Developmental changes of hypothalamic, pituitary and striatal tachykinins in response to testosterone: influence of prenatal melatonin.

Substance P (SP) and neurokinin A (NKA), members of the family of mammalian tachykinins, are involved in the regulation of many physiological functions and are widely distributed in mammalian tissues. In this report, the effects of prenatal melatonin on the postnatal developmental pattern of NKA, and SP, and on testosterone secretion were investigated. Also, tachykinin response to the administration of testosterone propionate (TP) was studied. The brain areas studied were medio-basal-hypothalamus, pituitary gland and striatum. Male rat offspring of control or melatonin treated mother rats were studied at different ages of the sexual development: infantile, juvenile or prepubertal periods, and pubertal period. Both groups received exogenous TP (control-offspring+TP and MEL-offspring+TP), or the vehicle (control-offspring+placebo and MEL-offspring+placebo). Hypothalamic concentrations of all peptides studied in control-offspring+placebo remained at low levels until the juvenile period, days 30-31 of age. After this age, increasing concentrations of these peptides were found, with peak values at puberty, 40-41 days of age, then declining until adulthood. In the MEL-offspring+placebo a different pattern of development was observed; hypothalamic concentrations of NKA and SP from the infantile period until the end of juvenile period were significantly higher than in control-offspring+placebo. TP administration exerted a more marked influence on MEL-offspring than on control-offspring and prevented the elevation in tachykinin concentrations associated with prenatal melatonin treatment. TP administration to control-offspring resulted in significantly reduced (P < 0.05) tachykinin concentration only at 40-41 days of age, and increased (P < 0.01) during infantile period as compared to control-offspring+placebo. Pituitary NKA concentrations were lower than in the hypothalamus. In control-offspring+placebo pituitary NKA levels did not show significant changes throughout sexual development. A different developmental pattern was observed in MEL-offspring+placebo, with significantly increased (P < 0.05) pituitary NKA concentrations at 35-36 days of age than in control-offspring+placebo. TP administration to control-offspring influenced pituitary NKA levels at the end of the infantile and pubertal periods, showing at both stages significantly higher (P < 0.05) NKA levels as compared to control-offspring+placebo. NKA levels in MEL-offspring+TP were only affected at 21-22 days of age, showing significantly increased (P < 0.01) values as compared to MEL-offspring+placebo. Striatal tachykinin concentrations in control-offspring did not undergo important modifications throughout sexual development, but during the prepubertal period they started to increase. Maternal melatonin and TP injections produced short-lived alterations during the infantile period. The results showed that prenatal melatonin delayed the postnatal testosterone secretion pattern until the end of the pubertal period and postnatal peptide secretion in brain structures. Consequently, all functions depending of the affected areas will in turn, be affected.     

Luteinizing hormone response to controlled-release deslorelin in estradiol benzoate primed ovariectomized gilts

Development of a controlled release formulation of gonadotropin releasing hormone that would stimulate a LH surge capable of reducing the time span of ovulations would greatly benefit reproductive management because a single timed insemination could be used. A dose-response study was conducted to determine if Deslorelin, a potent gonadotropin releasing hormone analogue, delivered via the SABER system, a biodegradable controlled release system, would stimulate an ovulatory-like LH surge in the pig. Twenty ovariectomized gilts, approximately 200 d old and 100 kg body weight (BW), received estradiol benzoate (15 microg/kg BW im) and 48 h later, the gilts were given deslorelin at 0, 12.5, 25.0, 50.0 or 100.0 microg im (n = 4 each treatment group). Compared to controls, mean blood deslorelin concentrations were still elevated at 30 h after deslorelin. Mean deslorelin magnitude, area under the curve and duration were sequentially greater (P<0.05) in a dose-dependent sequence. Compared to controls, serum LH concentrations were elevated (P<0.05) for 6 to 12 h after deslorelin. A dose-response relationship was absent for all parameters of LH secretion. Magnitude of the serum LH response was greatest (P<0.05) in the 12.5 microg and 50.0 microg groups, whereas area under the curve was lower (P<0.05) after 25.0 microg of deslorelin than after 12.5, 50.0 and 100.0 microg, which were not different from each other. Thus, no more than 12.5 microg of deslorelin is necessary to obtain maximum LH release in the model studied and doses less than 12.5 microg may also be effective.     

