Many important molecular events associated with implantation and development occur within the female reproductive tract, especially within the uterus endometrium, during pregnancy periods. The endometrium includes the mucosal lining of the uterus, which provides a suitable site for implantation and development of a fertilized egg and fetus. To date, the molecular cascades in the uterus endometrium during pregnancy periods in pigs have not been elucidated fully. In this study, we compared the functional regulated proteins in the endometrium during pregnancy periods with those in non-pregnant conditions and investigated changes in expression patterns during pregnancy (days 40, 70, and 93) using two-dimensional gel electrophoresis (2-DE) and western blotting. The functional regulated proteins were identified and discovered from differentially expressed proteins in the uterus endometrium during pregnancy. We discovered 820 protein spots in a proteomic analysis of uterus endometrium tissues with 2-DE gels. We identified 63 of the 98 proteins regulated differentially among non-pregnant and pregnant tissues (matched and unmatched spots). Interestingly, 10 of these 63 proteins are development-, cytoskeleton- and chaperon-related proteins such as transferrin, protein DJ-1, transgelin, galectin-1, septin 2, stathmin 1, cofilin 1, fascin 1, heat shock protein (HSP) 90β and HSP 27. The specific expression patterns of these proteins in the endometrium during pregnancy were confirmed by western blotting. Our results suggest that the expressions of these genes involved in endometrium function and endometrium development from early to late gestation are associated with the regulation of endometrium development for maintaining pregnancy.

Information obtained from peripheral blood could help us understand the underlying mechanisms in autoimmune diseases, cancer, pregnancy, and other conditions. In this paper, we present the protein map of porcine peripheral blood mononuclear cells (PBMC) to better understand the molecular expression changes that occur during pregnancy using proteomic analysis. We detected 94 differentially expressed proteins in pregnant vs. non-pregnant (NP) pigs, and a representative set of the proteins was subjected to LC-MS/MS analysis. Furthermore, the identified proteins were categorized according to their biological process and molecular function. By classifying the proteins according to their functions, a large number of differentially regulated proteins involved in anti-oxidant, detoxification and stress response pathways were found, including peroxiredoxin (PRX) 1, 2, and 6, glutathione-S-transferase (GST), annexin A2, and A6, and heat shock protein 27 (HSP 27) during pregnancy (pregnancy d of E40, embryonic day 40; E70, embryonic day 70; and E93, embryonic day 93) compared with non-pregnancy. In this study, a proteomic approach utilizing 2-DE and LC-MS/MS was applied to evaluate specific molecular expression changes during pregnancy compared with non-pregnancy. Together, these data offer new information about the proteome map and factors that are differentially regulated during maintenance of normal pregnancy.

While pregnancy-related proteins (PRP) are known to contribute to immunotolerance during pregnancy, their significance to development of invasive placenta is unclear. We compared PRP expression in humans and the common marmoset (Callithrix jacchus), a new-world monkey. Invasive placenta was observed at the maternal-foetal interface of marmoset placenta from green fluorescent protein (GFP)-expressing foetus and wild type mother. The pregnancy zone protein (PZP) and alpha-2 macroglobulin-like 1 (A2ML1) proteins exhibited the most prominent increase in expression during the second trimester in humans and marmoset, respectively. In humans, PZP accumulated at the maternal-foetal interface and A2ML1 accumulated in the amnion. Similarly, A2ML1 mRNA was detected in marmoset placenta. These proteins belong to the A2M family of protease inhibitors, and both PZP and A2ML1 share around 90% homology between human and marmoset and have highly conserved structures. However, the protease-reacting bait regions of the proteins had lower homology (56.8-60.7% in proteins) relative to the rest of the sequence. Notably, the cleavage site of a proinflammatory proline-endopeptidase was preserved in human PZP and marmoset A2ML1. These proteins contain multiple sites that are cleaved by proteases involving proline-endopeptidase. Systemic regulation of these A2M family proteins may be important in animals with invasive placenta.

