The development of activity of succinate dehydrogenase (EC.1.3.99.1) in the areas of the nigro-striatal pathway during the ontogenesis of a pig

The development of activity of succinate dehydrogenase was investigated by histochemical methods under the light and electron microscopes and also by biochemical methods. The studies were carried out in the areas of the nigro-striatal pathways of 7 foetal groups, two day-old piglets, three week-old piglets and adult pigs. It was demonstrated that the activity of succinate dehydrogenase appeared first in foetuses of about 86 days (180 mm). The highest activity in prenatal development appeared just before birth in about 112 days old (260 mm) foetuses. After birth in 2 day-old piglets the activity of the examined enzyme decreases, while in 3 week-old it increases however not reaching the activity level observed in adult pigs.     

Changes in the activity of various enzymes of carbohydrate metabolism in the liver and skeletal muscles of pigs during ontogenesis

Hexokinase, glucokinase, phosphofructokinase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activity was studied in the liver and musculus quadriceps femoris of 110-day foetuses 1, 2, 3, 30 and 60-day piglets and in adult pigs. The activity of all enzymes in the tissues of newborn piglets is considerably higher than in the tissues of foetuses. The activity of hexokinase in both tissues of piglets increases in the first days after birth and lowers by the one month age. The phosphofructokinase activity in the skeletal muscles and the glucokinase one in the pig liver increase during the postnatal development. The activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in both tissues of pigs increases after birth and then decreases. Glucose metabolism in the pig liver at all stages of odontogenesis proceeds more intensively by the pentose phosphate pathway, and in the skeletal muscles--by glycolytic one.     

Maturation of the human hypothalamo-hypophyseal neurosecretory system in prenatal ontogeny. A light-optical and electron microscopic study

The hypothalamic-hypophysial neurosecretory system (HHNS) was studied in the human foetuses from the age of 8 weeks till birth. The hypothalamus of 8 weeks old foetuses is weakly differentiated, no individual cell groups, so-called nuclei, are identified. The supraoptic (SON) and paraventricular (PVN) nuclei are identified from the age of 12 weeks on. The size of cell nuclei increases with the age. The Homori-positive granules were first found in some SON and PVN cell and in neurohypophysis in the 18 weeks old foetuses. It was shown under the electron microscope that the neurohypophysis of 8 weeks old foetuses consisted mainly of pituicytes with axons among the cytoplasmic processes of the latter. After the age of 10 weeks, the area of parenchyma of the neurohypophysis occupied by axons increased and typical elementary neurosecretory granules appeared in them. The data obtained are discussed with respect to the participation of HHNS in the regulation of water metabolism in the human foetuses.     

The time of appearance of blood group antigens in miniature pigs

Erythrocytes of foetuses and piglets of miniature pigs of different age were tested with 50 to 55 blood group reagents of 15 genetic systems. Out of 49 blood factors found to be present in parent animals, 40 were present in 34–46-day-old foetuses. Factor Kb was detected in 66-day-old foetuses, and other factors of the K system (Ka, Kc, Kd and Ke) at 77 days of age. Factor A was demonstrated in one day old piglets, factor 0 not earlier than in piglets aged 13 to 24 days.     

Critical periods for foetal mortality in gilts identified by analysing the length distribution of mummified foetuses and frequency of non-fresh stillborn piglets

The objective of this study was to investigate the timing of foetal mortality in gilts of a segregating F2 cross of Large White and Meishan pigs on the basis of the length distribution of mummified foetuses and the frequency of non-fresh stillborn piglets in order to establish whether critical periods for foetal mortality exist. All expelled conceptuses and placentae of 192 farrowing gilts with a normal health status were meticulously investigated to recover all mummified foetuses. The length of each mummified foetus was measured. The predicted number of foetuses present per gilt at the early foetal stage of gestation was calculated as the sum of numbers of mummified foetuses and non-fresh stillborn, fresh stillborn and liveborn piglets. Foetal loss was calculated as the sum of mummified foetuses and non-fresh stillborn piglets. The average foetal mortality rate per gilt was 8.7%. In total 162 mummified foetuses were found (average 0.84 per litter), ranging in length from 0.4 to 33.0 cm. This indicates a range in foetal age at death of approximately 35-100 days. Although mummified foetuses of all lengths within the above mentioned range were found, relatively many had a length of less than 4 cm or of 10-21 cm. The total number of non-fresh stillborn piglets (i.e. late foetal deaths) was 58 (average 0.30 per litter). It can be concluded that foetal mortality occurred in these gilts throughout the period from day 35 to term, with relatively high incidences at the early foetal stage (days 35-40), shortly after mid-pregnancy (days 55-75) and after approximately day 100 of gestation. These three periods coincide with reported periods of change in porcine placental growth.     

