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Biology of Reproduction 67, 196-203 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Stage-Specific Expression of the Mitochondrial Germ Cell Epitope PG2 During Postnatal Differentiation of Rabbit Germ Cells1

Albert M. Ricken3,a, and Christoph Viebahn2,a

a Institut für Anatomie und Zellbiologie, Martin-Luther-Universität Halle-Wittenberg, D-06097 Halle (Saale), Germany


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Structural and biochemical differentiation of germ cell mitochondria is supposed to determine the fate and integrity of mitochondria in the early embryo. Immunofluorescent labeling of the primordial germ cell epitope 2 (PG2), which is associated with the outer mitochondrial membrane and is germ cell specific from the time of germ cell segregation during gastrulation, was used to elucidate biochemical characteristics of mitochondrial differentiation leading to a functional gamete. The PG2 epitope is found in both mitotic and meiotic male and female postnatal germ cells, but PG2 expression ceases transiently in initial stages of meiosis, i.e., in the female during early stages of follicle formation and in the male during prespermatogenesis and initial phases of spermatogenesis. Because the PG2 epitope is detectable in germ cells at the time when structurally immature mitochondria are present, we speculate that PG2 immunoreactivity closely mirrors the progress of mitochondrial differentiation during gametogenesis.

follicular development, gamete biology, meiosis, oocyte development, sperm maturation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mitochondrial integrity is critical for the outcome of early embryonic development [1]. The embryo is endowed with mitochondria by the gametes under specifically regulated conditions at the time of fertilization. Whereas paternal mitochondria are readily degraded, maternal mitochondria are maintained and propagated [2, 3]. The difference in the fates of sperm and oocyte mitochondria in early embryogenesis has been suggested to be predetermined during germ cell maturation [4–6]. The course of germ cell maturation may influence the capacity of the mitochondria to be propagated in the embryo [7–10]. In both sexes, mitochondria modify their number, distribution, and structure during germ cell maturation [11–13]. Changes in structure are considerable at the beginning of meiosis. These changes culminate in a sequential transformation from the usual cristae-type mitochondria in male germ cells and the tubular cristae-type mitochondria in female germ cells into characteristic ovoid or spherical mitochondria almost devoid of cristae. Mitochondria with sparse cristae are almost unique to germ cells and are typically found in pachytene spermatocytes, early spermatids, and follicle-enclosed oocytes. Whereas in later stages of spermatogenesis the emergence of more cristae becomes evident again, the number of cristae remains low in mitochondria of more mature oocytes and of early embryos. Thus, sperm mitochondria can be distinguished from oocyte mitochondria in the zygote by structural features, including their cristae [14]. The changes in mitochondrial structure are accompanied by the stepwise expression of mitochondrial proteins. In the adult testis, some mitochondrial matrix proteins (e.g., heat shock protein 60) are largely localized in the cristae-type mitochondria of spermatogonia, whereas other proteins such as sulphydril oxidase (SOx) are almost exclusively found in the mitochondria of pachytene and early round spermatids [11, 12].

Little is known about whether the stepwise pattern of expression of mitochondrial proteins accompanies the structural changes observed in female germ cell mitochondria and what the relationship is between the expression of particular proteins and the structural changes observed. In the present study, we investigated the expression of the primordial germ cell epitope 2 (PG2) during the stages of gametogenesis in which the changes in mitochondrial structure occur. The PG2 epitope is recognized by a monoclonal antibody [15] and occurs in primordial germ cells (PGC) of the rabbit from the time of or shortly after their segregation from somatic cells [16]. During later stages of embryo development, the PG2 remains germ cell specific and occurs in the germ cells of both sexes. At the subcellular level, the PG2 is located close to the outer mitochondrial membrane. This subcellular localization and occurrence in both sexes [15] were features of the PG2 epitope that prompted us to investigate the characteristics of PG2 during postnatal germ cell differentiation. The PG2 epitope behaves similarly with regard to meiotic progression in both male and female germ cells. Here, we discuss this expression pattern in the context of the structural changes known to occur in the mitochondria.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissue and cells were obtained from male and female New Zealand rabbits at various time points from birth through to adulthood. Appropriate protocols for the care and use of laboratory animals were followed. The animals were housed under a 14L:10D light cycle and were kept with a nursing mother or allowed free access to standard feed. Sexually mature female animals were stimulated to ovulate with an i.m. injection of a GnRH analogue using (0.2 ml Receptal containing 0.8 µg buserelin acetate; Hoechst Roussel Vet, Unterschleissheim, Germany) or were mated to obtain secondary oocytes and ova. All animals were killed with Nacoren (Merial, Hallbergmoos, Germany) administered in a lethal dose i.v. or i.p. The reproductive system was dissected free, and gonads were excised and cut in half. Oocytes were obtained by flushing the fallopian tubes from the infundibulum with PBS (pH 7.2). One half of the gonads and the oocytes were snap frozen in liquid nitrogen either directly or following dehydration through an increment gradient of sucrose (3.2%, 35%, and 78%) in PBS. Six- to 10-µm-thick sections were cut, air dried, and used immediately or stored at -20°C prior to immunofluorescence staining. The other halves of the gonads were fixed in Bouin fluid overnight, embedded in paraffin, sectioned at 6 µm, and stained with hematoxylin and eosin (HE) for histological assessment of germ cell differentiation.

