Induction of mammary prolactin receptors and lactose synthesis after ovariectomy in the pregnant mouse.

The development of prolactin receptors in the mammary gland after ovariectomy was investigated in pregnant KA mice. Mice were ovariectomized on day 13 of pregnancy and used for the determination of the amount of specific binding of 125I-labelled prolactin to the mammary tissue, and the contents of lactose and nucleic acids in the mammary gland 0, 8, 24, and 72 hr after the operation. The specific binding of 125I-labelled prolactin, lactose and RNA contents in the mammary gland remained low until 8 hr, sharply increased 24 hr and decreased 72 hr after ovariectomy. When ovariectomized mice were treated with 0.2 mg progesterone, pregnancy was maintained and an increase (1.5-fold) in the amount of specific binding was observed with an increase of lactose content. Five mg progesterone completely inhibited lactose synthesis. Cortisol administered with progesterone did not show any specific change at the dose used (0.5 to 10 mg). Although the amount of specific binding was also increased after hysterectomy, this increase (2-fold) did not fully cover the increase after ovariectomy (3-fold). These results suggest that the recepter site for prolactin is induced before the initiation of lactose synthesis caused by ovariectomy during pregnancy.     

Lactogenesis induced by ovariectomy in pregnant rats and its regulation by oestrogen and progesterone

Ovariectomy on day 19 of pregnancy augmented galactosyl transferase activity 24 h after surgery preceding by 6 h the significant alpha-lactalbumin accumulation. Progesterone, injected immediately after ovariectomy showed a clear inhibitory effect on both galactosyl transferase and alpha-lactalbumin concentration, measured 30 h after ovariectomy. However, once the synthesis of lactose has been induced, progesterone is no longer inhibitory. Oestrogen induced a significant increase in lactose synthetase activity but no effect was obtained on galactosyl transferase activity. Progesterone, in a time and dose dependent relationship, was capable of preventing the effect of estrogen on lactogenesis. The lactogenic action of oestrogen in ovariectomized pregnant rats might be due to a direct effect at the mammary gland level facilitating the action of prolactin or through an indirect effect mediated via an increase on prolactin release.     

Effects of Ovariectomy and Prolactin on Mammary Gland Expressions of Transforming Growth Factor alpha and Epidermal Growth Factor Receptor mRNAs in Mice

Beginning 15 days after ovariectomy (OVX), a high mammary tumor strain of SHN virgin mice at 3 months of age received subcutaneous injections of danazol (0.5 mug / 0.1 ml olive oil, once a day), perphenazine (0.05 mg / 0.1 ml saline, twice a day) or ovine prolactin (oPRL: 0.25 mg / 0.05 ml buffer, twice a day) for 3 days to modulate their circulating PRL levels. The serum PRL level was significantly decreased by danazol and increased by perphenazine compared to the intact and OVX-control groups. The expression of both transforming growth factor alpha (TGFalpha) mRNA and epidermal growth factor receptor (EGFR) mRNA in the mammary gland was increased by danazol. However, TGFalpha mRNA expression was decreased by perphenazine. Meanwhile, mammary end-bud formation was inhibited in danazol-treated group. All findings suggest that the manifestation of the effect of TGFalpha on mammary gland is rather suppressed by PRL, while mammary gland growth needs the participation of PRL; in other words, PRL is dominant to TGFalpha on the mammary gland growth. OVX resulted in a significant decrease of TGFalpha mRNA expression in the mammary gland despite of little alteration in serum PRL, confirming the previous observations. The similar trend was observed in ICR mice; however, the response to hormonal modulation is generally less susceptible than SHN mice.     

Suckling-induced prolactin release potentiates mifepristone-induced lactogenesis in pregnant rats

Suckling, starting at 19:00 h on Day 18 of pregnancy, induced a significant increase in serum prolactin concentration at 20:00 h on Day 19 of pregnancy, but no increase in mammary gland casein or lactose content. Mifepristone (2 mg/kg) injection at 08:00 h on Day 19 of pregnancy induced significant increases in casein, but not in lactose, 24 h after administration. Mifepristone alone did not induce prolactin secretion, indicating that lactogenesis was induced by placental lactogen in the absence of progesterone action. When mifepristone was injected into suckling rats, serum prolactin concentrations were higher than in the untreated suckling rats. Casein in these rats increased significantly 12 h after mifepristone administration and lactose at 24 h after. If the suckling mifepristone-treated rats were given two injections of bromocriptine (1.5 mg/kg) at 12:00 h on Days 18 and 19 of pregnancy, serum prolactin concentrations were not increased by suckling, but casein and lactose concentrations in the mammary gland showed values similar to those obtained in the mifepristone-treated non-suckling rats. Mifepristone can therefore potentiate suckling-induced prolactin release in pregnant rats, demonstrating a direct central inhibitory action of progesterone on prolactin secretion. This suckling-induced prolactin secretion, unable to induce casein or lactose synthesis in the presence of progesterone, enhanced significantly synthesis of these milk components in the absence of progesterone action (rats treated with mifepristone). Fatty acid synthase, which is stimulated by the suckling stimulus in lactating rats, was not modified by mifepristone or suckling in pregnant rats.     

