Cintra, Thais G. et al. (2013) International Worm Meeting "PP1 phosphatases, GSP-3 and GSP-4, are required for chromosome segregation in sperm meiosis."

Beyene, Joseph, Chu, Diana, Wu, Jui-Ching, Cintra, Thais G.

[International Worm Meeting, 2013]

Proper meiotic chromosome segregation is crucial to maintaining the integrity of chromosomes and avoiding the inheritance of genetic disorders. Although oocyte and sperm both undergo meiosis during their development, each exhibit differences in molecular machinery and progression of meiotic divisions. In mouse and worms, key players in regulating sperm meiosis are sperm-specific PP1 phosphatases. For example, deletion of the genes encoding the C. elegans PP1 proteins, GSP-3 and GSP-4 (GSP-3/4), causes chromosome segregation defects during sperm meiosis. However, how GSP-3/4 regulates meiotic divisions is unclear. We have determined that GSP-3/4 localize in a manner similar to kinetochore proteins, which connect microtubules and chromosomes during meiotic divisions. Thus, we hypothesize that GSP-3/4 regulate chromosome-microtubule interactions and dynamics during spermatogenesis. Using live-imaging of strains expressing fluorescently-labeled histone and tubulin, we first defined the coordinated changes in chromosome and microtubule dynamics required for wild-type sperm meiotic divisions and then found specific features that are altered in gsp-3/4 mutants. These include defects in X-chromosome resolution and sister chromatid reorientation between meiosis I and II divisions, and sister chromatid segregation during meiosis II that result in multinucleate or anucleate sperm. We previously found the outer kinetochore CEN-P proteins, HCP-1 and HCP-2, are differentially localized in sperm and oocyte meiosis, suggesting HCP-2 may be important in mediating specific aspects of sperm meiotic divisions. Indeed, using immunolocalization we find that HCP-2 is mislocalized during the transition from meiosis I and II in gsp-3/4 mutants. Preliminary results also suggest other kinetochore components may be mislocalized. GSP-3/4 do not regulate the removal of the cohesin proteins REC-8 and COH-3/4 nor AIR-2, a key player in cohesin removal, as they are not mislocalized in gsp-3/4 mutants. Thus, GSP-3/4 specifically regulate changes in kinetochore localization required for the reorientation of sister chromatids during meiotic divisions necessary for successful sperm formation.

Cintra, Thais Godoy et al. (2011) International Worm Meeting "GSP-3 and GSP-4 are required for sperm meiotic chromosome segregation in C. elegans."

Go, Aiza, Cintra, Thais Godoy, Wu, Jui-Ching, Chu, Diana

[International Worm Meeting, 2011]

Errors in meiotic chromosome segregation during spermatogenesis have serious consequences: male infertility, embryonic lethality, and developmental abnormalities. Meiosis is distinctly regulated in spermatocytes and oocyes to control the developmental timing, size, and number of gametes produced. However, the molecular factors that differentiate chromosome segregation events during sperm meiosis from oocyte meiosis or mitosis are largely unknown. We have found that two nearly identical PP1 phosphatases in C. elegans, GSP-3 and GSP-4 (GSP-3/4) are required for male infertility and sperm meiosis. Using live-imaging of histone-GFP tagged strains deleted for both gsp-3/4, we find multiple defects in chromosome segregation during sperm meiosis. First, in the absence of GSP-3/4 during meiosis I, the partitioning of the X chromosome is delayed, causing the X chromosome to become stretched before finally resolving to one pole. Second, in the second meiotic division, sister chromatids are unable to segregate properly, forming bridged nuclei. These bridged nuclei either end up in the same spermatids or the residual body, a repository for cellular components, resulting in either multinucleated sperm or anucleated sperm, respectively. AIR-2 (Aurora kinase) and REC-8 (meiotic cohesin) are normally localized and removed in gsp-3/4 double mutant spermatids, confirming that cohesin removal does not contribute to chromosome segregation defects. Immunolocalization experiments show GSP-3/4 localization around sperm meiotic chromosomes in a pattern similar to that of kinetochore proteins, key connectors of chromosomes and microtubules. The chromosome segregation defects and localization pattern of GSP-3/4 suggest these PP1 phosphatases may regulate chromosome-microtubule dynamics. Thus, we are investigating microtubule dynamics during sperm meiosis in the absence of GSP-3/4. In addition, we are also characterizing the role of GSP-3/4 regulation of kinetochore components in sperm meiosis. In particular, we are investigating changes in HCP-2 and HCP-4 localization, as we have previously shown they are differentially enriched in sperm chromatin compared to oocyte chromatin.

