( J and K) At 17.5 dpc, the numbers of oocytes were comparable between Spdya +/+ ( J) and Spdya −/− ovaries ( K). ( H and I) By PD75, most of the Spdya −/− seminiferous tubes were depleted of spermatocytes (arrowhead). ( F and G) By PD18, the number of spermatogonia was comparable between Spdya +/+ ( F) and Spdya −/− testes ( G), but some seminiferous tubules showed depletion of spermatocytes in Spdya −/− testes ( G, arrowhead). ( D and E) At PD7, the number of spermatogonia was comparable between Spdya +/+ and Spdya −/− testes as indicated by DDX4 staining. ( C) Testes from PD35 Spdya +/+ and Spdya −/− male mice. ( B) Ovaries from PD35 Spdya +/+ and Spdya −/− female mice. β-Actin was used as the loading control, and 40-μg lysate from PD25 Spdya +/+ and Spdya −/− testes was loaded in each lane. ( A) Western blot showing that Speedy A was deleted in PD25 testes. Loss of Speedy A leads to depletion of male and female germ cells. The experiments were repeated more than three times.įig. ( D) RT-PCR detection of Spdya in female primordial germ cells and germ cells isolated from different stages of the embryonic ovary by FACS, showing that Speedy A was absent at 11.5 dpc, up-regulated at 14.5 and 17.5 dpc, and then down-regulated at 18.5 and 19.5 dpc.
The experiments were repeated more than three times each. For A–C, β-actin was used as the loading control, and 40-μg lysate was loaded in each lane. ( C) Western blots for Speedy A and Cdk2 in testes of different ages showing that Speedy A started to be highly expressed at PD12, which was concurrent with p39 Cdk2 up-regulation. ( B) Western blot of Speedy A and Cdk2 in isolated male germ cells, demonstrating that Speedy A was expressed at high levels in the preleptotene stage and decreased afterward, whereas the expression of the 39-kDa isoform of Cdk2 increased from the preleptotene to the pachytene stage. ( A) Western blot of Speedy A in different tissues indicating that Speedy A was specifically expressed in testes and embryonic ovaries. Speedy A is specifically expressed in meiotic germ cells at prophase I. Results Speedy A Is Specifically Expressed in Meiotic Germ Cells and Is Localized to Telomeres.įig. Rather, the binding between Speedy A and Cdk2 might mediate the initial assembly of the telomere–NE complex that is essential for meiotic prophase I progression.
NUCLEAR ENVELOPE FRAGMENTS MEANING ACTIVATOR
These results suggest that the function of Speedy A goes beyond that of a noncanonical activator of Cdk as previously suggested ( 14, 15, 18, 19).
Furthermore, we showed that the binding of Cdk2 to Speedy A is required for Cdk2 to localize onto telomeres. Moreover, we found that the proper attachment of telomeres to the NE is dependent on a telomere localization domain (TLD) on Speedy A, as defined by this study, whereas the C terminus of Speedy A, which is required for Cdk2 activation, is dispensible for the attachment of telomeres to the NE. Loss of Speedy A in mice impairs telomere–NE attachment in early meiosis, perturbs homologous recombination, and leads to infertility in both sexes. Instead, Speedy A is only specifically expressed in male and female germ cells at meiotic prophase I, and it is localized to the telomeres. We found that distinct from Xenopus, Speedy A in mice is not involved in oocyte maturation. In the present study, we generated Spdya knockout mice and studied the functional roles of Speedy A in mammals. Our results thus indicate that Speedy A and Cdk2 might mediate the initial telomere–NE attachment for the efficient assembly of the telomere complex that is essential for meiotic prophase I progression. However, Speedy A-Cdk2–mediated telomere–NE attachment is independent of Cdk2 activation. Furthermore, we found that the binding of Cdk2 to Speedy A is indispensable for Cdk2’s localization on telomeres, suggesting that Speedy A and Cdk2 might be the initial components that are recruited to the NE for forming the meiotic telomere complex. In addition, we have identified a telomere localization domain on Speedy A covering the distal N terminus and the Cdk2-binding Ringo domain, and this domain is essential for the localization of Speedy A to telomeres. Deletion of Spdya in mice disrupts telomere–NE attachment, and this impairs homologous pairing and synapsis and leads to zygotene arrest in male and female germ cells. Here we show that Speedy A, a noncanonical activator of cyclin-dependent kinases (Cdks), is specifically localized to telomeres in prophase I male and female germ cells in mice, and plays an essential role in the telomere–NE attachment. Telomere attachment to the nuclear envelope (NE) is a prerequisite for chromosome movement during meiotic prophase I that is required for pairing of homologous chromosomes, synapsis, and homologous recombination.