PREPARACIÓN DEL MÓDULO RASPBERRY

A theoretical model of meiotic drive during MI relies on three components: i) asymmetric cell division, ii) functional heterozygosity of a driving locus, and iii) asymmetric MI spindle (Pardo-Manuel de Villena and Sapienza, 2001a). Whereas asymmetric meiotic spindles were described in various species of plants and animals (Twell, 2011; Jones, 1991; Cortes et al., 2015), such asymmetry has not been reported in mammalian systems that exhibit meiotic drive (Pardo-Manuel de Villena and Sapienza, 2001; de Villena and Sapienza, 2001; Cattanach, 1962; Kaufman, 1972; LeMaire-Adkins and Hunt, 2000). It was thus not clear whether such asymmetry exists in mammals and if it does, how it contributes to meiotic drive. Studying meiotic spindles in mouse oocytes represents a system in which we can study the eventual mechanisms underlying the spindle asymmetry to better understand the meiotic drive mechanism.

Spindles can exhibit different types of asymmetries (differences in spindle pole- associated or MT-associated proteins, differences in MT length and dynamics) that can be tested by different assays. Since biased chromosome segregation depends on

interaction between centromeres and spindle MTs, we focus on kinetochore MTs that bind to centromeres region of chromosomes. To visualize these MTs, we performed cold stable assay by exposing oocytes from a standard laboratory strain (CF-1) to an ice cold media (Figure 25 A). Under such conditions only stable MTs are preserved whereas the

95

others depolymerize (Rieder, 1981). Analyzing tubulin signal in cold treated and fixed MI oocytes, we observed that MTs closer to the cortex are more stable (stronger tubulin signal) than MTs farther from the cortex (weaker tubulin signal). In contrast, oocytes that have centered spindles do not exhibit any asymmetry in MTs (Figure 25 A). To exclude the possibility that this result is a staining artifact, we treated the oocytes with taxol, a MT stabilizing drug. We observe that taxol treatment abolishes MT asymmetry,

demonstrating that the asymmetry is caused by differences in MT dynamics (Figure 25 A, B). We calculated the asymmetry for each spindle (Figure 25 C) and plotted it against the distance from the cortex. The results imply that spindles closer to the cortex exhibit stronger asymmetry in MTs than spindles farther from the cortex (Figure 25 D), consistent with our interpretation that such asymmetry is driven by proximity to the cortex.

Two parts of the meiotic drive model: i) formation of the asymmetric MI spindle, and ii) orientation of the asymmetric spindle towards the cortex, can be explained, if such asymmetry is induced by the cortex. Under that model, Rb fusion attaches to the more stable MTs from the spindle side that is closer to the cortex and becomes polar body. To test how the cortex affects spindle asymmetry, we manipulate spindle position by treating the oocytes with latrunculin, an actin depolymerizing drug. Since actin cytoskeleton drives spindle cortical positioning in unperturbed conditions (Schuh and Ellenberg, 2008), we predict that such treatment will generate mostly oocytes with centered spindles. Consistent with our prediction, we observe that spindles in latrunculin treated

96

oocytes are farther from the cortex and are mostly symmetrical (Figure 25 D). Our results are consistent with the model that MI spindle asymmetry depends on cortical proximity.

Cortical MI spindle positioning is maintained in oocytes with cumulus cells (COCs)

Understanding the importance of cortical spindle positioning relies on a system in which we can both experimentally perturb such spindle positioning, and test how that affects meiotic drive. It was previously shown that oocyte with cumulus cells (COCs) maintain the nucleus and MI spindle cortically localized through the entire MI. Removing the cumulus cells from COCs (denuding) leads to centered positioning of nuclei and MI spindles (Barrett and Albertini, 2010). Consistent with these previous findings, we observe that nuclei are cortically positioned in COCs, but they are centered in denuded oocytes (Figure 26 A). To assess spindle position, we measure the distance of nuclei or MI spindles from the cortex in oocytes fixed at various times in MI in vivo and in vitro with or without cumulus cells (denuded oocytes). Consistent with the results from nuclei positioning, MI spindles are positioned closer to the cortex in in vivo and in vitro matured COCs during MI. However, such spindle distance from the cortex is larger in denuded oocytes during the time when chromosomes congress to metaphase plate and form stable MT attachments, 4-6 h after nuclear envelope breakdown (Davydenko et al., 2013; Kitajima et al., 2011) (Figure 26 B, C). During that time, spindles are positioned close to the cortex and exhibit asymmetry in MTs that is likely to bias the orientation of the trivalent resulting in preferential segregation in MI. Overall, either keeping or

97

removing the cumulus cells from COCs provides us with an experimental system to manipulate the MI spindle position.

