Autophagy regulates organelle reorganization during spermiogenesis in the liverwort Marchantia polymorpha

ABSTRACT

Sperm mitochondria generally exhibit distinctive and diverse morphologies in animals and plants. Bryophytes, a plant group consisting of liverworts, mosses, and hornworts, produce motile male gametes, called spermatozoids, that possess a fixed number of two mitochondria in their cell bodies. Electron microscopy observations have revealed the detailed morphological aspects of plant spermatozoids, including mitochondrial morphology; however, the mechanism by which mitochondria are reorganized during spermiogenesis in bryophytes remains largely unknown. Our recent study using the liverwort, Marchantia polymorpha, revealed that the mitochondrial number is reduced to one via mitochondrial fission and macroautophagic/autophagic degradation, which subsequently becomes two via asymmetric division to form large anterior and small posterior mitochondria. Other cytoplasmic components, such as peroxisomes, are also degraded via autophagy; however, mitochondria are degraded at a time distinct from other cytoplasmic components. We also found that some cytoplasmic components were degraded in the vacuole independent of autophagy. Our study highlights the dynamic reorganization of organelles via multiple degradation pathways during spermiogenesis in M. polymorpha.

Mitochondria vary in shape, number, and distribution, depending on the cellular conditions and developmental status. Mitochondria in sperm cells exhibit distinct characteristics and significant morphological divergence compared to those in other types of eukaryotic cells. Among plant lineages, bryophytes produce motile flagellated male gametes, called spermatozoids, with a fixed number of two mitochondria: one at the anterior region and the other at the posterior region of the cell body. However, the specific mechanisms determining the fixed mitochondrial number and regulating the organelle reorganization process during spermiogenesis remain unclear.

Our recent study [1] indicated that mitochondria undergo dynamic reorganization during spermiogenesis in the liverwort, Marchantia polymorpha. Our confocal microscopy observation of the fluorescently-labeled mitochondria showed that the mitochondrial area and number are rapidly reduced and only one mitochondrion is retained at the basement of the flagella in the early stage of spermiogenesis, which then becomes two via mitochondrial fission to form the posterior mitochondrion (Figure 1). When mitochondrial fission is inhibited by the expression of a dominant-negative form of a mitochondrial dynamin-related protein (MpDRP3), spermatozoids with a single mitochondrion are formed.

Figure 1.

Schematic diagram of organelle reorganization during spermiogenesis in Marchantia polymorpha. During the early stage of spermiogenesis, mitochondria undergo fission and autophagic degradation, resulting in one mitochondrion at the base of the flagella. Subsequently, the mitochondrial number becomes two via mitochondrial fission. Other cytoplasmic components, such as peroxisomes, are also degraded via autophagy.

As autophagy is known to play crucial roles in mitochondrial degradation in various organisms, we investigated the mechanism by which the mitochondrial number was reduced during spermiogenesis, with a special focus on autophagy. As expected, spermatozoids formed in mutants lacking the core ATG (autophagy related) genes, MpATG5, MpATG7, MpATG13, and MpATG14, retain a larger number of mitochondria than wild-type spermatozoids. We also found that the translocation of mitochondria into the vacuole in transforming spermatids requires MpATG5. These results indicate that autophagy is responsible for the reduction in mitochondrial number during spermiogenesis. Cytoplasmic components other than mitochondria, including the cytosol, endoplasmic reticulum, and peroxisome, are also degraded via autophagy during spermiogenesis; these are not transported into the vacuole in the Mpatg5 mutant spermatid. Interestingly, however, these components are degraded at a specific time after mitochondrial degradation via autophagy (Figure 1). We further found that the mitochondria are selectively engulfed by phagophores, suggesting the involvement of mitophagy in mitochondrial clearance during spermiogenesis in M. polymorpha. We then investigated whether ubiquitination or loss of membrane potential in mitochondria precedes mitophagy during spermiogenesis, as seen in non-plant systems; however, this is not detected in M. polymorpha. This result suggests that mitophagy during M. polymorpha spermiogenesis occurs via a distinct mechanism.

Our study also indicated that non-autophagic degradation plays an important role during spermiogenesis; for example, the Golgi apparatus is degraded in the vacuole via both MpATG5-dependent and -independent pathways. Endocytic degradation is highly activated during spermiogenesis in M. polymorpha. These results suggest that multiple vacuolar degradation pathways coordinate the rapid and drastic reorganization of organelles during spermiogenesis.

Although autophagy is also involved in mammalian spermiogenesis, its role seems to differ in mammals and M. polymorpha. Unlike Mpatg mutant spermatozoids, excess or unnecessary cytoplasmic components are largely removed from the spermatozoa, even in the atg mutants of mice. In mammalian spermiogenesis, cytoplasmic removal occurs via phagocytosis by the neighboring Sertoli cells. However, phagocytosis cannot occur in plant cells as they are surrounded by a rigid cell wall; therefore, non-cell-autonomous removal of unnecessary cytoplasm, similar to that observed during mammalian spermiogenesis, is not practical in plants. This difference in the role of autophagy in mammalian and liverwort spermiogenesis may have arisen due to the distinct and/or specific restrictions in their cell architecture/systems. Diversification of the roles of autophagy during spermiogenesis has been detected even in bryophytes; For example, atg mutations affect spermiogenesis differently in the liverwort, M. polymorpha, and the moss, Physcomitrium patens. These plants are excellent models for unraveling the molecular mechanisms of organelle reorganization during spermiogenesis, including plant mitophagy. Furthermore, comparative analyses of plants and other systems can aid in elucidating how evolutionarily ancient mechanisms, such as autophagy, have been distinctly co-opted into sexual reproductive processes during the course of eukaryotic evolution.

Funding Statement

This work was supported by the JSPS [19J13751, 19H05670, 21H02515, 19H05670, 21H02515].

Disclosure statement

No potential conflict of interest was reported by the author(s).