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Background: MicroRNAs (miRNAs) are presented in the uterine lumen of many mammals, and in vitro experiments have determined that several miRNAs are important for the regulation of endometrial and trophoblast functions. Our aim was to identify and contrast the miRNAs present in extracellular vesicles (EVs) in the uterine lumen fluid (ULF) at the onset of attachment in cattle pregnancies (gestation d 18) initiated by artificial insemination (AI) or by the transfer of an in vitro-produced blastocyst (IVP-ET). A third group had no conceptus after the transfer of an IVP embryo. Results: The abundance of 263 annotated miRNAs was quantified in the EVs collected from ULF. There was an increase in the transcript abundance of 20 miRNAs in the ULF EVs from the AI pregnant group, while 4 miRNAs had a lower abundance relative to the group not containing a conceptus. Additionally, 4 miRNAs were more abundant in ULF EVs in the AI pregnant group relative to IVP-ET group (bta-mir-17, bta-mir-7-3, MIR7-1, MIR18A). Specific miRNAs in the ULF EVs were co-expressed with messenger RNAs expressed in extra-embryonic tissues and endometrium, including genes that are known to be their targets. Conclusions: The results provide biological insights into the participation of miRNAs in the regulation of trophoblast proliferation and differentiation, as well as in endometrium receptivity. The knowledge that in vitro cultured embryos can contribute to the altered abundance of specific miRNAs in the uterine lumen can lead to the development of corrective approaches to reduce conceptus losses during the first month of pregnancy in cattle. [ABSTRACT FROM AUTHOR]

The reproductive efficiency of dromedary camels is hindered by challenges such as early embryonic mortality, which may be attributed to a lack of synchronization between conceptus signalling and uterine receptivity. Understanding the intricate biological processes involved in feto‐maternal interactions during implantation is crucial to address these limitations. Osteopontin (OPN) is a protein involved in cell signalling and adhesion, playing a crucial role in embryonic implantation. Previous studies have shown the presence of OPN in the uterine endometrium of various mammalian species including dromedary camels. However, the expression pattern of OPN in dromedary conceptuses remains unexplored. Thus, the current study aimed, for the first time, to investigate the temporospatial expression of OPN in dromedary conceptuses during the peri‐implantation period at Days 8, 10, and 12 of pregnancy. Twelve conceptuses were recovered non‐surgically from pregnant females on Days 8, 10, 12 of pregnancy. Quantitative real‐time PCR (qrt‐PCR), immunohistochemistry (IHC), and immunofluorescence (IF) were employed for analysis of the expression of OPN mRNA and protein. The results revealed significant increases in both OPN mRNA and protein expression started on Day 10 and peaked at Day 12 of pregnancy. Immuno‐localization confirmed the presence of OPN protein in the trophectoderm and endoderm of dromedary conceptuses. In conclusion, the expression and localization of OPN during the peri‐implantation period in dromedary conceptuses imply its involvement as a crucial reproductive factor and its upregulation during this period, with a pronounced increase close to attachment time (Day 12 of pregnancy) further supports its role in embryo adhesion, implantation, and placentation. [ABSTRACT FROM AUTHOR]

Abstract Background MicroRNAs (miRNAs) are presented in the uterine lumen of many mammals, and in vitro experiments have determined that several miRNAs are important for the regulation of endometrial and trophoblast functions. Our aim was to identify and contrast the miRNAs present in extracellular vesicles (EVs) in the uterine lumen fluid (ULF) at the onset of attachment in cattle pregnancies (gestation d 18) initiated by artificial insemination (AI) or by the transfer of an in vitro-produced blastocyst (IVP-ET). A third group had no conceptus after the transfer of an IVP embryo. Results The abundance of 263 annotated miRNAs was quantified in the EVs collected from ULF. There was an increase in the transcript abundance of 20 miRNAs in the ULF EVs from the AI pregnant group, while 4 miRNAs had a lower abundance relative to the group not containing a conceptus. Additionally, 4 miRNAs were more abundant in ULF EVs in the AI pregnant group relative to IVP-ET group (bta-mir-17, bta-mir-7-3, MIR7-1, MIR18A). Specific miRNAs in the ULF EVs were co-expressed with messenger RNAs expressed in extra-embryonic tissues and endometrium, including genes that are known to be their targets. Conclusions The results provide biological insights into the participation of miRNAs in the regulation of trophoblast proliferation and differentiation, as well as in endometrium receptivity. The knowledge that in vitro cultured embryos can contribute to the altered abundance of specific miRNAs in the uterine lumen can lead to the development of corrective approaches to reduce conceptus losses during the first month of pregnancy in cattle.

We recently demonstrated that conceptus-derived interferon tau (IFNT), responsible for maternal recognition in cattle, acts on the uterus in a dose- and time-dependent manner by upregulating key interferon-stimulated genes (ISGs) in the endometrium. In high producing dairy cows, postpartum uterine infection is a major factor influencing fertility and pregnancy outcome. Lipopolysaccharide (LPS), an endotoxin of Gram-negative bacteria such as Escherichia coli , generates an altered uterine environment by inducing excessive inflammation at the maternal-conceptus interface. Thus, we aimed to investigate whether the endometrial response to IFNT is altered in the presence of LPS. Endometrial explants were isolated from uteri collected at a local abattoir from Holstein Friesian cows (n = 8) during the mid-luteal stage of the estrous cycle, and cultured in RPMI medium for 24 h in 5 % CO 2 in humidified air without (control), or with IFNT (100 ng/mL), a single Day 15 conceptus, LPS (1 μg/mL), both IFNT and LPS, or both a Day 15 conceptus and LPS. Incubation with IFNT and a Day 15 conceptus up-regulated (P < 0.05) well-known classical ISGs (ISG15 , OAS1 , MX1 and MX2) as well as other candidate ISGs (CMPK2 , IFI35 , TRIM38 and TNFSF10) and down-regulated expression of IL1B in endometrial explants. Incubation with LPS increased (P < 0.05) abundance of NFKB1 (a key transcription factor involved in inflammatory and immune response), TNFA , IL1B and IL6 (pro-inflammatory cytokines), IL10 (anti-inflammatory cytokine), IL8 , CXCL1 , CXCL3 and CCL2 (chemokines), and, to a lesser extent, classical ISGs in endometrial explants. However, LPS did not alter endometrial response to IFNT, irrespective of IFNT concentration (1, 10 or 100 ng/mL). Results suggest that the expression of ISGs, up-regulated by conceptus-derived IFNT, is not altered in the endometrium in the presence of LPS; however, the increased expression of inflammation-related genes induced by LPS indicate an altered endometrial immune response that may be associated with compromised pregnancy establishment or pregnancy failure. • Conceptus-derived interferon tau (IFNT) induces interferon-stimulated genes (ISGs) in the endometrium. • Bacterial lipopolysaccharide (LPS) does not influence endometrial ISG expression in response to conceptus-derived IFNT. • Inflammatory genes induced by LPS may be associated with compromised pregnancy establishment or pregnancy failure. [ABSTRACT FROM AUTHOR]