Nera, Bernadette et al. (2009) International Worm Meeting "Characterizing the roles of enhanced RNAi genes eri-1 and eri-3 in sperm development and function."

Ratner, Dmitry, Cintra, Thais, Nera, Bernadette, Chu, Diana

[International Worm Meeting, 2009]

Generation of functional sperm depends on timely control of the gene expression at specific stages of spermatogenesis. RNA interference (RNAi) pathways are involved in regulating gene expression and are required for male fertility in other organsims. In C. elegans, enhanced RNAi mutants, eri-1 and eri-3, are male sterile. Our lab is interested in investigating the role of these genes in proper sperm formation. Previous studies have found eri mutants are sterile. However, it is unknown if the sterility is caused by a lack of germ cell production or if germ cells are produced but not functional. We have found that eri mutant hermaphrodites produce less progeny than wild type at both permissive and restrictive temperatures. At 25 deg C eri mutants did not produce any viable progeny but were still able to produce oocytes and sperm. This indicates that though eri mutant sperm cannot fertilize, they are capable of generating a signal for oocyte maturation. In order to analyze how and where sperm formation is defective, temperature shifted eri mutant and wild type males were fixed and stained with the DNA dye DAPI. Overall the progression of germ cell nuclei appears to be normal; however, we observed chromosomes during late pachytene appear more diffusely compacted. In addition we observed defects in sperm meiotic chromosome segregation, with the presence of abnormally sized and shaped sperm nuclei, suggesting defects in chromosome segregation or integrity. We are currently analyzing the localization of components of the core chromosome segregation machinery to investigate how eri-1 and eri-3 mutants are defect in chromosome segregation. RNAi is important for genomic integrity in other organisms such as zebrafish, mice, and flies where small RNAs are important for transposon silencing. To investigate genomic integrity in C. elegans, we analyzed double strand break formation and germ cell apoptosis in eri mutants. Our preliminary results show that at 25C eri mutants do not generate more double stranded breaks than wild-type.Thus, unlike RNAi components in other organisms, eri mutants may not play a role in transposon silencing in C. elegans. The link between the enhanced RNAi phenotype of the eri mutants and chromosome defects resulting in abnormal sperm suggests that sperm-specific microRNAs may play an important role in sperm development.

Quintanilla, Luis et al. (2015) International Worm Meeting "Phosphoregulation balance allows successful segregation of chromosomes during male meiosis."

Cintra, Thais, Quintanilla, Luis, Cota, Vanessa, Chu, Diana

[International Worm Meeting, 2015]

Sperm meiosis, a highly regulated process, relies on proper microtubule attachments and timely removal of cohesion for chromosome segregation to take place. Phosphoregualtion has been demonstrated to play a role in the stabilizing and destabilizing of microtubule attachments at the kinetochore, a multi-layered structure connected to chromosomes. One example can be seen in human mitotic studies, where AIR-2 promotes microtubule destabilization while PLK-1 promotes microtubule stabilization. In another case, kinase AIR-2's destabilizing activity is opposed by PP1 Phosphatase GSP-2 in C. elegans oocyte meiosis. Although phosphoregulation plays a role in meiosis, little is understood about its regulatory mechanisms specifically in sperm meiosis. Our lab has discovered two 98% identical sperm-specific PP1 Phosphatases called GSP-3 and GSP-4 (GSP-3/4). They colocalize with the kinetochore and are essential for sperm meiotic chromosome segregation. Deletion of these phosphatases leads to delayed resolution of the single X chromosome, failure of chromosome segregation at meiosis II, and aneuploidy. We thus hypothesize that GSP-3/4 are dually responsible for regulating kinetochore microtubule interactions and directly or indirectly mediating the removal of cohesion proteins. Preliminary results show mislocalization of outer kinetochore kinase, BUB-1 during anaphase I in GSP-3/4 mutants. BUB-1 is prematurely released in the mid-zone, possibly explaining the lack of sister chromatic separation during the second part of meiosis. Indeed, we also see mislocalization of other outer kinetochore proteins like HCP-2, which supports role of GSP-3/4 regulating kinetochore microtubule interactions. Furthermore, we find that localization of AIR-2, the cohesion and microtubule destabilizing kinase, behaves normally in GSP3/4 mutant males. These results suggest that cohesion proteins are not impaired. In fact, our preliminary results indicate that REC-8, COH-3, and COH-4 are not mislocalized. Surprisingly, we see that LAB-1, a cohesion protector protein, is absent on the lagging X chromosome during anaphase 1, but remains intact between autosomes. Though LAB-1 absence suggests that cohesion would be cleaved early, we see a delay in X chromosome resolution. Thus, GSP-3/4 regulation of cohesion requires further investigation. Lastly, our preliminary results investigating the role of PLK-1, which mediates centriole duplication and kinetochore attachments, show that centrioles duplicate and separate normally during male meiosis in gsp-3/4 mutants. Overall the data presented begins to shed light on the relationship between phosphatases and kinases at the kinetochore in sperm meiosis.

