Changes in the activities of enzymes of the biosynthetic pathway of the nicotinamide nucleotides in rat mammary gland during the lactation cycle

1. The activities of NMN pyrophosphorylase, NMN adenylyltransferase and NAD kinase in the mammary glands of rats at different stages of pregnancy, lactation and involution were measured. 2. NMN pyrophosphorylase has a low activity early in pregnancy, but its activity increases at parturition and in early lactation to reach a maximum at the tenth day of lactation, after which it remains constant until it declines abruptly in involution. 3. NMN adenylyltransferase is already quite active by the tenth day of pregnancy and its activity does not rise further in the second half of gestation. After a sharp rise in activity at parturition, the activity of the enzyme declines slowly throughout the period of lactation and, more sharply, in involution. 4. NAD kinase has a low activity for most of pregnancy, but its activity rises at parturition to a value at 2 days of lactation that is maintained until the tenth day. Between the tenth and fifteenth days of lactation the activity almost doubles, but falls sharply in mammary involution. 5. The relation of the activities of these enzymes to the rates of synthesis of NAD and NADP is discussed.     

Calmodulin content of rat mammary tissue and isolated cells during pregnancy and lactation.

The calmodulin content of heat-treated extracts of rat mammary tissue and isolated cells was measured by using stimulation of cyclic nucleotide phosphodiesterase (PDE) activity and radioimmunoassay (r.i.a.) procedures. The calmodulin content of mammary tissue increased 2.5-fold near the time of parturition, remained at the elevated level during lactation, then, after the onset of involution, decreased to values similar to those measured from mammary tissue of pregnant rats. When tissue from 15 animals in different stages of pregnancy, lactation and involution were compared, the r.i.a. gave 2.6-fold higher results than the PDE assay. To investigate further the increase in calmodulin content of mammary tissue, secretory and myoepithelial cells were enzymically dissociated from rat mammary tissue during different stages of pregnancy, lactation and involution. Protein, DNA, lactose, glucose-6-phosphate dehydrogenase and alkaline phosphatase were assayed to characterize the cell fractions. By using r.i.a., the calmodulin content per mg of protein in isolated secretory-cell fractions was high near parturition, then decreased and remained relatively constant during lactation. The amount of calmodulin expressed per mg of DNA in secretory cells did not show a marked change near parturition, suggesting a constant amount of calmodulin per cell. The calmodulin content of myoepithelial cells dissociated from mammary tissue and measured by using r.i.a. was 6-fold lower than in secretory cells and remained relatively constant during the course of lactation. The changing levels of calmodulin in rat mammary tissue during development are suggested to be related to proliferation and destruction of secretory epithelial cells, events that occur near parturition and involution respectively.     

Differential expression of claudin 1, 3, and 4 during normal mammary gland development in the mouse

The claudins are a family of tight junction proteins that display varied tissue distribution and preferential specificity. We recently identified by microarray analysis, members of this family, particularly claudin 1 (cldn1), as highly upregulated genes in the mouse mammary gland during early involution. Gene expression was confirmed by immunohistochemistry and real-time PCR. We then examined gene and protein expression throughout normal mammary gland development. The cldn3 gene showed a steady increase in expression from pregnancy to involution, while cldn1 and cldn4 gene expression increased during pregnancy, but decreased sharply by D10 of lactation, and once again was significantly increased by D1 of involution (P < 0.001 for both genes). The different patterns of gene expression observed between cldn3, and cldn1, and 4 suggest that different family members may be functionally important at different times during mouse mammary gland development. All three genes exhibited a high level of expression at day 1 (D1) of involution, followed by a dramatic decrease in gene expression to day 10 of involution. Immunostaining with the cldn3 antibody showed intense staining of epithelial cells; however, a lesser degree of staining was evident with the cldn1 antibody. In addition to the lateral staining of epithelial cells, basal staining was evident at D1 and D2 of involution and cytoplasmic staining was evident during lactation. Since claudins are known to play a role as tight junction proteins, lateral and basal staining may suggest a role in other functions such as vesicle trafficking or remodeling of tight junctions at different stages of mammary gland development. Cldn1 and 3 antibodies also stained epithelial cells in mouse mammary tumors. In summary, cldn1, 3, and 4 are differentially expressed in the mammary gland during pregnancy, lactation, and involution, suggesting different roles for these proteins at different stages of mammary gland function. In addition, cldn1 and cldn3 are detected in mammary tumors and the wide distribution of cldn3 in particular, suggest specific roles for these proteins in mammary tumorigenesis.     

