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\DOI{10.5802/crbiol.155}
\datereceived{2024-03-18}
\daterevised{2024-05-13}
\dateaccepted{2024-05-23}
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\dateposted{2024-06-18}
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\title{Exploring the maternal inheritance transmitted by the oocyte to
its progeny}

\alttitle{H\'{e}ritage maternel transmis par l'ovocyte \`{a} sa descendance}

\author{\firstname{Marie-H\'{e}l\`{e}ne} \lastname{Verlhac}\CDRorcid{0000-0001-8377-9010}}
\address{Center for Interdisciplinary Research in Biology (CIRB),
Coll\`{e}ge de~France, Universit\'{e} PSL, CNRS, INSERM, 75005 Paris,
France}
\email{marie-helene.verlhac@college-de-france.fr}

\keywords{\kwd{Oocyte}
\kwd{Meiotic divisions}
\kwd{Biophysics}
\kwd{Actin microfilaments}}

\altkeywords{\kwd{Ovocyte}
\kwd{Divisions m\'{e}iotiques}
\kwd{Biophysique}
\kwd{Microfilaments d'actine}}

\begin{abstract} 
Fertility is declining worldwide and many couples are turning towards
assisted reproductive technologies (ART) to conceive babies. Organisms
that propagate via sexual reproduction often come from the fusion
between two gametes, an oocyte and a sperm, whose qualities seem to be
decreasing in the human species. Interestingly, while the sperm mostly
transmits its haploid genome, the oocyte transmits not only its haploid
set of chromosomes but also its huge cytoplasm to its progeny. This is
what can be defined as the maternal inheritance composed of
chromosomes, organelles, lipids, metabolites, proteins and RNAs. To
decipher the decline in oocyte quality, it is essential to explore the
nature of the maternal inheritance, and therefore study the last stages
of murine oogenesis, namely the end of oocyte growth followed by the
two meiotic divisions. These divisions are extremely asymmetric in
terms of the size of the daughter cells, allowing to preserve the
maternal inheritance accumulated during oocyte growth within these huge
cells to support early embryo development. Studies performed in
Marie-H\'{e}l\`{e}ne Verlhac's lab have allowed to discover the
unprecedented impact of original acto-myosin based mechanisms in the
constitution as well as the preservation of this maternal inheritance
and the consequences when these processes go awry. 
\end{abstract}

\begin{altabstract}
La f\'{e}condit\'{e} diminue mondialement et de nombreux couples se
tournent vers les techniques de procr\'{e}ation m\'{e}dicalement
assist\'{e}e (PMA) pour concevoir des b\'{e}b\'{e}s. Les organismes se
propageant par reproduction sexu\'{e}e sont souvent issus de la fusion
de deux gam\`{e}tes, un ovocyte et un spermatozo\"{i}de, dont les
qualit\'{e}s semblent diminuer dans l'esp\`{e}ce humaine. Si le
spermatozo\"{i}de transmet principalement son g\'{e}nome haplo\"{i}de,
l'ovocyte transmet \`{a} sa prog\'{e}niture non seulement son lot
haplo\"{i}de de chromosomes, mais aussi son immense cytoplasme. C'est
ce que l'on peut d\'{e}finir comme l'h\'{e}ritage maternel, compos\'{e}
de chromosomes, d'organelles, de lipides, de m\'{e}tabolites, de
prot\'{e}ines et d'ARNs. Pour comprendre la baisse de qualit\'{e} des
ovocytes, il est essentiel d'explorer la nature de cet h\'{e}ritage
maternel, et donc d'\'{e}tudier les derni\`{e}res \'{e}tapes de
l'ovogen\`{e}se murine, \`{a} savoir la fin de la croissance ovocytaire
suivie des deux divisions m\'{e}iotiques. Ces divisions sont
extr\^{e}mement asym\'{e}triques par la taille des cellules filles
engendr\'{e}es, ce qui permet de pr\'{e}server l'h\'{e}ritage maternel
accumul\'{e} pendant la croissance de cette \'{e}norme cellule,
l'ovocyte, pour soutenir le d\'{e}veloppement pr\'{e}coce de l'embryon.
Les \'{e}tudes men\'{e}es dans le laboratoire de Marie-H\'{e}l\`{e}ne
Verlhac ont permis de d\'{e}couvrir l'impact sans pr\'{e}c\'{e}dent de
m\'{e}canismes originaux d\'{e}pendant de l'acto-myosine dans la
constitution et la pr\'{e}servation de cet h\'{e}ritage maternel, ainsi
que les cons\'{e}quences des erreurs dans ces processus.
\end{altabstract}