Amnion is the innermost layer of the fetal membrane and has been suggested to regulate the volume of amniotic fluid via the amniotic epithelium. The transepithelial pathway is generally restricted by tight junctions (TJs). Thus far, human amniotic TJs have not been identified. In this study, we determined whether the human amniotic epithelium contains TJs. Reverse transcription polymerase chain reaction (RT-PCR) and western blotting analyses showed that the human amniotic epithelium has TJ components, such as occludin, ZO-1, and at least 2 types of claudins, i.e., claudin-4 and claudin-7. The TJ components were found to localize in the lateral membranes and cytoplasm at 35 weeks of gestation; these components disappeared from the lateral membrane at 37 weeks of gestation. Organ culturing of the amnion at 37 weeks gestation induced the relocalization of the TJ proteins from the cytoplasm to the lateral membranes. Furthermore, in cultured amniotic epithelial cells, dexamethasone induced the downregulation of the protein expression of TJs. These findings suggest that the human amniotic epithelium has TJs that disrupt during late pregnancy. The disruption may be induced by several factors such as glucocorticoids present in the amniotic fluid during late pregnancy.

Cronobacter sakazakii is a food-borne pathogen carried in milk powder that can cause severe bacteremia, enterocolitis, and meningitis in newborns, which can lead to death of newborns. Preventing infection by this pathogen is significant to the health of newborns. Since infants and young children are the main target group of C. sakazakii, it is considered that maternal immunity can enhance the protection of newborns. Previous studies showed that two proteins of C. sakazakii (GroEL and OmpX) exhibited high expression levels and elicited strong immune reactions, suggesting their potential as vaccine candidates. In this study, GroEL and OmpX were recombinantly expressed in Escherichia coli and purified as immunogens to immunize pregnant rats. Three days after birth, the progeny were challenged with C. sakazakii to determine the protective effect of maternal immunity on the offspring. The results showed that immunization during pregnancy decreased bacterial load in the brain and blood, reduced brain and intestine damage, and significantly increased specific antibody titers in the offspring. Immunization with the recombinant proteins significantly increased cytokine levels in the serum of the progeny. The group whose mothers were immunized with OmpX produced more IL-4, while the group whose mothers were immunized with GroEL produced more IFN-γ, indicating that the immunogens enhanced the Th2 and Th1 responses, respectively. However, although the immune response was induced by both proteins, only the offspring of the pregnant rats immunized with OmpX or OmpX/GroEL mixture showed delayed death, possibly because immunization with OmpX led to a stronger humoral immune response in the offspring, suggesting that OmpX was a better vaccine candidate than GroEL. This study first reported that exposure to C. sakazakii proteins during pregnancy could improve the offspring's ability to resist infection caused by this pathogen.

Two dimensional-fluorescence difference gel electrophoresis (2D DIGE) is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. The purpose of this study was to investigate bovine pregnancy-specific proteins in the proteome between bovine pregnant and non-pregnant serum using DIGE technique. Serums of 2 pregnant Holstein dairy cattle at day 21 after artificial insemination and those of 2 non-pregnant were used in this study. The pre-electrophoretic labeling of pregnant and non-pregnant serum proteins were mixed with Cy3 and Cy5 fluorescent dyes, respectively, and an internal standard was labeled with Cy2. Labeled proteins with Cy2, Cy3, and Cy5 were separated together in a single gel, and then were detected by fluorescence image analyzer. The 2D DIGE method using fluorescence CyDye DIGE flour had higher sensitivity than conventional 2D gel electrophoresis, and showed reproducible results. Approximately 1,500 protein spots were detected by 2D DIGE. Several proteins showed a more than 1.5-fold up and down regulation between non-pregnant and pregnant serum proteins. The differentially expressed proteins were identified by MALDI-TOF mass spectrometer. A total 16 protein spots were detected to regulate differentially in the pregnant serum, among which 7 spots were up-regulated proteins such as conglutinin precursor, modified bovine fibrinogen and IgG1, and 6 spots were down-regulated proteins such as hemoglobin, complement component 3, bovine fibrinogen and IgG2a three spots were not identified. The identified proteins demonstrate that early pregnant bovine serum may have several pregnancy-specific proteins, and these could be a valuable information for the development of pregnancy-diagnostic markers in early pregnancy bovine serum.

