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Among the therapeutic alternatives to orthotopic liver transplantation, hepatocyte transplantation (HT) offers the best potential in a number of liver diseases, mainly inborn errors of metabolism. Nevertheless, HT presents several inconveniences such as the scarce knowledge of the functionality of the transplanted hepatocytes, which has given rise to controversy about the specificity or unspecificity of the transplant, and the lack of a suitable system for preserving the cells. This study was designed to test a system for cryopreserving hepatocytes and to assess their functionality over prolonged periods after their ectopic transplantation. A medium and a freezing schedule which are reproducible and yield elevated viability have been used, and a number of hepatospecific parameters have been assessed: the activity of ornithine carbamoyltransferase--an enzyme of primary importance in the urea cycle--lipogenesis, gluconeogenesis, glucose-6-phosphatase and cytochrome oxidase activities, the presence of albumin--as an index of plasma protein synthesis--and IDA uptake and metabolism, showing the UDP-glucuronyl transferase activity. As dedifferentiation markers, gamma-glutamyl transpeptidase and alpha-fetoprotein have been studied. From the results, it can be deduced that hepatocytes can be cryopreserved and transplanted and that under these conditions they maintain hepatic features for a long time. Following transplantation, several specific liver functions appear or are enhanced in the spleen. Freshly isolated and cryopreserved transplanted hepatocytes have similar behaviors, although a difference in the expression of the function can be observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Whole-mounted preparations of the tunica albuginea of the rat testis were studied using light microscopy techniques for demonstration of cholinergic nerve fibres (Karnovski-Root method), catecholaminergic nerve fibres (De la Torre's method) and actin filaments (avidin-biotin-peroxidase method). An ultrastructural study of different regions of the albuginea was also performed. Cholinergic fibres were seen to penetrate into the albuginea with the testicular artery to form a broad network in the mediastinum testis, many fibres ending beneath the rete testis epithelium. Catecholaminergic fibres penetrated through the middle part of the cauda epididymis and formed a plexus in the albuginea covering the inferior testicular pole. This plexus gave rise to straight fibres which formed varicosities, some of them appeared related with mast cells. Actin-containing cells were only found beneath the rete testis epithelium. These cells were similar to myofibroblasts. The location of both cholinergic fibres and contractile cells among the rete testis channels suggests that these cells may be involved in the pumping of semen towards the ductuli efferentes and that their contractility may be regulated by cholinergic fibres.

To determine the mechanics of arteries forming the carotid rete mirabile of goat and cattle, the wall tension-circumference length relationship was recorded isometrically in isolated retial arteries of 350-600 microns in external diameter under relaxed and potassium-activated conditions. The results are compared to those of pial arteries of similar size. In relaxed retial and cerebral vessels, tension-length relationship had nonlinear characteristics and the incremental elastic modulus rose as the circumference length increased, the modulus being lower in retial than in pial arteries. However, the stiffness of the artery considered as a whole (stiffness parameter beta) was similar in the two types of vessels. In activated arteries, force development was a function of length and the magnitude of the response increased until an optimal length was reached after which the response decreased with further stretch. In all stretches studied the developed force was much lower in retial arteries. The calculated active stress at the various lengths was also smaller in retial than in cerebral arteries and the maximum active stress for retial arteries was about 30-50% of that for cerebral arteries. Therefore, the passive mechanical properties of retial arteries closely resemble those of cerebral arteries but retial arteries have a small contractility. This suggests that the carotid rete mirabile of Artiodactyla has a minor role in actively controlling blood supply to the brain. Comments on the hemodynamic significance of the carotid rete are included.

A new method for correlating testicular biopsies and spermiograms is proposed. The number of spermatogonia, round spermatids, and elongated spermatids per cross-sectioned tubule were calculated in the testes from 33 subfertile men and in 10 control normal testes. According to these quantitations, the testes in subfertile men were classified as testes with maturation arrest of spermatogenesis, testes with hypospermatogenesis, and testes with associated maturation arrest and hypospermatogenesis. A power regression curve correlating the number of elongated spermatids and sperm numbers in the spermiogram was performed.