Luteinizing hormone response to estradiol benzoate in cows postpartum and cows with ovarian cysts

Twenty-seven dairy cows were evenly assigned to one of three groups and given an intramuscular injection of 2 mg estradiol benzoate. Cows in group 1 were greater than 30 days postpartum at treatment and had been diagnosed via rectal palpation to have ovarian cysts. Cows in groups 2 and 3 were 12 to 14 and 30 to 40 days postpartum, respectively. Blood plasma was collected from all cows before treatment and then every three hours for 36 hours post-treatment. Concentrations of LH, estradiol-17 beta and progesterone in plasma were determined by radioimmunoassay. Four, zero and five cows in groups 1, 2 and 3, respectively, had concentrations of progesterone greater than 1.0 ng/ml before estradiol benzoate treatment. None of these cows had a peak LH release greater than 5 ng/ml following estradiol benzoate treatment. The numbers of cows with progesterone concentrations less than 1 ng/ml that released LH (>5 ng/ml) in response to estradiol benzoate were 3 of 5, 3 of 9, and 4 of 4 for groups 1, 2, and 3, respectively; the proportion for group 3 was higher (P<.05) than for group 2. Of the cows that released LH, mean peak LH concentrations were 33.3+/-5.4, 14.8+/-7.2 and 24.6+/-9.8 ng/ml for groups 1, 2 and 3, respectively, and the duration of the LH increase was 8.0+/-1.0, 8.0+/-2.0 and 13.0+/-4.0 hours. The time from estradiol benzoate treatment to peak LH release for cows with ovarian cysts (25+/-2 hours) was delayed (P<.05) compared with that for cows 30 to 40 days postpartum without ovarian cysts (16+/-1 hour). In summary, responsiveness to estradiol benzoate is regained between 2 to 4 weeks postpartum in most cows. In addition, some cows with ovarian cysts can release LH in response to estradiol benzoate, but peak LH release is delayed compared to cows at a comparable stage postpartum without ovarian cysts.     

Temporal changes in serum estradiol and prolactin in immature female rats given a single injection of estradiol benzoate

Serum concentrations of estradiol 17β(E₂) measured by immunoassay reached peak levels (100pg/ml) within one hour in immature female rats given a subcutaneous injection of lug 17βestradiol-3-benzoate (EB). Hypophysectomy did not alter the E₂ concentration, but lower peak levels were found in ovariectomized females. In animals with ovaries a secondary rise in serum E₂ was apparent 12 hours after the injection of EB; from 12 to 48 hours E₂ decreased linearly. A dose of Bug EB caused an abrupt rise in E₂ (180pg/ml) within 30 minutes, but during the next 24 hours rather wide fluctuations in serum levels were found. In animals acclimated to a reversed light schedule and given 5ug EB early in the dark period, E₂ decreased linearly over a period of 72 hours. Prolactin increased in response to the E₂: a rhythm was suggested by the occurrence of increases during the dark periods. The results indicate that statistically significant fluctuations in both E₂ and prolactin occur after a single injection of EB and that measurements at a single point in time are inadequate to determine the true pattern of hormonal changes.     

Prenatal melatonin exposure influences the maturation of gonadotropin and prolactin estradiol-benzoate feedback system.