Spindle cortical positioning drives preferential trivalent orientation and Rb fusion segregation

Preferential Rb fusion segregation is a result of biased trivalent orientation on an asymmetric MI spindle. Since such asymmetry correlates with the spindle proximity to the cortex, we manipulate the spindle position to test if it affects the trivalent orientation. To test the importance of spindle positioning in meiotic drive, we use mouse oocytes with a single Robertsonian (Rb) fusion chromosome that preferentially segregates to the polar body (Chmátal et al., 2014). The Rb chromosome pairs with two unfused homologous telocentric chromosomes to form a trivalent. There are two outcomes of balanced trivalent segregation in MI (Figure 27 A). Either the Rb fusion segregates to the polar body while two homologous telocentrics stay in the egg (i), or the Rb fusion stays in the egg and the two telocentrics are lost in the polar body (ii). We measure Rb fusion

segregation (Chapter 2 – Figure 6) under the conditions where we can manipulate spindle asymmetry (COCs and denuded oocytes). We detect drive of Rb fusion in in vivo and in vitro matured COCs, when MI spindles maintain the cortical position, but no drive is detected in denuded oocytes with more centered spindles (Figure 27 B).

We observe preferential trivalent orientation in COCs, with Rb fusion centromere facing the cortical pole and two centromeres of telocentrics facing the egg pole (Figure 27 C, D). In contrast, we observe a random orientation of the trivalent in denuded oocytes

98

(Figure 27 D), consistent with the measurement of Rb fusion segregation (Figure 27 B). Overall our data show that trivalent orientation on the MI spindle and segregation during anaphase I depends on cortically positioned MI spindles.

99

DISCUSSION

Our data provide experimental evidence for a model in which preferential Rb fusion segregation is driven by MI spindle asymmetry (Figure 27 E). Under that model, spindles maintain their cortical position in oocytes with COCs so that MTs closer to the cortex are more stable and MTs that are farther from cortex are less stable. Such

preferential trivalent orientation likely happens before 6 h after nuclear envelope

breakdown, before stable kinetochore-MT (kMT) attachments are formed (Davydenko et al., 2013, Kitajima et al., 2011). Consistently, a random trivalent orientation is achieved on symmetrical spindles. In summary, preferential segregation of Rb fusion is achieved when kMT-attachments form in an asymmetric MI spindle.

Nevertheless two intriguing questions still remain: i) how do cumulus cells regulate the cortically positioned spindles?, ii) how does the cortex induce the spindle asymmetry? It is possible that cumulus cells maintain spindle cortical position by controlling translation of specific maternal mRNA in oocytes (Chen et al., 2013), or alternatively by direct signaling through oocyte-cumulus cell junctions. Although it is unknown what regulates the MT asymmetry, it is likely independent of cumulus cells, because denuded oocytes have asymmetrical spindles after they migrate towards the cortex. MT asymmetry could be regulated by several complementary mechanisms: 1) the actin cap, a structure

mediating the contact between spindle and oocyte cortex, potentially binding modifiers of MT dynamics (tubulin tyrosinases and de-tyrosinases) (Barisic et al., 2015); 2)

100

kinase that might freely diffuse in oocyte center, but could be limited in proximity to the cortex (Gruss and Vernos, 2004; Lampson and Cheeseman, 2011). Although the role of these mechanisms in inducing spindle asymmetry is unknown, trivalent preferential orientation within such asymmetric spindle can be achieved by two mechanisms that are not mutually exclusive; 1) either capturing the stronger centromeres by less stable MTs (egg pole), or 2) by favoring the more stable trivalent orientation (Figure 28 C). Testing between these models will be a focus for future study.

