Pituitary DNA Research Articles

Cloning and sequence analysis of cDNA for bovine carboxypeptidase E.

Carboxypeptidase E (enkephalin convertase) was first identified as the carboxypeptidase B-like enzyme involved in the biosynthesis of enkephalin in bovine adrenal chromaffin granules. A similar enzyme is present in many brain regions and in purified secretory granules from rat pituitary and rat insulinoma. Within the secretory granules, carboxypeptidase E (CPE) activity is found in both a soluble and a membrane-bound form, which differ slightly in relative molecular mass (Mr). Here, to investigate whether the CPE activities in the various tissues are produced from a single gene, purified CPE was partially sequenced and oligonucleotide probes were used to isolate a clone encoding CPE from a bovine pituitary complementary DNA library. This cDNA hybridizes to bovine pituitary poly(A)+ RNAs of approximately 3.3, 2.6 and 2.1 kilobases (kb), with the 3.3-kb messenger RNA the predominant species. The predicted amino-acid sequence of the cDNA clone contains the partially determined sequences of CPE, several pairs of basic amino acids and displays some homology with both carboxypeptidases A and B. Restriction analysis of bovine genomic DNA suggests only one gene for CPE. This is consistent with a broad role for CPE in the biosynthesis of many neuropeptides.

ALPHA AND LUTEINIZING HORMONE BETA SUBUNIT MESSENGER RIBONUCLEIC ACIDS DURING THE RAT ESTROUS CYCLE

Alpha and LH beta subunit mRNAs were measured in pituitaries of 4-day cycling rats during the estrous cycle. A two-fold increase in alpha mRNA occurred between 0800-2000 h on diestrus, but alpha mRNA concentrations were stable during other days of the cycle. LH beta mRNA concentrations were low during estrus and metestrus (11-16 pg cDNA bound/100 micrograms pituitary DNA), but were elevated (27-30 pg) between 0800-2000 h on diestrus. A second increase in LH beta mRNA was observed on the afternoon of proestrus, prior to the onset of the LH surge with maximum values (45 pg) coincident with peak LH secretion. LH beta mRNA concentrations declined rapidly and had fallen to basal values by midnight on proestrus. These data show that alpha and LH beta mRNAs change in a similar manner during metestrus, diestrus and estrus, suggesting coordinate regulation of alpha and LH beta gene expression at these times. During the LH surge, however, LH beta mRNA alone is increased, suggesting that the LH beta gene can be differentially expressed at times when maximum LH secretion is occurring.

Enhancement of estradiol-induced DNA synthesis in the anterior pituitary gland by the peripheral-type benzodiazepine receptor ligand Ro 5-4864.

The effects of peripheral (Ro 5-4864) and central-type (Ro 15-1788) benzodiazepine receptor ligands on estrogen-induced DNA synthesis in the rat anterior pituitary gland was investigated. As expected, a single injection of estradiol (250 micrograms per rat) significantly increased the uptake of 3H-thymidine by anterior pituitary cells. Additional treatment with Ro 5-4864 potentiated the effects of estradiol on pituitary DNA synthesis. Under the same experimental conditions, no effect of Ro 15-1788 and sodium valproate, a GABA-transaminase inhibitor, was detected. These findings indicate the involvement of peripheral-type benzodiazepine receptors in the control of anterior pituitary cell proliferation.

Expression and Regulation of Proopiomelanocortin-Like Gene in the Ovary and Placenta: Comparison with the Testis*