The most of embryo losses occur before the day 16 after artificial insemination, but there is no low cost and easy operation that can detect pregnancy with high accuracy within three weeks post-insemination in cattle. In this study, blood samples were collected at day 18 of the estrous cycle, and days 18, 25 and 35 of pregnancy, and relative levels of interferon stimulated genes (ISGs), Toll-like receptor (TLRs), complement components, early pregnancy factor (EPF) and pregnancy-associated plasma protein A (PAPPA) proteins were analyzed through Western blot. In addition, a colloidal gold immunochromatographic test strip was developed using the selected antibody, and the test was used for early pregnancy diagnosis. The results showed that there were changes in relative levels of plasma ISGs, TLRs, complement components, EPF and PAPPA proteins during early pregnancy in cattle, and complement component 1q (C1q) could be used as an ideal marker to develop a colloidal gold immunochromatographic test strip for early pregnancy diagnosis. In addition, the accuracy of pregnancy diagnosis by this test strip was 91.67% (11/12) for pregnant cows and 80% (8/10) nonpregnant cows at day 18 after insemination. In conclusion, the changes in plasma ISGs, TLRs, complement components, EPF and PAPPA proteins may be related to the maternal systemic immune modulation during early pregnancy, and a colloidal gold immunochromatographic test strip was developed for early pregnancy diagnosis using C1q as the ideal marker in cows. However, this colloidal gold immunochromatographic test strip needs further studies to improve the accuracy.

Group B Streptococcus (GBS) recto-vaginal colonisation in pregnant women is the major risk factor for early-onset invasive GBS disease in their newborns. We aimed to determine the association between serum antibody levels against 11 GBS surface proteins and recto-vaginal acquisition of GBS colonisation during pregnancy. Sera collected from pregnant women at 20-25 weeks and ≥37 weeks of gestation age were measured for IgG titres against GBS surface proteins using  a multiplex immunoassay. Women were evaluated for recto-vaginal colonisation every 4-5 weeks. We observed that the likelihood of becoming colonised with GBS during pregnancy was lower in women with IgG titres ≥200 U/mL against gbs0233 (adjusted OR = 0.47 [95% CI: 0.25-0.89], p = 0.021) and ≥85 U/mL for gbs1539 (adjusted OR = 0.44 [95% CI: 0.24-0.82], p = 0.01) when comparing between women who acquired GBS colonisation and those that remained free of GBS colonisation throughout pregnancy. IgG titres (U/mL) specific to BibA and Sip were higher in pregnant women colonised with GBS (380.19 and 223.87, respectively) compared to women with negative GBS cultures (234.42 and 186.21, respectively; p < 0.01) at ≥37 weeks gestation. Antibodies induced by gbs0233 and gbs1539 were associated with a reduced likelihood of recto-vaginal GBS acquisition during pregnancy and warrant further investigation as vaccine targets.

Protein misfolding underlies the pathology of a large number of human disorders, many of which are age-related. An exception to this is preeclampsia, a leading cause of pregnancy-associated morbidity and mortality in which misfolded proteins accumulate in body fluids and the placenta. We demonstrate that pregnancy zone protein (PZP), which is dramatically elevated in maternal plasma during pregnancy, efficiently inhibits in vitro the aggregation of misfolded proteins, including the amyloid beta peptide (Aβ) that is implicated in preeclampsia as well as with Alzheimer's disease. The mechanism by which this inhibition occurs involves the formation of stable complexes between PZP and monomeric Aβ or small soluble Aβ oligomers formed early in the aggregation pathway. The chaperone activity of PZP is more efficient than that of the closely related protein alpha-2-macroglobulin (α2M), although the chaperone activity of α2M is enhanced by inducing its dissociation into PZP-like dimers. By immunohistochemistry analysis, PZP is found primarily in extravillous trophoblasts in the placenta. In severe preeclampsia, PZP-positive extravillous trophoblasts are adjacent to extracellular plaques containing Aβ, but PZP is not abundant within extracellular plaques. Our data support the conclusion that the up-regulation of PZP during pregnancy represents a major maternal adaptation that helps to maintain extracellular proteostasis during gestation in humans. We propose that overwhelming or disrupting the chaperone function of PZP could underlie the accumulation of misfolded proteins in vivo. Attempts to characterize extracellular proteostasis in pregnancy will potentially have broad-reaching significance for understanding disease-related protein misfolding.

Gap junctions between β-cells participate in the precise regulation of insulin secretion. Adherens junctions and their associated proteins are required for the formation, function and structural maintenance of gap junctions. Increases in the number of the gap junctions between β-cells and enhanced glucose-stimulated insulin secretion are observed during pregnancy. In contrast, protein restriction produces structural and functional alterations that result in poor insulin secretion in response to glucose. We investigated whether protein restriction during pregnancy affects the expression of mRNA and proteins involved in gap and adherens junctions in pancreatic islets. An isoenergetic low-protein diet (6% protein) was fed to non-pregnant or pregnant rats from day 1-15 of pregnancy, and rats fed an isocaloric normal-protein diet (17% protein) were used as controls.