Fonction catabolique de l'épithélium mammaire

Summary The histochemical (iron, lipopigments, acid phosphatase, leucine aminopeptidase) and cytologic (lysosomes) changes occuring during pregnancy, lactation and involution of mouse, rat, rabbit, guinea-pig mammary glands are studied by light microscopy and electron microscopy.In all the animals examined, the mammary epithelium has an intracellular digestive system which is adapted to subserve two functions. The first one is the segregation of cytoplasmic components which often precedes cellular involution. The second one is the regulation of secretory processes in the non lactating glands. This digestion of endogenous materials results in the formation of various lytic bodies: dense bodies sometimes containing ferritin, vacuolated dense bodies with membranous residues, autophagic vacuoles. The lysosomes can give large complex dense bodies like lipofuscin pigments with or without ferritin.Leucine aminopeptidase which always disappears in the mouse mammary epithelium during lactation is not present in rat, rabbit, guinea-pig mammary epithelium. In these species only the vascular tissue contains the enzyme. This observation indicates that leucine aminopeptidase does not take care of the overproduction of secretory products in the non-lactating glands.Acid phosphatase is concentrated in secretory granules and in lytic bodies: multivesicular bodies, dense bodies with ferritin, vacuolated dense bodies, lipopigments. This enzyme constitutes probably a mechanism for controlling and triggering the destruction of the secretory material with no active elimination.The iron of the mammary epithelium appears in virgin mice older than 30 weeks and in mice, rats, rabbits, guinea-pigs during glandular cells involution. This is a catabolic iron located in lysosomes. Its amount depends upon the iron content of the milk and upon the competitive secretory and catabolic activities of the glandular cells. An explanation of iron disappearance during a second pregnancy and lactation is discussed.     

Alkaline ribonuclease and ribonuclease inhibitor in mammary gland during the lactation cycle and in the R3230AC mammary tumour.

Alkaline RNAase (ribonuclease) and RNAase inhibitor were assayed to determine the potential role of the degradative process in regulating the amount of RNA in the mammary gland and mammary tumour. Very little free alkaline RNAase activity was found in the cytosol fraction of the mammary gland of virgin, pregnant, lactating or involuting Fischer rats. However, addition of p-chloromercuribenzoate to the assay medium revealed latent RNAase which, when expressed on a DNA basis, decreased during pregnancy and lactation. The cytosol latent RNAase is stable in 0.125 M-H2SO4. The non-cytosol RNAase activity also decreased during pregnancy and lactation. Addition of Triton X-100 produced slightly higher activity at all stages tested. The inhibitor activity in rat mammary gland was very low before pregnancy, increased gradually during pregnancy and more dramatically at parturition, continued to increase throughout lactation and returned to resting-gland values by the sixth day of involution. The increase during pregnancy may be due to the increased cellularity of the gland, whereas the gain during lactation was more than could be accounted for by increases in cell number. The R3230AC transplantable mammary tumour resembles the normal gland in early lactation with respect to both its cytosol and non-cytosol alkaline RNAase activities and its moderately high content of RNAase inhibitor. The relatively high inhibitor and low RNAase activities in both the gland of the lactating rat and in the tumour are of potential significance in maintaining high amounts of RNA and increased rates of protein synthesis in these tissues.     

Casein and alpha-lactalbumin messenger RNAs during the development of mouse mammary gland. Isolation, partial purification, and translation in a cell-free system

Messenger RNAs for the milk proteins, casein and α-lactalbumin, were isolated and partially purified from lactating mouse mammary glands by oligo(dT)cellulose chromatography followed by sucrose density gradient centrifugation. The translation of poly(A)⁺ mRNA in a wheat germ cell-free system yielded three casein polypeptides and a putative precursor form of α-lactalbumin which were precipitated by specific antibodies and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The casein polypeptides synthesized in vitro had a molecular weight that was no greater than that of the caseins in mouse milk. The presence of individual casein mRNAs coding for these polypeptides was demonstrated by the translation of various fractions of mRNA obtained by sucrose density gradient centrifugation of poly(A)⁺ mRNA. Casein mRNA activity increased about 250-fold between midpregnancy and the 10th–12th days of lactation, amounting to 50–60% of the total mRNA activity in that tissue. A similar study of α-lactalbumin mRNA showed an increase during lactation amounting to 0.2–0.4% of the total mRNA activity, which corresponds to the percentage of α-lactalbumin in total mouse milk protein.     