\def\thanksname{Note}

\thanks{Marie-H\'el\`ene Verlhac is the recipient of the prize Jaff\'e -
Fondation de l'Institut de France 2022}

\maketitle

\vspace*{.3pc}

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\section{Massaging the oocyte nucleus for a good quantitative and
qualitative transcriptome}\label{sec1}

\subsection{A non-specific gradient of pressure positions large objects
in the centre of the oocyte}\label{sec11} 

The position of the nucleus in a cell can instruct morphogenesis in
some cases, conveying spatial and temporal information. Abnormal
nuclear positioning can lead to disease~\cite{1}. In oocytes from
many species, nucleus position regulates embryo development. For
example, in \textit{Drosophila} it is located under the antero-dorsal
oocyte cortex and defines the future dorso-ventral axis of the embryo
and of the adult body plan. However, in mammals, the oocyte nucleus is
centrally located and does not instruct any future embryo axis. Yet an
off-centre nucleus correlates with a poor outcome for mouse and human
oocyte development~\cite{2,3}. This is surprising since oocytes
further undergo two extremely asymmetric divisions in terms of the size
of the daughter cells (enabling polar body extrusion), requiring
off-centring of their chromosomes (Figure~\ref{fig1}).

\begin{figure*}
\includegraphics{fig01}
\caption{\label{fig1}Scheme of the end of oocyte growth, oocyte
divisions and early development after fertilization. The nucleus (pink)
is centred at the end of oocyte growth thanks to the presence of
increasing acto-myosin-based forces. Oocytes are arrested in prophase~I
in the ovary and will resume meiosis upon hormonal trigger. Meiosis
resumption is marked by nuclear envelope breakdown (NEBD). The first
meiotic spindle assembles in the absence of canonical centrosomes. It
migrates towards the closest cortex, which permits a first asymmetric
division in size (extrusion of the first polar body, 1st PB), allowing
the oocytes to preserve the maternal inheritance accumulated during
growth. There is no DNA replication between the two meiotic divisions,
which permits the formation of a haploid gamete after second polar body
extrusion (2nd PB). Fertilization occurs after ovulation, in the
oviduct, when the oocyte is arrested in metaphase of the second meiotic
division (Metaphase~II) and at that stage the second meiotic spindle
stays anchored into the cortex. The arrow represents chromosome motion.}
\end{figure*}



We discovered that in the mouse, the nucleus is positioned at the
centre of the oocyte via an acto-myosin gradient of
pressure~\cite{4}. This gradient of pressure arises from a gradient
of velocities of actin-positive vesicles. These vesicles are positive
for Rab11a~\cite{5} and two essential actin nucleators,
Spire~1~\&~2 as well as Formin~2 are anchored onto their
membrane~\cite{6,7}. As a consequence, actin filaments grow from
the vesicles and this is why we name them actin-positive vesicles.
Furthermore, these vesicles are cargos for Myosin Vb~\cite{5,8}
and we showed that when the acto-myosin~Vb meshwork is dynamic
(growing and shrinking and containing vesicles that move along the
actin filaments via the Myosin~Vb motor activity), it propels the whole
cytoplasm by advection (Figure~\ref{fig2}). We demonstrated, using
biophysical and 3D agent-based simulations that this gradient is
non-specific and propels large objects towards the oocyte centre both
during prophase~I and meiosis~I, allowing the oocyte to preserve its
large organelle content, despite undergoing asymmetric
divisions~\cite{9} (Figure~\ref{fig3}).

