Interferon tau-dependent and independent effects of the bovine conceptus on the endometrial transcriptome†

Abstract

This study investigated bovine conceptus-induced modifications to the endometrial transcriptome related to effects of interferon tau (IFNT), conceptus origin (in vivo vs. in vitro), and conceptus sex. In vitro (IVF) or in vivo (superovulation and artificial insemination, AI) produced blastocysts were transferred into recipient heifers on day 7 of the estrous cycle. On day 15, IVF- or AI-derived conceptuses were obtained by uterine flushing and individually placed on endometrial explants in media for 6 h. Explants were also cultured with media alone as a control or media containing 100 ng/mL IFNT. Total explant RNA was analyzed by RNA-Seq. Incubation of endometrium with IFNT or IVF- or AI-derived conceptuses changed (P ≤ 0.001) expression of 491, 498, and 576 transcripts, respectively, compared to the control. Further, 369 differentially expressed genes (DEGs) were common between explants exposed to IFNT or a conceptus. A total of 240 DEGs were uniquely altered by conceptuses (IVF- and AI-derived) but not IFNT. Of these transcripts, 46 were shared between the IVF and AI groups, while 61 and 133 were specific to IVF and AI conceptuses, respectively. Five genes [melanophilin (MLPH), prominin-2 (PROM2), myeloid associated differentiation marker (MYADM), vomeronasal 1 receptor 4 like (VN1R4L) and 5-hydroxytryptamine receptor 1A (HTR1A)] were more abundant in endometrium exposed to female compared to male conceptuses (P < 0.001). A single gene [ADP-ribosylation factor like GTPase 4C (ARL4C)] was more abundant in response to male conceptuses (P < 0.001) than female conceptuses. These data support the hypothesis that conceptus regulation of gene expression in the endometrium is complex and involves factors other than IFNT that may have a biological role in pregnancy establishment.

Keywords: RNAseq; conceptus; endometrium; explant; interferon tau; transcriptome.

Figure 1.

Experimental design. In vivo -derived (A) and in vitro-produced (B) bovine blastocysts were transferred in groups of 10 to synchronized recipient heifers on day 7 and elongated conceptuses were recovered on day 15 (B) and co-cultured with endometrial explants (C) to detect conceptus-induced changes in gene expression. P4 = progesterone releasing device; GnRH = gonadotrophin releasing hormone; PG = prostaglandin F2α; FSH = follicle-stimulating hormone; AI = artificial insemination; ER = embryo recovery; ET = embryo transfer; IVM = in vitro maturation; IVF = in vitro fertilization; IVC = in vitro culture; D7 Blast = day 7 blastocyst; CR = conceptus recovery; Exp 6 h = in vivo and in vitro conceptuses cultured on top of an endometrial explant for 6 h. Day 0 was considered the day of fertilization (28–32 h after the start estrus behavior).

Figure 2.

A representative image of an elongated conceptus cultured on top of an endometrial explant. Conceptuses and endometrial explants were co-cultured in 1 mL of Roswell Park Memorial Institute (RPMI) media for 6 h before tissues were collected and snap-frozen for RNA (explant) and DNA (conceptus) extraction.

Figure 3.

A venn diagram (Venny 2.1.0 BioinfoGP) identifying differentially expressed genes (DEGs) in endometrium treated with ovine interferon tau (IFNT), in vitro fertilization (IVF), and artificially inseminated (AI)-derived conceptuses compared to control endometrium. The small black box within the venn diagram indicates genes differentially expressed in response to all three treatments and related to the maternal recognition of pregnancy signal, IFNT. The large box below the venn diagram lists the ensemble gene ID, gene name, and log fold change (LogFC) in expression (compared to control endometrium) for the 10 most upregulated (greatest to least) and 10 most downregulated (from least downregulated to greatest) genes identified within the small box. Of all the DEGs detected in this study, the 10 upregulated genes listed in the large box were also the most highly expressed transcripts in response to all three treatments. The DEGs identified within each section (indicated by roman numerals) of the venn diagram are listed in Supplementary Table S2. Numbers below roman numerals indicated the number of DEGs detect in that section. The hyphenated numbers indicate the number of DEGs that were up- or downregulated (up-down) within the total number of DEG identified in that section.

Figure 4.

The RT-qPCR relative gene expression of interferon stimulated genes: (A) interferon stimulated gene 15 (ISG15), (B) myxovirus resistance protein 2 (MX2), and (C) 2΄-5΄-oligoadenylate synthase 1 (OAS1) in endometrium treated with Roswell Park Memorial Institute (RPMI) media (control), ovine interferon tau (IFNT), in vitro fertilization (IVF), and artificial insemination (AI)-derived conceptuses. Data were normalized to the geometric mean expression of peptidylprolyl isomerase A (PPIA) and succinate dehydrogenase complex flavoprotein subunit A (SDHA) and are presented as least squares means ± standard error of the least squares means (LSM ± SEM). Letters over the bars indicate significant differences between treatment means (P ≤ 0.05).