Appearance of the liver form of pyruvate kinase in differentiating cultured foetal hepatocytes

Abstract. From about the 16th day of gestation three forms of pyruvate kinase are present in foetal rat liver (L, R, and M2). Hepatocytes isolated from 15-day-old foetuses do not possess the liver form of pyruvate kinase, but after three days in culture this enzyme can be detected. No effect on the appearance of the enzyme could be seen by administration of insulin and fructose.
Hepatocytes isolated from 19-day-old foetuses exhibit three forms of the enzyme (L, R, and M2) on day 1 of culture but thereafter only two forms are detectable (L and M2). A decrease in activity of the L form is observed. This could be retarded by administration of insulin and fructose.     

Appearance of the liver form of pyruvate kinase in differentiating cultured foetal hepatocytes

From about the 16th day of gestation three forms of pyruvate kinase are present in foetal rat liver (L, R, and M2). Hepatocytes isolated from 15-day-old foetuses do not possess the liver form of pyruvate kinase, but after three days in culture this enzyme can be detected. No effect on the appearance of the enzyme could be seen by administration of insulin and fructose. Hepatocytes isolated from 19-day-old foetuses exhibit three forms of the enzyme (L, R, and M2) on day 1 of culture but thereafter only two forms are detectable (L and M2). A decrease in activity of the L form is observed. This could be retarded by administration of insulin and fructose.     

Biochemical and Molecular Responses to Loss of Renal Mass: Atpase and Cyclic Nucleotides: Evidence for Altered Cyclic Nucleotide Metabolism During Compensatory Renal Hypertrophy and Neonatal Kidney Growth

In adult male Sprague-Dawley rats contralateral nephrectomy was followed by an initial fall of the concentration of cGMP in renal cortical tissue followed by a rise to a peak level of 300 percent of the initial concentration within two hours. cGMP concentration in the remaining renal cortex remained at about 300 percent of the initial value during the subsequent 72 hours and slowly declined to 150-200 percent in the following two weeks. The changes in cGMP concentration were due to exactly parallel changes in the soluble fraction of renal cortical guanylate cyclase activity, while cGMP-phosphodiesterase activity remained unchanged. cAMP concentration after contralateral nephrectomy fell significantly by about 25 percent within two hours and remained below baseline level for up to eight hours. In the kidneys of newborn rats the concentration of cAMP was approximately one-half that found in adult kidneys: it slightly fell between the fourth and the seventh day after birth and subsequently continuously rose to reach adult values approximately two weeks after birth. The concentration of cGMP was significantly greater four days after birth than in adult rats, further rose between the fourth and the seventh day after birth and subsequently gradually declined to adult levels. The increased cGMP concentration appears to be due to an increase of guanylate cyclase activity in total kidney homogenates which, in turn, was mainly due to an increase of the particulate (membrane-bound) fraction of the enzyme. cGMP-phosphodiesterase activity, however, was also increased in respect to adult levels, one or three weeks after birth. Renal growth from the seventh day after birth to adulthood is accompanied by a continuous increase of the ratio cAMP/cGMP. Removal of one kidney four to seven days after birth resulted in a slower increase of this ratio. The data suggest that cGMP may trigger renal growth and that increases of cGMP concentration in the kidneys are the result of a primary increase in the activity of guanylate cyclase.     