\begin{figure*}
{\vspace*{2pt}}
\includegraphics{fig02}
{\vspace*{2pt}}
\caption{\label{fig2}Scheme representing the acto-myosin based forces
which promote nucleus centering. ({A})~Global scheme of F-actin
(red filaments) and Myosin~Vb motor (violet) anchored into
Rab11a-positive vesicles (red circles) as shown in~\cite{4,5,6,7,8}.
({B}) Zoom on the effect of Rab11a-positive vesicle motion on
the cytoplasmic fluid, moving by advection.} 
{\vspace*{2pt}}
\end{figure*}

\begin{figure*}
{\vspace*{2pt}}
\includegraphics{fig03}
{\vspace*{2pt}}
\caption{\label{fig3}Confronting simulation to experiments to validate
the non-specific nature of the gradient of pressure. Left: Oocytes
arrested in prophase~I and microinjected with inert objects, such as
oil droplets (white arrows). These oil droplets mimic the nucleus
behavior, in terms of F-actin recruitment (right picture) and capacity
to become centralized with velocities comparable to the nuclear ones.
Right: Modelling and 3D-simulations of the motion of inert objects
subjected to the gradient of pressure of actin-positive vesicles allow
to confront theory and experiments~\cite{9}. Scale bar:
10~$\upmu$m.}
{\vspace*{2pt}}
\end{figure*}


\subsection{A mechano-transduction cascade controls gene expression in
the oocyte}\label{sec12}

Importantly, we showed that forces from the acto-myosin cytoskeleton,
which position the nucleus, increase in intensity during the last
stages of oocyte growth in the ovary, due to an increase in F-actin
density~\cite{10}. As a consequence, advection of the cytoplasm
rises during these last stages of growth and more forces impact the
nuclear envelope. We quantified the fluctuations of the nuclear
envelope and showed that they depend on the presence of
F-actin~\cite{11,12}. The oocyte nuclear envelope undergoes
prominent and important shape changes, arguing that it is massively
deformable and not extremely rigid. By performing RNA sequencing
comparing control versus oocytes that are devoid of a key oocyte
cytoplasmic F-actin nucleator, Formin~2~\cite{13,14,15}, we
observed a significant downregulation of about 200~genes in oocytes
devoid of acto-myosin based forces from the cytoplasm~\cite{11}. We
also observed a reduction in the motion of chromosomes inside the
nucleus in those mutant oocytes~\cite{11}. Since these genes are
distributed randomly in the mouse genome, we believe that a
mechano-transduction cascade agitates the whole nucleoplasm, promoting
the transcription of genes that are active at\break that stage.

\begin{figure*}
\includegraphics{fig04}
\caption{\label{fig4}Scale-crossing activity of forces from the
cytoskeleton inside mouse oocytes. Left: picture of a fully-gown
oocytes observed by transmitted light, where chromosomes appear in
pink. Scale bar: 10~${\upmu}$m. Right: scheme representing how
increasing acto-myosin based forces promote nucleus motion, nuclear
envelope fluctuations, nuclear bodies coarsening and increase of
molecular mobility and hence splicing activity inside nuclear speckles.
F-actin in red, nucleus in pink, nuclear bodies in various colors,
nuclear speckles in violet and molecules in green circles.}
\end{figure*}

\subsection{A mechano-transduction cascade controls splicing activity
in the oocyte}\label{sec13}