Characteristics of the early effect of prolactin on lactose biosynthesis in mouse mammary gland explants

These studies were carried out to characterize the early effect of prolactin (PRL) on lactose biosynthesis in cultured mammary gland explants derived from 12- to 14-day pregnant mice. The rate of lactose biosynthesis was assessed by the rate of radiolabeled glucose incorporation into lactose. For the rapid isolation of lactose, a new method which involves the use of thin-layer chromatography on cellulose-impregnated plastic sheets was employed. The onset of the PRL stimulation of [3H]glucose incorporation into lactose occurred 6-8 hr after exposing the explants to PRL. The response to PRL was essentially all or none with maximum responses occurring with PRL concentrations above 25 ng/ml. The lowest stimulatory concentration of PRL was 10 ng/ml. The action of PRL on lactose biosynthesis requires both ongoing RNA and protein synthesis since puromycin, cyclohexamide, and actinomycin D abolished the PRL effect.     

Ovariectomy during the luteal phase influences secretion of prolactin, growth hormone, and insulin-like growth factor-I in the bitch

A decline in circulating progesterone concentration plays an important role in the ethiopathogenesis of pseudopregnancy in the bitch. Because growth hormone (GH) and prolactin (PRL) are essential for normal mammogenesis and the secretion of these hormones is influenced by changes in the circulating progesterone concentration, the purpose of this study was to investigate the effects of mid-luteal phase ovariectomy on the 6-h pulsatile plasma profiles of GH and PRL and the basal plasma concentrations of GH, PRL, and insulin-like growth factor-I (IGF-I) in six beagle bitches. Ovariectomy was followed by only mild or covert signs of pseudopregnancy. The sharp decrease of the plasma progesterone concentration was accompanied by decreased basal plasma concentrations of GH and IGF-I and a rise in basal plasma PRL concentration. GH and PRL were secreted in a pulsatile fashion both prior to and after ovariectomy. The mean basal plasma GH concentration was significantly higher before ovariectomy than on days 1 and 7 after ovariectomy. The mean area under the curve above the zero level (AUC(0)) for GH was significantly higher before than at 7 days after ovariectomy. The mean area under the curve above basal level (AUC(b)) and the frequency of GH pulses at 7 days after ovariectomy were significantly higher than before and 1 day after ovariectomy. Both the mean basal plasma PRL concentration and the mean AUC(0) for PRL increased after ovariectomy. In conclusion, ovariectomy of bitches in the mid-luteal phase stops progesterone-induced GH release from the mammary gland, as evidenced by the lowering of basal plasma GH levels, the recurrence of GH pulsatility, and the lowering of circulating IGF-I levels. The sudden lowering of plasma progesterone concentration is probably a primary cause of a prolonged increase in PRL secretion. These observations underscore the importance of similar, albeit less abrupt, hormonal changes in the cyclical physiological alterations in the mammary gland and in the development of pseudopregnancy.     

Effect of estrogen and placental lactogen on lactogenesis in pregnant rats

The removal of the corpora lutea or ovariectomy on Day 18 of pregnancy induced a rise in serum prolactin 24 h after surgery with a rapid decline to control values 4 h after the surge, only in the ovariectomized group. When hysterectomy was performed in addition to luteectomy or ovariectomy a similar rise in prolactin was obtained. Lactose synthetase activity in mammary tissue was significantly higher in the luteectomized and luteectohysterectomized rats when compared with ovariectomized, ovariohysterectomized rats and the sham-operated group. Estrogen treatment 12 h after ovariectomy increased serum prolactin and lactose synthetase activity to values similar to those measured in luteectomized rats, but this increase was significantly greater when compared with the ovariectomized-nontreated group. Treatment with Tamoxifen did not decrease serum prolactin in the luteectomized rats but lactose synthetase was reduced to values similar to that obtained in ovariectomized rats. Treatment with 2 bromo-alpha-ergocryptine-mesylate (CB-154) prevented the rise in serum prolactin in the ovariectomized, luteectomized and luteectohysterectomized groups, but lactose synthetase activity was lowered to control values (sham-operated rats) only in the luteectohysterectomized rats. According to these findings, rat placental lactogen in the absence of prolactin and progesterone induces lactose synthesis. Estrogen facilitates prolactin but not placental lactogen action on lactose synthetase activity.     