We determined the role played by O-linked N-acetylglucosamine (O-GlcNAc) of proteins in systemic arteries during late pregnancy in normotensive and hypertensive rats.

In some species, female steroid hormones modify the profile of acute phase proteins (APPs) during the estrous cycle and pregnancy, according to the ovulation, embryonic implantation and placental development; however, nowadays there's no experimental evidence for equine species. Objectives of this study were: to compare the serum amyloid A (SAA), haptoglobin (Hp) and C-reactive protein (CRP) concentrations between cyclic and pregnant mares, and to analyze the influence of estradiol-17β (E2) during estrous cycle or estrone sulfate (E1) during pregnancy, and progesterone (P4) on these proteins to assess their potential role to identify the cyclicity or pregnancy in Spanish mares. Blood samples were taken from 20 Purebred Spanish mares on the day of ovulation (day 0), on days +5 and +16 post-ovulation, and then, monthly during the whole pregnancy. SAA, Hp and CRP did not change between day 0, +5 and +16 post-ovulation days. P4 concentrations were significantly higher on day +16 than on days +5 and 0; and E2 concentrations were significantly higher on day 0 than day +5. On the other hand, pregnancy was characterized by a progressive increase in the Hp, variable modifications of E1 and P4 concentrations, without changes in SAA and CRP. The absence of significant differences in the APPs between days 0, +5 and +16, suggested that these proteins cannot be used as biomarkers of diagnosis of heat or pregnancy in Spanish mares, at least early, since the Hp later increases during the gestation. Nevertheless, it is possible to use them for comparative purposes with other equine breeds, as supervisor instrument of health status in breeding females as diagnostic tools to monitor pregnancy's development and/or subclinical reproductive inflammations, that could lead to the early embryonic death.

The molecular mechanism underlying embryonic implantation is vital to understand the correct communications between endometrium and developing conceptus during early stages of pregnancy. This study's objective was to determine molecular changes in the uterine endometrial proteome during the preimplantation and peri-implantation between 9 days (9D), 12 days (12D), and 16 days (16D) of pregnant Polish Large White (PLW) gilts. 2DE-MALDI-TOF/TOF and ClueGOTM approaches were employed to analyse the biological networks and molecular changes in porcine endometrial proteome during maternal recognition of pregnancy. A total of sixteen differentially expressed proteins (DEPs) were identified using 2-DE gels and MALDI-TOF/TOF mass spectrometry. Comparison between 9D and 12D of pregnancy identified APOA1, CAPZB, LDHB, CCT5, ANXA4, CFB, TTR upregulated DEPs, and ANXA5, SMS downregulated DEPs. Comparison between 9D and 16D of pregnancy identified HP, APOA1, ACTB, CCT5, ANXA4, CFB upregulated DEPs and ANXA5, SMS, LDHB, ACTR3, HP, ENO3, OAT downregulated DEPs. However, a comparison between 12D and 16D of pregnancy identified HP, ACTB upregulated DEPs, and CRYM, ANXA4, ANXA5, CAPZB, LDHB, ACTR3, CCT5, ENO3, OAT, TTR down-regulated DEPs. Outcomes of this study revealed key proteins and their interactions with metabolic pathways involved in the recognition and establishment of early pregnancy in PLW gilts.

During maternal recognition of pregnancy (MRP), a conceptus-derived signal leads to the persistence of the corpus luteum and the maintenance of gestation. In the horse, the nature of this signal remains to be elucidated. Several studies have focused on the changes in gene expression during MRP, but little information exists at the protein level. The aim of this study was to identify the proteins at the embryo-maternal interface around signalling of MRP in the horse (day 13) by means of mass spectrometry. A distinct influence of pregnancy was established, with 119 proteins differentially expressed in the uterine fluid of pregnant mares compared to cyclic mares and with upregulation of several inhibitors of the prostaglandin synthesis during pregnancy. By creating an overview of the proteins at the embryo-maternal interface in the horse, this study provides a solid foundation for further targeted studies of proteins potentially involved in embryo-maternal interactions, MRP and pregnancy loss in the horse.