Proopiomelanocortin (POMC), a precursor protein for ACTH, beta-endorphin, and the MSHs, has been identified in the reproductive tracts of both male and female. With rat pituitary POMC complementary DNA (cDNA) as a hybridization probe, POMC-like messenger RNA (mRNA) was identified in the ovaries of rat, mouse, and monkey. The molecular size of POMC-like mRNA in the ovary was 150-200 bases smaller than in the pituitary and hypothalamus but identical to that in the testis and epididymis. The size heterogeneity of POMC mRNA observed in various tissues is not due to differences in the lengths of the poly(A) tail, as measured by RNase H digestion. S1 nuclease mapping analysis revealed that POMC mRNAs isolated from pituitary, testis, or ovary share the nucleotide sequences coding for ACTH, beta-lipotropin, and the 3'-untranslated region. The regulation of ovarian POMC-like mRNA was also investigated. Treatment of 25-day-old immature female rats with PMSG resulted in profound increases in the ovarian content of total RNA, poly(A) RNA, and POMC-like mRNA. The concentration of ovarian POMC-like mRNA during pregnancy increased increased to 3-4 times that in immature or normally cycling animals. POMC-derived peptides are present in the human placenta and are synthesized de novo in cultured placental cells. In this report we also demonstrate POMC-like mRNA in the placenta of rat, mouse, and human. The size of POMC-like mRNA in the placenta was similar to that observed in the testis, epididymis, and ovary and different from that found in the pituitary or hypothalamus. The concentration of placental POMC-like mRNA did not change throughout pregnancy. In conclusion, we have demonstrated that 1) POMC-like mRNA is present in the ovary and placenta of rodents and primates; 2) the size of POMC-like mRNA in the ovary and placenta, like that in the testis and epididymis, is smaller than that in the pituitary and hypothalamus, probably owing to a shortening of the 5'-ends; and 3) the expression of this gene is regulated by gonadotropins in the ovary but probably not in the placenta.

Relationship between release of LH and incorporation of tritiated thymidine in the anterior pituitary gland of the castrated male rat: effect of LHRH and its highly active analogue buserelin.

Castration of the adult male rat significantly (P less than 0.01) increased the concentration of LH in serum and the incorporation of (3H) thymidine into the pituitary DNA. The administration of a single dose of LHRH or its analogue buserelin stimulated the release of LH but it did not modify (3H) thymidine incorporation. When multiple doses of LHRH or buserelin were injected, there was a significant (P less than 0.01) inhibition of LH release and also the incorporation of (3H) thymidine into the DNA of the anterior pituitary gland was significantly (P less than 0.01) diminished. These observations are compatible with the idea of the close relationship between hormonal release and DNA synthesis in the anterior pituitary gland of the rat.

Relationship between release of LH and incorporation of tritiated thymidine in the anterior pituitary gland of the castrated female rat.

Castration of female rats during diestrus increases the concentration of circulating LH from days 3 and the incorporation of 3H thymidine into pituitary DNA from day 5. Both effects are completely abolished by the administration of dihydrotestosterone. Although the i.v. injection of LHRH markedly enhances the concentration of LH in serum, it does not modify the incorporation of 3H thymidine into pituitary DNA. Castration might produce a maximal stimulation in 3H thymidine incorporation and a further stimulation of LH release with LHRH is unable to enhance the incorporation of the radioactive precursor. The results suggest a relationship between LH secretion and DNA synthesis in the pituitary gland of the rat.

High-level expression of the bovine growth hormone gene in heterologous mammalian cells

The gene coding for bovine growth hormone (bGH) was isolated from a λ-phage library constructed using bovine pituitary DNA partially digested with MboI. Expression of this gene transfected into mouse and monkey cells was studied. CV-1 monkey cells transfected with simian virus 40 (SV40) vectors containing the intact bGH gene, including the putative promoter region, did not express bGH. However, replacement of the bGH promoter with the mouse metallothionein-I (MT) promoter resulted in high-level synthesis and secretion of bGH. These results show that the bGH promoter functions poorly in CV-1 cells but CV-1 cells process and translate the bGH mRNA accurately. The MT-bGH chimeric gene was used to establish permanent bGH-secreting mouse C127 cell lines using the 69% transforming fragment of bovine papilloma virus (BPV) as the vector. One such cell line produced high levels of bGH and secreted it into the medium efficiently. Secreted bGH is processed accurately and is bioactive as judged by its ability to bind to rabbit liver membrane preparations.

Effects of bromocriptine on pituitary organelle marker enzyme activities in lactating and postlactating rats: selective activation of lysosomal prolactin proteolytic activity.

