Ricardo S. Calandra

Number, distribution pattern, and identification of macrophages in the testes of infertile men

OBJECTIVE To investigate the number, location, and secretory products of macrophages in human testes showing normal and abnormal spermatogenesis. DESIGN Evaluation of testicular biopsies with the use of immunohistochemistry, laser capture microdissection, and reverse transcriptase polymerase chain reaction. SETTING University research and clinical institutes. PATIENT(S) Infertile men with germ cell arrest (n = 10), Sertoli cell only (n = 8), or mixed atrophy (n = 7) syndromes, and with cases of idiopathic infertility showing normal spermatogenesis (n = 8). INTERVENTION(S) Diagnostic testicular biopsy was performed on participants. MAIN OUTCOME MEASURE(S) We recorded the location, number, distribution, and cytokine expression of human testicular macrophages. RESULT(S) CD68-positive macrophages were found in the testes of all groups analyzed. These macrophages expressed the genes for interleukin 1 and tumor necrosis factor-alpha, and were located in the interstitium, tubular wall, and tubular lumen. In Sertoli cell only and germ cell arrest syndromes, the overall macrophage number was increased over twofold. In all pathologic states, there was a significant shift of these cells from the interstitium to the tubules. CONCLUSION(S) Our study suggests that increased numbers of CD68-positive macrophages directly (via phagocytosis) or indirectly (via paracrine actions exerted through their secretory products) are involved in the regulation of steroidogenesis, Sertoli cell activity, germ cell survival, and, in consequence, in the pathogenesis or maintenance of infertility states in the human testes.

Involvement of Tumor Necrosis Factor-α in the Pathogenesis of Autoimmune Orchitis in Rats

Abstract We studied the testicular macrophages of rats with experimental autoimmune orchitis (EAO) and analyzed whether the tumor necrosis factor-α (TNFα) is involved in germ cell apoptosis and in Leydig cell steroidogenesis. The EAO was induced in adult male Sprague-Dawley rats by active immunization with testicular homogenate and adjuvants. In the experimental group, a severe orchitis was observed 80 days after the first immunization. ED1- and ED2-positive macrophages were quantified by immunohistochemistry. The TNFα concentration of conditioned media from testicular macrophages (TMCM) was determined by ELISA. The number of apoptotic TNF receptor 1 (TNFR1)-positive germ cells was identified by combining in situ end labeling of apoptotic DNA and immunohistochemical techniques. The effect of TNFα on Leydig cell testosterone production was determined by RIA. In rats with EAO, we observed a significant increase in the number of TNFα-positive testicular macrophages, the TNFα concentration in TMCM, and the number of TNFR1-positive germ cells. Sixty percent of TNFR1-positive germ cells were apoptotic. These results suggest that TNFα could be involved in the pathogenesis of EAO. Acting together with other local factors such as Fas-FasL, TNFα could trigger germ cell apoptosis. We also demonstrated that TNFα inhibited in vitro testosterone production in basal and hCG-stimulated Leydig cells from rats with orchitis.

Evidence for a GABAergic System in Rodent and Human Testis: Local GABA Production and GABA Receptors

The major neurotransmitter of the central nervous system, gamma-aminobutyric acid (GABA), exerts its actions through GABAA, GABAB and GABAC receptors. GABA and GABA receptors are, however, also present in several non-neural tissues, including the endocrine organs pituitary, pancreas and testis. In the case of the rat testis, GABA appears to be linked to the regulation of steroid synthesis by Leydig cells via GABAA receptors, but neither testicular sources of GABA, nor the precise nature of testicular GABA receptors are fully known. We examined these points in rat, mouse, hamster and human testicular samples. RT-PCR followed by sequencing showed that the GABA-synthesizing enzymes glutamate decarboxylase (GAD) 65 and/or GAD67, as well as the vesicular GABA transporter vesicular inhibitory amino acid transporter (VIAAT/VGAT) are expressed. Testicular GAD in the rat was shown to be functionally active by using a GAD assay, and Western blot analysis confirmed the presence of GAD65 and GAD67. Interstitial cells, most of which are Leydig cells according to their location and morphological characteristics, showed positive immunoreaction for GAD and VIAAT/VGAT proteins. In addition, several GABAA receptor subunits (α1–3, β1–3, γ1–3), as well as GABAB receptor subunits R1 and R2, were detected by RT-PCR. Western blot analysis confirmed the results for GABAA receptor subunits β2/3 in the rat, and immunohistochemistry identified interstitial Leydig cells to possess immunoreactive GABAA receptor subunits β2/3 and α1. The presence of GABAA receptor subunit α1 mRNA in interstitial cells of the rat testis was further shown after laser microdissection followed by RT-PCR analysis. In summary, these results describe molecular details of the components of an intratesticular GABAergic system expressed in the endocrine compartment of rodent and human testes. While the physiological significance of this peripheral neuroendocrine system conserved throughout species remains to be elucidated, its mere presence in humans suggests the possibility that clinically used drugs might be able to interfere with testicular function.