Cota, Vanessa et al. (2015) International Worm Meeting "Sex-specific features of kinetochore function during sperm meiosis."

Cota, Vanessa, Chu, Diana, Quintanilla, Luis, Cintra, Thais, Dana, Byrd

[International Worm Meeting, 2015]

In meiosis, germ cells undergo two meiotic chromosome segregation events to generate haploid gametes. While many events during chromosome segregation are similar in both sperm and oocyte meiosis, there are key features that are unique to sperm. In spermatogenesis, cell division is symmetrical, and sperm spindle microtubules are organized by centrosomes. We report here that sperm have unique kinetochore composition, dynamics, and regulation in C. elegans. Another distinct feature are the sperm-specific PP1 phosphatases GSP-3/4 -key regulatory proteins during sperm meiotic chromosome segregation. gsp-3/4 mutant males have defects in anaphase I, which takes nearly twice as long to complete when compared to wildtype sperm. In meiosis II, sister chromatids fail to segregate resulting in sperm aneuploidy. It is unclear what targets GSP-3/4 are regulating, or the reason for the segregation defects. One clue is that GSP-3/4 localize to chromosomes in a pattern similar to that of kinetochore components. Interestingly, kinetochore composition is different in sperm when compared to oocytes. Sperm inner kinetochores lack HCP-3 (CENP-A) and are enriched with HCP-4 (CENP-C), while oocyte kinetochores are composed of both. Furthermore, outer kinetochores in oocytes have two CENP-F proteins, HCP-1 and HCP-2, while sperm only rely on HCP-2. Using immunostaining, we show the outer kinetochore protein HCP-2, as well as NDC-80 and KNL-3, have distinct dynamics during sperm chromosome segregation. In oocyte meiosis, outer kinetochore components move between segregating chromosomes in anaphase I. In sperm meiosis, kinetochore proteins remain localized to chromosomes as they segregate. Furthermore, we show that these kinetochore components are dependent on the regulation of the sperm-specific PP1 phosphatases. In gsp-3/4 mutant males, we see a mislocalization of HCP-2, NDC-80 and KNL-3 during anaphase I and meiosis II. This defect is consistent with the timing of the segregation defect previously described. Simultaneously, microtubule-associated protein CLASP-2 also shows a defect in gsp-3/4 mutants, further supporting outer kinetochore function is compromised. Taken together these results support that sperm-specific features of kinetochore components are regulated by the sperm-specific PP1 phosphatases. .

Black, Christopher et al. (2017) International Worm Meeting "Sex-specific kinetochore features in sperm meiosis."

Black, Christopher, Cota, Vanessa, Cintra, Thais, Wu, Jui-Ching, Chu, Diana

[International Worm Meeting, 2017]

