Embryos generated from oocytes lacking complex N- and O-glycans have compromised development and implantation

Female mice generating oocytes lacking complexN- andO-glycans (double mutants (DM)) produce only one small litter before undergoing premature ovarian failure (POF) by 3 months. Here we investigate the basis of the small litter by evaluating ovulation rate and embryo development in DM (Mgat1F/FC1galt1F/F:ZP3Cre) and Control (Mgat1F/FC1galt1F/F) females. Surprisingly, DM ovulation rate was normal at 6 weeks, but declined dramatically by 9 weeks.In vitrodevelopment of zygotes to blastocysts was equivalent to Controls although all embryos from DM females lacked a normal zona pellucida (ZP) and ∼30% lacked a ZP entirely. In contrast,in vivopreimplantation development resulted in less embryos recovered from DM females compared with Controls at 3.5 days post coitum (dpc) (3.2±1.3 vs 7.0±0.6). Furthermore, only 45% of mated DM females contained embryos at 3.5 dpc. Of the preimplantation embryos collected from DM females, approximately half were morulae unlike Controls where the majority were blastocysts, indicating delayed embryo development in DM females. Post-implantation development in DM females was analysed to determine whether delayed preimplantation development affected subsequent development. In DM females at 5.5 dpc, only ∼40% of embryos found at 3.5 dpc had implanted. However, at 6.5 dpc, implantation sites in DM females corresponded to embryo numbers at 3.5 dpc indicating delayed implantation. At 9.5 dpc, the number of decidua corresponded to embryo numbers 6 days earlier indicating that all implanted embryos progress to midgestation. Therefore, a lack of complexN- andO-glycans in oocytes during development impairs early embryo development and viabilityin vivoleading to delayed implantation and a small litter.

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293 EFFECT OF GUAIAZULENE ON IN VITRO BOVINE EMBRYO PRODUCTION

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab293 ◽

2007 ◽

Vol 19 (1) ◽

pp. 262 ◽

Cited By ~ 1

Author(s):

I. Dimitriadis ◽

E. A. Rekka ◽

E. Vainas ◽

G. S. Amiridis ◽

C. A. Rekkas

Keyword(s):

Lipid Peroxidation ◽

Embryo Development ◽

Antioxidant Properties ◽

Thiobarbituric Acid ◽

Early Embryo ◽

Bovine Embryo ◽

Embryo Production ◽

Reactive Material

The substrates used in in vitro embryo production (IVP) mimic the in vivo fluids in which oocytes mature, oocytes are fertilized, and the early embryos develop (follicular and oviductal fluid). It is well established that oxidative stress negatively affects in vitro culture (IVC) outcomes. Guaiazulene (G) is a component of chamomile species oil with known antioxidant properties. In the present study, all IVP media were modified by the addition of G solutions so that the former exhibited a total protection against induced lipid peroxidation (TPaLP) similar to that of the respective in vivo environment. The IVP outcomes were then compared between G-processed and control oocytes. Bovine preovulatory follicular (BF) and oviductal (BO) fluid samples were collected from 10 Holstein 4- to 5-year-old cows in estrus. TPaLP was assessed according to the samples' ability to inhibit rat hepatic microsomal lipid peroxidation, by determination of the 2-thiobarbituric acid reactive material. TPaLP (mean % � SEM) of the BF and BO were 70.63 � 10.03 and 16.33 � 4.33, respectively, whereas those of the IVP [in vitro-matured (IVM), in vitro-fertilized (IVF), and IVC] media were lower (17.94 � 1.66, -1.82 � 0.78, and 14.57 � 1.26, respectively). TPaLP of the 0.1 mM G-modified IVP medium increased to 67.2 � 5.85, 19.98 � 2.49, and 69.19 � 6.22, respectively. A total of 2041 class A oocytes were used. The proportion of cleavage, early embryo development (embryos with more than 4 cells), or both after IVP (18 h IVM–5% CO2 in air, and 18 h IVF, 48 h IVC–5% CO2, 10% O2, 85% N) in the presence of G (n = 1237) during each of the IVP phases or any possible combination of IVP phases was compared with the respective control (C, n = 804). Statistical analysis was performed by a chi-squared test; P < 0.05 was considered significant. G improved cleavage and embryo development rates when present during IVM (79.4 and 57.8% vs. 64.5 and 38.2% for C) or both IVM and IVC (78.0 and 60.7% vs. 57.8 and 36.5%, respectively). When present only during 18 h of IVF, G had no effect on embryo production. However, an increased embryo development rate resulted from the combined exposure to G during IVF and IVM (56.4 vs. 29.6%), during IVF and IVC (55.3 vs. 35.5%), or at all IVP phases (56.6 vs. 34.9%). The latter effect resembled the one obtained after G addition only to the IVC medium (62.5 vs. 39.7%, respectively). We concluded that the addition of G to IVP substrates, at concentrations that mimic the in vivo TPaLP conditions, could promote bovine IVP efficiency.

