Maternal Coordination of the Daily Rhythm of Malate Dehydrogenase Activity in Testes from Young Rats: Effect of Maternal Sympathetic Denervation of the Pineal Gland and Administration of Melatonin

Chronic sympathetic denervation of the pineal gland by bilateral removal of the superior cervical ganglia (SCG) was performed on female rats 30 days before impregnation. The offspring, maintained in the dark from birth, had disruption of the malate dehydrogenase circadian rhythm in the testes at 25 days of age. A daily injection of melatonin (1 mg/kg s.c. at 10:00 or 18:00 h) to denervated mothers from the 14th day of pregnancy up to the 10th day postpartum produced one daily phase in the enzyme activity of testes in the offspring. Entrainment of daily enzyme activity also was obtained when the hormone was administered orally to the pups during the postnatal period or when pups were reared by intact (not denervated) foster mothers. The results indicate the involvement of the maternal pineal gland in the maternal transfer of photoperiodic information necessary for the coordination of the circadian system in young rats.     

Prenatal Entrainment of Rat Testicular Malate Dehydrogenase Activity Circadian Rhythm

In mammals the photoperiodic synchronization of circadian system starts before birth. During fetal and neonatal period mothers relay the photoperiodic information to their litter. The maternal pineal melatonin 24 h cycle acts as a synchronizing signal. We have studied the effect of pineal maternal sympathetic denervation and administration of melatonin to mothers denervated during gestation on the prenatal synchronization of testicular malate dehydrogenase (MDH) activity circadian rhythm of the offspring 25 days after birth. When mothers were denervated at the 7th, 10th or 11th day of gestation, pups showed disruption of testicular MDH activity circadian rhythms. In contrast, no disruptive effect was observed when the mothers were denervated on the 12th or 14th day of gestation. When denervated mothers (7th day of gestation) were treated with a daily dose of melatonin from the 11th to the 14th day of gestation, pups showed a MDH activity circadian rhythm. The hormone failed to impose a daily phase when administered from the 9th to the 12th day of gestation. Results suggest that prenatal synchronization in the rat occurs very early in the development, before suprachiasmatic nuclei morphologic arrangement and functional activity begin.     

Prenatal Entrainment of Rat Testicular Malate Dehydrogenase Activity Circadian Rhythm

In mammals the photoperiodic synchronization of circadian system starts before birth. During fetal and neonatal period mothers relay the photoperiodic information to their litter. The maternal pineal melatonin 24 h cycle acts as a synchronizing signal. We have studied the effect of pineal maternal sympathetic denervation and administration of melatonin to mothers denervated during gestation on the prenatal synchronization of testicular malate dehydrogenase (MDH) activity circadian rhythm of the offspring 25 days after birth. When mothers were denervated at the 7th, 10th or 11th day of gestation, pups showed disruption of testicular MDH activity circadian rhythms. In contrast, no disruptive effect was observed when the mothers were denervated on the 12th or 14th day of gestation. When denervated mothers (7th day of gestation) were treated with a daily dose of melatonin from the 11th to the 14th day of gestation, pups showed a MDH activity circadian rhythm. The hormone failed to impose a daily phase when administered from the 9th to the 12th day of gestation. Results suggest that prenatal synchronization in the rat occurs very early in the development, before suprachiasmatic nuclei morphologic arrangement and functional activity begin.     

Prenatal and Neonatal Diazepam Administration Synchronizes Testicular Malate Dehydrogenase Circadian Rhythms in Young Rats

Rat or hamster pups exposed to constant light or darkness since birth exhibit many circadian rhythms synchronized with those of the mother. During early development, a number of cues derived from the maternal circadian system synchronize the fetal and neonatal circadian clock. Maternal pineal sympathetic denervation during early pregnancy disrupts maternal synchronization of parotid α-amylase and testicular malate dehydrogenase circadian rhythms in rat pups. Maternal pineal sympathetic denervation was used to study potential agents able to synchronize the fetal or neonatal circadian clock. Melatonin injection to denervated pregnant mothers prevents the pineal sympathetic denervation effect on those circadian rhythms. We now studied the synchronizing effect of a benzodiazepine compound, diazepam. This GABA_A agonist synchronized testicular malate dehydrogenase (MDH) activity of pups when it was injected to sympathetic denervated pregnant dams (a daily dose at 07:00 or 19:00 h from the 14 th to the 20 th day of gestation) or orally administered to the pups (a daily dose at 19:00 h from the 10 th to 24 th day of life). Co-injection of diazepam and GABA_A antagonist, flumazenil, blocked the synchronizing effect of diazepam. The results demonstrate that diazepam has a synchronizing effect on the development of the circadian clock in rats and suggest that modulation of maternal GABA_A could participate in mammalian maternal synchronization.     

