Early oogenesis in the bat Carollia perspicillata: Transient germ cell cysts and noncanonical intercellular bridges

Abstract

The ovaries of early embryos (40 days after fertilization) of the bat Carollia perspicillata contain numerous germ-line cysts, which are composed of 10 to 12 sister germ cells (cystocytes). The variability in the number of cystocytes within the cyst and between the cysts (that defies the Giardina rule) indicates that the mitotic divisions of the cystoblast are asynchronous in this bat species. The serial section analysis showed that the cystocytes are interconnected via intercellular bridges that are atypical, strongly elongated, short-lived, and rich in microtubule bundles and microfilaments. During the later stages of embryonic development (44–46 days after fertilization), the somatic cells penetrate the cyst, and their cytoplasmic projections separate individual oocytes. Separated oocytes surrounded by the single layer of somatic cells constitute the primordial ovarian follicles. The oocytes of C. perspicillata are similar to mouse oocytes and are asymmetric. In both species, this asymmetry is clearly recognizable in the localization of the Golgi complexes. The presence of germ-line cysts and intercellular bridges (although non-canonical) in the fetal ovaries of C. perspicillata indicate that the formation of germ-line cysts is an evolutionarily conserved phase in the development of the female gametes throughout the animal kingdom.

Introduction

In the 19th century, Brandt (1874) described two types of ovaries: panoistic and meroistic. In the panoistic ovary, all of the oogonia (with the exception of the ones that degenerate) become the oocytes. In the meroistic ovary, some of the oogonia become the oocytes, whereas the others form the nurse cells (trophocytes). The panoistic ovary is characteristic for vertebrates, majority of arachnids, crustaceans, pantopods (Miyazaki and Bilinski, 2006) and basal groups of insects (Buning, 1994, 1998; Bilinski, 1998). In addition, some highly specialized insect taxa (e.g., fleas, thrips and certain megalopterans) are characterized by secondary panoistic (neopanoistic) ovaries, which, according to current hypotheses, evolved from the meroistic ovaries through the secondary loss of nurse cells (see Stys and Bilinski, 1990; Buning, 1994; Bilinski, 1998, for a review).

In the majority of animals, oogenesis begins with the synchronous mitotic divisions of a specific oogonial cell, termed the cystoblast. The divisions of the cystoblast lead to the formation of the cyst of sister cells (cystocytes), which are interconnected by intercellular bridges. The bridges form as a result of incomplete cytokinesis and are reinforced by characteristic electron-dense rims (for further details see Buning, 1985). The final number of the cystocytes in a cyst depends on the number of mitotic divisions of the cystoblast and is governed by the Giardina N = 2ⁿ rule, where “N” represents the final cystocyte number, and “n” represents the number of consecutive divisions of the cystoblast (Giardina, 1901).

In meroistic ovaries, the oocytes are formed by only one or a few cystocytes, whereas the rest of the cystocytes differentiate into trophocytes. The function of the trophocyte is to provide nutrients and organelles for the developing egg cell and the subsequent embryo. In contrast, all of the cystocytes differentiate into functional oocytes in the panoistic ovaries of vertebrates (fish, amphibians, mammals; Dumont, 1972; Pepling and Spradling, 1998; Kloc et al., 2004) and invertebrates (basic groups of insects and pantopods; Stys and Bilinski, 1990; Buning, 1994; Bilinski, 1998; Miyazaki and Bilinski, 2006), as well as in secondary panoistic ovaries (Pritsch and Buning, 1989; Gottanka and Buning, 1990; Stys and Bilinski, 1990).

Among vertebrates, the most detailed descriptions of the formation of the germ-line cyst and of the differentiation of the oocyte exist for Xenopus laevis (Kloc et al., 2004) and mouse (Pepling and Spradling, 1998). In Xenopus, four synchronous divisions of the cystoblast result in the 16-cell germ-line cyst. Each cyst is eventually partitioned into 16 identical oocytes (Kloc et al., 2004). In Xenopus and in mice, the cytoplasmic projections of somatic cells of the gonad penetrate between the cystocytes, which causes the closure (breakage) of the intercellular bridges and the separation of the individual oocytes. As a result, the individual oocytes are surrounded by a single layer of somatic cells, and these structures constitute the primordial ovarian follicles.