Interrelationship of glycogen metabolism and lactose synthesis in mammary epithelial cells of mice.

Glycogen metabolism in mammary epithelial cells was investigated (i) by studying the conversion of glucose into glycogen and other cellular products in these cells from virgin, pregnant and lactating mice and (ii) by assaying the enzymes directly involved with glycogen metabolism. We find that: (1) mammary epithelial cells synthesized glycogen at rates up to over 60% that of the whole gland; (2) the rate of this synthesis was modulated greatly during the reproductive cycle, reaching a peak in late pregnancy and decreasing rapidly at parturition, when abundant synthesis of lactose was initiated; (3) glycogen synthase and phosphorylase activities reflected this modulation in glycogen metabolism; (4) lactose synthesis reached a plateau during late pregnancy, even though lactose synthase is reported to increase in the mouse mammary gland at this time. We propose that glycogen synthesis restricts lactose synthesis during late pregnancy by competing successfully for the shared UDP-glucose pool. The physiological advantage of glycogen accumulation during late pregnancy is discussed.     

Antiprogesterone and antiglucocorticoid actions of RU 486 on rabbit mammary gland explant cultures. Evidence for a persistent inhibitory action of residual progesterone upon the mammary tissue

The antiprogesterone and antiglucocorticoid compound RU 486 added to pregnant rabbit mammary gland explant cultures had no effect alone but significantly stimulated casein production in the presence of ovine prolactin (PRL) in a dose dependent manner. This stimulation was inhibited by progesterone (Pg) and the Pg agonist R5020. When the explants were cultured for 5 days with two changes of medium, to eliminate all steroids, and hormones added afterwards, the effect of PRL was potentiated, Pg was no longer inhibitory and RU 486 had no effect, RU 486 also could inhibit the stimulatory action of glucocorticoids added to the cultures along with PRL. The compound was able to displace [3H]dexamethasone and [3H]R 5020 from mammary gland glucocorticoid and Pg receptors respectively and proved to have a high relative binding affinity (RBA) for both receptors when compared with typical ligands for each receptor. The RBAs of RU 486 and the steroids used in this study to mammary gland glucocorticoid and Pg receptors correlated well with the ability of RU 486 to block their biological activities. These results demonstrate that RU 486 has both antiglucocorticoid and antiprogesterone activities in pregnant rabbit mammary glands as well as the existence of a strong inhibitory residual action of Pg in the gland that persists during the first 48 h of culture and that can be eliminated by RU 486 or after several days of culture with no hormones.     

Uterine specific antigens in the ewe

Uterine flushings from ewes on days 0, 3, 6, 9, 12 and 15 of the estrous cycle were analyzed for total protein content. Flushings from days 9, 12 and 15 had greater (P<.01) amounts of protein than those from 0, 3 and 6. Antisera to uterine fluids from ewes at day 10 to 12 or day 14 to 15 of pregnancy detected two uterine-specific antigens in uterine flushings at day 7, 11 and 15 but not at days 0 and 3 of the cycle. A third uterine antigen was also detected in kidney tissue extracts. All three antigens were present in endometrial extracts at each stage examined. Progesterone, or estrogen plus progesterone, administration to ovariectomized ewes induced the appearance of the two uterine-specific antigens. The third antigen was detectable even in ovariectomized ewes. No pregnancy-specific antigens were detected in flushings from days 7, 11 or 15 of gestation. The effect of pregnancy on endometrial protein synthesis was examined in vitro . No differences were seen in the incorporation of (3)H-leucine in day 11 pregnant or nonpregnant or in day 14 pregnant or nonpregnant endometrium. No differences in total uterine lumenal protein were observed. Endometrial secretions, obtained by conditioning media with endometrial explant cultures, were evaluated to assess their effect on protein synthesis in day 11 embryos cultured in vitro . No significant effects of endometrial secretions or serum were observed.     

Variation of the poly(A) size classes in the rat mammary gland during the lactation cycle

The sizes of the poly(A) tracts associated with rat mammary RNA were determined at several time points in the lactation cycle. The poly(A) tracts in the lactating gland displayed two predominant size class peaks at 80-85 and 45-47 residues. The 9S whey protein mRNA and the 15S casein mRNA purified from the 12 day lactating mammary gland both contained poly(A) tracts displaying a similar size distribution. The 45 residue tracts were a characteristic of lactation; they were not found at 8 days of pregnancy and only small amounts of these shorter poly(A) tracts were found in the 16 day pregnant gland. The poly(A) tracts of the involuted gland displayed the same size characteristics as those of late pregnancy. At all the developmental stages that were examined, the fraction of 45 residue poly(A) tracts was always proportional to the total poly(A) content of the mammary cells.     