The subcellular location, maturation and response to increased plasma glucagon of Ruthenium Red-insensitive calcium-ion transport in rat liver

1. The subcellular distribution and maturation of Ruthenium Red-insensitive Ca(2+) transport activity were determined in livers of rats ranging in age from 3 days pre-term to 10 weeks of adult life and compared with those of glucose 6-phosphatase, 5'-nucleotidase and Ruthenium Red-sensitive Ca(2+) transport. Initial rates of Ruthenium Red-insensitive Ca(2+) transport were highest in those fractions enriched in glucose 6-phosphatase, i.e. the microsomal fraction; this fraction was devoid of Ruthenium Red-sensitive Ca(2+) transport activity. Although the heaviest fraction (nuclear) contained significant amounts of 5'-nucleotidase activity it was devoid of Ruthenium Red-insensitive Ca(2+) transport activity. 2. Foetal rat liver contain minimal amounts of Ruthenium Red-insensitive Ca(2+) transport activity, glucose 6-phosphatase and 5'-nucleotidase activities. These begin to be expressed concomitantly soon after birth; Ruthenium Red-insensitive Ca(2+) transport is maximal by 3 to 4 days and remains so for up to at least 10 weeks of adult life. Glucose 6-phosphatase also reaches a peak at 3-4 days, but then rapidly decreases to approach adult values. Maximal activity of 5'-nucleotidase in the microsomal and nuclear fractions is seen about 4-6 days after birth; this enzyme activity remains increased for up to about 10 days and then falls, but not as rapidly as glucose 6-phosphatase. It is tentatively suggested that the bulk of the Ruthenium Red-insensitive Ca(2+) transport is attributable to the system derived from the endoplasmic reticulum. 3. Administration of glucagon to adult rats enhances by 2-3-fold the initial rate of Ruthenium Red-insensitive Ca(2+) transport in the intermediate but not the microsomal fraction. The hormone-induced effect is fully suppressed by co-administration of puromycin, is dose-dependent with half-maximal response at approx. 1mug of glucagon/100g body wt. and time-dependent exhibiting a half-maximal response about 1h after administration of the hormone. 4. Ruthenium Red-insensitive Ca(2+) transport in the post-mitochondrial fraction of foetal liver also responds to the administration in situ of glucagon. The response, which also is prevented by co-administration of puromycin, is maximal in those foetuses nearing term. The suggestion is made that these effects of the hormone on Ruthenium Red-insensitive Ca(2+) transport are an integral part of the physiological network in the liver cell.     

Ontogeny of the expression of some catecholamine synthesising enzymes in the female porcine preoptic area

Ontogeny of the catecholaminergic system of the preoptic area (PA) was studied in various animal species including mice, rats, cats and lower vertebrates. Until now, there has been no data about development of catecholaminergic structures in the porcine PA. To study this problem, hypothalami from six groups of animals were collected. Three groups of foetuses (70, 84 and 112 days old) and three groups of female pigs (1 day, 10 weeks and 7-8 months old) were used. Nerve structures immunoreactive for the studied substances: tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DbetaH) and phenylethanoloamine-N-metylthransferase (PNMT) were observed in different periods. In PA, TH-IR (immunoreactive) structures appeared before 70th day of foetal life, DbetaH-IR between 70th and 84th day of foetal live and PNMT-IR only in 10-week old and adult animals. In the PA of 70-day old foetuses, single smooth and varicose nerve fibres immunoreactive only to TH were found. In PA of 84-day old foetuses, additionally, single nerve cell bodies immunoreactive to TH were shown and some of them also contained immunoreactivity to DbetaH. In PA of 1-day old piglets, moderate numbers of nerve fibres immunoreactive to TH and only single TH/DbetaH-IR nerve terminals were observed. TH-IR nerve cell bodies were also moderate in number and many of them contained simultaneously immunoreactivity to DbetaH. In PA of 10-week old pigs, a moderate number of immunopositive nerve fibres was observed. They contained mainly TH, but part of them stained also for TH/DbetaH. Only very few nerve fibres containing exclusively DbetaH were found. These nerve terminals were observed in a close vicinity of blood vessels. In PA, moderate numbers of TH-IR nerve cell bodies were found, some of them contained also immunoreactivity to DH but never to PNMT. Perikarya containing PNMT were TH-negative. In the PA of sexually mature sows, additional, single, large nerve cell bodies (about 35 microm in a diameter) containing TH only were found. In many cases, TH- and DbetaH-IR "basket-like" structures surrounding nerve cell bodies were seen, suggesting an influence of those fibres on the neuronal activity.     