Although in mammals, the germ-line cysts and their partition into individual oocytes have been described only in mouse (Pepling and Spradling, 1998; Pepling, 2006; Pepling et al., 2007), the presence of such cysts in embryonic ovaries of other mammalian species can be easily deduced from the presence of intercellular bridges between young germ-line cells. At the electron microscopy level, these bridges have been described in fetal ovaries of mouse, hamster, rat, rabbit and human (Franchi and Mandl, 1962; Weakly, 1967; Zamboni and Gondos, 1968; Ruby et al., 1969; Gondos, 1973a, b; Spiegelman and Bennett, 1973). Here we show that in embryonic ovaries of the bat Carollia perspicillata, the germ-line cysts are present, although the intercellular bridges connecting the cyst cells are noncanonical and quickly degenerate. This leads to the separation of oocytes that associate with somatic cells and constitute primordial ovarian follicles. Above observations support the idea that the formation of germ-line cysts is the evolutionarily conserved phase in the development of the female gamete throughout the animal kingdom (see Cooley, 1995; Pepling et al., 1999; Matova and Cooley, 2001 for a review).

Materials and methods

Animals

The Carollia perspicillata colony was located in the Weill Medical College of Cornell University. All experiments were performed according to the Principles of Laboratory Animal Care and NIH standards as set forth in the “Guide for the Care and Use of Laboratory Animals” (DHHS publication No. (NIH) 85-23 Revised 1985), the PHS “Policy on Humane Care and Use of Laboratory Animals” and the NIH “Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research and Training. The female embryos (40, 44 and 46 days after the fertilization) of Carollia perspicillata (Chiroptera, Phyllostomidae) were used in the current study.

Fixation and processing

Isolated ovaries or whole embryos (in the earliest stages of development; after the removal of the gut and associated organs) were fixed in 2% formaldehyde and 3% glutaraldehyde (Ted Pella Inc., Redding, CA, USA) in PBS (pH 7.4) for 2 hr at room temperature. The fixed material was contrasted in 0.5% of uranyl acetate and postfixed in 1% osmium tetroxide (OsO₄) in PBS. After rinsing with water followed by dehydration using a series of ethanol and acetone, the samples were embedded in LX-112 (Ladd Research Industries, Burlington, VT, USA).

Histology and electron microscopy

Embedded samples were sectioned using the Tesla BS 490A (Brno, The Czech Republic) ultramicrotome. Serial or single semi-thin (0.7–1.0 μm) sections of ovaries or whole embryos were stained with a solution of 1% methylene blue in 1% borax and analyzed using Leica DMR (Heidelberg, Germany) or Nikon Eclipse E200 (Tokyo, Japan) light microscopes. The sections were used to locate the ovaries within the embedded samples and to select the fragments of embedded material for analysis by electron microscopy. Ultrathin (70–100 nm) sections were obtained using the Ultracut E Reichert Jung (Viena, Austria) ultramicrotome. Sections were contrasted with uranyl acetate and lead citrate (Reynolds, 1963) and analyzed using the transmission electron microscope Jeol JEM 100SX (Tokyo, Japan) at 80 kV.

Results

1. 40 days post-fertilization

The youngest embryos that were analyzed were at 40 days post-fertilization. At this stage, the paired, bean-shaped ovaries were located in the dorsal part of the trunk on the sides of the developing spine. The ovaries contained more or less spherical germ-line cysts that were surrounded by the somatic tissue of the ovary (Fig. 1A, B). The analysis of the serial sections showed that each cyst consisted of 10 to 12 morphologically identical, slightly elongated pear-shaped germ-line cells (Fig. 1A, B, and E). The basal region of these cells contained large, roughly spherical nucleus (Fig. 1F). In the apical cytoplasm, mitochondria, Golgi complexes, ribosomes, polyribosomes, elements of the rough endoplasmic reticulum (RER) and centrioles were present (Fig. 1G). The detailed analysis of serial and semi-serial ultrathin sections showed that germ cells of the cyst were interconnected by elongated and sometimes branched cytoplasmic processes (Figs. 1G and 2A). These processes originated from the apical portion of the cell (i.e., directed towards the cyst center) (Fig. 1G) and contained mitochondria, parallel-oriented microtubules and microfilaments (Figs. 1G and 2A). The electron-dense rims, which are characteristic of the intercellular bridges that connect germ-line cells in various vertebrates and invertebrates, were never found inside cytoplasmic processes.

