Improving sustainable hydrogen production from green waste: [FeFe]-hydrogenases quantitative gene expression RT-qPCR analysis in presence of autochthonous consortia.

BACKGROUND: Bio-hydrogen production via dark fermentation of low-value waste is a potent and simple mean of recovering energy, maximising the harvesting of reducing equivalents to produce the cleanest fuel amongst renewables. Following several position papers from companies and public bodies, the hydrogen economy is regaining interest, especially in combination with circular economy and the environmental benefits of short local supply chains, aiming at zero net emission of greenhouse gases (GHG). The biomasses attracting the largest interest are agricultural and urban green wastes (pruning of trees, collected leaves, grass clippings from public parks and boulevards), which are usually employed in compost production, with some concerns over the GHG emission during the process. Here, an alternative application of green wastes, low-value compost and intermediate products (partially composted but unsuitable for completing the process) is studied, pointing at the autochthonous microbial consortium as an already selected source of implementation for biomass degradation and hydrogen production. The biocatalysts investigated as mainly relevant for hydrogen production were the [FeFe]-hydrogenases expressed in Clostridia, given their very high turnover rates. RESULTS: Bio-hydrogen accumulation was related to the modulation of gene expression of multiple [FeFe]-hydrogenases from two strains (Clostridium beijerinckii AM2 and Clostridium tyrobutyricum AM6) isolated from the same waste. Reverse Transcriptase quantitative PCR (RT-qPCR) was applied over a period of 288 h and the RT-qPCR results showed that C. beijerinckii AM2 prevailed over C. tyrobutyricum AM6 and a high expression modulation of the 6 different [FeFe]-hydrogenase genes of C. beijerinckii in the first 23 h was observed, sustaining cumulative hydrogen production of 0.6 to 1.2 ml H2/g VS (volatile solids). These results are promising in terms of hydrogen yields, given that no pre-treatment was applied, and suggested a complex cellular regulation, linking the performance of dark fermentation with key functional genes involved in bio-H2 production in presence of the autochthonous consortium, with different roles, time, and mode of expression of the involved hydrogenases. CONCLUSIONS: An applicative outcome of the hydrogenases genes quantitative expression analysis can be foreseen in optimising (on the basis of the acquired functional data) hydrogen production from a nutrient-poor green waste and/or low added value compost, in a perspective of circular bioeconomy.

Behavioral effects of inhibition of cannabinoid metabolism: The amidase inhibitor AM374 enhances the suppression of lever pressing produced by exogenously administered anandamide.

Biochemical investigations have identified putative enzymatic pathways for the synthesis and metabolism of endogenous cannabinoids. Anandamide amidase is an enzyme that metabolizes anandamide into arachadonic acid and ethanolamine. Using in vitro methods, various inhibitors of amidase have been identified. The present studies were undertaken to determine if the amidase inhibitor AM 374 could enhance the effects of intraperitoneal (IP) injections of anandamide. Three studies were conducted to investigate the effects of various drug treatments on fixed ratio 5 operant lever pressing for food reinforcement. In the first study, the effects of different doses of anandamide were assessed, and it was demonstrated that 5.0 and 10.0 mg/kg anandamide IP significantly suppressed lever pressing, while 2.5 mg/kg produced very little effect. The second study tested the effects of intraventricular (ICV) injections of AM 374, and it was observed that doses up to 10.0, 20.0 and 40 microg AM 374 had no significant effect upon lever pressing. The third study investigated the combined effect of AM374 with a low dose of anandamide. Rats received two drug injections: one ICV and one IP. Four different drug treatments were assessed: 1) ICV vehicle + IP vehicle, 2) ICV vehicle + 2.5 mg/kg anandamide IP, 3) ICV 20.0 microg AM 374 + IP vehicle, and 4) ICV 20 microg AM 374 + 2.5 mg/kg anandamide IP. Combined administration of AM 374 plus anandamide led to a significant decrease in lever pressing compared to either AM374 or anandamide administered alone. Observations of the animals treated with the combination of AM374 plus anandamide indicated that the drug combination resulted in motor slowing, which is consistent with the notion that stimulation of cannabinoid receptors produced a motor deficit that interfered with lever pressing. Although AM374 produced no effect on its own, this amidase inhibitor did enhance the behavioral effect of a low dose of anandamide. These results are consistent with the notion that AM 374 inhibited the enzymatic breakdown of exogenously injected anandamide. This type of procedure can be used to assess a variety of different compounds for their ability to inhibit cannabinoid metabolism.