Influence of the pineal gland on testicular function in offspring of pinealectomized rats

Female Sprague-Dawley rats exposed to a short (6L:18D) photoperiod from 21 days of age were mated when they reached 55 days of age. On Day 2 of gestation animals were pinealectomized or sham-operated. On Day 5 after birth male pups of the two groups of dams were either pinealectomized or sham-operated. They were killed at 42 and 49 days of age. In offspring born to sham-operated dams and in those born to pinealectomized mothers, neonatal pineal ablation resulted in increased testicular testosterone and androstenedione content. In sham-operated and neonatally pinealectomized rats removal of the maternal pineal gland induced a decrease in testicular testosterone and androstenedione content. In contrast, after maternal pinealectomy there was a decrease in plasma testosterone and dihydrotestosterone values and testicular dihydrotestosterone content in sham-operated rats but not in those neonatally pinealectomized. We conclude that (1) the pineal glands of the mother and offspring are required to maintain normal testicular testosterone and androstenedione content in the rat, and (2) the pineal of the offspring influences the inhibitory effects of maternal pinealectomy on testicular dihydrotestosterone content and on plasma testosterone and dihydrotestosterone concentration in the offspring.     

Androgenic activity in 15-day-old male rats: role of the maternal pineal gland.

Female Sprague-Dawley rats, exposed to a long (18L:6D) or a short (6L:18D) photoperiod from 21 days of age, were mated when they reached 55 days of age. On Day 2 of gestation, dams were pinealectomized or sham-operated. Pre- and postnatal photoperiods were identical, and offspring were killed at 15 days of age. Maternal pinealectomy had no effect when rats were kept on 18L:6D. Rats born to sham-operated mothers and kept on 6L:18D had higher testicular testosterone and androstenedione content than offspring raised on the long photoperiod. This stimulatory effect of the short photoperiod was blocked by maternal pinealectomy and was not dependent on the offspring's own pineal since it was observed in both sham-operated and neonatally (on Day 5 after birth) pinealectomized rats. When sham-operated mothers housed on 18L:6D were treated daily during pregnancy and lactation by s.c. melatonin injection, there was an increase in the testicular testosterone content of offspring. It was concluded that when rats are maintained on a 6L:18D cycle the maternal pineal gland enhances the testicular testosterone and androstenedione content in 15-day-old offspring. This effect is probably mediated by maternally derived melatonin. At 15 days of age, the pineal of the offspring had no influence on testicular function.     

Maternal stress induces adult reduced REM sleep and melatonin level

We have previously reported that neonatal maternal deprivation (MD) resulted in a decrease of total sleep and an increase of orexin A in adult rats. Now, we characterized features of sleep, activity, and melatonin levels in rats neonatally treated with MD and control (MC) procedures. Adult male Sprague-Dawley rats were treated with either MD or MC procedures for 10 days starting at postnatal day 4. At 3 months of age, sleep was recorded for 48 h in one set of MD and MC rats, while another set of MD and MC rats was measured for locomotor activity (under LD = 12:12). Melatonin levels in the blood, pineal gland, and hypothalamus were measured as well as clock protein level in the hypothalamus. Compared to the MC rats, REM sleep in the MD rats was significantly reduced in the light periods but not in the dark periods. Both quiet wake and total wake in the MD rats were significantly increased during the light period compared to the MC rats. The weight of the pineal gland of the MD rats was significantly smaller than in MC rats. Melatonin levels of the MD group were significantly reduced in the pineal gland and hypothalamus compared to the MC group. No significant difference was identified between groups in the expression of the clock protein in the hypothalamus. Neonatal MD resulted in reduced REM sleep and melatonin levels, without changes of circadian cycle of locomotor activity and levels of clock protein.     