Steroidogenic Acute Regulatory Protein in Ovarian Follicles of Gonadotropin-Stimulated Rats Is Regulated by a Gonadotropin-Releasing Hormone Agonist

Abstract The aim of the present study was to examine the acute and chronic effects of the gonadotropin-releasing hormone agonist (GnRH-a) leuprolide acetate (LA) on the expression of the steroidogenic acute regulatory protein (StAR), the cytochrome P450 side-chain cleavage enzyme (P450scc), and steroid production in antral ovarian follicles obtained from prepubertal equine choriogonadotropin (eCG)-treated rats. Follicular contents of StAR and P450scc proteins were measured by Western blotting following in vivo injection of eCG (control) and eCG+LA (LA) to prepubertal rats. Treatment with eCG for 2 h resulted in no change in StAR protein content, but it was markedly increased at 4 and 8 h after hormone treatment. However, coadministration of eCG+LA produced a significant increase (P < 0.05) in StAR protein levels at 2, 4, and 8 h when compared with eCG treatment. Acute and chronic treatment with either eCG or eCG+LA did not alter the P450scc protein levels in freshly isolated follicles. The increase in StAR protein expression following LA treatment was qualitatively similar to StAR mRNA expression, as determined by quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis. Furthermore, administration of eCG demonstrated a time-dependent increase (2–8 h) in the levels of StAR mRNA, and these levels were markedly increased by eCG+LA. However, the temporal response pattern of StAR mRNA was much greater at 2 h following LA administration when compared with controls. In addition, 48 h of LA treatment in eCG-treated rats resulted in a significant increase (P < 0.05) in follicular progesterone levels, whereas significant decreases in androgen (testosterone and androsterone) and estradiol levels were observed. Similar results were obtained when serum androgens and estradiol were measured, but serum progesterone levels were unchanged. Collectively, these findings demonstrate that the inhibitory effect of LA on ovarian androgen and estradiol levels is related to changes in the follicular levels of StAR protein and steroid production.

Direct effect of ghrelin on leptin production by cultured rat white adipocytes.

Objective: Because ghrelin is known to stimulate adipogenesis, we tested whether ghrelin could contribute to the maintenance of homeostasis, directly affecting rat white adipocyte leptin production.

SPECIFIC PROLACTIN BINDING IN THE RAT ADRENAL GLAND: ITS CHARACTERIZATION AND HORMONAL REGULATION

Rat adrenal prolactin receptors possess the same hormonal specificity as those in the prostate gland and liver, but are less stable during storage and after freezing. There is a gradual decrease in specific prolactin binding to the adrenal during sexual maturation in male rats; maximum binding capacity of 980 fmol/mg protein is at 25 days of age decreasing to approximately 100 fmol/mg protein at day 90. Prolactin receptors in the prostate are high at 25 days of age (700 fmol/mg protein), decrease sharply by day 30 (180 fmol/mg protein) and then gradually increase. Ovariectomy resulted in a significant rise in total prolactin binding in the adrenal gland, while the administration of oestradiol or testosterone reduced the binding, the reverse of changes in prolactin binding in the liver. Only oestrogen increased serum levels of prolactin in female rats. Ovine prolactin (500 micrograms) given to female rats resulted in a rapid increase over a period of 2-8 h total prolactin receptors in the adrenal, and these then decreased to normal levels, indicating a possible positive regulation of prolactin receptors by homologous hormone.

Effect of bilateral denervation of the immature rat testis on testicular gonadotropin receptors and in vitro androgen production

We have studied the effect of superior spermatic nerve (SSN) section on testicular gonadotropin receptors and in vitro androgen production by immature rat testis. Bilateral testicular denervation had no effect on testicular weight, serum androgens, LH, FSH and PRL levels. Denervation resulted in a significant inhibition of hCG stimulated in vitro androgen production. A reduction in the number of testicular LH receptors was observed after SSN section, while FSH binding sites remained unchanged. These results indicate that the number of LH receptors and testicular steroidogenic response to hCG are influenced by nerves reaching the testis.