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Oviductal response to gametes and early embryos in mammals

Reproduction ◽

10.1530/rep-16-0120 ◽

2016 ◽

Vol 152 (4) ◽

pp. R127-R141 ◽

Cited By ~ 34

Author(s):

Veronica Maillo ◽

Maria Jesus Sánchez-Calabuig ◽

Ricaurte Lopera-Vasquez ◽

Meriem Hamdi ◽

Alfonso Gutierrez-Adan ◽

...

Keyword(s):

Embryo Development ◽

Reproductive Tract ◽

Early Embryo ◽

Secretory Cells ◽

Oocyte Fertilization ◽

Early Embryos ◽

Significant Research ◽

Oviductal Fluid

The oviduct is a complex and organized thin tubular structure connecting the ovary with the uterus. It is the site of final sperm capacitation, oocyte fertilization and, in most species, the first 3–4days of early embryo development. The oviductal epithelium is made up of ciliary and secretory cells responsible for the secretion of proteins and other factors which contribute to the formation of the oviductal fluid. Despite significant research, most of the pathways and oviductal factors implicated in the crosstalk between gametes/early embryo and the oviduct remain unknown. Therefore, studying the oviductal environment is crucial to improve our understanding of the regulatory mechanisms controlling fertilization and embryo development. In vitro systems are a valuable tool to study in vivo pathways and mechanisms, particularly those in the oviducts which in livestock species are challenging to access. In studies of gamete and embryo interaction with the reproductive tract, oviductal epithelial cells, oviductal fluid and microvesicles co-cultured with gametes/embryos represent the most appropriate in vitro models to mimic the physiological conditions in vivo.

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mRNA Expression and Role of PPARγ and PPARδ in Bovine Preimplantation Embryos Depending on the Quality and Developmental Stage

Animals ◽

10.3390/ani10122358 ◽

2020 ◽

Vol 10 (12) ◽

pp. 2358

Author(s):

Katarzyna Suwik ◽

Emilia Sinderewicz ◽

Dorota Boruszewska ◽

Ilona Kowalczyk-Zięba ◽

Joanna Staszkiewicz-Chodor ◽

...

Keyword(s):

Early Development ◽

Embryo Development ◽

Embryo Quality ◽

Early Embryo ◽

Developmental Competence ◽

Preimplantation Embryos ◽

Peroxisome Proliferator ◽

Early Embryo Development ◽

Quality Markers

Peroxisome proliferator-activated receptors (PPARs), a nuclear receptors for prostacyclin (PGI2) have been recognized as being essential for early embryo development. The objectives of the present study were to determine if the bovine early- and late-cleaved embryos in different stages of early development express PPARγ and PPARδ. Since embryo developmental competence depends on numerous biological factors, we evaluated if the expression of PPARγ and PPARδ correlate with selected embryo quality markers (SOX2, OCT4, PLAC8, IGF1R) in the in vitro produced embryos at different stages of their development. Developmental rates and embryo quality for early- and late-cleaved embryos were provided according to International Embryo Transfer Society (IETS; developmental stages: 2-, 4-, 16-cell embryo, morula, blastocyst (1—early, 2—developing, 3—expanded, 4—hatched); quality stages: A—high quality, B—moderate quality, C—low quality). We found that bovine embryos expressed mRNA of PPARδ and PPARγ at all stages of early development, independently of their quality. In addition, the expression of PPARδ and PPARγ correlated with the expression of quality markers in bovine blastocysts. Positive correlations were stronger and more frequent in the group of early-cleaved embryos, whereas the negative correlations were typical for the group of late-cleaved embryos. Obtained results and available literature reports may indicate the participation of PGI2, via PPARδ and PPARγ, in the processes related to the early embryo development, through the participation of this factor in the modulation of blastocyst hatching, implantation, and post-implantation development.

