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. 2015 Apr 20;10(4):e0124911.
doi: 10.1371/journal.pone.0124911. eCollection 2015.

Differences in the Kinetic of the First Meiotic Division and in Active Mitochondrial Distribution between Prepubertal and Adult Oocytes Mirror Differences in their Developmental Competence in a Sheep Model

Giovanni Giuseppe Leoni  1 , Maria Grazia Palmerini  2 , Valentina Satta  3 , Sara Succu  3 , Valeria Pasciu  3 , Angelo Zinellu  4 , Ciriaco Carru  4 , Guido Macchiarelli  2 , Stefania Annarita Nottola  5 , Salvatore Naitana  1 , Fiammetta Berlinguer  1
Affiliations

Differences in the Kinetic of the First Meiotic Division and in Active Mitochondrial Distribution between Prepubertal and Adult Oocytes Mirror Differences in their Developmental Competence in a Sheep Model

Giovanni Giuseppe Leoni et al. PLoS One. .

Abstract

Our aim is to verify if oocyte developmental potential is related to the timing of meiotic progression and to mitochondrial distribution and activity using prepubertal and adult oocytes as models of low and high developmental capacity respectively. Prepubertal and adult oocytes were incorporated in an in vitro maturation system to determine meiotic and developmental competence and to assess at different time points kinetic of meiotic maturation, 2D protein electrophoresis patterns, ATP content and mitochondria distribution. Maturation and fertilization rates did not differ between prepubertal and adult oocytes (95.1% vs 96.7% and 66.73% vs 70.62% respectively for prepubertal and adult oocytes). Compared to adults, prepubertal oocytes showed higher parthenogenesis (17.38% vs 2.08% respectively in prepubertals and adults; P<0.01) and polispermy (14.30% vs 2.21% respectively in prepubertals and adults; P<0.01), lower cleavage rates (60.00% vs 67.08% respectively in prepubertals and adults; P<0.05) and blastocyst output (11.94% vs 34.% respectively in prepubertals and adults; P<0.01). Prepubertal oocytes reached MI stage 1 hr later than adults and this delay grows as the first meiotic division proceeds. Simultaneously, the protein pattern was altered since in prepubertal oocytes it fluctuates, dropping and rising to levels similar to adults only at 24 hrs. In prepubertal oocytes ATP rise is delayed and did not reach levels comparable to adult ones. CLSM observations revealed that at MII, in the majority of prepubertal oocytes, the active mitochondria are homogenously distributed, while in adults they are aggregated in big clusters. Our work demonstrates that mitochondria and their functional aggregation during maturation play an active role to provide energy in terms of ATP. The oocyte ATP content determines the timing of the meiotic cycle and the acquisition of developmental competence. Taken together our data suggest that oocytes with low developmental competence have a slowed down energetic metabolism which delays later development.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Kinetics of in vitro maturation in prepubertal and adult oocytes.
Values are expressed as (A) percentages of prepubertal (n = 1186) and adult (n = 1139) oocytes that reached MI at 6 to 9 hrs of maturation culture and (B) percentages of prepubertal (n = 1026) and adult (n = 981) oocytes that reached MII stages between 19 and 22 hrs of maturation culture. *indicates statistical difference at each time point between the two experimental groups; (Chi square test: P<0.001).
Fig 2
Fig 2. Quantification of spots detectable in 2D electrophoresis gels of proteins extracted from 20 prepubertal and adult oocytes at different times of maturation culture.
(ANOVA; * indicates statistical difference between adult and prepubertal groups at each time; different upper case letters indicate statistical difference into adult group; different lower case letters indicate statistical difference into prepubertal group).
Fig 3
Fig 3. Representative electrophoretic gels of 20 oocyte proteins at 0, 7, 19 and 24 hrs of maturation culture.
Arrows represent one example of a spot detectable in both prepubertal and adult oocyte protein gels at 7 hrs of maturation and detectable only in prepubertals at 19 and 24 hrs of culture.
Fig 4
Fig 4. Fluctuation of ATP intracellular content in prepubertal and adult ewe oocytes during in vitro maturation.