Cryosections were postfixed in ice cold methanol for 10 min, immersed in ice cold acetone for 1 min, air dried, encircled with a hydrophobic pen (PAP PEN; Immunotech, Beckman Coulter, Unterschleissheim-Lohhof, Germany), then rehydrated in PBS for 5 min before indirect immunofluorescence staining. A 1:100 phosphate-buffered working dilution of mouse anti-PG2 supernatant [15] and a 1:10 working dilution of mouse anti-cytokeratin supernatant (RCK102; Progen, Heidelberg, Germany) or mouse anti-vimentin supernatant (Clone V9; Progen) were used. The monoclonal PG2 antibody is a mouse IgG raised after immunization with a cell suspension of sexually undifferentiated embryonic gonads of the rabbit, and this antibody reacts specifically with primordial germ cells in a screen on cryosections of immature rabbit gonads. Preembedment immunogold labeling of acetone-fixed cryosections confirmed the light microscopic impression of perimitochondrial immunolabeling by revealing gold particles close to germ cell mitochondria [15]. Cross-species immunoreactivity is limited to human and bovine germ cells, whereas germ cells from sheep, pig, goat, mouse, rat, golden hamster, chick, Xenopus, Medaka, and Drosophila have tested negative (unpublished results).

The secondary antibody employed was Cy3-conjugated goat anti-mouse IgG (2.5 µg/ml; Dianova, Hamburg, Germany). Primary antibodies were applied overnight at 4°C, and the flurochrome-conjugated secondary antibody was applied for 2 h at room temperature. After each incubation, the sections were washed thoroughly 3 times with PBS for 5 min. Sections were counterstained with a solution of 4',6-diamidino-2-phenylindole (DAPI, 1 µg/ml; Serva, Heidelberg, Germany), washed, and mounted in Mowiol (Hoechst, Frankfurt, Germany). Negative controls were created on adjacent sections by application of PBS diluent only, omitting the primary antibody, during the first incubation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Development of Postnatal Rabbit Testis

In the testes of newborn rabbits, the seminiferous cords contain prespermatogonia and Sertoli cells (Figs. 1–4). The germ cells are round and can be distinguished from the Sertoli cells, which are tall columnar cells generally oriented perpendicular to the tubular basal lamina. At Day 10, the germ cells proliferate and lie predominately in the center of the cords, having no contact with the basal lamina (Fig. 1). The cells attain contact with the basal lamina during the following weeks, with the majority of germ cells being in contact with the basal lamina by Week 5 (Fig. 2). The germ cells lining the basal lamina transform into transitional prespermatogonia before differentiating into spermatogonia [17]. Meiosis is initiated at 7–8 wk of age, as indicated by the appearance of young primary spermatocytes (germ cells in leptotene to zygotene) [18]. In testes of 3-mo-old animals, mature primary spermatocytes (germ cells in pachytene to diakinesis) are apparent, and the cords have developed into tubules (Fig. 4). In the tubules of 6-mo-old animals, the seminiferous epithelium has the microscopic appearance of cellular associations observed in the adult testis [18], as has been described in rodents [19].