101

EXPERIMENTAL PROCEDURE

Oocyte collection and culture

Mouse strains were purchased from the Jackson Laboratory [Rb(6.16)24Lub, stock# 000885] or Harlan NSA (CF-1, solicitation # DL3171940). For collecting the oocytes in meiosis I (MI), female mice (7-14 wk-old) were hormonally primed with 5U of Pregnant Mare Serum Gonadotropin (PMSG, Calbiochem, cat# 367222) 48 h prior to oocyte collection. To measure meiotic drive, MII eggs were collected 12 – 12.5 h after 5U of hCG (human chorionic gonadotropin, Sigma, cat# C1063-1VL) injection, 48 h after PMSG injection, and treated with 100 μM monastrol for 2 h (no oil cover) (Chmátal et al., 2014). Germinal vesicle (GV)-intact oocytes were collected in bicarbonate-free minimal essential medium with polyvinylpyrrolidone and Hepes (MEM-PVP)(Stein and Schindler, 2011), denuded from cumulus cells, and cultured in Chatot-Ziomek-Bavister (CZB) (Chatot et al., 1989) medium covered with mineral oil (Sigma, cat# M5310) in a humidified atmosphere of 5% CO2 in air at 37°C. To collect and culture oocytes with

COCs, IVM medium was used (10mL: 1mL 10% FBS; 0.005 g D-glucosamine, 12 μL 0.5M L-cystein, 20 μL 10μM ascorbatem 100 μL 2mM glutamine, 380 μL 5.25U/mL

horse FSH, 100 μL 1% insulin-transferrin-selenium, 8.388 mL CZB medium). Meiotic resumption was inhibited by addition of 2.5 µM milrinone. Milrinone was subsequently washed out to allow meiotic resumption 1.5 h after collecting. Oocytes were checked for nuclear envelope breakdown (GVBD) 1.5 h after the washout, and those that did not enter

102

or latrunculin (5 μM , 30 min) before the anaphase I, 7-7.5h after meiotic resumption (no oil cover). All animal experiments were approved by the Institutional Animal Care and Use Committee and were consistent with the National Institutes of Health guidelines.

Oocyte microscopy and live imaging

CF-1 oocytes at GV stage were denuded and imaged live every 10 min for 4 h using DIC optics. Oocytes were placed into 3.5 µl drops of CZB media with milrinon covered with mineral oil in a 35 mm tissue culture dish (FluoroDish FD35-100) in a heated environmental chamber with a stage top incubator (Incubator BL and Heating Insert P; PeCon GmBH) to maintain 5% CO2 in air and 37°C. Confocal images were

collected with microscope (DMI4000 B; Leica) equipped with a 63× 1.3 NA glycerol- immersion objective lens, an xy piezo Z stage (Applied Scientific Instrumentation), a spinning disk confocal scanner (Yokogawa Corporation of America), an electron

multiplier charge-coupled device camera (ImageEM C9100-13; Hamamatsu Photonics), and an LMM5 laser merge module with 488- and 593-nm diode lasers (Spectral Applied Research) controlled by MetaMorph software (Molecular Devices).

Oocyte immunocytochemistry

Oocyte with intact nuclei or MI oocytes (+/- COCs) in different stages during meiosis I were cultured for a given time (prior to nuclear envelope breakdown, 2, 4, 6 h after NEBD) and denuded from COCs prior to the fixation. Oocytes were fixed in freshly prepared 2% paraformaldehyde in PBS with 0.1% Triton X-100, pH 7.4, for 25 min at

103

room temperature, placed in blocking solution (PBS containing 0.3 % BSA and 0.01% Tween-20) overnight at 4°C, permeabilized in PBS with 0.3% BSA and 0.1% Triton X- 100 for 20 min, washed in blocking solution for 20 min, incubated 1 h with primary antibodies in blocking solution, washed 3 times for 15 min, incubated 1 h with secondary antibodies, washed 3 times for 15 min, and mounted in Vectashield (Vector, cat# H- 1400) with bisbenzimide (Hoechst 33342, Sigma-Aldrich) to visualize chromosomes. Primary antibodies were human CREST autoimmune serum (1:100 dilution,

PerkinElmer), rabbit anti-β-tubulin (9F3) monoclonal conjugated to Alexa Fluor 488 (1:75 dilution; Cell Signaling Technology), or rabbit anti-mouse AURKA (1:500, Bethyl, cat # A300-072A). To visualize actin a phalloidin-A633 (1:500) was used (Life

technologies, cat#: A12381). Secondary antibodies were Alexa Fluor 594–conjugated goat anti–human or donkey anti-rabbit (1:500, Invitrogen). Confocal images were collected as z-stacks at 0.4 µm intervals to visualize the entire meiotic spindle, using the spinning disc confocal microscope described above.

