Frank Talamantes

Prolactins of pregnancy and their cellular source.

Publisher Summary This chapter discusses the biochemistry, mechanism, and regulation of secretion along with the functions of the pituitary hormones prolactin (PRL) and growth hormone (GH). The placentas of numerous species produce hormones that are structurally and functionally similar to the PRL and GH, which are 20–25K molecular weight proteins that regulate various processes, including mammary gland differentiation, steroidogenesis, somatic growth, and intermediary metabolism. The most extensively studied placental PRL-like hormones are the placental lactogens (PLs). The PLs differ from one another in size and primary function, and some species produce more than one hormone that is a PL. The chapter summarizes the nomenclature used to describe PLs in various species. The maternal component of the placenta—the decidua—also produces PRL-like substances in some species. The primate decidua secretes a molecule that appears to be structurally identical to pituitary PRL. The rat decidua produces a substance designated decidual luteotropin, which has PRL-like biological activity but is not identical to rat pituitary PRL.

Ontogeny of placental lactogen-I and placental lactogen-II expression in the developing rat placenta

The purpose of this investigation was to identify the cellular origin, and the temporal and regional characteristics of placental lactogen-I (PL-I) and placental lactogen-II (PL-II) expression during placental development in the rat. PL-I and PL-II mRNA expression were assessed by Northern blot analysis and in situ hybridization. PL-I and PL-II protein expression were determined by Western blot and immunocytochemical analyses. PL-I mRNA was first detected by in situ hybridization at Day 6 of gestation in mural trophoblast giant cells and a day later, PL-I protein was first detected by immunocytochemistry. PL-I immunostaining extended to the polar trophoblast giant cells as gestation advanced. Polar trophoblast giant cell staining for PL-I was not as intense as the mural trophoblast giant cell staining. Northern and Western blot analyses confirmed the asymmetric distribution of PL-I expression. PL-I mRNA migrated as a 1-kb species and PL-I protein migrated as 30- and 36-40-kDa forms. PL-I expression abruptly declined at Day 12, and by Day 13, PL-I was not detectable. PL-II protein was first detectable at Day 11 of gestation and was localized to trophoblast giant cells. PL-II mRNA could be detected at Day 10 of gestation. Northern and Western blot analyses indicated that PL-II expression significantly increased as gestation advanced and that PL-II expression was asymmetrically distributed similar to PL-I. PL-II mRNA migrated as a 1-kb species and PL-II protein migrated as a 25-kDa species. Blastocysts recovered on Day 4 of gestation initially showed no detectable expression of PL-I or PL-II; however, after 2 days of culture PL-I protein expression was detectable. Biochemical characteristics of PL-I synthesized and secreted by blastocyst outgrowths were similar to PL-I synthesized and secreted by Day 10 placental explants. In summary, (1) PL-I and PL-II are produced by trophoblast giant cells of the developing placenta, (2) PL-I and PL-II exhibit distinct temporal and regional patterns of expression during placental morphogenesis, and (3) PL-I expression by blastocyst outgrowths can be induced in vitro, whereas a more complex array of signals appears necessary for induction of PL-II expression.

Short-term exposure to pregnancy levels of estrogen prevents mammary carcinogenesis

It is well established that pregnancy early in life reduces the risk of breast cancer in women and that this effect is universal. This phenomenon of parity protection against mammary cancer is also observed in rodents. Earlier studies have demonstrated that short-term administration of estradiol (E) in combination with progesterone mimics the protective effect of parity in rats. In this study, the lowest effective E dosage for preventing mammary cancer was determined. Rats were injected with N-methyl-N-nitrosourea at 7 weeks of age; 2 weeks later, the rats were subjected to sustained treatment with 20 μg, 100 μg, 200 μg, or 30 mg of E in silastic capsules for 3 weeks. Treatments with 100 μg, 200 μg, and 30 mg of E resulted in serum levels of E equivalent to those of pregnancy and were highly effective in preventing mammary cancer. E treatment (20 μg) did not result in pregnancy levels of E and was not effective in reducing the mammary cancer incidence. In another set of experiments, we determined the effect of different durations of E with or without progesterone treatments on mammary carcinogenesis. These experiments indicate that a period as short as one-third the period of gestation is sufficient to induce protection against mammary carcinogenesis. The pioneering aspect of our study in contrast to long-term estrogen exposure, which is thought to increase the risk of breast cancer, is that short-term sustained treatments with pregnancy levels of E can induce protection against frank mammary cancer.