Open field locomotor effects in rats after intraventricular injections of ethanol and the ethanol metabolites acetaldehyde and acetate.

The typical response to acute peripheral administration of low to high doses of ethanol in rats is a dose-dependent depression of motor activity. Nevertheless, recent studies indicate that intraventricular (ICV) injections of ethanol can produce signs of behavioral activation. In addition, considerable evidence indicates that brain metabolism of ethanol is involved in modulating some of the behavioral effects of this drug, which suggests that ethanol may have active metabolites with central actions. The present study was undertaken to investigate the effects of ICV ethanol, and its two major metabolites acetaldehyde and acetate, on open field locomotor activity in rats. Male Sprague-Dawley rats received different doses of ethanol, acetaldehyde or acetate ICV and immediately were placed in an open field chamber in which locomotion was measured. Rats injected with ICV ethanol or acetaldehyde showed an inverted U-shaped dose-response curve, with moderate doses increasing motor activity. In contrast, acetate produced a dose-dependent decrease in motor activity. These results demonstrate that central administration of low doses of ethanol can increase locomotor activity in rats, and suggest that acetaldehyde may be an active metabolite of ethanol that also can facilitate locomotor activity. Moreover, it is possible that some of the motor suppression or sedation produced by ethanol is due to the central actions of acetate.

Locomotor stimulant effects of intraventricular injections of low doses of ethanol in rats: acute and repeated administration.

RATIONALE: Low doses of ethanol stimulate locomotion in mice, but in rats the typical response to peripheral ethanol administration is a dose-dependent suppression of locomotion. Moreover, chronic ethanol administration fails to produce signs of locomotor sensitization in rats. OBJECTIVE: The present study was undertaken to determine whether intraventricular (i.c.v.) infusions of low doses of ethanol (as determined by comparisons with systemic doses, and by analyses of brain extract ethanol levels) could increase locomotor activity in rats after acute or repeated administration. METHODS: Male rats received acute doses of ethanol i.p. (0.0, 0.25, 0.5, 1.0, or 2.0 g/kg) or i.c.v. (0.0, 0.7, 1.4, or 2.8 micromol) and were tested for motor activity. In a third experiment, repeated i.c.v. vehicle or ethanol (2.8 micromol) was administered for 15 sessions over a 30-day period, and motor activity was recorded. This phase was followed by a single challenge session, in which a low dose of ethanol (0.7 micromol) was injected i.c.v. to both groups of rats. RESULTS: Rats injected with i.p. ethanol showed no increase in activity at low doses, with higher doses suppressing activity. In contrast, i.c.v. injections of low doses of ethanol increased motor activity. After repeated administration, ethanol-treated rats were more sensitive than control-treated rats to the locomotor stimulant effect of ethanol. CONCLUSIONS: These results demonstrate that central administration of low doses of ethanol can increase locomotor activity in rats and suggest that i.c.v. ethanol can produce some signs of motor sensitization, a characteristic that has been related to the potential addictive properties of many drugs.

Iodine supplementation restores fertility of sheep exposed to iodine deficiency.

The aims of the study were to monitor sheep iodine intake in different sheep breeding farms in Abruzzo and to evaluate the effects of iodine supplementation on ovine fertility. The urinary iodine concentrations (UIC) in animals of 8 out of the 11 breeding farms analyzed were borderline (UIC 100-150 microg/l) or very low (UIC < or = 50 microg/l). Only animals bred in 3 farms showed an adequate iodine intake with a mean UIC > or = 300 microg/l. Animals with very low iodine intake had lower T4 and T3 (p < 0.01) serum levels, compared to those with adequate iodine intake. To investigate the effects of iodine supplementation on ovine fertility, 32 ewes and 20 rams, characterized by low UIC, were randomly divided into 2 groups. One group (16 ewes and 10 rams) received a sc injection of 1 ml of Lipiodol, containing 480 mg of iodine, while the remaining animals were employed as control. This treatment was able to maintain UIC above 300 microg/l for 3 months and to increase T4 and T3 serum levels (p < 0.01). After 9 months, the fertility of control and treated animals was assessed by monitoring the rate of successful matings by ultrasonography. The results showed that 100% of treated ewes mated with treated rams were pregnant vs 37% of the control ewes mated with control rams (p = 0.007). The iodine content was 4-fold higher in milk from treated ewes (2393 +/- 453 microg/l), compared to controls (675 +/- 154 microg/l). The results demonstrated that iodine supplementation restores fertility of sheep living in iodine deficient areas and may represent a means to achieve a silent iodine prophylaxis of local populations.

