Small ubiquitin-related modifier (SUMO)-1, SUMO-2/3 and SUMOylation are involved with centromeric heterochromatin of chromosomes 9 and 1 and proteins of the synaptonemal complex during meiosis in men

Abstract

Background: Post-transcriptional modification by SUMOylation is involved in numerous cellular processes including human spermatogenesis. For human male meiosis, we previously showed that the small ubiquitin-related modifier-1 (SUMO-1) protein localizes to chromatin axes in early pachytene spermatocytes, then to kinetochores as meiosis progresses. Here, we delineate possible functional roles based on subcellular localization for SUMO-1 and SUMO-2/3.

Methods: Western and immunoprecipitation analyses were conducted on proteins isolated from the testis of two normal adult fertile men. Combinatorial immunofluorescence and chromosome-specific fluorescence in situ hybridization analyses were performed on male meiocytes obtained during testicular biopsy from four patients undergoing testicular sperm extraction for assisted reproduction technologies.

Results: The synaptonemal complex (SC) and SC proteins (SCP)-1 and SCP2, but not SCP3, are SUMOylated by SUMO-1 during the pachytene substage. Likewise, two distinct localization patterns for SUMO-1 are identified: a linear pattern co-localized with autosomal SCs and isolated SUMO-1 near the centromeric heterochromatin of chromosomes 9 and 1. In contrast to SUMO-1, which is not detectable prior to pachytene in normal tissue, SUMO-2/3 is identified as early as leptotene and zygotene and in some, but not all, pachytene cells; no linear patterns were detected. Similar to SUMO-1, SUMO-2/3 localizes in two predominant subnuclear patterns: a single, dense signal near the centromere of human chromosome 9 and small, individual foci co-localized with autosomal centromeres.

Conclusions: Our data suggest that SUMO-1 may be involved in maintenance and/or protection of the autosomal SC. SUMO-2/3, though expressed similarly, may function separately and independently during pachytene in men.

Figure 1:

During early pachytene, SUMO-1 is observed in three different patterns. Human spermatocytes were analyzed for SUMO-1 (green), SC protein (SCP)3 (red; B and C, F–H, J and K) and centromeres (blue). (A) SUMO-1 forms light, dotted linear structures similar to those identified as lateral SC elements by SCP3. (B) SUMO-1 seen in linear structures in early human pachytene spermatocytes. Merging of both SUMO-1 and SCP3 (C) showed co-localization on autosomal chromosomes. SUMO-1 was co-localized with the SC only at synapsed regions; asynapsed SCP3 negative regions are also SUMO-1 negative (inset of individual autosomal chromosome in C). The paired sex body is also devoid of SUMO-1 (arrowhead), except at the pseudo-autosomal region, where there is a small region of SUMO-1 (asterisk). (D) Another early pachytene spermatocyte that SUMO-1 displays linear structures in the absence of SCP3. X and Y chromosomes are labeled by BRCA-1 (red). In a second subset of human pachytene spermatocytes, SUMO-1 is consistently observed as a large, dense focus at or near the centromere of chromosome 9. (E) SUMO-1 has one large, extremely bright signal, which when merged with the SCP3 and centromere signals (G) is found to associate at the centromeric region of an autosomal chromosome. In this cell, SUMO-1 also has three lighter, secondary signals that also associate near autosomal centromeres and two small foci that are co-localized with the sex body. Identification of human chromosomes 1 and 9 by chromosome-specific FISH analysis demonstrate that the largest and brightest SUMO-1 signal in E is always associated at or near the centromeric region of chromosome 9 (magenta FISH signal in H). The second FISH signal, that of human chromosome 1 (white; H) shows a co-localization of chromosome 1 with the largest secondary SUMO-1 focal association. Additionally, merging of the SUMO-1 image with SCs and centromeres showed that lighter secondary SUMO-1 signals were associated with unidentified human acrocentric chromosomes (arrow). In a fourth mid-pachytene human spermatocyte (I–K), a third distinct SUMO-1 pattern can be resolved: linear structures co-localized with synapsed SCs, a single focal association at chromosome 9 and co-localized centromeric signals on all autosomal chromosomes. All images, ×1000.

Figure 2:

The human SC and proteins SCP1 and SCP2, but not SCP3, are SUMOylated in the testis of normal adult men. Individual protein extracts were prepared from the whole testis of two normal adult men (hT₁ and hT₂); Ø, no-protein extract but IP experimental procedures in parallel; neat extract: -, no IP; hT₁, normal human testis protein, 1 mg sample split for IP [500 µg with anti-SUMO-1 (Zymed) or anti-SCP1 (Novus) antisera]. Illustrated are the results following transfer of proteins electrophoresed using a 3–8% polyacrylamide gel. Human SCP1 (lanes 5 and 6: predicted/observed 114 kDa; ∼130 kDa observed, lanes 2 and 4); human SCP2 (predicted, 176 kDa; observed, ∼270 kDa, lanes 2, 4–6); human SCP3 (predicted/observed 34 kDa, lanes 2, 4–6). Lower inset: Western analysis, lanes 5 and 6; detection of SCP3 using Novus antisera in the non-IP samples hT₁ and hT₂, different exposure; companion lanes 2 and 4 data are not shown due to non-linear overexposure. Both β-actin and SCP-1 were equally detected in pre-IP samples (not shown).

Figure 3:

SUMO-2/3 is identified in human pachytene spermatocytes. Meiotic spermatocytes were triple-labeled for SUMO-2/3 (green), SCP3 (red) and centromere (blue). SUMO-2/3 is detected in a scattered pattern in meiosis as early as leptotene (A). In some pachytene cells (B–F), SUMO-2/3 (alone in B, merged with centromeres in C, and merged with SCs and centromeres in E) appears as a bright, dense cloud at, or near, the centromere of human chromosome 9 (magenta FISH signal in F); smaller and lighter signals associate with the sex body (arrowhead) and the centromeric region of an unidentified chromosome. In a second pachytene cell (G–L), SUMO-2/3 forms small punctate foci similar to meiotic centromeres (G and J); merging the two images (I and L) shows complete co-localization of both signals at all centromeres. This cell also has a small, brightly stained focal association (arrow). SCs and centromeres are shown in D and K, respectively. All images, ×1000.