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204 RELATIVE MESSENGER RNA ABUNDANCE OF HYALURONAN RECEPTORS AND SYNTHASES ON IN VITRO- AND IN VIVO-DERIVED DAY 7 AND DAY 13 BOVINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab204 ◽

2009 ◽

Vol 21 (1) ◽

pp. 200

Author(s):

M. Clemente ◽

A. T. Palasz ◽

J. de La Fuente ◽

P. Lonergan ◽

A. Gutierrez-Adan ◽

...

Keyword(s):

Embryo Development ◽

Messenger Rna ◽

Developmental Stages ◽

Transcript Abundance ◽

Early Embryo ◽

Bovine Embryos ◽

Cd44 Receptor ◽

Mrna Extraction

Hyaluronan (HA), which progressively increases during embryogenesis, is a glycosaminoglycan that plays a major role in oocyte/embryo development (Fenderson et al. 1993 Differentiation 54, 85–95). One of the main functions of HA is to participate in the cell proliferation and migration that are controlled by HA receptors, RHAMM and C44, and by the presence of different HA synthases, Has1, Has2, and Has3. All have very distinctive features and functions at different stages of embryo development. The objective of this study was to determine the relative mRNA abundance of HA receptors and synthases in Day 7 and 13 bovine embryos derived in vitro or in vivo. In vitro embryos were produced by standard oocyte maturation and fertilization procedures. Presumptive zygotes were cultured in groups of 25 in 25-μL droplets of synthetic oviduct fluid supplemented with 5% FCS at 39°C, 5% CO2, and 5%O2 with maximum humidity. In vivo blastocysts were collected from superovulated heifers on Day 7 (estrus = Day 0) by uterine flushing and on Day 13 immediately after slaughter by flushing the dissected reproductive tracts. All embryos were frozen in LN2 and stored at –80°C for mRNA extraction. Quantification of transcripts for RHAMM and CD44 receptors and Has2 and Has3 synthases was performed on groups of ten Day 7 blastocysts (3 groups for in vivo or in vitro) and individual Day 13 embryos (7 embryos in vivo or in vitro) by real-time quantitative RT-PCR. Data on differences in transcript abundance were analyzed by ANOVA. The relative abundance of the Has2 and Has3 synthases was similar between in vivo and in vitro embryos, irrespective of their developmental stage. The quantity of CD44 was significantly higher in in vitro compared with in vivo embryos only on Day 7. However, the quantity of RHAMM receptor was higher on Day 13 in in vitro compared with in vivo embryos. When the comparison was done between developmental stages (Day 7 v. Day 13) for in vivo and in vitro embryos, we found that in vivo-produced Day 7 blastocysts expressed significantly more RHAMM receptor than embryos on Day 13. The reverse pattern of expression was shown for CD44 receptor. For in vitro embryos, the only difference observed was for Has3, which was up-regulated on Day 13 compared with Day 7 embryos. In conclusion, the present study demonstrates, for the first time, developmental changes in the abundance of RHAMM and CD44 receptor mRNA and Has2 and Has3 synthase mRNA in in vivo and in vitro bovine-derived embryos on Day 7 and 13. We believe that our results will provide new insight into the potential role of this intriguing multifunctional molecule in bovine early embryo development.