Variation in the lack of polyadenylation of the rat milk protein mRNAs during the lactation cycle

Translationally active milk protein mRNAs were found as nonpolyadenylated mRNAs in the rat mammary gland during pregnancy, lactation and involution. Analyses of whey protein mRNA and casein mRNA with the corresponding cDNAs showed that the lack of polyadenylation of these mRNAs at different time points of the lactation cycle is not consistent with the hypothesis that polyadenylation may be incomplete in the mammary gland when large amounts of mRNA are synthesized. The fraction of whey protein mRNA and casein mRNA that lacked polyadenylation was inversely proportional to the concentration of each sequence in the tissue during pregnancy, lactation and involution. A model is proposed to explain the finding that in each animal the ratio of casein mRNA to whey protein mRNA was similar in polyadenylated RNA and in nonpolyadenylated RNA at all stages of the lactation cycle.     

Purification and characterization of mouse α-lactalbumin from lactating mammary glands

The whey protein, α-lactalbumin, was purified from lactating mammary glands of mice at high yields. It exists as two major charge forms (pI values of 6.2 and 5.8) with similar molecular weights (approx. 14 00). Antibodies prepared against these peptides precipitate newly synthesized and secreted α-lactalbumin from organ cultures of mid-pregnancy mammary glands. The antibody is specific for mouse α-lactalbumin as it does not react with mouse casein, mouse serum or purified bovine α-lactalbumin or galactosyl transferase. In addition, it blocks enzymatic activity of α-lactalbumin in mouse milk but has no effect on guinea pig or human milk. A very sensitive radioimmunoassay has been developed with this antibody which can detect α-lactalbumin levels as low as 0.25 ng.     

Developmental regulation of murine integrin β1 subunit- and Hsc73-encoding genes in mammary gland: sequence of a new mouse Hsc73 cDNA

A partial integrin β1 subunit-encoding cDNA (Itgβ1) and a new heat-shock protein 70-like-encoding cDNA (Hsc73) homologous to rat Hsc73 were cloned by differential display and RT-PCR from mouse mammary gland. Their developmental regulation during pregnancy, lactation and involution is reported. The Itgβl mRNA content was stable in the first half of gestation, decreased to a minimum during lactation and increased markedly in early involution. Hsc73 gene expression was high in the first half of gestation and decreased to a minimum during lactation. The possible significance of the two observed patterns of expression is discussed.     

Milk-protein mRNAs and poly(A) in the involuting rat mammary gland return to the levels found during late pregnancy

Analyses of the rat mammary gland show that the increase in the milk-protein mRNAs during the development of lactation and the rapid disappearance of these sequences during involution are not accompanied by similar changes in the poly(A) content. During the development of lactation the casein mRNA is initially in great excess to the whey-protein mRNA and this differential expression of the genes for the two types of milk proteins is again observed during early involution. Since the amounts of poly(A) and of both milk-protein mRNAs are also similar to the amounts found in the gland during late pregnancy, these results indicate that during early involution the mammary gland has reverted to the pattern of mRNA metabolism that occurs during late pregnancy.     

Histo-cytomorphologie de la glande mammaire de la souris C3H et de trois autres rongeurs

