The effect of Robertsonian translocations on the intranuclear positioning of NORs (nucleolar organizing regions) in human sperm cells

Abstract

Only a few studies have described sperm chromosome intranuclear positioning changes in men with reproductive failure and an incorrect somatic karyotype. We studied the influence of Robertsonian translocations on the acrocentric chromosome positioning in human sperm cells. The basis of the analysis was the localization of NORs (nucleolar organizing regions) in sperm nuclei from three Robertsonian translocation carriers, namely, rob(13;22), rob(13;15) and rob(13;14), with a known meiotic segregation pattern. All three carriers presented with a similar percentage of genetically normal sperm cells (i.e., approximately 40%). To visualize NORs, we performed 2D-FISH with directly labelled probes. We used the linear and radial topologies of the nucleus to analyse the NORs distribution. We found an affected positioning of NORs in each case of the Robertsonian translocations. Moreover, the NORs tended to group, most often in two clusters. Both in Robertsonian carriers and control sperm cells, NORs mostly colocalized in the medial areas of the nuclei. In the case of the Roberstonian carriers, NORs were mostly concentrated in the peripheral part of the medial area, in contrast to control sperm cells in which the distribution was more dispersed towards the internal area.

Figure 1

A scheme of the sperm cell nucleus areas used for the localization of FISH signals from NORs (nuclear organizing regions). (A) Linear division. The division of the sperm cell nucleus into three longitudinal areas was marked as follows: A = apical, closer to the acrosome; M = medial; and T = area close to tail. (B) Radial division. The division of the sperm cell nucleus into nine radial areas was marked as follows: (c1) = central; (c2 + c3) = internal; (p1 + p3) = peripheral, near the apical area; (p2 + p4) = peripheral, near the tail area; (a) = apical area; and (t) = tail area.

Figure 2

Percentage (%) of sperm cells with NORs localizations (recognized as FISH signals) according to schemes No. 1 – 23 (for representative images of FISH, see Suppl. Fig. S1). One-way ANOVA was used to compare results (values p ≤ 0.01 was considered to be significantly different). *Values were significantly different from the Control value (p < 0.01). ^HNr of the scheme, the frequency of sperm cells was the highest (p < 0.001) in R1, R2, R3 and Control. ¹Significantly different from the R1 and R2 results. ²Significantly different from the R1 result. ³All of the values of R1, R2 and R3 were significantly different. ⁴Significantly different from the R2 and R3 results. ⁵All values R1, R2 and R3 were significantly different. ⁶Significantly different from R1 result. ⁷All of the values of R1, R2 and R3 were significantly different. ⁸Significantly different from the R1 and R3 results. ⁹Significantly different from the R2 and R3 results. ¹⁰All of the values the R1, R2 and R3 were significantly different. ¹¹All of the values of R1, R2 and R3 were significantly different. ¹²All of the values of R1, R2 and R3 were significantly different. ¹³Significantly different from the R1 and R2 results. ¹⁴Significantly different from the R1 and R2 results.

Figure 3

Illustration of the results of the radial localization of NORs in sperm cells from Robertsonian translocation carriers (R1, R2 and R3) and the Control. Values based on the data from Table 6. Values in dark grey mark areas, including (p1 + p3) and in the case of the Control also (c2 + c3), were significantly higher, while the values in the white areas (t) were found to be the lowest (data in Table 6). The division of the sperm cell nucleus into radial areas according to the scheme in Fig. 1B.

Figure 4

Illustration of the differences in the radial localization of NORs in sperm cells from Robertsonian translocation carriers (R1, R2 and R3) compared to the Control (values based on the data from Table 6). The red background illustrates areas with results that were significantly different from the Control values (data in Table 6). The division of the sperm cell nucleus into radial areas according to the scheme shown in Fig. 1B.