Testicular biopsy specimens from adult patients affected with cryptorchidism, Klinefelter's syndrome, and Del Castillo's syndrome were examined by light and electron microscopy. The study revealed a high proportion of testes showing seminiferous tubules with hyperplasia of Sertoli cells (from 25 to 45 cells per transverse tubular section). These cells had an immature appearance and showed a pseudostratified distribution. The nucleus was round to ovoid and regular in outline, with a smaller nucleolus than that of mature Sertoli cells. The cytoplasm showed less development of the endoplasmic reticulum as well as of the secondary lysosomes and lipid droplets than that in mature Sertoli cells. Characteristic features of these immature Sertoli cells were abundant cytoplasmic microfilaments, elaborate interdigitations between adjacent cells, and extensive tight junctions, from basement membrane to lumen. In the cryptorchid testes, a more immature Sertoli cell was found to constitute the majority of the cells in hypoplastic zones. In Klinefelter's and Del Castillo's syndromes as well as in cryptorchid testes to a lesser degree, a transitional type of cell-from immature to mature-was also observed. These observations suggest that Sertoli cells in these primary testicular disorders reflect a congenital deficiency producing abnormal development.

The appearance of a liver DNA synthesis promoter (HP) in rat plasma after dimethylnitrosamine (DMNA) or thioacetamide injection was investigated. After 48 h, DMNA (30 mg kg-1 body weight) produced liver (centrilobular) necrosis and intense hepatic regeneration, as assessed by microscopic observations of liver slices, as well as augmented transaminase levels; HP was detectable under these conditions. After 5 days, transaminases and HP returned to normal values (the latter undetectable), coinciding with a lack of necrotic zones. At 60 mg DMNA kg-1 body weight, necrotic areas were more marked and transaminases and HP levels higher after 48 h than with the lower dose; these increases were even more pronounced at 90 mg DMNA kg-1 body weight. After thioacetamide injection (200 mg kg-1 body wt) the situation at 48 h was very similar, with focal, centrilobular necrosis, frequent regenerative signs, high transaminases and detectable HP. Rats recovered after 7 days in a similar fashion as with DMNA. At 400 mg thioacetamide kg-1 body weight, necrotic areas and regeneration zones were more widespread and transaminases and HP higher after 48 h than with the lower dose. On account of the differing modes of action of DMNA and thioacetamide in rat liver, it is proposed that the appearance of HP activity in plasma could be related to the regenerative process that follows hepatotoxic damage.

The reactivity of the dog cavernous carotid artery to stretch, field electrical stimulation, and norepinephrine was studied using arterial segments under isometric conditions. Light microscopy revealed that this artery is of muscular type and its external surface is covered by venous endothelium, and fluorescence microscopy showed a dense adrenergic innervation. On stretch, arteries exhibited an immediate, transient contraction (phasic response) and a late, maintained contraction (tonic response) that were unaffected by tetrodotoxin (10(-6) M) or endothelium removal but were reduced by the inhibitors of cyclooxygenase indomethacin (10(-6) M), acetylsalicylic acid (3 x 10(-5) M), or meclofenamate (10(-5) M). Electrical stimulation (0.5-4 Hz) contracted the arteries in a frequency-dependent manner, and the response was reduced by tetrodotoxin, phentolamine, (10(-6) M), or the inhibitors of cyclooxygenase used but was unaffected by endothelium removal. Norepinephrine (10(-9)-3 x 10(-4) M) caused dose-dependent contraction that was blocked by phentolamine and by the inhibitors of cyclooxygenase but was not modified by endothelium removal. The results indicate that the dog cavernous carotid artery develops myogenic tone on stretch and contracts on adrenergic stimulation. They also suggest that in these responses prostaglandins but not the endothelium are involved. Therefore, the cavernous carotid artery, because of its location and reactivity, could be of relevance in regulating blood flow or pressure within the cerebral circulation when arterial pressure or adrenergic activity increases.

Hepatocyte transplantation (HT) is among the most frequently employed approaches in the treatment of liver disease. This work deals with the occurrence of rejection when allogeneic hepatocytes are transplanted into the spleens of rats. Two strains of rats were used: Lewis and F344. The recipients were divided into two groups, one of which received ciclosporin (Cic), while the other received no treatment. After HT, the spleens were removed periodically. The animals that did not receive Cic suffered such intense rejection that 7 days after HT no hepatocytes could be identified in the splenic pulp. On the other hand, hepatocytes could be observed throughout the entire study in the spleens of rats treated with Cic. We conclude that HT is followed by intense rejection, which can be avoided by the administration of Cic.