Effects of the dopamine agonist 2-bromo-alpha-ergocryptine (bromocriptine) on plasma and pituitary PRL and enzyme activities in lactating and postlactating rats have been investigated. Lactating rats which had been suckling their young for 3 days were given a single sc injection of bromocriptine or solvent. The treated and control animals were divided into 2 further groups. One group (lactating rats) was permitted to suckle their pups for a further 12 or 24 h; the young were removed from the other group (postlactating rats). Homogenates were prepared from the anterior pituitaries and assayed for organelle marker enzyme activities. When 0.5-500 micrograms bromocriptine were administered to lactating rats for 24 h, pituitary PRL was increased by all doses, but only the 500-micrograms dose significantly reduced plasma PRL. Total protein was unchanged, lysosomal acid PRL proteolytic activity increased 8-fold, N-acetyl-beta-glucosaminidase and beta-glucuronidase (lysosomes) were unchanged, acid phosphatase (lysosomes and endoplasmic reticulum) was increased by three of four doses, 5'-nucleotidase and alkaline phosphatase (plasma membrane) were increased 4-fold, neutral-alpha-glucosidase (endoplasmic reticulum) and malate dehydrogenase (mitochondria) were unchanged, and catalase (peroxisomes) was significantly increased. Bromocriptine (500 micrograms) administration to lactating and postlactating rats for 12 and 24 h significantly decreased the pituitary DNA but not the total protein content of the pituitaries in all animals. The lysosomal acid PRL proteolytic activity and the lysosomal enzyme activities, N-acetyl-beta-glucosaminidase and beta-glucuronidase, were increased by suckling withdrawal alone. Acid PRL proteolytic activity was further increased (to 18-fold) by coadministration of bromocriptine, whereas the increase in the activities of the other lysosomal marker enzymes was blocked. Malate dehydrogenase activity (mitochondria) was also increased by litter removal and blocked by bromocriptine. The activity of the plasma membrane markers 5'-nucleotidase and alkaline phosphatase were increased by litter removal, and bromocriptine further increased both enzyme activities. The activity of neutral-alpha-glucosidase (endoplasmic reticulum) was unchanged by any treatment. The results demonstrate that bromocriptine produces significant changes in the activities of lysosomal marker enzymes, particularly acid PRL proteolytic activity, as well as marker enzymes of plasma membranes and other organelles in pituitaries of lactating and postlactating rats.

Correlation between LH secretion in castrated rats with cellular proliferation and synthesis of DNA in the anterior pituitary gland.

The relationship between the release of LH and the synthesis of DNA was studied in the anterior pituitary gland of castrated rats. Cell types were characterized immunocytochemically. Castration significantly (P less than 0.01) increased the concentration of LH in serum (1326%) and the incorporation of [3H]thymidine into pituitary DNA (72%). This was accompanied by an increment in the activity of the enzyme DNA polymerase-alpha (58%) and in the number of mitoses (from 2 +/- 0.1/mm2 in intact rats to 21 +/- 0.8/mm2 15 days after castration). Only 20% of the mitoses found in the pituitary gland of castrated rats were positively stained with the antiserum against the beta-subunit of LH. The other 80% did not stain either with LH antiserum or with antisera against the other pituitary hormones. There was a significant (P less than 0.01) increase in the number of LH cells in castrated rats (48%). All the changes produced in the anterior pituitary gland after castration were prevented by the administration of dihydrotestosterone. The results demonstrate that a stimulation of LH release is followed by an increase of DNA synthesis and cell proliferation of gonadotrophs in the anterior pituitary gland.

Characterization of a pineal-mediated inhibition of pubertal prolactin cell development in blind-anosmic female rats.

The purpose of this study was to define the peripubertal changes in prolactin (PRL) synthesis in blind-anosmic female rats. To do this prepubertal female rats were either rendered blind-anosmic (BA) or blind-anosmic-pinealectomized (BAP) or left intact (INT). Half the animals were killed after 1 wk (before puberty) while the remaining animals were terminated after 4 wk (after puberty). Pituitary PRL synthesis was measured by the in vitro incorporation of 3H-leucine into PRL. Anterior pituitary weight and DNA content did not differ between BA, BAP, and INT animals 1 wk after treatment. There was a 65% increase in weight and a 55% increase in DNA content in INT pituitaries between 1 and 4 wk. While these increases were also present in BAP rats, they were almost entirely prevented in BA animals. PRL synthesis followed a similar pattern with a 78% increase in synthesis in INT groups between 1 and 4 wk which was, once again, almost entirely absent in BA rats. Pinealectomy prevented this effect. Blinding and anosmia in postpubertal animals produced only a minor decrease in PRL synthesis and pituitary weight and no decrease in pituitary cell number. From these data we conclude that the pineal can inhibit the normal proliferation of PRL cells that occurs during puberty in blind-anosmic female rats.