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  		FIG. 1. FIGS. 1–4. Cross-sections of seminiferous cords and tubules of the postnatal and pubertal rabbit, respectively. x360. Testis at Day 10 p.p., showing prespermatogonia (arrow). a) Control section. The prespermatogonia are situated predominantly in the center of the seminiferous cords where they undergo division (arrowhead). b) Cryosection, immunostaining. PG2 immunoreactivity is concentrated at one pole of the cells as a crescent shape close to the nucleus. c) Cryosection, DAPI staining. FIG. 2. Testis at 5 wk p.p. a) Control section. Prespermatogonia are in intimate contact with the basal lamina (arrows). b) Cryosection, immunostaining. PG2 immunoreactivity appears diminished and more diffuse compared with Figure 1b. c) Cryosection, DAPI staining. FIG. 3. Testis at 7 wk p.p. a) Control section. Cords containing germ cells entering meiosis. b) Cryosection, immunostaining. No PG2 immunoreactivity is evident at this time. c) Cryosection, DAPI staining. FIG. 4. Testis at 12 wk p.p. a) Control section. Meiosis has been initiated. The cords are transformed into tubules. The seminiferous epithelium contains primary spermatocytes. The centrally located cells have reached the pachytene stage (arrow). b) Cryosection, immunostaining. PG2 immunoreactivity occurs in spermatocytes lying centrally. c) Cryosection, DAPI staining

PG2 Immmunoreactivity in the Testis

PG2 immunoreactivity is specific for germ cells in postnatal and adult testis, but supporting cells do not stain, as confirmed by double labeling using anti-intermediate filament antibodies in combination with the anti-PG2 antibody (data not shown) [15]. PG2 immunoreactivity is present in prespermatogonia at 10 days postpartum (p.p.). PG2 immunoreactivity is concentrated in a crescent shape eccentrically placed around the nuclei of cells lying in the center of the cords (Fig. 1b). In contrast, PG2 immunoreactivity is evenly distributed throughout the cytoplasm of cells that start to lie close to the basal lamina at 5 wk p.p. (Fig. 2b). PG2 immunoreactivity becomes progressively less detectable in the cords as the animals age, and when meiosis is initiated, PG2 immunoreactivity is entirely absent (7 wk p.p., Fig. 3b), although other epitopes, such as intermediate filament proteins, remain detectable (data not shown). PG2 immunoreactivity reemerges when the first spermatogenic wave reaches the pachytene stage at 12 wk p.p. (Fig. 4b). The germ cell population lining the basal lamina, however, continues to be devoid of PG2 immunoreactivity, and PG2 immunoreactivity is still absent in young primary spermatocytes, where there is intense nuclear hematoxylin and DAPI staining. In the adult testis, PG2 immunoreactivity occurs in mature primary spermatocytes, i.e., in germ cells in pachytene to diakinesis [18], which are located more centrally and have large nuclei (Figs. 5 and 6b). Frequently, several rows of positive cells can be observed when the first and the subsequent spermatogenic waves progress (Fig. 5b). PG2 immunoreactivity is markedly reduced in cells identified as spermatids based on their small round shape (Figs. 5 and 6b).



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  		FIG. 5. FIGS. 5 AND 6. Cross-sections of seminiferous tubules of the adult rabbit. x360. Stage 1 of the cycle of the rabbit seminiferous epithelium according to Swierstra and Foote [18]. a) Control section. Two generations of primary spermatocytes are present. A younger generation (arrow) is located near the basal lamina and comprises round cells with intensely staining nuclei. An older generation of primary spermatocytes (asterisk) is scattered between the younger generation of spermatocytes and spermatids (arrowhead). b) Cryosection, immunostaining. PG2 immunoreactivity is restricted to the older generation of spermatocytes (asterisk). c) Cryosection, DAPI staining. FIG. 6. Stage 8 of the cycle of the rabbit seminiferous epithelium, i.e., the last stage before stage 1 of the new cycle. a) Control section. The seminiferous epithelium consists mainly of type B spermatogonia, older spermatocytes, and round spermatids close to the lumen. Insert: Spermatozoa line the lumen (arrow). Normarski differential interference microscopy. b) Cryosection, immunostaining. PG2 immunoreactivity only appears in seminiferous epithelial cells close to the basal lamina. c) Cryosection, DAPI staining