Fig. 1.

Germ-line cysts and primordial follicles. A. 40 dpc. A germ-line cyst surrounded by the somatic tissue of the ovary (asterisks). Semithin section, methylene blue. B. 40 dpc. A germ-line cyst; its central part (asterisk) is filled with cytoplasmic processes. Semithin section, methylene blue. C, D. 46 dpc. Primordial follicles, oocytes and the ER aggregate (encircled). Semithin sections, methylene blue. E, F, G. 40 dpc. Electron micrographs of germ-line cysts. E. The fragment of a germ-line cyst. Note four cystocytes arranged around a central region (asterisk) filled with cytoplasmic processes. F. The basal cytoplasm and cystocytes’ nuclei (cn). G. The apical cytoplasm of neighboring cystocytes and the bases of their cytoplasmic processes (asterisk). Apical cytoplasm (ac); cystocyte nucleus (cn); nucleolus (arrow), oocyte nucleus (on); oocyte (oo). Scale bars: 10 μm in A – D, 1 μm in E – G.

Fig. 2.

Electron micrographs of germ-line cysts and the surrounding somatic cells. A. 40 dpc. Cytoplasmic processes in the center of germ-line cyst. B. 40 dpc. Basal parts of the cystocytes. C. 40 dpc. Somatic cells near a cyst, note mitotically dividing cell (scm). Adherens junctions (arrowheads); apical cytoplasm (ac); basal cytoplasm (bc), cystocyte nucleus (cn); mitochondrion (m); microtubules (arrows); somatic cell nucleus (scn); projection of a somatic cell (scp). Scale bars: 1 μm in A – C.

Spaces between germ-line cysts were occupied by numerous small somatic cells (Figs. 1F and 2B, C). Among them, mitotically dividing cells were frequently encountered (Fig. 2C). Initially, the distances between somatic cells and germ-line cysts were relatively large (Fig. 1F). During the later stages of development, these distances decreased (Fig. 2B). Simultaneously, the somatic cells formed very thin and elongated projections that contacted the plasma membrane of germ cells. In the places of contact between plasma membranes of germ and somatic cells, the intercellular junctions of the zonula adherens type, were often observed (Fig. 2B).

2. 44 and 46 days post-fertilization

At these stages, the ovaries were elongated and measured approximately 2 mm in length. The somatic cells of the gonad were equipped with thin, long cytoplasmic projections (Figs. 2C and 3A, B). The projections penetrated between the plasma membranes of the germ cells (Fig. 3C), which resulted in the formation of individual primordial ovarian follicles (Figs. 1C, D and 4A).

Fig. 3.

Electron micrographs of primordial follicles. A, 40 dpc. Golgi complexes (blue encircled) and secretory vesicles (sv) are located next to the oocyte nucleus (red encircled). B. 40 dpc. Secretory vesicles and Golgi complex at a higher magnification. C. 44 dpc. Two neighboring oocytes (oo) that are separated by projections of somatic cells (scp). Adherens junctions (arrowheads); Golgi complex (G); mitochondrion (m); oocyte nucleus (on); oocyte (oo); somatic cell nucleus (scn). Scale bars: 1 μm in A – C.

Fig. 4.