Effect of ovariectomy and clomiphene on the in-vitro incorporation of [3H]thymidine into pituitary DNA and on prolactin synthesis and release in rats.

In the anterior pituitary gland changes in prolactin synthesis and in the incorporation of [3H]thymidine into DNA are coincident under several experimental conditions. We investigated whether these changes are obligatory, thus indicating a regulatory mechanism common to the synthesis of both macromolecules. Alternatively, the parallel changes may represent similar responses to various stimuli operating through different pathways. The administration of alpha-methyl-p-tyrosine (alpha MpT) to rats stimulated the incorporation of [3H]leucine into prolactin and [3H]thymidine into DNA. When the effectiveness of oestrogen was suppressed by ovariectomy or by blockage of oestrogen receptors by the antioestrogen clomiphene, alpha MpT stimulated the synthesis of prolactin but not the incorporation of [3H]thymidine into pituitary DNA. The results clearly indicate two independent mechanisms regulating the synthesis of prolactin and DNA in the anterior pituitary gland.

Regulation of pituitary DNA synthesis during different reproductive states in the female rat: role of estrogens and prolactin

We studied DNA synthesis in the rat adenohypophysis during the estrous cycle, pregnancy and lactation. During the estrous cycle, DNA synthesis was 3 times higher on the morning of estrus than on the other days. This peak was abolished completely by ovariectomy or pentobarbital, which also blocked the preovulatory surges of LH and prolactin. Methallibure, which blocked the LH but not the prolaction surge, had a partial effect on DNA synthesis. An acute and significant decrease in pituitary DNA synthesis occurred between days 0 (estrus) and 1 of pregnancy, followed by a less pronounced diminution until parturition. After delivery, DNA synthesis increased steeply on day 1 of lactation, returning to low values by day 3, under normal suckling conditions. Thelectomy, which blocked suckling-induced prolactin release, or antiestrogen treatment, which did not decrease prolactin secretion, diminished pituitary DNA synthesis on day 1 of lactation. Estrogen administration to intact or ovariectomized rats on days 9–11 of lactation stimulated (100%) DNA synthesis. Ovariectomy had no effect. In conclusion, in the different reproductive states studied, pituitary DNA synthesis is related to prolactin release in the presence of estrogens.

Characterization of mRNA sequences encoding sheep somatotropin (growth hormone) by cloning of pituitary complementary DNA

Characterization of mRNA sequences encoding sheep somatotropin (growth hormone) by cloning of pituitary complementary DNA

Methylation of either cytosine in the recognition sequence CGCG inhibits ThaI cleavage of DNA.

ThaI (CGCG) sites which overlap HhaI (GCGC) sites in phi X174 and pBR322 DNA were methylated in vitro with HhaI methylase and S-adenosylmethionine to yield CGmCG, mCGCG or mCGmCG (5-methylcytosine, mC). Methylation of either cytosine in the ThaI recognition sequence rendered the DNA resistant to ThaI cleavage. Rat pituitary cell genomic DNA was digested with ThaI or 2 other known methylation-sensitive enzymes, AvaI or XhoI. After electrophoresis and ethidium bromide straining of the DNA, all 3 enzymes showed the infrequent DNA cleavage characteristic of methylation-sensitive enzymes. Comparison of pituitary growth hormone (GH) genes bearing strain-specific degrees of methylation showed the less methylated gene to be more frequently cut by either AvaI or ThaI. ThaI resistant sites in GH genes were cleaved by ThaI after exposing cells to 5-azacytidine, an inhibitor of DNA methylation. We conclude that ThaI is a useful restriction enzyme for the analysis of mC at CGCG sequences in eukaryotic DNA.

Effect of tamoxifen on growth and prolactin secretion of rat pituitary tumours.

