Meiotic cohesin complexes are essential for the formation of the axial element in mice

Abstract

Cohesin is a conserved multisubunit protein complex that participates in chromosome segregation, DNA damage repair, chromatin regulation, and synaptonemal complex (SC) formation. Yeast, but not mice, depleted of the cohesin subunit Rec8 are defective in the formation of the axial elements (AEs) of the SC, suggesting that, in mammals, this function is not conserved. In this paper, we show that spermatocytes from mice lacking the two meiosis-specific cohesin subunits RAD21L and REC8 were unable to initiate RAD51- but not DMC1-mediated double-strand break repair, were not able to assemble their AEs, and arrested as early as the leptotene stage of prophase I, demonstrating that cohesin plays an essential role in AE assembly that is conserved from yeast to mammals.

Figure 1.

Absence of RAD21L and REC8 arrest mouse spermatogenesis in early prophase I. (A) dKO-kls mice show a 70% reduction in testes size compared with wild type. Similar reductions are observed in Rad21l^−/− and Rec8^−/− males. (B) Mutation of both Rad21l and Rec8 elicits an arrest of spermatogenesis at stage IV characterized by intermediate spermatogonia (arrows) in a representative section of a seminiferous tubule. Massive apoptosis of spermatocytes (condensed nuclei indicated by asterisks) and absence of mature spermatozoa/spermatids are observed in the dKO-kls tubules. A similar arrest is observed in seminiferous tubules from singly mutant Rad21l and Rec8 mice. (C) Immunolabeling for SYCP3 in spermatocytes from a wild-type mouse at early and late leptonema and spermatocytes arrested at a leptotene-like stage from a dKO-kls mouse. (D) dKO-kls spermatocytes arrested at the leptotene-like stage show absence of chromosomal synapsis. Double immunolabeling for SYCP3 and SYCP2 or SYCP1 shows SYCP3/SYCP2 aggregates without synapsis as indicated by the lack of SYCP1 labeling in double mutant spermatocytes. Spermatocytes from Rad21l^−/− (zygotene-like arrest), Rec8^−/− (zygotene-like arrest), and wild-type (zygotene stage) mice show AEs and synapsed LEs with stretches of SYCP1. Bars: (A) 5 mm; (B) 25 µm; (C and D) 100 µm.

Figure 2.

Meiotic cohesins are dispensable for DSB formation but not for normal loading of RAD51. (A) Double immunolabeling of SYCP3 and γ-H2AX. In wild-type spermatocytes, γ-H2AX labels the chromatin from leptonema to pachynema when the signal disappears from autosomal chromosomes and remains only on sex body chromatin (not depicted). In the single and dKO-kls mutants, γ-H2AX also labels leptotene chromatin. (B) Double immunolabeling of SYCP3 and RAD51. In wild-type spermatocytes and spermatocytes of single Rad21l or Rec8 mutants, RAD51 localizes to AEs/LEs at leptonema. In Rec8^−/− Rad21l^−/− spermatocytes, there is an 8.5-fold reduction in the number of RAD51 foci. Bars, 100 µm.

Figure 3.

Normal loading of DMC1 and RPA accumulation at leptonema-arrested dKO-kls spermatocytes. Double immunolabeling of SYCP3 and DMC1 or RPA. (A) In wild-type and dKO-kls spermatocytes, DMC1 protein localizes to AEs/LEs at leptonema. (B) In dKO-kls spermatocytes, there is a threefold increase in the numbers of RPA foci at the leptotene-like arrest in comparison with wild-type spermatocytes. Bars, 100 µm.

Figure 4.

Cohesin complexes in the absence of RAD21L and REC8. Double immunofluorescence of SYCP3 and either RAD21, SMC3, SMC1β, or STAG3. In wild-type leptotene spermatocytes, the cohesins RAD21, SMC3, SMC1β, and STAG3 colocalize with SYCP3 along the AEs of the chromosomes. In Rad21l and Rec8 single mutant leptotene spermatocytes, RAD21, SMC3, SMC1β, and STAG3 colocalize also with SYCP3 along the AEs/LEs of the chromosomes. In spermatocytes from dKO-kls, arrested at leptonema, SMC1β and STAG3 are not detected by immunofluorescence, whereas immunolabeling for RAD21 and SMC3 renders robust and very faint fluorescence signals, respectively. Bar, 100 µm.

Figure 5.

dKO-kls spermatocytes are proficient for sister chromatid cohesion. Hybridization of two DNA probes to the X and Y chromosomes renders a single dot signal for each probe in both wild-type and dKO-kls spermatocytes. Clear FISH signals for both probes can be observed in 63% of the 90 leptonema-like cells analyzed. Bar, 100 µm.