Sphingomyelinase activity in mother's milk is essential for juvenile development: a case from lactating tsetse flies

Sphingosine is a structural component of sphingolipids. The metabolism of phosphoethanolamine ceramide (sphingomyelin) by sphingomyelinase (SMase), followed by the breakdown of ceramide by ceramidase (CDase) yields sphingosine. Female tsetse fly is viviparous and generates a single progeny within her uterus during each gonotrophic cycle. The mother provides her offspring with nutrients required for development solely via intrauterine lactation. Quantitative PCR showed that acid smase1 (asmase1) increases in mother's milk gland during lactation. aSMase1 was detected in the milk gland and larval gut, indicating this protein is generated during lactation and consumed by the larva. The higher levels of SMase activity in larval gut contents indicate that this enzyme is activated by the low gut pH. In addition, cdase is expressed at high levels in the larval gut. Breakdown of the resulting ceramide is likely accomplished by the larval gut-secreted CDase, which allows absorption of sphingosine. We used the tsetse system to understand the critical role(s) of SMase and CDase during pregnancy and lactation and their downstream effects on adult progeny fitness. Reduction of asmase1 by short interfering RNA negatively impacted pregnancy and progeny performance, resulting in a 4-5-day extension in pregnancy, 10%-15% reduction in pupal mass, lower pupal hatch rates, impaired heat tolerance, reduced symbiont levels, and reduced fecundity of adult progeny. This study suggests that the SMase activity associated with tsetse lactation and larval digestion is similar in function to that of mammalian lactation and represents a critical process for juvenile development, with important effects on the health of progeny during their adulthood.     

Analysis of milk gland structure and function in Glossina morsitans: milk protein production, symbiont populations and fecundity

A key process in the tsetse reproductive cycle is the transfer of essential nutrients and bacterial symbionts from mother to intrauterine offspring. The tissue mediating this transfer is the milk gland. This work focuses upon the localization and function of two milk proteins (milk gland protein (GmmMGP) and transferrin (GmmTsf)) and the tsetse endosymbionts (Sodalis and Wigglesworthia), in the context of milk gland physiology. Fluorescent in situ hybridization (FISH) and immunohistochemical analysis confirm that the milk gland secretory cells synthesize and secrete milk gland protein and transferrin. Knockdown of gmmmgp by double stranded RNA (dsRNA) mediated RNA interference results in reduction of tsetse fecundity, demonstrating its functional importance in larval nutrition and development. Bacterial species-specific in situ hybridizations of milk gland sections reveal large numbers of Sodalis and Wigglesworthia within the lumen of the milk gland. Sodalis is also localized within the cytoplasm of the secretory cells. Within the lumen, Wigglesworthia localize close to the channels leading to the milk storage reservoir of the milk gland secretory cells. We discuss the significance of the milk gland in larval nutrition and in transmission of symbiotic bacteria to developing offspring.     

Infections with immunogenic trypanosomes reduce tsetse reproductive fitness: potential impact of different parasite strains on vector population structure

The parasite Trypanosoma brucei rhodesiense and its insect vector Glossina morsitans morsitans were used to evaluate the effect of parasite clearance (resistance) as well as the cost of midgut infections on tsetse host fitness. Tsetse flies are viviparous and have a low reproductive capacity, giving birth to only 6-8 progeny during their lifetime. Thus, small perturbations to their reproductive fitness can have a major impact on population densities. We measured the fecundity (number of larval progeny deposited) and mortality in parasite-resistant tsetse females and untreated controls and found no differences. There was, however, a typanosome-specific impact on midgut infections. Infections with an immunogenic parasite line that resulted in prolonged activation of the tsetse immune system delayed intrauterine larval development resulting in the production of fewer progeny over the fly's lifetime. In contrast, parasitism with a second line that failed to activate the immune system did not impose a fecundity cost. Coinfections favored the establishment of the immunogenic parasites in the midgut. We show that a decrease in the synthesis of Glossina Milk gland protein (GmmMgp), a major female accessory gland protein associated with larvagenesis, likely contributed to the reproductive lag observed in infected flies. Mathematical analysis of our empirical results indicated that infection with the immunogenic trypanosomes reduced tsetse fecundity by 30% relative to infections with the non-immunogenic strain. We estimate that a moderate infection prevalence of about 26% with immunogenic parasites has the potential to reduce tsetse populations. Potential repercussions for vector population growth, parasite-host coevolution, and disease prevalence are discussed.     

The milk gland of the sheep ked, Melophagus ovinus: A comparison with Glossina

Structurally, the milk gland of the sheep ked, Melophagus ovinus, is quite similar to that of the tsetse fly, Glossina morsitans. In both insects the highly branched gland consists of two cell layers. An extracellular reservoir associated with each secretory cell initially receives the secreted milk. Milk then passes into the gland lumen through a dense cuticular rete. Gram-negative bacteria, presumably symbionts, are abundant in the lumen. Unlike tsetse, the secretory reservoir of the sheep ked is bi-lobed, and the secretory cell nucleus remains centrally located throughout the pregnancy cycle. Lipid droplets are much more abundant in the cytoplasm of the ked secretory cell, and analysis of larval milk shows 5–6% higher lipid content in the sheep ked. Results of histochemical analysis of ked milk are consistent with the analysis of tsetse milk. Four major milk proteins are detectable with polyacrylamide gel electrophoresis. Changes in abundance of ER and sizes of the secretory cell nucleus and secretory reservoir reflect a dramatic cycle of glandular activity during pregnancy. Unlike tsetse, the sheep ked remains constantly on its host and appears to take frequent, but small, blood meals. This strategy implies that the demand for nutrient storage is less than in tsetse.     

