Cytological observations and DNA microfluorometry of the hermaphrodite freshwater triploid clam Corbicula leana revealed unusual androgenetic development as follows: (1) the maternal genome of zygotes was extruded as two polar bodies just after karyokinesis of the first meiosis, (2) only chromosomes derived from one male pronucleus constituted the metaphase of the first cleavage of zygotes, (3) DNA content of 7-day-old veliger larvae was identical to the somatic cells of the parent. This spontaneous androgenetic process in C. leana zygotes is the first case in the phylum Mollusca and may be related to the specialized mode of reproduction; i.e. hermaphroditism and self-fertilization.

No abstract

In Mytilus mussels, paternal mitochondrial DNA (M type) from sperm is known to be transmitted to offspring. This phenomenon is called doubly uniparental inheritance (DUI). Under DUI, it has been reported that female mussels generally have only maternal mtDNA (F type). In this study, we examined the mode of mtDNA transmission in Mytilus galloprovincialis using M and F type-specific primer sets. The ratio of M and F types were measured in each sample by SNaPshot. The M type was detected in the adductor muscle and female gonad of all females. In unfertilized eggs spawned by 84.6% of females (22/26), M type was also detected. The F type was more abundant than the M type in all females. Although the ratio of M type in females was very low, all females contained the M type. From these results, we propose a new possibility about DUI inheritance. The presence of M type in unfertilized eggs indicates that the M type of eggs may also contribute to M type inheritance.

In Mytilidae, mitochondrial DNA (mtDNA) in the offspring is inherited from male and female parents. Sperm mitochondria are only incorporated into the testes. This phenomenon is called doubly uniparental inheritance (DUI). Sperm mitochondria should locate in the primordial germ cell during development to maintain DUI. However, the mechanism of sperm mitochondria localization is still unknown. To reveal the mechanism, we followed the location of sperm mitochondria in Mytilus galloprovincialis zygotes fertilized with sperm stained by MitoTracker. Just after fertilization, sperm mitochondria, which were found to enter eggs from various sites, remained at sperm entry point. Five sperm mitochondria located at the male pronucleus. After pronuclear expansion, sperm mitochondria migrated to the center of the egg together with the male pronucleus. At anaphase of cleavage-I, the distribution pattern of sperm mitochondria was divided into two patterns. In pattern A, sperm mitochondria located in the equatorial region of the eggs. In pattern B, sperm mitochondria migrated and divided into two groups with chromosomes. From observations of colchicine-treated eggs, we suggest that sperm mitochondria migration from fertilization to anaphase of cleavage-I depends on the microtubules. The difference between pattern A and pattern B may be caused by whether sperm mitochondria migrated or not by the microtubules at cleavage-I.

Hermaphroditic freshwater clams in the genus Corbicula produce non-reductional spermatozoa. The DNA content of spermatozoa was almost identical with that of somatic cells in C. leana from Mie Prefecture, Japan. Hermaphroditic C. aff. fluminea from Saga Prefecture and C. fluminea from Taiwan also produce non-reductional spermatozoa. On the other hand, spermatozoa of the dioecious C. sandai had half the DNA found in somatic cells. Analysis of chromosome numbers suggests that C. leana (3n = 54 in somatic cells and 18 in meiotic cells) from Mie Prefecture and C. aff. fluminea (2n = 36 in gills and 18 bivalents in meiotic cells) from Saga Prefecture are triploids and diploids, respectively. C. leana, C. aff. fluminea, and C. fluminea may lack either first or second meiosis, resulting in non-reductional spermatozoa. We assume that gynogenetic reproduction occurs in both species; maternal chromosomes are also nonreductional, and spermatozoa activate development of the eggs, but do not contribute to the offspring.

Two shell color types of the exotic bivalve Corbicula fluminea were collected in Kyoto city, Japan. DNA microfluorometry revealed that both types were diploids with non-reductional spermatozoa. Maternal chromosomes were found to be extruded as two polar bodies at the first meiosis, and the second meiosis could not be observed. Only the male pronucleus was present in the egg cytoplasm and became metaphase chromosomes at the first mitosis. The present study indicates that the diploid C. fluminea in Japan has the same mode of androgenetic reproduction as the triploid C. leana.

The developmental process of eu- and paraspermatozoa in the cottid fish, Hemilepidotus gilberti, was observed by electron microscopy. Euspermatozoa of H. gilberti consist of a thin disk-like sperm head (about 3 microm in length), a short middle piece, and a long flagellum, but lack an acrosome. On the other hand, during spermiogenesis, aberrant spermatids, rich in cytoplasm and possessing binuclei, develop into cysts containing spermatids. The developing aberrant spermatids connect with normal spermatids and euspermatozoa by intercellular bridges. The early phase of chromatin condensation in aberrant spermatids is almost identical to that in normal spermatids, but the nuclei in the later phase develop into a mass of highly electron-dense globules. Since the aberrant spermatids complete karyokinesis but not cytokinesis at telophase of the second meiotic division, they are considered to develop into hyperpyrenic cells due to incomplete cytokinesis of the second meiotic division. These spermatids are oval in shape (5-7 microm in diameter) and lack a flagellum. The aberrant spermatids of H. gilberti are shed along with euspermatozoa and amount to about 50% of semen in volume. Judging from their form and developmental process, aberrant spermatids produced in H. gilberti are considered hyperpyrenic paraspermatozoa.

To understand the unusual polar body formation in the androgenetic clam, Corbicula leana, whole-mount eggs stained with monoclonal antibodies against alpha-tubulin, gamma-tubulin, and 4'-6'-diamidino-2-phenylindole were examined. The meiotic spindle was located at the peripheral region of the egg at metaphase I, and its axis was parallel to the egg surface. After segregation of chromosomes at anaphase I, cytoplasmic bulges formed at both meiotic spindle pole sites. Centrosomes were located at the apical portion of the each bulge. From the apical portion of the bulge a bundle of astral microtubules radiated toward the bulge base in late anaphase resembling a half spindle. Maternal chromosomes and both centrosomes were all distributed in two "first polar bodies" and were eventually discarded. After the polar body formation only one male pronucleus existed in the egg cytoplasm. The present study showed that the anaphase microtubules originating from a single aster can induce the polar body formation without overlapping of microtubules from the opposing aster.