The current study investigated effects of dietary amino acid (AA) availability on lactational body condition loss and metabolic status, in relation to reproductive parameters after weaning up to Day 8 post-ovulation. Primiparous sows (n = 35) were allocated to one of two lactation diets containing either low crude protein (CP, 140 g/kg) with a low percentage (8%) of slow protein in total protein (LL, n = 18) or high CP (180 g/kg) with a high (16%) percentage of slow protein (HH, n = 17). The HH diet was expected to improve AA utilization by supplying more AA, in a more gradual fashion. The diets did not affect sow body condition loss during lactation, while the HH diet tended to increase litter weight gain during the week 3 of lactation (Δ = 1.3 kg, P = 0.09). On Day 14 post-farrowing, HH diet led to higher plasma urea both pre-feeding and post-feeding (Δ = 2.3 mmol/L, P < 0.01, Δ = 2.4 mmol/L, P < 0.01, respectively), whilst plasma creatinine, NEFA and IGF-1 were similar. No dietary effects on reproductive parameters were found, however several relationships were found between body condition and reproductive parameters. Sows with higher body weight on Day 1 or Day 21 post-farrowing had greater follicle size on Day 3 post-weaning (β = 0.03 mm/kg, P < 0.01, β = 0.04 mm/kg, P < 0.01, respectively). At Day 8 post-ovulation, plasma progesterone concentration was negatively related to loin muscle loss (β = −0.67 ng/ml · mm−1, P = 0.02), backfat loss (β = −2.33 ng/ml · mm−1, P = 0.02), and estimated body fat loss (β = −0.67 ng/ml · mm−1, P = 0.02). Both plasma progesterone and the number of corpora lutea were positively related to the energy balance during lactation (β = 0.03 ng/ml · ME MJ−1, P = 0.01, β = 0.01 CL/ME MJ, P = 0.02, respectively). The conceptus size at Day 8 post-ovulation was negatively related to body weight loss (β = −0.01 mm/kg, P = 0.01), estimated body fat loss (β = −0.02 mm/kg, P = 0.03) and estimated body protein loss (β = −0.06 mm/kg, P = 0.04), and was positively related to the energy balance during lactation (β = 5.2*10−4 mm/ME MJ, P = 0.01). In conclusion, body protein and fat losses during lactation reduced subsequent plasma progesterone concentration and conceptus development at Day 8 post-ovulation. • Lactational body condition loss impairs conceptus development at Day 8 post-ovulation. • A higher body condition loss relates to a lower plasma P4 at Day 8 post-ovulation. • A higher body weight on Day 1 or 21 post-farrowing relates to a larger follicle size. [ABSTRACT FROM AUTHOR]

Three experiments were conducted to evaluate the effects of long-acting injectable progesterone (iP4) in buffalo cows. In Experiment 1, ovariectomized buffaloes received 300 mg (iP300) or 600 mg (iP600) of iP4, and serum P4 concentrations were evaluated. In experiment 2, three groups were compared: control or administration of 300 mg of iP4 3 (iP4-D3) or 6 days (iP4-D6) after timed artificial insemination (TAI). On day 16, reproductive tract was recovered for conceptus, endometrium, and corpus luteum (CL) analysis. In experiment 3, pregnancy per AI (P/TAI) and proportion of pregnancy losses were evaluated after administration of 300 mg of iP4 3 (iP4-D3) or 6 days (iP4-D6) after TAI in lactating buffaloes. In experiment 1, serum P4 concentrations remained over 1 ng/mL for ~ 3 days in both groups. The 300 mg dose was used in subsequent experiments. In experiment 2, CL weight and endometrial glands density were decreased, and conceptus length was increased in iP4-D3 compared to control and to iP4-D6 (P < 0.05). Transcript abundance of Prostaglandin F Receptor (FP) and ISG15 in CL and of ISG15 and MX1 in endometrium was greater in iP4-D3 when compared to control and to iP4-D6 (P < 0.05). In experiment 3, there was no difference among experimental groups for P/TAI at D30 and pregnancy losses (P > 0.1); however, iP4-D3 presented a lower P/TAI at day 60 (41.7%) when compared to control (56.8%) and iP4-D6 (57.7%; P = 0.07). In conclusion, administration iP4 at 3 days after TAI affects CL development and consequently decreases final pregnancy outcome in buffaloes. [ABSTRACT FROM AUTHOR]

Introduction: Sex differences in prenatal growth may contribute to sexdependent programming effects on postnatal phenotype. Methods: We integrated for the first time phenotypic, histomorphological, clinico-chemical, endocrine and gene expression analyses in a single species, the bovine conceptus at mid-gestation. Results: We demonstrate that by mid-gestation, before the onset of accelerated growth, the female conceptus displays asymmetric lower growth compared to males. Female fetuses were smaller with lower ponderal index and organ weights than males. However, their brain:body weight, brain:liver weight and heart:body weight ratios were higher than in males, indicating brain and heart 'sparing'. The female placenta weighed less and had lower volumes of trophoblast and fetal connective tissue than the male placenta. Female umbilical cord vessel diameters were smaller, and female-specific relationships of body weight and brain:liver weight ratios with cord vessel diameters indicated that the umbilico-placental vascular system creates a growth-limiting environment where blood flow is redistributed to protect brain and heart growth. Clinico-chemical indicators of liver perfusion support this female-specific growth-limiting phenotype, while lower insulin-like growth factor 2 (IGF2) gene expression in brain and heart, and lower circulating IGF2, implicate female-specificmodulation of key endocrinemediators by nutrient supply. Conclusion: This mode of female development may increase resilience to environmental perturbations in utero and contribute to sex-bias in programming outcomes including susceptibility to non-communicable diseases. [ABSTRACT FROM AUTHOR]

In order to explore the suitable posture of giant pandas (Ailuropoda melanoleuca) during anorexia using B-ultrasound examination for conceptus,16 giant pandas at Chengdu Research Base of Giant Panda Breeding were investigated. Results showed that the conceptus (single and twins) was found in uterus of nine giant pandas by B-ultrasound examination. Moreover, we completed the B-ultrasound examination with the giant panda was either lying on its back, lying on its stomach, lying on its side,squatting, or sitting position of dog. We found that there was no difference in the time duration for detecting con-ceptus, as well as the length of conceptus in between lying on its back and non-lying on its back. The posture of non-lying on its back can reduce the specific trainings on animal behaviors when a B-ultrasound examination on conceptus is preformed, thereby vastly reduce the stress reaction and increase animal welfare, which can be advocated in the future B-ultrasound examination during giant panda’s reproductive process. [ABSTRACT FROM AUTHOR]

IntroductionSex differences in prenatal growth may contribute to sex-dependent programming effects on postnatal phenotype. MethodsWe integrated for the first time phenotypic, histomorphological, clinico-chemical, endocrine and gene expression analyses in a single species, the bovine conceptus at mid-gestation. ResultsWe demonstrate that by mid-gestation, before the onset of accelerated growth, the female conceptus displays asymmetric lower growth compared to males. Female fetuses were smaller with lower ponderal index and organ weights than males. However, their brain:body weight, brain:liver weight and heart:body weight ratios were higher than in males, indicating brain and heart ‘sparing’. The female placenta weighed less and had lower volumes of trophoblast and fetal connective tissue than the male placenta. Female umbilical cord vessel diameters were smaller, and female-specific relationships of body weight and brain:liver weight ratios with cord vessel diameters indicated that the umbilico-placental vascular system creates a growth-limiting environment where blood flow is redistributed to protect brain and heart growth. Clinico-chemical indicators of liver perfusion support this female-specific growth-limiting phenotype, while lower insulin-like growth factor 2 (IGF2) gene expression in brain and heart, and lower circulating IGF2, implicate female-specific modulation of key endocrine mediators by nutrient supply. ConclusionThis mode of female development may increase resilience to environmental perturbations in utero and contribute to sex-bias in programming outcomes including susceptibility to non-communicable diseases.