Summary The mammary glands of 185 C3H mice, ranging from 11 days post partum to old age multiparous animals in the period of involution, were studied by light- and electronmicroscopy.The secretion of milk begins at the 7th day of pregnancy. The precocity of this event is not in agreement with the classical endocrinological concept. During pregnancy the protein material is concentrated in microvesicular bodies and in small Golgi vacuoles.During lactation the concentration is initiated inside the terminal dilatations of the flattened Golgi sacs and continues in the big Golgi vacuoles.During the suckling lipid droplets are extruded together with bits of adherent cytoplasm. An explanation of the formation of cellular fragments is given.The results obtained in the mouse are compared with those of the rat, Guinea pig and rabbit during pregnancy, lactation and regression. The most important cytomorphological difference observed in the mammary glands of the 4 species rests in the intensity of the secretion process during the 2/3 of pregnancy. At this stage, the milk secretion is considerable in the mouse, well under way in the rat and only in its beginnings in the Guinea pig and the rabbit. The lipid synthesis appears in the prelactating mammary gland of the rabbit earlier than the protein synthesis.The protein granules are neither typical of lactation nor of pregnancy. They have been identified in mammary glandular cells of a few virgin mice and in rats, Guinea pigs and rabbits 3 months after the end of lactation. This fact and the catabolic activity of the mammary epithelium in prepubertal mice, show that the mammary cells are able to perform a few secretory activities also outside periods of intense hormonal stimulation (pregnancy and lactation).The cytolytic events during post-lactational and post-parturium regression in absence of lactation are compared with the results of other authors.The resorption of secretory material, which does not appear clearly recognizable in our studies, is discussed.

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Cet article recouvre en partie le travail d'une thèse de doctorat d'Etat ès sciences naturelles. La thèse sera présentée en 1968 à la Faculté des Sciences de Strasbourg.     

Polyamine biogenesis in the rat mammary gland during pregnancy and lactation

Polyamines and RNA accumulate in the rat mammary gland during pregnancy, but the major increases occur after parturition. Therefore the major increases occur after the gland has obtained its maximal complement of epithelial cells. During lactation, the spermidine concentration rises above 5mm and RNA content in the lactating mammary gland reaches a value 16 times that of the unstimulated mammary gland. The ratio of spermidine/spermine, an increase of which initially signals an elevation in biosynthetic activity, is near 1 in the normal mammary gland and is greater than 10 in the lactating mammary gland. Putrescine concentration is very low during the entire course of mammary-gland development, with the exception of early pregnancy. The low putrescine concentration probably reflects the very rapid conversion of putrescine into spermidine. Both ornithine decarboxylase, the enzyme that synthesizes putrescine, and putrescine-stimulated S-adenosyl-l-methionine decarboxylase, the enzyme that synthesizes spermidine, increase in activity during middle and late pregnancy; during lactation, both enzyme activities are elevated until the 21st day of lactation, and then decline. These declines are concomitant with involution. Also, it was found that the amount of ribonuclease activity in the mammary gland was very high during lactation, almost double that in the gland during pregnancy.     