Perinatal development of glucose-6-phosphatase and fructose-1,6-diphosphatase activities in pig liver

The activity of glucose-6-phosphatase (G6Pase) and fructose-1,6-bisphosphatase (FDPase) was determined in the homogenate of the liver of 69 pig fetuses during the last third of gestation (80th to 114th day), 47 piglets from birth to 4 weeks old (suckling period) and to slaughter pigs. G6Pase is evident in fetal liver at an early date and raises steadily during gestation. In newborn piglets, the enzyme activity increases rapidly during the first hours of life and remains at this high level during the first week of life. Afterwards the enzyme activity returns to birth level, which exists also in pigs at slaughtering. The activity of FDPase is constant during the fetal period. After birth enzyme activity rises at a lower rate than the G6Pase during the first week of life. This level remains constant during the suckling period and increases thereafter until the time of slaughtering of pigs. The role of hormones in the perinatal development of these enzymes is described. Probably, thyroxine causes the prenatal increase of the activity of both the enzymes. The rapid postnatal rise of G6Pase activity may be induced by the high level of hydrocortisone at parturition, and furthermore, glucagon may have a permissive effect.     

Cholesterol 7 alpha-hydroxylase activity and bile acid synthesis in hepatocytes of unweaned and weaned pigs in monolayer culture

Activity of cholesterol 7 alpha-hydroxylase (EC 1.14.13.17) in freshly isolated hepatocytes from unweaned piglets (2 to 3 weeks old) was 16-times lower as compared to hepatocytes from weaned piglets (7 to 8 weeks old). The monolayer culture activity of the enzyme remained low in unweaned piglet hepatocytes. In contrast, in cultured hepatocytes from weaned piglets, cholesterol 7 alpha-hydroxylase activity declined during the first day of culture, but was restored during the next 2 culture days, provided that fetal bovine serum (10%) was added to the culture medium. Addition of dexamethasone (50 nM) and insulin (135 nM) to the medium, further enhanced cholesterol 7 alpha-hydroxylase activity to values similar to those in freshly isolated hepatocytes and retarded the decline of enzyme activity after the 3rd culture day. Cultured hepatocytes from weaned and unweaned piglets synthesized similar types of bile acids from [14C]cholesterol, among which hyocholic acid (the most prominent), hyodeoxycholic acid, chenodeoxycholic acid, murocholic acid and lithocholic acid could be identified. 95% of radiolabelled bile acids synthesized was conjugated, mainly with glycine, but also with taurine, sulfate and glucuronic acid. The rate of mass production of bile acids by cultured hepatocytes of weaned piglets (as measured by gas-chromatography) parallelled cholesterol 7 alpha-hydroxylase activity, and was low in the absence of serum, but increased in medium containing fetal bovine serum, dexamethasone and insulin to a rate lying in the range of 75% of the in vivo bile acid production during the 3rd culture day. Bile acid production by unweaned piglet hepatocytes was 3-times lower under these conditions. It is concluded that hepatocytes from young weaned pigs cultured in medium containing 10% fetal bovine serum, offer a suitable in vitro model for the study of bile acid synthesis, in view of the high cholesterol 7 alpha-hydroxylase activities and bile acid production rates.     