The numbers of each different cell type in the human seminiferous epithelium were determined throughout the 6 stages of the cycle in both semithin and ultrathin sections obtained from 15 young adult men with normal testicular histology. Up to 4 types of A spermatogonia (Ad, Ap, Al and Ac) were distinguished. In addition, the DNA nuclear content of seminiferous epithelium cells was determined on Feulgen-stained sections. Both Ad and Ap spermatogonia showed a 2c DNA content and were present in the 6 stages of the cycle, though their numbers decreased in stages III-V. Both Al and Ac spermatogonia showed a DNA content varying from 2c to 4c. Al spermatogonia were observed in stages III-V; their numbers plus those of Ad spermatogonia in these stages were similar to the numbers of Ad spermatogonia in the other stages lacking in Al spermatogonia. Ac spermatogonia appeared in stages III-VI and their numbers plus those of Ap spermatogonia in stages III-V were similar to the numbers of Ap spermatogonia in the other stages lacking in Ac spermatogonia. The results suggest that Ad spermatogonia are the stem cells. Some of them replicate their DNA; during this replication they appeared as Al spermatogonia. Al spermatogonia divide, giving rise to both Ad and Ap spermatogonia. Some Ap spermatogonia replicate their DNA; during this process they are transformed into Ac spermatogonia which divide, giving rise to B spermatogonia.

The distribution of elastic fibres in the human ductus deferens from birth to senility was studied by light and electron microscopy. Elastic fibres are lacking in the ductus deferens in infants and children. In the adult ductus deferens, they form two layers in the lamina propria: (1) an inner layer of circumferentially oriented elastic fibres, and (2) an outer meshwork of elastic fibres. Elastic fibres are also present in the narrow intercellular spaces between the smooth muscle cells of the muscular coat, mainly in the inner muscular layer. A layer of elastic fibres surrounds the muscular coat. The ductus deferens of ageing subjects shows fragmentation and disorganisation of the elastic fibre layers of the lamina propria. Elastic fibres in the muscular coat are more abundant than in younger adults, forming larger bundles. Electron microscopy demonstrated the presence of immature elastic fibres at puberty as collections of microfibrils, some of them containing loci of amorphous substance (elastin). In the adult ductus most elastic fibres have a mature appearance. The amount of amorphous substance has increased and the number of microfibrils has decreased. Electron-dense inclusions are present within the amorphous substance. With advancing age the amorphous substance forms large, structureless masses showing abundant electron-dense inclusions and areas of rarefaction. A thin layer of microfibrils is present only at the periphery of the elastic fibres. Whether or not androgenic hormones are in any way involved in the formation of elastic fibres in the ductus deferens and testis is something which requires further study.

Mast cells are a constant cell-type in the connective tissues of the human testis and epididymis from birth to adulthood. Ultrastructural study shows that these cells are similar to those found in other connective tissues. Histometric studies revealed that the number of mast cells in the interstitium, mediastinum and albuginea of the testis as well as in the epididymal connective tissue increases slightly during infancy, decreases during childhood, and then increases again at puberty. Increases at puberty are particularly evident in both the testicular interstitium and the epididymis. During adulthood, the number of mast cells progressively decreases in all testicular and epididymal connective tissues. Changes in mast cell number may be related to changes observed in the development of testicular connective tissue which occurs primarily during infancy and puberty.

The development of a reproducible, effective system of cryopreservation of hepatocytes would create new possibilities for metabolic studies, as well as the clinical application of hepatocellular transplants in diverse hepatic disorders. For the purpose of dealing with both questions, we have studied different media and procedures of freezing, and have chosen that combination which afforded the best results for long-term studies of function. A series of intrasplenic transplants were performed with freshly isolated hepatocytes as well as cryopreserved ones, and several hepatospecific parameters (Alanine aminotransferase, ornithine carbamoyl transferase, lipogenesis, uptake of 99mTc-N-(p-butylphenylcarbamylmethyl)iminodiacetic acid, G6Pase, albumin, and gamma-glutamyl transpeptidase (gamma-GT) were quantified for 9 months. The histological study of the cells reveals that they acquired an architecture characteristic of the liver. All of the parameters indicative of hepatic function were detected throughout the 9-month period in the transplanted spleens. All the spleens, controls and transplanted, were found negative for the presence of the dedifferentiation marker gamma-GT. These results clearly indicate the possibility of recovering hepatocytes which maintain their specific functions after being subjected to a freezing-thawing process, with the corresponding implications for future clinical application as well as for hepatic biochemistry.

