mRNA expression pattern of selected candidate genes differs in bovine oviductal epithelial cells in vitro compared with the in vivo state and during cell culture passages

Abstract

Background: The mammalian oviduct provides the optimal environment for gamete maturation including sperm capacitation, fertilization, and development of the early embryo. Various cell culture models for primary bovine oviductal epithelial cells (BOEC) were established to reveal such physiological events. The aim of this study was to evaluate 17 candidate mRNA expression patterns in oviductal epithelial cells (1) in transition from in vivo cells to in vitro cells; (2) during three consecutive cell culture passages; (3) affected by the impact of LOW or HIGH glucose content media; and (4) influenced by different phases of the estrous cycle in vivo and in vitro. In addition, the release of a metabolite and proteins from BOEC at two distinct cell culture passage numbers was estimated to monitor the functionality.

Methods: BOEC from 8 animals were isolated and cultured for three consecutive passages. Total RNA was extracted from in vivo and in vitro samples and subjected to reverse transcription quantitative polymerase chain reaction to reveal mRNA expression of selected candidate genes. The release of prostaglandin E2 (PGE2), oviduct-specific glycoprotein 1 (OVGP1) and interleukin 8 (IL8) by BOEC was measured by EIA or ELISA after 24 h.

Results: Almost all candidate genes (prostaglandin synthases, enzymes of cellular metabolism and mucins) mRNA expression pattern differed compared in vivo with in vitro state. In addition, transcription of most candidate genes was influenced by the number of cell culture passages. Different glucose medium content did not affect mRNA expression of most candidate genes. The phase of the estrous cycle altered some candidate mRNA expression in BOEC in vitro at later passages. The release of PGE2 and OVGP1 between passages did not differ. However, BOEC in passage 3 released significantly higher amount of IL8 compared with cells in passage 0.

Conclusion: This study supports the hypothesis that candidate mRNA expression in BOEC was influenced by transition from the in vivo situation to the new in vitro environment and during consecutive passages. The consequence of cell culture passaging on BOEC ability to release bioactive compounds should be considered.

Keywords: Cell culture passage; Culture medium; Enzymes of cellular metabolism; Mucin; Oviduct; Prostaglandin synthase; mRNA expression.

Fig. 1

Cytokeratin immunostaining of cultured BOEC in different number of cell culture passages. Immunostaining with anti-cytokeratin antibody of cultured BOEC in: a passage 0; b passage 1; c passage 2; d passage 3; and e negative control. Goat anti-mouse-IgG DyLight 488 conjugate (green) was used for staining cytokeratin as a secondary antibody. DAPI (blue) was used to visualize nuclei. Magnification was set at 200 X. Bar in each figure represents 100 μm. Representative pictures of BOEC cultured in LOW glucose medium are shown

Fig. 2

WGA staining in sections of oviduct tissue and cultured BOEC. WGA-Alexa Fluor 594 (red) staining of: a oviductal cross-section of the ampulla collected during the luteal phase represents in vivo sample; b primary cultured BOEC in passage 0; c primary cultured BOEC in passage 3. DAPI (blue) was used to visualize nuclei. Magnification was set at 200 X. Bar in each figure represents 50 μm. Representative pictures of BOEC cultured in HIGH glucose medium are shown

Fig. 3

Effect of passage number or estrous cycle stage on PG synthesis enzyme mRNA expression pattern. Normalized mRNA expression of a PTGS1; b PTGS2; e PTGES2; and h PTGES3 in BOEC of in vivo samples and of cell culture passages P0, P1, P2 and P3 with LOW and HIGH glucose medium content (n = 8) as well as normalized mRNA expression depending of the phase of the estrous cycle on day of collecting cells of c PTGS2 in LOW glucose medium; d PTGS2 in HIGH glucose medium; f PTGES2 in LOW glucose medium; and g PTGES2 in HIGH glucose medium (n = 4). Different letters indicate significant difference (P < 0.05) between cell culture passages of the same glucose medium content. Asterisk on top of the line represents significant difference between different glucose content of medium at each cell culture passage or between the different estrous cycle phases on day of collection (P < 0.05)