Acute involution in the tammar wallaby: identification of genes and putative novel milk proteins implicated in mammary gland function

Marsupials provide a suitable alternative model to studying mammary gland involution. They have evolved a different reproductive strategy from eutherians, giving birth to an altricial young and secreting milk that changes in composition during lactation. In this study, we used a marsupial-specific EST microarray to identify 47 up-regulated genes during mammary gland involution in the tammar wallaby (Macropus eugenii). These include the pro-apoptotic tumour necrosis factor receptor superfamily 21 (TNFRSF21) gene, whose expression in the mammary gland has not previously been reported. Genes encoding putative novel milk proteins which may protect the mammary gland from infection were also found to be up-regulated, such as amiloride binding protein 1 (ABP1), complement component 1QB (C1QB), complement component 4A (C4A) and colony stimulating factor 2 receptor β (CSF2Rβ). Our results show that the marsupial reproductive strategy was successfully exploited to identify genes and putative novel milk proteins implicated in mammary gland involution.     

Vitamin B6 Generated by Obligate Symbionts Is Critical for Maintaining Proline Homeostasis and Fecundity in Tsetse Flies

The viviparous tsetse fly utilizes proline as a hemolymph-borne energy source. In tsetse, biosynthesis of proline from alanine involves the enzyme alanine-glyoxylate aminotransferase (AGAT), which requires pyridoxal phosphate (vitamin B₆) as a cofactor. This vitamin can be synthesized by tsetse''s obligate symbiont, Wigglesworthia glossinidia. In this study, we examined the role of Wigglesworthia-produced vitamin B₆ for maintenance of proline homeostasis, specifically during the energetically expensive lactation period of the tsetse''s reproductive cycle. We found that expression of agat, as well as genes involved in vitamin B₆ metabolism in both host and symbiont, increases in lactating flies. Removal of symbionts via antibiotic treatment of flies (aposymbiotic) led to hypoprolinemia, reduced levels of vitamin B₆ in lactating females, and decreased fecundity. Proline homeostasis and fecundity recovered partially when aposymbiotic tsetse were fed a diet supplemented with either yeast or Wigglesworthia extracts. RNA interference-mediated knockdown of agat in wild-type flies reduced hemolymph proline levels to that of aposymbiotic females. Aposymbiotic flies treated with agat short interfering RNA (siRNA) remained hypoprolinemic even upon dietary supplementation with microbial extracts or B vitamins. Flies infected with parasitic African trypanosomes display lower hemolymph proline levels, suggesting that the reduced fecundity observed in parasitized flies could result from parasite interference with proline homeostasis. This interference could be manifested by competition between tsetse and trypanosomes for vitamins, proline, or other factors involved in their synthesis. Collectively, these results indicate that the presence of Wigglesworthia in tsetse is critical for the maintenance of proline homeostasis through vitamin B₆ production.     

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.     

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.     

Serotonin (5-HT) Affects Expression of Liver Metabolic Enzymes and Mammary Gland Glucose Transporters during the Transition from Pregnancy to Lactation

The aim of this experiment was to demonstrate the ability of feeding serotonin (5-HT; 5-hydroxytryptamine) precursors to increase 5-HT production during the transition from pregnancy to lactation and the effects this has on maternal energy metabolism in the liver and mammary gland. Pregnant rats (n = 45) were fed one of three diets: I) control (CON), II) CON supplemented with 0.2% 5-hydroxytryptophan (5-HTP) or III) CON supplemented with 1.35% L-tryptophan (L-TRP), beginning on d13 of pregnancy through d9 of lactation (d9). Serum (pre and post-partum), milk (daily), liver and mammary gland tissue (d9) were collected. Serum 5-HT was increased in the 5-HTP fed dams beginning on d20 of gestation and remained elevated through d9, while it was only increased on d9 in the L-TRP fed dams. 5-HT levels were increased in mammary gland and liver of both groups. Additionally, 5-HTP fed dams had serum and milk glucose levels similar to the CON, while L-TRP had decreased serum (d9) and milk glucose (all dates evaluated). Feeding 5-HTP resulted in increased mRNA expression of key gluconeogenic and glycolytic enzymes in liver and glucose transporters 1 and 8 (GLUT-1, -8) in the mammary gland. We demonstrated the location of GLUT-8 in the mammary gland both in the epithelial and vascular endothelial cells. Finally, phosphorylated 5′ AMP-activated protein kinase (pAMPK), a known regulator of intracellular energy status, was elevated in mammary glands of 5-HTP fed dams. Our results suggest that increasing 5-HT production during the transition from pregnancy to lactation increases mRNA expression of enzymes involved in energy metabolism in the liver, and mRNA abundance and distribution of glucose transporters within the mammary gland. This suggests the possibility that 5-HT may be involved in regulating energy metabolism during the transition from pregnancy to lactation.     

