Constantine A. Simintiras

Amino acids in the uterine luminal fluid reflects the temporal changes in transporter expression in the endometrium and conceptus during early pregnancy in cattle.

In cattle, conceptus-maternal interactions are critical for the establishment and maintenance of pregnancy. A major component of this early interaction involves the transport of nutrients and secretion of key molecules by uterine epithelial cells to help support conceptus development during the peri-implantation period of pregnancy. Objectives were to: 1) analyze temporal changes in the amino acid (AA) content of uterine luminal fluid (ULF) during the bovine estrous cycle; 2) understand conceptus-induced alterations in AA content; 3) determine expression of AA transporters in the endometrium and conceptus; and 4) determine how these transporters are modulated by (Progesterone) P4. Concentrations of aspartic acid, arginine, glutamine, histidine, lysine, isoleucine, leucine, phenylalanine and tyrosine decreased on Day 16 of the estrous cycle but increased on Day 19 in pregnant heifers (P<0.05). Glutamic acid only increased in pregnant heifers on Day 19 (P<0.001). Asparagine concentrations were greater in ULF of cyclic compared to pregnant heifers on Day 7 (P<0.05) while valine concentrations were higher in pregnant heifers on Day 16 (P<0.05). Temporal changes in expression of the cationic AA transporters SLC7A1 SLC7A4 and SLC7A6 occurred in the endometrium during the estrous cycle/early pregnancy coordinate with changes in conceptus expression of SLC7A4, SLC7A2 and SLC7A1 (P<0.05). Only one acidic AA transporter (SLC1A5) increased in the endometrium while conceptus expression of SLC1A4 increased (P<0.05). The neutral AA transporters SLC38A2 and SLC7A5 increased in the endometrium in a temporal manner while conceptus expression of SLC38A7, SLC43A2, SLC38A11 and SLC7A8 also increased (P<0.05). P4 modified the expression of SLC1A1, -1A4, -1A5, -38A2, -38A4, -38A7, -43A2, -6A14, -7A1, -7A5 and -7A7 in the endometrium. Results demonstrate that temporal changes in AA in the ULF reflect changes in transporter expression in the endometrium and conceptus during early pregnancy in cattle, some of which are modified by P4.

Sexually Dimorphic Gene Expression in Bovine Conceptuses at the Initiation of Implantation

ABSTRACT In cattle, maternal recognition of pregnancy occurs on Day 16 via secretion of interferon tau (IFNT) by the conceptus. The endometrium can distinguish between embryos with different developmental competencies. In eutherian mammals, X-chromosome inactivation (XCI) is required to ensure an equal transcriptional level of most X-linked genes for both male and female embryos in adult tissues, but this process is markedly different in cattle than mice. We examined how sexual dimorphism affected conceptus transcript abundance and amino acid composition as well as the endometrial transcriptome during the peri-implantation period of pregnancy. Of the 5132 genes that were differentially expressed on Day 19 in male compared to female conceptuses, 2.7% were located on the X chromosome. Concentrations of specific amino acids were higher in the uterine luminal fluid of male compared to female conceptuses, while female conceptuses had higher transcript abundance of specific amino acid transporters (SLC6A19 and SLC1A35). Of note, the endometrial transcriptome was not different in cattle gestating a male or a female conceptus. These data support the hypothesis that, far from being a blastocyst-specific phenomenon, XCI is incomplete before and during implantation in cattle. Despite differences in transcript abundance and amino acid utilization in male versus female conceptuses, the sex of the conceptus itself does not elicit a different transcriptomic response in the endometrium.

Modelling aspects of oviduct fluid formation in vitro

© 2017 Society for Reproduction and Fertility. Oviduct fluid is the microenvironment that supports early reproductive processes including fertilisation, embryo cleavage and genome activation. However, the composition and regulation of this critical environment remain rather poorly defined. This study uses an in vitro preparation of the bovine oviduct epithelium to investigate the formation and composition of in vitro-derived oviduct fluid (ivDOF) within a controlled environment. We confirm the presence of oviduct-specific glycoprotein 1 in ivDOF and show that the amino acid and carbohydrate content resembles that of previously reported in vivo data. In parallel, using a different culture system, a panel of oviduct epithelial solute carrier genes and the corresponding flux of amino acids within ivDOF in response to steroid hormones were investigated. We next incorporated fibroblasts directly beneath the epithelium. This dual culture arrangement represents more faithfully the in vivo environment and impacts on ivDOF composition. Lastly, physiological and pathophysiological endocrine states were modelled and their impact on the in vitro oviduct preparation was evaluated. These experiments help clarify the dynamic function of the oviduct in vitro and suggest a number of future research avenues, such as investigating epithelial-fibroblast interactions, probing the molecular aetiologies of subfertility and optimising embryo culture media.