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313 PRE-IN VITRO MATURATION WITH CYCLIC AMP AND CYCLIC GMP MODULATORS IMPROVES DEVELOPMENTAL COMPETENCE OF MOUSE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab313 ◽

2015 ◽

Vol 27 (1) ◽

pp. 245 ◽

Cited By ~ 1

Author(s):

N. W. Santiquet ◽

A. F. Greene ◽

W. B. Schoolcraft ◽

R. L. Krisher

Keyword(s):

Embryo Development ◽

In Vitro Maturation ◽

Subsequent Development ◽

Developmental Competence ◽

Nuclear Maturation ◽

Oocyte Developmental Competence ◽

Oocyte Collection ◽

Short Period

In vitro maturation (IVM) of cumulus-oocyte complexes (COC) results in oocytes with reduced quality and is still not as efficient as in vivo maturation in most species. One hypothesis that could explain the low developmental competence of oocytes following IVM is that the oocytes resume meiosis too quickly after being retrieved from the follicles. Studies in mice and bovine have shown that a short period of prematuration in the presence of cAMP modulators, before IVM, enhances oocyte developmental competence. Moreover, other studies have recently demonstrated that cGMP is also a crucial molecule involved in meiotic resumption. Here, our objective was to examine the effect of a cGMP modulator in combination with a cAMP modulator during a short period of prematuration on mouse oocyte nuclear maturation and subsequent embryo development following IVF. The COC were collected (6 replicates) from 2-month-old outbred CF1 mice 48 h after PMSG (5 IU) injection in the presence (pre-IVM) or absence (control) of cGMP and cAMP modulators. Pre-IVM COC (n = 184) were then placed in prematuration medium that also contained these cGMP and cAMP modulators. After 2 h, pre-IVM COC were washed and transferred to our in-house prepared, completely defined IVM medium (Paczkowski et al. 2014 Reprod.) for the remaining 16 h of culture; 10 oocytes per 50 µL drop under oil, at 37°C in 7.5% CO2 and 6.5% O2 due to the increased altitude at our location. Control COC (n = 161) were matured in the same IVM medium under identical conditions for 18 h, without prematuration. After IVM, oocytes were fixed for assessment of nuclear maturation, or fertilized and cultured in vitro and subsequent development (96 and 112 h) was recorded (Paczkowski et al. 2014 Reprod.). Results were analysed by ANOVA. A short 2-h prematuration period in the presence of cGMP and cAMP modulators had no impact on oocyte nuclear maturation to metaphase II after IVM or on embryo cleavage after IVF. However, pre-IVM treatment improved the developmental competence of the oocyte, as demonstrated by increased embryo development. More (P < 0.02) blastocysts (96 h of culture) and hatched blastocysts (112 h of culture) developed in the pre-IVM treatment compared to control (31.0 ± 3.4 v. 19.9 ± 3.2%; 31.5 ± 3.4 v. 19.9 ± 3.2%, respectively). In conclusion, a combination of cGMP and cAMP modulators during oocyte collection and a subsequent short pre-IVM improves oocyte developmental competence and could therefore be a potential tool to improve embryo yield following IVM.

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Identification of a Novel Role for Endothelins within the Oviduct

Endocrinology ◽

10.1210/en.2009-1155 ◽

2010 ◽

Vol 151 (6) ◽

pp. 2858-2867 ◽

Cited By ~ 22

Author(s):

Myoungkun Jeoung ◽

Sungeun Lee ◽

Hee-kyung Hawng ◽

Yong-Pil Cheon ◽

Youn Kyung Jeong ◽

...

Keyword(s):

Epithelial Cells ◽

Embryo Development ◽

Early Embryo ◽

Receptor Subtypes ◽

Early Embryo Development ◽

Vitro Fertilization ◽

Protein Superfamilies ◽

Endothelin 3

Endothelins were first identified as potent vasoactive peptides; however, diversity in the biological function of these hormones is now evident. We have identified a novel role for endothelins: a requirement for these peptides within the oviduct during fertilization and/or early embryo development. In vivo, treatment after ovulation with a dual endothelin receptor antagonist (tezosentan) decreased the number of two-cell embryos that could be collected from within the oviducts. In vitro fertilization experiments showed that gamete viability and their ability to fertilize were not affected by treatment with this antagonist, suggesting that the effect observed in vivo was mediated by the oviduct itself. Expression of mRNA for all three isoforms of the endothelins and both receptor subtypes was detectable within the oviduct. Expression of mRNA for endothelin-3 was regulated by gonadotropins in epithelial cells of the oviduct and increased specifically within the isthmus of this structure. Immunostaining revealed localization of both endothelin receptors A and B to the columnar epithelial cells within the oviduct, suggestive of a local role for endothelins in the regulation of epithelial function and ultimately oviductal secretions. A microarray analysis revealed three likely endothelin-regulated protein networks for future analysis: the TGFβ, IL-10, and CCAAT/enhancer-binding protein superfamilies. Overall, these results suggest a novel and requisite role for endothelins within the oviduct during fertilization and/or early embryo development.

