Temporal and SUMO-specific SUMOylation contribute to the dynamics of Polo-like kinase 1 (PLK1) and spindle integrity during mouse oocyte meiosis.

During mammalian meiosis, Polo-like kinase 1 (PLK1) is essential during cell cycle progression. In oocyte maturation, PLK1 expression is well characterized but timing of posttranslational modifications regulating its activity and subcellular localization are less clear. Small ubiquitin-related modifier (SUMO) posttranslational modifier proteins have been detected in mammalian gametes but their precise function during gametogenesis is largely unknown. In the present paper we report for mouse oocytes that both PLK1 and phosphorylated PLK1 undergo SUMOylation in meiosis II (MII) oocytes using immunocytochemistry, immunoprecipitation and in vitro SUMOylation assays. At MII, PLK1 is phosphorylated at threonine-210 and serine-137. MII oocyte PLK1 and phosphorylated PLK1 undergo SUMOylation by SUMO-1, -2 and -3 as shown by individual in vitro assays. Using these assays, forms of phosphorylated PLK1 normalized to PLK1 increased significantly and correlated with SUMOylated PLK1 levels. During meiotic progression and maturation, SUMO-1-SUMOylation of PLK1 is involved in spindle formation whereas SUMO-2/3-SUMOylation may regulate PLK1 activity at kinetochore-spindle attachment sites. Microtubule integrity is required for PLK1 localization with SUMO-1 but not with SUMO-2/3. Inhibition of SUMOylation disrupts proper meiotic bipolar spindle organization and spindle-kinetochore attachment. The data show that both temporal and SUMO-specific-SUMOylation play important roles in orchestrating functional dynamics of PLK1 during mouse oocyte meiosis, including subcellular compartmentalization.

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.

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.

Sertoli cell expression of steroidogenic acute regulatory protein-related lipid transfer 1 and 5 domain-containing proteins and sterol regulatory element binding protein-1 are interleukin-1beta regulated by activation of c-Jun N-terminal kinase and cyclooxygenase-2 and cytokine induction.

In testicular Sertoli cells, IL-1beta regulates steroid, lactate, and transferrin secretion; although each influences germ cell development and spermatogenesis, little is known about the signaling mechanisms involved. In other cell types, IL-1beta potently induces reactive oxygen species and/or cyclooxygenase-2 (COX-2). In contrast, in Sertoli cells, IL-1beta does not generate reactive oxygen species, but rapidly phosphorylates c-Jun-NH(2)-terminal kinase (JNK), but not p44/42 or p38 MAPK. Phosphorylated JNK stimulates COX-2 activity, mediating the expression of ILs and steroidogenic acute regulatory (StAR)-related (StAR-related lipid transfer protein domain containing) proteins D1 and D5, but not D4. In a time- and dose-dependent manner, IL-1beta rapidly increases levels of COX-2 mRNA (2-fold); induction of COX-2 protein (50-fold) requires de novo protein synthesis. Concomitantly, increases in IL-1alpha, IL-6, and IL-1beta mRNAs (1-3 h) are observed. As StAR-related lipid transfer protein domain containing protein 1 (StARD1) mRNA decreases, StARD5 mRNA increases; substantial recovery phase induction of StARD1 mRNA above control is noted (24 h). Inhibition of JNK or COX-2 activities prevents IL-1beta induction of IL and StARD5 mRNAs and subsequent increases in StARD1 mRNA (24 h), indicating that these effects depend on the activation of both enzymes. StARD1 and D5 protein levels are significantly altered, consistent with posttranscriptional and posttranslational regulation. IL-1beta rapidly decreases levels of precursor and mature sterol regulatory element-binding protein-1, changes not altered by cycloheximide, suggesting coordinate regulation of StARD1 and -D5, but not StARD4, expression. These data demonstrate that JNK and COX-2 activities regulate Sertoli cytokines and particularly START domain-containing proteins, suggesting protective stress responses, including transcription and protein and lipid regulation, within this specialized epithelium.

Maintenance of spermatogenesis requires TAF4b, a gonad-specific subunit of TFIID.

The establishment and maintenance of spermatogenesis in mammals requires specialized networks of gene expression programs in the testis. The gonad-specific TAF4b component of TFIID (formerly TAF(II)105) is a transcriptional regulator enriched in the mouse testis. Herein we show that TAF4b is required for maintenance of spermatogenesis in the mouse. While young Taf4b-null males are initially fertile, Taf4b-null males become infertile by 3 mo of age and eventually exhibit seminiferous tubules devoid of germ cells. At birth, testes of Taf4b-null males appear histologically normal; however, at post-natal day 3 gonocyte proliferation is impaired and expression of spermatogonial stem cell markers c-Ret, Plzf, and Stra8 is reduced. Together, these data indicate that TAF4b is required for the precise expression of gene products essential for germ cell proliferation and suggest that TAF4b may be required for the regulation of spermatogonial stem cell specification and proliferation that is obligatory for normal spermatogenic maintenance in the adult.