Dopamine antagonists alter response allocation but do not suppress appetite for food in rats: contrast between the effects of SKF 83566, raclopride, and fenfluramine on a concurrent choice task.

RATIONALE: Dopamine is important for enabling organisms to overcome work-related response costs. One way of investigating this function has been with concurrent choice procedures using food reinforcement. In the present study, rats were given a choice between pressing a lever for preferred Bioserve pellets, or approaching and consuming a less-preferred laboratory chow that was concurrently available. In previous work with this task, dopamine antagonists and accumbens dopamine depletions decreased lever pressing but increased chow consumption. OBJECTIVE: The present study assessed three drugs (two dopamine antagonists and one appetite suppressant) using the lever pressing/chow feeding task. RESULTS: Under baseline conditions, rats pressed the lever at high rates (1,300-1,500 responses) to obtain the preferred food, and little of the laboratory chow was eaten (1-2 g). Selective D1 and D2 antagonists (SKF 83566 and raclopride) reduced fixed ratio 5 lever pressing, but substantially increased chow consumption. In contrast, the serotonergic appetite suppressant fenfluramine reduced both lever pressing and chow consumption. With the dopamine antagonists, lever pressing and chow consumption were inversely correlated across treatments, while these two measures were unrelated in the fenfluramine experiment. CONCLUSIONS: Dopamine antagonists and accumbens dopamine depletions do not simply reduce appetite. Rats with accumbens dopamine depletions, or rats treated with low doses of selective or non-selective dopamine antagonists, remain directed toward the acquisition and consumption of food. These results demonstrate that fundamental aspects of food reinforcement are left intact after treatment with low doses of dopamine antagonists.

D1 or D2 antagonism in nucleus accumbens core or dorsomedial shell suppresses lever pressing for food but leads to compensatory increases in chow consumption.

Although interference with dopamine (DA) systems can suppress lever pressing for food reinforcement, it is not clear whether this effect occurs because of a general disruption of food motivation. One way of assessing this has been a choice procedure in which a rat responds on an fixed ratio 5 (FR5) schedule for preferred Bioserve pellets while a less preferred lab chow is concurrently available in the operant chamber. Untreated rats consume little of the chow, preferring to respond for the Bioserve pellets. Previous studies have shown that depleting DA in the accumbens substantially decreased lever pressing while increasing chow consumption. In the present study, low doses (0.0625-1.0 microg) of the D1 antagonist SCH 23390 or the D2 antagonist raclopride were injected into the either the core or shell subregions of nucleus accumbens, and rats were tested on the concurrent lever pressing/feeding task. Analysis of the dose response curves showed that injections of SCH 23390 into the core were more potent than injections into the shell for suppressing lever pressing (i.e., the ED(50) was lower in the core). Nevertheless, injections of either drug into either site suppressed lever pressing and increased intake of the concurrently available chow. Across both drugs and at both sites, the amount of chow consumed was negatively correlated with the total number of responses. Neither drug significantly increased response duration, suggesting that accumbens DA antagonism did not produce the type of motor impairment that leads to severe alterations in the form of lever pressing. In summary, the blockade of D1 or D2 receptors in nucleus accumbens core or shell decreased lever pressing for food reinforcers, but rats remained directed toward the acquisition and consumption of food. These results indicate that accumbens D1 antagonism does not decrease lever pressing because of a general reduction in food motivation. Nevertheless, interference with accumbens DA does appear to set constraints upon which responses are selected for obtaining food, and may impair the ability of animals to overcome work-related response costs in order to obtain food.

Platelet-derived growth factor (PDGF) and PDGF receptors in rat corpus cavernosum: changes in expression after transient in vivo hypoxia.