Cholesterol 7α-hydroxylase activity and bile acid synthesis in hepatocytes of unwearied and weaned pigs in monolayer culture

Activity of cholesterol 7α-hydroxylase (EC 1.14.13.17) in freshly isolated hepatocytes from unweaned piglets (2 to 3 weeks old) was 16-times lower as compared to hepatocytes from weaned piglets (7 to 8 weeks old). The monolayer culture activity of the enzyme remained low in unweaned piglet hepatocytes. In contrast, in cultured hepatocytes from weaned piglets, cholesterol 7α-hydroxylase activity declined during the first day of culture, but was restored during the next 2 culture days, provided that fetal bovine serum (10%) was added to the culture medium. Addition of dexamethasone (50 nM) and insulin (135 nM) to the medium, further enhanced cholesterol 7α-hydroxyease activity to values similar to those in freshly isolated hepatocytes and retarded the decline of enzyme activity after the 3rd culture day. Cultured hepatocytes from weaned and unweaned piglets synthesized similar types of bile acids from [¹⁴C]cholesterol. among which hyocholic acid (the most prominent), hyodeoxycholic acid, chenodeoxycholic acid, murocholic acid and lithocholic acid could be identified. 95% of radiolabelled bile acids synthesized was conjugated, mainly with glycine, but also with taurine, sulfate and glucuronic acid. The rate of mass production of bile acids by cultured hepatocytes of weaned piglets (as measured by gas-chromatography) parallelled cholesterol 7α-hydroxylase activity, and was low in the absence of serum, but increased in medium containing fetal bovine serum, dexamethasone and insulin to a rate lying in the range of 75% of the in vivo bile acid production during the 3rd culture day. Bile acid production by unweaned piglet hepatocytes was 3-times lower under these conditions. It is concluded that hepatocytes from young weaned pigs cultured in medium containing 10% fetal bovine serum, offer a suitable in vitro model for the study of bile acid synthesis, in view of the high cholesterol 7α-hydroxylase activities and bile acid production rates.     

Decline with age in the rate of reduction of progesterone to 20 alpha-hydroxypregn-4-en-3-one in the blood of perinatal ruminants

The activity of the enzyme 20 alpha-hydroxysteroid dehydrogenase present in erythrocytes of foetal and new-born ruminants has been determined by incubating 0.1 ml blood with 0.16 mumol [4-14C]-progesterone for 15 min at 39 degrees C in a final volume of 2 ml buffered saline. It was found that the activity, measured as mumol 20 alpha-hydroxypregn-4-en-3-one produced from progesterone per millilitre of erythrocytes per hour, declined from levels at birth as high as 1.50 mumol for sheep, 0.50 mumol for goats and 0.43 mumol for cattle to levels of around 0.11, 0.08 and 0.04 mumol respectively by 30-60 days of age. This decline in activity was also apparent in blood taken from sheep foetuses in which longitudinal studies were possible and appeared to have begun prior to 35 days before term. The highest activity obtained was 2.59 mumol for foetal sheep blood taken at 115 days of gestation. It is suggested that the observed decline in 20 alpha-hydroxysteroid dehydrogenase activity is a function of the replacement of foetal erythrocytes with adult-type erythrocytes which begins around 120 days of gestational age and that the role of the enzyme is to maintain an appropriate progestational environment within the foetoplacental unit.     

Determination and postnatal development of fructokinase activity in swine liver]

Starting from the spectrophotometric method, described in ADELMAN et al., optimal reaction conditions for the measurement of fructokinase (ketohexokinase) in pig liver were systematically studied. It was necessary to increase the concentration of the substrate and further to lower the concentration of ATP for an optimal Mg: ATP-radio of 2:1. Using the optimized method fructokinase activity was determined in pig liver in relation to age, beginning from the last days of pregnancy to puberty. In liver of fetuses and newborn piglets during the first two days of life no or only a minute activity of the fructokinase was recorded. Therefore, the high level of fructose in fetal blood results from the inability of the fetus to metabolize fructose synthezised in the placenta or the fetal organs. At the end of the first week of life the activity of fructokinase was 10 times, after the second week 15-20 times higher than at birth. This high level remains constant during the suckling period and after weaning. For this reason, piglets after the first week of life are able to metabolize fructose and after the third week to form glucose and to release it into circulation. In adult pigs the activity of fructokinase in the liver decreases slightly. It corresponds-as in rat and human-to the elimination rate of experimentally applied fructose from the circulation. Therefore, this enzyme even in pigs is of significant importance for the utilization of fructose.     