Infection with endosymbiotic Spiroplasma disrupts tsetse (Glossina fuscipes fuscipes) metabolic and reproductive homeostasis

Tsetse flies (Glossina spp.) house a population-dependent assortment of microorganisms that can include pathogenic African trypanosomes and maternally transmitted endosymbiotic bacteria, the latter of which mediate numerous aspects of their host’s metabolic, reproductive, and immune physiologies. One of these endosymbionts, Spiroplasma, was recently discovered to reside within multiple tissues of field captured and laboratory colonized tsetse flies grouped in the Palpalis subgenera. In various arthropods, Spiroplasma induces reproductive abnormalities and pathogen protective phenotypes. In tsetse, Spiroplasma infections also induce a protective phenotype by enhancing the fly’s resistance to infection with trypanosomes. However, the potential impact of Spiroplasma on tsetse’s viviparous reproductive physiology remains unknown. Herein we employed high-throughput RNA sequencing and laboratory-based functional assays to better characterize the association between Spiroplasma and the metabolic and reproductive physiologies of G. fuscipes fuscipes (Gff), a prominent vector of human disease. Using field-captured Gff, we discovered that Spiroplasma infection induces changes of sex-biased gene expression in reproductive tissues that may be critical for tsetse’s reproductive fitness. Using a Gff lab line composed of individuals heterogeneously infected with Spiroplasma, we observed that the bacterium and tsetse host compete for finite nutrients, which negatively impact female fecundity by increasing the length of intrauterine larval development. Additionally, we found that when males are infected with Spiroplasma, the motility of their sperm is compromised following transfer to the female spermatheca. As such, Spiroplasma infections appear to adversely impact male reproductive fitness by decreasing the competitiveness of their sperm. Finally, we determined that the bacterium is maternally transmitted to intrauterine larva at a high frequency, while paternal transmission was also noted in a small number of matings. Taken together, our findings indicate that Spiroplasma exerts a negative impact on tsetse fecundity, an outcome that could be exploited for reducing tsetse population size and thus disease transmission.     

Pregnancy-Induced Noncoding RNA (PINC) Associates with Polycomb Repressive Complex 2 and Regulates Mammary Epithelial Differentiation

Pregnancy-induced noncoding RNA (PINC) and retinoblastoma-associated protein 46 (RbAp46) are upregulated in alveolar cells of the mammary gland during pregnancy and persist in alveolar cells that remain in the regressed lobules following involution. The cells that survive involution are thought to function as alveolar progenitor cells that rapidly differentiate into milk-producing cells in subsequent pregnancies, but it is unknown whether PINC and RbAp46 are involved in maintaining this progenitor population. Here, we show that, in the post-pubertal mouse mammary gland, mPINC is enriched in luminal and alveolar progenitors. mPINC levels increase throughout pregnancy and then decline in early lactation, when alveolar cells undergo terminal differentiation. Accordingly, mPINC expression is significantly decreased when HC11 mammary epithelial cells are induced to differentiate and produce milk proteins. This reduction in mPINC levels may be necessary for lactation, as overexpression of mPINC in HC11 cells blocks lactogenic differentiation, while knockdown of mPINC enhances differentiation. Finally, we demonstrate that mPINC interacts with RbAp46, as well as other members of the polycomb repressive complex 2 (PRC2), and identify potential targets of mPINC that are differentially expressed following modulation of mPINC expression levels. Taken together, our data suggest that mPINC inhibits terminal differentiation of alveolar cells during pregnancy to prevent abundant milk production and secretion until parturition. Additionally, a PRC2 complex that includes mPINC and RbAp46 may confer epigenetic modifications that maintain a population of mammary epithelial cells committed to the alveolar fate in the involuted gland.     

Post-lactational mammary gland regression: molecular basis and implications for breast cancer

During pregnancy, there is a massive increase in the number of luminal epithelial cells in the breast, which are destined to become the milk factories after birth. These cells are no longer required when the young are weaned, and are removed in a carefully orchestrated event called involution. In this process, the secretory epithelial cells die and are replaced by adipocytes, which redifferentiate as the epithelium is removed. It is essential that the gland is properly remodelled to a pre-pregnant state so that successful lactation can occur following a subsequent pregnancy. Furthermore, failure to remove unnecessary lactational alveoli during weaning could result in inflammation and tissue damage. Recently, it has been shown that components in the fatty stroma in involuting breast can promote metastasis. Thus, it is important to understand the molecular mechanisms that regulate involution, how these can fail, the consequences of the remodelling process, and how this knowledge can inform us about breast cancer. In this review, I discuss the roles of the JAK-STAT, NF-kappaB and other signalling pathways in the regulation of apoptosis and tissue remodelling during involution.