In addition, we showed that these nuclear fluctuations also affect
nuclear organization, in particular nuclear body organization. Nuclear
bodies are extremely dynamic membrane-less organelles which are
essential accelerators of mRNA processing. We demonstrated that growing
mouse oocytes experience an increase in cytoplasmic acto-myosin based
forces that boost nuclear condensate fusion
(Figure~\ref{fig4})~\cite{10}. When biomolecular condensates increase
their mobility, they have a greater chance of colliding with each other
and because they are liquid-like, the chances of fusion also increase.
This leads to a decrease in the number of biomolecular condensates and
to an increase in their size, as we observed in oocytes~\cite{10}
(Figure~\ref{fig4}).~Nuclear bodies are distinct membrane-less
organelles which play essential roles in the control of gene
expression. Among those best characterized so far, one can find the
nucleolus, which has essential functions in \mbox{ribosomal} subunit
biogenesis; Cajal bodies, which \mbox{participate} in the maturation of small
nuclear and small nucleolar ribonucleoprotein particles (snRNPs and
snoRNPs); nuclear speckles, essential for transcription and mRNA
maturation and processing; histone locus bodies, associated with the
biogenesis of histone mRNAs; PML (ProMyeloid Leukemia) \mbox{bodies,} involved
in the cell cycle and genome repair and maintenance; and paraspeckles,
which regulate gene expression through sequestration of specific
transcripts and protein components~\cite{16,17,18}. These
nuclear bodies, also named nuclear condensates, are composed of
specific RNAs and proteins and can be recognized using specific
markers. Importantly, these condensates cooperate globally inside the
nucleus to transcribe, process and export RNAs towards the cytoplasm,
accomplishing key functions in the cell. Nuclear speckles extensively
contribute to various RNA maturation and splicing~\cite{19}. The
\mbox{capacity} of the cytoskeleton forces to organize the nucleoplasm, and in
particular nuclear speckles, has been conserved during the evolution of
species being also observed in growing \textit{Drosophila}
oocytes~\cite{10}. We also showed that larger nuclear speckles
display increased splicing activity, suggesting the presence of a
cascade of energy transfer from the acto-myosin mechanical forces
towards motion of nuclear bodies and up to molecules inside the nuclear
speckles (Figure~\ref{fig4}). This energy transfer enhanced
condensate-associated activity such as active splicing, modulating mRNA
processing~\cite{10} (Figure~\ref{fig4}). Beyond reproduction, this
mechanism sheds new light on numerous anomalies linked to defects in
cytoplasmic forces correlating with defects in condensate architecture
observed in many cancers~\cite{20,21}, as is the case of PML
nuclear bodies in Acute Promyeloid Leukemia (APL-type of
Leukemia)~\cite{22}.

Our work in oocytes suggests that the cytoskeleton promotes
transcriptional activity but also promotes mRNA processing and thus
modulates the quantity and quality of maternal transcripts.

\section{A thickening of the cortex prevents oocyte cortical
contractions and preserves the architecture of organelles}\label{sec2}

\begin{figure*}
\includegraphics{fig05}
\caption{\label{fig5}Arp2/3 depleted oocytes display contractions which
impact  organelles distribution.
The absence of cortical microfilaments nucleated by the Arp2/3 complex
in mutant oocytes without branched actin induces a reorganization of
the oocyte cortex (compare bottom picture with top picture) and the
appearance of cortical contractions. These cortical contractions
promote the appearance of ectopic cytoplasmic flows, which impact on
the movement and architecture of organelles (mitochondria, cortical
granules). The progression of the oocyte cell cycle remains unaffected:
oocytes exit normally from their arrest in prophase of the first
division of meiosis (Prophase~I), followed by normal progression of the
first meiotic division (NEBD ${+}$2h and ${+}$6h) and an arrest in metaphase of
the second division of meiosis (Metaphase~II). However, fertilization
of mutant oocytes is reduced, as is their early development. The
fertility of mice producing these mutant oocytes is also reduced. The
cortex is labelled (in fire) with a specific probe for F-actin.}
\end{figure*}

A few decades ago, we also took an interest in studying the motion of
the first meiotic spindle to the \mbox{closest} cortex, a prerequisite for the
asymmetry in size of the first meiotic division (Figure~\ref{fig1}, see
arrow). Here again we demonstrated that biomechanics is instrumental is
this process. The first meiotic spindle forms where the nucleus was, in
the centre of the fully-grown mouse oocyte. Due to a high cytoplasmic
viscosity, the spindle migrates towards the closest cortex along its
long axis~\cite{23}. Indeed, the first meiotic spindle is never
perfectly centered when it assembles, and often displays one of its two
poles closer to one cortex. This initial slight imbalance in spindle
position inside the oocyte is sufficient to break the symmetry in a
cell that is otherwise non-polarized and with no obvious
asymmetry~\cite{24}. Surprisingly, most oocytes are devoid of
canonical centrosomes, made of a pair of centrioles surrounded by
pericentriolar material~\cite{25}. Hence, they assemble their
spindle a bit differently than cells in mitosis that use the two
centrosomes to produce bipolar spindles (Figure~\ref{fig1}). It takes
about four hours in the mouse and eight hours in human to assemble a
bipolar spindle~\cite{26,27}. Importantly also, due to the lack
of canonical centrosomes, meiotic spindles in oocytes are devoid of
astral microtubules connecting the two spindle poles to the cortex. In
somatic cells, these astral microtubules play key roles in controlling
spindle orientation inside the cell, and in transmitting forces from
the tissue towards the cell interior~\cite{28,29,30,31}.\looseness=-1