Interactions between Testicular Serotoninergic, Catecholaminergic, and Corticotropin-Releasing Hormone Systems Modulating cAMP and Testosterone Production in the Golden Hamster

We previously reported the presence of serotonin (5-HT) in testes from golden hamster, a photoperiodic species which is a useful model for the study of states of male (in)fertility. The aims of this study were to investigate: (1) the presence of intrinsic sources of 5-HT in the testis; (2) the role of 5-HT in in vitro androgen production; (3) the serotoninergic receptor subtypes in the testis, and (4) the existence of interactions among the 5-HT receptors and the testicular catecholaminergic and corticotropin-releasing hormone (CRH) systems. Immunohistochemical studies revealed the presence of tryptophan hydroxylase, a 5-HT-biosynthetic enzyme, in interstitial cells which show the characteristic punctate chromatin pattern of Leydig cells. We describe an inhibitory action of 5-HT on testosterone, dihydrotestosterone, and androstane-3α,17β-diol production from testes of peripubertal and adult hamsters maintained in a long photoperiod (14/10 h light/dark), and adult animals exposed to a short photoperiod (6/18 h light/dark). By using several agonists and antagonists of 5-HT receptors, we characterized 5-HT1A and 5-HT2A receptor subtypes involved in the inhibitory action of this neurotransmitter on human chorionic gonadotropin stimulated cyclic adenosine monophosphate and testosterone production. CRH also produced a negative modulation of both parameters, but epinephrine and norepinephrine, through α1/β1-adrenergic receptors, exerted a stimulatory action. 5-HT1A, 5-HT2, and CRH antagonists showed that the testicular activity of the serotoninergic system, but also the α1/β1-adrenergic receptor system, is mediated by CRH. Moreover, interactions between the 5-HT2A receptor system and α1/β-adrenergic receptors have been established. Thus, these data suggest that α1/β1-adrenergic receptors are involved in the local regulatory action exerted by 5-HT on steroidogenesis through a 5-HT2-receptor-mediated response and the CRH system.

Catecholamine distribution in adult rat testis

Summary Catecholamine distribution in the adult rat testis was examined using a sensitive radioenzymatic method. Norepinephrine was present in the capsule and the interstitial fluid, in higher concentrations than dopamine, while in the interstitial cell preparations only norepinephrine was found. Epinephrine was undetectable in all testicular compartments investigated. No catecholamines were found in the seminiferous tubules.

Modulatory Effects of Leptin on Leydig Cell Function of Normal and Hyperleptinemic Rats

Neonatal L-monosodium glutamate (MSG) administration in rats induces several neuroendocrine and metabolic disruptions. Leptin, the adipocyte product, modulates several neuroendocrine systems including the hypothalamic-pituitary-gonadal (HPG) axis in mammals. The aim of the present study was to determine whether MSG-induced chronic hyperleptinemia could play any relevant role in the hypogonadism developed by male rats when examined in adulthood. We found that 120-day-old MSG male rats displayed significant hyperleptinemia, hypogonadism, and undisturbed basic testis structure and spermatogenesis. In vitro studies in purified Leydig cells from normal (CTR) and MSG-damaged rats revealed that basal and human chorionic gonadotropin (hCG)-stimulated 17-hydroxy-progesterone (17-HO-P4), Δ4-androstenedione (Δ4A) and testosterone (T) secretions were significantly lower in MSG than in CTR cells. Exposure to murine leptin (mleptin, 10–8M) significantly inhibited hCG-elicited T secretion by CTR cells after 180 min incubation. While mleptin significantly inhibited hCG-stimulated Δ4A output and the Δ4A:17-OH-P4 ratio of secretion, conversely, it failed to modify the ratio T:Δ4A release by CTR Leydig cells. Interestingly, the effects of mleptin found on CTR Leydig cells were absent in MSG Leydig cells. Finally, endogenous hyperleptinemia was associated with a significant decrease in Leydig cell expression of Ob-Rb mRNA in MSG rats. In summary, this study demonstrates that: (1) mleptin inhibited testicular steroidogenesis in CTR rats; (2) MSG-treated rats showed lower in vitro 17-OH-P4, Δ4A and T production under basal and post-hCG stimulation conditions; (3) purified Leydig cells from MSG-treated rats displayed resistance to the inhibitory action of mleptin on T release, and (4) endogenous leptin exerts a modulatory effect on Leydig cell Ob-Rb mRNA expression. The inhibitory effect of leptin on testicular function is thus abrogated in MSG-damaged rats. The testicular leptin-resistance developed by MSG rats seems to be due to early chronic exposure of Leydig cells to high leptin circulating levels, which in turn down-regulate testicular Ob-Rb expression. It remains to be determined whether the testicular dysfunction of MSG rats can be reversed after correction of hyperleptinemia or whether it is an irreversible effect of the hypothalamic lesion.