Maternal LPS Exposure during Pregnancy Impairs Testicular Development,Steroidogenesis and Spermatogenesis in Male Offspring

Lipopolysaccharide (LPS) is associated with adverse developmental outcomes including embryonic resorption, fetal death, congenital teratogenesis and fetal growth retardation. Here, we explored the effects of maternal LPS exposure during pregnancy on testicular development, steroidogenesis and spermatogenesis in male offspring. The pregnant mice were intraperitoneally injected with LPS (50 µg/kg) daily from gestational day (GD) 13 to GD 17. At fetal period, a significant decrease in body weight and abnormal Leydig cell aggregations were observed in males whose mothers were exposed to LPS during pregnancy. At postnatal day (PND) 26, anogenital distance (AGD), a sensitive index of altered androgen action, was markedly reduced in male pups whose mothers were exposed to LPS daily from GD13 to GD 17. At PND35, the weight of testes, prostates and seminal vesicles, and serum testosterone (T) level were significantly decreased in LPS-treated male pups. At adulthood, the number of sperm was significantly decreased in male offspring whose mothers were exposed to LPS on GD 13–17. Maternal LPS exposure during gestation obviously diminished the percent of seminiferous tubules in stages I–VI, increased the percent of seminiferous tubules in stages IX–XII, and caused massive sloughing of germ cells in seminiferous tubules in mouse testes. Moreover, maternal LPS exposure significantly reduced serum T level in male mice whose mothers were exposed to LPS challenge during pregnancy. Taken together, these results suggest that maternal LPS exposure during pregnancy disrupts T production. The decreased T synthesis might be associated with LPS-induced impairments for spermatogenesis in male offspring.     

Neonatal Hormone Treatment and Maturation of the Pineal Noradrenergic System: Hydrocortisone and Thyroxine

Abstract The circadian release of norepinephrine from nerve terminals in the pineal gland drives acetyl-CoA:serotonin N -acetyltransferase (NAT; EC 2.3.1.5) activity in the adult pineal from a daytime low to a nighttime high. In the newborn, enzyme activity is intermediate between the adult's daily extremes and has only a small circadian fluctuation. With age, these fluctuations increase in amplitude until the adult pattern is attained at about days 10–12. Treatment of neonates with thyroxine for the first 3 days of life accelerated, whereas administration of hydrocortisone acetate at birth retarded the developmental decline in daytime serotonin-N-acetyltransferase activity. Maximal differences in daytime enzyme activity of controls and thyroxine-treated animals were seen at day 4 and between controls and steroid-treated pups at day 8. Desipramine treatment increased NAT activity in 8-day-old animals; hydrocortisone-treated animals were least affected. Freshly cultured pineals from steroid-treated animals were more responsive to low, and less responsive to high, concentrations of norepinephrine than glands from thyroxine-treated or control animals. They were also less responsive to isoproterenol both in acute and 48-h organ culture. Pineals from hydrocortisone-treated animals in culture accumulated less exogeneous norepinephrine than glands from controls but released a greater fraction of their content on transfer to fresh medium. Normal and steroid-treated animals released the same fraction of their norepinephrine contents into the medium when reuptake was blocked by desipramine (DMI).     

Maternal adrenalectomy affects development of adrenal medulla

This work investigates the effects of maternal adrenalectomy (ADX) on the development of the adrenal medulla. Adrenal catecholamines (AC) were measured at postnatal day (PN) 1, 8, 12 and 22 in rat offspring of ADX dams and in pups of control dams. The pups of ADX rats showed a reduction in AC concentrations in the adrenal medulla at PN 1, 12 and 22, although these were higher than in the pups of sham dams at PN 8. Further, in the pups of control mothers, there was an increase in ACs during the first two weeks of life whereas pups of ADX mothers only showed increases in noradrenaline, dopamine and adrenaline levels at day 8. These results suggest that maternal absence of corticosterone affects the medulla catecholamine content during development. These data support the idea that a maternal glucocorticoids are involved in the differentiation or/and maturation of the adrenal medulla.     

Maternal entrainment of tau mutant hamsters.