Platelet-derived growth factor (PDGF) overactivity has been implicated in atherosclerosis and several fibrotic conditions including lung and kidney fibrosis, liver cirrhosis and myelofibrosis. Low oxygen tension (hypoxia) is a known stimulus for transcriptional induction of PDGF ligand and receptor genes in different tissues. We studied the expression and localization of PDGF-A, PDGF-B, and PDGF receptor (PDGFR)-alpha and -beta subunits in adult rat isolated corpus cavernosum (CC) under generalized transient hypoxia (pO(2) 10%) in comparison with normoxic conditions. Semi-quantitative RT-PCR analysis of mRNA extracted from rat penis showed higher amounts of PDGF-A, PDGF-B and PDGFR-beta mRNA transcripts in hypoxic versus normoxic animals. The immunohistochemical analysis showed that the localization of PDGF subunits and PDGFR-beta was confined to the cytoplasm of the perivascular smooth muscle cells, endothelium and trabecular fibroblasts. Our findings indicate that transient low oxygen tension induces PDGF overexpression in rat CC, which in the long term may lead to an increase of connective tissue production. We suggest that a local impairment of the PDGF/PDGFR system may contribute to CC fibrosis, which is an established cause of erectile dysfunction in man.

Effects of sex and the estrous cycle on regulation of intravenously self-administered cocaine in rats.

RATIONALE: Previous research with both humans and animals suggests that there are sex differences in cocaine self-administration; in rodents, ovarian hormones may underlie these differences. OBJECTIVES: A two-lever drug self-administration procedure was used to compare regulation of intravenously self-administered cocaine in male and female rats and among females in different phases of the estrous cycle. METHODS: Eleven female and seven male age-matched Wistar rats were trained to self-administer nine doses of cocaine (0.0-2.4 mg/kg) during daily 5-h sessions. Experimental test chambers were equipped with two levers and associated stimulus lights. A response on the lever with stimuli signaling an increase in cocaine dose increased the infusion duration by 3 s, and a response on the other lever decreased the infusion duration by 3 s. RESULTS: After responding for cocaine stabilized, regulation was disrupted more in females than in males (r2=78.9, r2=92.6, respectively) with the greatest disruption observed in females during the estrus phase (r2=48.5). Mean dose size varied considerably for males and for females in the metestrus/diestrus and proestrus phases; however, estrus females responded almost exclusively on the lever associated with an increase in cocaine dose. CONCLUSIONS: These findings indicate sex differences in the regulation of cocaine self-administration, and they suggest that ovarian hormones may be responsible for the observed sex differences.

Fas and Fas ligand expression in fetal and adult human testis with normal or deranged spermatogenesis.

In mice, the Fas/Fas ligand (FasL) system has been shown to be involved in germ cell apoptosis. In the present study we evaluated the expression of Fas and Fas ligand (FasL) in fetal and adult human testis. Semiquantitative RT-PCR demonstrated the expression of Fas and FasL messenger ribonucleic acids in adult testis, but not in fetal testis (20-22 weeks gestation). In situ RT-PCR and immunohistochemistry experiments on adult human testis demonstrated the expression of FasL messenger ribonucleic acid and protein in Sertoli and Leydig cells, whereas the expression of Fas was confined to the Leydig cells and sporadic degenerating spermatocytes. The number of Fas-positive germ cells per 100 Sertoli cell nuclei was increased in 10 biopsies with postmeiotic germ cell arrest compared to 10 normal testis biopsies (mean, 3.82 +/- 0.45 vs. 2.02 +/- 0.29; P = 0.0001), but not in 10 biopsies with meiotic germ cell arrest (mean, 1.56 +/- 1.07). Fas and FasL proteins were not expressed in cases of idiopathic hypogonadotropic hypogonadism. Together, these findings may suggest that Fas/FasL expression in the human testis is developmentally regulated and under gonadotropin control. The increased germ cell expression of Fas in patients with postmeiotic germ cell arrest suggests that the Fas/FasL system may be involved in the quality control mechanism of the produced gametes.

[Incidental diagnosis of seminoma in male infertility: report of a clinical case]. / Diagnosi incidentale di seminoma nell'infertilità maschile: descrizione di un caso clinico.

