Centriolar defects, centrin 1 alterations, and FISH studies in human spermatozoa of a male partner of a couple that produces aneuploid embryos in natural and artificial fertilization

Abstract

Purpose: To study the potential paternal contribution to aneuploidies in the man of a couple who obtained trisomic embryos with natural and assisted fertilization.

Methods: Semen analysis, immunofluorescence for localization of tubulin and centrin 1, transmission electron microscopy (TEM), and fluorescence in situ hybridization (FISH) analysis for chromosomes 18 and 9 were performed. Sperm of fertile men were used as controls.

Results: The percentages of sperm motility and normal forms were decreased. The percentages of sperm with tail reduced in dimension, headless tails, coiled tails, and altered head-tail junction were significantly higher (P < 0.01) in the patient than in controls, whereas the percentage of sperm with a normal centrin 1 localization (two spots in the centriolar area) was significantly reduced (P < 0.01) in the patient. Immunofluorescence with anti-tubulin antibody showed that in most of the patient's sperm connecting pieces (83.00 ± 1.78%), two spots were present, indicating prominent proximal centriole/centriolar adjunct and evident distal centriole, whereas controls' sperm displayed a single spot, indicating the proximal centriole. The percentage of sperm with two spots was significantly higher (P < 0.01) in the patient than in controls. TEM analysis showed that centriolar adjuncts of the patient's sperm were significantly longer (721.80 ± 122.26 nm) than in controls' sperm (310.00 ± 64.11 nm; P < 0.001). The aneuploidy frequencies of the patient's sperm, detected by FISH analysis, were increased with respect to controls.

Conclusion: A paternal contribution to sperm aneuploidies cannot be excluded since the patient's sperm showed altered morphology, immature centriolar adjunct, presence of evident distal centriole, scarce presence of centrin 1, and high aneuploidy frequency.

Keywords: Aneuploidies; Centrin 1; Centriolar adjunct; FISH; Sperm; TEM.

Fig. 1

UV micrographs of the patient’s sperm (a, b, c) and control’s sperm (d) treated with a monoclonal antibody anti-tubulin. The patient’s sperm show normal length flagella and coiled flagella. The two spots (arrows) located in the connecting piece probably represent a prominent proximal centriole/centriolar adjunct (the spot closer to the nucleus and perpendicular to the tail midline) and an evident distal centriole. In figure c a spermatozoon with an evidently altered head-tail junction (asterisk) is shown. A normal spermatozoon of a fertile man shows a tiny spot representing the proximal centriole (d). The nuclei are stained with DAPI. Bars: 6 μm

Fig. 2

UV micrographs of control’s sperm (a) and the patient’s sperm (b) treated with a monoclonal antibody anti-centrin 1. The normal labelling of centrin 1 (a) consists in two spots localized at the base of the head in the centriolar area. Spermatozoa of the patient showed an almost totally lack of labelling (b). The nuclei are stained with DAPI. Bars: 6 μm

Fig. 3

TEM micrographs of longitudinal sections of the patient’s spermatozoa. A centriolar adjunct increased in length (arrow) and located in a wide cytoplasmic residue with coiled tail is shown in figure a. A spermatozoon with bent tail and broken head-tail junction (asterisk) is show in figure b. The asterisk in figure c indicates a perfect longitudinal section of a proximal centriole. In figure d the dotted outline follows the extension of a centriolar adjunct increased in length. The arrow indicates the area where some microtubules of distal centriole are visible. Bars: 2 μm (a,b); 200 nm (c, d)

Fig. 4

TEM micrographs of longitudinal sections of the patient (a) and controls’ spermatozoa (b). In figure a, the connecting piece is characterized by a centriolar adjunct increased in length (asterisk) and a concomitant defect in head-tail attachment (arrow). Figure b shows a section of a spermatozoon without centriolar adjunct of control sample. The section plausibly represents the area where the centriole adjunct can be located, if present. Bars: 700 nm (a), 400 nm (b)

Fig. 5

UV micrographs of the patient’s spermatozoa hybridized with probes for chromosome 18 (green) and 9 (red). Disomy of chromosome 18 (a) and chromosome 9 (b, c) are shown. Sperm nuclei are stained with DAPI (blue). Bars: 2 μm (a, b); 4 μm (c)