Development of Postnatal Rabbit Ovaries

In ovaries of rabbits 0–10 days of age, the ovarian cortex contains germ cells in tightly packed clusters (Figs. 7 and 8). At birth, mitotic oogonia are predominant but are soon reduced in number so that the overwhelming majority of the germ cell population consists of oocytes in various stages of meiotic prophase [20]. By Day 10, a few germ cells separate from the clusters located near the medulla (Fig. 8, insert). The cells become individually surrounded by flat follicle cells, and primordial follicles start to emerge. Subsequently, the number of primordial follicles progressively increases, while the extent of the cords diminishes. By Day 21, almost all germ cells lie within individual follicles (Fig. 9). The picture barely changes in ovaries over the next few weeks. In ovary sections of 4-mo-old animals, however, growing follicles at virtually all stages of development (e.g., preantral and antral) can be seen, in addition to the small individual follicles that form the pool of primordial follicles (Fig. 10). The antral follicles are latent but capable of attaining ovulatory maturity, because oocytes can be flushed out of the fallopian tube after the animals have been exposed to the ovulatory stimulus provided by either the injection of a GnRH analogue (Receptal) or mating with a fertile male. Regardless of whether the animals were hormonally stimulated or mated, oocytes could be collected 16 h poststimulation/mating. Oocytes from stimulated does had completed the first meiotic division (Fig. 11), and those from mated does had completed both meiotic divisions (Fig. 12), as defined by the extrusion of the first and secondary polar bodies [21].



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  		FIG. 9. FIGS. 7–9. Cross-sections of the ovarian cortex of the postnatal rabbit. x360. FIGS. 7 AND 8. Ovaries on the day of birth and at Day 10 p.p., respectively. a) Control sections. The ovarian cortex of both ovaries is packed with germ cells present as clusters (arrows). The cells leave the mitotic cycles and enter meiosis. By Day 10, germ cells with their associated follicle cells are seen in the medulla (insert). The cells have progressed beyond the transitory stages of meiotic prophase and are enclosed in a ring of follicle cells. b) Cryosections, immunostaining. PG2 immunoreactivity occurs in the germ cell clusters on the day of birth and in the most advanced medullary germ cell stages at Day 10 (insert). c) Cryosections, DAPI staining. Ovaries at Day 21 p.p. a) Control section. b) Cryosection, immunostaining. In general, germ cells are enclosed by follicle cells forming the rudiments of future primordial follicles (asterisk) and are PG2 immunoreactive. c) Cryosection, DAPI staining



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  		FIG. 10. Cryosections of the ovarian cortex of an adult rabbit. The cortex contains follicles at different stages of development. PG2 immunoreactivity is present in all oocytes. x86. a) DAPI staining. b) Immunostaining.FIGS. 11 AND 12. Cryosections of sucrose-infiltrated secondary oocytes. x360. FIG. 11. Combined immunostaining and DAPI staining of an unfertilized secondary oocyte arrested in metaphase II. The first polar body (arrow) and the metaphase plate (arrowhead) can be seen. PG2 immunoreactivity is present in the oocyte and polar body. In the oocyte, PG2 immunoreactivity is concentrated in a peripheral to inner cytoplasmic focus (compare the more even peripheral distribution seen in oocytes in Figs. 9b, 10b, and 12). FIG. 12. Immunostaining (a) and DAPI-staining (b) of a fertilized oocyte where the first and second polar bodies can be seen (arrow). PG2 immunoreactivity is maintained