The aims of the study were to evaluate the association between male infertility and risk of developing testis cancer and to establish guidelines for the early diagnosis of testis neoplasia in subfertile men. 32-year-old infertile man. The patient underwent random testicular biopsy to establish the exact cause of infertility. An incidental diagnosis of seminoma was made and the patient then underwent right testis excision. Anatomopathologic macroscopic examination revealed two nodules, the sizes of which were 0.8 x 0.4 and 0.3 x 0.2 cm, respectively. Histologic examination confirmed the diagnosis of typical seminoma, pT1, with copious lymphocytic struma infiltration. There appears to be a correlation between male infertility and occurrence of seminoma. Diagnosis of testis cancer is often incidental and sometimes occurs in men undergoing testicular biopsy to investigate infertility. Since the biopsy was not specifically targeted in our case, the diagnosis of seminoma was casual. This suggests the need for a careful follow-up, including testicular ultrasonography as a screening test to achieve an early diagnosis of testis cancer in all infertile men, because of their higher risk of developing testis cancer than the normal population.

Leydig cell loss and spermatogenic arrest in platelet-derived growth factor (PDGF)-A-deficient mice.

Platelet-derived growth factor (PDGF)- A-deficient male mice were found to develop progressive reduction of testicular size, Leydig cells loss, and spermatogenic arrest. In normal mice, the PDGF-A and PDGF-Ralpha expression pattern showed positive cells in the seminiferous epithelium and in interstitial mesenchymal cells, respectively. The testicular defects seen in PDGF-A-/- mice, combined with the normal developmental expression of PDGF-A and PDGF-Ralpha, indicate that through an epithelial-mesenchymal signaling, the PDGF-A gene is essential for the development of the Leydig cell lineage. These findings suggest that PDGF-A may play a role in the cascade of genes involved in male gonad differentiation. The Leydig cell loss and the spermatogenic impairment in the mutant mice are reminiscent of cases of testicular failure in man.

Regulation by thyroid hormone of the expression of basement membrane components in rat prepubertal Sertoli cells.

The present study reports the modulation of basement membrane (BM) components, laminin, entactin, and type IV collagen, expression in prepubertal rat Sertoli cell by the thyroid hormone T3. Immunocytochemical studies of permeabilized Sertoli cells in culture showed that T3 treatment (10[-7] M for 24 h) increased the number of cells staining positive for laminin and/or entactin (from 58 +/- 5.3% to 86.4 +/- 6.5%, P < 0.01). In contrast, a strong inhibition of type IV collagen immunopositivity was observed. Western blot analysis of Sertoli cell-conditioned media indicated that T3 treatment significantly (P < 0.01) increased the level of secreted entactin by 60-65% without affecting the levels of laminin A and B1/B2 chains. Moreover, thyroid hormone treatment of Sertoli cells significantly reduced type IV collagen secretion by 62% (P < 0.05). Slot blot analysis of poly-A RNA demonstrated a significant (P < 0.01) increase in the level of entactin messenger RNA (mRNA) by 140% (P < 0.01) and a 50% reduction of type IV collagen alpha1 chain mRNA after thyroid hormone treatment. No effect of the hormone was observed on the accumulation of the laminin B1 and B2 chain mRNAs in Sertoli cell cultures. These effects cannot be ascribed to changes in the degradation of BM components, because no effect of thyroid hormone was observed on plasminogen activators or metalloproteinase secretion by Sertoli cells. These observations indicate the Sertoli cell as a source of entactin within the testis, demonstrate the ability of T3 to differentially regulate the expression of BM components, and can be regarded as a part of the integrated mechanism by which thyroid hormone affects testicular development and differentiation.

Testicular development involves the spatiotemporal control of PDGFs and PDGF receptors gene expression and action.

Platelet-derived growth factors (PDGFs) are growth-regulatory molecules that stimulate chemotaxis, proliferation and metabolism primarily of cells of mesenchymal origin. In this study, we found high levels of PDGFs and PDGFs receptors (PDGFRs) mRNAs, and specific immunostaining for the corresponding proteins in the rat testis. PDGFs and PDGFRs expression was shown to be developmentally regulated and tissue specific. Expression of PDGFs and PDGFRs genes was observed in whole testis RNA 2 d before birth, increased through postnatal day 5 and fell to low levels in adult. The predominant cell population expressing transcripts of the PDGFs and PDGFRs genes during prenatal and early postnatal periods were Sertoli cells and peritubular myoid cells (PMC) or their precursors, respectively, while in adult animals PDGFs and PDGFRs were confined in Leydig cells. We also found that early postnatal Sertoli cells produce PDGF-like substances and that this production is inhibited dose dependently by follicle-stimulating hormone (FSH). The expression of PDGFRs by PMC and of PDGFs by Sertoli cells corresponds in temporal sequence to the developmental period of PMC proliferation and migration from the interstitium to the peritubulum. Moreover, we observed that all the PDGF isoforms and the medium conditioned by early postnatal Sertoli cells show a strong chemotactic activity for PMC which is inhibited by anti-PDGF antibodies. These data indicate that, through the spatiotemporal pattern of PDGF ligands and receptors expression, PDGF may play a role in testicular development and homeostasis.