Context: Arachidonic acid (AA) is the precursor of prostaglandins, which may play autocrine roles during early embryo development. Aims: To test the developmental effects of addition of AA to pre- and post-hatching culture media on in vitro –produced bovine embryos. Methods: Pre-hatching effects of AA were tested by culturing bovine zygotes in synthetic oviductal fluid (SOF) supplemented with 100 or 333 μM AA. Post-hatching effects of AA were tested by culturing Day 7 blastocysts in N2B27 supplemented with 5, 10, 20 or 100 μM AA up to Day 12. Key results: Pre-hatching development to blastocyst was completely abrogated at 333 μM AA, whereas blastocyst rates and cell numbers were not altered at 100 μM AA. Impaired post-hatching development was observed at 100 μM AA, whereas no effect on survival rates was noted at 5, 10 and 20 μM AA. However, a significant reduction in Day 12 embryo size was observed at 10 and 20 μM AA. Hypoblast migration, epiblast survival and formation of embryonic-disc-like structures were unaffected at 5–10 μM AA. AA exposure downregulated the genes PTGIS , PPARG , LDHA and SCD in Day 12 embryos. Conclusions: Pre-hatching embryos are mostly irresponsive to AA, whereas AA was observed to have negative effects during early post-hatching development. Implications: AA does not improve in vitro bovine embryo development and is not required up to early post-hatching stages. Arachidonic acid (AA) is the precursor of prostaglandins, which have been suggested to play autocrine roles during early development. In this study, we tested the developmental effect of the addition of AA to pre- and post-hatching bovine embryo culture, observing that pre-hatching embryos are mostly irresponsive to AA, whereas early post-hatching in vitro development is negatively affected by AA. [ABSTRACT FROM AUTHOR]

Abstract During initial maternal recognition of pregnancy (MRP), the equine embryo displays a series of unique events characterized by rapid blastocyst expansion, secretion of a diverse array of molecules, and transuterine migration to interact with the uterine surface. Up to date, the intricate transcriptome and proteome changes of the embryo underlying these events have not been critically studied in horses. Thus, the objective of this study was to perform an integrative transcriptomic (including mRNA, miRNAs, and other small non‐coding RNAs) and proteomic analysis of embryos collected from days 10 to 13 of gestation. The results revealed dynamic transcriptome profiles with a total of 1311 differentially expressed genes, including 18 microRNAs (miRNAs). Two main profiles for mRNAs and miRNAs were identified, one with higher expression in embryos ≤5 mm and the second with higher expression in embryos ≥7 mm. At the protein level, similar results were obtained, with 259 differentially abundant proteins between small and large embryos. Overall, the findings demonstrated fine‐tuned transcriptomic and proteomic regulations in the developing embryo associated with embryo growth. The identification of specific regulation of mRNAs, proteins, and miRNAs on days 12 and 13 of gestation suggested these molecules as pivotal for embryo development and as involved in MRP, and in establishment of pregnancy in general. In addition, the results revealed new insights into prostaglandin synthesis by the equine embryo, miRNAs and genes potentially involved in modulation of the maternal immune response, regulation of endometrial receptivity and of late implantation in the mare.

Ethical deliberations are unfolding for potentially controversial organoid‐entities such as brain organoids and embryoids. Much of the ethical deliberation centers on the questionable moral status of such organoid‐entities. However, while such work is important and appropriate, ethical deliberations may become too exclusively rooted in moral status and potentially overshadow other relevant moral dilemmas. The ethical discussion on organoid models can benefit from insights brought forth by both Judith Jarvis Thomson and Don Marquis in how they attempted to advance the abortion debate. To discuss other abortion ethical issues more fully, both Thomson and Marquis assumed differing moral status positions of the conceptus and followed lines of reasoning based on these moral status assumptions. We suggest a similar approach with controversial organoid‐entities like brain organoids and embryoids. To avoid overshadowing or overlooking other relevant ethical issues, ethicists ought to first assume an organoid‐entity moral status position (such as a high moral status or no moral status) and explore any possible arguments that may result from such a position. While we ought not to copy the content of Thomson and Marquis' arguments exactly for organoid‐entities, it is worthwhile to translate their arguments' overarching structures. This paper explores the relevant insights of Thomson and Marquis, how they can be translated into the organoid ethics debate, and what possible lines of inquiry may be worth exploring based on particular moral status assumptions. [ABSTRACT FROM AUTHOR]

Highly proliferative cells rely on one carbon (1C) metabolism for production of formate required for synthesis of purines and thymidine for nucleic acid synthesis. This study was to determine if extracellular serine and/or glucose and fructose contribute the production of formate in ovine conceptuses. Suffolk ewes (n = 8) were synchronized to estrus, bred to fertile rams, and conceptuses were collected on Day 17 of gestation. Conceptuses were either snap frozen in liquid nitrogen (n = 3) or placed in culture in medium (n = 5) containing either: 1) 4 mM D-glucose + 2 mM [U-13C]serine; 2) 6 mM glycine + 4 mM D-glucose + 2 mM [U-13C]serine; 3) 4 mM D-fructose + 2 mM [U-13C]serine; 4) 6 mM glycine + 4 mM D-fructose + 2 mM [U-13C]serine; 5) 4 mM D-glucose + 4 mM D-fructose + 2 mM [U-13C]serine; or 6) 6 mM glycine + 4 mM D-glucose + 4 mM D-fructose + 2 mM [U-13C]serine. After 2 h incubation, conceptuses in their respective culture medium were homogenized and the supernatant analyzed for 12C- and 13C-formate by gas chromatography and amino acids by high performance liquid chromatography. Ovine conceptuses produced both 13C- and 12C-formate, indicating that the [U-13C]serine, glucose, and fructose were utilized to generate formate, respectively. Greater amounts of 12C-formate than 13C-formate were produced, indicating that the ovine conceptus utilized more glucose and fructose than serine to produce formate. This study is the first to demonstrate that both 1C metabolism and serinogenesis are active metabolic pathways in ovine conceptuses during the peri-implantation period of pregnancy, and that hexose sugars are the preferred substrate for generating formate required for nucleotide synthesis for proliferating trophectoderm cells. [ABSTRACT FROM AUTHOR]

The endometrium is fundamentally required for successful pregnancy in ruminants and species where the posthatching conceptus undergoes a protracted elongation and peri-implantation phase of pregnancy. Moreover, there are substantial waves of pregnancy loss during this pre- and peri-implantation period of pregnancy the precise source of which has not been clearly defined i.e., the maternal uterine contribution to this loss. Understanding the molecular interactions required for successful pregnancy in cattle will allow us to intervene to support pregnancy success during this vulnerable window. The endometrium contributes to most key developmental milestones of pregnancy establishment, including (1) contributing to the regulation of the oestrus cycle, (2) nourishing the preimplantation conceptus, (3) responding to the conceptus to create a more receptive microenvironment, (4) providing essential biophysical support, and (5) signalling and producing factors which affect the mother systemically. This review will summarise what we currently know about conceptus-maternal interactions as well as identify the gaps in our knowledge that could be filled with newer in vitro model approaches. These include the use of microfluidics, organ-on-a-chip devices, and bioinformatic approaches. This will help maximise food production efficiency (both meat and dairy) and decrease the environmental burden, while enhancing our understanding of the fundamental processes required for successful implantation in cattle.

Lactating dairy cows have reduced chances for pregnancy survival following 1st parturition. It appears that timing of conceptus attachment, defined as an initial daily rise in pregnancy-specific protein B (PSPB) postartificial insemination (AI), is key to the survival of the embryo. PSPB is a protein produced from binucleate cells of the trophectoderm of the conceptus. Once the conceptus has attached to the uterine epithelial cells, this large protein is detectable in maternal circulation. Daily sampling of serum allows for detection of the initial increase of PSPB. A percent increase threshold for PSPB was determined from results from each study using a conservative three-day minimum increase. Thus far, a 10 or 12.5% daily increase for three consecutive days had the greatest sensitivities and specificities for studies reported in this review. Nulliparous heifers receiving AI following estrus appear to have a reduced time to conceptus attachment compared to lactating cows that received either Ovsynch or AI following estrus. Increasing progesterone post-AI did not reduce time to conceptus attachment in lactating cows. Multiparous cows treated post-AI with human chorionic gonadotropin had a reduced percent with conceptus attachment in addition to greater time to conceptus attachment. Increased time to conceptus attachment increased the chances of multiparous cows having embryonic death before 35 d post-AI. Concentrations of PSPB are consistently reduced from conceptus attachment until d 28 post-AI in cows with pregnancy loss compared to cows that maintain pregnancy on d 34 post-AI. This novel model to determine timing of conceptus attachment provides a new perspective on percent of cows pregnant around d 20–23 post-AI and the potential for a greater understanding of subsequent pregnancy loss.