PG2 Immunoreactivity in Ovaries and Ovulated Oocytes

As in the testis, PG2 immunoreactivity in the ovaries is germ cell specific. PG2 immunoreactivity is present in oogonia at birth (Fig. 7b). Thereafter, germ cells seem to become depleted of the epitope; no PG2 immunoreactivity can be detected in the cords of 6- and 10-day-old female animals (Fig. 8b), whereas intermediate filament proteins remain detectable in somatic cells (data not shown). At Day 10, however, PG2 immunoreactivity is again observed in a small number of oocytes located near the medulla, which are surrounded by follicle cells (Fig. 8b, insert). During the following weeks, the number of such follicles progressively increases, and PG2 immunoreactivity is present ubiquitously in germ cells again (Fig. 9b). Thereafter, PG2 immunoreactivity appears to be retained and can be detected in all oocytes of resting, growing, and postovulatory follicles (Fig. 10). One exception is the deformed, "unhealthy"-looking oocyte of atretic follicles, which do not stain for PG2 (data not shown). At no time point do sectioned oogonia or oocytes show a focal restriction of PG2 immunoreactivity, similar to that seen in prespermatogonia lying in the center of seminiferous cords. There are subtle changes in PG2 immunoreactivity in the cytoplasm of preovulatory and ovulated oocytes (compare Figs. 9b, 10b, 11, and 12a), but these changes could not be correlated with a specific state in the oocyte maturation process.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we investigated the presence and distribution of PG2 immunoreactivity in the postnatal germ cells of the rabbit. PG2 immunoreactivity is exclusively seen in the cytoplasm of germ cells but not in surrounding somatic cells. These results extend the previous finding that PG2 immunoreactivity is germ cell specific as soon as the segregation of PGC from somatic cells begins during gastrulation [16]. Postnatally, PG2 immunoreactivity is modulated in the male and female at different ages, but these changes occur at strikingly similar functional time points taking into consideration the specific schedules of postnatal germ cell differentiation in male and female rabbits [17, 20]. Thus, in both sexes PG2 immunoreactivity is present in proliferating germ cells before they enter the first meiotic division as oogonia or early prespermatogonia [22–25]. PG2 immunoreactivity disappears when germ cell proliferation stops and meiosis commences. Thus, the absence of PG2 immunoreactivity parallels the period of mitotic quiescence during which the stem cell pool is built up: the first 8 wk p.p. in the male, during which spermatogonia are accumulating in the basal compartment of the seminiferous chords/tubules, and the first 8 days p.p. in the female, during which the clusters of oogonial clones are broken up by the somatic cells to form primordial follicles, each containing a single oogonium [20]. As soon as germ cells are subjected to meiotic stimuli, PG2 immunoreactivity recurs: in the female after an interval of only a couple of days, i.e., as the primordial follicles form in the second and third postnatal week, and in the male at 12 wk p.p. in the differentiating spermatocytes half-way up the spermatogenic epithelium.

PG2 immunoreactivity most likely persists in germ cells of both sexes until the end of meiotic progression, i.e., after the pachytene-diplotene stage. In female germ cells, PG2 immunoreactivity is clearly evident in oocytes of all stages of follicular development and in oocytes with completed first and second meiotic divisions. In male germ cells, however, the only evidence to support this hypothesis is the definite change in PG2 immunoreactivity observed in spermatids, which are beyond the meiotic division. A reliable analysis of the PG2 status in the preceding stages of spermatogenesis, in particular the short-lived secondary spermatocytes, is difficult because of the limited preservation of tissue architecture in frozen sections. This problem is compounded by the difficulty involved in immunolabeling paraffin-embedded sections, which better preserve the architecture of the male germinal epithelium (unpublished data). The fact that PG2 immunoreactivity differs between female and male germ cells during gametogenesis may be accounted for by the different meiotic schedules of these germ cells. Although meiosis is a continuous process in the male, it is halted several times in the female and is completed only after penetration of sperm during fertilization.

The close association of the PG2 epitope with mitochondria [15] and with mitotic and meiotic cell division, as shown in the present study, raises the possibility of a role for the PG2 epitope in the generation of mitochondria or in mitochondrial redistribution. The generation of mitochondria does not, however, seem to be synchronized with cell division, at least as far as mitochondrial DNA (mtDNA) synthesis is concerned, because the mtDNA molecules may replicate several times or not at all during a given cell cycle [26]. Involvement in distribution of mitochondria seems unlikely because of the behavior of PG2 immunoreactivity in the oocytes of primordial follicles. PG2 immunoreactivity is consistently present in the oocytes when the primordial follicles form and when the pool of resting follicles is established in the adult ovary. Therefore, PG2 immunoreactivity most likely is associated with a constitutive mitochondrial characteristic, rather than a dynamic process (such as cytoskeletal reorganization) on which mitochondrial redistribution depends [27]. The changes from focal to dispersed or vice versa in the cytoplasmic location of PG2 immunoreactivity in both prespermatogonia and oocytes may be regarded as the result of passive rather than active involvement of the epitope in the redistribution process.

