Expression of vasoactive intestinal peptide receptor messenger RNA in the hypothalamus and pituitary throughout the turkey reproductive cycle

Vasoactive intestinal peptide (VIP) has been implicated in the regulation of avian reproductive activity and appears to act at the level of the hypothalamus and pituitary. This in situ hybridization histochemistry study describes the distribution of VIP receptor mRNA expression in the hypothalamus and the pituitary of reproductively active (laying) and quiescent (nonphotostimulated, incubating, and photorefractory) female turkeys and characterizes the differences observed in VIP receptor gene expression. VIP receptor mRNA, while expressed throughout the hypothalamus, was specifically expressed in areas known to contain GnRH-I neurons in the chicken, i.e., the lateral septum, medial preoptic area, anterior hypothalamus, and paraventricular nucleus. Significant differences in VIP receptor mRNA expression between different reproductive states was observed only within the infundibular nuclear complex. VIP receptor mRNA was markedly less in nonphotostimulated and photorefractory hens as compared with laying and incubating hens. The most dense VIP receptor mRNA was found in the anterior pituitary, where it was 2.4- and 3.0-fold greater in laying and incubating hens, respectively, as compared with that in nonphotostimulated ones. Hens that stopped incubating and became photorefractory displayed pituitary VIP receptor mRNA levels similar to those of nonphotostimulated birds. The changes in pituitary VIP receptor mRNA expression were positively correlated with known changes in pituitary prolactin (PRL) mRNA expression and PRL content and release. These findings indicate that the variations in PRL secretion observed across the turkey reproductive cycle are, in part, regulated by changes in VIP receptors at the pituitary level.     

Fluctuations of lactoferrin protein and messenger ribonucleic acid in the reproductive tract of the mouse during the estrous cycle.

The physiological role of lactoferrin (LF), the major estrogen-inducible protein in the murine uterus, is unclear; however, LF may be a useful marker for the study of estrogen action in the uterus. Thus, the expression of LF mRNA and the localization of the protein in genital tract tissues and secretions of female mice (6-8 wk old) at different stages of the estrous cycle were investigated. Uterine luminal fluid (ULF) was analyzed for LF by means of gel electrophoresis and Western blot techniques; LF mRNA and protein were identified in reproductive tract tissues through in situ hybridization and immunocytochemistry. At diestrus, the level of LF mRNA was low, and staining for the protein was very light in uterine epithelial cells; LF was undetectable in ULF. At proestrus, LF mRNA and protein increased in the uterine epithelium and LF was readily detectable in ULF. LF mRNA and protein reached the highest levels at estrus. At early metestrus as compared to estrus, LF mRNA and protein were detected in decreasing amounts in uterine epithelial cells; the protein was undetected in ULF. By late metestrus and diestrus, LF mRNA and protein returned to a low level, and the protein was undetectable in ULF. LF protein was also demonstrated by immunocytochemistry in the epithelium of the oviduct, cervix, and vagina. LF protein fluctuation similar to that observed in the uterus was seen in these tissues; however, the uterus demonstrated the most dramatic changes in the number of epithelial cells involved in LF production during the estrous cycle. In summary, LF mRNA and its expression in uterine epithelial cells of the mouse varied with the stage of the estrous cycle. These results, combined with previously reported findings that LF is a major constituent of mouse ULF under the influence of estrogen, suggest that LF may play an important role in normal reproductive processes.     

Cellular localization of estrogen receptor beta messenger ribonucleic acid in cynomolgus monkey reproductive organs.

There is now evidence that the recently identified estrogen receptor (ER) beta is more widely distributed in the body than is ER-alpha. In order to gain more information about the role of ER-beta in reproduction, we have investigated by in situ hybridization the localization of mRNA expression of this ER subtype in adult monkey reproductive organs. In the pituitary gland of animals of both sexes, in both the anterior and intermediate lobes, a large number of cells were positive. No specific signal was observed in the posterior lobe. In the ovary, granulosa cells in primary and growing follicles highly expressed ER-beta mRNA. The theca interna cells were also strongly labeled. In some corpora lutea, the luteal cells were strongly labeled, while in other ones, the signal was weak. A hybridization signal was also detected in the ovarian surface epithelium. In the uterus, ER-beta mRNA was found in high concentration in glandular epithelial cells and stromal cells of the endometrium, while weaker labeling was consistently observed in smooth muscle cells. In the mammary gland, labeling was detected in the epithelial cells of acini and interlobular ducts as well as stromal cells. In the testis, specific labeling was detected in the seminiferous epithelium whereas the interstitial Leydig cells were unlabeled. Although it was not possible to clearly identify all the positive cell types, it appears that Sertoli cells as well as the vast majority of germinal cells express ER-beta mRNA. In the prostate, the secretory epithelial cells exhibited a specific autoradiographic reaction while the stromal cells did not show mRNA expression. The epithelial cells of the prostatic urethra showed a strong labeling. No hybridization signal was detected in the seminal vesicles. It then appears quite clear that ER-beta is expressed in a cell-specific manner in all the monkey reproductive organs studied. In the female, the wide distribution of these receptors in the ovary and uterus suggests that ER-beta may play an important role in the mediation of the known effects of estrogen in reproduction functions. In the male testis and prostate, ER-beta has been found in cells that contain very little or no ER-alpha. The role of circulating or locally produced estrogens in the male reproductive system remains to be clarified.     