Gonadotropin and prolactin response to estrogen feedback in female rat offspring of control and melatonin treated (150 microg/100 g BW) mother rats during pregnancy (MEL-offspring) were studied at these periods: infantile, prepubertal and pubertal. In controls negative or absent LH feedback developed after estradiol benzoate (EB) injection up to 30 days of age indicating that the onset of puberty had not occurred. The positive feedback was established from day 33 on. However, in MEL-offspring the first activation of gonadotropin secretion during afternoon, 31 h after EB, was observed at 25 days of age, representing the first neuroendocrine sign of the onset of puberty. This positive response disappeared on day 30 in MEL-offspring. At 33 days of age, the LH positive response to EB was found in both groups, indicating a more advanced sexual development. In controls, this response increased at 35 days of age while in MEL-offspring it was highly depressed. FSH secretion in response to EB showed a negative feedback effect from infantile to the end of prepubertal period in both groups. The positive feedback was observed earlier in MEL-offspring (at 33 days of age) than in controls (at 35 days of age), but at this age it was absent in MEL-offspring. A positive prolactin response to EB at all ages in controls was observed. The typical pulsatility with higher values in the afternoon appeared by the first time at 30 days of age. However, in MEL-offspring no pulsatile response was observed throughout any age. These data suggest that prenatal melatonin administration altered gonadotropin and prolactin response to EB inducing precocious sensitivity during prepubertal period but depressed response during the pubertal period.     

DNA methylome changes by estradiol benzoate and bisphenol A links early-life environmental exposures to prostate cancer risk

Developmental exposure to endocrine-disrupting chemicals (EDCs), 17β-estradiol-3-benzoate (EB) and bisphenol A (BPA), increases susceptibility to prostate cancer (PCa) in rodent models. Here, we used the methylated-CpG island recovery assay (MIRA)-assisted genomic tiling and CpG island arrays to identify treatment-associated methylome changes in the postnatal day (PND)90 dorsal prostate tissues of Sprague-Dawley rats neonatally (PND1, 3, and 5) treated with 25 µg/pup or 2,500 µg EB/kg body weight (BW) or 0.1 µg BPA/pup or 10 µg BPA/kg BW. We identified 111 EB-associated and 86 BPA-associated genes, with 20 in common, that have significant differentially methylated regions. Pathway analysis revealed cancer as the top common disease pathway. Bisulfite sequencing validated the differential methylation patterns observed by array analysis in 15 identified candidate genes. The methylation status of 7 (Pitx3, Wnt10b, Paqr4, Sox2, Chst14, Tpd52, Creb3l4) of these 15 genes exhibited an inverse correlation with gene expression in tissue samples. Cell-based assays, using 5-aza-cytidine-treated normal (NbE-1) and cancerous (AIT) rat prostate cells, added evidence of DNA methylation-mediated gene expression of 6 genes (exception: Paqr4). Functional connectivity of these genes was linked to embryonic stem cell pluripotency. Furthermore, clustering analyses using the dataset from The Cancer Genome Atlas revealed that expression of this set of 7 genes was associated with recurrence-free survival of PCa patients. In conclusion, our study reveals that gene-specific promoter methylation changes, resulting from early-life EDC exposure in the rat, may serve as predictive epigenetic biomarkers of PCa recurrence, and raises the possibility that such exposure may impact human disease.     

Role of tachykinins in the host response to murine gammaherpesvirus infection

Tachykinins function not only as neurotransmitters but also as immunological mediators. We used infection of tachykinin-deficient (PPT-A(-/-)) mice and wild-type controls with murine gammaherpesvirus to assess the role of tachykinins in the host response to a virus infection. Although infection was ultimately controlled in PPT-A(-/-) mice, there were higher titers of infectious virus in the lungs, accompanied by a more rapid influx of inflammatory cells. Clearance of latently infected cells from the spleen was also delayed. This is the first report of the direct influence of tachykinins in the host response to a virus infection.     