Mammary Phenotypic Expression Induced in Epidermal Cells by Embryonic Mammary Mesenchyme

The goal of this research was to establish methods for inducing mammary epithelial differentiation from nonmammary epithelium. For this purpose, mid-ventral or dorsal epidermis (skin epithelium; SKE) from 13-day rat or mouse embryos was associated with 13-day embryonic mouse mammary mesenchyme (mammary gland mesenchyme; MGM) (mouse MGM+rat or mouse SKE). The resultant MGM+SKE recombinants as well as controls (homotypic mouse mammary recombinants, homotypic mouse skin recombinants and mouse mammary mesenchyme by itself) were grafted under the renal capsule of syngeneic or athymic female nude mouse hosts. Most female hosts were induced to undergo lactogenesis by grafting an adult pituitary which elicited a state of hyperprolactinemia. Tissue recombinants of mouse MGM+rat or mouse SKE grown for 1 month in vivo formed a hair-bearing keratinized skin from which mammary ductal structures extended into the mesenchyme. The ducts were composed of columnar luminal epithelial cells as well as basal, actin-positive myoepithelial cells. When grown in pituitary-grafted hosts, the ductal epithelial cells expressed casein and alpha-lactalbumin as judged by immunocytochemistry. The expression of caseins in MGM+SKE recombinants was confirmed by Western blot. The epithelial cells in mouse MGM+rat SKE recombinants expressing milk proteins were shown to be rat cells while the surrounding connective tissue was composed of mouse cells based upon staining with Hoechst dye 33258. Using mammary-specific markers, these studies confirmed the earlier morphological studies of Propper and unequivocally demonstrated for the first time that embryonic mammary mesenchyme can induce morphological and functional mammary differentiation from nonmammary epithelium.

Development of a homologous radioimmunoassay for secreted hamster prolactin.

Abstract A specific and sensitive homologous radioimmunoassay has been developed for secreted hamster prolactin. Hamster serum and pituitary extracts showed parallel dilution-response curves with hamster prolactin. The sensitivity of the assay ranged from 0.5 to 1.0 ng/ml, and the intra- and inter-assay coefficients of variation were 6 and 10%, respectively. Additionally we have demonstrated that the rat prolactin radioimmunoassay kit distributed by the National Institute of Arthritis, Metabolism, and Digestive Diseases is an inadequate method for the measurement of hamster prolactin.

Isolation and partial characterization of secreted mouse pituitary prolactin.

Prolactin secreted by mouse anterior pituitaries was purified by gel filtration on Sephadex G-100. Electrophoretic homogeneity of the purified hormone was demonstrated in several gel systems, and electrophoresis in the presence of sodium dodecyl sulfate indicated an apparent molecular weight of 21 000 +/- 2000. Mouse and ovine prolactin displayed parallel dose vs. response curves in radio-receptor binding studies, indicating that these two hormones compete for identical receptor sites on rabbit mammary membranes. Comparative peptide mapping studies carried out on tryptic digests of mouse and ovine prolactin suggested only partial homology between the two hormones. Internally labeled monomeric mouse prolactin was observed to undergo aggregation following storage at --20 degrees C for 2 months.

Antipeptide antibodies reveal structural and functional characteristics of rat placental lactogen-II☆☆☆

The purpose of this investigation was to develop specific immunologic probes to rat placental lactogen-II (PL-II) and to use the immunologic probes to further characterize rat PL-II. Five oligopeptides corresponding to different regions of rat PL-II (amino acids 1-13, 56-70, 89-103, 107-118, 150-164) were chemically synthesized by solid phase methods and purified to homogeneity by reverse phase high performance liquid chromatography. The synthetic peptides were coupled to keyhole limpet hemocyanin (KLH) and the peptide-KLH conjugates were used to immunize rabbits. Antibody production was monitored by enzyme-linked immunoassay (EIA), electrophoresis and immunoblotting analyses. Each of the antipeptide antisera showed reactivity with the entire rat PL-II protein; however, the extent of the reactivities of each antiserum with rat PL-II was dependent on the conformational state of rat PL-H. Antisera directed to amino acids 56-70 showed the best reactivity toward each of the conformational states of rat PL-II tested. Antibodies generated to the entire rat PL-II protein specifically recognized the 56-70 amino acid sequence but showed limited reactivity with synthetic peptide corresponding to amino acids 1-13, 89-103, 107-118, and 150-164 of rat PL-II. Antisera to amino acids 56-70 of rat PL-II were specific for PLs as demonstrated by their recognition of rat PL-II, mouse PL-II and human PL and by their lack of reactivity with rat pituitary prolactin and growth hormone and with a series of other synthetic peptides to rat PL-II and rat prolactin-like protein-A. The immunorecognition of human PL was restricted to antipeptide antibodies directed to amino acids 56-70 of rat PL-II. The chemically synthesized peptides representing various regions of rat PL-II did not show significant interactions with prolactin receptors, and antisera directed to the peptides failed to interfere with the binding of either rat PL-II or human PL to prolactin receptors. In summary, we have generated a series of immunologic probes for studying the structure of rat PL-II. The sequence comprising amino acids 56-70 of rat PL-II was shown to make up at least part of an epitope for rat PL-II and to be a region of significant structural homology with mouse PL-II and human PL.