Heterogeneity of the 5' terminus of hen ovalbumin messenger ribonucleic acid.

The 5'-terminal sequence of hen ovalbumin mRNA was investigated using a novel labeling method. Ovalbumin mRNA was purified by hybridization to complementary DNA coupled to cellulose. The mRNA thus purified was shown to be 97.9% pure by hybridization with plasmid DNA containing sequences to the messengers coding for conalbumin and ovomucoid, the next two most abundant messengers of oviduct. After digestion with RNase T1 and alkaline phosphatase, 5'-terminal capped oligonucleotides were selected by binding to anti-m7G-Sepharose. These were then labeled using RNA ligase and [5'-32P]pCp, separated by two-dimensional gel electrophoresis, and sequenced by partial digestion with base-specific ribonucleases. A nested set of three capped oligonucleotides was identified. Their structures and relative abundances were m7GpppAUACAG, 3% m7GpppACAUACAG, 61+; and m7GpppGUACAUACAG, 36%.     

Purification of brain D2 dopamine receptor.

D2 dopamine receptors have been extracted from bovine brain using the detergent cholate and purified approximately 20,000-fold by affinity chromatography on haloperidol-sepharose and wheat germ agglutinin-agarose columns. The purified preparation contains D2 dopamine receptors as judged by the pharmacological specificity of [3H]spiperone binding to the purified material. The sp. act. of [3H]spiperone binding in the purified preparation is 2.5 nmol/mg protein. The purified preparation shows a major diffuse band at Mr 95,000 upon SDS-polyacrylamide gel electrophoresis and there is evidence for microheterogeneity either at the protein or glycosylation level. Photoaffinity labelling of D2 dopamine receptors also shows a species of Mr 95,000. The D2 dopamine receptor therefore is a glycoprotein of Mr 95,000.     

Estrogen-induced decrease of glucocorticoid receptor messenger ribonucleic acid concentration in rat anterior pituitary gland

Using Northern blots and hybridization techniques, we have identified an approximately 6.5 kilobase glucocorticoid receptor mRNA species in rat anterior pituitary gland. Ovariectomy resulted in an approximately 2-fold increase in glucocorticoid receptor mRNA concentrations. This effect was maximal 8 days after surgery and glucocorticoid receptor mRNA levels remained elevated for at least up to 4 weeks. Administration of 17-beta-estradiol completely reversed the ovariectomy-induced increase in glucocorticoid receptor mRNA content of pituitary gland. Treatment of rats with corticosterone did not influence the ovariectomy-induced increase in glucocorticoid receptor mRNA content, indicating that this increase is not mediated via effects on circulating glucocorticoid levels or availability. In situ hybridization experiments confirmed the ovariectomy-induced increase in glucocorticoid receptor mRNA content and indicated that this action is widely distributed throughout the anterior pituitary gland.     

Effect of gonadotropin-releasing hormone on estrogen receptor messenger ribonucleic acid level in perifused pituitary cells

Summary 1. We examined the potential effect of GnRH pulses on pituitary estrogen receptor mRNA level.2. The treatment of perifused pituitary cell aggregates with four hourly pulses of GnRH (10 nM/1 min/h) resulted in a marked increase in the steady-state level of ER mRNA (25%vs unstimulated control, n = 3).3. No changes were observed for the LH ß mRNA. Data suggest, for the first time, that a cross-talk between the GnRH and nuclear ER may occur in the gonadotrope cells.     