Unusual outcome of in utero infection and subsequent postnatal super-infection with different PCV2b strains

VC2002, isolated from postweaning multisystemic wasting syndrome (PMWS)-affected pig, is a mixture of two porcine circovirus genotype 2b (PCV2b) viruses, K2 and K39. Preliminary experiments disclosed short-term adverse effects of K39, but not K2, on porcine foetuses. These findings led to the hypothesis that infection of immuno-incompetent foetuses with K2 confers a status of immunotolerance, and postnatal super-infection with K39 triggers PMWS. To explore this hypothesis, nine 55-day-old foetuses were inoculated in utero (three with K2-10^4.3TCID₅₀, three with K39-10^4.3TCID₅₀ and three with medium), and foeto-pathogenicity examined. At 21 days post-inoculation (dpi), K2 did not induce pathology, whereas pathological effects of K39 were evident. Twenty-four 45-day-old foetuses were subsequently inoculated to examine the long-term effect of K2, including six with K2-high dose-10^4.3TCID₅₀, six with K2-low dose-10^2.3TCID₅₀ and 12 mock-inoculated controls. Both doses resulted in five mummified foetuses and one live-born piglet each (69dpi). K2 was recovered from all mummies. K2 and K2-specific antibodies were not detected in serum of the two live-born piglets at birth, indicating full control of K2 infection. The K2-low dose-infected piglet was immunostimulated at day 2, but not the K2-high dose-infected piglet. Both non-stimulated and stimulated K2-infected piglets were super-inoculated with K39 at day 6 or 8 (taken as 0 days post super-inoculation). Low viral replication was observed in the non-stimulated K2-K39 piglet (up to 10^3.3TCID₅₀/g; identified as K39). In contrast, viral replication was extremely high in the stimulated K2-K39 piglet (up to 10^5.6TCID₅₀/g) and identified as K2, indicating that K2 infection is controlled during foetal life, but emerges after birth upon immunostimulation. However, none of the piglets showed any signs of PMWS.     

Peculiarities of proteasome pool formation in rat spleen and liver during postnatal development

Changes in the specific activity and amounts of 26S and 20S proteasome pools in rat spleen and liver during postnatal development and appearance in them of immune subunits were studied. Two decreases in chymotrypsin-like activity of the proteasome pools were recorded during the first three weeks after birth. The activity minimum fell on the 11th and 19th days, and the first decrease was more prolonged and pronounced than the second. The decrease in the specific activity of the 26S proteasome pools was associated with a reduction of their quantity. The 20S proteasome pools displayed no such decreases. Noticeable quantities of immune subunits LMP7 and LMP2 were revealed by Western blotting in the spleen on the 7th day and on the 19th day in the liver, concurrently with the beginning of the decrease in the proteasome activity. It was concluded that during the first three weeks of postnatal development the proteasome pools in rat spleen and liver were replaced twice, and in the spleen (a lymphoid organ) a qualitatively new pool containing immune subunits appeared nearly two weeks earlier than in the liver (a non-lymphoid organ). The appearance of immune proteasomes in different organs and tissues during some weeks after birth seems to explain the immune system inefficiency during embryogenesis and early postnatal development.     

Activity of selenium-dependent enzymes in erythroid cells of animals in the postnatal period

The age dynamics of selenium-dependent enzymes glutathione peroxidase and thyroxine-5'-deiodinases type I and II (D1 and D2 respectively) in bone marrow erythroblasts of new-born, 1-, 3-, 5- and 10-day old piglets as well as influences of hormones (thyroxine, cortisol) selenium and iron on enzyme activities were investigated. The enzyme activities in the pig erythroid cells were established to increase after birth. D1 activity increased in erythroblasts of 3-day old piglets, while augmentation of D2 activity was significant in the cells of 10-day old animals. Glutathione peroxidase and thyroxine-5'deiodinase activities in pig erythroblasts decreased under the influence of thyroxine and hydrocortisone in vivo, and increased after injections of sodium selenite. For comparison, activities of 5'-deiodinases in the cells of some other tissues were also investigated.