Study on the effects of heat stress on in vitro embryo production and the oviductal environment in cattle

The accomplishment of this PhD thesis required the conduction of five distinct experiments. The aim of these experiments was to study the effects of heat stress (HS) on the reproduction of cows. Particularly, the effects of HS on oocytes and early embryos, as well as on the oviduct environment, were studied. The experiments focused on studying: a) the effect of HS during the in vitro maturation (IVM) of bovine oocytes on blastocyst production rates and their quality, b) the effect of exogenous addition of heat shock protein 70 (Hsp70) in the maturation medium of oocytes exposed to HS, c) the differences in the developmental competence of Holstein and Limousine cows’ oocytes, after exposure to HS during the in vitro maturation, d) the effect of the addition of Hsp70 in the in vitro culture medium on the developmental competence and quality of bovine embryos which were exposed to HS, e) the oviductal epithelial cells transcriptome and extracellular vesicles characterization during thermoneutral and heat stress conditions in dairy cows.This PhD thesis is separated in six chapters.Chapter I includes the review of the international published literature for the scientific fields of the experiments.In Chapter II, the effects of short, moderate temperature elevation during IVM, on embryo yield, and on the expression of various genes were evaluated. In 8 replicates, cumulus oocyte complexes (COCs) were matured for 24 hours at 39°C (controls n=520) or at 41°C from hour 2 to hour 8 of IVM (treated, n =802), fertilized, and presumptive zygotes were cultured for 9 days at 39°C. Cleavage and embryo formation rates were evaluated 48 hours post insemination (pi) and on days 7, 8, 9 respectively. Cumulus cells, oocytes and blastocysts from 5 replicates were snap frozen for the relative expression analysis of genes related to metabolism, thermal and oxidative stress response, apoptosis, and placentation. In treated group, cleavage and embryo formation rates were statistically significantly lower compared with the control (cleavage 86.7% vs 74.2%; blastocysts: day 7, 29.9% vs 19.7%, day 8, 34.2% vs 22.9% and day 9 35.9% vs 24.5%). Relative mRNA abundance of three genes in cumulus cells (HSP90AA1, CPT1B, G6PD) and three genes in blastocysts (DNMT3A, PLAC8, GPX1) indicated significantly different expression between groups (p<0.05). The expression of G6PD, SOD2, GXP1 in oocytes and PTGS2 in blastocysts tended to differ among groups (0.05<p<0.08). Heat stress altered (p<0.05) the correlation of expression between HSPs and other genes in oocytes (G6PD, GPX1, CCNB1), cumulus cells (LDH, CCNB1) and blastocysts (AKR1B1, PLAC8).In Chapter III, the effects of supplementing Hsp70 in in vitro matured bovine oocytes under thermoneutral or heat stress conditions were examined, and its efficacy on in vitro embryo yield and quality was assessed; the latter was determined on the basis of the expression of various genes related to important cellular functions. Bovine oocytes were matured for 24h at 39°C without (group C39, n=471) or with Hsp70 (group H39, n=353) and at 41°C for the first 6h, followed by 16h at 39°C with (group H41, n=704) or without Hsp70 (group C41, n=405). After insemination, zygotes were cultured for 9 days at 39°C. Cleavage and embryo yield were assessed 48h pi and on days 7, 8, 9, respectively. Gene expression was assessed by RT-PCR in oocytes, cumulus cells and blastocysts. In C41, blastocysts formation rate was lower than in C39 and on day 9 it was lower than in H41. In oocytes, Hsp70 enhanced the expression of three HSP genes regardless of incubation temperature. Hsp70 at 39°C led to tight coordination of gene expression in oocytes and blastocysts, but not in cumulus cells.In Chapter IV, the effects of HS during in vitro maturation on the developmental potential of oocytes derived from Limousine