The functional significance of the focal perinuclear accumulation of PG2 immunoreactivity in the centrally located prespermatogonia is unclear. This localization of a mitochondria-associated protein may be indicative of cell degeneration or increased metabolic requirements of the nucleus [23, 28].

The PG2 epitope could also exert an effect on mitochondrial structure, which changes in premeiotic and meiotic germ cells [11–13]. Although there are differences among species and between the two sexes, mitochondria transform in general from a type with small vesicular cristae in PGC, via a usual cristae type or tubular-cristae type at the onset of meiosis, to an ovoid or spherical type with almost no cristae at the pachytene-diplotene stage. Whereas mitochondria with almost no cristae persist in oocytes thereafter, mitochondria revert to a type with more cristae later on in spermatogenesis, during spermatocytogenesis. These well-known changes in mitochondrial structure are correlated with the stage-specific occurrence of PG2 immunoreactivity in mitotically and meiotically dividing germ cells as outlined here; PG2 immunoreactivity is present when cristae are sparse, when the mitochondria are structurally immature.

In the absence of functional studies involving the PG2 epitope, the stage-specific expression of the PG2 epitope may indicate temporal correlations that may help to answer questions as to what the function of the PG2 epitope is and what factors influence its expression. At the time of the transient downregulation, meiotically dividing germ cells start to become sealed off in both sexes by Sertoli or follicular cells, respectively [20, 29]. Through this displacement, access and exposure of the germ cells to the factors derived from supporting cells and interstitial tissue are changed, and germ cells switch their energy metabolism to pyruvate and lactate rather than glucose [30–32]. These changes may also bring about the changes in mitochondrial structure (and the observed changes in PG2 immunoreactivity), a well-known features of malignant cells, in which mitochondria with sparse cristae are also seen most likely as a result of the predominance of anaerobic metabolism [33].

The results of this study also suggest that the previously described temporal pattern of expression of mitochondrial proteins in the male may also occur in the mitochondria of female germ cells. Among the proteins subject to modulation are components of the respiratory chain and molecules involved in protein import, folding, and breakdown [11, 12]. The PG2 epitope may be key to the process by which the embryo recognizes sperm mitochondria and prevents the inheritance of paternal mtDNA [4]. However, the PG2 epitope may have little to do with the specialized structure of germ cell mitochondria; although it is found close to the mitochondria, it may not be an integral part of mitochondria. Thus, further studies analyzing the biochemical nature of the PG2 epitope may or may not elucidate functional factors of mitochondrial differentiation, which in turn may be involved in germ cell maturation, early embryonic development, and experimental mechanisms such as nuclear reprogramming in germ cells [34].


   ACKNOWLEDGMENTS
 
The authors thank Elke Bernhard for her outstanding technical assistance, Rosemarie Rappold for her practical support, Dr. Gerd Hause (Biozentrum, Universität Halle-Wittenberg) for his patient help in preparing and sectioning the postovulatory oocytes, Dr. Uta Demus for her stimulating discussions and revision of the manuscript, and Dr. P. Lochhead for editing the English text.


   FOOTNOTES
 
First decision: 26 November 2001.

1 This work was supported by the Deutsche Forschungsgemeinschaft (grant Vi 151/6-1) and by the Deutsche Akademische Austauschdienst (grant D/9910412). Back

2 Correspondence: Christoph Viebahn, Institut für Anatomie und Zellbiologie, Martin-Luther-Universität Halle-Wittenberg, Grosse Steinstr. 52, D-06907 Halle (Saale), Germany. FAX: 49 345 551 1700;christoph.viebahn{at}medizin.uni-halle.de Back

3 Current address: Abteilung für Anatomie und Embryologie, Gebäude MA5/162, Ruhr-Universität Bochum, Universitätsstr. 150, D-44780 Bochum, Germany Back

Accepted: February 1, 2002.

Received: October 30, 2001.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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