Electron micrographs of primordial follicles. A – B. 46 dpc. Aggregates of endoplasmic reticulum cisternae (asterisks) in the ooplasm. A. Initial phase of formation. B. Fully formed aggregation; note lens-shaped accumulations of electron-transparent material (black arrowheads). C. 46 dpc. Elements of the smooth endoplasmic reticulum, which are surrounded by the cisternae of the rough endoplasmic reticulum (encircled) near the oocyte nucleus (on); fine granular material (intramitochondrial cement, ic) is present between closely opposed mitochondria (m). D. 46 dpc. Fragment of a somatic cell that surrounds a young oocyte; a centriole (c) and the base of a cilium (encircled) are observed. Adherens junction (white arrowhead); nucleolus (nu); oocyte nucleus (on); oocyte (oo); somatic cell (sc); projection of a somatic cell (scp) Scale bars: 1 μm in A – C.

Each primordial ovarian follicle consisted of the pachytene or post-pachytene oocyte, which was surrounded by a layer of flattened somatic cells (Fig. 1C, D). The oocyte nucleus (germinal vesicle) contained spherical reticulate nucleolus and electron-dense bodies (Figs. 1C, D and 4A). In the oocyte cytoplasm (ooplasm), ribosomes, mitochondria, elements of RER and Golgi complexes were observed (Fig. 3A, B and C). The latter organelles were preferentially located in the cortical ooplasm near the oocyte plasma membrane (Fig. 3C). The Golgi complexes were accompanied by polymorphic secretory vesicles that were filled with electron-dense granular material (Fig. 3A, B). The analysis of serial ultrathin sections showed that Golgi complexes and secretory vesicles were always concentrated in a relatively broad zone of the ooplasm, which occupied approximately one sixth of the oocyte volume (Fig. 3A). Mitochondria were spherical or cylindrical and were often interspaced by accumulations of nuage material (i.e., intramitochondrial cement; Fig. 4C). Peculiar aggregates of smooth and rough ER were present near the nuclear envelope. Within these aggregates, the smooth ER elements were surrounded by radially arranged RER cisternae (Fig. 3C, encircled). Numerous zonula adherens were observed in the contact zones between the plasma membranes of somatic and germ-line cells (Figs. 3C and 4A) and between the neighboring somatic cell projections (Fig. 3C). Our analyses also demonstrated that at least some somatic cells surrounding the primordial ovarian follicles were equipped with short cilia (Fig. 4D).

Aside from the typical organelles, each pachytene/postpachytene oocyte contained a single aggregate, which was composed of several elongated ER cisternae (Figs. 1D and 4A, B). In younger oocytes, these aggregates were relatively small and composed of loosely aggregated cisternae (Fig. 4A). In older oocytes, the ER cisternae were tightly packed, sometimes dilated and contained large lens-shaped accumulations of electron-transparent material (Fig. 4B, arrowheads).

Discussion

Although functionally identical, animal oocytes are extremely diverse between species in their morphology and ultrastructure. In mammals, the information on the structure and formation of the oocyte is limited to humans and model (mouse, rat) and economically relevant species (cow, horse, sheep, goat). Thus, the comparison between different mammalian species is important for our understanding of the general principles ruling the structure and formation of the oocyte. In this regard, bats offer a unique system to study the mechanisms that diversify oogenesis.

1. The formation of the primordial ovarian follicles during the embryogenesis of C. perspicillata

The formation of primordial follicles in C. perspicillata is identical to the general pattern of female gamete formation in mammals (mouse, hamster, rat, pig and cow). However, we have identified some strikingly novel peculiarities. The germ-line cysts in the embryonic ovaries of Carollia perspicillata contain 10 to12 sister cystocytes. The fact that the number of the cystocytes is not constant and varies between different cysts (i.e., does not follow the Giardina rule) suggests that the mitotic divisions of the cystoblast are asynchronous in this species. The cyst cells (cystocytes) are interconnected by slender and elongated processes. The morphology and the localization of these processes indicate that they represent atypical, intercellular bridges. These bridges are clearly morphologically different from the canonical bridges present between germ-line cells of other animals in the following ways: (1) they are not lined with electron dense rims, and (2) they are not stable and quickly degenerate. Therefore, the germ-line cysts of C. perspicillata are transient or even ephemeral compared to the cysts of other vertebrate and invertebrate species. These cysts become partitioned into individual germ cells as early as 40 days after fertilization. Consequently, the primordial follicles of C. perspicillata arise notably earlier than those in the ovaries of mice (Pepling and Spradling, 1998; Kloc et al., 2008).