and Holstein cows were investigated and the effect on the differential gene expression of important genes in oocytes, cumulus cells and blastocysts in the growth competence between the breeds was estimated. In seven replicates, COCs from Holstein and Limousine cows were matured for 24h at 39°C (controls C; HC, n=405, LC, n=370) or at 41°C from hour 2 to hour 8 of IVM (treated T; HT, n=652, LT, n=488), fertilized, and presumptive zygotes were cultured for 9 days at 39°C. Cleavage and embryo formation rates were evaluated 48h pi and on days 7, 8 and 9, respectively. From all groups, subsets of cumulus cells, oocytes and blastocysts were analyzed for the relative expression of genes related to metabolism, stress, apoptosis and placentation. No difference was detected in cleavage rate or in blastocyst formation rate among the control groups. In both breeds, HS reduced blastocyst yield, but at all days the suppression was higher in Limousines. In Holsteins, altered gene expression was detected in cumulus cells (G6PD, GLUT1) and blastocysts (PLAC8), while in Limousines, differences were found in oocytes (G6PD, HSP90AA1), in cumulus cells (CPT1B, HSP90AA1, SOD2) and blastocysts (DNMT, HSP90AA1, SOD2).The aims of Chapter V were to examine the effects of Hsp70 addition in the in vitro culture medium of day 3 embryos on their developmental competence and quality. Bovine oocytes (n=1442) were in vitro matured, inseminated and cultured for the first two days according to standardized methods. The presumptive zygotes were randomly allocated in three experimental groups: Control, C (embryos cultured at 39°C throughout the culture period=396), group C41 (temperature was raised to 41°C from the 48th to 72nd h pi and then it returned at 39°C for the remaining culture period, n=514), and group H41 (the temperature modification was the same as in C41 and during heat exposure, Hsp70 was added in the culture medium, n=532). Cleavage and embryo yield were assessed 48h pi and on days 7, 8, 9, respectively and gene expression in day 7 blastocysts was assessed by RT-PCR. Blastocyst yield was the highest in group C39 and higher in group H41 compared to group C41. From the gene expression analyses, altered expression of 11 genes was detected among groups. The analysis of the orchestrated patterns of gene expression differed between groups.In Chapter VI, the transcriptome of oviductal epithelial cells and certain characteristics of their extracellular vesicles (EVs) of dairy cows were described under thermoneutral and heat stress conditions. Twenty cows were compared in springtime at THI=65.6±0.90 and in summertime at THI=78.36±2.73. During each season, the estrous cycles of the cows were synchronized, and on day 3 of the ensuing cycle, a blood sample was collected for progesterone determination, while their oviducts were collected after slaughter. Epithelial cells and oviductal fluid were collected from the oviduct ipsilateral and contralateral to the corpus, respectively. For the gene expression study, a comparative transcriptomic approach, using RNA Sequencing (RNASeq), was performed on cells collected from the ipsilateral and the contralateral oviducts. The size and the concentration of EVs at both seasons were analyzed using Transmission Electron Microscopy and Nanoparticle tracking analysis and specific proteins were detected by Western blotting. Progesterone concentration was higher during the thermoneutral period. Between seasons, divergent expression of genes related to immune system, contractility, gamete protection and long non-coding RNAs (lncRNAs) was found. The size and the concentration of the EVs did not differ between seasons, however, the concentration in the ipsilateral oviduct tended to be lower (p=0.09) from the contralateral one in the summer, but not in the spring.The main findings of the present PhD thesis are summarized as following:1.The elevated temperature during oocyte maturation disrupts