During initial maternal recognition of pregnancy (MRP), the equine embryo displays a series of unique events characterized by rapid blastocyst expansion, secretion of a diverse array of molecules, and transuterine migration to interact with the uterine surface. Up to date, the intricate transcriptome and proteome changes of the embryo underlying these events have not been critically studied in horses. Thus, the objective of this study was to perform an integrative transcriptomic (including mRNA, miRNAs, and other small non‐coding RNAs) and proteomic analysis of embryos collected from days 10 to 13 of gestation. The results revealed dynamic transcriptome profiles with a total of 1311 differentially expressed genes, including 18 microRNAs (miRNAs). Two main profiles for mRNAs and miRNAs were identified, one with higher expression in embryos ≤5 mm and the second with higher expression in embryos ≥7 mm. At the protein level, similar results were obtained, with 259 differentially abundant proteins between small and large embryos. Overall, the findings demonstrated fine‐tuned transcriptomic and proteomic regulations in the developing embryo associated with embryo growth. The identification of specific regulation of mRNAs, proteins, and miRNAs on days 12 and 13 of gestation suggested these molecules as pivotal for embryo development and as involved in MRP, and in establishment of pregnancy in general. In addition, the results revealed new insights into prostaglandin synthesis by the equine embryo, miRNAs and genes potentially involved in modulation of the maternal immune response, regulation of endometrial receptivity and of late implantation in the mare. [ABSTRACT FROM AUTHOR]

Background: Methylphenidate hydrochloride (MPH) is a psychostimulant widely used in the treatment of attention‐deficit hyperactive disorder (ADHD), as well as a performance enhancer, for at least 60 years. Despite the notable effectiveness as a psychostimulant, ADHD is a chronic disorder and has a two‐third chance of accompanying the individual throughout life. Long‐term use of MPH has been associated not only with an increase in the development of neurodegenerative diseases, but it also causes side effects on male fertility in experimental animals. Objectives: To investigate whether methylphenidate poses a risk to sperm DNA structure and to the quality of embryos conceived after treatment during adolescence in rats. Materials and methods: Wistar rats at 38 days of age were treated either with 5 mg/kg body weight of MPH, in a single daily dose for 30 days, via gavage or with distilled water‐only protocol. Levels of oxidative stress in testicular and epididymal tissues were evaluated. Sperm chromatin quality and acrosome integrity was assessed under flow cytometry. From 107 days of age, animals were mated with untreated females. The effects of the paternal contribution at two different embryo development moments—cleavage stage (2.5 days post coitum) and late gestation (20 days post coitum) —were analyzed. Results: MPH caused high levels of sperm DNA damage, which was reflected in 40% of decrease in early embryo quality and a lower number of live pups at 20 dpc. Discussion: The high level of fragmentation seen in the embryos sired from the MPH group is consistent with the poor chromatin structure of the sperm and does not seem to be a result of oxidative stress in the reproductive tissues. Conclusions: The results presented here suggest that the subchronic use of MPH during male prepubertal phase may cause long‐term subfertility and compromise embryo survival. [ABSTRACT FROM AUTHOR]

In mammalian species, pregnancy is a complex process that involves the maternal recognition of pregnancy, implantation, decidualization, placentation, and parturition. The innate immune system is composed of cellular components, such as natural killer cells, neutrophils, monocytes, and macrophages, and effector molecules, such as cytokines, interferons, antimicrobial peptides, and complement components. The innate immune system plays a critical role as the first line of defense against infection or inflammation to maintain homeostasis and activate the adaptive immunity. During pregnancy, innate immune cells and effector molecules act on the regulation of innate immunity for host defense and processes such as embryo development, implantation, and placentation at the maternal-conceptus interface. In this review, we describe the components of the innate immune system and their functions at the maternal-conceptus interface to establish and maintain pregnancy in animal species that form hemochorial- or epitheliochorial-type placentas, including humans, rodents, ruminants, and pigs.

Lactate, an abundant molecule in fetal fluids and blood of mammalian species, is often overlooked as a metabolic waste product generated during pregnancy. Most of the glucose and fructose consumed by ovine conceptuses is converted to lactate, but proteins involved in lactate metabolism and transport have not been investigated. This study characterized total lactate produced by ovine conceptuses throughout gestation, as well as expression of mRNAs and proteins involved in lactate metabolism. Lactate increased in abundance in the uterine lumen during the preimplantation period and was more abundant than pyruvate. The abundance of lactate in allantoic and amniotic fluids increased with advancing days of gestation and most abundant on Day 125 of pregnancy (P < 0.05). Lactate dehydrogenase subunits A (converts pyruvate to lactate) and B (converts lactate to pyruvate) were expressed by conceptuses throughout gestation. Lactate is transported via monocarboxylic acid transporters SLC16A1 and SLC16A3, both of which were expressed by the conceptus throughout gestation. Additionally, the interplacentomal chorioallantois from Day 126 expressed SLC16A1 and SLC16A3 and transported lactate across the tissue. Hydrocarboxylic acid receptor 1 (HCAR1), a receptor for lactate, was localized to the uterine luminal and superficial glandular epithelia of pregnant ewes throughout gestation and conceptus trophectoderm during the peri-implantation period of gestation. These results provide novel insights into the spatiotemporal profiles of enzymes, transporters, and receptor for lactate by ovine conceptuses throughout pregnancy., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Fructose, the most abundant hexose sugar in fetal fluids and the blood of sheep and other ungulates and cetaceans, is synthesized from glucose via the polyol pathway in trophectoderm and chorion. However, the cell-specific and temporal expression of enzymes for the synthesis and metabolism of fructose in sheep conceptuses (embryo and placental membranes) and placentomes has not been characterized. This study characterized key enzymes involved in fructose synthesis and metabolism by ovine conceptuses throughout pregnancy. Day 17 conceptuses expressed mRNAs for the polyol pathway (SORD and AKR1B1) and glucose and fructose metabolism (HK1, HK2, G6PD, OGT, and FBP), but not those required for gluconeogenesis (G6Pase or PCK). Ovine placentomes also expressed mRNAs for SORD, AKR1B1, HK1, and OGT. Fructose can be metabolized via the ketohexokinase (KHK) pathway, and isoforms, KHK-A and KHK-C, were expressed in ovine conceptuses from Day 16 of pregnancy and placentomes during pregnancy in a cell-specific manner. The KHK-A protein was more abundant in the trophectoderm and cotyledons of placentomes, while KHK-C protein was more abundant in the endoderm of Day 16 conceptuses and the chorionic epithelium in placentomes. Expression of KHK mRNAs in placentomes was greatest at Day 30 of pregnancy (P < 0.05), but not different among days later in gestation. These results provide novel insights into the synthesis and metabolism of fructose via the uninhibited KHK pathway in ovine conceptuses to generate ATP via the tricarboxylic cycle, as well as substrates for the pentose cycle, hexosamine biosynthesis pathway, and one-carbon metabolism required for conceptus development throughout pregnancy., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In many species, the insulin-like growth factors (IGF1 and IGF2), their receptors and IGF binding proteins play important roles in preparing the endometrium for implantation, and regulating conceptus growth and development. To determine whether the IGF system may contribute to conceptus-maternal interaction during equine pre-implantation development, we evaluated mRNA expression for IGF system components in conceptuses, and endometrium recovered from pregnant and cycling mares, on days 7, 14, 21 and 28 after ovulation. We also investigated expression of IGF1, IGF2 and their receptors 6 and 11 days after transfer of day 8 embryos to synchronous (day 8) or asynchronous (day 3) recipient mares. Expression of IGF1 and IGF2, IGF1R, IGF2R, INSR and IGFBPs 1, 2, 4 and 5 was evident in endometrium and conceptus membranes during days 7–28. Endometrial IGF2, INSR, IGFBP1 and IGFBP2 expression increased between days 7 and 28 of pregnancy. In conceptus membranes, expression of all IGF system components increased with developmental stage. Immunohistochemistry revealed strong expression of IGF1, IGF2 and IGF1R in both endometrium and conceptus membranes, whereas INSR was highly expressed in endometrium but barely detectable in the conceptus. Finally, a negatively asynchronous uterine environment retarded IGF1, IGF2 and INSR expression in the conceptus, whereas in the endometrium only INSR expression was altered by asynchrony. The presence of IGFs, their receptors and IGFBPs in the endometrium and conceptus during early equine pregnancy, and down-regulation in the conceptus following asynchronous embryo transfer, suggest a role in conceptus-maternal communication during the preparation for implantation.

