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ABSTRACT

Liposome-entrapped dichloromethylene diphosphonate was injected locally into the right testes of adult rats. This treatment, which has been found to deplete resident macrophages in some other organs, reduced the number of testicular macrophages by at least 90%. Testicular weight and seminiferous tubule morphology were unaffected by liposome treatment. Leydig cell testosterone secretion gradually declined in the macrophage-depleted testes, and there was a compensatory increase in Leydig cell size and testosterone secretion in the contralateral saline-injected testes. These observations suggest that macrophages influence Leydig cell function locally. It is concluded that liposome-mediated depletion of testicular macrophages may serve as an experimental model with which to study the physiological role of these cells.

Journal of Endocrinology (1993) 136, 407–413

ABSTRACT

Macrophages are constituents of all normal connective tissue including the murine uterus. Macrophages have been identified previously in endometrium and myometrium of pregnant and non-pregnant murine uterus using antibodies against macrophages. In the current study immunohistochemical analysis of murine uterus demonstrated that there were not significant quantitative differences in uterine macrophages between the dioestrous, pro-oestrous and oestrous stages. However, distributional changes occurred during the oestrous cycle. Macrophages were evenly distributed throughout uterine tissue during dioestrus, while, during pro-oestrus and oestrus, their concentration was highest in the subepithelial stroma. Because the oestrous cycle is hormonally regulated, we asked whether or not oestrogen and/or progesterone might influence macrophage distribution. Ovariectomy, which eliminates cyclical production of oestrogen and progesterone, resulted in a significant decrease in both the relative and the absolute number of uterine macrophages within 6 days. Injections of progesterone or oestrogen to ovariectomized mice resulted in restoration of uterine macrophage numbers. Injection of oestrogen plus progesterone in a regimen known to prepare the uterus for receptivity for blastocyst implantation increased the number of macrophages to levels which were consistently higher than those seen during oestrus. Moreover, following hormone administration macrophages were more concentrated in the subepithelial stroma, a distributional pattern which was most evident following injection of both hormones. The results suggest that both oestrogen and progesterone promote quantitative and distributional changes in the uterine macrophage population.

Journal of Endocrinology (1990) 126, 417–424

ABSTRACT

Liposome-entrapped dichloromethylene diphosphonate (Cl₂MDP) was injected locally into the right testes of adult rats in order to deplete testicular macrophages. The number of testicular macrophages in the treated testes was reduced by at least 90% at 7 and 14 days after treatment. Unilaterally testicular macrophage-depleted animals were treated with 100 IU human chorionic gonadotrophin (hCG) subcutaneously and the inflammatory response was compared in the macrophage-depleted and intact contralateral testis. Four hours after hCG treatment, intratesticular testosterone was similarly increased in intact and macrophage-depleted testes. In macrophage-depleted testes there was a large increase in the number of leukocytes in testicular blood vessels and numerous leukocytes had migrated into the interstitial tissue. This response was greater than in the intact contralateral testis. It was concluded that testicular macrophages are probably not the origin of the inflammatory mediator secreted in the rat testis after hCG treatment. On the contrary, it appears that testicular macrophages may secrete factors inhibiting hCG-induced testicular inflammation.

Journal of Endocrinology (1993) 136, 415–420

Abstract

The Leydig cells of young hypophysectomized rats are highly sensitive to the stimulatory effects of exogenous pituitary hormones. The aim of this study was to analyse the role of testicular macrophages in the response of Leydig cells to different hormones. Male rats were hypophysectomized at 28 days of age and 10 days later they were injected intratesticularly with dichloromethylene diphosphonate-containing liposomes (right testis) to deplete testicular macrophages, and with 0·9% NaCl (left testis). One week later, the animals were treated daily with 1 IU rat GH (rGH)/rat, 5 IU recombinant human FSH (recFSH)/rat, 10 IU human chorionic gonadotrophin (hCG)/rat, or vehicle for 7 days. The animals were killed on the day after the last injection. The animals treated with rGH showed increased body weight and increased number and size of testicular macrophages in the left testes, but no significant effects on Leydig cells were found. Treatment with recFSH induced a significant increase in testicular weight and tubular diameter in both testes. In the left testes, the number and size of macrophages were increased; the number of Leydig cells was not changed, although they showed a significantly increased cross-sectional area. This effect was abolished in the right (macrophage-depleted) testes. However, the effect of recFSH on the growth of the seminiferous tubules was not modified by the absence of macrophages. Rats treated with hCG showed increased testicular weight and serum testosterone levels, as well as an increased weight of the ventral prostate. In the left testes, the number and size of both macrophages and Leydig cells were increased. Otherwise, the number of Leydig cells was unchanged in the absence of macrophages, whereas the increase in the size of Leydig cells was partially abolished. These data indicate that testicular macrophages are needed for the response of Leydig cells to gonadotrophin treatment.