Data are expressed as mean ± SEM. Lower case letters indicate statistical difference among different time points in the adult group: ANOVA p<0.01. Upper case letters indicate statistical difference among different time points in the prepubertal group: ANOVA p<0.01. * Indicates statistical difference at each time point between adult and prepubertal groups (ANOVA: p<0.01).
Fig 5
Fig 5. Morphology of prepubertal ovine oocytes (control group).
(a) Representative micrograph by TEM, showing the oocyte surrounded by a continuous zona pellucida (ZP) and a multilayer of granulosa cells (GCs). A normal distribution of vacuoles (V) and lipid droplets (LD), typical for the ovine oocyte, is seen. Note also the presence of numerous mitochondria (m). Bar: 5 μm. (b) Rosette-like arrangement of mitochondria. TEM, bar: 1 μm. (c) High magnification TEM micrograph of mitochondria. Arrows indicated hooded mitochondria. Asterisks: mitochondria containing a clear vesicle. Arrowheads: mitochondrial cristae. V: vacuoles; m: mitochondria; LD: lipid droplets.
Fig 6
Fig 6. Morphology of adult ovine oocytes (control group).
(a) Clusters of mitochondria (m) in close proximity to tubular element of the smooth endoplasmic reticulum (SER) and clear vacuoles (V). TEM, bar: 1 μm. (b) Groups of round-to-ovoid mitochondria (m) near to tubular elements of SER and a Golgi apparatus (G). TEM, bar: 1 μm. (c). N: nucleus, NE: nuclear envelope; V: vacuoles; m: mitochondria. Arrows: hooded mitochondria; asterisks: mitochondria containing a clear vesicle.
Fig 7
Fig 7. Morphology of prepubertal (a) and adult (b) ovine oocytes at 7 hrs of IVM.
Representative low magnification TEM micrograph, showing the oocytes surrounded by a continuous zona pellucida (ZP), with clusters of mitochondria (m) located in the cortex. Bar: 5 μm. Inset: high magnification of a rosette-like arrangement of mitochondria (m). GCs: granulosa cells. ZP: zona pellucida. LD: lipid droplets. V: vacuoles. Bar: 1 μm.
Fig 8
Fig 8. Morphology of prepubertal (a) and adult (b) ovine oocytes at 19 hrs of IVM.
Representative low magnification TEM micrograph, showing the oocytes surrounded by a continuous zona pellucida (ZP), with small clusters and isolated mitochondria (m). Bar: 2 μm. Insets: high magnification of a small cluster of mitochondria (m). Secretory vacuoles are visible in the inset of Fig 4a (arrowheads). Bar: 0.5 μm. CGs: cortical granules; ZP: zona pellucida; V: vacuoles.
Fig 9
Fig 9. Morphology of prepubertal (a) and adult (b) ovine oocytes at 24 hrs of IVM.
Representative low magnification TEM micrograph, showing the oocytes surrounded by a continuous zona pellucida (ZP), with clusters of mitochondria (m) bigger in adults than in prepubertals. Bar: 2 μm. Insets: high magnification of a cluster of mitochondria (m). Bar: 0.5 μm. ZP: zona pellucida; V: vacuoles. Arrows: hooded mitochondria; asterisks: mitochondria containing a clear vacuole.
Fig 10
Fig 10. Quantification of active mitochondrial-specific fluorescence intensity of the Mitotracker stain in prepubertal and adult oocytes at GV and MII stage.
Values are expressed as arbitrary units (Mean ± SE); Different letters indicate a statistical difference (ANOVA: a vs b = P<0.05; a,b vs c = P<0.01).
Fig 11
Fig 11. Representative CLSM patterns of active mitochondria distribution at GV and MII stages in prepubertal and adult ovine oocytes.
(a) fine distribution of mitochondria in GV-stage oocytes; (b) fine distribution in MII-stage oocytes; (c) a granular distribution in GV-stage oocytes; (d) granular distribution in MII-stage oocytes; (e) clustered distribution in MII-stage oocytes. On the right are particulars of fine (f), granular (g) and clustered (h) distributions.
Fig 12
Fig 12. Schematic representation of data obtained by TEM and CLSM on morphological and morphometric distribution of mitochondria related to their activity in prepubertal and adult ovine oocytes.
A) schematic representation of mitochondria (light blue) distribution as seen at TEM; B) superimposition of total mitochondria distribution as seen at TEM (light blue) with active mitochondria distribution as seen at CLSM (red).
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This study was supported by the Fondazione Banco di Sardegna (www.fondazionebancodisardegna.it) and the Sardinian Region Council (www.regione.sardegna.it). SS received a grant from the Fondazione Banco di Sardegna (www.fondazionebancodisardegna.it). The Sardinian Region Council (www.regione.sardegna.it) gave a grant to VS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.