A major unresolved issue in the cloning of mammals by somatic cell nuclear transfer (SCNT) is the mechanism by which the process fails after embryos are transferred to the uterus of recipients before or during the implantation window. We investigated this problem by using RNA sequencing (RNA-seq) to compare the transcriptomes in cattle conceptuses produced by SCNT and artificial insemination (AI) at day (d) 18 (preimplantation) and d 34 (postimplantation) of gestation. In addition, endometrium was profiled to identify the communication pathways that might be affected by the presence of a cloned conceptus, ultimately leading to mortality before or during the implantation window. At d 18, the effects on the transcriptome associated with SCNT were massive, involving more than 5,000 differentially expressed genes (DEGs). Among them are 121 genes that have embryonic lethal phenotypes in mice, cause defects in trophoblast and placental development, and/or affect conceptus survival in mice. In endometria at d 18,

RNA sequencing reads and isobaric tags for a relative and absolute quantification (iTRAQ)-Based Proteomic Data were used to determine the impact of conceptus presence and preovulatory estradiol concentration on function of the d16 uterus in beef cattle. Conceptuses and endometrial biopsies were collected from the uterine horn ipsilateral to the corpus luteum. Total cellular RNA was extracted from endometrium for RNA sequencing across two lanes of a NovaSeq S2, 2 × 50-bp run. Two independent uterine luminal fluid pools (ULF) were made for each group: highE2/conceptus, highE2/noconceptus, lowE2/conceptus, and lowE2/noconceptus. Peptides were labeled with iTRAQ reagents and analyzed using 2-dimensional liquid chromatography mass spectrometry. Transcript abundances were determined using DESeq2 (FDR 2). Scaffold Q+ was used to quantitate peptide and protein identifications in ULF. Datasets include uterine transcript and protein abundances among highE2/conceptus vs highE2/noconceptus and lowE2/conceptus vs lowE2/noconceptus groups. This information can be useful for further investigating the role of specific transcripts and proteins in the maintenance of early pregnancy in beef cattle. This dataset is related to the article ‘Influence of conceptus presence and preovulatory estradiol exposure on uterine gene transcripts and proteins around maternal recognition of pregnancy in beef cattle’ by E.J. Northrop-Albrecht, J.J.J. Rich, R.A. Cushman, R. Yao, X. Ge, G.A. Perry. Molecular and Cellular Endocrinology.

Maternal recognition of pregnancy (MRP) is a term utilized in mammals to describe pathways in which the conceptus alters the endometrial environment to prevent regression of corpora lutea to ensure continued production of progesterone (P4) required for establishment and maintenance of pregnancy. For nearly 40 years after publication of the endocrine/exocrine theory, conceptus estrogen (E2) was considered the primary maternal recognition signal in the pig. Conceptus production of prostaglandin E2 (PGE2) was also considered to be a major factor in preventing luteolysis. An addition to E2 and PGE2, pig conceptuses produce interleukin 1B2 (IL1B2) and interferons (IFN) delta (IFND) and gamma (IFNG). The present review provides brief history of the discovery of E2, PGs and IFNS which led to research investigating the role of these conceptus secreted factors in establishing and maintaining pregnancy in the pig. The recent utilization of gene editing technology allowed a more direct approach to investigate the in vivo roles of IL1B2, E2, PGE2, AND IFNG for establishment of pregnancy. These studies revealed unknown functions for IFNG and ILB2 in addition to PGE2 and E2. Thus, pregnancy recognition signal is via a servomechanism in requiring sequential effects of P4, E2, IL1B2, PGE2 and IFNG. Results indicate that the original established dogma for the role of conceptus E2 and PGs in MRP is a far too simplified model that involves the interplay of numerous mechanisms for inhibiting luteolysis, inducing critical elongation of the conceptuses and resolution of inflammation in pigs. • Review of the history of estrogen, prostaglandin and interferon discovery and function. • Models for maternal recognition of pregnancy in the pig. • Role of estrogen and prostaglandin E in CL maintenance in the pig. • Utilization of conceptus gene editing to establish the role conceptus factors in establishment of pregnancy. [ABSTRACT FROM AUTHOR]

In ruminants, RNA-sequence analyses have revealed many characteristics of transcripts expressed in conceptuses (embryo and extraembryonic membrane) during peri-implantation periods; however, lncRNA profiles are yet characterized. In this study, we aimed to characterize the lncRNA expression profile in conceptuses during peri-implantation periods in sheep. We analyzed the RNA-sequence data of ovine conceptuses and endometria obtained from pregnant animals on days 15, 17, 19 and 21 (day 0 = day of estrus, n = 3 or 4/day). We predicted the protein coding ability of the assembled transcripts to identify the lncRNA candidates. This analysis identified 8808 lncRNAs, 3423 of which were novel lncRNAs. Gene ontology analysis revealed that lncRNA target genes were enriched for biological processes involved in the respiratory electron transport chain (RETC). qPCR analysis demonstrated that the expression levels on transcripts encoding RETC such as mitochondrially encoded cytochrome c oxidase II (MTCO2) and mitochondria DNA copy number in conceptuses were not increased on P21, although western blotting analysis and immunohistochemistry demonstrated that MTCO2 protein in conceptuses was increased on P21. NAD/NADH assay revealed that NADH level in conceptuses was increased on P21. These results indicate that lncRNAs could regulate the RETC through post-transcriptional levels in the conceptuses. Therefore, lncRNA is a potential new regulator in ovine conceptus development during peri-implantation periods. • lncRNAs play a role in ovine conceptus development and endometrial function. • Our analyses identified 8808 lncRNAs in ovine conceptuses and endometria. • The lncRNA target genes were enriched in the respiratory electron transport chain. • MTCO2 protein was increased on pregnant day 21 after conceptus attachment. [ABSTRACT FROM AUTHOR]

Horse is a seasonal breeder with reproductive activity being associated with long days, i.e. occurring in spring and early summer. Early equine pregnancy shares many features with that of more intensively assessed domestic animals species, but there are also characteristic differences. In mares, early diagnosis of pregnancy can be done due to the spherical shape. The ultrasonic visualization of conceptus is possible from day 9 of equine pregnancy. In spite of numerous, substantial advances in equine reproduction, many stages of embryonic and foetal morphological development are poorly understood, with no apparent single source of comprehensive information. Hence, in the present study we aimed at studying the dynamics of early embryonic developments in equines, particularly for Marwari breed of horses. The results from the current study revealed that, embryonic vesicles (EV) were first observed on 11th day, fixation occurred after 16.25±0.25 days of ovulation, EV lost spherical shape on 15.83±0.38 days, became oval on 17–18th days and irregular on 19–20th days. Heartbeat, detection of allantoic sac, start of foetal ascent, start of foetal descend and end of foetal descend were detected on 23.91±0.41, 23.58±0.35, 38.66±0.28, 40.66±0.33 and 47.33±0.28 days, respectively. In conclusion, early detection of embryonic vesicles and continuous changes observed in conceptus structure indicate positive pregnancy, viability of embryo and are much beneficial in preventing economical loss of equine owner and stable farm.