Structure of the 5' terminus of hen oviduct lysozyme messenger ribonucleic acid

Lysozyme mRNA (mRNAlys) was purified from hen oviduct poly(A)-containing RNA by hybridization, labeled with NaB[3H]4 and digested with RNase T1. This revealed the presence of equal amounts of two major oligonucleotides having structures of m7Gppp(Np)7 and m7Gppp(Np)4 plus minor amounts of m7Gppp(Np)2 and m7GpppNp. The total mRNAlys contained the cap structures m7Gpppm6Am, m7GpppGm, m7GpppAm, m7GpppCm, m7GpppA, and m7GpppG, in decreasing order of abundance. The m7Gppp(Np)7 oligonucleotide contained only A-caps and the m7Gppp(Np)4, only G-caps. 32P-labeled 5'-terminal T1-oligonucleotides were prepared, and at least 12 different types were observed, the most abundant being m7Gppp(Np)7 and m7Gppp(Np)4. Their sequences were determined to be m7Gppp(m6)AmNmUCCCG and m7GpppGmNmAG. Taken together with the findings of Grez et al. (Grez, M., Land, H., Giesecke, K., Schutz, G., Jung, A., and Sippel, A. E. (1981) Cell 25, 743-752), these results indicate that in the genomic sequence AGCTTGCAGTCCCGT, 52% of the mRNAlys molecules begin at the underlined A residue and 38% at the underlined G residue.     

Expression of D2 dopamine receptor in the mouse brain

The neurotransmitter, dopamine, binds to dopamine receptor (DR), and is involved in several functions of the brain, such as initiation and execution of movement, emotion, prolactin secretion, etc. Of all the five DRs, D2 dopamine receptor has maximal affinity for dopamine. D2 has a short isoform, D2S, and a long isoform D2L. D2L is longer than D2S by 29 amino acid residues. We studied the expression of the gene and protein of D2 receptor in the cerebral and cerebellar cortices of the brain of new born, developing, adult, and old male mice to find out: (i) at what stage of development, expression of the gene peaks and (ii) if it undergoes any changes as the animal ages, which may account for the neurodegenerative changes and symptoms of Parkinson's and other diseases seen in old age. RT-PCR and Western blot studies show that peak expression of D2 gene occurs in the cerebral and cerebellar cortices around 15-day after birth. We speculate that the majority of dopaminergic synapses are established and possibly become functional in the brain around 15-day after birth. The expression of D2 receptor is upregulated in the cerebral cortex in old mice. However, it is down-regulated in the cerebellar cortex.     

Divergent changes in the concentrations of gonadotropin beta-subunit messenger ribonucleic acid during the estrous cycle of sheep

Cyclic changes in the production of the pituitary gonadotrophic hormones, LH and FSH are essential events in the maintenance of the reproductive system of female mammals. While studies have examined changes in the secretion of LH and FSH during the estrous cycle and demonstrated the importance of these hormones in regulation of ovarian development and gametogenesis, considerably less is known concerning the regulation of the biosynthesis of these hormones. Although initial studies have examined changes in LH subunit mRNA concentrations during the rat and ovine estrous cycles, no information concerning the physiological regulation of FSH beta mRNA concentrations has been available. In the present study we have examined the relationship between pituitary concentrations of LH and FSH subunit mRNAs and the serum concentrations of these gonadotropins. The results demonstrate a very different pattern of change for FSH beta subunit mRNA than that observed for alpha and LH beta subunit mRNAs. In fact, FSH beta mRNA concentration decline substantially during the preovulatory period, reaching minimal values at a time when alpha and LH beta mRNA levels are near maximal. Furthermore, this decline in FSH beta mRNA amounts occurs when serum FSH concentrations are maximal. Thus, FSH beta mRNA concentrations follow a very different pattern than that of serum FSH. In contrast, LH beta mRNA and serum LH concentrations tend to increase at the same time. These findings provide evidence that concentrations of LH beta and FSH beta mRNAs are likely regulated by different mechanisms.     

Prolactin messenger ribonucleic acid concentrations throughout the ovine estrous cycle: assessment relative to prolactin serum and pituitary amounts

Prolactin (PRL) mRNA concentrations were assessed by nucleic acid hybridization assays in pituitaries of ewes representing the defined stages of the ovine estrous cycle. Concomitantly, pituitary and serum PRL concentrations were measured in these ewes using radioimmunoassays. It was observed that PRL serum, pituitary and mRNA concentrations tended to increase near the time of the gonadotropin preovulatory surge, particularly between 24 hrs before behavioral estrus to 5 hours after estrus. However, the changes in PRL mRNA, serum and pituitary concentrations were shown not to be statistically significant. These data suggest that PRL production during the sheep estrous cycle is maintained without dramatic changes in synthesis or secretion.     