Effect of metabolic status on conceptus-maternal interactions on day 19 in dairy cattle: II. Effects on the endometrial transcriptome

Abstract The aim of this study was to test the hypothesis that the metabolic stresses associated with lactation alter the ability of the endometrium to respond appropriately to the conceptus by examining endometrial gene expression on day 19 of pregnancy. Immediately after calving, primiparous Holstein cows with similar production and fertility estimated breeding values were randomly divided into two groups and either dried off (i.e. never milked) immediately or milked twice daily. Approximately 65–75 days postpartum, grade 1 blastocysts recovered from superovulated Holstein heifer donors (n = 5) were transferred (1 per recipient) into lactating (n = 11) and nonlactating (n = 11) recipients. Control nulliparous Holstein heifers (n = 6) were artificially inseminated. RNAsequencing was performed on intercaruncular endometrial samples recovered at slaughter from confirmed pregnant animals on day 19 (n = 5 lactating and nonlactating cows; n = 4 heifers). Differentially expressed genes (DEGs) were identified between both postpartum groups compared to heifers and between lactating and nonlactating cows. Functional annotation of DEGs between cows and heifers revealed over-representation of categories, including endosome, cytoplasmic vesicle, endocytosis, regulation of exocytosis, and cytokine receptor activity. Functional categories including transcription factor binding sites, cell motility, and cell migration were enriched for DEGs between endometria from lactating and nonlactating cows. In conclusion, while the evidence for a major effect of lactation on the endometrial transcriptome is relatively weak, these data suggest that the metabolic status of the animal (heifer vs cow) modulates the response of the endometrium to the developing conceptus. Summary Sentence The endometrial response to a high quality embryo following transfer is minimal in lactating dairy rows compared to non-lactating cows but differs in its response to a conceptus in lactating cows compared to heifers.

Genistein crosses the bioartificial oviduct and alters secretion composition

The dietary derived isoflavone and oestrogen analogue, genistein, is known to perturb fundamental reproductive events such as implantation and embryo cleavage. However the question of whether genistein is able to traverse the oviduct epithelial monolayer and impact oviduct fluid secretion remains unclear. This study tests these research questions using a bioartificial oviduct to show that genistein permeates the oviduct lumen in vitro with a biphasic (burst and plateau) kinetic profile, faster than spontaneous diffusion, and alters the amino acid composition of in vitro derived oviduct fluid (ivDOF) but not as an oestrogen analogue. In addition to offering insights into the potential mechanisms of these findings, this manuscript demonstrates the potential to use the bioartificial oviduct model to characterise the transport or barrier properties of the oviduct towards a range of circulating xenobiotics.

Temporally differential protein expression of glycolytic and glycogenic enzymes during in vitro preimplantation bovine embryo development

Proteomic analyses are useful for understanding the metabolic pathways governing embryo development. This study investigated the presence of enzymes involved in glycolysis and glycogenesis in in vitro-produced bovine embryos at five developmental stages leading up to blastocyst formation. The enzymes examined were: (1) glycolytic: hexokinase-I (HK-I), phosphofructokinase-1 (PFK-1), pyruvate kinase mutase 1/2 (PKM-1/2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and (2) glycogenic: glycogen synthase kinase-3 isoforms α/ β (GSK-3α/β). Glucose transporter-1 (GLUT-1) was also analysed. The developmental stages examined were: (1) 2-4-cell, (2) 5-8-cell, (3) 16-cell, (4) morula and (5) expanded blastocyst. The enzymes HK-I, PFK-1, PKM-1/2, GAPDH and GLUT-1 were differentially expressed throughout all stages (P<0.05). GSK-3α and β were also differentially expressed from the 2-4-cell to the expanded blastocyst stage (P<0.05) and GLUT-1 was identified throughout. The general trend was that the abundance of PFK1, GAPDH and PKM-1/2 decreased whereas HK-I, phospho-GSK3α (P-GSK3α) and P-GSK3β levels increased as the embryo advanced. In contrast, GLUT-1 expression peaked at the 16-cell stage. These data combined suggest that in vitro bovine embryo metabolism switches from being glycolytic-centric to glycogenic-centric around the 16-cell stage, the developmental window also characterised by embryonic genome activation.