Chromosome segregation in sperm has unique features that make it different from oocyte meiosis. Oocytes undergo two rounds of asymmetrical chromosome segregation without centrosomes, while sperm have symmetrical divisions and utilize centrosomes to organize microtubule attachment to chromosomes. This attachment is mediated by a protein complex called the kinetochore, but the role of the kinetochore during sperm meiosis remains poorly understood. We report here that sperm-specific phosphatases GSP-3/4 may regulate kinetochore-microtubule connection to organize sister chromatid orientation from segregating to the same pole in meiosis I to opposite poles in meiosis II. gsp-3/4 mutant sperm exhibit delayed resolution of a univalent lagging X chromosome and sisters fail to separate. Therefore, I hypothesize that sperm uniquely require kinetochore-microtubule attachment/detachment and GSP-3/4 regulate detachment. Interestingly, GSP-3/4 localize in a kinetochore-like pattern, which suggests these phosphatases may regulate kinetochore function. Sperm also have distinct outer kinetochore localization patterns (KNL-1, KNL-3, and NDC-80), compared to oocytes. For example, similar to oocyte meiosis, KNL-1 and KNL-3 localize in a cup shape on chromosomes, but NDC-80 surrounds chromosomes only in sperm. A bigger difference between oocyte and sperm meiosis is kinetochore presence at anaphase. In oocytes, immunofluorescence results depicted the kinetochore disassembles from chromosomes during anaphase I. However, the kinetochore is retained during anaphase in sperm suggesting kinetochore importance for chromosome segregation. The question still remains, do kinetochores mediate the connection between chromosomes and microtubules in sperm? Recent super-resolution images of immunostained sperm in anaphase I show there is a gap between chromosomes and microtubules where NDC-80 localizes, indicating the kinetochore functions in sperm meiosis to connect chromosomes to microtubules during anaphase separation. Further, live imaging of H2B labeled DNA and GFP labeled tubulin show that sperm meiosis exhibits pole-chromosome shortening (anaphase A) in anaphase I, which contrast to oocyte meiosis and mitosis, which rely on anaphase B mechanisms. This result taken together with the gsp-3/4 mutant phenotypes of delayed lagging X resolution and sister separation failure, support GSP-3/4 role in detaching microtubules from kinetochores. GSP-3/4-regulated detachment would allow X resolution and sisters to shift orientation towards opposite poles. Microtubule detachment from kinetochores may be unique in sperm to reconcile the combination of centrosome organized microtubules and chromosomes going through two rounds of segregation.

Wu, Jui-ching et al. (2011) International Worm Meeting "Independent evolution of PP1 phosphatases for regulation of diverse sperm motility structures."

Chu, Diana, Mirosoian, Susan, Cintra, Thais, Wu, Tammy, Wu, Jui-Ching, Estrada, Rodrigo, Go, Aiza, Routman, Eric, Samson, Mark, Jow, Margaret

[International Worm Meeting, 2011]

The rapid evolution of male reproductive proteins has generated distinctive sperm motility structures, including tubulin-based flagella in mammals and Major Sperm Protein (MSP)-based pseudopods in nematodes. Despite such molecular divergence, we have found that sperm-specific PP1 phosphatases, which function in mouse sperm motility, are required for male fertility in C. elegans. We were thus interested to determine how GSP-3/4 can function in motility in species with morphologically distinct sperm. First, using genetic, molecular, and cell biological approaches in C. elegans, we find the sperm-specific PP1 phosphatases GSP-3 and GSP-4 (GSP-3/4) are functionally redundant and required for male fertility in worms. During meiosis, GSP-3/4 are required for proper sperm meiotic chromosome segregation and localize to sperm chromatin. Post-meiosis, GSP-3/4 shift localization from chromatin to pseudopods, suggesting GSP-3/4 are also required for motility. Consistent with this, gsp-3/4 mutant spermatids develop shorter pseudopods with greatly reduced pseudopod treadmilling activity and are thus immotile. GSP-3/4 spatially regulate MSP: they concentrate at the pseuodpodial base and regulate MSP disassembly during morphogenesis. Thus, GSP-3/4 regulate sperm motility through modulating MSP cytoskeleton dynamics during sperm activation. How have sperm-specific PP1 phosphatases come to play a central role in sperm motility function in mouse and nematodes given that GSP-3/4 regulate aspects of motility specific to nematode sperm? To determine if sperm-specific PP1 phosphatases in nematodes and mammals arose from a common ancestor that adapted sperm-specificity, we conducted a phylogenetic analysis of PP1 proteins in species from yeast to humans. We determined that sperm-specific PP1 phosphatases evolved in separate lineages and not from a common ancestor. Therefore, animals from a broad range of species, regardless of their sperm morphology, have separately adapted PP1 phosphatases as critical regulators of male reproduction.