Journal of Endocrinology (1995) 147, 463–471

ABSTRACT

A method involving centrifugal elutriation followed by density gradient centrifugation and incubation with a macrophage monoclonal antibody has been investigated to separate and characterize Leydig cells and macrophages from adult rat testes. After dispersion of the testes with collagenase, the isolated interstitial cells were found to contain 18% Leydig cells and 12% macrophages. These cells were then separated by centrifugal elutriation into eight fractions (F1–F8) (9 to 74 ml/min at 386 g ). Each of these fractions was then further purified by density gradient centrifugation on 0–90% Percoll gradients. After centrifugal elutriation, the macrophages were mainly eluted in the first three fractions (F1–F3), whereas the Leydig cell percentage increased in each fraction with increasing flow rate. After further purification of each fraction on Percoll gradients, high percentages of macrophages (11–20%) were found in fractions F1–F3 (average density 1·045 g/ml), containing 11–37% Leydig cells. Less than 3% of the cells in fraction F4–F8 (average density 1 ·075 g/ml) were macrophages and more than 95% were Leydig cells. Heterogeneity of Leydig cells with respect to sedimentation velocities and function was found. Leydig cells from elutriated-and Percoll-purified fractions F4–F8 were heterogeneous with respect to testosterone and cyclic AMP (cAMP) production but showed a similar binding capacity for ¹²⁵I-labelled human chorionic gonadotrophin. Leydig cells with the highest sedimentation velocity (35·7 mm/h-g) from fractions F7 and F8 were approximately twofold more responsive to LH (3·3 nmol/l) with respect to testosterone and cAMP production compared with Leydig cells with the lowest sedimentation velocity (20·7 mm/h-g).

The elutriated and Percoll-purified cells (corresponding to fractions F4–F8) were further purified by incubation with magnetic beads coated with a macrophage monoclonal antibody; this yielded very pure Leydig cells containing <0·3% macrophages. The incubation temperature (room temperature or 4 °C) during the purification with magnetic beads did not affect the degree of purity or the responsiveness of the Leydig cells to LH. The removal of the remaining macrophages with magnetic beads did not have any significant effect on the Leydig cell responsiveness to LH.

It was concluded that Leydig cells purified by elutriation and density gradient centrifugation are heterogeneous with respect to their sedimentation velocities and responses to LH; the higher the sedimentation velocity, the higher is their capacity to respond to LH. Leydig cells free from macrophages can be prepared by further purification using magnetic beads coated with a macrophage monoclonal antibody.

Journal of Endocrinology (1991) 130, 357–365

Abstract

Testicular macrophages are a relevant cell type for the regulation of Leydig cell steroidogenesis. The availability of liposome technology allows in vivo manipulation of macrophages in order to analyze their role in the regulation of the hypothalamic-pituitary-testicular axis. In this study, adult (70 days of age) and prepubertal (22 days of age) rats were injected intratesticularly with liposomes containing either dichloromethylene diphosphonate (C12MDP) to deplete testicular macrophages or muramyl tripeptide (MTP-PE) to activate them. Control rats were injected with the corresponding volumes of 0·9% NaCl. Animals were killed 10 days after treatment. Adult rats injected bilaterally or unilaterally with C12MDP liposomes showed increased serum LH and testosterone concentrations, as well as increased testosterone concentrations in the testicular interstitial fluid. In unilaterally injected rats, testosterone concentrations in the interstitial fluid were higher in the macrophage-containing testes than in the contralateral, macrophage-depleted testes. Adult rats treated bilaterally with MTP-PE liposomes showed increased numbers of testicular macrophages, whereas the number of Leydig cells was unchanged. Serum LH concentrations were decreased, but no changes were found in testosterone concentrations. Prepubertal rats treated bilaterally with C12MDP liposomes showed decreased numbers of Leydig cells. However, serum LH and testosterone concentrations were increased. Otherwise, prepubertal rats treated bilaterally with MTP-PE liposomes showed increased numbers of macrophages and Leydig cells, as well as increased serum testosterone concentrations. These data suggest that testicular macrophage-derived factors act at two different levels in the pituitary-testicular axis: first, at a central level by inhibiting LH secretion, and secondly, at a local level by stimulating Leydig cell steroidogenesis.

Journal of Endocrinology (1996) 150, 57–65

We undertook the present studies to determine if clodronate-containing liposomes have direct effects on Leydig cells. Macrophages and Leydig cells were isolated and maintained separately in culture. Following treatment with clodronate-containing liposomes, macrophages were killed in a dose-response fashion over a range of 5-200 microliters liposomes. By comparison, a 500 microliters dose was required to kill Leydig cells, but this was not dependent upon clodronate since liposomes containing buffer elicited an identical response. The concentration of testosterone in medium from Leydig cells treated with clodronate-containing liposomes was significantly reduced compared with untreated cells. However, we subsequently found that liposomes can adsorb testosterone. Therefore, testosterone production was determined at various times following removal of liposomes from Leydig cells, thereby circumventing this complication. It was found that testosterone production was not altered by liposomes under these conditions. Finally, free clodronate had no effect on testosterone production, even at doses representing the amount present within the 500 microliters dose of liposomes. In summary, clodronate-containing liposomes killed testicular macrophages at a far smaller dose than required to kill Leydig cells. Most importantly, neither liposomes no free clodronate had a direct effect on testosterone production. Thus, clodronate-containing liposomes represent a valuable tool to study Leydig cell-macrophage interactions.