Maternal entrainment of the circadian wheel-running activity rhythm was examined in Syrian hamsters heterozygous for a single gene mutation (tau) that affects the free-running period of circadian rhythms. Heterozygous tau pups were born to and raised by wild-type mothers under constant dim light. The pups' wheel-running activity was recorded after weaning on postnatal day 18 or 24. Pups weaned on day 18 had an average free-running period of 21.70 hr, demonstrating that the tau phenotype was fully expressed at this age. Using the activity onset of the postnatal free-running rhythms as a phase reference, we estimated the phase relationships between the pups and their mothers on days 18 and 24. In contrast to results with wild-type pups, the activity rhythms of tau pups were not in phase with the rhythms of their wild-type mothers; that is, activity onsets of mothers and pups did not coincide. The pups did, however, show synchrony among themselves, indicating that they had been exposed to a synchronizing signal sometime during development. It is likely that this synchronizing signal was provided by the mothers, since pups from different litters showed phase relationships similar to those of their mothers. Thus the mothers provided a signal that was sufficient to cause entrainment, despite the 2-hr difference in free-running period between the mothers and pups. Although the pups' activity rhythms appeared to have been entrained by the mothers, they were clearly free-running by postnatal day 18. The mechanism for entrainment is lost during the course of development, despite continued interaction between the mothers and pups.     

Maternal melatonin influences rates of somatic and reproductive organs postnatal development of male rat offspring

Female rat dams, housed in 12L:12D photoperiod, were pinealectomized or injected daily 1(1/2) h before onset of darkness with 250 mg melatonin/100 g BW., during pregnancy; control and pinealectomized dams received a placebo. Somatic, reproductive organs and gonadotropins levels luteinizing hormone (LH) and follicle stimulating hormone (FSH) of male offspring were examined at the following phases of their sexual development: neonate, infantile, juvenile or prepubertal and pubertal periods. Pinealectomy of the mother produced an altered developmental pattern in the offspring (PIN-X offspring). During the infantile period when pups are lacking maternal melatonin and their own melatonin rhythm is not yet established, a delayed growth of body and testis weights was observed. After the second week of life, from 15 to 25 days of age, coinciding with the initiation of the melatonin rhythm, a speed-up growth of body and testes was observed, followed by a delayed growth from 25 to 30 days, in the juvenile period; this also coinciding with reduced LH levels observed at 30 days of age. Indeed, in PIN-X offspring significantly greater growth rate was observed during the pubertal period than in control offspring, which could be due to the increase in LH secretion up to normal values observed in the PIN-X offspring. Seminal vesicles of the PIN-X offspring also showed delayed growth, which was overcome at the pubertal period. Melatonin (MEL) treatment during pregnancy produced minor alterations in postnatal development of the reproductive tract. Only increased pituitary gland weight was observed at 15 and decreased at 25 days of age. At 25 days of age, MEL offspring reached the highest LH values, and at 30 days of age, PIN-X offspring still show low values. Which suggests that other factors than the endocrine activity of the gland are affecting the somatic growth of the pituitary gland. Seminal vesicles weight was delayed at 25 days of age in the MEL offspring. These results indicate that maternal melatonin is necessary for a normal somatic growth and postnatal development of reproductive organs of the offspring.     

Maternal transfer of melatonin alters the growth and sexual maturation of young Indian palm squirrel Funambulus pennanti

To date, the phenomenon of maternal transfer of hormones to the young is an enigma. The present study explains for the first time the maternal transfer of melatonin (MEL) to the young, affecting neonatal growth and sexual maturation. The suckling pups of MEL-treated mothers exhibited significant decreases in body, testicular, vas deferens (male pups), ovarian and uterine (female pups) weights and increases in pineal gland activity along with high plasma MEL levels. The plasma level of testosterone decreased significantly in male pups, while estradiol increased and progesterone decreased in female pups of MEL-treated mothers. These results clearly suggest that MEL could be transported from the mothers to their young postnatally via the milk in order to influence neonatal growth and sexual maturation. Our results support the earlier concept and show for the first time that MEL can be transported from the mother to the young either prenatally through the placenta or postnatally via the milk. Therefore, maternal MEL can act as a biological signal for neonatal growth and sexual maturation.     