In mouse oocytes, spindle positioning to the cortex cannot depend on
astral microtubules, which are absent, but we and others have shown
that it depends on F-actin~\cite{14,15,23,32,33}. Our
lab has also shown that cortical properties, and in particular the
thickening of the oocyte cortex during meiotic divisions is essential
to amplify the initial imbalance of forces acting on meiotic spindle
poles to promote first meiotic spindle off-centring~\cite{34,35}.
The cortical thickening depends on the nucleation of microfilaments via
the Arp2/3 complex~\cite{34}. Recently, we used a conditional
knockout of a key subunit of the Arp2/3 complex, Arpc4, to induce a
depletion of this complex specifically in oocytes at the end of their
growth~\cite{36}. This depletion was efficient in suppressing cortex
thickening during oocyte divisions (Figure~\ref{fig5}). Unexpectedly,
Arp2/3 complex-depleted oocytes displayed important F-actin cortex
reorganization (Figure~\ref{fig5} compare the two pictures), which
subsequently triggered prominent contractions starting shortly after
entry into meiosis. The energy of these cortex contractions, that
lasted for a few hours, induced an increase in the motion of objects
inside the cytoplasm and defects in the distribution and organization
of different organelles, such as lipid droplets, mitochondria and
cortical granules. Cortical granules have an important role in the
prevention of polyspermy~\cite{37}. Not surprisingly, Arp2/3
complex-depleted oocytes displayed massive polyspermy, associated with
poor embryo development after fertilization. As a consequence, female
mice producing these mutant oocytes harboured a reduced
fertility~\cite{38}. Interestingly, using an
AI-pipeline~\cite{39} to automatically quantify oocyte morphological
features on a collection of about 250~movies of developing human
oocytes, we could observe that they presented similar cortex
contractions, correlating nicely with increased cytoplasmic motion.
However, in both mouse and human oocytes, the presence of cortex
contractions during the first meiotic division did not correlate with
aneuploidy, a major factor impacting the female gamete
fitness~\cite{40,41,42}. The occurrence of contractions in
developing human oocytes may correspond to a novel factor contributing
to cytoplasmic infertility, different from the well-described
aneuploidy. Previous studies have indicated a correlation between the
developmental potential of oocytes and factors such as organelle size,
quantity, and distribution, particularly concerning mitochondria, lipid
droplets, and cortical granules~\cite{43}. In the future, we will
investigate whether contracting human oocytes exhibit an altered
organelle status, potentially linked to a reduced likelihood of
successful development. While these oocytes are not used in routine in
ART centres, certain patients may lack better alternatives. In such
instances, the presence or absence of cortical contractions could aid
in identifying oocytes with a higher probability of favorable
outcomes~\cite{38} for these patients.

Altogether, our work has highlighted the impact of actin meshes in the
control of maternal inheritance, in particular on chromosomes and
organelle positioning, which are essential factors contributing to the
developmental potential of the oocyte. Interestingly, both nucleus
position and cortex properties constitute non-invasive predictors of
early embryo development quality that could be used easily in Assisted
Reproduction Technologies, where embryo selection currently consists of
highly subjective morphological assessments.

\section*{Declaration of interests}

The authors do not work for, advise, own shares in, or receive funds
from any organization that could benefit from this article, and have
declared no affiliations other than their research organizations.

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