Prolactin binding in rat mammary gland during pregnancy and lactation.

Lactogenic hormones from the placenta and pituitary are primarily responsible for the growth and function of the mammary gland during pregnancy and lactation. In the present study we described the optimal conditions for the measurement of 125I-labeled ovine prolactin binding to mammary gland slices of pregnant and lactating rats. Prolactin binding is saturable (Kd approx. 2.36 - 10(-9) M), hormone specific and destroyed by proteases. The hormonal environments of pregnancy and lactation dramatically influence the availability and measurement of prolactin binding sites. Whereas binding consistently appears to be low in mammary glands removed from rats during pregnancy, binding levels rise 7--8-fold shortly after birth and remain high during the 22 days of lactation. However, the removal of the ovaries and gravid uteri at specific times during pregnancy results in a prompt 3--6-fold increase in prolactin binding. Elevated levels in potential prolactin binding capacity appear in mammary tissue coincident with the reported rise in serum rat placental lactogen between the eighth and eleventh days. We suggest that high levels of this lactogenic hormone promote the appearance of prolactin binding sites during pregnancy and mask the sites such that they are not available for measurement in vitro.     

Prolactin stimulation of iodide uptake and incorporation into protein is polyamine-dependent in mouse mammary gland explants

Previous studies have demonstrated that the prolactin stimulation of most lactational processes (casein, lactose, and triglyceride synthesis) requires an earlier stimulating effect of prolactin on the synthesis of the polyamines. Spermidine appears to be the specific polyamine required for prolactin to enhance milk product synthesis. Inorganic iodide is present in milk at more than an order of magnitude higher concentration than that of the maternal plasma. Since prolactin stimulates iodide accumulation in milk, the goal of these studies was to determine the role of the polyamines in this hormone response. Two drugs were employed in these studies: DFMO (difluoromethylornithine), which inhibits ornithine decarboxylase, and MGBG [methylglyoxal bis(guanyl-hydrazone)], which inhibits S-adenosyl methionine decarboxylase. In mammary gland explants from midpregnant (10-14 days of pregnancy) mice, MGBG at 100 microM abolished the prolactin stimulation of iodide uptake and incorporation into milk proteins, whereas DFMO caused a concentration-dependent inhibition of the PRL response. Selected sensitivity of the MGBG and DFMO inhibitions was validated by a reversal of the drug inhibitions with the addition of 1 mM spermidine to the culture medium. These data suggest that the polyamine signaling pathway is involved in the prolactin stimulation of iodide uptake into milk.     

Effect of ovariectomy on pregnancy in mares.

Pony mares were bilaterally ovariectomized at different stages of pregnancy between Days 25 and 210. Abortion or fetal resorption occurred within 2 to 6 days after operations in all 14 mares ovariectomized between Days 25 and 45 and after an interval of 10 to 15 days in 9 of 20 other ovariectomized between 50 and 70 days. All 12 mares ovariectomized on either 140 or 210 days carried their foals to normal term. The termination of early pregnancy was preceded by a loss of uterine tone and of a palpable uterine bulge. The mean length of gestation in all mares in which pregnancy was not interrupted by ovariectomy was not significantly different from that in a group of contemporary control mares. Plasma progestagen concentrations dropped to less than 2 ng/ml after ovariectomy, whether or not pregnancy was maintained. Mares ovariectomized on Day 25 and injected with 100 mg progesterone daily for 10 or 20 days remained pregnant during treatment but showed a loss of uterine tone and the fetal bulge disappeared within 4 to 6 days after the end of treatment. Non-pregnant ovariectomized or intact seasonally anoestrous mares injected i.m. with 50 or 100 mg progesterone daily for 8 weeks showed changes in uterine tone, length and thickness similar to those occurring in mares during early pregnancy.     