We have reported that a liver growth factor isolated from plasma of partially hepatectomized rats is an albumin-bilirubin complex. In this paper, we characterize the liver growth factor purified from subjects with hepatitis (h-LGF). This factor increases synthesis of DNA in a dose-dependent manner both in vivo in mouse hepatocytes, with a dose of maximal stimulation of 150 ng of h-LGF/mouse, and in vitro in rat liver cell culture, with maximal effect at 7.5 to 10 ng of h-LGF/ml. In vivo, h-LGF increases the mitotic index of mouse hepatocytes, its action being organ-specific, acting on liver, but not on spleen, kidney, lung or brain. In vitro, h-LGF stimulates the uptake of 22Na+ by hepatocytes. In addition, we carried out a study comparing it with human serum albumin in terms of absorbance, fluorescence, circular dichroism spectra, amino acid composition, tryptic maps and antigenic determinants (Ouchterlony immunodiffusion). All these tests suggested that human serum albumin is a constituent of h-LGF. Moreover, when albumin isolated from humans without hepatic pathology is incubated with bilirubin, the albumin-bilirubin complex formed mimics the activity of the human liver growth factor with respect to stimulation of DNA synthesis and the effects on the mitotic index of mouse hepatocytes in vivo. We propose that this human liver growth factor is an albumin-bilirubin complex.

The carotid rete of Artiodactyla, an intracranial arterial plexus which supplies blood to the brain, has intrigued investigators for a long time. This study was designed to examine the responsiveness of isolated retial arteries (250-700 microns in external diameter) of goat, pig, and cattle. The findings in these arteries were compared to those observed in cerebral arteries (250-650 microns in external diameter) of the same animal species. The magnitude of the arterial responses to potassium chloride varied with the resting tension applied to the tissue. The two types of vessels exhibited similar resting tension values (0.3 g) for maximal tension development in response to potassium chloride; however, the ability of retial vessels to contract in the presence of potassium chloride was consistently smaller than that of cerebral arteries. The contractile response of retial arteries to norepinephrine (10(-9) to 10(-4) M), tyramine (10(-8) to 10(-3) M), and field electrical stimulation (2-16 Hz) was negligible. The same retial arteries exhibited dose-dependent contractions in response to 5-hydroxytryptamine (10(-9) to 10(-5) M) and histamine (10(-9) to 3 X 10(-4) M). Cerebral arteries exhibited larger responses to the vasoactive agents than retial arteries. Our findings indicate that retial arteries have a small vasomotor activity in response to adrenergic stimulation or to vasoactive agents. Therefore, the carotid rete of Artiodactyla may have a low ability to change resistance to blood flow under neural or hormonal influences.

We have reported the purification and characterization of a protein that behaves as a liver growth factor, showing activity either in vivo or in vitro [Díaz-Gil et al. (1986) Biochem. J. 235, 49-55]. In the present paper, we identify this liver growth factor (LGF) as an albumin-bilirubin complex. This conclusion is supported by the results of chemical and spectroscopic characterization of this protein as well as by experiments in vivo. Incubation of albumin isolated from normal rats with bilirubin/albumin molar ratios (r) resulted (when r = 1 or 2) in a complex with liver DNA synthesis promoter activity identical with that of LGF. The exact amount of bilirubin bound to albumin was assessed by fluorescence and c.d. spectra. This albumin-bilirubin complex showed the same dose-dependence profile as LGF either at low or high dose of protein injected per mouse. Both LGF and albumin-bilirubin complex produced similar increases in the mitotic index of mouse hepatocytes in vivo. A new mechanism for the onset of the hepatic regenerative process is proposed.

A liver DNA synthesis promoter activity was detected in human plasma from subjects with hepatitis. The assay procedure consisted of intraperitoneal injection into mice of aliquots of plasma, previously chromatographed on Sephadex G-25. After 24 hr, [3H]thymidine was injected and its incorporation into liver DNA measured. The increase in [3H]thymidine uptake of injected mice was not detected in those administered plasma from normal subjects (basal [3H]thymidine incorporation was that corresponding to saline-injected mouse values). At a maximal effective dose (0.3 mg protein per mouse), plasma from subjects with hepatitis increased the mitotic index of mouse liver hepatocytes; at the same dose, plasma from normal subjects had no effect. This DNA synthesis promoter activity appears to be a protein, as it is sensitive to trypsin digestion and heat.