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Early embryonic development of bovine oocytes challenged with LPS in vitro or in vivo

Reproduction ◽

10.1530/rep-19-0316 ◽

2019 ◽

Vol 158 (5) ◽

pp. 453-463

Author(s):

Joao Alveiro Alvarado Rincón ◽

Patricia Carvalho Gindri ◽

Bruna Mion ◽

Ferronato Giuliana de Ávila ◽

Antônio Amaral Barbosa ◽

...

Keyword(s):

Embryonic Development ◽

Embryo Development ◽

Cumulus Cells ◽

Early Embryo ◽

Cleavage Rate ◽

Early Embryo Development ◽

Lps Challenge ◽

Bovine Oocytes

The aim of this study was to evaluate the effect of exposing bovine oocytes to lipopolysaccharides (LPS) in vivo and in vitro on early embryo development. In experiment 1, cumulus oocyte complexes (COCs, n = 700/group) were challenged with 0, 0.1, 1.0 or 5.0 μg/mL of LPS during in vitro maturation (IVM). Later, in vitro fertilization (IVF) and in vitro culture (IVC) were performed. In experiment 2, COCs (n = 200/group) matured and in vitro fertilized without LPS were subjected to IVC with the same doses of LPS from experiment 1. In experiment 3, heifers received two injections of saline solution (n = 8) or 0.5 μg/kg of LPS (n = 8) 24 h apart, and 3 days later, COCs were recovered and submitted to IVM, IVF, and IVC. In experiments 1 and 3, the expression of TLR4, TNF, AREG and EREG genes in cumulus cells was evaluated. Exposure to 1 and 5 μg/mL of LPS during IVM decreased nuclear maturation (39.4 and 39.6%, respectively) compared with control (63.6%, P < 0.05). Despite that, no effect on cleavage and blastocyst rates were observed. Exposure to LPS during IVC did not affect embryonic development. In vivo exposure to LPS decreased the in vitro cleavage rate (54.3 vs 70.2%, P = 0.032), but cleaved embryos developed normally. Number of cells per embryo and gene expression were not affected by the LPS challenge in any experiment. In conclusion, although in vitro exposure to LPS did not affect early embryo development, in vivo LPS exposure reduced cleavage rate.

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Early cleavage of preimplantation embryos is regulated by tRNAGln-TTG–derived small RNAs present in mature spermatozoa

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013003 ◽

2020 ◽

Vol 295 (32) ◽

pp. 10885-10900 ◽

Cited By ~ 1

Author(s):

Xiaoxu Chen ◽

Yi Zheng ◽

Anmin Lei ◽

Hanxue Zhang ◽

Huimin Niu ◽

...

Keyword(s):

Embryo Development ◽

Small Rnas ◽

Early Embryo ◽

Preimplantation Embryos ◽

Rna Seq ◽

Epigenetic Factors ◽

Early Cleavage ◽

Preimplantation Embryo Development ◽

Mature Spermatozoa

tRNA-derived small RNAs (tsRNAs) from spermatozoa could act as acquired epigenetic factors and contribute to offspring phenotypes. However, the roles of specific tsRNAs in early embryo development remain to be elucidated. Here, using pigs as a research model, we probed the tsRNA dynamics during spermatogenesis and sperm maturation and demonstrated the delivery of tsRNAs from semen-derived exosomes to spermatozoa. By microinjection of antisense sequences into in vitro fertilized oocytes and subsequent single-cell RNA-seq of embryos, we identified a specific functional tsRNA group (termed here Gln-TTGs) that participate in the early cleavage of porcine preimplantation embryos, probably by regulating cell cycle–associated genes and retrotransposons. We conclude that specific tsRNAs present in mature spermatozoa play significant roles in preimplantation embryo development.