Enzyme Histochemistry as a Link between Biochemistry and Morphology

A study has been made of the progress of involution of the mouse and rat mammary gland using histologic, electron microscopic, histochemical and autoradiographic methods. Particular emphasis has been placed on the morphology, metabolic alte-rations and activities of histochemically identifiable enzymes, and on the pharmacologic effects of lactation inhibiting agents and cytostatic drugs on lactation and involution. In order to allow a systematic investi-gation, involution was initiated in rats and mice by ligation of individual gland ducts at various time intervals. Both lactating glands and glands in different phases of involution were thus available in a given animal.The most important observation was that involution, which altogether takes approximately 2 weeks to be complete, involves a three-phase process, each phase being clearly distinguishable by morphologic and histochemical criteria. The first phase comprises approximately 4 days during which production of milk may be reinitiated. The second phase starts on day 5 of involution and constitutes the period of involution per se characterized by appreciable parenchymal cell degradation. The third phase, which starts around day 10, is the period of reorganization to the resting mammary gland.Early in the first phase of involution, substantial alveolar enlargement due to engorgement with milk, together with epithelial flattening, are prominent features. By day 3, the glandular contents decrease again in volume, the number of glandular cells and the constituent cytoplasmic organelles remaining unchanged during this period, except for the diminished appearance of fat droplets. In addition to normal appearing vacuoles with only occasional or sparse protein granules, giant vacuoles containing, in part, several hundred casein granules are found. Their formation appears to be due to increased stacking of granules in distended vacuoles prior to dissociation from the Golgi apparatus. In addition, however, the enhanced reactions of a1P (alkaline phosphatase) and ATPase, which are found in the apical plasmalemma, are suggestive of resorptive activities. Protein particles absorbed from the glandular lumen equally appear to have a capacity for fusing into large vacuoles. The large protein granule-containing vacuoles regularly exhibit intense (3-Glu activity. This enzyme would appear to contribute actively to the degradation of excess milk during the first phase of involution.Autoradiographic studies reveal that the synthesis and release of proteins into the secretion is maintained for 3 days. While 3H-tyrosine uptake by the alveolar cells continues unchanged, the incorporation of 3H-palmitic acid into glandular lipoids, and of 3H-fucose into glandular polysaccharides is virtually blocked completely. An immediate reaction of the lipoid metabolism is also indicated by the decrease in 3HBDH activity on the first day of involution.The activities of the histochemically detected oxidoreductases (LDH, MDH, SDH, G6PDH, 3HBDH) show a sharp fall on day 1 of involution, reaching levels approximately one half of the activity observed during lactation, as shown on micro densitometry. The activities remain unchanged during the following 4 days.No degradation of glandular parenchyma is noted during the-first phase of involution. The glandular cells rather take a -wait-and-see- attitude which enables them to participate again in the secretion of milk, as need arises. At this time the activities of the enzymes implicated in energy metabolism have reached approximately the resting mammary gland level. Only protein synthesis is maintained virtually unrestricted and this results in the production of excess milk constituents that are degraded as soon as they are being formed.In the second phase of involution, large seg-ments of the glandular epithelium undergo invo-lution, a process which involves the destruction of glandular epithelial cells and the removal of the resulting cellular debris from the mammary gland. The glandular cells remaining are transformed into resting cells. The lysosomes of the glandular epithelial cells, with maximum numbers being attained between days 7 to 9, contribute decisively to this degradative process. Ultra-structurally, this stage is initially characterized by the appearance in the alveolar cell cytoplasm of segregated cytoplasmic areas which stain negatively for acP (acid phosphatase) and are rich in organelles. These cytoplasmic areas change to membrane-bordered lysosomes which possess intense acP activity. The lysosomes are obviously required for the autophagic degradation of cytoplasmic segments. At the same time the activities of other lysosomal enzymes, involving acP, N-A-Gase, AMPase and AS, show a sharp increase. ATPase and TPPase likewise exhibit considerably increased activity during the second phase of involution. It is seen on microdensitometry that during this phase the acP attains approximately three times the lactation activity. In contrast, the activity of (3-Glu, after having shown a very high increase during the first phase, reverts again to the resting mammary gland level.During the second phase of involution, the oxidoreductases are subject to a further drastic decline of their activities. This process, which consistently affects all segments of the mammary gland,,comes to completion within a few hours. The reaction is found to be minimal around day 5 of involution, at a time when the enzyme activities are approximately one quarter of the lactation levels.At this time, many alveolar cells are destroyed and released into the glandular lumen, the acP retaining its activity in the lysosomes of sloughed cells or cell debris. The resulting gaps in the alveolar epithelium are either bridged immediately or remain detectable on histology. Yet the glandular contents do not pass into the interstitial tissue. The adherence of the glandular tree and the glandular epithelium is ensured by myoepithelial cells. The basement membrane effects the complete segregation of the parenchyma from the interstitial tissue:Macrophages which at this stage occur increa-singly near the alveoli, in the alveolar epithelium and in regional lymph nodes, and which are conspicuous due to numerous acP-laden lysosomes participate essentially in glandular regression.The third phase of involution, which takes place approximately 10 to 14 days following the onset of milk stasis, is the period of reorganization to the resting mammary gland, a period during which glandular cells containing little cytoplasm and sparse organelle lining make their appearance. However, the activities of acP and other lysosomal enzymes remain elevated compared to the pregestational level. Histochemically, the reaction of the oxidoreductases is more intense than during the second involutionary phase, corresponding roughly to the level of the mammary gland in the resting state. The formation of the glandular epithelial cells of the resting mammary gland is not due to renewed mitotic activity, but results from the reduction of actively secreting epithelial cells.Throughout the period of involution the myo-epithelium consistently changes its shape. How-ever, cytoplasmic alterations are not discernible on electron microscopy, nor do these cells undergo degradation during glandular involution. The alP reaction is of particular value for the identification of the myoepithelial cells. No alteration in enzyme activities is demonstrable at the histochemical level throughout the process of involution.Alterations in the interstitial tissue affect particularly the adipose tissue. During lactation, the interalveolar space exhibits only a narrow connective tissue layer which changes insignificantly during the first phase of involution, whereas the subsequent incorporation of lipids results in the formation of plurilocular lipid cells and the reappearance of unilocular adipose tissue as involution advances. During this period the vessels move away from the alveoli.The response of the mammary gland to lactation inhibiting and cytostatic drugs varies, depending on the agent administered. Estrogencontaining drugs lead to an involutionary process which in its initial phase differs from that observed during normal glandular regression. Due to the fact that the milk continues to be suckled by the young, milk production however ceasing very rapidly, the (3-Glu activity is not found to increase greatly during the first phase of involution. The behavior of the lysosomal enzymes during the second phase resembles that seen during normal involution.After administration of the ergot alkaloid 2-Br-a-ergokryptine methane sulfonate (CB 154), the process of involution is initiated only in individual mammary gland cells if the young are left with the mother. In these areas involution takes a normal course. Altogether, milk production changes only insignificantly.The cytostatic drug 5-fluorouracil does not induce direct involution. Milk production apparently is not arrested. However, the pups, when left with the mother, die after 5 to 6 days. Death would appear to be due to the cytotoxic action of 5-fluorouracil.     

