Characterisation of an in vitro system to study maternal communication with spermatozoa

Abstract

In vivo, gamete maturation, fertilisation and early embryonic development take place inside the oviduct. Several studies have indicated that local responses towards gametes and embryos are generated by the maternal reproductive tract. However, no defined in vitro model currently exists to allow detailed and systematic investigation of maternal communications with gametes and embryos. Therefore, we characterised an in vitro model based on the interaction of boar spermatozoa with an immortalised porcine oviduct epithelial cell line to evaluate different factors that may affect this model. The factors tested were sperm viability, source of spermatozoa, cell passage effect and the effect of reproductive and non-reproductive epithelial cells in the interaction with spermatozoa. After 24 h of co-incubation, RNA was extracted and used to synthesise cDNA for quantitative real-time PCR. Alteration in the expression of genes such as adrenomedullin, heat-shock 70-kDa protein 8 and prostaglandin E synthase was considered as the end point of this assay. The results showed that sperm viability and cell passage number had an effect on oviductal gene expression in response to spermatozoa. Oviductal cells showed significant alterations in gene expression when compared with non-reproductive epithelial cells. The simple in vitro system described here has potential application for further studies in our understanding of mechanisms involved in maternal interactions with spermatozoa.

Viable and morphologically normal boar spermatozoa alter the expression of heat‐shock protein genes in oviductal epithelial cells during co‐culture in vitro

“…The observation made herein, that HSP gene expression is only markedly upregulated when spermatozoa are in direct contact with OECs rather than when they are kept separate using diffusible membrane inserts, tends to support this view. In agreement with other studies (Aldarmahi et al, 2012, 2014), we thus propose that spermatozoa binding directly to OECs in co‐culture activates a specific signal transduction pathway within the OECs that results in the upregulation of HSP gene expression. HSPs synthesized de novo in response to sperm appear to translocate from within OECs to the oviductal lumen (Georgiou et al, ) and interact directly with the sperm membrane, probably through cholesterol molecules and/or lipid rafts that are present/accessible in uncapacitated but not in capacitated spermatozoa (Moein‐Vaziri et al, ).…”

“…or nonbiological (e.g., glass‐beads) entities on chaperone/HSP gene expression by OECs. Notwithstanding, existing evidence suggests that OECs do respond to the presence of both spermatozoa and oocytes by altering the abundance of the proteins they secrete, although the exact alterations are cell‐type specific (Georgiou et al, 2005, 2007; Kodithuwakku et al, ; Aldarmahi et al, 2012, 2014). The observation made herein, that HSP gene expression is only markedly upregulated when spermatozoa are in direct contact with OECs rather than when they are kept separate using diffusible membrane inserts, tends to support this view.…”

Effects of spermatozoa–oviductal cell coincubation time and oviductal cell age on spermatozoa–oviduct interactions

“…All primers were tested for specificity using National Center for Biotechnology Information (NCBI) blast (http://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed 1 January 2011) and for primer-dimer and secondary structure using the DNA analysis tools on the integrated DNA technology website (http://eu.idtdna.com/scitools/scitools.aspx, accessed 1 January 2011). Primers for b-actin (BACT), ADM, HSPA8 and PGES were designed from the DNA sequence of Sus scrofa species, as described previously (Aldarmahi et al 2012). The efficiencies of the reference and targets genes were assessed and determined.…”

“…Some studies report that the level of mRNA expression is altered with increasing culture passage number and presume that dedifferentiation of differentiated cells takes place in vitro after several cell culture passages (Neumann et al 2010). In a previous study, we demonstrated that expression of adrenomedullin (ADM), heat shock 70 kDa protein 8 (HSPA8) and prostaglandin E synthase (PGES) in an immortalised oviductal epithelial cell line decreased as the number of oviductal culture passages increased (Aldarmahi et al 2012). However, it was not clear whether the variation between different cell passages was affected by the handling of cells on different experimental days and/or altered in response to coculture with spermatozoa.…”

Effect of a pre-freezing treatment with cholesterol-loaded cyclodextrins on boar sperm longevity, capacitation dynamics, ability to adhere to porcine oviductal epithelial cells in vitro and DNA fragmentation dynamics

“…Several authors have demonstrated that these cells could be used to study spermatozoa-oviduct epithelial cell interactions because this cell line is not dedifferentiated and has not lost the expression of functional receptors (Hombach-Klonisch et al 2006). Additionally, when these cells are incubated with spermatozoa, the alteration in the expression of some genes (ADM, HSPA8 and PGES) is similar to what was described for primary oviductal epithelial cells (Aldarmahi et al 2012). Spermatozoa-oviductal epithelium binding assays evaluate the plasma membrane for multiple functions before the fertilisation process (Waberski et al 2005), and this in vitro binding test has proven to be very useful for the assessment of sperm quality given its high sensitivity for detecting differences (De Pauw et al 2002;Petrunkina et al 2007).…”