the developmental competence of the oocytes, suppresses blastocyst yield and significantly alters the coordinated pattern of gene expressions.2.The external supplementation of Hsp70 protects heat stressed maturing bovine oocyte and restores its developmental competence, by preventing apoptosis, supporting signal transduction, and increasing antioxidant protection of the embryo.3.The Holstein COCs are more tolerant than Limousine COCs, possibly due to compulsory, production driven selection.4.The temperature rise is detrimental for the developmental potential of the early embryos. The Hsp70 can partly mitigate the harmful effects of HS, by improving the embryo yield and by reducing the adverse effects of HS on some embryo quality characteristics.5.The altered oviductal environment during HS could be related with the suppressed summer fertility in dairy cows.Για την ολοκλήρωση της παρούσας Διδακτορικής Διατριβής διενεργήθηκαν πέντε διακριτοί πειραματισμοί που είχαν ως στόχο τη μελέτη της επίδρασης της θερμικής καταπόνησης (ΘΚ) στο περιβάλλον του ωοθυλακίου και στον ωαγωγό των αγελάδων. Ειδικότερα, σε in vitro συνθήκες, μελετήθηκαν οι επιπτώσεις της ΘΚ στην ωρίμανση του συμπλέγματος ωαρίου – κυττάρων ωοφόρου δίσκου, στη γονιμοποιητική ικανότητα του ωαρίου, στην ποιότητα των παραγόμενων εμβρύων, ενώ σε in vivo συνθήκες μελετήθηκαν οι επαγόμενες από τη ΘΚ αλλαγές στο περιβάλλον του ωαγωγού. Οι πειραματισμοί εστίασαν: α) στην επίδραση της ΘΚ κατά τη διάρκεια της in vitro ωρίμανσης ωαρίων βοοειδών στα ποσοστά παραγωγής βλαστοκύστεων και στην ποιότητα τους, β) στη διερεύνηση της επίδρασης της εξωγενούς προσθήκης heat shock protein 70 (Hsp70) στο υπόστρωμα ωρίμανσης ωαρίων που εκτέθηκαν σε ΘΚ, γ) στη συγκριτική μελέτη της ικανότητας ανάπτυξης ωαρίων αγελάδων των φυλών Holstein και Limousine, μετά από έκθεση σε ΘΚ κατά τη διάρκεια της in vitro ωρίμανσης, δ) στη μελέτη της επίδρασης της προσθήκης Hsp70 στο υπόστρωμα της in vitro καλλιέργειας στην ανάπτυξη και την ποιότητα εμβρύων βοοειδών που εκτέθηκαν σε ΘΚ, ε) στη συγκριτική μελέτη του μεταγραφώματος των επιθηλιακών κυττάρων του ωαγωγού και των εξωκυτταρικών κυστιδίων σε θερμοουδέτερες συνθήκες και σε συνθήκες ΘΚ σε αγελάδες γαλακτοπαραγωγής.Η Διατριβή χωρίζεται σε έξι κεφάλαια.Το Κεφάλαιο Ι αποτελεί ανασκόπηση της διεθνούς βιβλιογραφίας των επιστημονικών πεδίων στα οποία εκτείνονται οι πειραματισμοί της Διατριβής. Στο Κεφάλαιο II, αξιολογήθηκαν οι επιδράσεις της μικρής διάρκειας, ήπιας αύξησης της θερμοκρασίας κατά τη διάρκεια της IVM, στην παραγωγή εμβρύων και στην έκφραση διαφόρων γονιδίων. Σε 8 επαναλήψεις, τα συμπλέγματα ωαρίων-κυττάρων ωοφόρου δίσκου (COCs) ωρίμασαν για 24 ώρες στους 39°C (μάρτυρες, C, n=520) ή στους 41°C από την ώρα 2 έως την ώρα 8 της IVM (ομάδα έκθεσης σε αυξημένη θερμοκρασία, T, n=802), γονιμοποιήθηκαν, και τα πιθανά ζυγωτά καλλιεργήθηκαν για 9 ημέρες στους 39°C. Η εκτίμηση του ποσοστού αυλάκωσης στα πρώιμα έμβρυα και του ποσοστού παραγωγής βλαστοκύστεων πραγματοποιούνταν 48 ώρες και 7, 8, και 9 ημέρες μετά τη γονιμοποίηση (post insemination, pi), αντίστοιχα. Κύτταρα του ωοφόρου δίσκου, απογυμνωμένα ωάρια και βλαστοκύστεις από 5 επαναλήψεις καταψύχθηκαν για την ανάλυση της σχετικής έκφρασης γονιδίων, που σχετίζονται με το μεταβολισμό, την απόκριση στο θερμικό και οξειδωτικό στρες, την απόπτωση και τη δυνατότητα ανάπτυξης πλακούντα. Στην ομάδα T, τα ποσοστά αυλάκωσης και παραγωγής βλαστοκύστεων ήταν σημαντικά (p<0.05) χαμηλότερα σε σύγκριση με την ομάδα C (αυλάκωση 86.7% έναντι 74.2%, βλαστοκύστεις: ημέρα 7, 29.9% έναντι 19.7%, ημέρα 8, 34.2% έναντι 22.9% και ημέρα 9, 35.9% έναντι 24.5%). Η έκφραση τριών γονιδίων στα κύτταρα του ωοφόρου δίσκου (HSP90AA1, CPT1B, G6PD) και τριών γονιδίων στις βλαστοκύστεις (DNMT3A, PLAC8, GPX1) εμφάνισε σημαντικές διαφορές μεταξύ των ομάδων (p<0.05). Η έκφραση των G6PD, SOD2, GXP1 στα ωάρια και του PTGS2 στις βλαστοκύστεις έτεινε να διαφέρει μεταξύ των ομάδων (0.05<p<0.08). Η ΘΚ μετέβαλε (p