Increased knowledge on serum protein profiles during early pregnancy in dogs would be valuable for several reasons, including animal welfare. Inflammatory changes during this period have been described. Today, mass spectrometry (MS) is a well-established technique to perform unbiased qualitative and quantitative studies of proteins in body fluids regardless of species. In the present study, a shotgun proteomic analysis based on nano-liquid chromatography-MS was performed to identify proteins of altered abundance during canine pregnancy, and, thereafter, a targeted parallel reaction monitoring (PRM)-method was developed and applied to absolutely quantify the concentrations of a selection of these proteins. Among the 32 proteins found altered between pregnant and non-pregnant dogs in the initial analysis, 12 were selected based on their changes in concentration and known biological importance, and these were analyzed using the PRM method. The PRM method showed good linearity, repeatability and sensitivity, and confirmed the higher concentration of Fibrinogen A, protein S alpha and C-reactive protein at early time points in pregnant bitches. In conclusion, the combination of both methods allowed the identification of several altered proteins, and the quantification and description of the concentration patterns for a selection of them during the early stage of dog pregnancy. SIGNIFICANCE: MS is a powerful technique that allows the investigation of protein variations in samples from different origin, such as serum from dogs. The application of a shotgun proteomic analysis as a screening method has revealed the alteration of several proteins after fifteen days of pregnancy in dogs. The complementary development of a PRM MS-based method for several of these proteins has enabled the absolute quantification of their concentrations at five different time points during early pregnancy. With the MS technique, a combination of proteins can be studied with lower limits of detection than with immunoassays. Care should be taken not to interpret the observed changes in pregnant dogs as signs of disease.

The placenta is a unique pregnancy-related tissue and plays a key role in occurrence of unexplained recurrent pregnancy loss (URPL). Abnormal placentation might play a key role in occurrence of URPL. Therefore, the purpose of this study was to compare the human placental proteome between URPL placentas and normal placental matched for gestational week. Total placental proteins were extracted, and the two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) technique was used for separation of the placental proteomes. Protein spots differentially expressed between URPL and normal placentas were selected and identified by the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF/TOF) technique after being digested in the gel. Moreover, quantitative real-time PCR and Western blot techniques were used to confirm the differential expression mass results for some differentially expressed proteins. The results indicated that at least 19 protein spots were differentially expressed between URPL and normal placentas (P < 0.05), and twelve of them were successfully identified. While only two proteins were downregulated (calumenin and enolase 1), the remaining ten spots (actin gamma 1 propeptide, cathepsin D prepropeptide, heat shock protein gp96, tubulin beta, tubulin alpha 1, glutathione S-transferase, vitamin D binding protein, prohibitin, actin beta, apolipoprotein A-I) showed increased expression in URPL cases in comparison with normal placentas. Real-time PCR also confirmed the downregulation of calumenin and upregulation of prohibitin and apolipoprotein A-I at the mRNA levels. In conclusion, the results of the present study showed that alteration in the expression of proteins involved in proliferation and migration of endothelial cells as well as control of coagulation by these cells might play an important role in the pathogenesis of URPL.

Early pregnancy in jennies is routinely determined by palpation per rectum or ultrasonography and also by detecting steroid hormone and chorionic gonadotropin levels in the blood, plasma, and serum. Herein we applied label-free mass spectrometry-based quantitative proteomics to identify serum proteins that were differentially expressed between early pregnant (day 45 after ovulation) and non-pregnant jennies. Bioinformatics analysis allowed illustration of pathways potentially involved in early pregnancy. We identified 295 proteins from a total of 2,569 peptides. Twenty-five proteins (22 upregulated and three downregulated) were significantly differentially expressed between the early pregnant and non-pregnant groups. The majority of the differentially expressed proteins were involved in defense response, early embryonic development, and hormone signaling pathways. Furthermore, functional protein analyses suggested that proteins were involved in binding, enzyme inhibitor activity, and enzyme regulator activity. Five serum proteins-granulin precursor/acrogranin, transgelin-2, fibronectin, fibrinogen-like 1, and thrombospondin 1-can be considered as novel, reliable candidates to detect pregnancy in jennies. To the best of our knowledge, this is the first study to use label-free mass spectrometry-based quantitative proteomics to analyze serum proteins during early pregnancy in jennies. Our results should facilitate the identification of valuable pregnancy diagnostic markers in early pregnant jennies.