Coenzyme Q: potentially useful index of bioenergetic and oxidative status of spermatozoa.

The concentration of coenzyme Q10 (CoQ10), a key intermediate of the mitochondrial respiratory chain, was determined in spermatozoa of 13 fertile subjects, 8 potentially fertile patients, and 12 infertile patients. CoQ10 concentrations were significantly higher (P < 0.001) in infertile patients than in fertile and potentially fertile subjects. The difference between potentially fertile and fertile subjects was also significant (P < 0.001). We propose that a decrease in consumption of CoQ10 in both infertile and potentially fertile populations is due to an autoregulatory mechanism of ATP production.

Immunohistochemical localization of growth hormone-releasing hormone in human gonads.

Human gonads were examined for the presence of ir growth hormone-releasing-hormone. We demonstrated the presence of immunostainable cells both in ovarian and in testicular tissue using a non-crossreactive anti-GRH antiserum and the immunoperoxidase detection technique. In ovaries from ovulating women, GRH immunoreactivity was localized in the corpora lutea; granulosa cells, theca cells and cells of primary follicles did not stain. In premenopausal ovaries, staining was detectable in scattered luteinized stromal cells. In testes from post-puberal men GRH immunoreactivity was localized in the Leydig cells; cells of the germinal epithelium did not stain. These results demonstrate the presence of GRH in human gonads and suggest that this peptide may exert regulatory function at the testicular and ovarian levels.

Gastrin-releasing peptide-like immunoreactivity in porcine follicular fluid and ovary.

Porcine follicular fluid was found to contain gastrin-releasing peptide (GRP) by radioimmunoassay. High pressure liquid chromatography of the extracted material showed two peaks of GRP immunoreactivity that closely resembled the C-terminal fragments of GRP, GRP18-27 and GRP14-27. Immunohistochemical studies revealed specific staining for GRP in the granulosa cells of adult porcine ovary. These results demonstrate the presence of substances which behave like authentic GRP-like peptides in porcine ovary and follicular fluid and suggest that these peptides may play a paracrine and/or autocrine role in the regulation of the ovarian function.

Acetylcholinesterase and pseudocholinesterases in developing chick adrenal.

The distribution of acetylcholinesterase (AChE) and pseudocholinesterases (PsChE) in chick adrenal gland during the first phases of organogenesis was studied. Acetylthiocholine iodide and butyrylthiocholine iodide were used as substrates for the two enzymes, respectively, whereas BW284c51 (1,5 bis (4-allyldimethylammonium-phenyl)pentan-3-one-dibromide) and ISO-OMPA (tetraisopropylpyrophosphoramide) were used as respective inhibitors of AchE and PsChE. AchE was present on the plasma membrane, in the perinuclear cisterna and in some cisternae of the rough endoplasmic reticulum of both interrenal and chromaffin cells; moreover enzymatic activity was found in the same sites of ganglion cells and mesenchymatic undifferentiated cells, i.e. on the inside and in the proximity of the glandular anlage. PsChE activity was localized in the perinuclear space and in the rough endoplasmic reticulum of all types of cells in the anlage. It is suggested that these enzymatic activities may be implicated in morphogenetic mechanisms.

Active cell movements and embryonic cholinesterase in the postmetamorphic completion of the adrenal morphogenesis in frogs.

The adrenal gland in Rana esculenta complex, as in other advanced anurans, is not yet in its definitive position at the end of the metamorphosis and reaches it subsequently, before sexual maturity. The displacement takes place by various means, among which active cellular movements prevail. These are demonstrated by the presence of acetylcholinesterase (AChE) in noninnervated cells (embryonic AChE). The disintegration of the connective tissues which delimit the cords of the adrenal gland, the passive transport of cells by the blood vessels and the movements of adjacent renal and mesenchymal cells collaborate with the active movements of the adrenal cells.