Pre-Treatment of Swine Oviductal Epithelial Cells with Progesterone Increases the Sperm Fertilizing Ability in an IVF Model

Abstract

Mammalian spermatozoa are infertile immediately after ejaculation and need to undergo a functional modification, called capacitation, in order to acquire their fertilizing ability. Since oviductal epithelial cells (SOECs) and progesterone (P4) are two major modulators of capacitation, here we investigated their impact on sperm functionality by using an IVF swine model. To that, we treated SOECs with P4 at 10, 100, and 1000 ng/mL before the coincubation with spermatozoa, thus finding that P4 at 100 ng/mL does not interfere with the cytoskeleton dynamics nor the cells’ doubling time, but it promotes the sperm capacitation by increasing the number of spermatozoa per polyspermic oocyte (p < 0.05). Moreover, we found that SOECs pre-treatment with P4 100 ng/mL is able to promote an increase in the sperm fertilizing ability, without needing the hormone addition at the time of fertilization. Our results are probably due to the downregulation in the expression of OVGP1, SPP1 and DMBT1 genes, confirming an increase in the dynamism of our system compared to the classic IVF protocols. The results obtained are intended to contribute to the development of more physiological and efficient IVF systems.

Keywords: DMBT1; OVGP1; SPP1; cytoskeleton; in vitro fertilization; oviduct; oviductal epithelial cells; progesterone; sperm capacitation; spermatozoa.

Figure 1

Experimental design for IVF. (A) Experimental groups to identify the most effective P4 concentration. (B) Experimental groups using the most effective P4 concentration and sperm capacitation with and without P4 (100 ng/mL) in the presence of SOECs previously treated or not with P4.

Figure 2

Effect of the different P4 concentrations on SOECs doubling time. The histograms show a normal proliferation rate of the samples treated with different concentrations of P4 (10, 100 and 1000 ng/mL), similar to the control (CTRL) group (p > 0.05). Three independent experiments were performed.

Figure 3

(A) Microtubules organization of SOECs. Immunofluorescence analysis of α-tubulin of SOECs after P4 treatment at different confluence time points (50% e 100%) and control condition (CTRL). Monoclonal anti-mouse-α-tubulin antibody and secondary antibody anti-mouse cy3 TRICT-conjugated (red) were used to detect α-tubulin; DAPI was used to stain the nuclei (blue). Three independent experiments were performed. (B) Actin filaments of SOECs. Phalloidin staining was used to study the F-actin of SOECs after P4 treatments at different confluence time points (50% e 100%) and control condition (CTRL). DAPI (blue, nuclei), TRITC-conjugated phalloidin (red, F-actin). Three independent experiments were performed.

Figure 4

Graphical representation of the effects of P4 at different concentrations. Results are ex-pressed as the difference in percentage (Δ IVF%) of the fertilization rates, the number of polyspermic oocytes and the number of spermatozoa per polyspermic oocyte, comparing the groups of spermatozoa capacitated in the presence of SOEC treated with P4 (at 10,100 and 1000 ng/mL) to the control group without P4. The best IVF results were obtained when SOECs were treated with P4 at 100 ng/mL, due to the higher values obtained with this concentration in terms of difference of percentage in the number of polyspermic oocytes and in the number of spermatozoa/polyspermic oocyte. The data are presented as mean of four independent experiments. Data were analyzed using a Tukey’s test ** p < 0.01.

Figure 5

IVF outcomes: comparison between pre-treatment of SOECs with P4 and addition of P4 during sperm capacitation. The graph shows the effects of co-incubation of sperm during capacitation with P4 (100 ng/mL) and SOECs pre-treated with P4 (100 ng/mL) on IVF outcomes. By analyzing the graph, it arises that the best IVF outcomes are obtained when SOECs are pre-treated with P4 at 100 ng/mL and sperm capacitation is performed without the addition of P4. The data are presented as mean of four independent experiments. Data were analyzed using a Dunnett’s test. * p < 0.05 versus control.

Figure 6

The effect of progesterone and estradiol on relative gene expression (2^−ΔΔCt) of (A) OVGP1, (B) SPP1, and (C) DMBT1. Results are represented as median value with 95 % confidence interval. Data were analyzed using the Kruskal-Wallis test. For each qPCR analysis, each sample was performed in triplicate, and values were normalized to the GAPDH endogenous reference gene. P4, Progesterone; E2, Estradiol; ** = p < 0.001, * = p < 0.01.