Dexamethasone and estradiol benzoate induced parturition in dairy cattle

Fifteen 2-year-old Holstein cows and 21 mature Holstein cows were assigned to one of three groups. Cows in Group I calved spontaneously. Cows in Groups II and III received single intramuscular injections of 20 mg dexamethasone and 25 mg estradiol benzoate to induce parturition prematurely. In addition, cows in Group III received a single intramuscular injection of 12.5 mg estradiol benzoate 48 hr prior to dexamethasone and estradiol benzoate. The objective of the experiment was to determine the effectiveness of estradiol benzoate in combination with dexamethasone on traits at parturition and on productive and reproductive characteristics following parturition. Induction of parturition shortened gestation length and increased the incidence of retained placentas (both P < .01). All induced cows calved between 21 and 59 hr postinjection with less (P < .05) udder edema when compared to control cows. Mean plasma estrogen concentrations, using an assay system which does not measure estradiol benzoate, were not different among groups following injections of estradiol benzoate. Mean estradiol-17β concentrations in induced cows, however, using an assay system which does recognize estradiol benzoate (70.8% crossreactivity), were higher (P < .01) following estradiol benzoate injection, tended to be higher through parturition, and remained elevated (P < .01) at 12 and 24 hr following parturition when compared to cows calving spontaneously. Mean monthly milk production and the 2x, 305-ME records for milk, fat and FCM were not different among groups.     

Macrophages sequentially change their functional phenotype in response to changes in microenvironmental influences

Recent studies have described the development of distinct functional subsets of macrophages in association with cancer, autoimmune disease, and chronic infections. Based on the ability of Th1 vs Th2 cytokines to promote opposing activities in macrophages, it has been proposed that macrophages develop into either type 1 inflammatory or type 2 anti-inflammatory subsets. As an alternative to the concept of subset development, we propose that macrophages, in response to changes in their tissue environment, can reversibly and progressively change the pattern of functions that they express. As demonstrated herein, macrophages can reversibly shift their functional phenotype through a multitude of patterns in response to changes in cytokine environment. Macrophages display distinct functional patterns after treatment with IFN-gamma, IL-12, IL-4, or IL-10 and additional functional patterns are displayed depending on whether the cytokine is present alone or with other cytokines and whether the cytokines are added before or concomitantly with the activating stimulus (LPS). Sequential treatment of macrophages with multiple cytokines results in a progression through multiple functional phenotypes. This ability to adapt to changing cytokine environments has significant in vivo relevance, as evidenced by the demonstration that macrophage functional phenotypes established in vivo in aged or tumor-bearing mice can be altered by changing their microenvironment. A concept of functional adaptivity is proposed that has important implications for therapeutic targeting of macrophages in chronic diseases that result in the dominance of particular functional phenotypes of macrophages that play a significant role in disease pathology.     

Melatonin or a melatonin agonist corrects age-related changes in circadian response to environmental stimulus

The effects of a melatonin agonist, S-20098, included in the diet were tested on a specific effect of aging in hamsters: the marked decline in the phase shifting effects of a 6-h pulse of darkness on a background of constant light. In contrast to young hamsters, old hamsters fed with the control diet showed little or no phase shifts in response to a dark pulse presented in the middle of their inactive or active period. Old hamsters fed with S-20098 showed phase shifts that were ~70% of the ones in young animals and significantly greater than those in old controls. The phase advancing response to a dark pulse presented during the inactive period was dose dependent and reversed after S-20098 discontinuation. Melatonin included in the diet showed comparable restorative effects on the phase shifting response to a dark pulse in old hamsters. Replacement therapy with melatonin or melatonin-related compounds could prove useful in treating, preventing, or delaying disturbances of circadian rhythmicity and/or sleep in older people.     

Adaptation to developmental diet influences the response to selection on age at reproduction in the fruit fly

Experimental evolution (EE) is a powerful tool for addressing how environmental factors influence life‐history evolution. While in nature different selection pressures experienced across the lifespan shape life histories, EE studies typically apply selection pressures one at a time. Here, we assess the consequences of adaptation to three different developmental diets in combination with classical selection for early or late reproduction in the fruit fly Drosophila melanogaster. We find that the response to each selection pressure is similar to that observed when they are applied independently, but the overall magnitude of the response depends on the selection regime experienced in the other life stage. For example, adaptation to increased age at reproduction increased lifespan across all diets; however, the extent of the increase was dependent on the dietary selection regime. Similarly, adaptation to a lower calorie developmental diet led to faster development and decreased adult weight, but the magnitude of the response was dependent on the age‐at‐reproduction selection regime. Given that multiple selection pressures are prevalent in nature, our findings suggest that trade‐offs should be considered not only among traits within an organism, but also among adaptive responses to different—sometimes conflicting—selection pressures, including across life stages.     