Tubulin signal in MI spindles was quantified from maximal intensity z-projection images by drawing a line-scan (thickness = 50) from the cortical to the egg pole. A minimum corresponding to the DNA at metaphase plate was calculated and integrated tubulin signal for each part of the spindle was calculated. An asymmetrical index for each spindle was calculated as a fraction between the integrated signals from cortical vs. the egg spindle part. GV or MI spindle distance from the cortex was measured as the shortest distance of the nucleus (DNA signal) or spindle (tubulin signal) from the cortex (phalloidin signal) at the individual z-stack when such distance was shortest.

104

Trivalent orientation was scored in MI oocytes with COCs (mature in vivo, n = 25) and in denuded oocytes (matured in vitro, n = 23). There were two possible orientation of trivalent in balanced conformation: i) fusion centromere closer to the cortex, ii)

telocentric centromeres close to the cortex. Only oocytes with detectable trivalent in MI spindles close to the cortex were selected for the analysis.

105 Figure 25

106

Figure 25. Meiosis I spindle is asymmetric in microtubule stability that correlates with the proximity to the cortex. (A)CF-1 oocytes with MI spindles cortically

localized, centered, and treated with taxol (10 μM, 10 min), were subjected to cold stable assay, fixed and stained for tubulin, actin (phalloidin) and DNA. (B) Tubulin signal represented by a line scan profile for control or taxol treated MI spindles. (C-D) Spindle asymmetry was calculated for control or latrunculin treated oocytes (5 μM, 30 min) and plotted against the distance from the cortex. Asterisk shows the cortical spindle pole; A.U., arbitrary units; R2, correlation coefficient; d, distance from the cortex; scale bar, 5

107 Figure 26

108

Figure 26. Cortical MI spindle positioning is maintained in oocytes with cumulus cells (COCs). CF-1 oocytes with or without cumulus cells were (A) imaged live in 10 min intervals and nucleus position was calculated at each 1 h time point, or (B) were fixed and stained for tubulin, actin, and DNA in 2 h time intervals after nuclear envelope breakdown, and (C) spindle or nucleus distance from the cortex was calculated. Dashed

109 Figure 27

110

Figure 27. Spindle cortical positioning drives preferential trivalent orientation and Rb fusion segregation. (A) Two possible outcomes of balanced trivalent segregation in MI (i, ii), schematics of trivalent, telocentrics and Rb fusion. (B) Rb(6.16) x CF-1 eggs were collected in vivo or oocytes with intact nuclei were collected and matured in vitro with or without cumulus cells, fixed and stained for kinetochores (CERST) and DNA, and segregation of Rb fusion was calculated (in vivo COCs, n = 168, segregation ration 60:40, X2 = 6.48, p = 0.0087; in vitro COCs, n = 141, segregation ration 60:40, X2 = 5.64, p = 0.017; in vitro denuded, n = 120, segregation ration 50:50, X2 = 0.033, p = 0.855). (C-D) Trivalent orientation (inset) was assessed before anaphase in (C) in vivo matured COCs (X2 = 8.52, p = 0.0017), or in denuded oocytes (X2 = 0.64, p = 0.32), fixed and stained for spindle poles (AURKA), kinetochores (CREST), and DNA. (E) Model of preferential trivalent orientation and Rb fusion segregation in COCs and denuded oocytes. Numbers in each bar represent numbers of oocytes in each group,

arrows show the Rb fusion centromere; white asterisk, p < 0.05; scale bar, 5 μm; NS, not

significant; N, oocyte nucleus; COCs, oocytes with cumulus cells; 60:40 and 50:50 represent Rb fusion segregation to polar body vs. to the egg.

