Bovine herpesvirus 1 can cross the intact zona pellucida of bovine oocytes after artificial infection

Abstract

Bovine herpesvirus 1 (BHV1) is an important bovine pathogen, responsible for respiratory diseases and reproductive problems. This study investigated the penetration capacity of BHV1 into oocytes after co-incubation for either 1 h or 24 h. Immunofluorescence assays in cumulus-oocyte complexes (COCs) and denuded oocytes (without the presence of cumulus cells) were performed and evaluated using confocal laser scanning microscopy. Blood samples and ovaries from BHV1 seronegative cows were used. The oocytes recovered were divided into two groups. Group I comprised COCs (n = 312) and denuded oocytes (n = 296), which were experimentally infected with BHV1 and incubated for 1 h at 38.5°C and 5% CO2. Group II comprised COCs (n = 425) and denuded oocytes (n = 405), which were co-incubated with BHV1 under the same conditions for 24 h. The negative control of these two groups was respectively subjected to the same protocol, except for exposure to BHV1. To our knowledge, this study provides the first evidence of BHV1 detection within COCs and denuded oocytes exhibiting intact zona pellucida when co-incubated with the virus for 24 h. Immunolocalization also confirmed the presence of BHV1 in the cytoplasm of the cumulus cells of all COCs exposed to the virus after both incubation periods. In conclusion, detection of BHV1 inside oocytes has a great meaning for the field of animal reproduction. The detection of BHV1 in different layers of cumulus cells also demonstrates that these cells are sources of viral infection.

Fig 1. Image of partial disintegration of cumulus cells obtained by stereoscope microscopy.

Experimental infection of bovine oocytes (O) with BHV1 showing partial disintegration of cumulus cells (CC). Magnification of 40x.

Fig 2. Images of COCs and denuded oocytes obtained by confocal laser scanning microscopy.

Experimental infection of bovine oocytes with BHV1 incubated for one and 24 h. (A) confocal image of COC negative control, (B) immunolabelling of BHV1 (green) in the cytoplasm of cumulus cells (CC) of co-incubated COCs for 1 h (Scale bar = 20 μm) and (C) immunolabelling of BHV1 in the cytoplasm of COC cumulus cells co-incubated for 24 h (Scale bar = 50 μm). O: oocyte. The nuclei of the cumulus cells were stained with TO-PRO 3 iodide (red). (D) denuded oocyte control. (E) denuded oocyte co-incubated with BHV1 for 1 h and (F) denuded oocyte co-incubated with BHV1 for 24 h (Scale bar = 20 μm).

Fig 3. Cumulus oocyte complex in a sequence of confocal Z-stack images.

Images of COC co-incubated with BHV1 for 24 h in a series of focal planes (step size of 1μm) evidencing the presence of BHV1 (green) inside the oocyte.

Fig 4. Confocal Z-stack image sequence of a denuded oocyte.

Images of a denuded oocyte co-incubated with BHV1 for 24 h in a sequence of different focal planes (step size of 1μm) elucidating the presence of BHV1 (bright green dots) within the oocyte and on the outer face of the zona pellucida.