Synergistic effect of estradiol benzoate and dihydrotestosterone on aggression in mice

Orchiectomized CD-1 mice were treated with several steroids, alone and in combination, and then tested for aggressiveness against group-housed, intact “stimulus” mice. Estradiol benzoate (EB) in combination with dihydrotestosterone (DHT) elicited aggression equivalent to that shown by intact, sham-operated animals and by testosterone-treated subjects. EB alone was less effective than EB with DHT. DHT alone elicited no more aggression than that observed in control animals. The data suggest a synergism between EB and DHT within the central nervous system in the induction of aggressive behavior.     

Pituitary response to LHRH, LH pulsatility and plasma melatonin and prolactin changes in ewe lambs treated with melatonin implants to delay puberty

Prepubertal ewe lambs were treated with empty or filled melatonin implants. The implants were placed s.c. at birth and pituitary responsiveness to various doses of LHRH, LH/FSH pulsatility and prolactin and melatonin secretion were examined at 10, 19, 28, 36 and 45 weeks of age. Control animals (N = 10) showed no consistent alteration in pituitary responsiveness to LHRH during development. Ewes treated with melatonin (N = 10) had puberty onset delayed by 4 weeks (P less than 0.03) but no effect of melatonin on LH or FSH response to LHRH injection was observed at any stage of development. In the control and melatonin-treated ewe lambs the responses to LHRH injection were lower during darkness than during the day at all stages of development. No consistent differences in LH or FSH pulsatility were observed between treatment groups or during development. Prolactin concentrations, however, failed to decrease at the time of puberty (autumn) in the melatonin-treated group. Melatonin-treated ewe lambs maintained normal rhythmic melatonin production which was superimposed on a higher basal concentration and showed the same increase in melatonin output with age as the control ewes. These results indicate that the delayed puberty caused by melatonin implants is not due to decreased pituitary responsiveness to LHRH or to dramatic changes in basal LH or FSH secretion.     

Influence of season on estrous and luteinizing hormone responses to estradiol benzoate in ovariectomized sows

The objective of this experiment was to determine whether seasonal differences existed in estrous and LH responses to estradiol benzoate (EB) in ovariectomized sows. Sows were ovariectomized after weaning their first litter, and treatment was begun 120 d after ovariectomy. Sows were given 400 mug EB intramuscularly (i.m.) on July 24, 1982 (summer), October 24, 1984 (fall), January 29, 1985 (winter), and March 27, 1985 (spring). Beginning 24 h after EB, sows were checked for estrus four times daily. Proportion in estrus was affected by season, with all sows exhibiting estrus within 5 d after EB in summer, winter, and spring. Only three of five sows exhibited estrus within 5 d after EB in fall. Interval (h) to estrus was delayed in fall (80 h) compared to other seasons (62.6 h; SEM = 4.5). Concentrations of LH were suppressed within 6 h after EB in all seasons but rebounded to pre-injection levels more slowly in fall and spring than in winter and summer. Frequency of LH peaks (3.2 +/- .4 4 h ) was not affected by season, but amplitude (1.9 vs 0.9 ng/ml) and baseline (2.7 vs 1.6 ng/ml) were greater (P < 0.05) for summer than for the other seasons combined. At 6 h after injection, concentrations of estradiol-17beta (pg/ml) were greater in summer (58.3) than in fall (19.0), winter (32.4), or spring (16.6; SEM = 10.4). We conclude that environmental factors associated with season alter responsiveness of the brain to estradiol, thereby controlling sexual behavior and LH secretion.