Summary: Serial ultrasound examinations of the gravid uterus of a Jenny donkey (Equus asinus) carrying a transferred extraspecies horse (Equus caballus) conceptus between 11 and 33 days of gestation demonstrated that the embryo died before Day 33 even though the conceptus persisted in the uterus and the horse chorionic girdle invaded the maternal endometrium to form a circle of typically large horse endometrial cups in the right uterine horn. The high concentrations of horse CG secreted by these cups with its higher than usual ratio of FSH‐like to LH‐like biological activities of 1.4 compared to the 0.1 ratio of normal donkey CG hyper‐stimulated the donkey's ovaries to develop multiple large accessory follicles and resulting secondary corpora lutea which secreted high concentrations of progestagens that persisted in her bloodstream for the next 90 days. This case illustrated that, in all likelihood, girdle invasion can happen after the death of the embryo indicating autonomy of the chorionic girdle. [ABSTRACT FROM AUTHOR]

The mouse placenta accumulates and possibly produces serotonin (5-hydroxytryptamine; 5-HT) in parietal trophoblast giant cells (pTGC) located at the interface between the placenta and maternal deciduum. However, the roles of 5-HT in placental function are unclear. This lack of information is unfortunate, given that selective serotonin-reuptake inhibitors are commonly used to combat depression in pregnant women. The high affinity 5-HT transporter SLC6A4 (also known as SERT) is the target of such drugs and likely controls much of 5-HT uptake into pTGC and other placental cells. We hypothesized that ablation of the Slc6a4 gene would result in morphological changes correlated with placental gene expression changes, especially for those involved in nutrient acquisition and metabolism, and thereby, provide insights into 5-HT placental function. Placentas were collected at embryonic age (E) 12.5 from Slc6a4 knockout (KO) and wild-type (WT) conceptuses. Histological analyses, RNAseq, qPCR, and integrative correlation analyses were performed. Slc6a4 KO placentas had a considerable increased pTGC to spongiotrophoblast area ratio relative to WT placentas and significantly elevated expression of genes associated with intestinal functions, including nutrient sensing, uptake, and catabolism, and blood clotting. Integrative correlation analyses revealed upregulation of many of these genes was correlated with pTGC layer expansion. One other key gene was dopa decarboxylase (Ddc), which catalyzes conversion of L-5-hydroxytryptophan to 5-HT. Our studies possibly suggest a new paradigm relating to how 5-HT operates in the placenta, namely as a factor regulating metabolic functions and blood coagulation. We further suggest that pTGC might be functional analogs of enterochromaffin 5-HT-positive cells of the intestinal mucosa, which regulate similar activities within the gut. Further work, including proteomics and metabolomic studies, are needed to buttress our hypothesis. • Parietal trophoblast giant cells (pTGC) accrue and/or produce serotonin (5-HT). • Ablation of Slc6a4 , the 5-HT transporter, causes expansion of the pTGC layer. • Slc6a4 KO placentas display elevation of genes linked to metabolite transport. • Placental 5-HT may have a broad regulatory role unrelated to neurotransmitter function. [ABSTRACT FROM AUTHOR]

The present study aimed to investigate the conceptus-related changes during early pregnancy in the Black Bengal breed of goat. A total of 22 gravid genitalia of the Black Bengal goats were collected from local slaughterhouses. The crown-rump lengths (CRL) of the conceptuses were determined to estimate the gestational age (GA). The length and diameter of uterine horn and amniotic sac were measured, and volume of amniotic and allantoic fluid formed by individual conceptus were recorded. The results reveal that the CRL is positively correlated with GA of the conceptus (R2 = 0.89, p < 0.05); however, CRL was not influenced by number of conceptus. Both the left and the right uterine horn gradually increased in size with the advancement of pregnancy irrespective of conceptus number present in the horn. The size of the amniotic sac of conceptus gradually increased with the conceptus age but maintained spherical shape from 5 to 7 weeks of pregnancy. The amniotic fluid formed by individual conceptus rapidly increased from 5 weeks (3.4 ± 0.3 mL) to 7 weeks (21.0 ± 2.0 mL) and 9 weeks (111.5 ± 4.0 mL). The volume of allantoic fluid formed by individual conceptus was steadily increased until 7 weeks (60.0 ± 5.0 mL) and began to decline slowly thereafter (50.0 ± 5.0 mL at 9 weeks). Notably, there was no effect of conceptus number per pregnancy on individual amniotic and allantoic fluid volume. The cotyledons have first appeared on the allanto-chorionic surface from 4 to 5 weeks of pregnancy. The closed eye, nostril and hooves of the conceptus became visible at 7 weeks of pregnancy. The present study has shown the basic information on conceptus-related developmental changes during early pregnancy up to 9 weeks in Black Bengal goat.

Abstract Background Implantation failure limits the success of in vitro fertilization and embryo transfer (IVF-ET). Well-organized embryo-maternal crosstalk is essential for successful implantation. Previous studies mainly focused on the aberrant development of in vitro fertilized (IVF) embryos. In contrast, the mechanism of IVF-induced aberrant embryo-maternal crosstalk is not well defined. Results In the present study, using ewes as the model, we profiled the proteome that features aberrant IVF embryo-maternal crosstalk following IVF-ET. By comparing in vivo (IVO) and IVF conceptuses, as well as matched endometrial caruncular (C) and intercaruncular (IC) areas, we filtered out 207, 295, and 403 differentially expressed proteins (DEPs) in each comparison. Proteome functional analysis showed that the IVF conceptuses were characterized by the increased abundance of energy metabolism and proliferation-related proteins, and the decreased abundance of methyl metabolism-related proteins. In addition, IVF endometrial C areas showed the decreased abundance of endometrial remodeling and redox homeostasis-related proteins; while IC areas displayed the aberrant abundance of protein homeostasis and extracellular matrix (ECM) interaction-related proteins. Based on these observations, we propose a model depicting the disrupted embryo-maternal crosstalk following IVF-ET: Aberrant energy metabolism and redox homeostasis of IVF embryos, might lead to an aberrant endometrial response to conceptus-derived pregnancy signals, thus impairing maternal receptivity. In turn, the suboptimal uterine environment might stimulate a compensation effect of the IVF conceptuses, which was revealed as enhanced energy metabolism and over-proliferation. Conclusion Systematic proteomic profiling provides insights to understand the mechanisms that underlie the aberrant IVF embryo-maternal crosstalk. This might be helpful to develop practical strategies to prevent implantation failure following IVF-ET.