Maternal Depression Model: Long-Lasting Effects on the Mother Following Separation from Pups

This study was carried out to ascertain the effects of maternal separation (3 h per day) of mothers from their pups in the neonatal period in rats, which has been suggested to induce a depressive-like state, would have long lasting effects on different parameters including hippocampal Na(+), K(+)-ATPase activity, NO production, free radical production and antioxidant enzymes activities in dams. Fourty-eight Wistar rats were divided into 3 groups: control, brief separation (10 min) and long separation (3 h). The neonatal interventions were done on postpartum days 1-10. At 35 days post-partum the dams were killed and the hippocampal Na(+), K(+)-ATPase activity were measured, as well as the activity of the antioxidant enzymes catalase, glutathione peroxidase, superoxide dismutase, free radicals production, and the production of nitric oxide. Hippocampal Na(+), K(+)-ATPase activity was decreased in the brief separated group and in dams subjected to 3 h separation from their pups. A reduction in nitric oxide levels in the hippocampus in dams of the long separated group was also observed. It is concluded that the withdrawal of pups from their mothers make the mothers more susceptible to the development of neurochemical alterations that could be related to depressive features.     

Seasonal variations of gonadotropins and prolactin in the laboratory rat. Role of maternal pineal gland

The laboratory rat, a non-photoperiodic rodent, exhibits seasonal fluctuations of melatonin. Melatonin has been found to be readily transferred from the maternal to the fetal circulation. No data exist on the possible influence of maternal pineal gland upon seasonal variations of the offspring. The aim of the present study was to asses the influence of the maternal melatonin rhythm on the offspring postnatal development of the reproductive hormones LH, FSH and prolactin. Male offspring from control, pinealectomized (PIN-X) and PIN-X + melatonin (PIN-X+MEL) mother Wistar rats were studied at 21, 31, and 60 days of age. Seasonal age-dependent variations were found for all hormones studied in control offspring but PIN-X offspring showed a tendency to have reduced duration or altered seasonal variations. Maternal melatonin treatment to PIN-X mothers partially restored the effect of pinealectomy. The chronological study of LH, FSH, and prolactin in PIN-X offspring also showed an altered pattern as compared to control-offspring. Melatonin treatment to the mothers partially restored the developmental pattern of reproductive hormones. Results of this study indicate that maternal pineal gland of the laboratory rat is involved in the seasonal postnatal development variations of reproductive hormones of the offspring.     

Effects of prolactin deficiency during the early postnatal period on the development of maternal behavior in female rats: Mother's milk makes the difference

During early life, prolactin (PRL) ingested by the pups through the milk participates in the development of neuroendocrine, immunological and reproductive systems. The present study tested whether a deficiency in PRL in the dam's milk during early lactation affected the offspring in terms of the maternal responsiveness in the sensitization paradigm and behavioral response to a novel environment in the offspring. Thus, lactating rats were injected (sc) on postnatal days (PND) 2–5 with bromocriptine (125 μg/day), bromocriptine + ovine PRL (125 μg + 300 μg/day), or vehicle. As juveniles (at PND 24) or adults (PND 90–100), one female from each litter was exposed to 5 foster pups continuously for 8 days and their maternal responsiveness was recorded. Female offspring were also tested in an open field arena. Adult, but not juvenile, female offspring of bromocriptine-treated mothers showed an increased latency to become maternal, in comparison to latencies displayed by the offspring of control mothers. Furthermore, the proportion of adult, but not juvenile, offspring of bromocriptine-treated mothers that became maternal was lower than that showed by the offspring of vehicle-treated mothers. In comparison to female offspring of vehicle-treated mothers, female offspring of bromocriptine-treated mothers spent less time hovering over the pups (as juvenile females), body licking (as both juvenile and adult females), and in close proximity to pups (as adult females) during the maternal behavior test. Simultaneous administration of ovine PRL and bromocriptine reversed almost all the negative effects of bromocriptine. These data suggest that maternally-derived PRL participates during the early postnatal period in the development of neural systems that underlie the control of maternal behavior.     

Different uptake and handling of oleoyl-estrone by fetuses and neonatal rats.

To determine whether oleoyl-estrone can be transferred from mothers to their offspring either during pregnancy or lactation, a gavage of tracer dose of (3)H-Oleoyl-estrone was given to 21-day pregnant rats and to lactating rats on day 15 after delivery. In pregnant rats, the label was found in maternal blood as well as in liver and fetal serum, the latter showing the highest specific activity observed. In lactating rats, oleoyl-estrone label was found both in the mammary gland and maternal serum; in the pups, label was found in their stomach contents (i.e., clotted milk) and serum. The results suggest that the placenta effectively blocks the passage of oleoyl-estrone to the fetuses probably because of its high esterase activity. On the other hand, oleoyl-estrone is easily transferred from dams to pups, as a component of milk.     