The antioestrogenic drug tamoxifen was administered to rats bearing transplanted prolactin-secreting tumours derived from spontaneously occurring pituitary adenomas in Wistar-Furth rats. Some inhibition of tumour growth was observed but this was accompanied by an increase in plasma prolactin concentrations. Bromocriptine, however, consistently inhibited both growth and prolactin secretion of these tumours. The addition of tamoxifen to bromocriptine treatment produced no increased response to the dopamine agonist. Tamoxifen increased prolactin secretion by tumour cells in vitro but did not affect DNA synthesis. Normal rats responded to tamoxifen with a moderate increase in plasma prolactin concentrations and there was no change in pituitary DNA synthesis.

Hyperprolactinaemia and DNA synthesis in the pituitary gland of the rat.

Hyperprolactinaemia produced in rats by the transplanted prolactin-secreting tumours MtTW15 and 7315a significantly (P less than 0.01) inhibited by 70% the incorporation of [3H]thymidine into the pituitary DNA of the host animals. The weight and the DNA content of the glands were significantly (P less than 0.01) reduced by 30%. The administration of haloperidol, a dopamine receptor blocking agent, to the tumour-bearing rats increased the suppressed DNA replication in the anterior pituitary glands by approximately 560% in the MtTW15-bearing rat and by 100% in the 7315a-bearing animals. Furthermore, injection of drugs which stimulate prolactin release either by blocking the synthesis of dopamine (alpha-methyl-p-tyrosine) or the re-uptake of dopamine (reserpine) stimulated DNA synthesis by 800 and 100% respectively in the anterior pituitary gland of rats bearing the MtTW15 tumour. In contrast, lisuride, a dopamine agonist, significantly inhibited the incorporation of [3H]thymidine into the DNA of the pituitary gland of normal but not hyperprolactinaemic rats. Chronically administered oestrogens to hyperprolactinaemic rats increased the weight (100%). DNA content (31%), incorporation of [3H]thymidine into DNA (680%) and synthesis and release of prolactin (300%) in the pituitary gland. The incorporation of [3H]thymidine into tumour DNA was several times higher than in the pituitary gland of the host animal and was not significantly modified by any of the above treatments. Likewise the hyperprolactinaemia of the tumour-bearing rats was not significantly changed. In conclusion, we have shown that hyperprolactinaemia inhibits DNA synthesis in the anterior pituitary gland and this inhibition can be reversed completely by a dopamine receptor blocking agent and by hypothalamic dopamine depleting drugs. We propose that dopamine regulates, either directly or indirectly, DNA synthesis in the lactotrophs of the pituitary gland, which may be responsive to negative feedback mechanisms.

Relationships among release of prolactin, synthesis of DNA and growth of the anterior pituitary gland of the rat: effects of oestrogen and sulpiride

The effect of daily injections of sulpiride was compared with that of a single injection of the drug in male rats which had been treated with oestradiol diundecenoate for various periods of time. We studied the effect of the different treatments on weight of the pituitary gland, concentration of prolactin and incorporation of [3H]thymidine into DNA in the pituitary gland and on serum levels of prolactin. Administration of the oestrogen produced a marked increase in the synthesis of DNA at day 7. The stimulation diminished at day 21 and was not significant at day 45. The maximum increase in the concentration of prolactin in serum and pituitary glands was observed during the first 7 days (approximately 400 and 150% respectively) and in the weight of the anterior pituitary gland after 21 days of treatment (approximately 107%). A single injection of sulpiride markedly stimulated the release of prolactin and the synthesis of DNA at day 7. Both these effects diminished at day 21 and disappeared by day 45. Daily injections of sulpiride also produced similar changes in the release of prolactin and in the replication of DNA. The growth of the anterior pituitary gland was greater in this group than in the rats which had been treated with oestradiol diundecenoate only. After the end of treatment with oestrogen and sulpiride the pituitary weight and the concentration of prolactin in the anterior pituitary gland diminished together with levels of prolactin and oestrogen in serum. There was a good correlation between weight of the gland and serum levels of prolactin. The results further support the idea of a mechanism which controls the proliferation of lactotrophs in which the release of the hormone is accompanied by an increase in pituitary DNA synthesis.

Nuclear diameter in the anterior pituitary gland of the rat: effects of estrogen, bromocriptine, and haloperidol.