Figure 5.

A venn diagram (Venny 2.1.0 BioinfoGP) identifying DEGs in endometrium treated with ovine interferon tau (IFNT), in vitro fertilization (IVF), and artificial insemination (AI)-derived conceptuses compared to control endometrium. The broken oval circle within the venn diagram indicates genes differentially expressed in response to AI-derived conceptuses but independent of IFNT. The solid oval circle indicates genes differentially expressed in response to IVF but not AI-derived conceptuses. The large broken and solid boxes below the venn diagram include the Ensemble gene ID, gene name, and log fold change (LogFC) in expression (compared to control endometrium) for the 10 most upregulated (greatest to least) and 10 most downregulated (from least downregulated to greatest) genes identified within the corresponding ovals. The DEGs identified within each section (indicated by roman numerals) of the venn diagram are listed in Supplementary Table S2. Numbers below roman numerals indicate the number of DEGs detect in that section. The hyphenated numbers indicate the number of DEGs that were up- or downregulated (up-down) within the total number of DEG identified in that section.

Figure 6.

The RT-qPCR relative gene expression of (A) interleukin 1 beta (IL1B), (B) interleukin 6 (IL6), and (C) nuclear factor kappa B1 (NFKB1) in endometrium treated with Roswell Park Memorial Institute (RPMI) media (control), ovine interferon tau (IFNT), in vitro fertilization (IVF), and artificial insemination (AI)-derived conceptuses. Data were normalized to the geometric mean expression of peptidylprolyl isomerase A (PPIA) and succinate dehydrogenase complex flavoprotein subunit A (SDHA) and are presented as least squares means ± standard error of the least squares means (LSM ± SEM). Letters over the bars indicate significant differences between treatment means (P ≤ 0.05). The IL1B residual data were not normal distributed; therefore, the data were corrected by log transformation (see the statistical analysis section of Materials and Methods); untransformed data are presented in the figure.

Figure 7.

A venn diagram (Venny 2.1.0 BioinfoGP) identifying differentially expressed genes (DEGs) in endometrium treated with male and female conceptuses compared to control endometrium. The boxes below the venn diagram include the Ensemble gene ID, gene name, and log fold change (LogFC) in expression (compared to control endometrium) for the 10 most upregulated (greatest to least) and 10 most downregulated (from least downregulated to greatest) genes identified within the corresponding venn diagram section. The DEGs identified within each section (indicated by roman numerals) of the venn diagram are listed in Supplementary Table S6. Numbers below roman numerals indicated the number of DEGs detected in that section. The hyphenated numbers indicate the number of DEGs that were up- or downregulated (up-down) within the total number of DEG identified in that section. The location and direction of change (up- or downregulated) for five of the six DEGs (MLPH, MYADML, HTR1A, VN1R4L, ARL4C) revealed by a direct comparison of RNA Seq data from endometrium exposed to male vs. female conceptuses is indicated.

Figure 8.

Relative gene expression (log fold change, LogFC) in endometrial explants exposed to male vs. female conceptuses for ADP-ribosylation factor-like 4C (ARL4C), melanophilin (MLPH), prominin 2 (PROM2), myeloid-associated differentiation marker like (MYADML), vomeronasal 1 receptor 4 like protein (VN1R4L), and 5-Hydroxytryptamine receptor 1A (HTR1A). Expression of ARL4C was greater (P < 0.001) in male compared to female conceptus-treated endometrium. Expression of MLPH, PROM2, MYADML, VN1R4L, and HTR1A was greater (P < 0.001) in female compared to male conceptus-treated endometrium.

Figure 9.

The RT-qPCR relative gene expression of endometrial Lectin, galactose binding, soluble 1 (LGALS1; also known as GAL1) in response to Roswell Park Memorial Institute (RPMI) media (control) and male and female conceptuses. Data were normalized to the geometric mean expression of peptidylprolyl isomerase A (PPIA) and succinate dehydrogenase complex flavoprotein subunit A (SDHA) and are presented as least squares means ± standard error of the least squares means (LSM ± SEM). Letters over the bars indicate significant differences between treatment means (P ≤ 0.05). There was a tendency (P = 0.070) for an effect of treatment on endometrial expression of LGALS1. Male conceptuses induced greater endometrial expression of LGALS1 compared to female conceptuses.