大豆黄酮促进妊娠大鼠乳腺发育和泌乳的实验研究

研究大豆黄酮（Ｄａ）对妊娠大鼠乳腺发育和泌乳的作用及其与神经内分泌的关系，结果表明；妊娠期口服大豆黄酮，能显著提高泌乳前期大鼠乳腺重量、ＤＮＡ、ＲＮＡ含量和ＲＮＡ／ＤＮＡ值，同时极显著地提高大鼠的泌乳量、血清ＧＨ和ＰＲＬ含量及乳腺胞浆雌二醇受体的数目和亲和力；相反．妊娠后期口服多巴胺激动剂溴隐亭，能显著降低泌乳前期乳腺重量、ＤＮＡ和ＲＮＡ含量及泌乳量，并显著降低血清ＧＨ和ＰＲＬ含量及乳腺雌二醇受体的数目和亲和力；但连续两周口服Ｄａ后再口服澳隐亭一周，溴隐亭的上述作用被阻断。结果提示：Ｄａ促进大鼠乳腺发育和泌乳的作用与其抑制或站抗多巴胺对垂体ＰＲＬ、ＧＨ分泌的抑制作用有关。     

Prolactin binding in rat mammary gland during pregnancy and lactation

Lactogenic hormones from the placenta and pituitary are primarily responsible for the growth and function of the mammary gland during pregnancy and lactation. In the present study we describe the optimal conditions for the measurement of ¹²⁵I-labeled ovine prolactin binding to mammary gland slices of pregnant and lactating rats. Prolactin binding is saturable (K_{d approx. 2.36 · 10^?9 M)}, hormone specific and destroyed by proteases. The hormonal environments of pregnancy and lacation dramatically influence the availability and measurement of prolactin binding sites. Whereas binding consistently appears to be low in mammary glands removed from rats during pregnancy, binding levels rise 7–8-fold shortly after birth and remain high during the 22 days of lactation. However, the removal of the ovaries and gravid uteri at specific times during pregnancy results in prompt 3–6-fold increase in prolactin binding. Elevated levels in potential prolactin binding capacity appear in mammary tissue coincident with the reported rise in serum rat placental lactogen between the eight and eleventh days. We suggest that high levels of this lactogenic hormone promote the appearance of prolactin binding sites during pregnancy and mask the sites such that they are not available for measurement in vitro.     

Depressed pituitary prolactin mRNA, prolactin synthesis, and prolactin storage after light-deprivation in female hamsters is not due to loss of estrous cyclicity alone

Pituitary prolactin (PRL) cell activity (i.e. PRL messenger ribonucleic acid [mRNA] levels, PRL synthesis, and radioimmunoassayable [RIA]-PRL), and serum RIA-PRL were measured in female golden Syrian hamsters that were (1) light-deprived and then ovariectomized before loss of estrous cyclicity, (2) light-deprived but not yet acyclic, and (3) light-deprived and ovariectomized simultaneously. The results indicate that light-deprivation can decrease PRL cell activity in ovariectomized hamsters but not in animals that continue to cycle. Thus, estrous cyclicity can be said to largely protect PRL cell activity from depressions due to light deprivation. After acyclicity/ovariectomy, however, PRL cell activity is no longer protected and light-deprivation leads to large depressions in PRL mRNA levels, PRL synthesis, and RIA-PRL beyond that caused by acyclicity/ovariectomy alone. As seen in previous studies of total light-deprivation in nonovariectomized female hamsters, we found that removing the pineal gland in conjunction with light-deprivation in ovariectomized hamsters can completely, partially, or fail to restore various measures of PRL cell activity.     

Cortactin-binding protein 90 (CBP90) expression in the mouse mammary glands during prolactin-induced lobuloalveolar development

We have previously performed suppression subtractive hybridization to identify genes that were induced during prolactin (PRL)-driven lobuloalveolar development of the mammary gland. This suggested that cortactin-binding protein 90 (CBP90), which is known to be a brain-specific protein that binds to cortactin, was expressed under the regulation of PRL in the mammary glands (preliminary observation). In this study, the expression of CBP90 was examined in the mammary glands of mice under manipulated hormonal circumstances. PRL treatment by 9 days of pituitary grafting induced CBP90 expression in the normal mammary glands but not in the cleared fat pads, while cortactin was expressed constitutively in both the normal mammary glands and the cleared fat pads. Unlike milk proteins, longer treatment with PRL (36 days of pituitary grafting) did not increase the expression level of CBP90 mRNA, while it slightly increased the cortactin mRNA level. Mammary CBP90 mRNA expression was induced by pituitary grafting but not by progesterone treatment in PRL-deficient mice, while pituitary grafting induced mammary CBP90 expression in ovariectomized PRL-deficient mice only when estrogen and progesterone were appropriately supplemented to permit the formation of alveolar buds. The CBP90 protein was detected by immunohistochemistry in the luminal epithelium of the alveolar buds and more faintly in the ductal epithelium. Thus, from the unique expression pattern, CBP90 may be useful as a molecular marker for the hormone-stimulated development of mammary alveolar buds.