Induction of glutathione S-transferases A, B and C in rat liver cytosol by trans-stilbene oxide

RNAase H, which catalyzes the hydrolysis of the RNA moiety of an RNA-DNA hybrid, was measured in the mammary gland of virgin, pregnant, lactating, and weaning Fischer rats and in the R3230AC mammary tumor grown in the same animals. In the normal mammary gland when DNA levels were low, as in the virgin state or during involution, RNAase H activity was also low. During pregnancy and lactogenesis when DNA levels increased, RNAase H activity, either on the basis of mammary gland weight or DNA content, also increased. During lactation when cellular proliferation ceases but rates of RNA and protein synthesis continue to reach peak values, RNAase H activity decreased. Compared to the corresponding enzyme from host glands, RNAase H from the R3230AC mammary tumor grown in pregnant and lactating hosts changes similarly, but to a lesser extent. The RNAase H activity which, ona tissue weight basis, was higher than in normal tissue also increased during pregnancy and directly after parturition, but decreased during lactation. During pregnancy these changes were accompanied by an increase in tumor DNA values. During lactation the tumor DNA values returned to the level seen in virgin hosts. These results are consistent with a role for RNAase H in DNA replication in rat mammary gland and in R3230Ac mammary tumor.     

Regulation of casein messenger RNA during the development of the rat mammary gland.

Casein mRNA was isolated and partially purified from RNA extracts of rat lactating mammary glands and translated in a teterologous cell-free protein synthesizing system derived from wheat germ. Casein mRNA activity was assayed by immunoprecipitation using a specific antiserum prepared against a mixture of the purified rat caseins. Properties of rat casein mRNA were examined using a variety of sizing techniques, including chromatography on Sepharose 4B, sedimentation on sucrose gradients after heat denaturation, and electrophoresis on 2.5% agarose gels in 6 M urea. Casein mRNA activity was found in an 8-16S region after gradient centrifugation with the peak occurring at 10.5 S. In addition, the binding of rat casein mRNA to dT-cellulose was examined. Only 40% of the total casein mRNA activity was selectively retained. A partial purification of casein mRNA was accomplished by a combination of these sizing and affinity chromatography techniques. In the purified preparations casein mRNA activity comprises approximately 90% of the total mRNA activity. Characterization of this material by agarose gel electrophoresis revealed two main bands of RNA at approximately 12 and 16 S, both containing casein mRNA activity. These mRNAs were of the correct size to code for two of the principal rat caseins of approximately 25,000 and 42,000 molecular weights. Casein mRNA and total mRNA activities were then compared in total RNA extracts at various stages of normal mammary gland development in the rat, i.e. during pregnancy, lactation, and involution following weaning. A selective induction of casein mRNA activity compared to total mRNA activity was found to occur during pregnancy and lactation. Moreover, a selective loss of activity was also observed during mammary gland involution. A surprisingly high level of casein mRNA activity was found in RNA extracts from early and midpregnant mammary glands.