Fig. 4

Effect of passage number or estrous cycle stage on selected mucin mRNA expression pattern. Normalized mRNA expression of a MUC1; c MUC4; and e MUC6 in BOEC of in vivo samples and of cell culture passages P0, P1, P2 and P3 with LOW and HIGH glucose medium content (n = 8) as well as normalized mRNA expression depending of the phase of the estrous cycle on day of collecting cells of b MUC1 in HIGH glucose medium; d MUC4 in LOW glucose medium; and f MUC6 in HIGH glucose medium (n = 4). Different letters indicate significant difference (P < 0.05) between cell culture passages of the same glucose medium content (ab and de for LOW and HIGH glucose medium content, respectively). Asterisk on top of the line represents significant difference between the different estrous cycle phases on day of collection (P < 0.05)

Fig. 5

Effect of passage number or estrous cycle stage on MUC16, OVGP1 and IL8 mRNA expression. Normalized mRNA expression of a MUC16; c OVGP1; and e IL8 in BOEC of in vivo samples and of cell culture passages P0, P1, P2 and P3 with LOW and HIGH glucose medium content (n = 8); and d magnified inset of OVGP1 in BOEC in cell culture passages P0, P1, P2 and P3 with LOW and HIGH glucose medium content as well as normalized mRNA expression depending of the phase of the estrous cycle on day of collecting cells of b MUC16 in LOW glucose medium; and f IL8 in HIGH glucose medium (n = 4). Different letters indicate significant difference (P < 0.05) between cell culture passages of the same glucose medium content (ab and de for LOW and HIGH glucose medium content, respectively). Asterisk on top of the line represents significant difference between different glucose content of medium at each cell culture passage or between the different estrous cycle phases on day of collection (P < 0.05)

Fig. 6

Effect of passage number or estrous cycle stage on enzymes of cellular metabolism mRNA expression. Normalized mRNA expression of a BDH2; and d OXCT2 in BOEC of in vivo samples and of cell culture passages P0, P1, P2 and P3 with LOW and HIGH glucose medium content (n = 8); and e magnified inset of OXCT2 in BOEC in cell culture passages P0, P1, P2 and P3 with LOW and HIGH glucose medium content as well as normalized mRNA expression depending of the phase of the estrous cycle on day of collecting cells of b BDH2 in LOW glucose medium; c BDH2 in HIGH glucose medium; f OXCT2 in LOW glucose medium; and g OXCT2 in HIGH glucose medium (n = 4). Different letters indicate significant difference (P < 0.05) between cell culture passages of the same glucose medium content (ab and de for LOW and HIGH glucose medium content, respectively). Asterisk on top of the line represents significant difference between the different estrous cycle phases on day of collection (P < 0.05)

Fig. 7

Effect of passage number or estrous cycle stage on GAPDH, SDHA and HK1 mRNA expression. Normalized mRNA expression of a GAPDH; d SDHA; and g HK1 in BOEC of in vivo samples and of cell culture passages P0, P1, P2 and P3 with LOW and HIGH glucose medium content (n = 8) as well as normalized mRNA expression depending of the phase of the estrous cycle on day of collecting cells of b GAPDH in LOW glucose medium; c GAPDH in HIGH glucose medium; e SDHA in LOW glucose medium; and f SDHA in HIGH glucose medium (n = 4). Different letters indicate significant difference (P < 0.05) between cell culture passages of the same glucose medium content. Asterisk on top of the line represents significant difference between different glucose content of medium at each cell culture passage or between the different estrous cycle phases on day of collection (P < 0.05)

Fig. 8

Effect of passage number on PGE₂, OVGP1 and IL8 release from BOEC. Release estimated in P0-HIGH and P3-HIGH BOEC of a PGE₂ (n = 3); b OVGP1 (n = 3); and c IL8 (n = 4). Each bar chart illustrates data as means ± SEM. Asterisk depicts significant difference (P < 0.05) between P0 and P3