Endocrine disrupting compounds (EDCs) are ubiquitous environmental pollutants that alter endocrine system function, induce birth defects, and a myriad of other negative health outcomes. Although the mechanism of toxicity of many EDCs have been studied in detail, little work has focused on understanding the mechanisms through which pregnant dams and fetuses protect themselves from EDCs, or if those protective mechanisms are sexually dimorphic in fetuses. In this study, we examined proteomic alterations in the livers of mouse dams and their male and female fetuses induced by vinclozolin, a model antiandrogenic EDC. Dam livers upregulated nine phase I and phase II detoxification pathways and pathway analysis revealed that more pathways are significantly enriched in dam livers than in fetal livers. Phase I and II detoxification proteins are also involved in steroid and steroid hormone biosynthesis and vinclozolin likely alters steroid levels in both the dam and the fetus. The response of the fetal liver proteome to vinclozolin exposure is sexually dimorphic. Female fetal livers upregulated proteins in xenobiotic metabolism pathways, whereas male fetal livers upregulated proteins in oxidative phosphorylation pathways. These results suggest that female fetuses increase protective mechanisms, whereas male fetuses increase ATP production and several disease pathways that are indicative of oxidative damage. Females fetuses upregulate proteins and protective pathways that were similar to the dams whereas males did not. If this sexually dimorphic pattern is typical, then males might generally be more sensitive to EDCs.

The objective of this study was to evaluate the associations of 92 maternal blood-based proteins with increased infant birth size. The study was performed at the Uppsala University Hospital, Sweden, and included 857 mother and child dyads. The mean age of the women was 30.3 years, and 53.2% were nulliparous. Blood samples were collected at mean 18 + 2 weeks' gestation, and the Olink cardiovascular II panel was used to measure 92 proteins, either known to be or suspected to be markers of cardiovascular and inflammatory disease in humans. Multiple linear regression models adjusted for maternal age, parity, pre-conception BMI, height, and smoking were performed to evaluate the association of each individual protein with infant birth size. We also performed sex-stratified analyses. Eight proteins (Matrix metalloproteinase-12 (MMP-12), Prostasin (PRSS8), Adrenomedullin (ADM), Pappalysin-1 (PAPP-A), Angiotensin-converting enzyme 2 (ACE2), Sortilin (SORT1), Lectin-like oxidized LDL receptor 1 (LOX-1), and Thrombomodulin (TM)) were associated with infant birth size after false discovery rate adjustment. In the analyses including only female infants, ten proteins (MMP-12, Growth/differentiation factor 2 (GDF-2), PRSS8, SORT1, ADM, Interleukin-1 receptor antagonist protein (IL-1ra), Leptin (LEP), ACE2, TM, and Tumor necrosis factor receptor superfamily member 11A (TNFRSF11A)) were associated with infant birth size. Two proteins (PAPP-A and PRSS8) were associated with infant birth size among male infants. Our study suggests several proteins as potential biomarkers for increased birth weight, and our findings could act as a base for future research to identify new potential markers that could be added to improve screening for large infants.

Regulation of tissue and development specific gene expression patterns underlies the functional specialization of organs in multi-cellular organisms. In the viviparous tsetse fly (Glossina), the female accessory gland is specialized to generate nutrients in the form of a milk-like secretion to support growth of intrauterine larva. Multiple milk protein genes are expressed specifically in the female accessory gland and are tightly linked with larval development. Disruption of milk protein synthesis deprives developing larvae of nutrients and results in extended larval development and/or in abortion. The ability to cause such a disruption could be utilized as a tsetse control strategy. Here we identify and delineate the regulatory sequence of a major milk protein gene (milk gland protein 1:mgp1) by utilizing a combination of molecular techniques in tsetse, Drosophila transgenics, transcriptomics and in silico sequence analyses. The function of this promoter is conserved between tsetse and Drosophila. In transgenic Drosophila the mgp1 promoter directs reporter gene expression in a tissue and stage specific manner orthologous to that of Glossina. Analysis of the minimal required regulatory region of mgp1, and the regulatory regions of other Glossina milk proteins identified putative homeodomain protein binding sites as the sole common feature. Annotation and expression analysis of Glossina homeodomain proteins identified ladybird late (lbl) as being accessory gland/fat body specific and differentially expressed between lactating/non-lactating flies. Knockdown of lbl in tsetse resulted in a significant reduction in transcript abundance of multiple milk protein genes and in a significant loss of fecundity. The role of Lbl in adult reproductive physiology is previously unknown. These results suggest that Lbl is part of a conserved reproductive regulatory system that could have implications beyond tsetse to other vector insects such as mosquitoes. This system is critical for tsetse fecundity and provides a potential target for development of a reproductive inhibitor.