Although atypical in their structure, the intercellular bridges of C. perspicillata contain cytoskeletal components such as microfilaments and bundles of microtubules, which are frequent in the bridges of other species. We hypothesize that these cytoskeletal components are the remnants of the midbody. The midbody is a transient structure found between dividing mammalian cells (Flemming, 1891). The midbody is present during cytokinesis and immediately precedes the separation of sister cells. The midbody is composed of microtubules, which originate from the mitotic spindle and disperse at the end of the cytokinesis (Mullins and McIntosh, 1982). The midbody contains the proteins necessary for chromosome segregation and cytokinesis, although the exact function of the midbody remains unknown (Skop et al., 2004).

The presence of intercellular bridges and germ-line cysts (although atypical and short-lived) in the fetal ovaries of C. perspicillata supports the idea that the formation of germ-line cysts is the evolutionarily conserved phase in the development of the female gamete throughout the animal kingdom.

2. Oocyte organelles

The analysis of ultrathin serial sections from the oldest oocytes (46 days post fertilization) showed that all Golgi complexes were located in a relatively broad zone of the ooplasm. This observation clearly indicates that developing bat oocytes are asymmetric, which is similar to mouse oocytes. In both species the asymmetry is related to the localization of the Golgi complexes. In mouse oocytes, the Golgi complexes are organized around a pair of centrioles (the centrosome) and constitute a specific structure called the Balbiani body (Pepling et al., 2007; Kloc et al., 2008). The 3D reconstruction of the distribution of organelles in mouse oocytes showed that the Balbiani body was always located near the intercellular bridge connecting sister oocytes.

The oocytes of C. perspicillata do not have a typical Balbiani body. The Golgi complexes in this species are rather loosely arranged and are not organized around the centrosome. Because the intercellular bridges of C. perspicillata are transient and are only found between the youngest germ-line cells, the localization of Golgi complexes in relation to the bridges remains elusive.

In the oocytes of C. perspicillata, the zone of the ooplasm that contains the Golgi complex is also enriched in polymorphic secretory vesicles that contain granular material. We suggest that the secretory vesicles contain the macromolecules necessary for the formation of the zona pellucida similar to the mouse oocytes (Kloc et al., 2008).

In the oldest stage that was analyzed, each oocyte contained a single large aggregate of the ER cisternae. These aggregates contain lens-shaped accumulations of electron-transparent material. The fertilized egg cells and the blastomeres of the bat species Macrotus californicus were shown to contain large paracrystalline inclusions of unknown origin, composition and function (Bleier, 1975). The morphological criteria, the shape and size of ER aggregates (in the oocytes of C. perspicillata) and the paracrystalline inclusions (in early embryos of M. californicus) suggest that these two structures are related. We hypothesize that the accumulations of electron-transparent material that were observed inside the ER aggregate of C. perspicillata represents the early phase of the formation of paracrystalline inclusions.

Morphological differences between mouse and C. perspicillata germ-line cysts, cystocytes and primordial ovarian follicles are summarized in Table 1.

Table 1.

Comparison of mouse and Carollia perspicillata germ-line cysts, cystocytes and primordial follicles.

	mouse	Carollia perspicillata
Intercellular bridges connecting cystocytes	relatively short, stable and lined with electron dense rims.	strongly elongated, ephemeral, devoid of electron dense rims; contain parallel–oriented microtubules (presumably remnants of a mitotic spindle) and microfilaments.
Balbiani body in cystocytes and early oocytes	present; consists of several Golgi complexes and secretory vacuoles, always organized around a pair of centrioles.	absent or “dispersed”; Golgi complexes and secretory vacuoles aggregated at one pole of a cystocyte/oocyte near their plasma membranes
ER aggregate	absent	present; consists of numerous ER cisternae.