst corresponding morphological changes in the testicular interstitium were studied with light and electron microscopy. No discernible effect on ¹²⁵I-labelled hCG binding compared with controls was observed until 24 h after treatment. Between 24 and 32 h a sharp decline in binding occurred which was correlated with extensive Leydig cell destruction. By 48 h the ¹²⁵I-labelled hCG binding was negligible and no morphologically recognizable Leydig cells were found at this time. The specific binding remained low until 21 days after treatment and then a marked increase occurred to give nearly normal levels by 49 days. This was consistent with a generalized repopulation of the interstitium with Leydig cells, seemingly the result of differentiation of fibroblast-like precursor cells.

Summary. The importance of the pulsatility of luteinizing hormone (LH) secretion in maintaining key enzymes in the testosterone biosynthetic pathway in Leydig cells was studied using rats in which LH secretion was suppressed by passive immunization against gonadotrophin-releasing hormone (GnRH) and replaced by continuous or pulsatile i.v. infusions of exogenous LH, all delivering the same daily dose of the hormone (300 ng per 100 g NIDDK-ovine LH-24). Continuous infusions (12·5 ng per 100 g h⁻¹) were compared with infusions of 1 min pulses every 2 h (25 ng per 100 g) and every 4 h (50 ng per 100 g). After 5 days of treatment in vivo with sheep anti-GnRH serum (or normal sheep serum) and LH (or vehicle), Leydig cells were purified and assayed in vitro for maximum production of testosterone stimulated by human chorionic gonadotrophin (hCG) and supported by 25-hydroxycholesterol and for the activities of cholesterol side-chain cleavage, Δ⁵-3β-hydroxysteroid dehydrogenase-Δ^5–4-isomerase (3β-HSD-isomerase) and 17α-hydroxylase. Relative contents of cholesterol side-chain cleavage and 17α-hydroxylase were also quantified by western and immunoblotting analysis. Activity of 3β-HSD-isomerase was reduced by about 40% by anti-GnRH treatment and was increased by all LH regimens in anti-GnRH-treated animals, with no consistent pattern in the effects of the different LH regimens. Results for testosterone-producing capacity and the other two enzymes differed in several respects. Treatment with anti-GnRH serum markedly reduced basal, hCG-stimulated and 25-hydroxycholesterol-supported testosterone production (by 80–90%) and the activities of cholesterol side-chain cleavage (about 80%) and 17α-hydroxylase (about 65%). Infusion of exogenous LH in any of the regimens tested prevented these changes or increased the activities to values greater than those in normal serum-treated controls. Differences in immunodetectable contents of the two enzymes generally paralleled those in enzyme activities. There was a consistent trend in the effects of LH replacement regimens on these parameters of steroidogenic activity: continuous infusions were more effective than pulses at 2 h intervals and these in turn were more effective than pulses at 4 h intervals, suggesting that the frequency of LH exposure is more important than the amplitude of individual exposures in maintaining Leydig cell steroidogenic function. Consistent differences among groups in hCG-stimulated relative to 25-hydroxycholesterol-supported testosterone production suggest that some constituents of Leydig cells prior to cholesterol side-chain cleavage enzyme are more sensitive to LH withdrawal and deviations from 'optimal' LH exposure than are the side-chain cleavage and subsequent enzymes in the testosterone biosynthetic pathway.

Keywords: Leydig cells; testosterone; steroidogenesis; P-450; luteinizing hormone; rat

Several workers have studied various parameters as an index of Leydig cell differentiation and attempted to correlate them with the growth of the accessory sex organs. In the prepuberal rat, little correlation seems to have been achieved (see Niemi & Ikonen, 1963; Clegg, 1966). Others have examined testosterone production in vitro by the immature testis and attempted to correlate this with the increase in weight of the seminal vesicles and prostate gland. In this connection, a good deal of attention has been paid to the production of testosterone in vitro and its apparent regulation by 5α-reductase activity. Nayfeh, Barefoot & Baggett (1966) reported an increase in testosterone production per unit weight of tissue at about the time of sexual maturity and suggested that this might be due mainly to reduced metabolism to 5α-androstane-3α,17β-diol (androstanediol). Inano, Hori & Tamaoki (1967) found a remarkable increase in the activity of various enzymes

Summary. Rat testes were exposed to heat (43°C) for 15 or 30 min to induce moderate or severe disruption of spermatogenesis, respectively. Over 3–42 days after treatment, testicular morphology and weight, the serum concentrations of FSH and the concentrations in interstitial fluid of testosterone, androgen-binding protein (ABP) and a factor(s) capable of stimulating Leydig cell testosterone secretion were monitored. Moderate seminiferous tubule damage induced by 15 min heat exposure caused a small decrease (20%) in testicular weight, but did not affect the other measures, other than transiently. In contrast, after exposure of testes to heat for 30 min there was a major and progressive decline in testicular weight throughout the experimental period, reaching 39% of control values by 42 days. In these animals, the serum concentrations of FSH were significantly increased (P < 0·01) throughout the period of study as also where the serum and interstitial fluid concentrations ABP (P < 0·05–0·01) and levels of interstitial fluid factor (P < 0·01).

It is concluded that the activity of the interstitial fluid factor(s) can be increased by inducing severe but selective disruption of spermatogenesis, whereas moderate disruption has no effect. Moreover, as ABP secretion into interstitial fluid was increased after severe but not moderate disruption, this suggests that in such animals proportionately more ABP may be secreted via the base of the Sertoli cell. The parallel changes in activity of the interstitial fluid factor(s) and concentrations of ABP in interstitial fluid also provides further circumstantial evidence that these products may have a common (Sertoli cell) origin.