Maternal obesity disrupts circadian rhythms of clock and metabolic genes in the offspring heart and liver

Early life nutritional adversity is tightly associated with the development of long-term metabolic disorders. Particularly, maternal obesity and high-fat diets cause high risk of obesity in the offspring. Those offspring are also prone to develop hyperinsulinemia, hepatic steatosis and cardiovascular diseases. However, the precise underlying mechanisms leading to these metabolic dysregulation in the offspring remain unclear. On the other hand, disruptions of diurnal circadian rhythms are known to impair metabolic homeostasis in various tissues including the heart and liver. Therefore, we investigated that whether maternal obesity perturbs the circadian expression rhythms of clock, metabolic and inflammatory genes in offspring heart and liver by using RT-qPCR and Western blotting analysis. Offspring from lean and obese dams were examined on postnatal day 17 and 35, when pups were nursed by their mothers or took food independently. On P17, genes examined in the heart either showed anti-phase oscillations (Cpt1b, Pparα, Per2) or had greater oscillation amplitudes (Bmal1, Tnf-α, Il-6). Such phase abnormalities of these genes were improved on P35, while defects in amplitudes still existed. In the liver of 17-day-old pups exposed to maternal obesity, the oscillation amplitudes of most rhythmic genes examined (except Bmal1) were strongly suppressed. On P35, the oscillations of circadian and inflammatory genes became more robust in the liver, while metabolic genes were still kept non-rhythmic. Maternal obesity also had a profound influence in the protein expression levels of examined genes in offspring heart and liver. Our observations indicate that the circadian clock undergoes nutritional programing, which may contribute to the alternations in energy metabolism associated with the development of metabolic disorders in early life and adulthood.     

Malate Dehydrogenase Activity of Rat Testis: Circadian Rhythm and its Ontogeny

Malate dehydrogenase activity and soluble protein content in testes from rats exposed to a 14:00 h light:10:00 h dark photoperiod, have been determined every two or four hours over a 24 hour period in 5, 15, 25 and 120 day-old rats. By using the Cosinor method, the ontogeny of an unimodal rhythm was studied for MDH activity and soluble protein content in testis. In 5 and 15 day-old rats, the MDH acrophases were recorded around 19:00 h and 17:00 h, respectively. Rats aged 25 and 110 days showed the MDH acrophases during the dark period. An inversion of the MDH circadian rhythms was detected in 25 day-old compared to those of 5 and 15 day-old rats. An inversion of the protein circadian rhythm was also detected at 15 days compared to that at 5 days. These inversions persist in the adult rats. The amplitude of the MDH and protein rhythms reached the lowest value in adulthood. The mean daily value of testicular MDH increased between day 5 and 15, decreasing at day 35 and remaining unchanged until adulthood. The variation of malate dehydrogenase activity, soluble protein content levels, and the circadian rhythm parameters during the maturation process may be related to gonad development.     

Malate Dehydrogenase Activity of Rat Testis: Circadian Rhythm and its Ontogeny

Malate dehydrogenase activity and soluble protein content in testes from rats exposed to a 14:00 h light:10:00 h dark photoperiod, have been determined every two or four hours over a 24 hour period in 5, 15, 25 and 120 day-old rats. By using the Cosinor method, the ontogeny of an unimodal rhythm was studied for MDH activity and soluble protein content in testis. In 5 and 15 day-old rats, the MDH acrophases were recorded around 19:00 h and 17:00 h, respectively. Rats aged 25 and 110 days showed the MDH acrophases during the dark period. An inversion of the MDH circadian rhythms was detected in 25 day-old compared to those of 5 and 15 day-old rats. An inversion of the protein circadian rhythm was also detected at 15 days compared to that at 5 days. These inversions persist in the adult rats. The amplitude of the MDH and protein rhythms reached the lowest value in adulthood. The mean daily value of testicular MDH increased between day 5 and 15, decreasing at day 35 and remaining unchanged until adulthood. The variation of malate dehydrogenase activity, soluble protein content levels, and the circadian rhythm parameters during the maturation process may be related to gonad development.