Insulin-like growth factor (IGF)-I obliterates the pregnancy-associated protection against mammary carcinogenesis in rats: evidence that IGF-I enhances cancer progression through estrogen receptor-α activation via the mitogen-activated protein kinase pathway

IntroductionPregnancy protects against breast cancer development in humans and rats. Parous rats have persistently reduced circulating levels of growth hormone, which may affect the activity of the growth hormone/insulin-like growth factor (IGF)-I axis. We investigated the effects of IGF-I on parity-associated protection against mammary cancer.MethodsThree groups of rats were evaluated in the present study: IGF-I-treated parous rats; parous rats that did not receive IGF-I treatment; and age-matched virgin animals, which also did not receive IGF-I treatment. Approximately 60 days after N-methyl-N-nitrosourea injection, IGF-I treatment was discontinued and all of the animal groups were implanted with a silastic capsule containing 17β-estradiol and progesterone. The 17β-estradiol plus progesterone treatment continued for 135 days, after which the animals were killed.ResultsIGF-I treatment of parous rats increased mammary tumor incidence to 83%, as compared with 16% in parous rats treated with 17β-estradiol plus progesterone only. Tumor incidence and average number of tumors per animal did not differ between IGF-I-treated parous rats and age-matched virgin rats. At the time of N-methyl-N-nitrosourea exposure, DNA content was lowest but the α-lactalbumin concentration highest in the mammary glands of untreated parous rats in comparison with age-matched virgin and IGF-I-treated parous rats. The protein levels of estrogen receptor-α in the mammary gland was significantly higher in the age-matched virgin animals than in untreated parous and IGF-I-treated parous rats. Phosphorylation (activation) of the extracellular signal-regulated kinase-1/2 (ERK1/2) and expression of the progesterone receptor were both increased in IGF-I-treated parous rats, as compared with those in untreated parous and age-matched virgin rats. Expressions of cyclin D1 and transforming growth factor-β3 in the mammary gland were lower in the age-matched virgin rats than in the untreated parous and IGF-I-treated parous rats.ConclusionWe argue that tumor initiation (transformation and fixation of mutations) may be similar in parous and age-matched virgin animals, suggesting that the main differences in tumor formation lie in differences in tumor progression caused by the altered hormonal environment associated with parity. Furthermore, we provide evidence supporting the notion that tumor growth promotion seen in IGF-I-treated parous rats is caused by activation of estrogen receptor-α via the Raf/Ras/mitogen-activated protein kinase cascade.

The amino acid composition of secreted mouse prolactin, growth hormone, and hamster prolactin: the presence of one tryptophan in mouse prolactin.

Abstract The amino acid compositions and the electrophoretic properties of secreted mouse prolactin (mPRL), mouse growth hormone (mGH), and hamster prolactin (haPRL) were determined. The amino acid compositions of secreted mPRL and haPRL were similar to the compositions of other rodent prolactins, except that secreted mPRL contained only one tryptophan residue rather than the usual two. The composition of secreted mGH was similar to that of rat growth hormone. On 10% alkaline polyacrylamide gels, mPRL, mGH, and haPRL migrated with R f s of 0.54, 0.21, and 0.69, respectively. The molecular weights of mPRL, mGH, and haPRL, determined by SDS-gel electrophoresis, were 23,000, 21,000, and 22,000, respectively.

Prevention of mammary carcinogenesis by short-term estrogen and progestin treatments

IntroductionWomen who have undergone a full-term pregnancy before the age of 20 have one-half the risk of developing breast cancer compared with women who have never gone through a full-term pregnancy. This protective effect is observed universally among women of all ethnic groups. Parity in rats and mice also protects them against chemically induced mammary carcinogenesis.MethodsSeven-week-old virgin Lewis rats were given N-methyl-N-nitrosourea. Two weeks later the rats were treated with natural or synthetic estrogens and progestins for 7–21 days by subcutaneous implantation of silastic capsules.ResultsIn our current experiment, we demonstrate that short-term sustained exposure to natural or synthetic estrogens along with progestins is effective in preventing mammary carcinogenesis in rats. Treatment with 30 mg estriol plus 30 mg progesterone for 3 weeks significantly reduced the incidence of mammary cancer. Short-term exposure to ethynyl estradiol plus megesterol acetate or norethindrone was effective in decreasing the incidence of mammary cancers. Tamoxifen plus progesterone treatment for 3 weeks was able to confer only a transient protection from mammary carcinogenesis, while 2-methoxy estradiol plus progesterone was effective in conferring protection against mammary cancers.ConclusionsThe data obtained in the present study demonstrate that, in nulliparous rats, long-term protection against mammary carcinogenesis can be achieved by short-term treatments with natural or synthetic estrogen and progesterone combinations.