Conceptus estrogens and prostaglandins have long been considered the primary signals for maternal recognition of pregnancy (MRP) in the pig. However, loss-of-function studies targeting conceptus aromatase genes (CYP19A1 and CYP19A2) and prostaglandin-endoperoxide synthase 2 (PTGS2) indicated that conceptuses can not only signal MRP without estrogens or prostaglandins but can maintain early pregnancy. However, complete loss of estrogen production leads to abortion after day 25 of gestation. Although neither conceptus estrogens nor prostaglandins had a significant effect on early maintenance of CL function alone, the two conceptus factors have a biological relationship. To investigate the role that both conceptus estrogens and prostaglandins have on MRP and maintenance of pregnancy, a triple loss-of function model (TKO) was generated for conceptus CYP19A1, CYP19A2 and PTGS2. In addition, a conceptus CYP19A2-/- model (A2KO) was established to determine the role of placental estrogen during later pregnancy. Estrogen and prostaglandin synthesis were greatly reduced in TKO conceptuses which resulted in a failure to inhibit luteolysis after day 15 of pregnancy despite the presence of conceptuses in the uterine lumen. However, A2KO placentae not only maintained functional CL but were able to maintain pregnancy to day 32 of gestation. Despite the loss of placental CYP19A2 expression, the allantois fluid content of estrogen was not affected as the placenta compensated by expressing CYP19A1 and CYP19A3, which are normally absent in controls. Results suggest conceptuses can signal MRP through production of conceptus PGE or stimulating PGE synthesis from the endometrium through conceptus estrogen. Failure of conceptuses to produce both factors results in failure of MRP and loss of pregnancy., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In pigs, the majority of embryonic mortality occurs when free-floating conceptuses (embryos/fetuses and associated placental membranes) elongate and the uterine-placental interface undergoes folding and develops areolae. Both periods involve proliferation, migration, and changes in morphology of cells that require ATP. We hypothesize that insufficient ATP in conceptus and uterine tissues contributes to conceptus loss in pigs. Creatine is stored in cells as phosphocreatine (PCr) for ATP regeneration through the creatine (Cr)-creatine kinase (CK)-PCr pathway. However, the expression of components of this pathway in pigs has not been examined throughout gestation. Results of qPCR analyses indicated increases in AGAT, GAMT, CKM, CKB, and SLC6A8 mRNAs in elongating porcine conceptuses and immunofluorescence microscopy localized GAMT, CKM, and CKB proteins to the trophectoderm of elongating conceptuses, to the columnar chorionic epithelial cells at the bottom of chorioallantoic troughs, and to endometrial luminal epithelium (LE) at the tops of the endometrial ridges of uterine-placental folds on Days 40, 60, and 90 of gestation. GAMT protein is expressed in endometrial LE at the uterine-placental interface, but immunostaining is more intense in LE at the bottoms of the endometrial ridges. Results of this study indicate that key elements of the pathway for creatine metabolism are expressed in cells of the conceptus, placenta, and uterus for potential production of ATP during two timepoints in pregnancy with a high demand for energy; elongation of the conceptus for implantation and development of uterine-placental folding during placentation., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Abstract Background Embryonic and fetal exposure to maternal obesity causes several maladaptive morphological and epigenetic changes in exposed offspring. The timing of these events is unclear, but changes can be observed even after a short exposure to maternal obesity around the time of conception. The hypothesis of this work is that maternal obesity influences the ovine preimplantation conceptus early in pregnancy, and this exposure will affect gene expression in embryonic and extraembryonic tissues. Results Obese and lean ewe groups were established by overfeeding or normal feeding, respectively. Ewes were then bred to genetically similar rams. Conceptuses were collected at day 14 of gestation. Morphological assessments were made, conceptuses were sexed by genomic PCR analysis, and samples underwent RNA-sequencing analysis. While no obvious morphological differences existed between conceptuses, differentially expressed genes (≥ 2-fold; ≥ 0.2 RPKM; ≤ 0.05 FDR) were detected based on maternal obesity exposure (n = 21). Also, differential effects of maternal obesity were noted on each conceptus sex (n = 347). A large portion of differentially expressed genes were associated with embryogenesis and placental development. Conclusions Findings reveal that the preimplantation ovine conceptus genome responds to maternal obesity in a sex-dependent manner. The sexual dimorphism in response to the maternal environment coupled with changes in placental gene expression may explain aberrations in phenotype observed in offspring derived from obese females.

Prenatal mortality remains a significant concern to the pig farming industry around the world. Spontaneous fetal loss ranging from 20 to 45% by term occur after fertilization, with most of the loss happening during the implantation period. Since the factors regulating the high mortality rates of early conceptus during implantation phases are poorly understood, we sought to analyze the overall gene expression changes during this period, and identify the molecular mechanisms involved in conceptus development. This work employed Illumina’s next-generation sequencing (RNA-Seq) and quantitative real-time PCR to analyze differentially expressed genes (DEGs). Soft clustering was subsequently used for the cluster analysis of gene expression. We identified 8236 DEGs in porcine conceptus at day 9, 12, and 15 of pregnancy. Annotation analysis of these genes revealed rRNA processing (GO:0006364), cell adhesion (GO:1904874), and heart development (GO:0007507), as the most significantly enriched biological processes at day 9, 12, and 15 of pregnancy, respectively. In addition, we found various genes, such as T-complex 1, RuvB-like AAA ATPase 2, connective tissue growth factor, integrins, interferon gamma, SLA-1, chemokine ligand 9, PAG-2, transforming growth factor beta receptor 1, and Annexin A2, that play essential roles in conceptus morphological development and implantation in pigs. Furthermore, we investigated the function of PAG-2 in vitro and found that PAG-2 can inhibit trophoblast cell proliferation and migration. Our analysis provides a valuable resource for understanding the mechanisms of conceptus development and implantation in pigs.

Prenatal mortality remains a significant concern to the pig farming industry around the world. Spontaneous fetal loss ranging from 20 to 45% by term occur after fertilization, with most of the loss happening during the implantation period. Since the factors regulating the high mortality rates of early conceptus during implantation phases are poorly understood, we sought to analyze the overall gene expression changes during this period, and identify the molecular mechanisms involved in conceptus development. This work employed Illumina's next-generation sequencing (RNA-Seq) and quantitative real-time PCR to analyze differentially expressed genes (DEGs). Soft clustering was subsequently used for the cluster analysis of gene expression. We identified 8236 DEGs in porcine conceptus at day 9, 12, and 15 of pregnancy. Annotation analysis of these genes revealed rRNA processing (GO:0006364), cell adhesion (GO:1904874), and heart development (GO:0007507), as the most significantly enriched biological processes at day 9, 12, and 15 of pregnancy, respectively. In addition, we found various genes, such as T-complex 1, RuvB-like AAA ATPase 2, connective tissue growth factor, integrins, interferon gamma, SLA-1, chemokine ligand 9, PAG-2, transforming growth factor beta receptor 1, and Annexin A2, that play essential roles in conceptus morphological development and implantation in pigs. Furthermore, we investigated the function of PAG-2 in vitro and found that PAG-2 can inhibit trophoblast cell proliferation and migration. Our analysis provides a valuable resource for understanding the mechanisms of conceptus development and implantation in pigs. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the intra‐ and interovarian relationships among the corpus luteum (CL), the largest follicle (LF) and follicular population in non‐pregnant and between the conceptus and ovarian structures in pregnant ewes. In experiment 1, the follicular and luteal structures were examined in 538 reproductive systems of non‐pregnant Awassi ewes. The follicular population was categorised into small (SF), medium (MF) and large (LF) groups. Inter‐relationships between CL and follicular population and between LF and subordinate follicles were determined. In experiment 2, the location and number of conceptuses were identified and correlated with the ovarian structures in 58 reproductive systems of pregnant ewes. Effects of pregnancy status, stage of pregnancy, pregnancy side and conceptual number on follicular population were determined. The results showed that the right ovary was more active than the left ovary. CL had intraovarian positive effect on the number of medium and large follicles. LF had no local suppressive effect on the subordinate follicles. Side and stage of pregnancy and the conceptual number did not affect the follicular population. Accordingly, it can be concluded that the LF has no local suppressive effect on the subordinate follicles. The CL has intraovarian positive effect on the follicular population. Follicular population does not show remarkable changes during the first term of pregnancy. The present study probably provides information which may help in the understanding of the ovarian dynamics during pregnancy in sheep. [ABSTRACT FROM AUTHOR]