Nuclear diameter, DNA synthesis, and mitotic index in the pituitary cells of male rats and serum prolactin were measured after a period of 8 days of treatment with a dopamine agonist and an antagonist given with and without estrogen. In the absence of estrogen, the dopamine agonist, bromocriptine, diminished the mean nuclear diameter of the pituitary cells and lowered pituitary DNA synthesis, and the dopamine-blocking agent haloperidol had no effect. Estrogen increased the mean nuclear diameter, pituitary mitotic index, and DNA synthesis. Bromocriptine prevented the estrogen-induced increase in mean nuclear diameter and pituitary DNA synthesis and mitotic index were lowered. Haloperidol augmented the estrogen-induced increase in mean nuclear diameter, pituitary DNA synthesis, and mitotic index. Positive correlations were obtained between mean nuclear diameter and DNA synthesis and serum prolactin. It was concluded that nuclear diameter was influenced by both DNA synthesis and secretory activity in pituitary cells.

Suckling withdrawal increases pituitary lysosomal enzyme activities and prolactin protease in lactating rats.

We have investigated the physiological importance of enzymic alterations in the rat pituitary during lactotroph involution induced by cessation of lactation. Lactating rats that had been suckling their young for 3 days were divided into two groups. One group was permitted to suckle their pups for up to 2 more days; the young were removed from the other group. At successive times, homogenates were prepared from their anterior pituitaries. The postlactating animals showed a rapid fall in plasma PRL levels, and this was accompanied by a transient rise in pituitary PRL content maximal 12 h after pup removal, which returned to control levels 36 h later. The pituitary homogenates were assayed for organelle marker enzyme activities. Both groups showed no significant alteration in pituitary protein or DNA content. The lactating group showed no change in the specific activity (milliunits per mg protein) of the following enzymes (organelle shown between parentheses): N-acetyl-β- glucosaminidase, acid PRL protease, β-glucuronidase, acid phosphatase, and cathepsin C (lysosomes); neutral α-glucosidase, (endoplasmic reticulum); 5′-nucleotidase and alkaline phosphatase (plasma membrane); malate dehydrogenase (mitochondria); lactate dehydrogenase (cytosol). The postlactating animals showed a transient increase in lysosomal enzyme activities, with the exception of cathepsin C, between 12 and 36 h after pup removal. This was particularly striking for PRL protease, which showed a 16-fold rise at 24 h. There were no significant alterations in neutral α-glucosidase, but both 5′-nucleotidase and alkaline phosphatase showed increased activities between 12 and 36 h after pup removal. There was no consistent alteration in the activities of lactate and malate dehydrogenases. These data provide biochemical evidence for the role of lysosomes in the degradation of PRL in the lactotroph involution after cessation of lactation.

Pineal gland inhibition of prolactin cell activity is independent of gonadal regression.

In order to determine whether the inhibitory effect of the pineal gland on the PRL cells of blind-anosmic female rats is a result of the attendant gonadal regression observed in these animals, mammotroph activity was compared between intact and ovariectomized rats eight weeks after prepubertal blinding and olfactory bulbectomy. PRL synthesis was evaluated by measuring the amount of 3H-leucine incorporated into PRL by interior pituitaries in vitro. PRL synthesis was reduced by 47% in blind-anosmic rats, an effect which was reversed by pinealectomy. Although ovariectomy itself led to a 64% decrease in PRL synthesis, dual-sensory deprivation in these animals resulted in a further 59% suppression of PRL production. PRL storage, as estimated by measuring total immunoreactive PRL in vitro also appeared to be significantly depressed in blind-anosmic female rats. Once again, ovariectomy had no effect on this reduction. PRL release, as assessed by monitoring serum levels of the hormone, was decreased in blind-anosmic rats. Ovariectomy also caused a reduction in serum PRL levels; however, the combination of blinding and olfactory bulbectomy had no further depressive effect on circulating PRL titers in these animals. Correlated with the decrease in PRL cell activity in both intact and ovariectomized blind-anosmic rats was a pineal-induced decrease in anterior pituitary weight and DNA content in both sets of animals. From these data we conclude that the pineal's inhibition of the PRL cell in blind-anosmic female rats is largely independent of the gonads and, therefore, could not be secondary to gonadal involution.