Quantification of prolactin messenger ribonucleic acid, pituitary content and plasma levels of prolactin, and detection of immunoreactive isoforms of prolactin in pituitaries from turkey embryos during ontogeny.

The content of prolactin mRNA as well as total prolactin content and type of isoforms of prolactin were measured in single pituitary glands from turkey embryos and poults. Levels of mRNA and pituitary content of prolactin remained low until 5 days before hatching, while plasma concentrations remained low until 2 days before hatching. Levels of prolactin mRNA then increased until the day of hatch, stayed stable during the 3 first days of age, and significantly increased until 2 wk of age. Similar changes were observed in pituitary content and plasma levels of prolactin. Two immunoreactive bands of apparent molecular masses of 24 and 27 kDa, corresponding to the nonglycosylated and glycosylated form of prolactin, respectively, were visualized on Western blots. In pituitary glands from embryos at 22 days of incubation, 31.5% of the protein was glycosylated, whereas in embryos at 27 days of incubation and poults at 1 and 7 days of age, 48.6%, 48.0%, and 56. 0% of prolactin was glycosylated, respectively. The results indicate that the increases in the synthesis and the release of prolactin occur mainly around and after the time of hatching in the turkey embryo. Higher percentages of glycosylated isoforms were associated with increasing levels of total prolactin in the pituitary gland. Thus, the synthesis of prolactin and its post-translational modifications may be important factors involved in the physiologic changes occurring around the time of hatching.     

Effect of 9-cis retinoic acid on dopamine D2 receptor expression in pituitary adenoma cells

The dopamine receptor subtype 2 (D2R) promoter contains a functional retinoic acid response element involved in the control of D2R expression. The aim of the study was to evaluate the effect of 9-cis retinoic acid (9-cis RA) on D2R protein expression in human pituitary adenomas and GH3 cell line. Treatment with 9-cis RA (100 nM for 48 hrs) caused a 109 +/- 32% increase of basal D2R levels in five of eight growth hormone (GH)-secreting adenomas (GH-omas), a 129 +/- 28% increase in 7 of 11 nonfunctioning adenomas, and no effect in two resistant prolactinomas by Western blotting. The lack of D2R induction in some tumors was not associated with a different pattern of retinoid x receptor (RXR) and retinoic acid receptor (RAR) isoform expression that was similar in all tumors by immunohistochemistry. While the induction of D2R did not affect the slight but significant inhibitory effect exerted by dopamine (10 nM) on in vitro GH release by GH-oma cultured cells, in pituitary GH3 cell lines cis-9 RA enhanced the dopamine-induced inhibition of in vitro GH release (% inhibition: 16 +/- 2 versus 26 +/- 5, P < 0.05), cell proliferation (25 +/- 2% versus 44 +/- 5%, P < 0.05) and cell viability (16 +/- 0.8% versus 29 +/- 1%, P < 0.05), likely by activating caspase-3 (28 +/- 3% versus basal, P < 0.05). In conclusion, this study provides novel evidence for a permissive role of retinoids on the expression of D2R in a good proportion of pituitary tumors and on the generation of pro-apoptotic signals in GH3 cell line.     

Pituitary luteinizing hormone and prolactin messenger ribonucleic acid levels are inversely related in laying and incubating turkey hens.

The relationships of prolactin (PRL) and LH messenger (m) RNA to serum and pituitary content were determined for turkey hens at different phases of the reproductive cycle. In the nonphotostimulated, reproductively inactive hen, serum and pituitary PRL content and pituitary PRL mRNA levels were low. All three PRL values rose after photostimulation and peaked during the incubation phase. Relative to nonphotostimulated hens, hyperprolactinemic incubating hens showed 220-, 11-, and 57-fold increases in serum PRL, pituitary PRL content, and pituitary PRL mRNA levels, respectively. These peak levels declined 80-, 3-, and 6-fold, respectively, in photorefractory hens. In contrast to PRL levels, serum LH, pituitary LH, and pituitary LH beta-subunit mRNA levels did not change as dramatically. Serum LH showed no significant changes for the different reproductive phases. Pituitary LH peaked after photostimulation and declined to its lowest level in incubating hens. Pituitary LH-beta mRNA abundance was highest in photostimulated and laying hens and lowest in incubating and photorefractory hens. These results demonstrate that the abundance of LH-beta and PRL mRNA shows an inverse relationship in photostimulated/laying and incubating turkey hens.