A central determinant of pregnancy success is proper development of the conceptus (embryo/fetus and associated extraembryonic membranes including the placenta). Although the gross morphology and histology of the bovine placenta have been well studied, the cellular and molecular mechanisms regulating placenta development and trophoblast differentiation and function remain essentially undefined. Here, single-cell transcriptome (scRNA-seq) analysis was performed on the day 17 bovine conceptus and chorion of day 24, 30, and 50 conceptuses (n = 3-4 samples per day) using the 10X Genomics platform. Bioinformatic analyses identified cell types and their ontogeny including trophoblast, mesenchyme, and immune cells. Loss of interferon tau-expressing trophoblast uninucleate cells occurred between days 17 and 30, whereas binucleate cells, identified based on expression of placental lactogen (CSH2) and specific pregnancy-associated glycoprotein genes (PAGs), first appeared on day 24. Several different types of uninucleate cells were present in day 24, 30, and 50 samples, but only one (day 24) or two types of binucleate cells (days 30 and 50). Cell trajectory analyses provided a conceptual framework for uninucleate cell development and binucleate cell differentiation, and bioinformatic analyses identified candidate transcription factors governing differentiation and function of the trophoblasts. The digital atlas of cell types in the developing bovine conceptus reported here serves as a resource to discover key genes and biological pathways regulating its development during the critical periods of implantation and placentation., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Interferon-τ (IFNT), IFN stimulated genes (ISG15, CTSL1, RSAD2, SLC2A1, CXCL10, and SLC27A6), Peroxisome proliferator activated receptors (PPARA, D, and G), Retinoic acid receptors (RXRA, B, and G), and Mucin-1 (MUC1) play decisive roles in embryo elongation. The objective was to elucidate expressions of these genes in day 16 embryo [tubular (n = 4) vs. filamentous (n = 4)] and corresponding endometrium [without (n = 4) vs. with subclinical endometritis (SCE; n = 4)] of repeat breeder Holstein cows (2 × 2 factorial design). Results showed that the mRNA abundances (except PPARA and RXRB) were greater (P < 0.05) in filamentous embryo and endometrium without SCE compared with tubular embryo and endometrium with SCE, respectively. Overall, the mRNA abundances (except RSAD2, PPARA and RXRA) in filamentous embryo and corresponding endometrium of cows without SCE were greater (P < 0.05) than tubular embryo and corresponding endometrium of cows with SCE. Proteins IFNT, ISGs, PPARs and RXRs (except RXRB) were greater (P < 0.05) and protein MUC1 was lower (P < 0.01) in filamentous embryo and corresponding endometrium of cows without SCE compared to tubular embryo and corresponding endometrium of cows with SCE. On pairwise comparison, mRNA and protein abundances of MUC1 significantly differed between tubular embryo in uterus with or without SCE, and corresponding endometrium with or without SCE (P < 0.05). In conclusion, the mRNA and protein abundances of IFNT, ISG15, CXCL10, PPARG and MUC1 differed among filamentous and tubular conceptuses, and endometrium with or without SCE of repeat breeder cows on Day 16, indicating that these genes and their downstream signaling cascades play important roles in embryo elongation. Perhaps, interruptions in cross-talk between endometrium and conceptus impaired conceptus elongation in repeat breeder cows with SCE. In addition to disrupted signaling, the tubular conceptus (compared to filamentous conceptus) was unable to downregulate MUC1 (anti-adhesive glycocalyx) in repeat breeder cows with or without SCE, resulting in early embryonic demise. [ABSTRACT FROM AUTHOR]

The implantation process requires precisely controlled interactions between the maternal uterine endometrium and the implanting conceptus. Conceptus-derived secretions affect endometrial cells to facilitate the adhesion and attachment of trophoblasts, and endometrial secretions support the growth and development of the conceptus. In pigs, the conceptus secretes a large amount of type II interferon, interferon-γ (IFNG), during the implantation period. However, the role of IFNG in the implantation process has not been fully understood in pigs. Thus, to determine the role of IFNG in the endometrium during early pregnancy in pigs, we treated endometrial explant tissues with increasing doses of IFNG and analyzed the transcriptome regulated by IFNG using an RNA-sequencing analysis. Data analyses identified 276 differentially regulated genes, their Gene Ontology terms, and 94 signature genes in a Gene Set Enrichment Analysis. Furthermore, we analyzed the expression of IFNG-regulated genes, including CIITA , KYNU , IDO1 , WARS , and MHC class II molecules, in the endometrium throughout pregnancy and found that levels of those genes in the endometrium were highest on Day 15 of pregnancy, corresponding to the time of peak IFNG secretion by porcine conceptuses. In addition, immunohistochemical analyses revealed that CIITA, KYNU, and IDO proteins were expressed in a cell type- and pregnancy status-specific manner in the endometrium. These results show that genes overrepresented in endometrial tissues in response to IFNG were mainly related to immune responses, suggesting that conceptus-derived IFNG could play critical roles in regulating the maternal immune response for the establishment of pregnancy in pigs. • Increasing doses of IFNG induced the expression of many genes in endometrial tissue. • The IFNG-regulated DEGs related mostly to immune response in the KEGG pathway and GO enrichment analyses. • GSEA revealed that IFNG response was the most abundant and significant enrichment mechanism. • The expression of the select IFNG-regulated DEGs was greatest on Day 15 of pregnancy in the endometrium. • The expression of the select IFNG-regulated DEGs was localized in the endometrium in a pregnancy stage- and cell type-specific fashion. Summary: IFNG of conceptus origin induces the expression of many immune-related genes in the endometrium during early pregnancy in pigs. [ABSTRACT FROM AUTHOR]

Abstract The establishment of pregnancy is a complex process that requires a well-coordinated interaction between the implanting conceptus and the maternal uterus. In pigs, the conceptus undergoes dramatic morphological and functional changes at the time of implantation and introduces various factors, including estrogens and cytokines, interleukin-1β2 (IL1B2), interferon-γ (IFNG), and IFN-δ (IFND), into the uterine lumen. In response to ovarian steroid hormones and conceptus-derived factors, the uterine endometrium becomes receptive to the implanting conceptus by changing its expression of cell adhesion molecules, secretory activity, and immune response. Conceptus-derived estrogens act as a signal for maternal recognition of pregnancy by changing the direction of prostaglandin (PG) F2α from the uterine vasculature to the uterine lumen. Estrogens also induce the expression of many endometrial genes, including genes related to growth factors, the synthesis and transport of PGs, and immunity. IL1B2, a pro-inflammatory cytokine, is produced by the elongating conceptus. The direct effect of IL1B2 on endometrial function is not fully understood. IL1B activates the expression of endometrial genes, including the genes involved in IL1B signaling and PG synthesis and transport. In addition, estrogen or IL1B stimulates endometrial expression of IFN signaling molecules, suggesting that estrogen and IL1B act cooperatively in priming the endometrial function of conceptus-produced IFNG and IFND that, in turn, modulate endometrial immune response during early pregnancy. This review addresses information about maternal-conceptus interactions with respect to endometrial gene expression in response to conceptus-derived factors, focusing on the roles of estrogen and IL1B during early pregnancy in pigs.

This review focuses on extracellular vesicles (EV) in the uterus and their potential biological roles as mediators of conceptus-uterine interactions essential for implantation and pregnancy establishment. Growing evidence supports the idea that EV are produced by both the endometrium and conceptus during pregnancy. Exosomes and microvesicles, collectively termed EV, mediate cell-cell communication in other tissues and organs. EV have distinct cargo, including lipids, proteins, RNAs, and DNA, that vary depending on the cell of origin and regulate processes including angiogenesis, adhesion, proliferation, cell survival, inflammation, and immune response in recipient cells. Molecular crosstalk between the endometrial epithelium and the blastocyst/conceptus, particularly the trophectoderm, regulates early pregnancy events and is a prerequisite for successful implantation. Trafficking of EV between the conceptus and endometrium may represent a key form of communication important for pregnancy establishment. Increased understanding of EV in the uterine environment and their physiological roles in endometrial-conceptus interactions is expected to provide opportunities to improve pregnancy success. • Growing evidence supports the idea that EV are produced by both the endometrium and conceptus in women and domestic animals. • Extracellular vesicles (EV), including exosomes and microvesicles, mediate cell-cell communication in other tissues. • EV cargo regulate processes including angiogenesis, adhesion, proliferation, and cell survival in recipient cells. • Molecular crosstalk between the endometrium and the conceptus regulates early pregnancy events and successful implantation. [ABSTRACT FROM AUTHOR]