73 SPATIAL DIFFERENCES IN METABOLITES AND ENERGY SUBSTRATES IN THE BOVINE OVIDUCT

Knowledge of the biochemical composition of the bovine oviduct in the presence of an embryo is lacking. We have recently reported the detection of alterations to the bovine oviduct transcriptome when multiple embryos, but not a single embryo, were present (Maillo et al. 2015 Biol. Reprod. 92, 144). Thus, we hypothesised that the presence of an embryo would modify the concentration of metabolites and energy substrates in the oviducal fluid. Thus, the aim of the present study was to examine how the presence of an embryo affects metabolites and energy substrates within the oviduct. Cross-bred beef heifers were synchronized, and those in standing oestrus were randomly allocated to cyclic, not bred (n=7), or pregnant, artificially inseminated (n=11), groups. All heifers were slaughtered on Day 3 after oestrus. The oviducts from each animal were isolated, straightened, and cut, separating ampulla and isthmus. Each portion was flushed with 500μL of PBS, enabling recovery of any oocyte/embryo. Recovered unfertilized oocytes (cyclic group) and embryos (pregnant group) were located in the isthmus of the oviduct ipsilateral to the corpus luteum. Samples of flushing medium from the isthmus and ampulla from oviducts ipsilateral to the corpus luteum from 5 cyclic and 5 pregnant animals (8-cell embryos) were used for metabolites and energy substrates analysis, together with PBS as blank control. Amino acid content in the oviducal fluid was determined by high performance liquid chromatography. Glucose, lactate, and pyruvate were quantified using microfluorometric enzyme-linked assays. Statistical analysis was carried out by 2-way ANOVA with a Bonferroni post hoc for multiple comparisons. No differences were found either in the concentration of metabolites or in the energy substrates between cyclic and pregnant heifers. However, significant differences were detected between ampulla and isthmus in cyclic and pregnant heifers, which is in agreement with our previous findings on the transcriptome between isthmus and ampulla (Maillo et al. 2016 Reproduction 152, 37-46). In pregnant and cyclic heifers, glycine and alanine were more abundant in the ampulla compared with the isthmus (P<0.01). In addition, in pregnant heifers, arginine was less abundant in the ampulla than in the isthmus (P<0.01). Energy substrates were also similar between pregnant and cyclic heifers. However, lactate was significantly different between ampulla and isthmus, being present in the ampulla of cyclic and pregnant heifers and being absent from the isthmus (P<0.05). In conclusion, the content of metabolites and energy substrates in the oviduct varies between the ampulla and isthmus independent of the presence of an embryo.

Effect of lactation on conceptus-maternal interactions at the initiation of implantation in cattle: I. Effects on the conceptus transcriptome and amino acid composition of the uterine luminal fluid

Abstract Approximately 65–75 days postpartum (dpp), the estrous cycles of nonlactating (dried off immediately postpartum: n = 12) and lactating (n = 13) Holstein Friesian cows were synchronized and on day 7 a single blastocyst derived from superovulated nulliparous Holstein Friesian heifers was transferred to each cow. A control group of nulliparous heifers (n = 8) were synchronized, inseminated to a standing heat, and slaughtered on the same day as nonlactating and lactating recipients (day 19; estrus = day 0). The uterine horn ipsilateral to the corpus luteum was flushed with 10 ml phosphate-buffered saline and the conceptus, and uterine luminal fluid (ULF) was snap-frozen in liquid nitrogen. Gene expression analysis of the conceptus was performed by RNA sequencing, while amino acid composition of ULF was determined by high-performance liquid chromatography. No differentially expressed genes (DEGs) were observed between conceptuses recovered from nonlactating and lactating cows. Eight DEGs were identified between conceptuses recovered from nonlactating cows and heifers. A total of 269 DEGs (100 up- and 169 downregulated) were identified between conceptuses recovered from lactating cows compared to heifers. Alanine, glycine, serine, threonine, arginine, leucine, and valine were significantly lower in abundance in ULF recovered from heifers compared to nonlactating or lactating cows. This study demonstrates that the environment in which the embryo develops post the blastocyst stage can have an effect on the conceptus transcriptome and amino acid composition of the ULF but this was mainly observed between the two extreme groups in terms of metabolic status (nulliparous heifers vs postpartum lactating cows). Summary Sentence Exposure of embryos from a common pool to a uterine environment under different metabolic statuses alters conceptus transcriptome and composition of uterine fluidmainly between extremes of metabolic status.

Embryo–Uterine Interactions During Implantation: Potential Sites of Interference by Environmental Toxins

Synchronous development of the embryo to the blastocyst stage and its communication with the receptive uterus is crucial to successful implantation. The process of embryo implantation involves a complex series of molecular dialogues between the embryo and the uterus. Estrogen plays an essential role for the onset of embryo–uterine interactions during implantation and involves molecular interplay of numerous growth and differentiation regulating signaling molecules, but the hierarchical molecular relationships downstream of estrogen signaling remain poorly understood. It is well established that any aberration of normal signaling by estrogenic mimics (xenoestrogens) poses a major threat to the process of implantation and therefore the pregnancy outcome. In this review, we highlight our current knowledge about estrogen (or its mimics) regulated molecular events in implantation and in general uterine signaling, and the future challenges of the field. Basic understanding of estrogen-mediated signaling mechanisms during the onset of implantation will help alleviate problems of infertility, develop novel contraceptives, and to better manage uterine cancer problems.