111

CHAPTER 5

112

Unifying model of meiotic drive of Rb fusions

De Villena et al. proposed a theoretical unifying model of meiotic drive, consisting from three major parts: i) functional heterozygosity of the drive locus, mediating or influencing the attachment of the chromosome to meiotic spindle, ii) functional asymmetry in meiotic spindle observed in numerous systems (Twell, 2011; Jones, 1991; Hewitt, 1976; Cortes et al., 2015), and iii) asymmetry in meiosis in respect to the cell fate (Pardo-Manuel de Villena and Sapienza, 2001a). In this model the driving locus interacts differently with MTs from the opposite spindle poles resulting in

preferential orientation or favorable position within the meiotic spindle that segregates such locus into the functional gamete. Consistent with such model, we observed

asymmetry of the MI spindle in mouse oocytes and found that MTs closer to the cortex are more stable (less dynamic) than MTs far from the cortex (less stable, more dynamic) (unpublished results) (Figure 28 A).

Mechanisms that could explain the spindle asymmetry include: 1) spindle MTs are stabilized by unknown cortical signal, or 2) differences in MT stability are due to differences in spindle pole associated proteins detected in previous studies (Carabatsos et al., 2000; Vinot et al., 2004). Whereas the second model relies on additional mechanism of preferential spindle orientation towards the cortex before anaphase I, the first model (1) is simpler and does not need to invoke any additional step. Thus we favor the first model, suggesting that the spindle asymmetry is established while the spindle is getting assembled in proximity of the cortex in MI.

113

How do bivalents orient in such asymmetrical spindle? In our previous study, we show that crossing a strong centromere species (CF-1) with a weak centromere species (CHPO) produces an offspring with bivalents having one strong and one weak

centromere (Chmátal et al., 2014). There are two possible orientations of such

asymmetrical bivalent within the asymmetrical MI spindle: (1) the weaker centromere towards the more stable MTs at the cortical pole and the stronger centromere towards the more dynamic MTs at the egg pole, or (2) the weaker centromere towards more dynamic MTs and the stronger centromere towards the more stable MTs (Figure 28 B). The first orientation should be preferred if stronger centromeres preferentially remain in the egg, consistent with the centromere drive hypothesis (Malik, 2009) and with our observations (Chmátal et al., 2014). Two mechanisms, which are not mutually exclusive, could explain this preference. More dynamic MTs from the egg pole may initially bind the stronger centromeres, which have larger kinetochores with more MT-binding proteins, with the weaker centromeres subsequently binding MTs from the cortical pole to establish tension across the bivalent. Alternatively, the bivalent may sample both orientations, and one is preferred because it is relatively more stable than the other. In this model, the interaction of the weak centromere with the more dynamic MTs may be unstable and will tend to re- orient, so that the interaction of the weak centromere with the more stable MTs is

preferred. Further observations from live imaging during MI may discriminate between these models.

The same models can explain the preferential orientation and segregation of a trivalent. Either the stronger centromeres attach first to the more dynamic egg pole

114

(Figure 28 C-1), or the interaction of weak centromeres with more dynamic MTs is unstable. In either case, the Rb fusion centromere orients preferentially to the egg pole if it is stronger relative to the homologous telocentrics, or preferentially to the cortical pole if it is relatively weaker (Figure 28 C).

Based on various meiotic drive systems we propose a single unifying model for drive in MI and MII (Figure 28 D). MII drive, represented by Ab10 system, consists from two major parts: i) functional heterozygosity of the drive locus that influences attachment of the chromosome to meiotic spindle, and ii) asymmetry in meiosis in respect to the cell fate. Under that model, the locus exhibiting nonrandom segregation interacts with MTs from opposite spindle poles, resulting in a favorable position within the cell that becomes a functional gamete. This model does not require spindle asymmetry, because the

preferential position of the driving locus is achieved by the unique architecture of plant female meiosis. Nevertheless meiotic spindle asymmetry is an important aspect of drive in MI because preferential segregation of a driving locus is achieved by favored MT- attachment to a given spindle pole.

115

DISCUSSION

Chromosomal rearrangements are frequent events involving partial or whole