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Distribution of tubulointerstitial nephritis antigen-like 1 and structural matrix proteins in mouse embryos during preimplantation development in vivo and in vitro

Zygote ◽

10.1017/s0967199412000469 ◽

2012 ◽

Vol 22 (2) ◽

pp. 259-265 ◽

Cited By ~ 6

Author(s):

Masahiro Sakurai ◽

Yusuke Sato ◽

Kuniaki Mukai ◽

Makoto Suematsu ◽

Emiko Fukui ◽

...

Keyword(s):

Tubulointerstitial Nephritis ◽

Preimplantation Development ◽

Matrix Proteins ◽

Preimplantation Embryos ◽

Collagen Type Iv ◽

Matricellular Protein ◽

Ecm Proteins ◽

Structural Matrix

SummaryTubulointerstitial nephritis antigen-like 1 (TINAGL1) is a novel matricellular protein that interacts with structural matrix proteins and promotes cell adhesion and spreading. We have previously reported unique localization of TINAGL1 to the trophectoderm (TE) of mouse blastocysts. TINAGL1 was found to be upregulated in implantation-competent blastocysts after estrogen treatment using progesterone-treated delayed-implantation models. Moreover, colocalization of TINAGL1 and extracellular matrix (ECM) protein laminin 1 was detected in the Reichert membrane on embryonic days 6.5 and 7.5. Although these data suggested a role for TINAGL1 in the embryo development at postimplantation, its relevance to other ECM proteins during preimplantation development is not clear. In this study, we examined the expression of TINAGL1 and its relevance to other ECM proteins fibronectin (FN) and collagen type IV (ColIV) during in vivo development of preimplantation embryos, particularly at blastocyst stage in detail. Localizations of TINAGL1, FN, and ColIV were similar. In 1-cell to 8-cell embryos, they were expressed in cytoplasm of blastomeres, and in morulae they were localized in the outer cells. FN and ColIV were expressed primarily on outer surface of the cells. In blastocysts, FN and ColIV were distributed in the cytoplasm of TE, but, just prior to implantation, they became localized uniquely to the blastocoelic surface of TE. In in vitro fertilized (IVF) blastocysts, expression levels of TINAGL1 and FN were lower than in in vivo blastocysts. These results suggest that, during preimplantation development, TINAGL1 may be involved in roles of structural matrix proteins, whose expression in blastocysts may be affected by in vitro culture.

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Involvement of Nlrp9a/b/c in mouse preimplantation development

Reproduction ◽

10.1530/rep-19-0516 ◽

2020 ◽

Vol 160 (2) ◽

pp. 181-191 ◽

Cited By ~ 2

Author(s):

Satoko Kanzaki ◽

Shiori Tamura ◽

Toshiaki Ito ◽

Mizuki Wakabayashi ◽

Koji Saito ◽

...

Keyword(s):

Asymmetric Cell Division ◽

Culture Conditions ◽

Preimplantation Development ◽

Preimplantation Embryos ◽

Dependent Manner ◽

Blastocyst Development ◽

Triple Mutant ◽

Cell Stage

Nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing proteins (NRLPs) are central components of the inflammasome. Accumulating evidence has shown that a reproductive clade of NRLPs is predominantly expressed in oocyte to cleavage stage embryos and participates in mammalian preimplantation development as a component of a multiprotein complex known as the subcortical maternal complex (SCMC). Nlrp9s belong to the reproductive class of NLRPs; Nlrp9b is unique in acting as an inflammasome against rotavirus in intestines. Here we generated mice carrying mutations in all three members of the Nlrp9a/b/c gene (Nlrp9 triple mutant (TMut) mice). When crossed with WT males, the Nlrp9 TMut females were fertile, but deliveries with fewer pups were increased in the mutants. Consistent with this, blastocyst development was retarded and lethality to the preimplantation embryos increased in the Nlrp9 TMut females in vivo. Under in vitro culture conditions, the fertilized eggs from the Nlrp9 TMut females exhibited developmental arrest at the two-cell stage, accompanied by asymmetric cell division. By contrast, double-mutant (DMut) oocytes (any genetic combination) did not exhibit the two-cell block in vitro, showing the functional redundancy of Nlrp9a/b/c. Finally, Nlrp9 could bind to components of the SCMC. These results show that Nlrp9 functions as an immune or reproductive NLRP in a cell-type-dependent manner.

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